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In the Name of Allah, the Most Beneficent

And The Most Merciful!

Oh, Allah Almighty open our eyes,To see what is beautiful,

Our minds to know what is true,Our heart to love what is Allah.

I

Alleviation of Terminal Heat Stress in Wheat (Triticum aestivum L.)

Through Potassium and Selenium Nutrition

BY

MUHAMMAD SHAHIDM.Sc. (Hons.) Agriculture

2008-ag-2268

By

A thesis submitted in partial fulfillment of the requirement for the

degree

of

DOCTOR OF PHILOSOPHY

in

AGRONOMY

DEPARTMENT OF AGRONOMY,FACULTY OF AGRICULTURE,

UNIVERSITY OF AGRICULTURE, FAISALABAD-

PAKISTAN

2018

II

The Sublime

LoveOf

My Beloved and Kind ParentsWho taught me,

The first step to take,The first word to speak,

VII

Dedicated To

The first alphabet to write,Inspired me to higher ideas of life,

Whose hands always raise in prayer for me,

Who are with me to feel the bud of their wishes and prayers blooming into a flower

andUnder whose feet my heaven lies.

AND My Beloved BROTHER and

SISTERSAcknowledgements

Bounteous praise for ALMIGHTY ALLAH, the magnificent, the merciful, the propitious, the supreme, the omnipotent, the omnipresent, the omniscient and sovereign whose blessing and glories flourish my cogitation and all the eulogies for the HOLY PROPHET MUHAMMAD (SAW) for edifying our conscience of faith in ALLAH, converging all his kindness and mercy upon him.

I feel much honor to express my deepest sense of gratitude, philanthropy and magnanimity to my honorable supervisor, Dr. Muhammad Farrukh Saleem, Associate Professor, Department of Agronomy, University of Agriculture Faisalabad from the core of my heart for his dynamic supervision, marvelous guidance, keen interest and encouraging behavior. With humble, profound and deepest sense of devotion I wish to record my sincere appreciation to Dr. Shakeel Ahmad Anjum, Assistant Professor, Department of Agronomy, University of Agriculture Faisalabad and Dr. Irfan Afzal, Associate Professor, Department of Agronomy, University of Agriculture, Faisalabad for their sincere help, dynamic supervision and inspiring guidance throughout the course of this research work. I am genially thankful to Dr. Muhamad Shahid, Associate Professor, Department of Biochemistry, University of Agriculture Faisalabad for abetting in the conduct of biochemical analysis during the whole research work.

I feel inordinate appreciativeness for Higher Education Commission of Pakistan for economic backing to conduct this research work. I cordially applaud the

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facilities provided by Analytical Laboratory, Department of Agronomy, University of Agriculture Faisalabad and Medicinal Plants Biochemistry Laboratory, Department of Biochemistry, University of Agriculture Faisalabad for assistance in conduct of biochemical analysis.

I want to express my great appreciation and sincerest gratitude to my friends and juniors; Abdul Shakoor, Ubaid-Ur-Rehman and Siraj Ahmed for their dexterous, dynamic, untiring help, friendly behavior and moral support during my whole study.

Round out the picture, no acknowledgement could ever adequately express my obligation to my affectionate Parents whose endless efforts and best wishes sustained me at all stages of my life and encouraged me for achieving higher ideas of life. Just as importantly, I want to express my everlasting love for my loving brother Muhammad Faisal and caring Sisters they offered irreplaceable endorsement and my Nephews and Nieces whom countenances have bestowed me the blisses of life.

May ALLAH bless all these people with long, happy and peaceful lives (Aameen)!

Muhammad Shahid

IX

LIST OF TABLESTable Title Page

3.1 Physio-chemical analysis of experimental site during 2014-15, 2015-16 and 2016-17 15

3.2 Monthly averages of weather elements during growing season of crop in 2014-15, 2015-16 and 2016-17 16

3.3 Varying mean temperatures (°C) 2014-15 for experiment 1 17

3.4 Varying mean temperatures (°C) during heat imposition for experiment 2, 2015-16 and 2016-17 17

3.5 Varying mean temperatures (°C) during heat imposition for experiment 3, 2015-16 and 2016-17 18

4.1.1 Effect of heat stress on fertile tillers of wheat varieties 31

4.1.2 Effect of heat stress on grains per spike and 1000-grain weight of wheat varieties 32

4.1.3 Effect of heat stress on grain yield of wheat varieties 34

4.1.4 Effect of heat stress on grain filling rate (GFR) and grain filling duration (GFD) of wheat varieties 38

4.1.5 Effect of heat stress on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of wheat varieties 41

4.1.6 Effect of heat stress on superoxide dismutase (SOD) and peroxidase (POD) of wheat varieties 42

4.1.7 Effect of heat stress on catalase (CAT) and total phenolic contents (TPC) of wheat varieties 43

4.1.8 Effect of heat stress on leaf proline and glycine betaine of wheat varieties 47

4.1.9 Effect of heat stress on total soluble proteins of wheat varieties 484.1.10 Effect of heat stress on malondialdehyde of wheat varieties 50

4.1.11 Correlation analyses showing strength of association among recorded attributes of different wheat varieties under no heat stress (H0)

52

4.1.12Correlation analyses showing strength of association among recorded attributes of different wheat varieties under heat from spike to grain filling (H1)

53

4.2.1 Effect of foliar applied potassium on fertile tillers and grains per spike of heat stressed wheat 57

4.2.2 Effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat 59

4.2.3 Effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat 64

4.2.4 Effect of foliar applied potassium on straw yield and plant height of heat stressed wheat 66

4.2.5 Effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat 71

4.2.6 Effect of foliar applied potassium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat 73

4.2.7 Effect of foliar applied potassium on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of heat stressed wheat 79

Table Title Page

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4.2.8 Effect of foliar applied potassium on superoxide dismutase (SOD) and peroxidase (POD) of heat stressed wheat 81

4.2.9 Effect of foliar applied potassium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat 83

4.2.10 Effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat 89

4.2.11 Effect of foliar applied potassium on total soluble proteins (TSP) and malondialdehyde (MDA) of heat stressed wheat 91

4.2.12 Effect of foliar applied potassium on osmotic (ΨS) and water potential (ΨW) of heat stressed wheat 96

4.2.13 Effect of foliar applied potassium on turgor potential (ΨP) and shoot potassium (K) contents of heat stressed wheat 98

4.2.14 Effect of foliar applied potassium on grain crude proteins of heat stressed wheat 100

4.2.15 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2015-16

106

4.2.15 (b)


107

4.2.15 (c)


108

4.2.15 (d)


109

4.2.16 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2015-16

110

4.2.16 (b)


111

4.2.16 (c)


112

4.2.16 (d)


113

4.2.17 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2015-16

114

4.2.17 (b)


115

4.2.17 (c)


116

Table Title Page

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4.2.17 (d)


117

4.3.1 Effect of foliar applied selenium on fertile tillers and grains per spike of heat stressed wheat 120

4.3.2 Effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat 122

4.3.3 Effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat 128

4.3.4 Effect of foliar applied selenium on straw yield and plant height of heat stressed wheat 130

4.3.5 Effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat 135

4.3.6 Effect of foliar applied selenium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat 137

4.3.7 Effect of foliar applied selenium on chlorophyll a (Chl a) and on chlorophyll b (Chl b) contents of heat stressed wheat 143

4.3.8 Effect of foliar applied selenium on superoxide dismutase (SOD) and peroxidase (POD) contents of heat stressed wheat 145

4.3.9 Effect of foliar applied selenium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat 147

4.3.10 Effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat 154

4.3.11 Effect of foliar applied selenium on total soluble proteins (TSP) and malondialdehyde (MDA) contents of heat stressed wheat 156

4.3.12 Effect of foliar applied selenium on osmotic (ΨS) and water potential (ΨW) of heat stressed wheat 162

4.3.13 Effect of foliar applied selenium on turgor potential (ΨP) and grain crude protein contents of heat stressed wheat 164

4.3.14 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2015-16

168

4.3.14 (b)


169

4.3.14 (c)


170

4.3.14 (d)


171

4.3.15 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2015-16

172

4.3.15 (b)


173

Table Title Page4.3.15 Correlation analyses showing strength of association among 174

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(c) recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2016-17

4.3.15 (d)


175

4.3.16 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2015-16

176

4.3.16 (b)


177

4.3.16 (c)


178

4.3.16 (d)


179

LIST OF FIGURES

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Figure Title Page

4.2.1 Regression analysis for effect of foliar applied potassium on grains per spike of heat stressed wheat 58

4.2.2 Regression analysis for effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat 60

4.2.3 Regression analysis for effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat 65

4.2.4 Regression analysis for effect of foliar applied potassium on straw yield and plant height of heat stressed wheat 67

4.2.5 Regression analysis for effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat 72

4.2.6 Regression analysis for effect of foliar applied potassium on grain filling rate and grain filling duration of heat stressed wheat 74

4.2.7 Regression analysis for effect of foliar applied potassium on chlorophyll a and chlorophyll b contents of heat stressed wheat 80

4.2.8 Regression analysis for effect of foliar applied potassium on superoxide dismutase and peroxidase contents of heat stressed wheat 82

4.2.9 Regression analysis for effect of foliar applied potassium on catalase and total phenolic contents of heat stressed wheat 84

4.2.10 Regression analysis for effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat 90

4.2.11 Regression analysis for effect of foliar applied potassium on total soluble proteins and malondialdehyde of heat stressed wheat 92

4.2.12 Regression analysis for effect of foliar applied potassium on osmotic and water potential of heat stressed wheat 97

4.2.13 Regression analysis for effect of foliar applied potassium on turgor potential and shoot potassium contents of heat stressed wheat 99

4.2.14 Regression analysis for effect of foliar applied potassium on grain crude proteins of heat stressed wheat 101

4.3.1 Regression analysis for effect of foliar applied selenium on grains per spike of heat stressed wheat 121

4.3.2 Regression analysis for effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat 123

4.3.3 Regression analysis for effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat 129

4.3.4 Regression analysis for effect of foliar applied selenium on straw yield and plant height of heat stressed wheat 131

4.3.5 Regression analysis for effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat 136

4.3.6 Regression analysis for effect of foliar applied selenium on grain filling rate and grain filling duration of heat stressed wheat 138

4.3.7 Regression analysis for effect of foliar applied selenium on chlorophyll a and on chlorophyll b contents of heat stressed wheat 144

4.3.8 Regression analysis for effect of foliar applied selenium on superoxide dismutase and peroxidase contents of heat stressed wheat 146

4.3.9 Regression analysis for effect of foliar applied selenium on catalase and total phenolic contents of heat stressed wheat 148

Figure Title Page

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4.3.10 Regression analysis for effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat 155

4.3.11Regression analysis for effect of foliar applied selenium on total soluble proteins and malondialdehyde contents of heat stressed wheat

157

4.3.12 Regression analysis for effect of foliar applied selenium on osmotic and water potential of heat stressed wheat 163

4.3.13 Regression analysis for effect of foliar applied selenium on turgor potential and grain crude protein contents of heat stressed wheat 165

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ABSTRACTCoincidence of high temperature at the terminal phenological stages of the wheat crop is a prime constraint to reach full yield potential in Pakistan. The present research work was conducted to determine the thermo-sensitivity of Pakistani wheat genotypes and alleviation of negative implications of heat through exogenous application of potassium and selenium. All research work was performed at the Agronomic Research Area, University of Agriculture Faisalabad, Pakistan from November 2014 to May 2017. In the first year, wheat genotypes were screened for terminal heat tolerance under field conditions. The experiment was laid out in a Randomized Complete Block Design (RCBD) in a split plot arrangement and was replicated 4 times. Treatments were comprised of heat stress in main plots viz. H0 = no heat imposition; H1 = Heat imposition from complete emergence of spike to grain filling initiation (Feekes Scale = 10.50 to 11.00) and wheat genotypes in sub plots viz. Punjab-2011, AARI-2011, Galaxy-2013, Millat-2011, Aas-2011, Fareed-2006, Chakwal-50, Mairaj-2008, Pakistan-2013, NIBGE-NIAB-1 and Kohistan-97. Imposition of heat stress deleteriously impacted the metabolism of all genotypes. The synthesis of antioxidants and osmo-protectants were enhanced in genotypes ‘AAS-2011, Chakwal-50 and Mairaj-2008’ under the high temperature environment compared to ‘no heat stress’. While, in all other genotypes biosynthesis of antioxidants and osmo-protectants was suppressed under heat compared to control. Likewise, adverse impacts of heat on spike growth, stay green trait, grain yield and yield components were relatively lesser in genotypes ‘AAS-2011, Chakwal-50 and Mairaj-2008’ than other genotypes. Statistically similar and relatively more grain yields compared to other genotypes were recorded for ‘Aas-2011’ (3.71 t ha-1), ‘Chakwal-50 (3.36 t ha-1) and ‘Mairaj-2008 (3.04 t ha-1) under heat stress. In the second year, two independent field experiments were conducted with the objective of mitigating heat stress using potassium and selenium as beneficial nutrients. The experimental design for both experiments was randomized complete block design (RCBD) with split plot arrangement having three replications. In both experiments, the main plot factor was comprised of three heat stress treatments viz. H0 = No heat imposition; H1 = Heat stress imposition from complete emergence of spike to grain filling initiation (Feekes scale = 10.50 to 11.0); H2 = Heat stress imposition from flowering initiation to grain filling initiation (Feekes scale = 10.5.1 to 11.0). In the second experiment, potassium was supplied via foliar application in sub plots at K0 = Control/ water spray; K15 = 15 g L-1; K30 = 30 g L-1; K45 = 45 g L-1 and K60

= 60 g L-1 to mitigate heat stress. In the third experiment, selenium was foliar applied at Se0 = Control/ water spray; Se25 = 25 mg L-1; Se50 = 50 mg L-1; Se75 = 75 mg L-1 and Se100 = 100 mg L-1 to alleviate heat stress. During the third year, heat stress mitigating experiments were repeated as described in the second year. Negative implications of heat were more pronounced under ‘heat from spike to grain filling’ compared to ‘heat from flowering to grain filling’. Grain yield in second experiment was decreased by 42-45% under ‘heat from spike to grain filling’ and 25-31% under ‘heat from flowering to grain filling’ compared to ‘no heat stress’. While, in third experiment, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’ caused decrease in grain yield compared to ‘no heat stress’ by 43-44% and 33-36%, respectively. Whereas, varying concentrations of foliar potassium and selenium differed significantly from each other and remarkably improved response variables compared to control/water spray. Application of potassium at 45 and 60 g L-1 and selenium at 75 and 100 mg L-1 depicted statistically similar and relatively more grain yield, yield components, spike growth attributes, chlorophyll content and quality attributes compared to other concentrations under all treatments of heat stress. Likewise, statistically alike and comparatively more

antioxidants, osmo-protectants and water relations attributes and statistically similar and relatively lesser malondialdehyde were observed with 45 and 60 g L -1 foliar potassium and 75 and 100 mg L-1 foliar selenium under ‘no heat stress’. However, application of 60 g L-1 potassium and 100 mg L-1 selenium showed significantly more antioxidants, osmo-protectants and water relations attributes and significantly lesser malondialdehyde under ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’. Conclusively, genotypes ‘AAS-2011, Chakwal-50 and Mairaj-2008’ displayed terminal heat tolerance while genotypes ‘Fareed-2006’ and ‘Punjab-2011’ exhibited medium tolerance. In contrast, all other genotypes tested did not produce remarkable responses under heat and were characterized as terminal heat susceptible based on recorded parameters. Under ‘no heat stress’ application of exogenous potassium at 45 g L-1 and selenium at 75 mg L-1

effectively alleviated the adverse impacts of heat. Whereas, application of potassium at 60 g L-1 and selenium at 100 mg L-1 provided more promising morphological and biochemical responses under ‘heat from spike to grain filling and ‘heat from flowering to grain filling’. While, foliar applied potassium and selenium proved more important under heat treatments compared to ambient conditions. Moreover, biochemical attributes modulated regulations in growth, yield components and grain yield were significant under varying temperatures.

INTRODUCTION CHAPTER-1

Wheat is extensively grown all over the world and is an important source of starch

and protein for humans (Ang and Fredriksson, 2017). Wheat is cultivated on more than

218 million hectares with 743 million tons annual production around the globe (FAO,

2017). The share of wheat in value addition in agriculture is 9.6% while it supplements

1.9% in gross domestic product of Pakistan. Its area of cultivation is 9. 052 million

hectares and production is 25.75 million tons (Govt. of Pakistan, 2017). Each 100 g

serving of wheat provides 247 calories, 58-62 g bioavailable carbohydrates, 12-13 g

proteins, 1.8 g lipids, 13-14 g fiber and 1.7 g minerals (400 mg sodium, 248 mg

potassium). Besides, 1 kg wheat of grains also provide 4.6-5.0 mg vitamin-B1 (thiamine),

0.9-1.2 mg vitamin-B2 (riboflavin), 51-55 mg nicotinamide, 12-13 mg pantothenic acid,

2.7-3.0 mg vitamin-B6 and 41 mg tocopherols (Koehler and Wieser, 2013).

Productivity of wheat in Pakistan is lagging far behind than the potential owing to

numerous factors. Different factors that are responsible for low productivity of wheat in

Pakistan are delayed soil preparation after rice and cotton, late sowing, low input use

efficiency, unavailability of quality seed, fertilizers, irrigation water and terminal heat

stress (Rehman et al., 2015).

Heat stress at reproductive stages (terminal heat stress) of wheat is one of the chief

constraints hampering the full attainment of yield potential. The upper threshold

temperature above which terminal stages of wheat are deleteriously impacted is 26°C

(Shahid et al., 2017). Terminal heat stress might be a consequence of rapid

industrialization, deforestation, burning of fossil fuels, emission of chloro flouro carbons,

rapid changes in land utilization and injudicious use of synthetic fertilizers in agriculture

(Szymańska, 2017). In addition, the decline in rainfall over sub tropics including

Pakistan has further intensified extreme temperature events (Rahut and Ali, 2017).

Moreover, the rate of increase of temperature in the last decade (2000-2010) had been

2.2% higher than the rate of increase of temperature in previous 30 years (1970-2000)

(IPCC, 2014). While, late sowing of wheat in rice-wheat, cotton-wheat and hybrid

maize-wheat cropping systems leads to high temperature stress at reproductive stages of

wheat (Mumtaz et al., 2015).

Wheat is a C3 and temperate plant and therefore is very susceptible to high

temperature stress. High temperature stress at reproductive and grain filling stages of

1

wheat is called terminal heat stress (Alghabari et al., 2016). It is usually 10-15°C higher

temperature than ambient temperature (Dwivedi et al., 2017). It is anticipated to increase

in the near future due to global warming. Temperature will rise by 2.6-4.8°C during the

period of 2016-2035 (IPCC, 2014) while optimum temperature for reproductive stages of

wheat is 12-22°C (Dwivedi et al., 2017).

Damages due to high temperature stress depends on the duration of high

temperature, the magnitude of rise of temperature and the rate of increase in temperature

(Prasad et al., 2017). Temperature may rise slowly, rapidly or in cyclic pattern (increases

during day while decreases during night). Cyclic increase is the most damaging while

slow rise of temperature is the least damaging for wheat productivity (Rezaei et al.,

2015).

The temperature optima for spikelet, anthesis and grain filling for wheat are 12, 23

and 21ºC, respectively (Innes et al., 2015). According to a previous assessment, an

increase of 1ºC during the growing season declines grain yield by 3-17% in Pakistan and

India (Mondal et al., 2013).

High temperature stress reduces wheat productivity by dehydration, pollen

sterility, shortening of phenology, decreased CO2 assimilation, increased photorespiration

and decreased growth rate (Altenbach, 2012). Under heat stress, photosynthesis is the

most sensitive process. High temperature dissociates oxygen evolving complex of PS-II

and initiates photorespiration (Mathur et al., 2014).

Chlorophyll enzymatic activity is also disturbed at higher temperature. Activity of

adenosine diphosphate glucose pyro phosphatase (ADPG-PPase) is particularly reduced.

It downregulates the synthesis of starch (Dwivedi et al., 2017). Diurnal fluctuations of

temperature are more damaging to promote senescence (Laza et al., 2015).

Grain growth and development is also affected at higher temperature. Spike

initiation stage is the most sensitive stage to high temperature as at this stage ridges

development on spike rachis takes place. The number of ridges determines the number of

spikelets in the spike (Iqbal et al., 2017).

Under heat stress excessive generation of reactive oxygen species (ROS)

overcome scavenging mechanisms. Excessive ROS results in increased membrane

damages, lipid peroxidation, protein carbonylation and damage to DNA by insertion,

deletion, mutation and affecting nitrogen bases of DNA. High temperature stress

increases superoxide radical (O2●-) while hydrogen peroxide (H2O2) generation also rises

above normal level. Other ROS that are excessively produced and aggravate lipid

2

peroxidation at sub cellular level are singlet oxygen (1O2*) and hydroxyl radical (OH●-)

(Czégény et al., 2016).

To manage heat stress different strategies are available. These are breeding for

heat stress tolerance and selection of tolerant genotypes (Mondal et al., 2016). While,

agronomic management comprises of reduced tillage and stubble management, pre-

sowing heat treatment, manipulation of sowing time and foliar sprays of various

substances (Gouache et al., 2012).

Different types of compounds that can be foliar applied to mitigate heat stress

include osmo-protectants, osmolytes, inorganic salts, compatible solutes, signaling

molecules, plant growth regulators and oxidants (Farooq et al., 2011; Hu et al., 2016).

Foliar application of mineral nutrients is one of solutions to the problem. Exogenous

application of mineral nutrients augments tolerance against extreme temperature stresses

(Waraich et al., 2012).

Potassium (K) is the most important osmoticum in the plant cell cytosol. The

availability of K improves heat stress tolerance in plant. Potassium also helps the plant to

make osmotic adjustments as it is the safest osmoticum (Zahoor et al., 2017a). It is an

osmolyte and thus depresses the cellular water potential more than the apoplast. Net

movement of water takes place into cell that helping them to maintain turgor and creating

a favorable environment to maintain cellular enzymatic activities under heat stress (Jan

et al., 2017; Xiaokang et al., 2017).

Potassium maintains the electrical charge balance at site of ATP synthesis and

photophosphorylation remains continuous under stress conditions (Kanai et al., 2011).

Potassium activates ATP for utilization by H+-ATPase pumps. Hydrogen pump ATPases

exclude H+ out of the cell and create a favorable electrochemical gradient known as

proton motive force (Ahmad and Maathuis, 2014). Most nutrient uptake utilizes proton

motive force. Thus, K also helps in maintaining nutrient uptake under stressed conditions

(Anschütz et al., 2014).

Potassium enhances dry matter accumulation by maintaining activities of different

enzymes involved in starch and protein deposition. Exogenously applied potassium

enhances activation of RuBisCO, sucrose phosphate synthase, sucrose synthase and

soluble acid invertase under stressed environments. Increase in activities of these

enzymes escalates the sucrose and starch accumulation in reproductive and vegetative

organs under abiotic stress conditions. Moreover, easily available foliar applied

potassium also increases stomatal conductance and gaseous exchange with the

3

environment. Ultimately, dry matter accumulation of vegetative and reproductive parts,

net assimilation rate, partitioning of starch and sucrose towards reproductive parts

improves under stress conditions (Zahoor et al., 2017b). It also maintains activity of

hydrolases (Pectinases, Cellulases) under stress condition. Activation of hydrolases

loosens cell wall and concurrently K mediated depression of cell water potential causes

influx of water into cell. Cell is able to expand and maintain growth under heat stress (Jin

et al., 2011).

Foliar applied K enhanced net photosynthesis, stomatal conductance, yield and

growth attributes of wheat under stress conditions (Zareian et al., 2013). Availability of

K reduced photo oxidative damage, increased leaf potassium contents, water and osmotic

potential, enhanced CO2 fixation, transpiration rate, maximum and actual quantum yield

of photosystem-II (PS-II), non-photochemical quenching and increased utilization of

light use efficiency under stressed conditions. Moreover, foliar application of potassium

boosted the activities of superoxide dismutase, catalase, peroxidase and proline and

consequence into decreased lipid peroxidation of bio-membranes (Zahoor et al., 2017c).

Potassium availability under stressed conditions improved root hydraulic by increasing

expression of aquaporin (Wang et al., 2013). Consequences of modulation in potassium

balance and in physiochemical attributes are improved growth and yield under stress

conditions. Availability of K reduced oxidative stress by reducing NADH oxidase

activity, K deficiency augmented O2●- generation and thus aggravated oxidative stress

(Jiménez-Quesada et al., 2016).

Selenium (Se) down regulates ROS production under stress by upregulating the

activity of antioxidants. It increases the activity of ascorbate peroxidase that is a key

enzyme in detoxification of H2O2. It upregulates activity of catalase and glutathione

peroxidase under heat stress (Cheng et al., 2016). Selenium compounds under heat stress

quench 1O2* and OH●-. It promotes stability of membranes as OH●- is most damaging for

lipid peroxidation and 1O2* causes mutation by reacting with nitrogen bases of DNA

(Feng et al., 2013). It accelerates the non-enzymatic detoxification of O2●- to H2O2 and

protects cellular membranes. Selenium also acts as an activator of glutathione

peroxidase, which detoxifies H2O2 (Huang et al., 2017).

In photosynthesis, elemental Se replaces sulfur from Fe-S cluster and reduces

ROS synthesis through regulation of electron flow. Selenium enhances PS-I ability to

produce reductants at the end of light reactions and promotes CO2 reduction under high

temperature stress (Gupta and Gupta, 2017). Selenium reduces damage to PS-II light

4

harvesting complex by excessive UV and high light intensity under heat stress (Feng et

al., 2013).

Exogenous application of Se augmented synthesis of catalase, superoxide

dismutase, peroxidase, glutathione and ascorbate reductase in wheat. Moreover, water

retention capability of tissues was also enhanced with foliar applied selenium under

stressed conditions over control in wheat (Nawaz et al., 2015). Improvement in

accumulation of proline under exogenous selenium resulted in detoxification of reactive

oxygen species and upregulated the biosynthesis of chlorophyll, total soluble sugars and

phenyl ammonia lyase contents under stressed conditions (Manaf, 2016). Exogenous Se

enhanced accumulation of ascorbate, carotenoids, anthocyanin, ascorbate peroxidase,

chlorophyll a, b and reduced malondialdehyde (MDA) in wheat under high temperature

stress (Iqbal et al., 2015). Selenium augmented antioxidant defense system under high

temperature by increasing synthesis of glutathione reductase, dehydro ascorbate

reductase and by maintaining high reducing power of NADH (Sieprawska et al., 2015).

Selenium at low concentration acts as reductant for ROS. At higher concentration, it

functions as pro antioxidant that improves signaling for upregulation of the antioxidant

defense system (Ahmad et al., 2016). Moreover, application of selenium increased

accumulation of anthocyanin, ascorbic acid, antioxidants and nutrients. Alleviation of

stress under Se application can be attributed to selenium mediated improvements in

redox buffering capacity of plant, phyto hormone regulations, antioxidant regeneration,

ROS scavenging and enhanced cell division (Shekari et al., 2015).

Selenium reduced protochlorophyllide oxidoreductase contents, enhanced

activities of starch biosynthesis enzymes and maintained normal function and shape of

chloroplast (Kaur et al., 2014). Selenium improved the staygreen trait and maintained

carbohydrates supply for longer duration of time (Haghighi et al., 2015). Selenium

mediated synthesis of chlorophyll a and b, increased stomatal conductance, transpiration

rate and exchange of gases with atmosphere under heat stress (Mora et al., 2015).

Different wheat cultivars depict assortment and heterogeneity in response to high

temperature (Siebert and Ewert, 2014). Furthermore, numerous quantitative trait loci

exist for a single targeted trait having complex inheritance pattern (Mwadzingeni et al.,

2016). Therefore, selection of polygenic target traits can be accomplished indirectly

employing biochemical markers closely related to heat tolerance (Sadat et al., 2013).

Likewise, diversity among wheat cultivars combined with polyploidy and genes

profusion makes it challenge to select a suitable genotype using morphological traits

5

under high-temperature environment (Dube et al., 2016). Selection of wheat genotypes

merely on the basis of morphological traits often leads to faulty inferences (Reynolds and

Langridge, 2016). While, physiochemical markers assisted screening of genotypes

depicts higher efficacy of selection than mere morphological markers-based selection for

polygenic traits (Sadat et al., 2013).

Previous experiments were mainly comprised of heat imposition under controlled

environments of glasshouse. Although, studies regarding manipulation of sowing dates

are abundantly available to observe adverse effects of high temperature. Relatively little

information is available regarding the imposition of heat stress under field conditions.

Moreover, studying potassium and selenium mediated transformations in biochemical

attributes in correlation with morphological traits might prove advantageous for

agronomic management of heat stress. Information regarding the correlation of

biochemical attributes with growth and yield parameters at terminal stages predisposed to

heat are also scarce. Moreover, most of previous studies quantified biochemical attributes

only at seedling stages without considering yield and other phenotypic traits at terminal

stages.

In this context, a compendious understanding and boost of biochemical

mechanisms using exogenous potassium and selenium is indispensable to induce heat

tolerance. Moreover, distinctive biochemical response of varying heat stressed terminal

pheno-stages leads us to a closer inspection of the problem and its management through

exogenous potassium and selenium. Since, improvements in physiochemical traits might

prove a potent tool to alleviate adversities on morphological attributes of wheat crop.

Hence, elucidation of biochemical attributes in correlation with grain growth and yield

will improve the efficacy of agronomic management of terminal heat.

It can be inferred that terminal heat stress in wheat badly impacts various growth,

yield, biochemical and physiological attributes. As a consequence of negative

implications of high temperature stress grain shriveling takes place under agro-

climatological conditions of Pakistan. It reduces yield of wheat each year by sudden rises

in temperature and increases the costs of wheat production. It is the hour of need to

manage heat stress by devising strategies that are economical, everlasting and alleviate

heat stress effectively. Foliar applied K and Se may have potential to regulate various

physiological, biochemical, growth and yield related processes under high temperature

stress.

Objectives

6

The study was conducted with the following objectives

1- Screening of Pakistani wheat genotypes for tolerance to terminal heat

2- Studying the comparative vulnerability of terminal phenological stages of wheat to

high temperature

3- Exploring the morphological responses of wheat in relation to physiochemical

perturbations under varying temperatures

4- Optimizing foliar potassium (K) and selenium (Se) to alleviate negative impacts of

terminal heat in wheat

7

REVIEW OF LITERATURE CHAPTER-2

Wheat is among the widest grown cereals around the globe. Wheat chip in 21% to

the world’s calorie intake and is grown on an area of 221 million-hectare worlds widely

(Tao et al., 2015). Food security in Pakistan is affiliated with wheat production and

consumption. Increasing prevalence of extreme temperatures is becoming a limiting

factor for crop production specifically for cereals (Wang et al., 2015). Wheat production

under changing climate has been an arduous task (Trnka et al., 2014).

The increasing accumulation of greenhouse gases will further intensify warm

temperature together with the disturbance in water resources (Harris et al., 2015).

Excessive emission of carbon dioxide from burning of fuels has increased the frequency

of heat waves on wheat (Fernando et al., 2014). Carbon dioxide and other greenhouse

gases are expected to increase by 50% of the current concentrations in atmosphere by

2050 due to incessant increasing demands for energy (OECD, 2012). Late sowing of

wheat is one of the major reasons leading to grain shriveling in wheat by the abrupt rise

of temperature during grain filling (Ihsan et al., 2016).

Heat stress negatively influences innumerable plant processes. High temperature

increased catalytic activity of RuBisCO while its affinity for CO2 was decreased. Oxygen

solubility into mesophyll cells of wheat was little affected while CO2 solubility decreased

at higher temperature (Mathur et al., 2014). RuBisCO started to act as an oxygenase

enzyme and photo respiration decreased yield. During photorespiration consumption of

ATPs using assimilated carbohydrates promoted grain shriveling. RuBisCO sensitivity to

higher temperature was more than any other enzyme in photosynthesis (Perez et al.,

2011). RuBisCO activase (RCA) enzyme removes inhibitory sugar phosphates from

active site of RuBisCO and makes it to react with CO2. At higher temperature, the

activity of RCA was also reduced as well as photosynthesis (Carmo-Silva et al., 2012).

Photosystem-II (PS-II) is more labile to higher temperature than Photosystem-I

(PS-I). Increase of temperature above 40°C disrupted light harvesting complex of PS-II

by separation of manganese (Mn) from the D1D2 complex (Ashraf and Harris, 2013). It

inhibited the photolysis of water at start of photosynthesis, so electron flow was

8

disturbed and generation of reductants at the end of light reaction for CO2 reduction were

also reduced. Rise of temperature further disrupted the plastoquinone in electron pool in

the transport chain of light reactions (Mathur et al., 2014).

High temperature stress reduced water potential and relative water content of

leaves (Hasanuzzaman et al., 2013). Heat stress promoted respiration and water loss from

leaves (Duan et al., 2017). Most species tend to close stomata and conserve water rather

than regulation of temperature by transpiration. It impaired gaseous exchange with the

atmosphere, thus photosynthesis was negatively affected (Marias et al., 2017).

The rise in temperature caused a rapid grain filling rate and reduced the duration

of grain filling. The increased rate of grain filling could not compensate for the decreased

duration of grain filling as assimilate partitioning towards the grain was less leading to

the consequence of grain shriveling (Barlow et al., 2015).

Temperature above 30°C caused completely infertile pollen grains and reduced

the size of ovaries. Reduced size of ovaries was due to reduced activity of the acid

invertase enzyme and partitioning of carbohydrates towards reproductive organs. Acid

invertase governs the upper limit of sink size, so small sized grains were produced at

high temperature stress (Dwivedi et al., 2017). Grain size was reduced due to shortening

of phenology between anthesis and physiological maturity of grains (Hatfield and

Prueger, 2015). Changes in the aleuron layer around the endosperm of wheat grains

decreased starch deposition due to different enzymes involved in starch assimilation in

endosperm (Iqbal et al., 2017).

Temperatures greater than 25°C at grain filling stages reduced activity of starch

synthase, granule bound starch synthase, sucrose fructosyltransferase, fructan

fructosyltransferase and sucrose synthase. Reduced sucrose synthase activity dwindled

phloem sucrose loading (Dwivedi et al., 2017). Diminished translocation of

carbohydrates towards grain caused assimilate accumulation in the phloem that

introduced a feedback mechanism to down regulate photosynthesis (Wang et al., 2012).

Different wheat cultivars display an assortment and heterogeneity in response

towards high temperature (Siebert and Ewert, 2014). Diversity among wheat cultivars

combined with polyploidy and genes profusion makes it challenging to select suitable

genotypes under high temperature environment. Therefore, phenological and

biochemical markers assisted screening of wheat cultivars increases cultivar selection

efficacy (Sharma et al., 2014a).

9

Moreover, different management strategies are available to alleviate the adversity

of heat stress in wheat. Soil application of minerals is an energy consuming process

regarding plant metabolism. Most nutrients are taken up through secondary active

transport that requires ATP. Plants under stress conditions with activated defense

mechanisms are not able to extract nutrients from soil solution (Ma et al., 2017). Foliar

application can resolve this problem under these hostile conditions of heat stress. Foliar

applied nutrients are taken through diffusion that is driven by concentration gradient of

nutrient across leaf epicuticular waxes (Wasaya et al., 2017).

Different agronomic strategies that can alleviate heat stress are water

conservation, conservation tillage practices and timely sowing of crops (Farooq et al.,

2011). Early sowing of wheat in different cropping systems may allow the wheat to

escape from terminal heat stress (Suryavanshi and Buttar, 2016). Different foliar sprays

i.e. compatible solutes, signaling molecules, plant growth substances and osmolytes

enhance tolerance against heat stress. Application of mineral nutrients helps to mitigate

high temperature stress in wheat. Nitrogen, phosphorous, potassium, zinc and boron are

important in this regard (Hemantaranjan et al., 2014).

Foliar application of potassium (K) and selenium (Se) assists the plant to

acclimatize under heat stress by regulation of various biochemical processes. Potassium

regulates stomatal opening and closing under heat stress and aids the plant in gas

exchange with the atmosphere. Thus, plants are able to uphold sufficient CO2 for

RubisCO to act as carboxylase enzyme under heat stress (Wang et al., 2013; Nawaz et

al., 2015).

Potassium mediated activation of ATP proved helpful for phloem sucrose loading

and unloading. It sustained assimilate partitioning towards grain under heat stress

(Marschner, 2012). Potassium diminished diffusible resistance of CO2 into leaf

mesophyll by stomatal regulation that made RuBisCO to act as carboxylase enzyme and

photorespiration was reduced (Jan et al., 2017).

Potassium enabled plants to make osmotic adjustments under heat stress by

promoting accumulation of proline and glycine betaine. Proline acts as an

osmoprotectant and alternate electron donor to PS-I and PS-II activity when photolysis of

water was lessened at higher temperature (Hayat et al., 2012). Potassium declined

malondialdehyde production under stressed conditions, which is an indication of

membrane stability (Oosterhuis et al., 2013). Potassium enhanced activity of catalase that

is involved in detoxification of excessive H2O2 produced under heat stress (Ahmad et al.,

10

2016). Glycine betaine is a quaternary nitrogen compound, its accumulation was

enhanced in presence of K as K is involved in activation of nitrate reductase and

glutamine synthase. Glycine betaine also protects membranes from ROS damage under

heat stress. Application of potassium improved glycine betaine accumulation,

chlorophyll contents and yield related attributes of wheat under stress (Raza et al., 2014).

Potassium improved growth and photosynthetic rate by regulating stomatal movement

under stress conditions (Ahmad et al., 2014). Potassium application under stressed

conditions enhanced dry matter content and relative leaf water content over control

(Zahoor et al., 2017b).

Potassium enhanced grain quality by improving protein contents as well as protein

quality (Zorb et al., 2014). Potassium is involved in each step of protein synthesis from

nitrogen uptake by secondary active transport, activation of nitrate reductase, glutamine

synthase, reading of genetic codes and binding of tRNA to ribosomes at ribosomal site of

protein synthesis (Sharma et al., 2013).

Exogenous application of Se is more effective for improving plant selenium

contents than soil application (Nawaz et al., 2014). Selenium is a beneficial element, but

non-essential for growth. It improved relative water contents and water potential of cell

under stress condition. Starch deposition in grain was increased under selenium

application in high temperature environment (Malik et al., 2012). Selenium delayed the

senescence and improved stay green trait under high UV light stress. Application of

selenium improved δ- aminolevulinic acid dehydratase and porphobilinogin deaminase.

These enzymes promoted chlorophyll biosynthesis under heat stress. Selenium

application reduced protochlorophyllide oxidoreductase activity. Protochlorophyllide

oxidoreductase converts protochlorophyllide (precursor of chlorophyll biosynthesis) to

chlorophyllide (inactive chlorophyll), thus hindered chlorophyll deprivation in wheat

(Yao et al., 2011). Selenium enhanced chlorophyll biosynthesis and reduced degradation.

Maintenance of high chlorophyll content under high intensity of UV improved the

staygreen trait. In addition, it maintained carbohydrate synthesis in high temperature

environment (Yildiztugay et al., 2017).

Selenium assimilation boosted synthesis of glutathione reductase (GSH). It

detoxified H2O2 and upgraded antioxidant defense mechanism of plant (Mehdi et al.,

2013). Application of Se reduced oxidative stress by slowing down the synthesis of O2●-

and enhancing detoxification of H2O2 (Feng et al., 2013). Selenium improved superoxide

dismutase activity in heat stressed wheat and alleviated oxidative stress significantly as

11

compared to controls (Tedeschini et al., 2015). Selenium declined the reduction of

tocopherol under stress conditions that improved glutathione peroxidase activity

(Klusonova et al., 2015). Foliar application of Se improved uptake of Na, Fe, Ca and Zn.

Increased antioxidant activity under heat stress might be due to enhanced uptake of

micronutrients that act as cofactor for activation of enzymatic antioxidants (Nawaz et al.,

2015).

Selenium enhanced non-enzymatic dismutation of O2●- to H2O2. Selenium

mediated synthesis of proteins act as reductants, which promoted non-enzymatic

dismutation of O2●- (Kaur et al., 2014). Together with non-enzymatic dismutation of O2

●-,

Se also enhanced activity of superoxide dismutase. Different enzymes that are involved

in detoxification of ROS are dehydro-ascorbate reductase, mono-dehydro-ascorbate

reductase and glutathione reductase. For activation of these enzymes reductants are

required. Selenium compounds-maintained reductants for activity of these enzymes

(Nawaz et al., 2015).

Selenium improved PS-II stability of heat stressed wheat crop by regulating

multiple processes. These processes include decreased excitation energy of PS-II, light

absorption by antenna molecules, electron flux, energy quanta of PS-II and impairment

of oxygen evolving complex (Labanowska et al., 2012). Selenium augmented cell

membrane stability by increasing lipid to protein ratio and degree of unsaturation of

lipids under stressed conditions (Feng et al., 2014). Selenium is useful to reduce lipid

peroxidation of membranes as it reduces malondialdehyde production under stress

conditions (Jiang et al., 2017). Selenium promoted lipid unsaturation and breaks ROS

chain to reduce oxidative stress (Malik et al., 2012).

Application of Se improved starch accumulation and the stay green trait under UV

light stress (Mostafa and Hassan, 2015). Selenium enhanced water uptake by roots under

stressed conditions (Nawaz et al., 2014). Application of Se enhanced total soluble sugars,

antioxidant activities, chlorophyll contents and yield in wheat under stressed conditions

(Nawaz et al., 2015). Application of Se enhanced biosynthesis of chlorophyll,

carotenoids and improved yield (Dong et al., 2013). Selenium alleviated oxidative stress

by enhancing super oxide dismutase, catalase, glutathione peroxidase, ascorbate and

tocopherol activities under stressed conditions (Lin et al., 2012). Selenium improved

phenolic contents in stressed wheat by boosting phenylalanine ammonia lyase activity

(Iqbal et al., 2015).

12

Furthermore, existence of numerous quantitative trait loci for a single targeted

trait depicted complex inheritance pattern (Mwadzingeni et al., 2016). Hence, selection

of wheat genotypes merely based on response of morphological traits often leads to

faulty inferences (Reynolds and Langridge, 2016). While, biochemical markers assisted

selection of genotypes exhibited more efficacy of selection than mere morphological

markers-based selection for polygenic traits. Selection of genotypes using morphological

attributes leads to poor selection efficacy studies (Jacoby et al., 2016). Selection of

genotypes on basis of biochemical attributes in association to morphological attributes is

lacking in previous experimentation.

The crux of the issues is that, high temperature negatively affects innumerable

physiological, growth and yield attributes of wheat. Minor variations in ambient

temperature affect physiochemical attributes of wheat crop. While, availability of

potassium and selenium improves biochemical attributes that ultimately confer heat

tolerance at morphological level. However, heat mediated changes and potassium and

selenium triggered regulations in physiochemical attributes are not disclosed copiously

so far. Moreover, data regarding potassium and selenium instigated regulations in

physiochemical attributes of terminal heat stressed wheat are scarce. Hence, elucidation

of thermo-tolerance at biochemical level is crucial for food security since improvements

in biochemical attributes confer tolerance in growth and yield components. In addition,

better understanding of the relation between biochemical attributes and yield components

of heat stressed wheat provides sound basis for agronomic management of heat stress.

Likewise, knowledge about heat caused deteriorations and potassium and selenium

trigged improvements in in grain quality is also scarce.

It can be hypothesized that different genotypes and terminal growth stages will

perform distinctly under high heat stress. While, varying concentrations of exogenous

potassium and selenium might prove a potent tool to alleviate adversities of heat at

biochemical and morphological level. Besides, foliar potassium and selenium instigated

biochemical regulations will confer tolerance in growth and yield components of heat

stressed wheat crop.

13

MATERIALS AND METHODS CHAPTER-3

The present research wok was carried out to alleviate deleterious impacts of

terminal heat stress on wheat. Three years of field-based experiments were performed to

accomplish this objective. For the 1st year (2014-15), wheat varieties were characterized

for heat tolerance and a medium heat tolerant wheat genotype was selected for further

experimentation. In the 2nd year (2015-16), two independent field experiments were

performed whereby heat stress was alleviated through exogenous spray of potassium in

one and selenium in other experiment. During the 3rd year (2016-17), the same

experiments were repeated as in 2015-16. Variables such as grain yield, yield

components, biomass accumulation, the stay green trait, antioxidants activities, osmo-

protectants water relations and quality attributes were used as potential indicators of

thermo-tolerance.

3.1. Experimental site

All research activities were carried out at Agronomic Research Area, University

of Agriculture Faisalabad Pakistan during the period of November 2014 to May 2017.

The site is located at latitude of 31°-26’N, longitude 73°-06’E and altitude of 184.4 m.

3.2. Physio-chemical analyses of soil

Soil samples were randomly taken from various points of the field at depths of 15

and 30 cm. Soil samples were mixed separately for the depths of 15 and 30 cm to record

electrical conductivity (Rhoades, 1996), pH (Thomas, 1996), organic matter (Moodie et

al., 1959), total nitrogen (Jackson, 1962), available phosphorous using 0.5 M sodium

bicarbonate (NaHCO3) as extraction solution (Kuo, 1996) and available potassium using

1 N ammonium acetate (NH4OAc) as extraction solution (Helmke and Sparks, 1996).

Textural class of experimental soil was loam (Table 3.1).

3.3. Weather elements

14

Data of different weather elements were collected from Meteorological

Observatory, University of Agriculture Faisalabad Pakistan during the growing season of

wheat. Data on average temperature, relative humidity, rainfall, pan evaporation, sunshine

duration, evapotranspiration and wind speed were recorded on daily basis and averaged

each month (Table 3.2).

3.4. Plant material

Numerous genotypes were collected from different institutes to determine thermo-

tolerance and sensitivity for ‘Experiment 1’.

15

Table 3.1: Physio-chemical analyses of experimental site during 2014-15, 2015-16 and 2016-17

Soil characteristics Depth of sample (cm)

Experiment I Experiment II Experiment III2014-15 2015-16 2016-17 2015-16 2016-17

Sand (%) 0-15 45 45 44 43 4615-30 43 44 43 45 44

Silt (%) 0-15 23 25 26 24 2215-30 24 26 28 25 24

Clay (%) 0-15 29 27 29 31 3315-30 28 26 28 29 31

Textural class 0-15 Loam Loam Loam Loam Loam15-30

EC (dS m-1) 0-15 2.06 2.10 1.99 2.01 1.9615-30 1.98 1.96 1.97 2.03 1.98

pH 0-15 7.7 7.5 7.6 7.8 7.915-30 7.6 7.8 7.9 7.7 7.8

Organic matter (g kg-1) 0-15 9.2 5.9 5.3 5.8 5.115-30 9.4 5.8 5.5 5.8 5.2

Total nitrogen (g kg-1) 0-15 0.44 0.46 0.45 0.46 0.4415-30 0.41 0.45 0.43 0.45 0.42

Available phosphorous (mg kg-1) 0-15 7.7 8.02 7.7 7.8 7.315-30 8.04 7.9 7.4 7.7 7.1

Available potassium (mg kg-1) 0-15 177 179 162 177 15915-30 165 176 159 177 155

Latitude = 31° - 26’N; Longitude = 73°- 06’E; Altitude = 184.4 m

16

Table 3.2: Monthly averages of weather elements during growing season of crop in 2014-15, 2015-16 and 2016-17

Weather elements Years November December January February March April MayAverage temperature (°C)

2014-15

18.9 12.2 11.7 16.5 19.1 27.0 31.8Relative humidity (%) 61.7 75.0 75.3 66.0 64.0 43.9 27.5Rainfall (mm) 10.0 0.0 12.2 20.5 67.9 32.8 17.0Pan evaporation (mm) 1.8 1.5 1.0 2.1 13.0 5.3 7.6Sunshine duration (hours) 7.6 4.7 5.0 5.6 4.9 9.1 10.4Evapotranspiration (mm) 1.5 1.3 0.7 1.8 2.8 3.7 5.3Wind speed (km h-1) 3.1 2.0 3.6 5.3 5.6 6.2 5.7Average temperature (°C)

2015-16

19.6 14.5 12.5 16.3 21.2 27.2 32.8Relative humidity (%) 61.5 62.6 74.4 58.1 59.7 34.2 28.8Rainfall (mm) 8.8 0.0 13.1 7.8 66.7 5.6 25.0Pan evaporation (mm) 2.4 1.9 3.5 2.3 2.7 6.1 9.5Sunshine duration (hours) 6.6 7.0 1.2 8.5 6.6 8.3 10.4Evapotranspiration (mm) 2.1 1.6 0.8 1.6 1.9 4.3 6.4Wind speed (km h-1) 2.6 2.3 27.6 3.8 4.7 5.2 5.4Average temperature (°C)

2016-17

20.1 16.4 12.9 16.8 23.7 29.3 33.5Relative humidity (%) 60.1 68.7 72.0 53.0 49.5 30.6 29.8Rainfall (mm) 0.0 0.0 11.5 4.1 16.2 28.3 10.1Pan evaporation (mm) 2.4 2.1 3.6 2.7 3.9 7.5 9.2Sunshine duration (hours) 6.4 6.7 1.3 6.6 7.2 9.2 10.4Evapotranspiration (mm) 1.8 1.7 0.9 1.9 2.7 5.2 5.7Wind speed (km h-1) 2.6 2.8 3.5 4.0 3.9 5.8 5.4


17

Table 3.3: Varying mean temperatures (°C) 2014-15 for experiment 1

Heat stress YearMarch

21

March

22

March

23

March

24

March

25

March

26

March

27

March

28

March

29

March

30

March

31

No heat stress (H0)2014-15

30.02 30.20 31.90 32.60 32.15 31.55 31.40 33.40 31.30 30.50 32.40

Heat from spike to grain filling (H1) 39.37 39.77 38.76 38.82 38.23 40.40 41.60 40.80 41.20 39.30 39.70


Table 3.4: Varying mean temperatures (°C) during heat imposition for experiment 2, 2015-16 and 2016-17

Heat stress YearMarc

h 1

March

2

Marc

h 3

Marc

h 4

Marc

h 5

Marc

h 6

Marc

h 7

March

8

Marc

h 9

March

10

Marc

h 11

Marc

h 12

March

13

Marc

h 14

No heat stress (H0)

2015-16

26.0 27.0 29.0 27.0 26.5 26.0 27.0 25.0 26.0 25.0 25.5 25.5 26.0 26.5

Heat from spike to grain filling (H1) 33.3 34.1 34.6 33.9 33.0 33.4 34.0 32.0 32.5 32.0 31.7 31.0 31.4 32.8

Heat from flowering to grain filling (H2) - - - - - - - 32.2 32.4 32.3 31.5 31.0 31.5 32.5

No heat stress (H0) 29.0 28.0 30.5 29.0 28.5 28.0 29.0 28.0 28.5 28.0 28.5 27.5 28.0 27.5

Heat from spike to grain filling (H1) 2016-17 35.1 34.0 36.8 35.2 34.3 34.0 36.2 35.4 36.0 35.9 34.2 34.0 35.3 34.6



18

Table 3.5: Varying mean temperatures (°C) during heat imposition for experiment 3, 2015-16 and 2016-17

Heat stress YearMarc

h 1

March

2

Marc

h 3

Marc

h 4

Marc

h 5

Marc

h 6

Marc

h 7

March

8

Marc

h 9

March

10

Marc

h 11

Marc

h 12

March

13

Marc

h 14

No heat stress (H0)

2015-16

26.0 27.0 29.0 27.0 26.5 26.0 27.0 25.0 26.0 25.0 25.5 25.5 26.0 26.5

Heat from spike to grain filling (H1) 32.9 34.3 34.2 33.3 32.8 33.1 34.6 32.6 32.1 32.3 31.3 31.4 31.8 33.0


No heat stress (H0) 29.0 28.0 30.5 29.0 28.5 28.0 29.0 28.0 28.5 28.0 28.5 27.5 28.0 27.5

Heat from spike to grain filling (H1) 2016-17 34.8 34.2 36.1 35.9 34.8 34.4 36.7 34.6 35.7 35.5 33.0 33.7 34.8 34.1



19

Plant material of genotypes ‘Aas-2011’ and ‘Fareed-2006’ was procured from

‘Regional Agriculture Research Institute Bahawalpur, Pakistan’. While, seeds of

genotypes ‘Mairaj-2008’, ‘AARI-2011’, ‘Punjab-2011’, ‘Millat-2011’ and ‘Galaxy-2013’

were obtained from ‘Ayub Agriculture Research Institute (AARI) Faisalabad, Pakistan’.

Whereas, seeds of genotype ‘Pakistan-2013’ were obtained from ‘National Agriculture

Research Center Islamabad, Pakistan’. Seeds of genotypes ‘Chakwal-50’ and ‘Kohistan-

97’ were procured from ‘University of Agriculture Faisalabad (UAF), Pakistan’. Plant

material of genotype ‘NIBGE-NIAB-1’ was procured from ‘Nuclear Institute for

Agriculture and Biology (NIAB) Faisalabad, Pakistan’.

3.5. Agronomic practices

Wheat was sown with the help of single row hand drill with R × R of 22.5 cm.

Seed was sown at the rate of 100 kg ha-1. During the 1st, 2nd and 3rd year, sowing was done

on 17th November 2014-15, 25th November 2015-16 and 29th November 2016-17,

respectively. Fertilizer was applied at the rate of 120:75:60 kg NPK ha-1 in ‘Experiment

1’. While, in ‘Experiment 2’ and ‘Experiment 3’ 120:75 kg NP ha -1 was applied. Half of

nitrogen fertilizer (urea) and all the phosphorus (SSP) and potash fertilizers (SOP) were

applied as basal dose. While, remaining half nitrogen fertilizer was applied with first

irrigation at crown root initiation. Fertilizers were band placed in inter row spaces with

the help of single row hand drill. Irrigations were applied at four critical growth stages

viz. crown root initiation, tillering, spike initiation and flowering. Two manual hoeings

were performed in all treatments to maintain weeds population below economic threshold

level; first after 40 days of sowing and second after 60 days of sowing.

3.6. 1st year (2014-15) trial

Experiment I: Biochemical markers assisted screening of wheat cultivars for

terminal heat stress tolerance

Treatments:

Factor A: Heat stress (Main plot)

H0 = No heat stress imposition (Plots without polythene sheet)

H1 = Heat stress imposition from complete emergence of spike to grain filling initiation

(early milk stage) (Feekes scale = 10.50 to 11.0)

Factor B: Varieties (Sub plot)

V1 = Punjab-2011

V2 = AARI-2011

V3 = Galaxy- 2013

20

V4 = Millat-2011

V5 = Aas-2011

V6 = Fareed-2006

V7 = Chakwal-50

V8 = Mairaj-2008

V9 = Pakistan-2013

V10 = NIBGE-NIAB-1

V11 = Kohistan-97

(a) Experimental design

Experiment was conducted using a Randomized Complete Block Design (RCBD)

with split plot arrangement having 4 replications. Heat stress was imposed in main plots

and genotypes were randomized in sub plots. Gross plot size of each experimental unit

was 3.0 m × 1.35 m.

(b) Imposition of heat stress

Five plants were randomly selected and tagged in each plot to notice for 50%

‘complete emergence of spike’ and ‘grain filling initiation’. Heat stress was imposed

when 50% of plants reached the ‘complete emergence of spike’ and removed when 50%

of plants had achieved ‘grain filling initiation’ growth stage. The heat stressed main plot

was covered with transparent polythene sheet from complete emergence of spike to grain

filling initiation (Feekes Scale= 10.50 to 11.0) (Javed et al., 2014; Kamal et al., 2017;

Shahid et al., 2017). Whereas, control (no heat stress) plots were left in ambient

environment. Relative humidity under polythene sheet was maintained as in ambient

conditions by making large number of small sized holes in the polythene sheet.

Temperature of heat stress and control/no heat stress main plots was recorded three times

a day (morning, noon and evening) and averaged. Temperature was recorded with the

help of digital temperature and humidity probe (Digital Multimeter-50302). Comparative

temperatures under ‘no heat stress’ and ‘heat from spike to grain filling’ are given as

tabulated form (Table 3.3). Leaves were collected randomly from each experimental unit

1 day after removing stress, stored in liquid nitrogen and processed to record various

biochemical response variables.

(c) Parameters recorded

Yield components and grain yield

1. Number of fertile tillers per m2

2. Number of grains per spike

21

3. 1000-grain weight (g)

4. Grain yield (t ha-1)

Growth of spike

1. Grain filling rate (g per day) (Hunt, 1978)

2. Grain filling duration (days) (Hunt, 1978)

Stay green and antioxidants

1. Chlorophyll a contents (mg g-1 FW) (Arnon, 1949)

2. Chlorophyll b contents (mg g-1 FW) (Arnon, 1949)

3. Superoxide dismutase (U mg-1 protein) (Giannopolitis and Ries, 1977)

4. Peroxidase (U mg-1 protein) (Liu et al., 2009)

5. Catalase (U mg-1 protein) (Liu et al., 2009)

6. Total phenolic contents (mg GAE g-1) (Ainsworth and Gillespie, 2007)

Osmo-protectants and lipid peroxidation

1. Proline (µmol g-1) (Bate et al., 1973)

2. Glycine betaine (µmol g-1) (Grieve and Grattan, 1983)

3. Total soluble proteins (mg g-1) (Bradford, 1976)

4. Malondialdehyde contents (µmol g-1) (Cakmak and Horst, 1991)

(d) Statistical analysis

Data of recorded attributes were analyzed statistically (p ≤ 0.05) using the Fisher’s

analysis of variance technique (Steel et al., 1997) and Tukey’s Honestly Significant

Difference (Tukey’s HSD) test was employed to compare the means of different

genotypes at 5% probability level. While type and strength of relationship among the

recorded parameters was determined calculating correlation among these parameters

using STATISTIX 8.1 software (Gomez and Gomez, 1984).

A medium heat tolerant genotype (Punjab-2011) was selected on the basis of

recorded parameters and used in further experimentation (Van Esbroeck et al., 1998; Van

Deynze et al., 2009; Conaty et al., 2012).

3.7. 2nd year (2015-16) trials

Experiment II: Exploring role of foliar applied potassium to induce terminal heat

stress tolerance in wheat

Treatments:


H0 = No heat imposition (Plots without polythene sheet)

22



H2 = Heat stress imposition from flowering initiation to grain filling initiation (early milk

stage) (Feekes scale = 10.5.1 to 11.0)

Heat stress was imposed by covering the plots with perforated, transparent polythene

sheet (Javed et al., 2014; Kamal et al., 2014; Shahid et al., 2017).

Factor B: Potassium foliar application (subplot)

K0 = Control (0 kg K ha-1)

K15 = 15 g L-1 (4.5 kg K ha-1)

K30 = 30 g L-1 (9 kg K ha-1)

K45 = 45 g L-1 (13.5 kg K ha-1)

K60 = 60 g L-1 (18 kg K ha-1)

Experiment III: Alleviation of terminal heat stress in wheat through foliar

application of selenium

Treatments:


H0 = No heat imposition (Plots without polythene sheet)



H2 = Heat stress imposition from flowering initiation to grain filling initiation (early milk

stage) (Feekes scale = 10.5.1 to 11.0)

Heat stress was imposed by covering the plots with perforated, transparent polythene

sheet (Javed et al., 2014; Kamal et al., 2017; Shahid et al., 2017).

Factor B: Selenium foliar application (subplot)

Se0 = Control (0 g Se ha-1)

Se25 = 25 mg L-1 (7.5 g Se ha-1)

Se50 = 50 mg L-1 (15 g Se ha-1)

Se75 = 75 mg L-1 (22.5 g Se ha-1)

Se100 = 100 mg L-1 (30 g Se ha-1)

3.8. 3rd year (2016-17) trials

Experiment II and experiment III were repeated as in 2015-16.

(a) Experimental design

Both the experiments were laid out in Randomized Complete Block Design

(RCBD) with split plot treatments arrangement in 3 blocks. Heat was imposed in main

23

plots whereas exogenous potassium was applied in split plots. Each experimental unit was

comprised of 3.0 m × 1.35 m gross area.

(b) Imposition of heat stress and foliar application of potassium and selenium

Five plants were randomly selected in each experimental unit and were observed

for 50% ‘complete emergence of spike’, ‘flowering initiation’ and ‘grain filling

initiation’. When 50% plant reached the complete emergence of spike, heat stress was

imposed by covering the plots with perforated polythene sheet (Javed et al., 2014; Kamal

et al., 2017; Shahid et al., 2017). While, in the other main plot, heat was imposed in the

same way on the 50% completion ‘initiation of flowering’. Polythene sheets (heat stress)

in both main plots were removed at the same time i.e. on 50% ‘initiation of grain filling’.

One main plot was also left in open environment as ‘control/no heat stress’. Recorded

temperatures are given in tabulated form (Table 3.4 and Table 3.5).

Different concentrations of potassium and selenium as per treatments were applied

after the imposition of heat stress on ‘flowering initiation’. Potassium and selenium were

foliar applied with the help of a hand sprayer at the rate of 300 liter per hectare.

Potassium was foliar applied using source ‘potassium nitrate (KNO3) (K = 36.52%, K2O

= 44%) and selenium was applied using ‘sodium selenate (Na2SeO4) (Se= 41.79%). Leaf

samples were collected 1 day after removing of heat stress, stored in liquid nitrogen and

processed to record various attributes.

(c) Parameters recorded


1. Number of fertile tillers per m2

2. Number of grains per spike

3. 1000-grain weight (g)

4. Grain yield (t ha-1)

Biomass accumulation

1. Biological yield (t ha-1)

2. Harvest index (%)

3. Straw yield (t ha-1)

4. Plant height (cm)

Growth of spike

1. Spike length (cm)

2.Spikelets per spike

3. Grain filling rate (g per day) (Hunt, 1978)

24

4. Grain filling duration (days) (Hunt, 1978)


1. Chlorophyll a contents (mg g-1 FW) (Arnon, 1949)

2. Chlorophyll b contents (mg g-1 FW) (Arnon, 1949)

3. Superoxide dismutase (U mg-1 protein) (Giannopolitis and Ries, 1977)

4. Peroxidase (U mg-1 protein) (Liu et al., 2009)

5. Catalase (U mg-1 protein) (Liu et al., 2009)

6. Total phenolic contents (mg GAE g-1) (Ainsworth and Gillespie, 2007)


1. Proline (µmol g-1) (Bate et al., 1973)

2. Glycine betaine (µmol g-1) (Grieve and Grattan, 1983)

3. Total soluble proteins (mg g-1) (Bradford, 1976)

4. Malondialdehyde contents (µmol g-1) (Cakmak and Horst, 1991)

Water relations and quality attributes

1. Osmotic potential (-MPa) (Scholander et al., 1964)

2. Water potential (-MPa)

3. Turgor potential (MPa)

4. Shoot potassium contents (µg g-1) (Chapman and Pratt, 1961; Gupta, 1999) (Only for

Experiment II)

5. Grain crude protein contents (%) (Bremner and Mulvaney, 1982; Ryan et al., 2001)

(d) Statistical analysis

Data of recorded attributes were analyzed statistically (p ≤ 0.05) using the Fisher’s

analysis of variance technique (Steel et al., 1997) and Tukey’s Honestly Significant

Difference (Tukey’s HSD) test was employed to compare the means of different

genotypes at 5% probability level. While, type and strength of relationship among the

recorded parameters was determined by calculating correlation among these parameters

using STATISTIX 8.1 software (Gomez and Gomez, 1984). Moreover, regression

analysis was performed to determine trends of response variables and improvements in

different attributes towards different concentrations of foliar spray under varying

treatments of heat stress. Years means were determined for each studied response variable

without pooling of data for two years study period. Microsoft Excel-2016 was used for

graphical work.

(e) Methodologies to record parameters

25


Number of fertile tillers was counted in 30 cm row length at five different places

of each experimental unit and converted into fertile tillers for 1 m2 area through unitary

method. Ten spikes were manually harvested, threshed and average number of grains per

spike was calculated. Five samples of 1000 seeds were randomly taken from the seed lot

of each experimental unit and averaged to calculate thousand seed weight. The crop in

each experimental unit was harvested, threshed and grain yield was weighed and

converted into tons per hectare.

Biomass accumulation

Ten plants in each experimental unit were randomly selected and plant height was

measured from the base of plant to tip of spike with the help of meter rod at maturity. The

biological yield of each experimental plot was weighed using a weighing balance and

converted into tons per hectare. Harvest index was calculated by dividing the grain yield

of each plot by respective biological yield (Gardner et al., 1985).

Harvest index (% )= Grain yieldBiological yield × 100

Straw yield of each treatment was computed by subtracting grain yield from the

respective biological yield.

Growth of spike

Ten spikes were randomly selected in each plot, their length was measured and

averaged. Similarly, spikelets per spike were counted and averaged for ten spikes. To

determine grain filling rate, five spikes were randomly harvested from each plot on

initiation of grain filling at interval of 5 days and their dry weight was recorded. Grain

filling rate was calculated using formula described by Hunt (1978).

Grain filling rate(g per day )=W 2−W 1

t2−t 1

Whereas, ‘W1’ and ‘W2’ represent ‘dry weight’ of spike at the time of ‘first

harvest (t1) and second harvest (t2). Grain filling duration was determined by tagging five

plants in each plot and days taken from grain filling initiation to physiological maturity

were counted (Hunt, 1978).


Green leaf samples were collected randomly from each experimental plot, 0.5 g

sub sample was taken and soaked overnight in 80% acetone. Leaves extracts of 1.5 µL

26

were taken in ELISA plate and absorbance was recorded at 663 and 645 nm. Final

readings of chlorophyll a and b were computed using formulae given by Arnon (1949)

Chla (mg g−1 FW )=[ 12.7 × A 663−2.69× A 645 ] × V1000

×W

Chlb (mg g−1 FW )=[ 22.9 × A 645−4.68 × A 663 ] × V1000

×W

Where ‘A’ indicates ‘absorbance’, ‘V’ ‘volume of extract (mL)’ and ‘W’ ‘weight of

fresh leaves tissue’.

Superoxide dismutase (SOD) contents were quantified as enzyme units that

inhibited photochemical reduction of nitro blue tetrazolium (NBT). Leaf tissues were

extracted using potassium phosphate buffer (pH 4) prepared by dissolving KH2PO4 (7.45

g) + K2HPO4 (1.74 g) + KCl (7.45 g) + EDTA (0.58 g) in 1000 mL DI water. The

reaction mixture was comprised of potassium phosphate buffer (pH 5) + 200 µL

methionine + 200 µL triton X + 100 µL NBT + 800 µL distilled water. Enzyme extracts

of 100 µL volume was mixed with reaction mixture in Eppendorf tubes, placed under

ultraviolet light for 15 minutes and added 100 µL riboflavin and took 100 µL in ELISA

plate and recorded absorbance at 560 nm. Absorbance for blanks (standards) was also

recorded using reaction mixture and riboflavin without adding enzyme extract

(Giannopolitis and Ries, 1977). Regression equation was formed plotting blanks on x-axis

and respective absorbance on y-axis and thus, finalized readings of SOD were computed

from a calibration curve

Y=aX+b

Where, ‘Y’ specifies ‘absorbance of blanks solutions’, ‘X’ ‘final concentration of

SOD of unknown sample, ‘a’ ‘slope between blank and ‘unknown (SOD) sample’ and ‘b’

‘intercept’.

Peroxidase (POD) contents were estimated as enzyme units that oxidize guaiacol.

The same enzyme extracts as used for SOD contents was also used to quantify POD

contents. Reaction mixture for determination of POD was comprised of 800 µL potassium

phosphate buffer (pH 5) + 100 µL H2O2 (40 mM) + 100 µL guaiacol (20 mM). Added

100 µL enzyme extract + 100 µL reaction mixture in Eppendorf tubes, took 150 µL in

ELISA plate and recorded absorbance at 470 nm (Liu et al., 2009).

Catalase (CAT) contents were measured as enzyme units that detoxified H2O2 to

H2O and O2. Same enzyme extracts as prepared to quantify SOD were also used for

determination of CAT contents. Enzyme extract of volume 100 µL + 100 µL H2O2 (5.9

27

mM) were mixed in cuvettes, took 150 µL of mixture in ELISA plate and recorded

absorbance at 240 nm (Liu et al., 2009).

Total phenolic contents were measured by extracting 0.5 g leaf tissues in 10 mL

80% acetone using Folin-Ciocalteu reagent method. Supernatant of 20 µL volume was

shifted in cuvettes. Then added 1.50 mL DI water + 100 µL Folin-Ciocalteu (Rover and

Brown, 2013) reagent, vortexed the cuvettes. Then added 300 µL Na2CO3 (700 mM)

solution in cuvettes and incubated for 2 hours at room temperature (25°C), 150 µL from

cuvettes was shifted to ELISA plate and recorded the absorbance at 760 nm. Gallic acid

(10-100 ppm) was used as standard to develop calibration curve for determination of TPC

and results were reported as gallic acid equivalent (GAE) (Ainsworth and Gillespie,

2007).


Proline was determined by extracting 0.5 g leaf tissues with 3% 5 mL of

sulfosalicylic acid. Obtained leaf extract was centrifuged for 15 minutes. Ninhydrin

solution of concentration 3% was prepared in equal volumes of glacial acetic acid and 6

M orthophosphoric acid. Added 1 mL centrifuged leaf extract + 1 mL glacial acetic acid +

1mL 3% ninhydrin solution prepared in glacial acetic acid and orthophosphoric acid in

cuvettes. The mixture was mixed and incubated at 100°C for 1 hour. Afterwards, the

mixture was cooled in an ice bath, added 1 mL toluene in mixture and vortexed it for 5

minutes. The upper aqueous layer was discarded after vortex and organic layer was

retained. Volume of 150 µL was placed in an ELISA plate and recorded absorbance at

520 nm using toluene as blank for standard curve (Bate et al., 1973).

Glycine betaine was measured homogenizing leaf tissues weighing 0.5 g with 5

mL distilled water and centrifuged the extracts for 5 minutes. Potassium tri-iodide

solution was prepared dissolving 7.5 g iodine + 10 g potassium iodide in 10 mL, 1 M HCl

solvent. Then 1 mL leaf tissue extract + 1 mL HCl (2 M) + 0.1 mL potassium tri-iodide

solution were thoroughly mixed and incubated at 4°C for 1 hour. After it, 5 mL chilled DI

water + 10 mL 1,2- di-di-chloroethane was added and vortexed for 5 minutes. Upper

aqueous layer was discarded, and absorbance was recorded at 365 nm using organic layer

(Grieve and Grattan, 1983).

Total soluble proteins were analyzed by using same enzyme extract of leaves as

was used for SOD determination. Enzyme extract of volume 40 µL + 160 µL Bradford

Reagent was added in ELISA plate and recorded absorbance at 595 nm (Bradford, 1976).

28

To quantify malondialdehyde (MDA), leaf samples of weight 0.5 g were

homogenized with 3 mL 0.1% (w/v) trichloroacetic acid (TCA). Then the samples were

centrifuged for 15 minutes and the supernatant of 0.5 mL volume was transferred in a test

tube. Then 3 mL 20% TCA solution containing 0.5% thiobarbituric acid was added to the

supernatant. Afterwards, the mixture was incubated at a temperature of 70°C for30

minutes and cooled with an ice water bath. Mixture containing leaf extracts and blanks

were added in ELIZA plate taking volume of 150 µL each and recorded absorbance at

532 nm and 600 nm (Cakmak and Horst, 1991).

Water relations and quality attributes

Leaves were collected early in the morning between 6-8 am randomly from each

plot. Leaves were placed in Scholandar pressure gauge (ARIMAD-2, ELE, International)

and pressure was applied until drop of sap appeared on midrib. Pressure applied from

pressure gauge was considered equal to water potential (ΨW) of leaf organs. Leaves used

in water potential determination were frozen at -4°C and ground to obtain cell sap which

was taken to osmometer (Wescor 5520) and recorded osmotic potential (ΨS). Turgor

potential was determined by subtracting osmotic potential from water potential

(Scholander et al., 1964).

Ψ P=Ψ W−Ψ S

Wheat shoots were collected at physiological maturity, sun dried, oven dried and

ground to powder form. Ground powder weighing 0.5 was digested with nitric acid per-

chloric acid (HNO3: HCLO4 in 2:1 ratio). The mixture was heated at 60°C to complete

reaction until synthesis of fumes from reaction mixture was stopped. Then the mixture

was heated in a digestion chamber at temperature of 120°C until clear aliquot was

obtained. DI water was added to make volume of a 100 mL. Stock solution of

concentration 1000 ppm was diluted to make concentrations of 0, 25, 50, 75 and 100

ppm. A regression (calibration) curve was developed plotting different concentrations on

x-axis and respective absorbances on y-axis. Leaf samples were also loaded in a Flame

photometer and recorded absorbances for different samples. Final readings of shoot

potassium contents (µg g-1) were computed from the regression equation (Chapman and

Pratt, 1961; Gupta, 1999).

Grain crude proteins were quantified by using the method of Gunning and

Hibbard. Sulphuric acid was used for digestion of wheat flour and it was followed by

distillation of NH3 in boric acid with the help of Kjeldhal apparatus. Grains were milled to

29

form flour and 1 g flour was taken in digestion tubes. Together with it, 25 mL

concentrated H2SO4 + 5 g digestion mixture (K2SO4 + FeSO4 + CuSO4 in 85: 10: 5 ratio)

were added. Digestion tubes were heated on digestion block at 400°C until clear liquid

was obtained. Then DI water added to make total volume 250 mL and 10 mL from

digested and clear aliquot was taken to distillation unit. In the receiver flask of distillation

unit, 10 mL 4% boric acid was taken and added a few drops of methyl red indicator.

Upon distillation, the colour of boric acid in receiver flask was changed from purple to

golden yellow. After it, boric acid was titrated against 0.1 N H2SO4 to get purple endpoint

from golden yellow colour of boric acid and computed grain crude proteins (Jackson,

1962).

Nitrogen (%) =

0.0014 ×Titrant for sample (mL )−Titrant for blank (mL ) 0.1N H2 SO4 ×250 (DF)Sample weight (g )× Aliquot volume used∈distillation

Whereas DF represents dilution factor if there is any.

Graincrude proteins contents ( %)=%N ×5.83

Whereas 5.83 is constant for wheat (Bremner and Mulvaney, 1982; Buresh et al.,

1982; FAO, 2003).

30

RESULTS AND DISCUSSION CHAPTER-4

Experiment I: Biochemical markers assisted screening of wheat cultivars for

terminal heat stress tolerance

Heat stress had an overall deleterious effect at reproductive stages of wheat.

However, cultivars specific response was evident on different growth, yield, and

biochemical attributes. However, heat stress, genotypes and their interaction unveiled

distinctive response under controlled and stressed conditions and resulted in significant

heat × genotypes effect for various parameters.

4.1.1. Yield components and grain yield

(a) Results

Number of fertile tillers did not differ significantly in control and heat stress main

plots. However, cultivars significantly varied from each other. All cultivars showed

undistinguishable trend in both main plots to record non-significant interaction. The

highest number of fertile tillers was observed for ‘Punjab-2011’ (377.13 m-2) and it was

statistically alike to genotypes ‘AARI-2011’, ‘Galaxy-2013’, ‘AAS-2011’ and ‘Pakistan-

2013’. Genotype ‘Kohistan-97’ produced minimum number of fertile tillers (278.88 m -2)

(Table 4.1.1).

Heat stress and genotypes manifested significant distinction for number of grains

per spike. The heat × variety interaction was significant as varieties revealed unlike

response in ambient and heat imposed conditions. Negative implications of high

temperature were apparent for number of grains per spike. Different cultivars exhibited

diverse performance when assayed for number of grains per spike. Maximum decrease in

the number of grains per spike under high temperature over control was obtained for

31

‘Kohistan-97’ (21%). Minimum decline in number of grains per spike in stressed

conditions was revealed by genotypes ‘Aas-2011’ and ‘Chakwal-50’ (10% for both)

(Table 4.1.2).

Weight of thousand grains was significantly diminished owing to adverse

consequences of high temperature while varied response of cultivars was also evident.

Nonetheless, all genotypes depicted incompatibility in control and heat stress to cause

significant interaction of heat and varieties. Highest decline in 1000-grain weight under

heat stress over ambient conditions was recorded for genotype ‘AARI-2011’ (35%) and

‘Pakistan-2013’ (33%) (Table 4.1.2).

Table 4.1.1: Effect of heat stress on fertile tillers of wheat varieties

A. Mean sum of square

Source of variation DF Fertile tillers

Blocks 3 10854.7

Heat (H) 1 4756.9NS

Error I 3 588.6

Genotypes (V) 10 10817.4**

H × V 10 125.5NS

Error II 60 570.4** = Significant (p ≤ 0.01); NS = Non-significant

B. Comparison of treatments’ means

Treatments Fertile tillers per m2

Heat stress (H)

No heat stress (H0) 334

Heat from spike to grain filling (H1) 320

Tukey’s HSD (p ≤ 0.05) NS

Genotypes (V)

Punjab-2011 377 A

AARI-2011 343 AB

Galaxy-2013 367 AB

Millat-2011 301 CD

32

Aas-2011 367 AB

Fareed-2006 286 D

Chakwal-50 288 D

Mairaj-2008 335 BC

Pakistan-2013 353 AB

NIBGE-NIAB-1 300 CD

Kohistan-97 279 D

Tukey’s HSD (p ≤ 0.05) 39.95Any two means not sharing a letter in common differ significantly at p ≤ 0.05; NS = Non-significant

Table 4.1.2: Effect of heat stress on grains per spike and 1000-grain weight of wheat varieties


Source of variation DF Grains per spike 1000-grain weight

Blocks 3 483.10 2074.75Heat (H) 1 1071.01** 1902.78*Error I 3 8.28 76.55**Genotypes (V) 10 247.07** 96.25H × V 10 9.51* 9.49**Error II 60 4.45 3.42

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01)


Treatments Grains per spike 1000-grain weight (g)

No heat stress (H0)Punjab-2011 55.7 a 31.3 eAARI-2011 41.7 cde 35.3 cdeGalaxy-2013 42.5 cde 33.2 deMillat-2011 43.5 cd 39.8 abAas-2011 53.5 ab 33.9 cdeFareed-2006 44.0 c 40.8 abChakwal-50 49.2 b 41.9 aMairaj-2008 52.2 ab 33.7 cdePakistan-2013 39.0 de 37.8 abcNIBGE-NIAB-1 38.5 e 37.0 bcdKohistan-97 44.2 c 37.4 bcdHeat from spike to grain filling (H1)Punjab-2011 45.0 ab 22.1 f

33

AARI-2011 37.0 d 23.1 efGalaxy-2013 37.0 d 23.8 defMillat-2011 38.2 cd 29.0 bcAas-2011 48.0 a 27.2 cdeFareed-2006 38.0 cd 32.0 abChakwal-50 44.2 ab 34.9 aMairaj-2008 42.0 bc 27.5 cdPakistan-2013 32.0 e 25.1 cdefNIBGE-NIAB-1 31.0 e 26.1 cdefKohistan-97 35.0 de 28.9 bcTukey’s HSD (p ≤ 0.05) 4.99 4.37

Any two means not sharing a letter in common within a column differ significantly at p ≤ 0.05

Lowest diminishment in 1000-grain weight under heat compared to control was

observed for ‘Chakwal-50’ (14%). Genotypes ‘Millat-2011’, ‘Fareed-2006’ and ‘Pakistan

2013’ were statistically alike to ‘Chakwal-50’ in non-stressed environment. Contrarily,

cultivars ‘Millat-2011’ and ‘Pakistan 2013’ produced significantly lesser 1000-grain

weight than ‘Fareed-2006’ and ‘Chakwal-50’ under high temperature stress (Table 4.1.2).

Grain yield (GY) was significantly decreased in heat treatment relative to control.

Distinctive genetic makeup of genotypes was statistically obvious denoting their different

capability to produce yield in ambient and high temperature environment. Among the

cultivars, ‘Aas-2011’ exhibited highest GY (3.71 t ha-1) under high temperature

environment. ‘Aas-2011’ was statistically identical to ‘Mairaj-2008’ and ‘Chakwal-50’

under heat stressed conditions. Cultivars ‘Pakistan 2013’, ‘NIBGE-NIAB-1’ and

‘Kohistan-97’ manifested 2.21, 2.03 and 2.24 t ha-1 GY, respectively under heat

representing heat vulnerability of these cultivars. All other cultivars produced GY

midway between described cultivars signifying medium tolerance to heat (Table 4.1.3).

(b) Discussion

Diverse response regarding number of fertile tillers can be attributed to distinct

genetic makeup of genotypes. Different genotypes depicted diverse genetic pattern of

nodal roots (Gulnaz et al., 2011). Varying number of productive tillers among genotypes

can be ascribed to different carbohydrates partitioning capability of genotypes towards

nodal roots (Albokari et al., 2016).

Diminution in number of grains per spike under heat stress can be elucidated in

context of rapid development of ridges of wheat spike under aggravated membrane

damage. Increment in growth rate might have reduced proline and glycine betaine

contents. Subsequently, aggravated malondialdehyde might decline number of grains per

34

spike. While, highest decrement in number of grains per spike for ‘Kohistan-97’ under

heat over control can be ascribed to adverse implications of heat on accumulation of

proline, glycine betaine and total phenolic contents. Declined proline contents might

reduce pollen viability and germination. Reduced accumulation of proline and glycine

betaine might aggravate membrane damage in Kohistan-97 that adversely affected

number of grains per spike. Likewise, lower accumulation of total phenolic contents

might have aggravated malondialdehyde (MDA) accumulation resulting into

diminishment in number of grains. It can be attributed to capability of these genotypes to

accumulate higher glycine betaine, proline and total phenolic contents under heat over

control.

Table 4.1.3: Effect of heat stress on grain yield of wheat varieties


Source of variation DF Grain yield

Blocks 3 21.31Heat (H) 1 60.40**Error I 3 0.17Genotypes (V) 10 2.49**H × V 10 0.26**Error II 60 0.09

** = Significant (p ≤ 0.01)


Treatments Grain yield (t ha-1)

No heat stress (H0)Punjab-2011 5.09 abAARI-2011 4.22 cGalaxy-2013 4.15 cMillat-2011 4.06 cAas-2011 5.22 aFareed-2006 4.19 cChakwal-50 4.95 abMairaj-2008 4.46 bcPakistan-2013 4.10 cNIBGE-NIAB-1 3.35 dKohistan-97 3.31 d

35

Heat from spike to grain filling (H1)Punjab-2011 2.71 bcdAARI-2011 2.20 dGalaxy-2013 2.26 dMillat-2011 2.40 cdAas-2011 3.71 aFareed-2006 2.73 bcdChakwal-50 3.36 abMairaj-2008 3.04 abcPakistan-2013 2.21 dNIBGE-NIAB-1 2.03 dKohistan-97 2.24 dTukey’s HSD (p ≤ 0.05) 0.709


Moreover, a strong positive association of leaf proline, glycine betaine and total

phenolic contents was observed with number of grains per spike. Whereas, more negative

correlation of malondialdehyde was observed under heat compared to control. Hence,

malondialdehyde induced a decrease in the number of grains per spike under heat was

also confirmed (Table 4.1.11 and Table 4.1.12). Enhanced accumulation of

malondialdehyde declined biosynthesis of proline and other osmo-protectants.

Subsequently, diminution in pollen fertility diminished number of grains (Paupière et al.,

2014). Enhanced proline biosynthesis eased pollen germination and micro-sporogenesis

(Mattioli et al., 2012). Our results are comparable to those of Ghaffari et al. (2015) that

high temperature impaired photosystem-II, enhanced photorespiration, lipid peroxidation,

chlorophyll degradation and eventually resulted in reduced number of grains per spike.

High temperature stress might have promoted pollen sterility and thus reduced the

number of grains per spike (Ghaffari et al., 2015). Furthermore, dissimilar response of

genotypes can be ascribed to genetic differences. According to Nawaz et al. (2015),

different genotypes depicted varied response for number of grains per spike.

The capability of ‘Chakwal-50’ to sustain 1000-grain weight can be ascribed to

lower malondialdehyde synthesis. Lower malondialdehyde might decrease lipid

peroxidation. Consequently, partitioning of carbohydrates was sustained for longer time

and hence 1000-grain weight was improved. Genotypes with capability to hamper

malondialdehyde depicted higher 1000-grain weight, water potential and membrane

integrity (Jain, 2013). Likewise, capability to accumulate GB enhanced tolerance for

stressed environments and it differed among genotypes (Raza et al., 2015). Highest

diminishment in 1000-grain weight of cultivars ‘AARI-2011’ and ‘Pakistan-2013’ under

36

high temperature environment can be elucidated in the context of reduced accumulation

of glycine betaine and other secondary metabolites. The decline in glycine betaine might

be a consequence of impaired photosynthesis and enhanced lipid peroxidation under heat

stress (Reguera et al., 2012). Enhanced phenolic might have contributed to sustain

temperature, thus alleviated heat mediated denaturation of starch accumulating enzymes.

Declined accumulation of proline and glycine betaine may disturb water relations of

plants and consequently cause a decrease in grain weight (Almeselmani, 2009).

‘Chakwal-50’ showed the smallest decline in grain weight under heat stress, which may

be ascribed to reduced peroxidation of membrane lipids and augmented proline, glycine

betaine and total phenolic contents. Diminished malondialdehyde synthesis confirmed

boosted membrane stability for ‘Chakwal-50’. Genotype ‘Chakwal-50’ might have

established tolerance against heat interceded disintegration of mesophyll membranes. It

eventually augmented carbohydrates availability for grain filling (Ghaffari et al., 2015).

Moreover, significant negative association of malondialdehyde with 1000-grain weight

under heat established enhanced lipid peroxidation and associated adversities. Similarly,

strong negative association of leaf proline and glycine betaine with malondialdehyde

further accomplished proline and glycine betaine role in maintaining 1000-grain weight

under heat stress (Table 4.1.11 and Table 4.1.12).

Degradation of chlorophyll under high-temperature environment might reduce

yield by decreasing assimilate availability for grain filling. The decrement in antioxidant

activity under heat stress might be related to enhanced oxidative stress causing more

reduction in yield in cultivars AARI-2011, Galaxy-2013 and Punjab-2011 than tolerant

genotypes Aas-2011, Chakwal-50 and Mairaj-2008. Less heat mediated reduction in yield

for cultivars Aas-2011, Chakwal-50, and Mairaj-2008 might be due to their capability to

maintain higher CHL a and b contents under heat than other cultivars. Moreover, strong

positive and significant association of antioxidants and chlorophyll contents further

confirmed the role of these attributes in grain yield in different genotypes (Table 4.1.11

and Table 4.1.12). Our results correspond to those of Innes et al. (2015), where they

recorded a 15% reduction in grain yield (GY) per annum due to the higher temperature.

Every 1ºC rise in temperature declined GY by 5.3%. The decrement in GY is also alike to

findings of Tao et al. (2015); a three-decade long experiment was conducted. Wheat

varieties were exposed to the temperatures above 34ºC from booting to maturity. High

temperature decreased GY and reduced the growing period in all wheat genotypes.

37

High grain yields of genotypes ‘Aas-2011’, ‘Mairaj-2008’ and ‘Chakwal-50’ can

be attributed to higher number of grains per spike. The role of grains number per spike in

enhancing yield under heat and control was confirmed from their strong positive

correlation of grain yield with grains per spike (Table 4.1.11 and Table 4.1.12).

Significantly lower malondialdehyde synthesis was manifested by genotypes ‘Aas-2011’,

‘Mairaj-2008’ and ‘Chakwal-50’. A strong negative association of malondialdehyde with

grain yield further established malondialdehyde triggered decline in grain yield (Table

4.1.11 and Table 4.1.12). Reduced lipid peroxidation and oxidative stress might

contribute to enhanced yield (Wormuth et al., 2007). Membrane integrity under stressed

environment is a direct indicator of heat tolerance. Wheat seedlings with lower

malondialdehyde were less prone to oxidative stress and ultimately produced higher yield

(Savicka and Skute, 2010). Higher yield of genotypes ‘Aas-2011’, ‘Mairaj-2008’ and

‘Chakwal-50’ can be attributed to capability of these genotypes to manifest increment in

proline, glycine betaine and total phenolic contents under high temperature. Enhanced

phenolic contents in genotypes ‘Aas-2011’, ‘Mairaj-2008’ and ‘Chakwal-50’ might

increase heat tolerance. Cultivars ‘Pakistan-2013’, ‘NIBGE-NIAB-1’ and ‘Kohistan-97’

manifested comparatively more decrement in proline and total phenolic contents under

heat over the control. It could be another reason for heat susceptibility of these cultivars.

Moreover, strong positive correlation of total phenolic contents, glycine betaine and

proline with grain yield under ambient and heat stress conditions confirmed role of these

metabolites in sustaining grain yield (Table 4.1.11 and Table 4.1.12). Genotypes capable

to accumulate proline, glycine betaine and diminish malondialdehyde under stress

compared to no stress were heat tolerant (Fardus et al., 2014).

4.1.2. Growth of spike

(a) Results

Heat stress (H) and varied performance of genotypes (V) significantly affected

grain filling rate (GFR) and grain filling duration (GFD). Whereas, the same trend

amongst all cultivars was observed in both main plots resulting in non-significant

interactions (H × V) for GFR and GFD. Heat stress accelerated GFR by 33% while

diminished GFD by 27%. Concerning GFR, Galaxy-2013 recorded the highest GFR (0.16

g per day) and it was statistically similar to Punjab-2011, AARI-2011, Pakistan-2013,

NIBGE-NIAB-1 and Kohistan-97. Significantly lower GFR was noted for cultivars Aas-

2011 and Chakwal-50. Regarding GFD, the cultivar Aas-2011 recorded highest value

(37.59 days). Aas-2011 was statistically comparable to Chakwal-50 and Mairaj-2008. The

38

genotype NIBGE-NIAB-1 recorded the lowest GFD (24.72 days) and it was statistically

alike to Millat-2011, Pakistan-2013 and Kohistan-97. The high-temperature environment

caused rapid grain filling rate (GFR) and diminished grain filling duration (GFD). Even

though, genotypes Aas-2011 and Chakwal-50 maintained significantly lower GFR than

all other cultivars. Genotypes Aas-2011 and Chakwal-50 manifested significantly lowest

decline in GFD against maximum in cultivars Punjab-2011 and AARI-2011 (Table 4.1.4).

(b) Discussion

Minimum GFR and maximum GFD in Aas-2011 and Chakwal-50 can be

explained in the context of high chlorophyll content and enhancement of antioxidant

defense system under heat. Higher chlorophyll contents might maintain assimilate

partitioning for the longer duration of time. Declined chlorophyll degradation might be a

consequence of detoxification of ROS in cultivars AAS-2011 and Chakwal-50.

Table 4.1.4: Effect of heat stress on grain filling rate (GFR) and grain filling duration (GFD) of wheat varieties


Source of variation DF Grain filling rate Grain filling duration

Blocks 3 0.00038 7.14

Heat (H) 1 0.05600** 2132.53**

Error I 3 0.00038 7.99

Genotypes (V) 10 0.00519** 178.51**

H × V 10 0.00035NS 4.34NS

Error II 60 0.00030 4.60** = Significant (p ≤ 0.01); NS = Non-significant


Treatments GFR (g per day) GFD (days)

Heat stress (H)

No heat stress (H0) 0.10 B 35.86 A

Heat from spike to grain filling (H1) 0.15 A 26.01 B

Tukey’s HSD (p ≤ 0.05) 0.013 1.918

Genotypes (V)

Punjab-2011 0.14 AB 32.29 CD

AARI-2011 0.14 AB 29.10 DE

39

Galaxy-2013 0.16 A 31.67 CD

Millat-2011 0.12 B 27.36 EF

Aas-2011 0.08 C 37.59 A

Fareed-2006 0.12 B 32.89 BC

Chakwal-50 0.08 C 36.69 A

Mairaj-2008 0.12 B 36.46 AB

Pakistan-2013 0.14 AB 25.22 F

NIBGE-NIAB-1 0.14 AB 24.71 F

Kohistan-97 0.14 AB 26.29 EF

Tukey’s HSD (p ≤ 0.05) 0.029 3.586Any two means not sharing a letter in common differ significantly at p ≤ 0.05

The highest grain yields obtained in AAS-2011 and Chakwal-50 further confirmed

the contribution of chlorophyll contents in maintaining GFR and GFD. A strong positive

correlation (0.93) between GFD and grain yield under heat established contribution of

longer GFD in grain yield (Table 4.1.11 and Table 4.1.12). Furthermore, enhanced

antioxidants and TSPs were observed for AAS-2011 and Chakwal-50 under heat over

control. Thus, improved defense might have maintained growth rate of grains and

consequence in higher grain yield. Under heat stress, higher flux intensity and greater

difference in maximum day temperature and minimum night temperature caused rapid

morphogenesis. The increment in GFR can also be considered an adaptive behavior to

complete growing cycle rapidly and produce seed for the upcoming generation. Each 5ºC

rise of temperature above 20ºC resulted in increased GFR and reduced GFD by 12 days in

wheat. Moreover, every 1ºC increase in temperature declined GFD by 2.8 days, enhanced

ROS, lipid peroxidation and decreased chlorophyll contents (Talukder et al., 2014).

Enhanced GFR and diminished GFD might lead to a decline grain yield in

susceptible varieties. Thus, enhanced GFR could not compensate for diminished

phenology. Rapid GFR and declined GFD might have adversely affected grain yield.

Negative correlation of grain yield with GFR (- 0.82) under high temperature

environment further accomplished the adverse effects of rapid grain filling (Table 4.1.12).

Reduction in phenology might have reduced assimilates partitioning towards grains.

Pollen grains in wheat were not able to produce heat shock proteins and thus are highly

sensitive to rise in temperature. Consequently, decreased grain setting negatively affected

grain yield under high temperature environments (Hasanuzzaman et al., 2013).

40

4.1.3. Stay green and antioxidants

(a) Results

Different wheat genotypes manifested significant difference for chlorophyll

(CHL) contents. High-temperature stress had the negative impact on chlorophyll pigment.

Heat stress mediated diminishment compared to control in CHL a and CHL b contents

was 33% and 38% (averaged across 11 genotypes), respectively. All cultivars showed a

similar trend under heat and no heat-induced conditions to depict a non-significant

interaction. Cultivars varied response was apparent as Aas-2011 recorded maximum CHL

contents (2.08 mg g-1) and it was statistically alike the cultivars Chakwal-50 and Mairaj-

2008. Similarly, the wheat cultivar Aas-2011 recorded highest CHL b contents (0.58 mg

g-1) and was statistically like Chakwal-50. Conversely, minimum CHL contents was

observed for cultivar NIBGE-NIAB-1 (CHL a 1.01 mg g-1) (CHL b 0.15 mg g-1). Wheat

cultivar NIBGE-NIAB-1 depicted greater reduction than other genotypes in CHL a (52%)

and CHL b (53%) contents. Contrarily, genotypes Chakwal-50, Mairaj-2008, Aas-2011,

and Fareed-2006 recorded almost similar response and lesser decline in CHL a (15-22%)

and CHL b (27-35%) content than other genotypes were observed (Table 4.1.5).

Dissimilar performance of different wheat genotypes for antioxidant activities was

observed in main plots and resulted in significant interaction of genotypes and heat stress.

Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and total phenolic

contents (TPC) activities of Aas-2011, Chakwal-50 and Mairaj-2008 was enhanced under

heat over ambient conditions. Whereas, all other cultivars recorded diminishing trend in

enzymatic activity under stressed environment over control (Table 4.1.6 and Table 4.1.7).

Regarding SOD activity, Aas-2011, Chakwal-50 and Mairaj-2008 depicted an

increase of 12, 14 and 14%, respectively under heat stress over control. In control Aas-

2011 and Chakwal-50 were statistically alike while in heat stress Aas-2011, Chakwal-50

and Mairaj-2008 remained at par. Galaxy-2013 and Millat-2011 depicted maximum

decline (38 and 40%, respectively) in SOD activity under heat compared to no heat

environment. The cultivars Aas-2011, Chakwal-50 and Mairaj-2008 exhibited 17, 15 and

24% enhancement in POD activity, respectively under heat-induced conditions over no

heat imposition. Under high-temperature maximum diminishment (52%) in POD activity

was observed for Punjab-2011 and Galaxy-2013. Under high-temperature stress, Aas-

2011, Chakwal-50 and Mairaj-2008 recorded significantly higher POD activity than all

other genotypes (Table 4.1.6).

41

Cultivars Aas-2011, Chakwal-50, and Mairaj-2008 depicted an increase of 19, 15

and 16% respectively in CAT activity under high-temperature stress environment over

control. The highest reduction in CAT activity was recorded for genotypes Punjab-2011

(50%) and Galaxy-2013 (46%). Aas-2011, Chakwal-50, and Miaraj-2008 manifested

significantly higher SOD, POD and CAT activity than all other cultivars in heat stressed

main plots. Likewise, almost similar trend was observed in control. Regarding total

phenolic contents (TPC), a boost of 22, 20 and 24% was manifested by genotypes ‘Aas-

2011’, ‘Chakwal-50’ and ‘Mairaj-2008’ respectively. Under ‘no heat stress’, ‘Aas-2011’

and ‘Chakwal-50’ depicted significantly higher TPC and were statistically alike to one

another. Under stressed conditions, ‘Aas-2011’ and ‘Chakwal-50’ were statistically

similar whereas ‘Mairaj-2008’ was statistically analogous to ‘Chakwal-50’. Maximum

decline in TPC in stressed environment over control was depicted by ‘Kohistan-97’

(67%) and ‘Pakistan-2013’ (65%) (Table 4.1.7).

Table 4.1.5: Effect of heat stress on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of wheat varieties


Source of variation DF Chlorophyll a Chlorophyll b

Blocks 3 7.76 0.117

Heat (H) 1 7.35** 0.482**

Error I 3 0.19 0.009

Genotypes (V) 10 1.10** 0.212**

H × V 10 0.04NS 0.002NS

Error II 60 0.04 0.002** = Significant (p ≤ 0.01); NS = Non-significant


Treatments Chl a (mg g-1 FW) Chl b (mg g-1 FW)

Heat stress (H)

No heat stress (H0) 1.75 A 0.39 A

Heat from spike to grain filling (H1) 1.18 B 0.24 B

Tukey’s HSD (p ≤ 0.05) 0.298 0.064

Genotypes (V)

Punjab-2011 1.53 CDE 0.32 C

42

AARI-2011 1.28 EFG 0.24 DE

Galaxy-2013 1.40 DEF 0.28 CD

Millat-2011 1.18 FG 0.19 EF

Aas-2011 2.08 A 0.58 A

Fareed-2006 1.71 BCD 0.40 B

Chakwal-50 1.92 AB 0.57 A

Mairaj-2008 1.82 ABC 0.46 B

Pakistan-2013 1.15 FG 0.15 F

NIBGE-NIAB-1 1.01 G 0.15 F

Kohistan-97 1.07 G 0.16 F

Tukey’s HSD (p ≤ 0.05) 0.320 0.072Any two means not sharing a letter in common differ significantly at p ≤ 0.05

Table 4.1.6: Effect of heat stress on superoxide dismutase (SOD) and peroxidase (POD) of wheat varieties


Source of variation DF Superoxide dismutase Peroxidase

Blocks 3 15226.7 55.72Heat (H) 1 7103** 128.87**Error I 3 142.3 3.74Genotypes (V) 10 15882.3** 342.18**H × V 10 1673.9** 47.09**Error II 60 35.9 1.13



Treatments SOD (U mg-1 protein) POD (U mg-1 protein)

No heat stress (H0)Punjab-2011 143.8 b 17.0 cAARI-2011 108.5 d 11.5 efGalaxy-2013 124.4 c 13.9 deMillat-2011 95.5 de 10.2 fgAas-2011 168.9 a 21.3 aFareed-2006 144.4 b 15.5 cdChakwal-50 166.0 a 19.8 ab

43

Mairaj-2008 155.4 ab 17.8 bcPakistan-2013 84.0 ef 8.0 ghNIBGE-NIAB-1 79.2 f 7.0 hKohistan-97 81.4 ef 8.1 ghHeat from spike to grain filling (H1)Punjab-2011 96.5 c 8.1 bcAARI-2011 69.1 de 6.7 cdGalaxy-2013 76.8 d 6.7 cdMillat-2011 57.3 ef 5.3 dAas-2011 192.1 a 25.8 aFareed-2006 112.9 b 10.1 bChakwal-50 192.8 a 23.3 aMairaj-2008 179.9 a 23.5 aPakistan-2013 62.2 ef 4.6 dNIBGE-NIAB-1 54.3 f 4.2 dKohistan-97 59.8 ef 5.3 dTukey’s HSD (p ≤ 0.05) 14.17 2.51


Table 4.1.7: Effect of heat stress on catalase (CAT) and total phenolic contents (TPC) of wheat varieties


Source of variation DF Catalase Total phenolics

Blocks 3 224.34 87.14Heat (H) 1 375.8* 93.26**Error I 3 23.27 0.78Genotypes (V) 10 1170.58** 488.87**H × V 10 143.30** 51.09**Error II 60 2.55 0.74



Treatments CAT (U mg-1 protein) TPC (mg GAE g-1)

No heat stress (H0)Punjab-2011 34.4 c 13.4 cdAARI-2011 24.6 e 8.8 efGalaxy-2013 29.7 d 12.6 dMillat-2011 20.6 f 11.1 deAas-2011 38.6 ab 21.4 aFareed-2006 35.1 bc 16.0 bChakwal-50 39.0 a 20.8 a

44

Mairaj-2008 37.7 abc 18.0 bPakistan-2013 15.9 g 7.5 fgNIBGE-NIAB-1 14.1 g 4.6 hKohistan-97 15.6 g 5.9 ghHeat from spike to grain filling (H1)Punjab-2011 18.9 c 7.8 dAARI-2011 15.6 cd 3.9 efgGalaxy-2013 16.1 c 6.3 deMillat-2011 11.9 de 4.8 efAas-2011 47.1 a 27.3 aFareed-2006 26.0 b 11.0 cChakwal-50 46.4 a 26.1 abMairaj-2008 45.1 a 23.8 bPakistan-2013 11.3 e 2.6 fghNIBGE-NIAB-1 10.3 e 1.9 hKohistan-97 11.0 e 2.0 ghTukey’s HSD (p ≤ 0.05) 3.78 2.03


(b) Discussion

The reduction in chlorophyll contents under the elevated temperature can be

explained in the context of increased rate of chlorophyll degradation over its biosynthesis.

High-temperature stress-induced chlorophyll degradation might be an outcome of

impaired biosynthesis of total soluble proteins and antioxidants. Similar performance of

cultivars Chakwal-50, Aas-2011 and Mairaj-2008 might be related to the stay green trait

under heat. Higher chlorophyll a and b established the stay green trait. Capability to

maintain chlorophyll structure and function might improve the heat tolerance of cultivars.

A strong positive correlation of chlorophyll a and b contents with number of grains per

spike, grain yield and other biochemical attributes further accomplished role of stay green

in enhancing heat tolerance (Table 4.1.11 and Table 4.1.12). Declining

protochlorophyllide reduced the rate of chlorophyll biosynthesis over degradation

because of depressed total soluble proteins (TSP) and antioxidant under heat

(Hemantaranjan et al., 2014). Decline in chlorophyll contents under stress might be due to

the accelerated production of reactive oxygen species (ROS) that increased thylakoid

membrane leakiness of photosystem-II and disrupted chlorophyll structure (Raza et al.,

2015). Prolonged high temperature suppressed chloroplast enzymatic activity and

restricted rate of de novo chlorophyll synthesis (Wang et al., 2015).

Antioxidant defense system of varieties Aas-2011, Chakwal-50, and Mairaj-2008

manifested improvement against decline for other cultivars. Heat stress might have

45

aggravated oxidative stress and thus antioxidants activities were improved in Aas-2011,

Chakwal-50, and Mairaj-2008 as an adaptation to detoxify excessive reactive oxygen

species (ROS). Contrarily, antioxidant activities were not strong enough to scavenge

excessive ROS in all other cultivars. Consequently, increased biosynthesis of ROS

overcame defensive mechanism and thus heat impacts were more pronounced. High

temperature might increase production of superoxide (1O2●-) radical. Superoxide radical

might act as the substrate for SOD. Moreover, strong association of SOD and POD (0.99)

under high temperature environment confirmed accelerated synthesis of 1O2●- (Table

4.1.11 and Table 4.1.12). The increment in SOD activity in tolerant cultivars might be an

adaptive response to high temperature. For susceptible cultivars, decrement in SOD

activity under high temperature can be attributed to lower efficacy of scavenging

mechanism of susceptible wheat cultivars. Decline in SOD activity might be due to

declined chlorophyll a and b contents. Thus, diminished chlorophyll a and b contents

confirmed the diminished SOD activity. While, Aas-2011, Chakwal-50, and Mairaj-2008

still maintained higher chlorophyll contents than other cultivars.

Enhancement in POD and CAT activity under high temperature over control was

recorded for genotypes Aas-2011, Chakwal-50 and Mairaj-2008. It can be attributed to

enhanced SOD activities of genotypes Aas-2011, Chakwal-50 and Mairaj-2008.

Enhanced generation of 1O2●- might have enhanced H2O2 levels in leaves under high-

temperature environment. Subsequently, detoxification of H2O2 to H2O and O2 in plants

was mediated by POD and CAT. Greater H2O2 production in heat stress might have

resulted in the increment of POD and CAT activity as the defensive mechanism against

stress in tolerant cultivars. Strong positive and highly significant association (0.99)

between POD and CAT under stressed conditions confirmed the escalated generation of 1O2

●- and H2O2 (Table 4.1.11 and Table 4.1.12). The varied response of susceptible

genotypes under high-temperature stress might be due to the dissimilar genetic capability

for heat tolerance. Decline in SOD, POD and CAT activity in cultivars Punjab-2011,

AARI-2011, Galaxy-2013, Millat-2011, Fareed-2006, Pakistan-2013, NIBGE-NIAB-1,

and Kohistan-97 can be defined in the context of poor antioxidant defense system due to

excessive generation of reactive oxygen species (ROS). Cultivars Galaxy-2013, Punjab-

2011, and Millat-2011 depicted the highest decline in antioxidant activities under stressed

environment over non-stressed environment. It can be interrelated to the inability of these

cultivars to counteract production of superoxide (1O2●-), hydroxyl (OH●-), singlet oxygen

(1O2٭) and hydrogen peroxide (H2O2) under high-temperature environment.

46

Moreover, higher SOD activity for Aas-2011, Chakwal-50, and Mairaj-2008 and

decreased SOD for other cultivars confirmed excessive generation of 1O2●-. It can also be

elucidated in terms of autocatalytic peroxidation of membrane lipid and degradation of

chlorophyll together with other pigments resulting into significant cell damage. Declined

CHL a and b contents under heat have confirmed enhanced lipid peroxidation.

Furthermore, chlorophyll mediated boost in SOD activity under heat was further

confirmed from strong association of CHL a (0.96) and b (0.97) with SOD activities

(Table 4.1.11 and Table 4.1.12). Naderi et al. (2014) observed that heat tolerant cultivars

recorded enhancement while susceptible cultivars depicted decline in SOD activity.

Tolerant spring wheat genotypes depicted increasing while susceptible recorded

diminishing trends in antioxidant activities under heat over ambient conditions (Iqbal et

al., 2015). These results are also analogous to Khaliq et al. (2015), they observed increase

in POD and CAT activity in salt stress environment for tolerant cultivars of wheat.

However, decline or no change in CAT activity was also recorded for stress tolerant

cultivars (Wang et al., 2014).

Higher TPC in ‘Chakwal-50’, Mairaj-2008 and ‘Aas-2011’ can be ascribed to

more proline, glycine betaine and lesser malondialdehyde accumulation under heat over

control. Higher proline and glycine betaine might have promoted assimilate partitioning

to grains and hence yield decline was lesser under heat for these varieties. Furthermore,

strong positive correlation of TPC with proline and glycine betaine accomplished role of

TPC in sustaining proline and glycine betaine contents (Table 4.1.11 and Table 4.1.12).

Increment in phenolic contents enhanced stress tolerance. Improved stress tolerance was

consequence of escalated proline and glycine betaine accumulation (Saleem et al., 2016).

4.1.4. Osmo-protectants and lipid peroxidation

(a) Results

‘Cultivars Aas-2011’, ‘Chakwal-50’ and ‘Mairaj-2008’ manifested increment of

21% in leaf proline contents under high temperature environment over control. Inclined

proline contents characterized heat tolerance of these cultivars. Highest decrement (51%)

in stressed conditions over control was revealed for genotypes ‘NIBGE-NIAB-1’ and

‘Kohistan-97’. Decline in proline content under stressed conditions represented heat

susceptibility of these genotypes. The genotypes ‘Aas-2011’, ‘Chakwal-50’ and ‘Mairaj-

2008’ exhibited 17, 12 and 13% boost in glycine betaine contents, respectively under heat

stress over control. While, statistically alike and more glycine betaine contents were

quantified for ‘Aas-2011’, ‘Fareed-2006’, ‘Chakwal-50’, ‘Punjab-2011’, ‘AARI-2011’,

47

‘Galaxy-2013’, ‘Millat-2011’ and ‘Mairaj-2008’ under ambient conditions. Whereas,

under high temperature environment genotypes ‘Aas-2011’, ‘Chakwal-50’ and ‘Mairaj-

2008’ exhibited significantly higher GB contents than other cultivars (Table 4.1.8).

Differential response of wheat genotypes resulted in significant heat × variety

effect for total soluble proteins (TSP). Higher temperature enhanced accumulation of TSP

in cultivars Aas-2011 (16%) and Chakwal-50 (17%) and both cultivars were statistically

similar. In controls, Aas-2011 and Chakwal-50 were also statistically similar to Mairaj-

2008 for TSP. The highest diminishment in TSP under heat over control was observed for

cultivars AARI-2011 (40%) and Millat-2011 (64%) (Table 4.1.9). Different wheat

genotypes significantly varied for Malondialdehyde (MDA) biosynthesis. High

temperature environments significantly augmented MDA contents symbolizing boosted

lipid peroxidation under stressed conditions. Indistinguishable response of genotypes in

stressed and non-stressed environment resulted in non-significant interaction.

Table 4.1.8: Effect of heat stress on leaf proline and glycine betaine of wheat varieties


Source of variation DF Proline Glycine betaine

Blocks 3 2.189 29574.2Heat (H) 1 3.656** 37756.8*Error I 3 0.059 1860.4Genotypes (V) 10 2.345** 15420.1**H × V 10 0.756** 4987.7**Error II 60 0.038 512.9



Treatments Proline (µmol g-1) Glycine betaine (µmol g-1)

No heat stress (H0)Punjab-2011 2.19 abcd 225.7 abAARI-2011 2.02 abcd 205.9 abGalaxy-2013 2.05 abcd 217.1 abMillat-2011 1.99 bcd 197.8 abAas-2011 2.48 a 243.2 aFareed-2006 2.30 abc 233.7 abChakwal-50 2.44 ab 245.2 aMairaj-2008 2.35 abc 239.5 ab

48

Pakistan-2013 1.89 cd 188.0 bNIBGE-NIAB-1 1.82 d 188.8 bKohistan-97 1.78 d 187.6 bHeat from spike to grain filling (H1)Punjab-2011 1.50 bc 143.5 bcAARI-2011 1.26 cd 134.8 cGalaxy-2013 1.47 bc 138.4 cMillat-2011 1.14 cd 130.1 cAas-2011 3.02 a 285.8 aFareed-2006 1.91 b 160.0 bChakwal-50 2.96 a 276.8 aMairaj-2008 2.84 a 272.1 aPakistan-2013 0.99 d 126.4 cNIBGE-NIAB-1 0.89 d 123.4 cKohistan-97 0.86 d 125.8 cTukey’s HSD (p ≤ 0.05) 0.461 53.56


Table 4.1.9: Effect of heat stress on total soluble proteins of wheat varieties


Source of variation DF Total soluble proteins

Blocks 3 0.027Heat (H) 1 0.130*Error I 3 0.004Genotypes (V) 10 0.304**H × V 10 0.026**Error II 60 0.003



Treatments Total soluble proteins (mg g-1)

No heat stress (H0)Punjab-2011 0.57 cdAARI-2011 0.47 deGalaxy-2013 0.39 eMillat-2011 0.41 eAas-2011 0.75 aFareed-2006 0.62 bcChakwal-50 0.74 abMairaj-2008 0.69 abcPakistan-2013 0.38 e

49

NIBGE-NIAB-1 0.37 eKohistan-97 0.37 eHeat from spike to grain filling (H1)Punjab-2011 0.42 cdAARI-2011 0.31 deGalaxy-2013 0.33 deMillat-2011 0.27 eAas-2011 0.89 aFareed-2006 0.49 bcChakwal-50 0.89 aMairaj-2008 0.55 bPakistan-2013 0.26 eNIBGE-NIAB-1 0.23 eKohistan-97 0.25 eTukey’s HSD (p ≤ 0.05) 0.129


Significantly lesser MDA was recorded for genotypes ‘AAS-2011’ (0.91 µmol g-1)

and ‘Chakwal-50’ (0.96 µmol g-1). Highest MDA biosynthesis was observed for

‘Pakistan-2013’ (1.24 µmol g-1). Genotype ‘Pakistan-2013’ was statistically comparable

to ‘Punjab-2011’, ‘AARI-2011’, ‘Galaxy-2013’, ‘Millat-2011’, ‘Fareed-2006’, ‘NIBGE-

NIAB-1’ and ‘Kohistan-97’ (Table 4.1.10).

(b) Discussion

Increment in proline and glycine betaine in genotypes ‘Aas-2011’, ‘Chakwal-50’

and ‘Mairaj-2008’ can be defined in context of higher TPC of these genotypes over

control. Accumulation of TPC enhanced thermotolerance in sensitive genotypes also.

Furthermore, a strong association of glycine betaine and proline with TPC confirmed

positive influence of proline and glycine betaine on TPC (Table 4.1.11 and Table 4.1.12).

A positive correlation was observed between TPC and thermotolerance (Mahmood et al.,

2014). Increment in GB and proline is also alike to findings of Raza et al. (2015); they

showed accumulation of proline and glycine betaine improved stress tolerance. Tolerant

genotypes manifested increase while susceptible genotypes depicted decrease in proline

and glycine betaine under stressed conditions. Further proline and glycine betaine reduced

MDA and consequence in high yield under stress.

The interactive effect of heat and varieties was significant for total soluble

proteins (TSP). Cultivars Aas-2011 and Chakwal-50 manifested increment while other

cultivars recorded decrement in TSP under heat stress over control. Augmented

production of TSP for genotypes Aas-2011 and Chakwal-50 might be due to enhancement

50

of antioxidant enzymes. Stronger correlation between TSP and antioxidants under heat

and control further accomplished antioxidant-mediated augmentation in TSP (Table

4.1.11 and Table 4.1.12). Higher TSP might be a consequence of chlorophyll mediated

carbohydrate biosynthesis. Higher grain yield under heat stress for Aas-2011 and

Chakwal-50 was observed. Therefore, contribution of TSP and chlorophyll for

maintaining assimilate partitioning towards grains under heat stress was confirmed in

form of higher grain yield. The decrease in TSP in cultivars Punjab-2011, AARI-2011,

Galaxy-2013, Millat-2011, Fareed-2006, Mairaj-2008, Pakistan-2013, NIBGE-NIAB-1,

and Kohistan-97 might be due to denaturation of proteins and inability of these cultivars

to enhance heat shock proteins (HSPs) under heat stress. Declined chlorophyll contents

and antioxidant activities established escalation in protein denaturation. Our results are

like those of Li et al. (2013), under high-temperature environment, glutenin biosynthesis

declined, gliadin remained stable resulting in deteriorated grain proteins and quality.

Table 4.1.10: Effect of heat stress on malondialdehyde of wheat varieties


Source of variation DF Malondialdehyde

Blocks 3 8.540

Heat (H) 1 2.940*

Error I 3 0.207

Genotypes (V) 10 0.092**

H × V 10 0.015NS

Error II 60 0.009* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant


Treatments Malondialdehyde (µmol g-1)

Heat stress (H)

No heat stress (H0) 0.94 B

Heat from spike to grain filling (H1) 1.31 A

Tukey’s HSD (p ≤ 0.05) 0.308

Genotypes (V)

Punjab-2011 1.13 AB

51

AARI-2011 1.20 AB

Galaxy-2013 1.16 AB

Millat-2011 1.20 AB

Aas-2011 0.91 C

Fareed-2006 1.12 AB

Chakwal-50 0.96 C

Mairaj-2008 1.06 BC

Pakistan-2013 1.24 A

NIBGE-NIAB-1 1.19 AB

Kohistan-97 1.21 AB

Tukey’s HSD (p ≤ 0.05) 0.158Any two means not sharing a letter in common differ significantly at p ≤ 0.05

52

Wheat genotypes with higher antioxidants depicted heat tolerance at physiological

maturity that consequently enhanced TSPs (Sharma et al., 2014a). Low MDA for ‘Aas-

2011’, ‘Chakwal-50’ and ‘Mairaj-2008’ can be attributed to higher proline, TPC and

glycine betaine accumulating capability of these genotypes under high temperature

environment. Highest lipid peroxidation in ‘Pakistan-2013’ and ‘Kohistan-97’ genotypes

might have decreased capability of these genotypes to accumulate proline, TPC and

glycine betaine. A strong negative association of glycine betaine, proline and phenolic

contents with MDA under heat stress confirmed negative impacts of MDA on proline and

glycine betaine accumulation (Table 4.1.11 and Table 4.1.12). Wheat genotypes with

lower MDA were better able to survive under stress conditions (Khaliq et al., 2015).

53

Table 4.1.11: Correlation analyses showing strength of association among recorded attributes of different wheat varieties under no heat stress (H0)

Parameters GPS TGW GY GFR GFD Chl a Chl b SOD POD CAT TPC LP GB TSPTGW - 0.42NS

GY 0.80** - 0.27NS

GFR - 0.54NS - 0.35NS - 0.54NS

GFD 0.80** - 0.25NS 0.85** - 0.50NS

Chl a 0.77** - 0.14NS 0.86** - 0.67* 0.96**Chl b 0.74** - 0.007NS 0.81** - 0.73* 0.94** 0.98**SOD 0.80** - 0.16NS 0.86** - 0.58NS 0.98** 0.97** 0.97**POD 0.83** - 0.19NS 0.89** - 0.62NS 0.98** 0.98** 0.97** 0.99**CAT 0.79** - 0.17NS 0.84** - 0.51NS 0.98** 0.95** 0.94** 0.99** 0.98**TPC 0.74** - 0.002NS 0.84** - 0.70* 0.94** 0.98** 0.97** 0.96** 0.97** 0.95**LP 0.75** - 0.03NS 0.86** - 0.68* 0.95** 0.98** 0.98** 0.98** 0.97** 0.96** 0.98**GB 0.76** - 0.10NS 0.81** - 0.58NS 0.98** 0.96** 0.97** 0.99** 0.98** 0.99** 0.96** 0.98**TSP 0.76** - 0.08NS 0.82** - 0.66* 0.97** 0.98** 0.99** 0.99** 0.98** 0.97** 0.97** 0.98** 0.99**MDA - 0.77** 0.09NS - 0.79** 0.67* - 0.95** - 0.95** - 0.97** - 0.96** - 0.97** - 0.95** - 0.95** - 0.95** - 0.96** - 0.97**

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant; GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyden (number of pairs of observations) = 88

54

Table 4.1.12: Correlation analyses showing strength of association among recorded attributes of different wheat varieties under heat from spike to grain filling (H1)

Parameters GPS TGW GY GFR GFD Chl a Chl b SOD POD CAT TPC LP GB TSPTGW 0.17NS

GY 0.89** 0.44NS

GFR - 0.60NS - 0.67* - 0.82**GFD 0.86** 0.42NS 0.93** - 0.71*Chl a 0.85** 0.44NS 0.95** - 0.76** 0.98**Chl b 0.83** 0.46NS 0.96** - 0.82** 0.98** 0.98**SOD 0.81** 0.46NS 0.95** - 0.80** 0.96** 0.96** 0.98**POD 0.79** 0.43NS 0.94** - 0.80** 0.93** 0.92** 0.96** 0.99**CAT 0.79** 0.47NS 0.94** - 0.81** 0.96** 0.95** 0.98** 0.99** 0.99**TPC 0.81** 0.47NS 0.96** - 0.82** 0.95** 0.95** 0.97** 0.99** 0.99** 0.99**LP 0.81** 0.45NS 0.94** - 0.80** 0.97** 0.97** 0.99** 0.99** 0.98** 0.99** 0.99**GB 0.76** 0.46NS 0.93** - 0.82** 0.92** 0.91** 0.95** 0.98** 0.99** 0.99** 0.99** 0.98**TSP 0.83** 0.51NS 0.97** - 0.90** 0.91** 0.94** 0.97** 0.95** 0.93** 0.94** 0.95** 0.94** 0.93**MDA - 0.78** - 0.45NS - 0.95** 0.87** - 0.87** - 0.89** - 0.94** - 0.93** - 0.94** - 0.93** - 0.94** - 0.92** - 0.94** - 0.98**

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant; GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyden (number of pairs of observations) = 88

55

Experiment II: Exploring role of foliar applied potassium to induce terminal heat

stress tolerance in wheat

Generally, high temperature environments deleteriously impacted grain yield,

yield components, biomass accumulation and spike growth compared to no heat stress

(H0). Moreover, antioxidants enzymes, the stay green trait, osmo-protectants, water

relations and grain quality were also disturbed under both treatments of heat stress over

the normal environment. However, ‘heat from spike to grain filling (H1)’ proved more

detrimental than ‘heat from flowering to grain filling (H2)’ for all the recorded

parameters. Whereas, exogenously applied potassium (K) and selenium (Se) remarkably

improved biochemical attributes which ultimately modulated agronomic and other

morphological regulations under control and heat.

4.2.1. Soil physio-chemical attributes

(a) Results

Temporal and soil variations were pronounced for various parameters and more

promising response was observed during the year 2015-16 than 2016-17 for most of

instances. Relatively more organic matter, total nitrogen, available phosphorous and

potassium were recorded during 2015-16 than 2016-17 (Table 3.1).

(b) Discussion

Better soil nutrient status during 2015-16 might have improved activation of

antioxidant enzymes, accumulation of osmo-protectants, which ultimately improved the

expression of morphological attributes. The increased availability of nitrogen,

phosphorous and potassium triggered the biosynthesis of leaf proteins, alleviated the

negative implications of oxidative stress and improved the utilization of light in

biosynthesis of carbohydrates and ultimately biomass and grain yield were more

promising (Waraich et al., 2012; Vimal et al., 2017). Likewise, more organic matter in

soil enhanced the water holding capacity of soil and thereby improved nutrient and water

availability in the soil profile (Bastida et al., 2017).

4.2.2. Weather elements

(a) Results

Comparatively more rainfall in 2015-16 than 2016-17 was recorded during

months of November, January, February and March. Whereas, higher rainfall in April

2016-17 (28.3 mm) was observed than in April 2015-16 (5.6 mm). Average temperature

was higher throughout growing season in year 2016-17 than 2015-16 (Table 3.1).

56

(b) Discussion

More rainfall during vegetative growth stages and lesser temperature during 2015-

16 than 2016-17 might have favorably enhanced biomass accumulation, grain yield, yield

components and biochemical response variables of the wheat crop. While, more rainfall

during April 2016-17 after grain filling stage aggravated lodging which eventually

decreased the grain yield and yield components. Moreover, overall rise of temperature

during growing period in 2016-17 might have negatively influenced all the studied

attributes. Coincidence of rainfall with vegetative stages was highly associated with the

yield and yield components. Contrarily, prolongation of rainy seasons up to reproductive

growth either depicted moderate or negative correlation with yield and yield components

of wheat crop (Bekele et al., 2017). While, slight rise of temperature during growing

season accelerated the progression of pheno-stages, adversely affecting pollination and

grain quality (Hatfield and Prueger, 2015).


(a) Results

High temperature either from ‘spike to grain filling’ or ‘flowering to grain filling’

significantly reduced grain yield and components. Although, ‘heat from spike to grain

filling’ depicted more negative effects than ‘‘heat from flowering to grain filling’.

Conversely, exogenous potassium effectively alleviated negative perturbations of heat

and significant effect of varying potassium concentrations was apparent. While, similar

trend was observed for foliar applied potassium in all main plots that resulted into non-

significant heat × potassium effect. However, number of fertile tillers did not vary

significantly under varying temperatures as well as exogenously applied potassium.

Significantly lower number of grains per spike and 1000-grain weight was

observed under ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’

as compared to control over the years. Whereas, ‘heat from spike to grain filling’

produced significantly lesser yield compared to ‘heat from flowering to grain filling’ and

non-stressed conditions over the years. Moreover, the number of grains per spike was

diminished by 35-37% and 25-26% under ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’, respectively over the years. Whereas, ‘heat from spike to grain

filling’ and ‘heat from flowering to grain filling’ induced decrease in grain yield was 42-

45% and 25-31%, respectively compared to ambient conditions over the temporal

variability.

57

The number of grains per spike, 1000-grain weight and grain yield were enhanced

with increasing concentration of potassium. However, statistically similar number of

grains per spike was recorded for 30, 45 and 60 g L-1 potassium over the years. While,

statistically alike 1000-grain weight and grain yield were recorded for 45 and 60 g L-1

foliar potassium. Whereas, comparatively lesser and statistically similar number of grains

per spike, 1000-grain weight and grain yield were observed for control, 15 and 30 g L -1

exogenous potassium for both the studied years (Table 4.2.1 and Table 4.2.2).

Moreover, number of grains per spike was enhanced by 0.19-0.20 under control,

0.11-0.16 under ‘heat from spike to grain filling’ and 0.10-0.13 under ‘heat from

flowering to grain filling’ with each addition of 15 g L-1 potassium, over the two years.

While each 15 g L-1 increase of foliar potassium enhanced 1000-grain weight by 0.08-0.11

g under ‘no heat stress’, 0.15 g under ‘heat from spike to grain filling’ and 0.12-0.15 g

under ‘heat from flowering to grain filling’ over the years. Likewise, the increase of

potassium by each 15 g L-1 enhanced grain yield by 0.011-0.014 t ha-1 under the no ‘heat

stress’, 0.009-0.011 t ha-1 under ‘heat from spike to grain filling’ and 0.010 t ha-1 under

‘heat from flowering to grain filling’. Moreover, potassium modulated enhancement in

1000-grain weight and grain yield was higher under both treatments of heat as compared

to control over the years. Therefore, dependence of 1000-grain weight and grain yield (R2

values) on availability of potassium was enhanced under stress conditions over the control

(Figure 4.2.1 and Figure 4.2.2).

(b) Discussion

Heat was imposed from ‘spike to grain filling’ and ‘flowering to grain filling’

hence number of spike bearing tillers, that had already been determined, and therefore did

not vary significantly afterwards with heat and foliar potassium. Heat stress might have

escalated the degradation of chlorophyll and rate of biosynthesis of chlorophyll was lesser

as compared to degradation. Hence, aggravated senescence under heat over control might

have deleteriously affected yield components and yield. Moreover, senescence might

have downregulated the carbohydrates partitioning towards grains and resulted in

diminishment of number of grains, 1000-grain weight and eventually grain yield under

heat compared to control. Moreover, significant positive associations of chlorophyll a and

b contents with grains per spike, 1000-grain weight and grain yield further accomplished

the adverse impacts of chlorophyll degradation on grain yield and components under the

‘no heat stress’ (Table 4.2.15 a, c), ‘heat from spike to gain filling’ (Table 4.2.16 a, c) and

58

‘heat from flowering to grain filling’ (Table 4.2.17 a, c) over the years 2015-16 and 2016-

17.

Table 4.2.1: Effect of foliar applied potassium on fertile tillers and grains per spike of heat stressed wheat

A. Mean sum of squares

Source of

variationDF

Fertile tillers Grains per spike

2015-16 2016-17 2015-16 2016-17

Blocks 2 152179 141942 4367.64 3622.50

Heat (H) 2 1398NS 1949NS 1225.21** 1071.67**

Error I 4 10367 4296 31.50 34.49

Potassium (K) 4 3299NS 939NS 151.76** 113.42**

H × K 8 769NS 205NS 7.98NS 5.03NS

Error II 24 1299 669 22.36 16.51

** = Significant (p ≤ 0.01); NS = Non-significant


TreatmentsFertile tillers per m2 Grains per spike

2015-16 2016-17 2015-16 2016-17

Heat stress (H)

No heat stress (H0) 280 281 48.5 A 46.7 A

Heat from spike to grain filling (H1) 300 300 31.1 B 30.3 B

Heat from flowering to grain filling (H2) 280 301 35.8 B 35.1 B

Tukey’s HSD (p ≤ 0.05) NS NS 7.30 7.65

Potassium foliar spray (K)

Control/ water spray (K0) 303 301 35.6 BC 33.8 B

15 g L-1 potassium (K15) 312 303 34.1 C 34.5 B

30 g L-1 potassium (K30) 295 299 37.1 ABC 36.4 AB

45 g L-1 potassium (K45) 267 289 43.5 A 40.5 A

60 g L-1 potassium (K60) 274 278 42.1 AB 41.7 A

Tukey’s HSD (p ≤ 0.05) NS NS 6.57 5.64

Year mean 290 294 38.5 37.4

Tukey’s HSD (p ≤ 0.05) NS NSAny two means not sharing a letter in common differ significantly at p ≤ 0.05; NS = Non-significant

59

Figure 4.2.1: Regression analysis for effect of foliar applied potassium on grains per spike of heat stressed wheat

H0 = No heat stress; H1 = Heat from spike to grain filling; H2 = Heat from flowering to grain filling

60

Table 4.2.2: Effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat


Source of

variationDF

1000-grain weight Grain yield

2015-16 2016-17 2015-16 2016-17

Blocks 2 1713.29 998.30 3.389 4.660

Heat (H) 2 1217.21** 961.59** 28.918** 18.553**

Error I 4 25.12 34.75 0.191 0.219

Potassium (K) 4 77.20** 99.59** 0.654** 0.641**

H × K 8 4.16NS 2.02NS 0.041NS 0.015NS

Error II 24 7.21 7.54 0.064 0.059** = Significant (p ≤ 0.01); NS = Non-significant


Treatments1000-grain weight (g) Grains yield (t ha-1)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)

No heat stress (H0) 56.7 A 54.3 A 6.01 A 5.25 A

Heat from spike to grain filling (H1) 38.9 B 39.3 B 3.29 C 3.04 C

Heat from flowering to grain filling (H2) 45.0 B 42.0 B 4.17 B 3.96 B

Tukey’s HSD (p ≤ 0.05) 6.53 7.676 0.569 0.609


Control/ water spray (K0) 43.8 C 41.8 B 4.20 C 3.78 B

15 g L-1 potassium (K15) 44.9 C 42.6 B 4.28 C 3.87 B

30 g L-1 potassium (K30) 45.8 BC 44.2 B 4.42 BC 4.05 AB

45 g L-1 potassium (K45) 49.2 AB 48.4 A 4.72 AB 4.34 A

60 g L-1 potassium (K60) 50.6 A 49.1 A 4.82 A 4.37 A

Tukey’s HSD (p ≤ 0.05) 3.73 3.81 0.352 0.337

Year mean 46.8 45.2 4.49 4.08


61

Figure 4.2.2: Regression analysis for effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat


62

Imposition of heat stress at reproductive stages decreased the number of grains per

spike by 39%, 1000-grain weight by 27% and grain yield by 79% compared to no heat

stress conditions (Pimentel et al., 2015).

The heat induced decrease in yield and yield components can also be ascribed to

lipid peroxidation of bio-membranes at cellular level. Heat stress might have accelerated

the synthesis of reactive oxygen species (ROS) and thus caused oxidative stress.

Oxidative stress down regulated the biosynthesis of superoxide dismutase, which

ultimately slowed down the conversion of superoxide radicals to hydrogen peroxide and

water. While, diminished activities of catalase and peroxidase enzymes under heat further

accomplished the accelerated production of superoxide radicals and worsening of

oxidative stress under heat. Faster production of ROS might have lessened the plant

capability to make osmotic adjustments under high temperature environment. So, a

significant positive association of antioxidants and phenolics with yield and yield

components for most of instances accomplished the negative impacts of lesser

biosynthesis of antioxidants on yield and components (Table 4.2.15-4.2.17 a, c).

Similarly, positive and significant correlation of antioxidants with osmo-protectants

further established the perturbations in water relations owing to lesser synthesis of

antioxidants and phenolics (Table 4.2.15-4.2.17 b, d). Negative impacts of heat caused the

grain yield reduction by 6-11% in different genotypes (Feng et al., 2014). Similarly,

imposition of heat decreased the activities of superoxide dismutase, peroxidase, catalase

and glutathione reductase in heat sensitive and moderately sensitive genotypes (Wang et

al., 2014).

Potassium availability under heat might have sustained the allocation of

carbohydrates to spike development for longer duration, protracted grain filling duration

and ultimately enhanced number of grains per spike. Availability of potassium might

have improved the capacity of cells to retain water leading to activation of hydrolases and

growth. Therefore, grain filling rate was enhanced which resulted in increase of 1000-

grain weight and grain yield under ambient and heat stress conditions. Enhancement of

grain filling rate and duration owing to potassium was also confirmed from the

significant, strong and positive association of shoot potassium contents with grain filling

rate and duration for most of times over the temporal variability (Table 4.2.15-4.2.17 a,

c). Availability of potassium enhanced grain filling rate by 0.8% and duration by 1.6% in

maize crop. Prolongation of grain filling time ultimately enhanced grain yield (Liu et al.,

2011). Imposition of stress after the anthesis adversely affected the activities of catalase,

63

superoxide dismutase and glutathione reductase while application of potassium alleviated

adverse impacts of stress (Xiaokang et al., 2017).

Increase in superoxide dismutase under higher doses of potassium over control

might have enhanced the detoxification superoxide to hydrogen peroxide, which acts as a

substrate for catalase and peroxidase. Besides, higher shoot potassium contents under

potassium conferred heat tolerance through regulations in antioxidants, phenolics, lipid

peroxidation and water relations of wheat and the same was confirmed from positive and

strong association of shoot potassium contents with these attributes over the years (Table

4.2.15-4.2.17 b, d). Foliar application of potassium enhanced photosynthetic pigments,

gaseous exchange, accumulation of proline, superoxide dismutase, glutathione reductase

and peroxidase. While, improvements in biochemical attributes depicted strong and

positive correlation with morphological attributes (Jan et al., 2017). Similarly, availability

of potassium under stress environment enhanced photosynthetic carbohydrates, regulated

stomatal conductance, improved the antioxidants activities and accumulation of osmo-

protectants (Zahoor et al., 2017b).

4.2.4. Biomass accumulation

(a) Results

Heat stress significantly decreased biological, straw yield, harvest index and plant

height compared to ‘no heat stress’. Whereas, foliar application of potassium remarkably

improved biomass accumulating attributes over water spray (control). While, similar

potassium mediated improvements were observed under ‘no heat stress’, ‘heat from spike

to grain filling’ and ‘heat from flowering to grain filling’. Therefore, interaction of heat

stress and foliar applied potassium was non-significant for biological, straw yield, harvest

index, and plant height.

Significantly higher biological, straw yield, harvest index and plant height were

recorded under ‘no heat stress’ compared to ‘heat from spike to grain filling’ and ‘heat

from flowering to grain filling’ over the years. Moreover, ‘heat from spike to grain

filling’ and ‘heat from flowering to grain filling’ induced diminution in biological yield

was 34% and 19-22% respectively compared to ‘no heat stress’ over the 2015-16 and

2016-17. While, harvest index was diminished by 12-17% under ‘heat from spike to grain

filling’ and 6-10% under ‘heat from flowering to grain filling’ compared to ‘no heat

stress’ over the years. Likewise, ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filing’ caused decline in straw yield was 26-18% and 16% respectively,

compared to non-stressed conditions. Similarly, plant height was decreased by 20-21%

64

under ‘heat from spike to grain filling’ and 11-13% under ‘heat from flowering to grain

filling’ over the temporal variability.

Biological, straw yield, harvest index and plant height response was linear with

enhancing foliar concentration of potassium. Though, foliar potassium modulated

improvements were dissimilar for different biomass accumulating attributes of heat

stressed wheat crop. Relatively more and statistically comparable biological yield and

plant height were recorded with 45 and 60 g L-1 foliar potassium over the years. Whereas,

15, 30, 45 and 60 g L-1 exogenous potassium depicted statistically alike harvest indices

during 2015-16 and straw yield during 2016-17. While, statistically similar harvest index

and straw yield during 2016-17 and 2015-16 respectively were recorded with 30, 45 and

60 g L-1 foliar potassium. Moreover, relatively lesser and statistically alike response of

biomass accumulating variables was observed with exogenous application of water spray

(control), 15 and 30 g L-1 potassium over the two years study period (Table 4.2.3 and

Table 4.2.4).

Foliar potassium modulated improvements in biological yield were 0.021-0.022,

0.014-0.015 and 0.017-0.018 t ha-1 with each increment of 15 g L-1 exogenous potassium

under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling, respectively over the two years. While, each unit application of potassium

enhanced harvest index by 0.02-0.03, 0.05-0.07 and 0.03% under ‘no heat stress’, ‘heat

from spike to grain filling’ and ‘heat from flowering to grain filling’ respectively over the

two years period. Likewise, potassium modulated enhancement in straw yield with the

increments in exogenous potassium rates was 0.007-0.010 t ha-1 under ‘no heat stress’,

0.004-0.005 t ha-1 under ‘heat from spike to grain filling’ and 0.007-0.008 t ha-1 under

‘heat from flowering to grain filling’ over the years. While, plant height was boosted by

0.13, 0.14-0.15 and 0.12-0.16 cm under ambient conditions, ‘heat from spike to grain

filling’ and ‘heat from ‘flowering to grain filling’ respectively with each one-unit

application of exogenous potassium. Moreover, improvements were generally more

dependent on potassium under the stress conditions than in non-stressed environments

since R2 was enhanced under heat compared to control (Figure 4.2.3 and Figure 4.2.4).

(b) Discussion

Heat stress might have enhanced the degradation while enhanced the biosynthesis

of photosynthetic pigments owing to disruption in activities of biosynthesizing enzymes.

Thus, decreased photosynthetic capability caused decreased accumulation of

carbohydrates and thus reduced biological yield, straw yield and plant height.

65

Table 4.2.3: Effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat


Source of

variationDF

Biological yield Harvest index

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.270 6.365 173.19 131.19

Heat (H) 2 91.14** 67.015** 191.62* 101.02**

Error I 4 0.250 0.958 13.78 1.63

Potassium (K) 4 1.629** 1.777** 7.97** 8.49**

H × K 8 0.086NS 0.076NS 0.84NS 1.56NS

Error II 24 0.207 0.132 1.51 1.00* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant


TreatmentsBiological yield (t ha-1) Harvest index (%)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 9.26 C 8.10 C 35.53 B 37.53 C

Heat from flowering to grain filling (H2) 10.94 B 9.91 B 38.12 AB 39.96 B

Tukey’s HSD (p ≤ 0.05) 0.651 1.274 4.834 1.664


Control/ water spray (K0) 11.02 C 9.57 C 38.11 B 39.50 B

15 g L-1 potassium (K15) 11.12 BC 9.81 C 38.48 AB 39.45 B

30 g L-1 potassium (K30) 11.30 BC 10.05 BC 39.11 AB 40.30 AB

45 g L-1 potassium (K45) 11.70 AB 10.53 AB 40.34 A 41.22 A


Tukey’s HSD (p ≤ 0.05) 0.632 0.505 1.706 1.386

Year mean 11.43 A 10.11 B 39.28 40.36

Tukey’s HSD (p ≤ 0.05) 0.844 NSAny two means not sharing a letter in common differ significantly at p ≤ 0.05; NS = Non-significant

66

Figure 4.2.3: Regression analysis for effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat


67

Table 4.2.4: Effect of foliar applied potassium on straw yield and plant height of heat stressed wheat


Source of

variationDF

Straw yield Plant height

2015-16 2016-17 2015-16 2016-17

Blocks 2 49.443 1.902 465.56 2515.94

Heat (H) 2 17.370** 14.544** 1665.10** 1712.40**

Error I 4 0.091 0.060 47.25 32.91

Potassium (K) 4 0.244* 0.280** 122.24** 98.80**

H × K 8 0.026NS 0.039NS 1.64NS 1.91NS



TreatmentsStraw yield (t ha-1) Plant height (cm)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 5.97 C 5.06 C 85.1 C 80.2 C


Tukey’s HSD (p ≤ 0.05) 0.393 0.319 8.95 7.47


Control/ water spray (K0) 6.82 B 5.79 B 92.7 BC 87.0 C

15 g L-1 potassium (K15) 6.84 B 5.94 AB 91.9 C 87.4 BC

30 g L-1 potassium (K30) 6.88 AB 6.00 AB 93.5 BC 88.7 BC

45 g L-1 potassium (K45) 6.98 AB 6.19 A 96.8 AB 91.9 AB


Tukey’s HSD (p ≤ 0.05) 0.382 0.326 4.58 4.64

Year mean 6.94 A 6.03 B 95.1 90.0


Figure 4.2.4: Regression analysis for effect of foliar applied potassium on straw yield and plant height of heat stressed wheat

68


Moreover, strong, positive and significant association of these parameters with

chlorophyll pigments was observed for most of instances under ‘no heat stress’ (Table

69

4.2.15 a, c), ‘heat from spike to grain filling’ (Table 4.2.16 a, c) and ‘heat from flowering

to grain filling’ over years (Table 4.2.17 a, c). High temperature impaired activities of

amino-levulinic acid dehydratase and porphobilinogen deaminase which eventually

downregulated the biosynthesis of chlorophyll and assimilates availability for vegetative

growth (Hemantaranjan et al., 2014).

In this regard, lesser vegetative growth might have reduced the light interception

and photosynthesis under heat. Thereafter, decrease in carbohydrates availability for grain

filling caused lesser harvest index under heat over control. Eventually, the number of

grains per spike and 1000-grain weight were decreased as outcome of poor biological

yield and lesser plant height. Moreover, heat induced decline in grain yield was more than

decrease in biological yield leading to decreased harvest index. Decrease of harvest index

because of poor biomass accumulation was also established from strong, positive and

highly significant correlation of biological yield and plant height with number of grains

per spike and 1000-grain weight for most of instances under varying heat stressed pheno-

stages over the years (Table 4.2.15-4.2.17 a, c). Heat stress during flowering stage of

wheat escalated grain filling rate, reduced grain filling duration and assimilate

partitioning towards grains. Ultimately, adverse impacts of heats instigated a significant

reduction in grain yield and components (Stratonovitch and Semenov, 2015).

Decrease in biological and straw yield under heat can also be elucidated in context

of adverse impacts of heat on plant height. Heat stress might have deprived the capability

of plant to make osmotic adjustments and thus water relation attributes were disturbed.

Therefore, cell elongation was impaired due to depressed water potential and growth was

affected. This explanation was also confirmed from strong, positive and pronouncing

association of these parameters with leaf proline, glycine betaine and total soluble

proteins under varying temperatures over the two studied years (Table 4.2.15-4.2.17 a, c).

Coincidence of heat stress during flowering and grain filling stages decreased the plant

height growth and dry matter accumulation in wheat (Liu et al., 2016a).

Exogenous potassium mediated improvements in biological yield, straw yield,

harvest index and plant height can be explained in terms of potassium mediated

improvements in accumulation of osmo-protectants. Availability of potassium under heat

increased the plant capability to accumulate proline, glycine betaine and total soluble

proteins. Availability of potassium further improved the biosynthesis of proline and

proline quenches singlet oxygen and hydroxyl radicals. Therefore, the most devastating

ROS (singlet oxygen and hydroxyl radical) regarding lipid peroxidation of bio-

70

membranes might have been detoxified through potassium mediated accumulation of

proline. Thereby, catalase and peroxidase activities were increased. Consequently,

membrane stability and water retaining capacity at cellular level might have improved

which decreased the sensitivity to heat. Proline mediated detoxification of hydroxyl

radical and singlet oxygen was also established from the strong positive and highly

significant correlation of proline with the catalase and peroxidase activity under different

heat stressed growth stages of wheat over the years (Table 4.2.15-4.2.17 b, d). Whereas,

strong, negative and significant association of biomass accumulating attributes with

malondialdehyde for most of cases was recorded. Hence, potassium mediated

improvement in membrane stability was accomplished (Table 4.2.15-4.2.17 a, c).

Availability of potassium under the stressed conditions enhanced water potential,

membrane stability and gaseous exchange of rice but decrease of malondialdehyde was

negatively associated with accumulation of proline. Hence, improved proline contents

under stress resulted into improved growth of rice (Zain and Ismail, 2016). Likewise,

enhancing potassium concentration in flag leaf in barley caused more activation of

hydrolases, which ultimately improved vegetative growth (Hosseini et al., 2016).

Moreover, potassium modulated enhancements in accumulation of total soluble

proteins and glycine betaine might have conferred heat tolerance by preserving water

under heat. The role of osmo-protectants in enhancing water potential and membrane

integrity was established from strong positive correlation of biological yield, harvest

index and plant height with total soluble proteins, proline and glycine betaine under

different growth stages and temperature over the years (Table 4.2.15-4.2.17 a, c).

Exogenous application of potassium enhanced the glycine betaine accumulation and

water potential in wheat under stressed conditions and thus imparted tolerance (Raza et

al., 2014).


(a) Results

Wheat spike length, spikelets per spike, grain filling rate and duration were

seriously affected by heat. Moreover, longer heat duration proved more deleterious over

the two years study period. Whereas, exogenous potassium effectively alleviated negative

impacts of high temperature and significantly improved spike traits and growth. Yet,

interaction of heat and foliar potassium had a non-significant impact on spike growth

related parameters.

71

Significantly smaller spikes were observed under ‘heat from spike to grain filling’

compared to ‘no heat stress’ and ‘heat from flowering to grain filling’ over years. While,

statistically similar and significantly fewer spikelets per spike and statistically prolonged

and alike grain filling rate were recorded under both heat treatments compared to ‘no heat

stress’ over the years. Whereas, grain filling duration was reduced with the increasing

duration of heat imposition.

Furthermore, ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling’ triggered decline in spike length was 45-53% and 25-29% and in spikelets per

spike was 29-36% and 20-27% respectively compared to no heat stress over the two years

study duration. An acceleration of grain filling rate by 50-57% under ‘heat from spike to

grain filling’ and 42-43% under ‘heat from flowering to grain filling’ was quantified over

years. Whereas, heat induced diminishment in grain filling duration was 36% (2015-16)

and 45% (2016-17) under ‘heat from spike to grain filling’. Whereas, ‘heat from

flowering to grain filling’ triggered shortening in grain filling duration was 21% (2015-

16) and 27% (2016-17). Application of foliar applied potassium remarkably reduced the

negative impacts of heat. Concerning this, significantly more and statistically alike spike

length, spikelets per spike, grain filling rate and duration were observed with 45 and 60 g

L-1 exogenous potassium compared to other potassium concentrations over the years

(Table 4.2.5 and Table 4.2.6).

A linear response was obvious with the enhancing concentrations of foliar

potassium for all spike related attributes. An increase of 0.09-0.14, 0.05-0.07 and 0.05 cm

in spike length was recorded with each unit enhancement of foliar potassium under ‘no

heat stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’,

respectively over two years. Whereas, each 15 g L-1 mediated improvements in spikelets

per spike were 0.06-0.07 cm under ‘no heat’, 0.04-0.06 cm under ‘heat from spike to

grain filling and 0.04 cm under ‘heat from flowering to grain filling’ over temporal

variability. While, exogenous potassium triggered improvements in grain filling rate were

almost negligible over the years but 45 and 60 g L-1 were still high enough to produce

significant enhancement in grain filling rate. Whereas, prolongation of 0.8-0.9 days under

‘no heat stress’, 0.09 days under ‘heat from spike to grain filling’ and 0.06-0.07 days

under ‘heat from flowering to grain filling’ with each 15 g L-1 addition of foliar potassium

was observed over the 2015-16 and 2016-17 (Figure 4.2.5 and Figure 4.2.6).

72

Table 4.2.5: Effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat


Source of

variationDF

Spike length Spikelets per spike

2015-16 2016-17 2015-16 2016-17

Blocks 2 124.64 152.63 200.13 209.27

Heat (H) 2 334.88** 427.81** 110.58** 150.93**

Error I 4 3.77 3.20 3.96 3.81

Potassium (K) 4 27.75** 32.64** 15.68** 13.10**

H × K 8 1.79NS 5.11NS 0.48NS 0.77NS



TreatmentsSpike length (cm) Spikelets per spike

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 11.5 C 9.4 C 12.9 B 10.9 B


Tukey’s HSD (p ≤ 0.05) 2.53 2.33 2.59 2.54


Control/ water spray (K0) 14.6 BC 12.8 C 14.0 C 12.0 C

15 g L-1 potassium (K15) 14.1 C 13.0 C 13.8 C 12.5 C

30 g L-1 potassium (K30) 15.8 B 14.1 BC 14.9 BC 13.1 BC

45 g L-1 potassium (K45) 17.6 A 15.8 AB 16.4 AB 14.5 AB


Tukey’s HSD (p ≤ 0.05) 1.30 2.18 1.72 1.49

Year mean 16.0 14.6 15.1 13.4


Figure 4.2.5: Regression analysis for effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat

73


Table 4.2.6: Effect of foliar applied potassium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat

74


Source of

variationDF

Grain filling rate Grain filling duration

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.00200 0.00645 177.76 200.94

Heat (H) 2 0.02614** 0.01463** 659.09* 956.28**

Error I 4 0.00057 0.00040 44.69 37.98

Potassium (K) 4 0.00402** 0.00233** 45.74** 34.56**

H × K 8 0.00020NS 0.00006NS 1.98NS 2.10NS



TreatmentsGFR (g per day) GFD (days)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)

No heat stress (H0) 0.14 B 0.12 B 36.27 A 34.87 A

Heat from spike to grain filling (H1) 0.22 A 0.18 A 23.07 B 19.00 B

Heat from flowering to grain filling (H2) 0.20 A 0.17 A 28.60 AB 25.43 B

Tukey’s HSD (p ≤ 0.05) 0.031 0.026 8.704 8.024


Control/ water spray (K0) 0.17 C 0.14 B 28.11 B 24.10 D

15 g L-1 potassium (K15) 0.16 C 0.14 B 26.67 B 24.99 CD

30 g L-1 potassium (K30) 0.19 B 0.15 B 28.56 B 26.33 BC

45 g L-1 potassium (K45) 0.20 AB 0.17 A 31.00 A 27.96 AB


Tukey’s HSD (p ≤ 0.05) 0.020 0.017 1.972 1.949

Year mean 0.19 A 0.16 B 29.31 26.43


Figure 4.2.6: Regression analysis for effect of foliar applied potassium on grain filling rate and grain filling duration of heat stressed wheat

75


(b) Discussion

76

Heat stress might have impaired the capability of plant to utilize excessive solar

radiations for biosynthesizing of carbohydrates. Consequently, biological yield was

decreased which eventually downregulated the assimilate partitioning towards

reproductive parts. Hence, carbohydrates availability for development of rachis might

have declined. This, decrease in number of ridges on rachis of spike ultimately lead to

consequence of decreased spike length and spikelets per spike. Decrease of spike length

and spikelets per spike were attributable to biological yield was also accomplished from

their strong, positive and significant association under varying heat stress treatments over

the years (Table 4.2.15-4.2.17 a, c) Improvement in plant height increases the

photosynthetically active area for partitioning of assimilates to reproductive parts of

wheat. Ultimately, the number of ridges on the rachis, number of grains and spikelets per

spike enhance (Slafer et al., 2015). Increase of biological yield was highly correlated to

spike length, spikelets per spike, grain filling period and grain yield (Wolde et al., 2016).

In addition to biological yield, disruption in water, osmotic and turgor potential

under heat stress might be another reason for decreased spike length and spikelets per

spike. Heat stress depressed the capability of plant to accumulate osmo-protectants and

thus diminished water potential. It caused lesser extension of cells and spike growth was

not up to the mark. The aggravated damage to membranes because of lipid peroxidation

might have promoted sterility of pollens and development of ovules. Consequently, the

number of spikelets per spike were decreased. Moreover, water potential mediated

decrease in spike length and spikelets per spike was accomplished from their strong,

positive and significant correlation over the temporal variability. A positive and

reasonably strong correlation of osmo-protectants with spike traits was also recorded over

the years. This also provided the evidence for negative impacts of depressed

accumulation of osmo-protectants on spike length and spikelets per spike (Table 4.2.15-

4.2.17 a, c). High temperature stress (2.7-5.2°C more temperature than ambient

conditions) decreased the spike length and spikelets per spike and ultimately grain yield

was decreased by 21% in heat tolerant genotype and 36% in heat susceptible genotype

(Dwivedi et al., 2016). Imposition of heat stress at anthesis for 7 days decreased spike

productivity, length and spiklets per spike of wheat (Hlaváčová et al., 2017). Decreased

spikelets and spike length was due to impaired pollen variability under heat stress

(Paupière et al., 2014).

Acceleration of grain filling rate and shortening of duration might be an adaptive

response to produce seeds for the next generation under heat stress environment.

77

However, rapid senescence under heat might have decreased capability of source organs

to satiate carbohydrate needs of rapidly growing spikes. Hence, it resulted in decreased

number of grains per spike and 1000-grain weight under heat stress. Such relationship

was also established from strong, positive and significant correlation of these attributes

with chlorophyll a and b. Shortening of phenology and rapid grain filling rate can also be

a consequence of aggravated oxidative stress. Excessive synthesis of superoxide radicals

might have initiated a cascade of reactions for even more burst of ROS and thus

destabilized photosystem and biosynthesis of chlorophyll. Ultimately, senescence was

rapid relative to carbohydrates supply. Whereas, strong, significant and positive

association of antioxidants with grain filling rate and duration further accomplished the

oxidative stress mediated damages for grain filling rate and duration (Table 4.2.15-4.2.17

a, c). Stress conditions aggravated oxidative stress, downregulated biosynthesis of

glutathione reductase and other antioxidants and significantly reduced grain filling

duration in wheat. Accelerated senescence of the flag leaf was highly associated with

shortening of phenology of wheat under stressed environments (Gallé et al., 2013).

Potassium mediated improvements in grain filling rate and extension of duration

of filling can be attributed to improvements in membrane stability. Enhancing potassium

enhanced accumulation of osmo-protectants and osmotic potential and thereby retained

cellular water under heat. Ultimately, sensitivity of photosynthesis to heat might have

decreased and partitioning of carbohydrates to grains was sustained for relatively longer

duration compared to water spray. Improvement in biological yield due to potassium

application also provided indication of extension of grain filling duration and acceleration

of grain filling rate with photosynthesis. Such type of explanation was also supported by

correlation analyses indicating a positive and strong correlation of these attributes with

shoot potassium contents over the years (Table 4.2.15-4.2.17 a, c). Application of

potassium enhanced grain filling rate, duration and ultimately yield (Liu et al., 2011).

Foliar application of potassium improved quality traits of terminal heat stressed wheat,

spike length and spikelets per spike (Rahman et al., 2014).

Exogenous potassium induced improvements in water relations might also have

alleviated oxidative stress enhanced biosynthesis of chlorophyll and wheat remained

green for longer duration. Hence, availability of source might have sustained

carbohydrates partitioning for a longer duration. Additionally, enhancement of 1000-grain

weight and grains per spike under heat stress because of potassium confirmed the

potassium modulation in transport of sucrose to grains. Besides, significant and strong

78

positive association of shoot potassium contents with chlorophyll a and b under ‘no heat

stress’ (Table 4.2.15 a, c), ‘heat from spike to grain filling’ (Table 4.2.16 a, c) and ‘heat

from flowering to grain filling’ (Table 4.2.17 a, c) established the potassium role in

enhancing the staying green trait. Supplementation of potassium enhanced the

accumulation of proline, soluble sugars, free amino acids and biosynthesis of chlorophyll

a and b under the stressed environment (Ahanger and Agarwal, 2017). The improved

cellular water status of wheat under the potassium availability improved the antioxidant

defense system which ultimately enhanced grain filling rate and duration (Xiaokang et al.,

2017).


(a) Results

Heats stress adversely affected the biosynthesis of chlorophyll and antioxidants

compared to the control. Yet, relatively more degradation of chlorophyll and inhibition in

biosynthesis of antioxidants was observed under ‘heat from spike to grain filling’

compared to ‘heat from flowering to grain filling’. Increasing concentrations of

exogenous potassium significantly improved chlorophyll content and antioxidant

activities compared to water spray (control). However, similar responses under different

heat imposition treatments were recorded regarding potassium modulated improvements

in chlorophyll biosynthesis to result a non-significant ‘heat × foliar potassium’ effect.

Whereas, a significant interaction of heat stress and foliar applied potassium was

observed for antioxidants activities.

‘Heat from spike to grain filling’ and ‘heat from flowering to grain filling’ caused

degradation by 43-44% and 28-30% compared to ‘no heat stress’ in chlorophyll a

contents over the 2015-16 and 2016-17. While, chlorophyll b content was reduced by 51-

54% under ‘heat from spike to grain filling’ and 41-45% under ‘heat from flowering to

grain filling’ relative to ‘no heat stress’ over the temporal variability.

Foliar applied potassium significantly enhanced the biosynthesis of chlorophyll a

and b contents compared to water spray (control). Statistically similar and significantly

more chlorophyll a and b contents were quantified with 45 and 60 g L-1 foliar potassium

compared to other application rates over the two years study period. While, statistically

similar and significantly lesser chlorophyll a and b contents were measured for water

spray, 15 and 30 g L-1 exogenous potassium with the slight inconsistencies over the years

(Table 4.2.7). Moreover, each 15 g L-1 foliar potassium modulated improvements in

chlorophyll a contents were 0.0094-0.012 mg g-1 FW under ‘no heat stress’, 0.007 mg g-1

79

FW under ‘heat from spike to grain filling’ and 0.0068-0.0082 mg g-1 FW under ‘heat

from flowering to grain filling’ over 2015-16 and 2016-17. While, chlorophyll b contents

were improved by 0.0022-0.0035, 0.0019-0.0024 and 0.0019-0.0028 mg g-1 FW under ‘no

heat stress’, ‘heat from spike grain filling’ and ‘heat from flowering to grain filling’,

respectively over the years. Moreover, increase in chlorophyll contents with each unit

foliar application of potassium were generally more under stressed environment (with

little discrepancies) compared to ‘no heat stress’ over the years (Figure 4.2.7).

Heat stress, foliar potassium as well as interaction of heat and foliar potassium

significantly affected the activities of superoxide dismutase (SOD), peroxidase (POD),

catalase (CAT) and total phenolics (TPC). Under ambient conditions, significantly more

and statistically similar activities of SOD and CAT activities were recorded for 45 and 60

g L-1 foliar potassium over the years. Likewise, application of potassium at 45 and 60 g L -

1 during 2015-16 and 30, 45 and 60 g L-1 during 2016-17 depicted statistically alike and

significantly higher activities of POD and TPC under ‘no heat stress’ conditions.

Whereas, significantly higher activities of SOD, CAT, POD and TPC were recorded with

60 g L-1 foliar potassium under the heat stressed conditions over the two study years

(Table 4.2.8 and Table 4.2.9).

Upregulation in SOD activity due to per unit application of potassium were 1.13-

1.28 U per mg protein under ‘no heat stress’, 0.49-0.54 U per mg protein under ‘heat from

spike to grain filling’ and 0.51-0.55 U per mg protein under ‘heat from flowering to grain

filling’ was recorded over the years. While, improvements in POD activities with the per

unit application of potassium were 0.13-0.16 U per mg protein under ‘no heat stress’,

0.09-0.10 U per mg protein under ‘heat from spike to grain filling’ and 0.07-0.12 U per

mg protein under ‘heat from flowering to grain filling’ over the two years study period

(Figure 4.2.8).

Application of exogenous potassium enhanced CAT activity by 0.26-0.32 U per

mg protein under ‘no heat stress’, 0.12-0.16 U per mg protein under ‘heat from spike to

grain filling’ and 0.15-0.18 U per mg protein under ‘heat from flowering to grain filling’

over the years. Potassium triggered improvements in catalase activity were higher under

‘heat from flowering to grain filling’ during 2015-16 and under ‘no heat stress’ during

2016-17 compared to other treatments. Whereas, per unit enhancement in foliar

potassium improved TPC by 0.16-0.21 mg GAE g-1 under ‘no heat stress’, 0.08-0.10 mg

GAE per g-1 under ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling’ (Figure 4.2.9).

80

(b) Discussion

Degradation of chlorophyll contents in response to heat can be attributed to

damaged permeability of membranes under heat stress.

Table 4.2.7: Effect of foliar applied potassium on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of heat stressed wheat


Source of

variationDF

Chlorophyll a Chlorophyll b

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.953 0.349 0.0255 0.0289

Heat (H) 2 2.819** 1.708** 0.3939** 0.3829**

Error I 4 0.024 0.017 0.0021 0.0014

Potassium (K) 4 0.341** 0.443** 0.0482** 0.0213**

H × K 8 0.006NS 0.013NS 0.0007NS 0.0002NS



TreatmentsChl a (mg g-1 FW) Chl b (mg g-1 FW)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 1.10 C 0.86 C 0.30 C 0.26 C


Tukey’s HSD (p ≤ 0.05) 0.202 0.171 0.059 0.048


Control/ water spray (K0) 1.33 B 0.92 C 0.36 BC 0.33 B

15 g L-1 potassium (K15) 1.29 B 0.96 C 0.35 C 0.34 B

30 g L-1 potassium (K30) 1.44 B 1.11 B 0.40 B 0.36 B



Tukey’s HSD (p ≤ 0.05) 0.161 0.143 0.045 0.042

Year mean 1.48 A 1.15 B 0.42 0.38


81

Figure 4.2.7: Regression analysis for effect of foliar applied potassium on chlorophyll a and chlorophyll b contents of heat stressed wheat


82

Table 4.2.8: Effect of foliar applied potassium on superoxide dismutase (SOD) and peroxidase (POD) contents of heat stressed wheat


Source of variation

DFSuperoxide dismutase Peroxidase

2015-16 2016-17 2015-16 2016-17Blocks 2 3987.8 1000.7 27.13 16.00Heat (H) 2 24951.8** 39494.5** 843.32** 622.95**Error I 4 225.4 76.1 2.58 0.91Potassium (K) 4 3263.4** 2723.8** 92.31** 48.15**H × K 8 481.2** 341.9** 5.31** 4.63**Error II 24 47.1 23.8 0.66 0.78



TreatmentsSOD

(Unit per mg protein)POD

(Unit per mg protein)2015-16 2016-17 2015-16 2016-17

No heat stress (H0)Control/ water spray (K0) 135.0 c 134.0 d 26.0 d 20.0 c15 g L-1 potassium (K15) 133.7 c 138.0 cd 25.0 cd 23.0 b30 g L-1 potassium (K30) 157.7 b 147.3 bc 27.8 bc 25.8 a45 g L-1 potassium (K45) 196.0 a 190.0 a 33.0 a 27.0 a60 g L-1 potassium (K60) 200.0 a 192.7 a 34.4 a 27.5 aHeat from spike to grain filling (H1)Control/ water spray (K0) 70.3 c 48.7 c 13.0 d 11.2 c15 g L-1 potassium (K15) 82.7 bc 52.0 bc 14.1 cd 11.9 bc30 g L-1 potassium (K30) 85.7 bc 59.0 bc 15.3 bc 12.1 bc45 g L-1 potassium (K45) 88.3 b 63.7 b 16.1 b 13.7 b60 g L-1 potassium (K60) 108.3 a 79.3 a 19.7 a 17.0 aHeat from flowering to grain filling (H2)Control/ water spray (K0) 88.7 c 78.7 c 14.3 c 12.4 b15 g L-1 potassium (K15) 97.0 bc 81.0 c 14.6 c 12.5 b30 g L-1 potassium (K30) 100.0 bc 86.0 bc 16.6 b 13.2 b45 g L-1 potassium (K45) 106.3 b 93.3 b 17.0 b 14.0 b60 g L-1 potassium (K60) 125.3 a 111.0 a 22.2 a 17.2 aTukey’s HSD (p ≤ 0.05) 16.48 11.72 1.95 2.12Year mean 118.3 103.6 20.6 A 17.2 BTukey’s HSD (p ≤ 0.05) NS 2.72

Any two means not sharing a letter in common within a column differ significantly at p ≤ 0.05; NS = Non-

significant

83

Figure 4.2.8: Regression analysis for effect of foliar applied potassium on superoxide dismutase and peroxidase contents of heat stressed wheat


84

Table 4.2.9: Effect of foliar applied potassium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat


Source of variation

DFCatalase Total phenolics

2015-16 2016-17 2015-16 2016-17Blocks 2 858.76 427.09 84.12 14.18Heat (H) 2 2443.59* 2537.71** 717.93** 709.25**Error I 4 161.95 6.25 15.98 2.04Potassium (K) 4 270.77** 160.68** 111.98** 62.34**H × K 8 20.00** 15.78** 14.80** 9.41**Error II 24 3.76 1.19 0.61 0.56



TreatmentsCAT (Unit per mg protein) TPC (mg GAE g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (K0) 41.0 b 33.7 c 22.7 c 18.9 b15 g L-1 potassium (K15) 40.6 b 34.7 c 21.5 c 19.4 b30 g L-1 potassium (K30) 44.7 b 41.0 b 25.3 b 25.7 a45 g L-1 potassium (K45) 55.4 a 46.7 a 32.9 a 27.1 a60 g L-1 potassium (K60) 57.3 a 47.3 a 33.1 a 27.2 aHeat from spike to grain filling (H1)Control/ water spray (K0) 19.7 b 12.3 c 12.3 c 8.6 b15 g L-1 potassium (K15) 20.0 b 13.7 bc 12.9 bc 8.9 b30 g L-1 potassium (K30) 22.0 b 15.0 b 13.2 bc 9.5 b45 g L-1 potassium (K45) 23.4 b 15.3 b 14.7 b 10.0 b60 g L-1 potassium (K60) 30.3 a 20.7 a 19.2 a 14.0 aHeat from flowering to grain filling (H2)Control/ water spray (K0) 25.7 c 19.0 d 14.0 b 12.8 b15 g L-1 potassium (K15) 26.8 c 20.4 cd 14.2 b 13.0 b30 g L-1 potassium (K30) 28.5 bc 22.0 bc 14.9 b 14.2 b45 g L-1 potassium (K45) 31.7 b 23.7 b 15.3 b 14.5 b60 g L-1 potassium (K60) 37.0 a 28.3 a 21.0 a 18.3 aTukey’s HSD (p ≤ 0.05) 4.66 2.62 1.87 1.80Year mean 33.6 A 26.2 B 19.1 A 16.2 BTukey’s HSD (p ≤ 0.05) 5.53 2.84


significant

85

Figure 4.2.9: Regression analysis for effect of foliar applied potassium on catalase and total phenolic contents of heat stressed wheat


86

High temperature has aggravated generation of ROS and lipid peroxidation. The

same was confirmed from strong negative and significant correlation of chlorophyll a and

b contents with malondialdehyde under ‘no heat stress’ (Table 4.2.15 a, c), under ‘heat

from spike to grain filling’ (Table 4.2.16 a, c) and under ‘heat from flowering to grain

filling’ (Table 4.2.17 a, c) over the 2015-16 and 2016-17. Excessive ROS might have

impaired the activities of aminolevulinic acid dehydratase and porphobilinogen

deaminase. Moreover, heat stress induced boost in activities of protochlorophyllide

oxidoreductase might have accelerated the conversion of protochlorophyllide to

chlorophyllide and thus reduced chlorophyll contents under heat stress (Hemantaranjan et

al., 2014).

High temperature stress caused lesser translocation of sucrose towards grains from

source organs (leaves) leading to negative feedback for photosynthesis and chlorophyll

pigments. Since, grain filling duration was decreased so 1000-grain weight was also

decreased under heat. Whereas, lesser time availability for grain filling might have

transduced signal to leaves to downregulate the synthesis of carbohydrates and ultimately

biosynthesis of chlorophyll pigments was also slow under heat owing to disturbed

enzymes that regulated chlorophyll biosynthesis. Moreover, positive and significant

association of chlorophyll a and b contents with 1000-grain weight and grain filling

duration was recorded under ‘no heat stress’ (Table 4.2.15 a, c), ‘heat from spike to grain

filling’ (Table 4.2.16 a, c) and ‘heat from flowering to grain filling’ (Table 4.2.17 a, c)

over the years.

Improvement of chlorophyll a and b contents under foliar applied potassium

might be a consequence of potassium mediated enhancement in grain filling rate and

prolongation of grain filling duration. Concurrent enhancement of grain filling rate and

duration might have enhanced the time availability to accumulate sucrose in grains and

thus improved sink (grains) capacity. Improved sink capacity might have transduced

signal to vegetative parts to partition carbohydrates for longer duration. Improvement in

sink capacity was also confirmed from increasing 100-grain weight and number of grains

per spike under potassium compared to control (water spray). Moreover, strong positive

and significant association of chlorophyll a and b with grain filling rate and duration

established the grain filling rate and duration mediated delaying of senescence and

staying green for longer duration (Table 4.2.15-4.2.17 a, c). Under terminal stress,

application of potassium enhanced the stay green trait and carbohydrates partitioning to

grains (Hosseini et al., 2016).

87

Increase in chlorophyll a and b contents can also be explained in context of lesser

peroxidation of lipids under potassium availability. Potassium itself acts as an osmolyte

and enhances membrane integrity. Availability of potassium increases the accumulation

of osmo-protectants and thereby improves water potential. Thereafter, enhancement of

cellular water may inhibit the activities of chlorophyll degrading enzymes. Furthermore,

strong positive and significant relationship of shoot potassium contents with chlorophyll

a and b accomplished the potassium triggered regulations of chlorophyll biosynthesis

(Table 4.2.15-4.2.17 a, c). Application of foliar potassium enhanced the biosynthesis of

phenolics and chlorophyll under stress conditions. While, accumulation of osmolytes

enhanced the water potential under stress and ultimately improved growth (Jan et al.,

2017).

Diminishment of activities of SOD, POD, CAT and TPC under heat can be

attributed to excessive generation of ROS under heat stress. Heat stress might have

destabilized reaction center of photosystem-II whereby excessive light triggered

photolysis of water generated plethora of free electrons. These free electrons might react

with triplet oxygen (atmospheric oxygen) (1O2) at reaction center of photosystem-II and

reduce it to superoxide radical (1O2●-). Moreover, chlorophyll in excited state after

accepting electron from reaction center might react with triplet oxygen and release singlet

oxygen (1O2*) under heat stressed environments. Therefore, excessive generation of 1O2

●-

might have initiated a cascade of reactions for generation of other ROS as well. Excessive

generation of 1O2●- might have augmented substrate concentration for SOD. Hence, more

hydrogen peroxide (H2O2) was produced by POD and CAT mediated reduction of 1O2●-.

Concurrently, heat mediated generation of 1O2●- and H2O2 was high enough to impair the

activities of SOD, POD, CAT and TPC since potassium modulated improvement in

enzyme activities was lesser compared to heat triggered decrease in activities. Therefore,

inhibition of SOD, POD, CAT and TPC activities under heat might be a consequence

saturation of enzymes by excessive substrate concentration. Moreover, strong positive

and remarkable association of SOD, POD, CAT and TPC was observed under ‘no heat

stress’ (Table 4.2.15 b, d), under ‘heat from spike to grain filling’ (Table 4.2.16 b, d) and

under ‘heat from flowering to grain filling’ (Table 4.2.17 b, d) over the years. Association

of these attributes accomplished the enhancement/diminishment with each other based on

substrates availability. Enzymes activities generally enhance with enhancing

concentrations of substrates up to a point where enzymes are saturated by substrates and

activities start to diminish afterwards due to substrate saturation (Das and Roychoudhury,

88

2014). Furthermore, excessive hydroxyl radical (OH●-) under heat might have reacted

with hydroxyl group of phenolics and thus aggravated the degradation of phenolics under

heat (Yamauchi, 2015).

Upregulations in SOD, POD, CAT and TPC might be an outcome of potassium

mediated improvements in water relations at the cellular level due to enhanced capability

of plant to accumulate proline, glycine betaine and total soluble proteins which led to

increased detoxification of ROS. Hence, antioxidant activities were enhanced because of

substrates concentration that remained lower than the enzyme-saturation point under

availability of potassium. Moreover, strong positive and significant correlations of SOD,

POD, CAT and TPC with shoot potassium contents and proline were recorded under ‘no

heat stress’ (Table 4.1.15 b, d), ‘heat from spike to grain filling’ (Table 4.1.16 b, d) and

‘heat from flowering to grain filling’ for most of instances (Table 4.1.15 b, d) Likewise,

correlation of SOD, POD, CAT and TPC with proline and glycine betaine was also strong

positive and significant under varying temperatures over the two years (Table 4.2.15-

4.2.17 b, d). Proline biosynthesis under stress environments not only enhanced precursors

availability for biosynthesis of proteins but also enhanced stress tolerance by improving

signal transduction, regulation of turgor and water potential and scavenging of ROS

(Hayat et al., 2012). While, foliar applied potassium under stress conditions improved the

accumulation of glycine betaine and proline in wheat (Raza et al., 2014).


(a) Results

Heat stress, foliar applied potassium and their interaction negatively impacted leaf

proline, glycine betaine, total soluble proteins and malondialdehyde. Comparatively

higher proline, glycine betaine, total soluble proteins and lesser malondialdehyde were

recorded under ‘no heat stress’ than heat stress environment. Moreover, ‘heat from spike

to grain filling was more destructive than ‘heat from flowering to grain filling’ regarding

the biosynthesis of osmo-protectants and lipid peroxidation of bio-membranes. Under ‘no

heat stress’, statistically similar and significantly more proline, glycine betaine and total

soluble proteins and were recorded with 45 and 60 g L-1 foliar potassium than water

spray/control, 15 and 30 g L-1 foliar potassium over the years. Whereas, in heat stressed

wheat 60 g potassium L-1 resulted in higher concentrations of proline, glycine betaine and

total soluble proteins than lower concentrations. Comparatively more malondialdehyde

contents were computed with ‘control/water spray’ and 15 g L-1 exogenous potassium

than 30, 45 and 60 g L-1 potassium over 2015-16 and 2016-17 under ‘no heat stress’, ‘heat

89

from spike to grain filling’ and ‘heat from flowering to grain filling’ (Table 4.2.10 and

Table 4.2.11).

Moreover, an increment of 0.02 µmol g-1 under ‘no heat stress’, 0.009-0.01 µmol

g-1 under ‘heat from spike to grain filling’ and 0.009-0.01 µmol g-1 in proline contents was

recorded with each 15 g L-1 application of foliar potassium over the two years. Whereas,

each 15 g L-1 application of exogenous potassium enhanced the leaf glycine betaine

contents by 1.45-1.47, 0.90-0.93 and 0.77-0.89 µmol g-1 under ‘no heat stress’, ‘heat from

spike to grain filling and ‘heat from flowering to grain filling’, respectively over the two

years of study. Likewise, per unit foliar potassium modulated improvements in total

soluble proteins were 0.0041-0.0051 mg g-1 under ‘no heat stress’, 0.0013-0.0025 mg g-1

under ‘heat from spike to grain filling’ and 0.0020-0.0029 mg g-1 under ‘heat from

flowering to grain filling’ over the years. Whereas, each per unit potassium mediated

decrease in malondialdehyde was 0.0025-0.0029 µmol g-1 under ‘no heat stress’, 0.008

µmol g-1 under ‘heat from spike to grain filling’ and 0.005-0.007 µmol g-1 under ‘heat

from flowering to grain filling’ over the years. Moreover, potassium mediated

improvements in osmo-protectants and decrease in malondialdehyde were generally more

under stressed environments compared to ‘no heat stress’ over the years, however some

discrepancies were observed during 2016-17. (Figure 4.2.10 and Figure 4.2.11).

(b) Discussion

Heat stress triggered the degradation of chlorophyll and thus decreased the

sucrose availability for biosynthesis of osmo-protectants. Consequently, availability of

carbon chain might have decreased which ultimately downregulated the biosynthesis of

osmo-protectants. Furthermore, strong positive and significant association of leaf proline,

glycine betaine and total soluble proteins was recorded under varying temperatures which

accomplished the dependence of osmo-protectants on chlorophyll (Table 4.2.15-4.2.17 a,

c). High temperature negatively impacted the accumulation of osmo-protestants and

photosynthetic pigments while, the ability of plant to accumulate osmo-protectants was

enhanced with enhancing chlorophyll pigments under the high temperature stress

(Awasthi et al., 2015). Decrease of photosynthetic pigments under the stressed

environment decreased the water potential of cells since plant accumulated lesser osmo-

protectants (Moharramnejad et al., 2015).

Heat stress might have accelerated the production of ROS and excessive ROS

might have overcome defensive mechanism of plant. Thereafter, plethora of 1O2●-, 1O2

*

and H2O2 might have dominated the activities of SOD, POD and CAT. While, excessive

90

generation of OH●- might have reacted with hydrogen of carbon chain and convert carbon

chain itself into carbon centered radical (another ROS). Thereafter carbon centered

radicals might have initiated a cascade of reaction to synthesize alkoxyl radicals (RO●-)

and peroxyl radical (ROO●-).

Table 4.2.10: Effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat


Source of variation

DFLeaf proline Leaf glycine betaine




TreatmentsProline (µmol g-1) Glycine betaine (µmol g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (K0) 3.02 c 2.65 c 223.5 c 201.7 c15 g L-1 potassium (K15) 2.98 c 2.68 c 218.8 c 210.0 c30 g L-1 potassium (K30) 3.47 b 3.13 b 248.5 b 231.7 b45 g L-1 potassium (K45) 3.87 a 3.52 a 291.7 a 275.0 a60 g L-1 potassium (K60) 3.90 a 3.57 a 296.0 a 279.3 aHeat from spike to grain filling (H1)Control/ water spray (K0) 1.28 c 1.04 b 111.7 d 105.0 c15 g L-1 potassium (K15) 1.33 c 1.10 b 118.7 cd 109.3 c30 g L-1 potassium (K30) 1.57 b 1.23 b 128.2 c 120.7 bc45 g L-1 potassium (K45) 1.63 b 1.27 b 145.0 b 128.3 b60 g L-1 potassium (K60) 1.97 a 1.63 a 168.0 a 163.0 aHeat from flowering to grain filling (H2)Control/ water spray (K0) 1.80 c 1.47 c 128.3 d 111.7 d15 g L-1 potassium (K15) 1.98 bc 1.48 c 133.5 cd 118.2 cd30 g L-1 potassium (K30) 2.08 b 1.66 bc 144.3 bc 133.3 bc45 g L-1 potassium (K45) 2.12 b 1.79 b 153.1 b 141.5 b60 g L-1 potassium (K60) 2.39 a 2.17 a 176.7 a 166.7 aTukey’s HSD (p ≤ 0.05) 0.215 0.304 13.26 18.05Year mean 2.36 2.03 179.0 166.4

91

Tukey’s HSD (p ≤ 0.05) NS NSAny two means not sharing a letter in common within a column differ significantly at p ≤ 0.05; NS = Non-

significant

Figure 4.2.10: Regression analysis for effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat

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Table 4.2.11: Effect of foliar applied potassium on total soluble proteins (TSP) and malondialdehyde (MDA) of heat stressed wheat


Source of variation

DFTotal soluble proteins Malondialdehyde




TreatmentsTSP (mg g-1) MDA (µmol g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (K0) 0.52 b 0.45 c 0.83 a 0.87 a15 g L-1 potassium (K15) 0.49 b 0.47 c 0.84 a 0.86 a30 g L-1 potassium (K30) 0.55 b 0.53 b 0.72 b 0.79 ab45 g L-1 potassium (K45) 0.75 a 0.64 a 0.70 b 0.74 b60 g L-1 potassium (K60) 0.77 a 0.67 a 0.68 b 0.73 bHeat from spike to grain filling (H1)Control/ water spray (K0) 0.27 b 0.16 b 1.55 a 1.60 a15 g L-1 potassium (K15) 0.27 b 0.16 b 1.52 a 1.57 ab30 g L-1 potassium (K30) 0.32 b 0.17 b 1.33 b 1.48 bc45 g L-1 potassium (K45) 0.33 b 0.17 b 1.32 b 1.44 c60 g L-1 potassium (K60) 0.42 a 0.25 a 1.06 c 1.10 dHeat from flowering to grain filling (H2)Control/ water spray (K0) 0.31 c 0.25 b 1.42 a 1.47 a 15 g L-1 potassium (K15) 0.31 c 0.26 b 1.35 a 1.40 ab30 g L-1 potassium (K30) 0.34 bc 0.28 b 1.25 b 1.30 bc45 g L-1 potassium (K45) 0.40 b 0.29 b 1.17 b 1.28 c60 g L-1 potassium (K60) 0.49 a 0.39 a 1.02 c 1.12 dTukey’s HSD (p ≤ 0.05) 0.065 0.042 0.090 0.107Year mean 0.44 A 0.34 B 1.11 1.18

93

Tukey’s HSD (p ≤ 0.05) 0.071 NSAny two means not sharing a letter in common within a column differ significantly at p ≤ 0.05; NS = Non-

significant

Figure 4.2.11: Regression analysis for effect of foliar applied potassium on total soluble proteins and malondialdehyde of heat stressed wheat

94


Thus, burst of ROS might have degenerated lipids (ground structure) of bio-

membranes and thus decreased the capability of plant to synthesize osmo-protectants.

Furthermore, strong negative and significant correlation of proline, glycine betaine and

95

total soluble proteins with malondialdehyde both under no heat and heat-imposed

conditions established the aggravated lipid peroxidation caused negative impacts on

osmo-protectants (Table 4.2.15-4.2.17 a, c). High temperature stress enhanced ROS,

which overcame the antioxidant defense mechanism of plant and aggravated lipid

peroxidation. Moreover, the capability of plants to accumulate total soluble sugars,

trehalose, free amino acids, soluble proteins and proline was decreased under heat stress

(Asthir, 2015).

Availability of potassium from exogenous application might have improved the

activation of ATPs and synthesis of reductants at the end of light reactions of

photosynthesis. Henceforth, sucrose loading from leaves and unloading from phloem and

subsequent translocation towards grains might have enhanced under potassium

availability. This not only enhanced carbohydrates availability to develop grains but also

enhanced carbon chain availability for biosynthesis of amino acids (proline, glycine

betaine) and succeeding soluble proteins. The availability of potassium under stress

conditions enhanced the synthesis of osmo-protectants, antioxidants, carbohydrates

translocation to reproductive parts and reduced lipid peroxidation (Zahoor et al., 2017b).

Foliar application of potassium under heat stress in wheat enhanced carbohydrates

partitioning to grains, photosynthetic efficiency, regulated stomatal opening and closing

and ultimately enhanced grain yield (Kajla et al., 2015).

Potassium-mediated improvements in carbohydrate availability and osmo-

protectants might have enhanced water potential of cell and alleviated adversities of

oxidative stress. Biosynthesis of ROS under potassium availability might have diminished

to a concentration lower enough to not impair enzyme activities because of substrate

saturation. Therefore, comparatively lesser 1O2●-, 1O2

*, H2O2 and OH●- concentrations

under foliar potassium might have elicited activities of SOD, POD and CAT which

ultimately alleviated oxidative stress and enhanced capability of plant to synthesize osmo-

protectants. Furthermore, antioxidant triggered improvements in synthesis of osmo-

protectants was established from strong positive and significant association of

antioxidants and osmo-protectants under varying temperature regimes and over the

temporal variations (Table 4.2.15-4.2.17 b, d). Exogenous potassium enhanced

biosynthesis of proline, total soluble proteins, antioxidants and photosynthetic pigments

in wheat under stress environment (Wei et al., 2013). Deficiency of potassium enhanced

vulnerability to stress in tolerant and susceptible genotypes of wheat (Ruan et al., 2015).

96

Wheat might have compartmentalized excessive potassium in vacuole under ‘no

heat stress’ conditions. While, under heat stress, vacuolar potassium might have

mobilized from vacuole and re-translocated to the cytosol at sub-cellular level.

Henceforth, potassium presence in cytosol might have acted as osmolyte, maintained

water potential, activated antioxidants and eventually regulated morphological responses

of wheat under heat. Excessive application of potassium did not show negative impacts,

yet plant accumulated excessive potassium in vacuoles where it mostly regulated

homeostasis and water relations (Andrés et al., 2014; Shin, 2017). Whereas, plants

remobilized and redistributed vacuolar compartmentalized potassium under stress

conditions into the cell cytosol where it activated enzymes and improved photosynthesis

(Wang and Wu, 2017).

4.2.8. Water relations and quality attributes

(a) Results

Imposition of heat depressed water, osmotic and turgor potential compared to ‘no

heat stress’. ‘Heat from spike to grain filling’ was more deleterious than ‘heat from

flowering to grain filling’ in this regard. Moreover, exogenous potassium significantly

enhanced osmotic, water and turgor potential compared to control/ water spray. Varying

concentrations of foliar potassium exhibited dissimilar response to produce a significant

‘heat × foliar potassium’ effect for water relation attributes and shoot potassium contents.

However, the interaction was non-significant for grain crude protein contents.

Significantly more osmotic, water, turgor potential and shoot potassium contents

was observed with 60 g L-1 exogenous potassium compared to control/ water spray, 15, 30

and 45 g L-1 foliar potassium under ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’ over years. However, under no heat stress both 45 and 60 g L -1

potassium performed equally well in improving water relations and shoot potassium

contents (Table 4.2.12 and Table 4.2.13).

Heat imposition caused a notable decrease in grain crude protein contents, so a

decrease of 22-27% and 14-19% in grain crude protein contents was recorded under heat

from ‘spike to grain filling’ and ‘heat from flowering to grain filling’, respectively over

the years. Furthermore, statistically similar and comparatively more grain crude protein

contents were quantified with 45 and 60 g L-1 foliar potassium compared to other

concentrations of potassium in 2015-16. Whereas, statistically alike and relatively higher

grain crude protein contents were observed with 30, 45 and 60 g L-1 foliar potassium

97

during 2016-17. Conversely, control/water spray and 15 g L-1 exogenous potassium

exhibited comparatively lesser grain crude protein contents (Table 4.2.14).

Each 15 g L-1 application of exogenous potassium improved osmotic potential by

0.004-0.006 MPa under ‘no heat stress’, 0.007-0.008 MPa under ‘heat from spike to grain

filling’ and 0.006 MPa under ‘heat from flowering to grain filling’ over the two study

years. Likewise, each unit application of foliar potassium enhanced water potential by

0.005-0.008, 0.008-0.009 and 0.007-0.008 MPa under ‘no heat stress’, ‘heat from spike to

grain filling’ and’ heat from flowering to grain filling’, respectively over the temporal

variability. While, each unit application of potassium improved the turgor potential by

0.001-0.002 MPa under ‘no heat stress’, 0.0007-0.0010 under ‘heat from spike to grain

filling’ and 0.0007-0.002 MPa under ‘heat from flowering to grain filling’ over the years.

Similarly, shoot potassium contents was improved by 11.7-11.9 µg g -1 under ‘no heat

stress’, 8.45-10.6 µg g-1 under ‘heat from spike to grain filling’ and 7.5-7.9 µg g -1 under

‘heat from flowering to grain filling’ with each 15 g L-1 of foliar potassium over the years.

Moreover, potassium modulated improvements in osmotic, water potential and shoot

potassium contents were generally more under stress environment than ‘no heat stress’

over the years. However, some anomalies were also observed during 2016-17 where

dependence on foliar potassium was decreased under stress compared to ‘no heat stress’.

Whereas, dependence of turgor potential on potassium was lesser under stress compared

to ‘no heat stress’ over the years (Figure 4.2.12 and Figure 4.2.13).

Grain crude protein contents were enhanced by 0.028-0.029% under ‘no heat

stress’ and 0.024% under both heat imposition treatments with each unit (15 g L-1)

enhancement in foliar potassium over the 2015-16 and 2016-17. Moreover, the

importance of foliar applied potassium in improving grain crude protein contents was

more under heat than no heat over the years (Figure 4.2.14).

(b) Discussion

Decrease of osmotic, water and turgor potential under heat can be attributed to

degradation of chlorophyll. Heat stress might have aggravated degradation of chlorophyll

and ultimately carbohydrate biosynthesis was also impaired. Decrease in carbohydrates

availability might have negatively impacted carbon chain availability for the synthesis of

amino acids such as soluble proteins, glycine betaine and proline contents. Henceforth,

diminished capability to synthesize osmo-protectants not only decreased the capability of

plant to withhold water under stress but also reduced potent antioxidative characteristics

of proline and glycine betaine.

98

Table 4.2.12: Effect of foliar applied potassium on osmotic (ΨS) and water potential (ΨW) of heat stressed wheat


Source of variation

DFOsmotic potential Water potential

2015-16 2016-17 2015-16 2016-17Blocks 2 0.0727 0.1434 0.1091 0.1117Heat (H) 2 0.9054** 1.2661** 1.8181** 1.8637**Error I 4 0.0020 0.0230 0.0080 0.0040Potassium (K) 4 0.2250** 0.1956** 0.3561** 0.2498**H × K 8 0.0106** 0.0085* 0.0129** 0.0089*Error II 24 0.0017 0.0035 0.0029 0.0034



TreatmentsΨS (-MPa) ΨW (-MPa)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (K0) 1.22 b 1.24 b 0.93 c 1.04 d15 g L-1 potassium (K15) 1.22 b 1.24 b 0.92 c 1.01 cd30 g L-1 potassium (K30) 0.98 a 1.19 b 0.67 b 0.93 bc45 g L-1 potassium (K45) 0.93 a 1.15 ab 0.54 a 0.88 a60 g L-1 potassium (K60) 0.93 a 1.01 a 0.52 a 0.74 aHeat from spike to grain filling (H1)Control/ water spray (K0) 1.73 d 1.93 c 1.60 d 1.81 c15 g L-1 potassium (K15) 1.60 c 1.79 bc 1.46 c 1.67 bc30 g L-1 potassium (K30) 1.57 c 1.76 b 1.43 c 1.63 b45 g L-1 potassium (K45) 1.42 b 1.71 b 1.28 b 1.58 b60 g L-1 potassium (K60) 1.25 a 1.44 a 1.04 a 1.28 aHeat from flowering to grain filling (H2)Control/ water spray (K0) 1.58 c 1.71 b 1.45 c 1.58 b 15 g L-1 potassium (K15) 1.57 c 1.67 b 1.42 c 1.53 b30 g L-1 potassium (K30) 1.45 b 1.65 b 1.29 b 1.50 b45 g L-1 potassium (K45) 1.39 b 1.61 b 1.22 b 1.47 b60 g L-1 potassium (K60) 1.19 a 1.28 a 0.94 a 1.10 aTukey’s HSD (p ≤ 0.05) 0.099 0.142 0.129 0.140Year mean 1.33 A 1.49 B 1.11 A 1.32 BTukey’s HSD (p ≤ 0.05) 0.117 0.144


99

Figure 4.2.12: Regression analysis for effect of foliar applied potassium on osmotic and water potential of heat stressed wheat


100

Table 4.2.13: Effect of foliar applied potassium on turgor potential (ΨP) and shoot potassium (K) contents of heat stressed wheat


Source of variation

DFTurgor potential Shoot potassium contents

2015-16 2016-17 2015-16 2016-17Blocks 2 0.00240 0.00138 99473 1138381Heat (H) 2 0.15788** 0.05928** 7600945** 8374906**Error I 4 0.00012 0.00011 8639 35174Potassium (K) 4 0.01595** 0.00381** 563187** 476563**H × K 8 0.00145** 0.00046** 25319** 18697*Error II 24 0.00011 0.00009 6706 7529



TreatmentsΨP (MPa) Shoot K (µg g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (K0) 0.29 b 0.20 c 2326 b 2225 c15 g L-1 potassium (K15) 0.30 b 0.23 b 2320 b 2229 c30 g L-1 potassium (K30) 0.31 b 0.26 a 2473 b 2473 b45 g L-1 potassium (K45) 0.39 a 0.27 a 2910 a 2793 a60 g L-1 potassium (K60) 0.41 a 0.27 a 2923 a 2823 aHeat from spike to grain filling (H1)Control/ water spray (K0) 0.13 b 0.12 b 1000 c 933 c15 g L-1 potassium (K15) 0.14 b 0.12 b 1026 c 936 c30 g L-1 potassium (K30) 0.14 b 0.13 b 1085 c 1001 c45 g L-1 potassium (K45) 0.14 b 0.13 b 1358 b 1213 b60 g L-1 potassium (K60) 0.21 a 0.16 a 1628 a 1428 aHeat from flowering to grain filling (H2)Control/ water spray (K0) 0.13 c 0.13 b 1389 c 1228 c 15 g L-1 potassium (K15) 0.15 bc 0.14 b 1408 c 1232 c30 g L-1 potassium (K30) 0.16 b 0.15 b 1577 bc 1239 c45 g L-1 potassium (K45) 0.17 b 0.14 b 1608 b 1464 b60 g L-1 potassium (K60) 0.25 a 0.18 a 1883 a 1678 aTukey’s HSD (p ≤ 0.05) 0.025 0.023 196.7 208.4Year mean 0.22 A 0.17 B 1794 1660Tukey’s HSD (p ≤ 0.05) 0.033 NS


significant

101

Figure 4.2.13: Regression analysis for effect of foliar applied potassium on turgor potential and shoot potassium contents of heat stressed wheat


102

Table 4.2.14: Effect of foliar applied potassium on grain crude proteins of heat stressed wheat


Source of variation DFGrain crude proteins

2015-16 2016-17

Blocks 2 25.84 26.46

Heat (H) 2 25.99* 42.52*

Error I 4 1.92 2.71

Potassium (K) 4 3.45** 3.49**

H × K 8 0.08NS 0.07NS

Error II 24 0.27 0.48* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant


TreatmentsGrain crude proteins (%)

2015-16 2016-17

Heat stress (H)

No heat stress (H0) 11.69 A 12.20 A

Heat from spike to grain filling (H1) 9.10 B 8.93 B

Heat from flowering to grain filling (H2) 10.02 AB 9.85 B

Tukey’s HSD (p ≤ 0.05) 1.802 2.144


Control/ water spray (K0) 9.67 C 9.67 B

15 g L-1 potassium (K15) 9.73 C 9.84 B

30 g L-1 potassium (K30) 10.12 BC 10.17 AB

45 g L-1 potassium (K45) 10.81 AB 10.87 A

60 g L-1 potassium (K60) 11.02 A 11.08 A

Tukey’s HSD (p ≤ 0.05) 0.726 0.967

Year mean 10.27 10.33

Tukey’s HSD (p ≤ 0.05) NSAny two means not sharing a letter in common differ significantly at p ≤ 0.05; NS = Non-significant

Figure 4.2.14: Regression analysis for effect of foliar applied potassium on grain crude proteins of heat stressed wheat

103


These disturbances might have worsened oxidative stress and aggravated

membrane leakage and ultimately disturbed water relations of wheat. Moreover, strong

104

positive and pronouncing relationship of osmotic, water and turgor potential with

chlorophyll a and b accomplished the chlorophyll triggered decrease in synthesis of

osmo-protectants. Accumulation of lesser osmo-protectants and antioxidants enhanced

lipid peroxidation, which ultimately overwhelmed the antioxidant defense system in heat

and drought tolerant and susceptible genotypes (Rehman et al., 2016). Enhancement in

biosynthesis of osmo-protectants improved dry matter accumulation, chlorophyll

contents, photosynthesis and decreased lipid peroxidation in terminal heat stressed wheat

(Suryavanshi et al., 2016).

Depressed osmotic, water and turgor potential under heat might be a consequence

of lesser shoot potassium content. According to Jan et al. (2017) deficiency of potassium

stress reduced photosynthetic efficiency and capability of plant to make osmotic

adjustments. While according to Zahoor et al. (2017a) diminished capability of plant to

synthesize osmo-protectants impaired the photosynthesis and decreased soluble sugars.

Henceforward, decreased shoot potassium might have adversely influenced osmo-

protectants (proline, glycine betaine and total soluble proteins) by affecting carbon chain

availability for synthesis of amino acid on one side. On the other side, decrease of

potassium itself might have reduced osmolyte concentration of cytosol and thus disturbed

the homeostasis. Resultantly, capability of cells to retain water was decreased and hence

water, osmotic and turgor potentials were also depressed accordingly. Decreased

availability of potassium under stress conditions reduced the capability of plant to

synthesize osmo-protectants and sustain water and turgor and ultimately growth and yield

was deleteriously impacted (Hassan et al., 2017).

Likewise, heat stress. Heat stress might have accelerated the generation of 1O2●-,

1O2*, OH●- and H2O2 react with hydroxyl group of carbon chain of fatty acids (ground

structure of membranes) of bio-membranes. It augments the synthesis of carboxyl and

lipid peroxyl radical and thus enhanced membrane leakage. Hence, wheat under heat

might have lost its capability to withheld cellular constituents along with water owing to

aggravated lipid peroxidation of bio-membranes at sub-cellular level. Besides, strong

negative and significant association of malondialdehyde with water relations attributes

was calculated under ‘no heat stress’ (Table 4.2.15 b, d), under ‘heat from spike to grain

filling’ (Table 4.2.16 b, d) and under ‘heat from flowering to grain filling’ (Table 4.2.17

b, d) over the years. This negative correlation further confirmed lipid peroxidation

triggered depressions in water relations. High temperature and light intensity aggravated

lipid peroxidation and downregulated the activities of ascorbate peroxidase, glutathione

105

peroxidase, SOD, POD and CAT under heat. Decrease in biosynthesis of antioxidants

depressed water potential and relative leaf water contents under (Chen et al., 2017).

Improvements in water relations (osmotic, water and turgor potential) under foliar

applied potassium can be attributed to enhanced synthesis of osmo-protectants (total

soluble proteins, proline and glycine betaine contents). These enhanced solutes

concentration of cells improved the capability of cells to withheld water under stress.

Moreover, strong positive and pronouncing association of water, osmotic and turgor

potentials was recorded with proline, glycine betaine and total soluble proteins (Table

4.2.15- 4.2.17 b, d). Improvements in water relations of plant was a consequence of

improved accumulation of osmo-protectants (Zahoor et al., 2017a).

Likewise, foliar applied potassium boosted the SOD, POD, CAT and TPC

activities, which ultimately accelerated the detoxification of ROS and thus decreased the

lipid peroxidation. Improvement in stability of membranes might have enhanced the

capability of cells to retain water and turgor under high temperature stress. Strong

negative and significant correlation of water relations with malondialdehyde. It

established the negative impacts of water relations with lipid peroxidation of bio-

membranes (Table 4.2.15-4.2.17 b, d). Foliar application of potassium improved

antioxidant defense system and water relations while decreased the biosynthesis of

malondialdehyde (Xiaokang et al., 2017).

Lesser shoot potassium contents under heat stress over ‘no heat stress’ were

ascribed to heat induced changes in antioxidants activity. Decreases of antioxidants

activities might have exposed the plant to water loss and oxidative stress. Deteriorated

defensive system of plant led to decreased shoot potassium and it was confirmed from

strong, positive and significant association of shoot potassium contents with SOD, POD,

CAT and TPC over two years of study under varying temperatures (Table 4.2.15-4.2.17 b,

d). Slightly high temperature decreased the accumulation of potassium in root, shoot and

leaves (Benlloch-González et al., 2016). Potassium contents of wheat were decreased

under stressed environment due to oxidative stress and diminished activities of

antioxidant enzymes compared to control (Krishnasamy et al., 2014).

Enhancement of shoot potassium with the enhancing potassium application might

be consequence of lesser lipid peroxidation. Application of potassium improved

antioxidant defense system of plant, which triggered the scavenging of ROS and

ultimately enhanced capacity of cells to retain osmolytes (potassium) and osmo-

protectants (soluble proteins, glycine betaine and proline). Moreover, strong negative and

106

significant association of shoot potassium contents with malondialdehyde was recorded

under all the heat stress treatments (Table 4.2.15-4.2.17 b, d). Foliar application of

potassium under stress condition in wheat enhanced the shoot potassium contents of

wheat while lesser shoot potassium contents were recorded under stressed environments

(Shabbir et al., 2015).

Significantly more shoot potassium content and water relation attributes under

stress compared to ‘no heat stress’ were recorded with higher level (60 g L -1) of foliar

potassium. It can be attributed to the capability of plant to accumulate excessive

potassium in the vacuole. While, under the stress plant might have triggered

transportation of potassium from vacuole to cell cytosol where it might have enhanced

osmo-protectants and osmolytes and thus improved water relations of plant under stress

(Andrés et al., 2014; Shin, 2017).

Decrease in grain crude protein contents under heat might be a consequence of

lesser potassium availability under heat. Decreases of shoot potassium might have

negatively affected photosynthesis and translocation of carbohydrates to grains decreased

under heat. Lesser partitioning of carbohydrates might be consequence of impaired

sucrose loading and unloading in phloem sieve tube complex. Hence, sucrose might have

accumulated in phloem and thus induced a negative feedback for photosynthesis.

Eventually, carbon chain availability for biosynthesis of amino acids and thus grain

proteins were lesser under heat compared to ‘no heat stress’. While, strong positive and

significant association of shoot potassium contents with grain crude proteins under

different conditions of heat stress over the years was recorded (Table 4.2.15-4.2.17 b, d).

It established the role of shoot potassium in enhancing carbon chain availability for

synthesis of amino acids. Potassium was required to activate ATP at site of loading of

sucrose in phloem, to maintain high pH in phloem-sieve complex regulating homeostasis

to continue sucrose movements from source to sink organs, to main maintain water

potential of phloem necessary for flow of sucrose in phloem and unloading of sucrose to

translocate it towards sink organs (Geiger, 2011; Lemoine et al., 2013).

The heat induced decrease in proline and glycine betaine decreased disturbed

water relations of plant and aggravated oxidative stress. Excessive synthesis of ROS

impaired activities of antioxidants and thus degraded proteins. Moreover, strong positive

and significant association of grain crude protein contents with proline and glycine

betaine was observed under ‘no heat stress (Table 4.2.15 b, d), under ‘heat from spike to

grain filling’ (Table 4.2.16 b, d) and under ‘heat from ‘flowering to grain filling’ (Table

107

4.2.17 b, d) over the years. It accomplished the role of decreased amino acids in reducing

the grain crude protein contents. While, strong positive and significant association of

grain crude protein contents with SOD, POD, CAT and TPC under varying temperature

regimes over the temporal variations also accomplished aggravated oxidative stress

induced degradation of grain crude proteins (Table 4.2.15-4.2.17 b, d).

Improvement of grain crude proteins owing to potassium application might be

consequence of improved carbohydrates translocation. Availability of potassium might

have triggered the translocation of carbohydrates from phloem to reproductive organs,

ultimately carbon chain availability for synthesis of amino acids was enhanced.

Consequently, grain crude protein contents were enhanced. Moreover, strong positive and

significant association of shoot potassium with grain crude proteins was observed under

varying temperatures over the years (Table 4.2.15-4.2.17 b, d). It further accomplished

the shoot potassium mediated regulations in biosynthesis of grain proteins. Application of

potassium reduced degradation if grain crude protein contents under the stress conditions,

improved antioxidants and decreased the biosynthesis of malondialdehyde (Jan et al.,

2017).

Likewise, foliar applied potassium might have triggered the activities of

antioxidants, which ultimately detoxified ROS and enhanced membrane stability. All

these regulations enhance the synthesis of carbohydrates in photosynthesis and

chlorophyll contents. Therefore, it not only enhanced carbohydrates availability for

synthesis of amino acids but also decreased the degradation of protein due to excessive

generation of ROS. Moreover, strong positive and remarkable association of grain crude

proteins with antioxidants (SOD, POD, CAT and TPC), osmo-protectants (soluble

proteins, proline and glycine betaine) and strong negative and pronouncing association

with malondialdehyde was observed under varying temperatures over the two years of

study (Table 4.2.15-4.2.17 b, d). Availability of potassium under stress conditions

improved quality of protein, protein contents and antioxidant activities in wheat

(Anschütz et al, 2014).

108

Table 4.2.15 (a): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2015-16

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl b

TGW 0.71NS

GY 0.98** 0.76NS

BY 0.96** 0.87NS 0.97**HI 0.91* 0.51NS 0.93* 0.82NS

SY 0.78NS 0.95* 0.77NS 0.90* 0.50NS

PH 0.72NS 0.90* 0.70NS 0.85NS 0.41NS 0.99**SL 0.98** 0.68NS 0.97** 0.95* 0.90* 0.77NS 0.74NS

SPS 0.98** 0.85NS 0.98** 0.99** 0.85NS 0.88* 0.82NS 0.95*GFR 0.98** 0.60NS 0.94* 0.91* 0.90* 0.71NS 0.67NS 0.98** 0.93*GFD 0.92* 0.67NS 0.87NS 0.90* 0.73NS 0.83NS 0.83NS 0.95* 0.91* 0.94*Chl a 0.88* 0.90* 0.87NS 0.96** 0.64NS 0.98** 0.96** 0.88* 0.94* 0.83NS 0.92*Chl b 0.96** 0.88* 0.95* 0.99** 0.79NS 0.92* 0.87NS 0.93* 0.99** 0.91* 0.91* 0.97**SOD 0.97** 0.86NS 0.97** 0.99** 0.83NS 0.90* 0.84NS 0.95* 0.99** 0.92* 0.91* 0.96* 0.99**POD 0.95* 0.89* 0.94* 0.99** 0.76NS 0.93* 0.89* 0.92* 0.99** 0.90* 0.91* 0.97** 0.99**CAT 0.94* 0.91* 0.95* 0.99** 0.77NS 0.93* 0.88* 0.91* 0.99** 0.88* 0.88* 0.97** 0.99**TPC 0.97** 0.87NS 0.96** 0.99** 0.81NS 0.90* 0.84NS 0.93* 0.99** 0.91* 0.89* 0.95* 0.99**LP 0.99** 0.79NS 0.98** 0.99** 0.87NS 0.84NS 0.80NS 0.99** 0.99** 0.96** 0.94* 0.93* 0.98**GB 0.97** 0.85NS 0.97** 0.99** 0.83NS 0.89* 0.84NS 0.95* 0.99** 0.93* 0.92* 0.95* 0.99**TSP 0.94* 0.91* 0.94* 0.98** 0.76NS 0.92* 0.86NS 0.89* 0.98** 0.87NS 0.86NS 0.95* 0.99**

MDA - 0.95* - 0.65NS - 0.94* - 0.92* - 0.87NS - 0.76NS - 0.74NS - 0.99** - 0.92* -0.96* - 0.95* - 0.88* - 0.90*ΨS 0.96** 0.63NS 0.95* 0.92* 0.90* 0.72NS 0.69NS 0.99** 0.93* 0.97** 0.94* 0.85NS 0.90*ΨW 0.98** 0.73NS 0.98** 0.97** 0.90* 0.80NS 0.76NS 0.99** 0.97** 0.97** 0.94* 0.90* 0.95*ΨP 0.93* 0.92* 0.95* 0.99** 0.79NS 0.92* 0.86NS 0.91* 0.99** 0.86NS 0.85NS 0.96* 0.99**

SKC 0.95* 0.90* 0.96** 0.99** 0.81NS 0.90* 0.84NS 0.92* 0.99** 0.88* 0.87NS 0.95* 0.99**GCPC 0.94* 0.90* 0.94* 0.98** 0.77NS 0.91* 0.85NS 0.89* 0.98** 0.87* 0.86NS 0.94* 0.99**

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; ΨS = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; SKC = Shoot potassium contents; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

109

Table 4.2.15 (b): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2015-16

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.99**CAT 0.99** 0.99**TPC 0.99** 0.99** 0.99**LP 0.99** 0.97** 0.97** 0.98**GB 0.99** 0.99** 0.99** 0.99** 0.99**TSP 0.99** 0.99** 0.99** 0.99** 0.96* 0.99**

MDA - 0.92* - 0.89* - 0.88* - 0.89* - 0.97** - 0.92* - 0.85NS

ΨS 0.92* 0.89* 0.88* 0.90* 0.97** 0.93* 0.85NS - 0.99**ΨW 0.97** 0.94* 0.93* 0.94* 0.99** 0.97** 0.91* - 0.99** 0.99**ΨP 0.99** 0.98** 0.99** 0.98** 0.96** 0.98** 0.99** - 0.88* 0.88* 0.93*

SKC 0.99** 0.99** 0.99** 0.99** 0.97** 0.99** 0.99** - 0.88* 0.88* 0.94* 0.99**GCPC 0.98** 0.99** 0.99** 0.99** 0.99** 0.98** 0.99** - 0.85NS 0.85NS 0.91* 0.98** 0.99**

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; SKC = Shoot potassium contents; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

110

Table 4.2.15 (c): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2016-17


TGW 0.95*GY 0.97** 0.98**BY 0.97** 0.98** 0.99**HI 0.96** 0.98** 0.99** 0.99**SY 0.92* 0.99** 0.98** 0.98** 0.98**PH 0.89* 0.89* 0.88* 0.88* 0.89* 0.89*SL 0.96* 0.94* 0.94* 0.94* 0.94* 0.93* 0.98**SPS 0.99** 0.91* 0.93* 0.93* 0.93* 0.86NS 0.85NS 0.93*GFR 0.99** 0.91* 0.92* 0.92* 0.92* 0.87NS 0.92* 0.97** 0.98**GFD 0.99** 0.90* 0.88* 0.89* 0.87NS 0.84NS 0.89* 0.95* 0.97** 0.99**Chl a 0.99** 0.97** 0.98** 0.99** 0.99** 0.95* 0.88* 0.95* 0.97** 0.96** 0.93*Chl b 0.94* 0.96** 0.96* 0.96** 0.95* 0.96** 0.98** 0.99** 0.91* 0.95* 0.92* 0.95*SOD 0.97** 0.98** 0.99** 0.99** 0.99** 0.98** 0.88* 0.94* 0.94* 0.93* 0.89* 0.99** 0.96*POD 0.94* 0.85NS 0.84NS 0.84NS 0.82NS 0.77NS 0.75NS 0.84NS 0.96** 0.94* 0.96** 0.91* 0.82NS

CAT 0.99** 0.92* 0.95* 0.95* 0.95* 0.89* 0.87NS 0.94* 0.99** 0.98** 0.95* 0.99** 0.92*TPC 0.95* 0.80NS 0.85NS 0.85NS 0.85NS 0.75NS 0.78NS 0.87NS 0.98** 0.96** 0.94* 0.92* 0.83NS

LP 0.99** 0.92* 0.95* 0.95* 0.95* 0.89* 0.87NS 0.94* 0.99** 0.98** 0.95* 0.99** 0.92*GB 0.99** 0.97** 0.99** 0.99** 0.98** 0.95* 0.89* 0.95* 0.98** 0.97** 0.94* 0.99** 0.95*TSP 0.99** 0.97** 0.99** 0.99** 0.98** 0.95* 0.91* 0.97** 0.97** 0.97** 0.94* 0.99** 0.97**

MDA -0.99** - 0.89* - 0.93* - 0.93* - 0.93* - 0.85NS - 0.83NS - 0.91* - 0.99** - 0.97** - 0.94* - 0.97** - 0.89*ΨS 0.88* 0.88* 0.87NS 0.87NS 0.86NS 0.88* 0.99** 0.98** 0.85NS 0.92* 0.91* 0.87NS 0.97**ΨW 0.94* 0.91* 0.90* 0.90* 0.89* 0.89* 0.98** 0.99** 0.92* 0.97** 0.97** 0.92* 0.98**ΨP 0.92* 0.82NS 0.81NS 0.81NS 0.79NS 0.73NS 0.70NS 0.82NS 0.95* 0.92* 0.94* 0.88* 0.78NS

SKC 0.99** 0.94* 0.98** 0.98** 0.98** 0.93* 0.89* 0.95* 0.98** 0.97** 0.93* 0.99** 0.94*GCPC 0.97** 0.98** 0.99** 0.99** 0.99** 0.98** 0.87NS 0.93* 0.93* 0.91* 0.88* 0.99** 0.95*


111

Table 4.2.15 (d): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2016-17

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.85NS

CAT 0.96** 0.94*TPC 0.86NS 0.95* 0.97**LP 0.96** 0.93* 0.99** 0.97**GB 0.99** 0.91* 0.99** 0.92* 0.99**TSP 0.99** 0.90* 0.98** 0.91* 0.98** 0.99**

MDA - 0.94* - 0.94* - 0.99** - 0.98** - 0.99** - 0.97** - 0.97**ΨS 0.87NS 0.77NS 0.86NS 0.77NS 0.86NS 0.88* 0.90* - 0.82NS

ΨW 0.90* 0.87NS 0.92* 0.86NS 0.92* 0.92* 0.94* - 0.89* 0.99**ΨP 0.82NS 0.99** 0.92* 0.94* 0.91* 0.89* 0.87NS - 0.93* 0.73NS 0.83NS

SKC 0.98** 0.90* 0.99** 0.94* 0.99** 0.99** 0.99** - 0.98** 0.87NS 0.92* 0.88*GCPC 0.99** 0.85NS 0.95* 0.84NS 0.95* 0.99** 0.98** - 0.93* 0.85NS 0.89* 0.82NS 0.97**


112

Table 4.2.16 (a): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2015-16


TGW 0.84NS

GY 0.75NS 0.92*BY 0.69NS 0.92* 0.99**HI 0.80NS 0.91* 0.99** 0.96**SY 0.55NS 0.87NS 0.92* 0.97** 0.87NS

PH 0.92* 0.88* 0.90* 0.84NS 0.95* 0.68NS

SL 0.95* 0.88* 0.87NS 0.81NS 0.92* 0.65NS 0.99**SPS 0.88* 0.96* 0.87NS 0.84NS 0.90* 0.73NS 0.94* 0.94*GFR 0.86NS 0.97** 0.90* 0.87NS 0.91* 0.78NS 0.93* 0.93* 0.99**GFD 0.95* 0.83NS 0.82NS 0.75NS 0.89* 0.58NS 0.98** 0.99** 0.92* 0.91*Chl a 0.90* 0.91* 0.93* 0.88* 0.97** 0.74NS 0.99** 0.99** 0.95* 0.95* 0.97**Chl b 0.92* 0.95* 0.89* 0.85NS 0.92* 0.73NS 0.97** 0.98** 0.99** 0.99** 0.96** 0.98**SOD 0.61NS 0.85NS 0.98** 0.98** 0.96* 0.94* 0.82NS 0.77NS 0.79NS 0.82NS 0.72NS 0.86NS 0.80NS

POD 0.69NS 0.88* 0.98** 0.97** 0.98** 0.88* 0.89* 0.85NS 0.86NS 0.88* 0.82NS 0.93* 0.88*CAT 0.69NS 0.81NS 0.95* 0.92* 0.97** 0.80NS 0.91* 0.87NS 0.83NS 0.85NS 0.85NS 0.93* 0.86NS

TPC 0.69NS 0.79NS 0.95* 0.91* 0.97** 0.80NS 0.90* 0.86NS 0.79NS 0.81NS 0.84NS 0.92* 0.83NS

LP 0.72NS 0.90* 0.97** 0.95* 0.97** 0.86NS 0.91* 0.88* 0.91* 0.93* 0.85NS 0.94* 0.92*GB 0.81NS 0.92* 0.99** 0.96** 0.99** 0.87NS 0.95* 0.92* 0.91* 0.93* 0.89* 0.97** 0.94*TSP 0.72NS 0.87NS 0.96** 0.93* 0.98** 0.82NS 0.92* 0.89* 0.89* 0.91* 0.86NS 0.95* 0.90*

MDA - 0.68NS - 0.89* - 0.96* - 0.94* - 0.96** - 0.85NS - 0.89* - 0.86NS - 0.90* - 0.92* - 0.83NS - 0.93* - 0.90*ΨS 0.78NS 0.94* 0.99** 0.99** 0.99** 0.92* 0.91* 0.88* 0.88* 0.90* 0.84NS 0.94* 0.91*ΨW 0.76NS 0.91* 0.99** 0.98** 0.99** 0.91* 0.91* 0.88* 0.87NS 0.89* 0.84NS 0.94* 0.89*ΨP 0.51NS 0.62NS 0.86NS 0.82NS 0.88* 0.70NS 0.79* 0.74NS 0.65NS 0.67NS 0.72NS 0.81NS 0.69NS

SKC 0.85NS 0.89* 0.97** 0.92* 0.99** 0.80NS 0.97** 0.95* 0.89* 0.90* 0.93* 0.98** 0.93*GCPC 0.84NS 0.97** 0.95* 0.93* 0.96** 0.84NS 0.95* 0.94* 0.98** 0.99** 0.91* 0.97** 0.98**


113

Table 4.2.16 (b): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2015-16

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.98**CAT 0.95* 0.99**TPC 0.95* 0.97** 0.99**LP 0.96* 0.99** 0.97** 0.95*GB 0.95* 0.98** 0.97** 0.96** 0.98**TSP 0.95* 0.99** 0.99** 0.97** 0.99** 0.98**

MDA - 0.95* - 0.99** - 0.97** - 0.94* - 0.99** - 0.97** - 0.99**ΨS 0.97** 0.97** 0.94* 0.94* 0.96* 0.99** 0.95* - 0.94*ΨW 0.98** 0.99** 0.96** 0.96** 0.97** 0.99** 0.97** - 0.96* 0.99**ΨP 0.90* 0.92* 0.96* 0.97** 0.88* 0.87NS 0.92* - 0.88* 0.83NS 0.88*

SKC 0.92* 0.96** 0.96** 0.97** 0.95* 0.99** 0.96** - 0.94* 0.97** 0.97** 0.88*GCPC 0.89* 0.94* 0.91* 0.88* 0.97** 0.97** 0.95* - 0.96* 0.96* 0.95* 0.76NS 0.95*


114

Table 4.2.16 (c): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2016-17


TGW 0.94*GY 0.90* 0.99**BY 0.88* 0.99** 0.99**HI 0.91* 0.99** 0.99** 0.98**SY 0.74NS 0.88* 0.89* 0.94* 0.85NS

PH 0.99** 0.93* 0.89* 0.88* 0.89* 0.77NS

SL 0.99** 0.97** 0.95* 0.93* 0.96* 0.78NS 0.97**SPS 0.99** 0.93* 0.90* 0.87* 0.91* 0.70NS 0.97** 0.99**GFR 0.99** 0.97** 0.95* 0.94* 0.94* 0.83NS 0.98** 0.99** 0.98**GFD 0.90* 0.96** 0.96** 0.96** 0.96* 0.87NS 0.87NS 0.95* 0.91* 0.95*Chl a 0.94* 0.99** 0.99** 0.98** 0.98** 0.89* 0.94* 0.97** 0.93* 0.97** 0.95*Chl b 0.97** 0.98** 0.96** 0.96* 0.95* 0.87* 0.98** 0.98** 0.96* 0.99** 0.94* 0.99**SOD 0.91* 0.90* 0.89* 0.88* 0.86NS 0.81NS 0.95* 0.89* 0.87NS 0.90* 0.78NS 0.93* 0.95*POD 0.91* 0.84NS 0.80NS 0.79NS 0.79NS 0.70NS 0.96** 0.88* 0.88* 0.88* 0.72NS 0.87NS 0.92*CAT 0.84NS 0.82NS 0.79NS 0.80NS 0.77NS 0.76NS 0.91* 0.81NS 0.78NS 0.82NS 0.67NS 0.86NS 0.88*TPC 0.83NS 0.76NS 0.72NS 0.72NS 0.72NS 0.65NS 0.89* 0.79NS 0.78NS 0.79NS 0.61NS 0.81NS 0.84NS

LP 0.86NS 0.85NS 0.83NS 0.83NS 0.81NS 0.77NS 0.92* 0.84NS 0.81NS 0.85NS 0.71NS 0.89* 0.91*GB 0.88* 0.86NS 0.83NS 0.83NS 0.82NS 0.76NS 0.94* 0.86NS 0.84NS 0.87NS 0.73NS 0.90* 0.92*TSP 0.76NS 0.67NS 0.63NS 0.62NS 0.63NS 0.55NS 0.83NS 0.71NS 0.71NS 0.71NS 0.50NS 0.73NS 0.77NS

MDA - 0.85NS - 0.82NS - 0.78NS - 0.78NS - 0.77NS - 0.71NS - 0.92* - 0.82NS - 0.81NS - 0.83NS - 0.67NS -0.86NS -0.88*ΨS 0.86NS 0.83NS 0.80NS 0.82NS 0.77NS 0.80NS 0.92* 0.83NS 0.81NS 0.86NS 0.72NS 0.87NS 0.91*ΨW 0.86NS 0.82NS 0.79NS 0.81NS 0.77NS 0.80NS 0.92* 0.82NS 0.80NS 0.85NS 0.71NS 0.87NS 0.90*ΨP 0.80NS 0.77NS 0.74NS 0.75NS 0.72NS 0.72NS 0.88* 0.92** 0.75NS 0.78NS 0.62NS 0.82NS 0.85NS

SKC 0.97** 0.91* 0.89* 0.86NS 0.88* 0.73NS 0.99** 0.95* 0.95* 0.94* 0.82NS 0.93* 0.96**GCPC 0.94* 0.99** 0.98** 0.97** 0.97** 0.87NS 0.96* 0.96** 0.91* 0.96** 0.92* 0.99** 0.99**


115

Table 4.2.16 (d): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2016-17

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.98**CAT 0.99** 0.98**TPC 0.96** 0.98** 0.99**LP 0.99** 0.98** 0.99** 0.98**GB 0.99** 0.99** 0.99** 0.99** 0.99**TSP 0.92* 0.95* 0.96** 0.99** 0.95* 0.95*

MDA -0.98** -0.99** -0.99** -0.99** -0.99** -0.99** -0.97**ΨS 0.98** 0.97** 0.99** 0.97** 0.98** 0.98** 0.93* -0.98**ΨW 0.98** 0.98** 0.99** 0.97** 0.99** 0.98** 0.93* -0.98** 0.99**ΨP 0.97** 0.97** 0.99** 0.99** 0.99** 0.99** 0.97** -0.99** 0.98** 0.98**

SKC 0.97** 0.98** 0.94* 0.94* 0.95* 0.97** 0.89* -0.95* 0.93* 0.93* 0.92*GCPC 0.96* 0.91* 0.90* 0.86NS 0.92* 0.93* 0.78NS -0.90* 0.90* 0.90* 0.86NS 0.95*


116

Table 4.2.17 (a): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2015-16


TGW 0.91*GY 0.91* 0.99**BY 0.84NS 0.97** 0.98**HI 0.92* 0.91* 0.92* 0.82NS

SY 0.71NS 0.89* 0.89* 0.97** 0.65NS

PH 0.86NS 0.99** 0.98** 0.99** 0.85NS 0.95*SL 0.98** 0.97** 0.97** 0.93* 0.94* 0.82NS 0.94*SPS 0.96** 0.98** 0.98** 0.95* 0.94* 0.84NS 0.96** 0.99**GFR 0.89* 0.99** 0.96** 0.94* 0.87NS 0.87NS 0.97** 0.95* 0.96**GFD 0.96* 0.95* 0.96** 0.95* 0.88* 0.87NS 0.94* 0.98** 0.98** 0.92*Chl a 0.90* 0.97** 0.92* 0.90* 0.86NS 0.81NS 0.94* 0.95* 0.95* 0.99** 0.89*Chl b 0.94* 0.99** 0.96** 0.94* 0.90* 0.85NS 0.96** 0.98** 0.98** 0.99** 0.95* 0.99**SOD 0.73NS 0.94* 0.94* 0.95* 0.83NS 0.89* 0.96* 0.85NS 0.88* 0.92* 0.83NS 0.86NS 0.89*POD 0.70NS 0.93* 0.92* 0.96** 0.73NS 0.95* 0.97** 0.83NS 0.86NS 0.93* 0.83NS 0.87NS 0.89*CAT 0.82NS 0.98** 0.98** 0.98** 0.86NS 0.92* 0.99** 0.92* 0.94* 0.96** 0.90* 0.92* 0.95*TPC 0.63NS 0.88* 0.89* 0.95* 0.66NS 0.97** 0.93* 0.77NS 0.81NS 0.85NS 0.80NS 0.77NS 0.81NS

LP 0.69NS 0.92* 0.90* 0.89* 0.74NS 0.82NS 0.91* 0.81NS 0.84NS 0.90* 0.76NS 0.86NS 0.86NS

GB 0.78NS 0.97** 0.96** 0.96** 0.84NS 0.91* 0.98** 0.89* 0.92* 0.96** 0.87* 0.92* 0.94*TSP 0.83NS 0.98** 0.98** 0.99** 0.85NS 0.94* 0.99** 0.92* 0.95* 0.96* 0.92* 0.91* 0.94*

MDA - 0.81NS - 0.98** - 0.95* - 0.93* - 0.87NS -0.86NS - 0.96** - 0.90* - 0.92NS - 0.97** - 0.86NS - 0.94* - 0.95*ΨS 0.78NS 0.97** 0.95* 0.96** 0.81NS 0.92* 0.98** 0.89* 0.91* 0.97** 0.87NS 0.93* 0.94*ΨW 0.76NS 0.96** 0.95* 0.97** 0.80NS 0.93* 0.98** 0.87NS 0.90* 0.96* 0.86NS 0.91* 0.93*ΨP 0.68NS 0.92* 0.92* 0.95* 0.74NS 0.94* 0.95* 0.81NS 0.85NS 0.89* 0.82NS 0.83NS 0.86NS

SKC 0.74NS 0.95* 0.92* 0.95* 0.77NS 0.92* 0.97** 0.85NS 0.88* 0.96* 0.83NS 0.92* 0.92*GCPC 0.88* 0.97** 0.95* 0.90* 0.94* 0.78NS 0.94* 0.94* 0.95* 0.97** 0.87NS 0.96* 0.96**


117

Table 4.2.17 (b): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2015-16

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.97**CAT 0.99** 0.97**TPC 0.95* 0.98** 0.94*LP 0.98** 0.95* 0.96** 0.90*GB 0.99** 0.98** 0.99** 0.94* 0.97**TSP 0.98** 0.97** 0.99** 0.95* 0.93* 0.99**

MDA - 0.98** - 0.96* - 0.98** - 0.89* - 0.98** - 0.99** - 0.97**ΨS 0.98** 0.99** 0.99** 0.94* 0.96** 0.99** 0.98** - 0.99**ΨW 0.98** 0.99** 0.99** 0.96* 0.96** 0.99** 0.98** - 0.98** 0.99**ΨP 0.98** 0.99** 0.97** 0.99** 0.95* 0.98** 0.97** - 0.94* 0.97** 0.98**

SKC 0.97** 0.99** 0.98** 0.95* 0.96** 0.99** 0.97** - 0.98** 0.99** 0.99** 0.97**GCPC 0.94* 0.89* 0.96* 0.81NS 0.94* 0.96* 0.94* - 0.98** 0.94* 0.93* 0.88* 0.93*


118

Table 4.2.17 (c): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2016-17


TGW 0.98**GY 0.94* 0.93*BY 0.91* 0.91* 0.99**HI 0.96* 0.95* 0.98** 0.95*SY 0.87NS 0.87NS 0.99** 0.99** 0.91*PH 0.99** 0.99** 0.95* 0.93* 0.95* 0.89*SL 0.97** 0.96** 0.92* 0.91* 0.91* 0.88* 0.99**SPS 0.94* 0.95* 0.99** 0.99** 0.95* 0.98** 0.95* 0.93*GFR 0.97** 0.99** 0.94* 0.93* 0.91* 0.91* 0.99** 0.98** 0.97**GFD 0.96* 0.92* 0.87NS 0.86NS 0.85NS 0.84NS 0.96** 0.99** 0.89* 0.96*Chl a 0.99** 0.99** 0.94* 0.92* 0.95* 0.87NS 0.99** 0.98** 0.95* 0.98** 0.94*Chl b 0.99** 0.99** 0.95* 0.94* 0.94* 0.91* 0.99** 0.99** 0.97** 0.99** 0.96** 0.99**SOD 0.95* 0.90* 0.85NS 0.84NS 0.83NS 0.83NS 0.95* 0.98** 0.87NS 0.94* 0.99** 0.93* 0.95*POD 0.90* 0.85NS 0.78NS 0.78NS 0.76NS 0.76NS 0.90* 0.95* 0.80NS 0.90* 0.98** 0.88* 0.90*CAT 0.95* 0.90* 0.87* 0.89* 0.86NS 0.87NS 0.95* 0.99** 0.90* 0.95* 0.99** 0.93* 0.96*TPC 0.90* 0.83NS 0.78NS 0.78NS 0.76NS 0.77NS 0.89* 0.95* 0.80NS 0.87NS 0.97** 0.86NS 0.89*LP 0.96* 0.91* 0.86NS 0.84NS 0.85NS 0.82NS 0.95* 0.99** 0.86NS 0.93* 0.99** 0.93* 0.95*GB 0.97** 0.92* 0.91* 0.90* 0.89* 0.88* 0.96** 0.99** 0.91* 0.94* 0.98** 0.94* 0.96*TSP 0.90* 0.83NS 0.77NS 0.76NS 0.75NS 0.75NS 0.89* 0.95* 0.79NS 0.88* 0.98** 0.87NS 0.89*

MDA -0.94* -0.88* -0.92* -0.91* -0.89* -0.90* -0.94* -0.97** -0.91* -0.92* -0.96* -0.92* -0.94*ΨS 0.84NS 0.77NS 0.72NS 0.73NS 0.67NS 0.72NS 0.84NS 0.91* 0.75NS 0.85NS 0.96* 0.81NS 0.85NS

ΨW 0.84NS 0.77NS 0.72NS 0.73NS 0.67NS 0.73NS 0.84NS 0.91* 0.75NS 0.84NS 0.95* 0.80NS 0.84NS

ΨP 0.84NS 0.74NS 0.72NS 0.72NS 0.70NS 0.70NS 0.82NS 0.89* 0.72NS 0.79NS 0.93* 0.79NS 0.81NS

SKC 0.94* 0.93* 0.82NS 0.81NS 0.81NS 0.78NS 0.95* 0.97** 0.86NS 0.96* 0.98** 0.94* 0.95*GCPC 0.98** 0.98** 0.98** 0.97** 0.97** 0.94* 0.99** 0.98** 0.98** 0.98** 0.95* 0.98** 0.99**


119

Table 4.2.17 (d): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2016-17

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP SKCPOD 0.99**CAT 0.99** 0.98**TPC 0.99** 0.99** 0.98**LP 0.99** 0.99** 0.99** 0.99**GB 0.98** 0.96* 0.99** 0.97** 0.99**TSP 0.99** 0.99** 0.98** 0.99** 0.99** 0.96*

MDA -0.97** -0.94* -0.99** -0.96** -0.97** -0.99** -0.94*ΨS 0.97** 0.99** 0.96* 0.99** 0.96* 0.93* 0.99** -0.92*ΨW 0.97** 0.99** 0.96* 0.99** 0.96* 0.93* 0.99** -0.92* 0.99**ΨP 0.95* 0.97** 0.94* 0.99** 0.95* 0.94* 0.98** -0.94* 0.97** 0.97**

SKC 0.98** 0.96* 0.96* 0.94* 0.96* 0.94* 0.95* -0.90* 0.93* 0.92* 0.87NS

GCPC 0.93* 0.88* 0.95* 0.88* 0.94* 0.96* 0.87NS -0.95* 0.82NS 0.82NS 0.81NS 0.92** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

120

Experiment III: Alleviation of terminal heat stress in wheat through foliar

application of selenium


(a) Results

Wheat grain yield and components were adversely affected due to heat however,

exogenous selenium remarkably alleviated these adverse impacts. Selenium mediated

improvements were similar in all main plots and thus non-significant interaction of heat

and foliar spray was observed for these parameters.

Treatments’ effects were non-significant on fertile tillers over the two study years.

While, significantly lesser number of grains per spike was recorded under ‘heat from

spike to grain filling’ compared to ‘no heat stress’ and ‘heat from flowering to grain

filling’ over the years. Whereas, some discrepancies were observed in 1000-grain weight

and grain yield over the years under varying heat imposition treatments. Number of grains

per spike was decreased by 41-43% under ‘heat from spike to grain filling’ and 23%

under ‘heat from flowering to grain filling’ compared to ‘no heat stress’ over the two

years of study. Whereas, ‘heat from spike to grain filling’ and ‘heat from flowering to

grain filling’ caused a decrease in 1000-grain weight by 21-22% and 12-19%,

respectively over the years. Likewise, grain yield was decreased by 43-44% under ‘heat

from spike to grain filling’ and 33-36% under ‘heat from flowering to grain filling’

compared to no heat stress’ over the years.

Significant improvements in grain yield and yield components were recorded with

the varying concentrations of exogenous selenium compared to control/water spray.

Statistically similar and relatively more number of grains per spike were observed with

25, 50, 75 and 100 mg L-1 exogenous selenium compared to control/water spray over

temporal variations. Whereas, statistically alike and relatively more 1000-grain weight

was observed with 50, 75 and 100 mg L-1 foliar selenium compared to control or lower

level of selenium (25 mg L-1) selenium during 2015-16 and 2016-17. While, statistically

similar and more grain yield was recorded with 75 and 100 mg L -1 exogenous selenium in

2015-16 and with 50, 75 and 100 mg L-1 foliar selenium in 2016-17 compared to other

selenium concentrations (Table 4.3.1 and Table 4.3.2).

Furthermore, number of grains per spike was enhanced by 0.13-0.14 under ‘no

heat stress’, 0.07 under ‘heat from spike to grain filling’ and 0.06 under ‘heat from

flowering to grain filling’ with each 25 mg L-1 increase in exogenous selenium over the

years. While, each 25 mg L-1 addition of exogenous selenium enhanced 1000-grain

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weight by 0.05-0.06 g, 0.05-0.07 g and 0.03-0.05 g under ‘no heat stress’, ‘heat from

spike to grain filing’ and ‘heat from flowering to grin filling’ respectively, over the years.

Whereas, grain yield was enhanced by 0.011 t ha-1 under ‘no heat stress’, 0.005 t ha-1

under ‘heat from spike to grain filling’ and 0.009-0.011 t ha-1 under ‘heat from flowering

to grain filling’ during 2015-16 and 2016-17 with each unit addition of selenium.

Moreover, the selenium modulated improvements in grain yield and components were

more dependent on exogenous selenium under heat compared to ‘no heat stress’ over the

years (Figure 4.3.1 and Figure 4.3.2).

(b) Discussion

Deleterious impacts of high temperature on grain yield and components can be

elucidated in the context of heat-triggered decrease in biological yield of wheat.

Imposition of heat might have aggravated photorespiration and reduced the carbohydrates

availability for the vegetative growth. Ultimately, lesser green area of plant under heat

might have intercepted lesser light in photosynthesis and resulted into poor partitioning of

carbohydrates for the development of grains and related components. Moreover, strong

positive and significant correlation of grain yield, grains per spike and 1000-grain weight

with biological yield was recorded under ‘no heat stress (Table 4.3.14 a, c), under ‘heat

from spike to grain filling’ (Table 4.3.15 a, c) and under ‘heat from flowering to grain

filling (Table 4.3.16 a, c) over the temporal variations. High temperature stress decreased

the light interception, triggered photorespiration and ultimately decreased carbohydrates

availability for the development of vegetative and reproductive parts (Szymańska et al.,

2017). While, coincidence of high temperature with reproductive stages of wheat reduced

the carbohydrates synthesis for vegetative growth, enhanced grain filling rate and

ultimately decreased grain yield and components (Dwivedi et al., 2017).

Decrease in yield components and grain yield under heat can be attributed to

adverse impacts of heat on spike growth. High temperature mediated diminishment in

carbohydrates availability might have deleteriously reduced carbohydrate partitioning

towards spike and ultimately impaired ovule, pollens and pollen tube development.

Hence, decreased number of grains per spike might be a consequence of lesser ovules in

each spike. While, impaired pollen viability might have diminished the number of

fertilized ovules. Consequently, grains per spike might have decreased because of lesser

spike length and number of spikelets under heat.

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Table 4.3.1: Effect of foliar applied selenium on fertile tillers and grains per spike of heat stressed wheat


Source of

variationDF

Fertile tillers Grains per spike

2015-16 2016-17 2015-16 2016-17

Blocks 2 381588 274930 535.0 226.7

Heat (H) 2 844NS 6911NS 1598.4** 1643.7**

Error I 4 1292 15641 38.3 42.1

Selenium (Se) 4 1490NS 525NS 117.5** 117.2**

H × Se 8 849NS 772NS 11.6NS 11.7NS

Error II 24 1276 1792 20.6 19.9** = Significant (p ≤ 0.01); NS = Non-significant


TreatmentsFertile tillers per m2 Grains per spike

2015-16 2016-17 2015-16 2016-17

Heat stress (H)

No heat stress (H0) 258 239 50.3 A 48.7 A

Heat from spike to grain filling (H1) 273 277 29.7 C 27.8 C

Heat from flowering to grain filling (H2) 265 240 38.8 B 37.3 B

Tukey’s HSD (p ≤ 0.05) NS NS 8.06 8.45

Selenium foliar spray (Se)

Control/ water spray (Se0) 280 262 34.8 B 33.1 B

25 mg L-1 selenium (Se25) 276 258 37.1 AB 35.3 AB

50 mg L-1 selenium (Se50) 260 247 40.5 AB 38.9 AB

75 mg L-1 selenium (Se75) 248 246 42.9 A 41.2 A

100 mg L-1 selenium (Se100) 261 247 42.9 A 41.2 A

Tukey’s HSD (p ≤ 0.05) NS NS 6.30 6.20

Year mean 265 252 39.6 37.9


Figure 4.3.1: Regression analysis for effect of foliar applied selenium on grains per spike of heat stressed wheat

123


Table 4.3.2: Effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat

124


Source of

variationDF

1000-grain weight Grain yield

2015-16 2016-17 2015-16 2016-17

Blocks 2 368.51 251.8 8.36 1.84

Heat (H) 2 606.92** 391.4* 17.63* 18.08**

Error I 4 18.40 45.5 1.23 0.87

Selenium (Se) 4 52.22** 34.6** 1.17** 1.13**

H × Se 8 2.69NS 3.3NS 0.10NS 0.06NS



Treatments1000-grain weight (g) Grain yield (t ha-1)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 40.6 B 38.3 B 2.91 B 2.63 B

Heat from flowering to grain filling (H2) 42.0 B 42.5 AB 3.87 AB 3.18 B

Tukey’s HSD (p ≤ 0.05) 5.58 8.78 1.443 1.214


Control/ water spray (Se0) 41.9 B 40.8 B 3.54 C 3.10 C

25 mg L-1 selenium (Se25) 43.4 B 41.4 B 3.63 C 3.21 BC

50 mg L-1 selenium (Se50) 44.8 AB 43.5 AB 3.98 B 3.56 AB

75 mg L-1 selenium (Se75) 47.8 A 45.3 A 4.26 AB 3.88 A

100 mg L-1 selenium (Se100) 46.8 A 44.7 A 4.34 A 3.84 A

Tukey’s HSD (p ≤ 0.05) 2.98 2.92 0.355 0.423

Year mean 44.9 43.2 3.95 3.52


Figure 4.3.2: Regression analysis for effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat

125


Likewise, high temperature might have enhanced the growth rate of spike while

lesser carbohydrates were available relative to accelerated growth of spike under heat.

Hence, sucrose availability was not enough to satiate the needs of rapidly growing spike

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under heat stressed environment. Resultantly, 1000-grain weight might have reduced

because of more sink demand and lesser availability of carbohydrates from source which

ultimately decreased grain yield also. Besides, strong positive and pronouncing

association of spike length, spikelets per spike, grain filling rate and duration with grain

yield, grains per spike and 1000-grain weight under varying temperature stress

accomplished the spike growth meditated damages in grain yield and yield components

over the years (Table 4.3.14-4.3.16 a, c). Heat stress at terminal stages reduced the

duration of phenology, enhanced the growth, caused infertile pollen and ultimately

decreased grain yield and components (Iqbal et al., 2017). High temperature stress

triggered the grain filling rate, decreased pollen fertility, number of rains per spike and

grain yield (Barlow et al., 2015).

Another explanation is that heat stress caused excessive biosynthesis of ROS.

High temperature stress might have triggered the biosynthesis of ROS, which might have

outcompeted defensive mechanism of wheat leading to increased lipid peroxidation.

Oxidative stress might have reduced the carbohydrates partitioning towards reproductive

parts and thus grain yield and related components were adversely affected under heat.

Moreover, negative impacts of excessive ROS were also established form strong negative

and significant association of malondialdehyde with grains per spike, 1000-grain weight

and grain yield under ‘no heat stress’ (Table 4.3.14 a, c), under ‘heat from spike to grain

filling’ (Table 4.3.15 a, c) and under ‘heat from flowering to grain filling’ (Table 4.3.16 a,

c) over the years. Heat stress reduced the activities of superoxide dismutase, catalase,

peroxidase, glutathione reductase and enhanced membrane leakage in wheat seedlings

(Wang et al., 2014). Heat stress at terminal stages damaged the photosystem and

decreased grains per spike, 1000-grain weight and grain yield of wheat (Chen et al.,

2017).

Improvement in grain yield and components was ascribed to selenium modulated

improvements in chlorophyll a and b. Exogenous selenium improved the activities of

chlorophyll biosynthesizing enzymes and thus wheat stayed green for comparatively

longer duration. Hence, carbohydrates partitioning towards grains sustained for longer

duration and grain yield and components were improved. Strong positive and significant

association of chlorophyll contents with grain yield and yield components was recorded

which further accomplished the stay green role in the improvement of grain yield and

components over the years (Table 4.3.14-4.3.16 a, c) over the years. Likewise, selenium

127

enhanced chlorophyll contents and stay green trait under the stressed conditions which

ultimately improved yield (Nawaz et al., 2015).

Foliar applied selenium boosted the biosynthesis of osmo-protectants which

ultimately enhanced osmotic potential. Depression of osmotic potential ultimately caused

the water movements from apoplast to cells and thus cells retained turgor under heat and

conferred heat tolerance. Moreover, strong positive and pronouncing association of grain

yield and components with osmo-protectants and water relation attributes was recorded

under distinct temperature regimes over temporal variations. It accomplished the role of

osmo-protectants in improving grain yield and components (Table 4.3.14-4.3.16 a, c).

Foliar applied selenium enhanced the osmotic potential and improved turgor.

Enhancement in capability of plant to retain water under stress and improved antioxidants

conferred tolerance against stress (Naz et al., 2015).

Improvement in grain yield and components under exogenous selenium can also

be defined in terms of improved antioxidants activities. Exogenous selenium might have

triggered the SOD mediated dismutation of 1O2●- into H2O2. Resultantly, increased

availability of H2O2 might have triggered CAT and POD activities. Detoxification of ROS

might have alleviated oxidative stress and enhanced membrane integrity and thus induced

heat tolerance. Alleviation of adversities of heat might have improved yield and yield

components. Besides, strong positive and pronouncing correlation of antioxidants and

phenolics with grain yield and yield components was recorded under ‘no heat stress’

(Table 4.3.14 a, c), under ‘heat from spike to grain filling’ (Table 4.3.15 a, c) and under

‘heat from flowering to grain filling’ (Table 4.3.16 a, c) over the years which

accomplished the antioxidants modulated improvements in grain yield and yield

components. Application of selenium enhanced activities of SOD, POD, CAT,

glutathione peroxidase, proline contents and improved chlorophyll fluorescence under

stress environment (Cheng et al., 2016). Application of selenium enhanced pollen

viability, alleviated adverse impacts of oxidative stress and boosted antioxidant activities

under stressed environment (Tedeschini et al., 2015). Likewise, exogenously applied

selenium effectively alleviated the adverse impacts of stress and enhanced proline

biosynthesis, improved gaseous exchange attributes, photosynthetic efficiency and water

relations of wheat (Hajiboland et al., 2015).

4.3.2. Biomass accumulation

(a) Results

128

Imposition of heat stress significantly decreased biological yield, harvest index,

straw yield and plant height compared to ‘no heat stress’. While, deleterious impacts of

heat were more pronouncing under ‘heat from spike to grain filling’ compared to ‘heat

from flowering to grain filling’ over years. Yet, exogenously applied selenium depicted

significant improvements in biomass accumulation attributes compared to control/water

spray over temporal erraticism. Similar trends of selenium-modulated improvements

under all main plots were observed which resulted into non-significant interaction over

the years.

More reduction in plant height, straw yield, biological yield and harvest index was

observed in plots where crop remained under heat stress for relatively longer duration

(spike to grain filling) than in plots where crop faced heat for shorter time (flowering to

grain filling). However, crop under ‘no heat stress’ gained maximum values for these

parameters. So, ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling’ induced decrease in biological yield compared to ‘no heat stress’ was 34-35% and

18-23%, respectively. While, a diminishment of 12-16% under ‘heat from spike to grain

filling’ and 7-13% under ‘heat from flowering to grain filling’ compared to ‘no heat

stress’ was observed over the years. Similarly, straw yield was decreased by 27-31%

under ‘heat from spike to grain filling’ and 15-17% under ‘heat from flowering to grain

filling’ compared to ‘no heat stress’ over the two study years. Whereas, plant height was

reduced by 21-22% and 12-15% under ‘heat from spike to grain filling’ and under ‘heat

from flowering to grain filling’ over temporal variations.

Biomass accumulation attributes were improved significantly under exogenous

selenium application. Statistically similar and relatively more biological yield and harvest

index were recorded with 50,75 and 100 mg L-1 foliar selenium compared to other

concentrations over the years. Whereas, statistically similar and comparatively more

straw yield was observed with 50, 75 and 100 mg L-1 in 2015-16 and 25, 50, 75 and 100

mg L-1 application of foliar selenium in 2016-17. While, plant height was significantly

more with 75 mg L-1 selenium in 2015-16 whereas relatively more and statistically alike

under the 50, 75 and 100 mg L-1 foliar selenium in 2016-17 compared to other doses of

exogenous selenium (Table 4.3.3 and Table 4.3.4).

Besides, each 25 mg L-1 increment in exogenous selenium, biological yield was

increased by 0.012-0.016 t ha-1, 0.007-0.008 t ha-1 and 0.017-0.022 t ha-1 under ‘no heat

stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’,

respectively over the years. While, per unit foliar selenium modulated improvements in

129

harvest index were 0.04-05% under ‘no heat stress’, 0.02-0.04% under ‘heat from spike to

grain filling’ and 0.003% under’ heat from flowering to grain filling’ during 2015-16 and

2016-17. While, straw yield was enhanced by 0.001-0.004 t ha-1 under ‘no heat stress’,

0.002-0.003 t ha-1 under ‘heat from spike to grain filling’ and 0.008-0.011 t ha-1 under

‘heat from flowering to grain filling’ over the two years study period. Similarly, with each

addition of 25 mg L-1 of selenium dose, plant height was enhanced by 0.05-0.07 cm, 0.11

cm and 0.07 cm under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘hat from

flowering to grain filling’ over the years. While, selenium mediated improvements in

biomass accumulating attributes were more under heat compared to ambient conditions

(Figure 4.3.3 and Figure 4.3.4).

(b) Discussion

High temperature environment might have impaired the activities of enzymes that

regulate chlorophyll biosynthesis. While, heat stress might have boosted the activities of

chlorophyll degrading enzymes and thus further escalated the senescence. It led to the

interception of light and thereby reduced the availability of carbohydrates and thus

decreased harvest index, plant height, straw and biological yield. Moreover, strong

positive and pronouncing association of biomass accumulation attributes with chlorophyll

contents was recorded under ‘no heat stress’ (Table 4.3.14 a, c), ‘heat from spike to grain

filling’ (Table 4.3.15 a, c) and ‘heat from flowering to grain filling’ (Table 4.3.16 a, c)

over the years. It accomplished the negative impacts of accelerated degradation of

chlorophyll on biomass accumulating attributes. High temperature stress aggravated

photoinhibition, disintegrated Mn-D1D2 complex at light harvesting complex of

photosystem-II, damaged photosynthetic machinery and inhibited the electron transfer in

electron transport chain of light reactions (Mathur et al., 2014). Consequently,

biosynthesis of reductants (NADH and NADPH) was decreased under heat which

ultimately downregulated the photosynthesis (Szymańska et al., 2017).

Similarly, lesser accumulation of proline under heat might have enhanced the

accumulation of singlet oxygen (1O2*), which aggravated chlorophyll degradation and

lipid peroxidation. While, impaired biosynthesis of osmo-protectants due to heat might

have depressed osmotic, water and turgor potential. Thereafter, hydrolases activities were

decreased which ultimately restricted the extension of cells under limitations of water

potential. Hence, cells could not sustain growth and resulted in lesser plant height,

biological and straw yield. Moreover, strong positive and remarkable correlation of osmo-

protectants with biomass accumulating attributes was observed under varying

130

temperatures over the temporal discrepancies. It established the damages in biomass

accumulation due to lesser biosynthesis of osmo-protectants.

Table 4.3.3: Effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat


Source of

variationDF

Biological yield Harvest index

2015-16 2016-17 2015-16 2016-17

Blocks 2 20.93 16.51 726.15 565.91

Heat (H) 2 87.54** 66.94** 68.13* 163.71**

Error I 4 1.48 0.84 6.15 7.43

Selenium (Se) 4 3.37** 2.33** 13.40** 26.46**

H × Se 8 0.36NS 0.14NS 1.01NS 0.91NS



TreatmentsBiological yield (t ha-1) Harvest index (%)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)



Heat from flowering to grain filling (H2) 11.22 B 9.34 B 34.49 AB 34.05 B

Tukey’s HSD (p ≤ 0.05) 1.584 1.194 3.228 3.548


Control/ water spray (Se0) 10.60 C 9.20 C 33.40 C 33.70 B

25 mg L-1 selenium (Se25) 10.71 BC 9.40 BC 33.89 BC 34.15 B

50 mg L-1 selenium (Se50) 11.40 AB 9.93 ABC 34.91 ABC 35.85 AB

75 mg L-1 selenium (Se75) 11.80 A 10.33 A 36.10 AB 37.56 A

100 mg L-1 selenium (Se100) 11.93 A 10.27 AB 36.38 A 37.39 A

Tukey’s HSD (p ≤ 0.05) 0.722 0.894 2.233 2.617

Year mean 11.29 A 9.83 B 34.99 35.81


131

Figure 4.3.3: Regression analysis for effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat


132

Table 4.3.4: Effect of foliar applied selenium on straw yield and plant height of heat stressed wheat


Source of

variationDF

Straw yield Plant height

2015-16 2016-17 2015-16 2016-17

Blocks 2 12.81 0.07 5198.7 3435.6

Heat (H) 2 26.61* 15.64** 1562.4* 2101.8*

Error I 4 1.85 0.12 131.7 137.0

Selenium (Se) 4 0.57** 0.22* 140.4** 121.2**

H × Se 8 0.13NS 0.07NS 9.9NS 6.7NS



TreatmentsStraw yield (t ha-1) Plant height (cm)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 6.00 B 5.37 C 84.7 B 83.4 B

Heat from flowering to grain filling (H2) 7.35 AB 6.16 B 94.3 AB 90.9 B

Tukey’s HSD (p ≤ 0.05) 1.773 0.452 14.94 15.24


Control/ water spray (Se0) 7.06 C 6.10 B 91.7 C 89.1 C

25 mg L-1 selenium (Se25) 7.08 BC 6.19 AB 93.4 BC 90.8 BC

50 mg L-1 selenium (Se50) 7.42 ABC 6.37 AB 95.6 BC 94.3 ABC

75 mg L-1 selenium (Se75) 7.54 AB 6.45 A 101.9 A 97.9 A

100 mg L-1 selenium (Se100) 7.59 A 6.43 AB 97.3 B 96.1 AB

Tukey’s HSD (p ≤ 0.05) 0.473 0.339 4.44 5.50

Year mean 7.34 A 6.31 B 95.97 93.6


133

Figure 4.3.4: Regression analysis for effect of foliar applied selenium on straw yield and plant height of heat stressed wheat


134

Likewise, strong negative and pronouncing correlation of biological, straw yield,

harvest index and plant height with malondialdehyde was recorded under varying

temperature regimes during 2015-16 and 2016-17 (Table 4.3.14-4.3.16 a, c). High

temperature stress reduced the pollen viability, caused the abortion of flowers and thus

reduced harvest index of wheat by affecting grains (Prasad et al., 2017). While, 5°C

higher temperature than ambient environment deleteriously impacted gaseous exchange

attributes, photosynthetic efficiency, plant height, harvest index and grain yield of

terminal heat stressed wheat crop (Dwivedi et al., 2017).

Improvement of biomass accumulation attributes under exogenous selenium can

be ascribed to selenium modulated improvements in water relations of wheat under heat.

Exogenously applied selenium induced the biosynthesis of proline and other osmo-

protectants leading to the detoxification of 1O2* which ultimately alleviated oxidative

stress. As a result, ability of cells to retain water was enhanced under heat. This greater

amount of water might have transferred from apoplast to cell cytosol, which enhanced

turgor and cells continued to grow under stressed environment. Moreover, strong positive

and pronouncing correlation of water relations attributes with biological, straw yield,

harvest index and plant height was recorded under different temperatures over the

temporal variability (Table 4.3.14-4.3.16 a, c). It further established the water relations

modulated enhancements in biomass accumulation. Foliar application of selenium

improved antioxidant defensive mechanism of plant, alleviated ROS, improved

membrane stability and water relations under stress condition (Ahmad et al., 2016).

Whereas, exogenous selenium modulated alleviation of oxidative stress decreases lipid

peroxidation under UV light stress (Mostafa and Hassan, 2015).

Likewise, selenium might have boosted the enzymatic (SOD) scavenging of 1O2●-

to H2O2. Concurrently, improved water relations might have decreased the sensitivity of

wheat to ROS and antioxidants were not saturated from excessive substrates (ROS).

Consequently, alleviation of oxidative stress might have slowed down the degradation of

chlorophyll and augmented activities of enzymes involved in synthesis of chlorophyll.

More chlorophyll ultimately enhanced the availability of assimilates to produce more

biological yield, harvest index and plant height. Furthermore, strong positive and

significant association of chlorophyll contents with biomass accumulating attributes over

the temperature and temporal variations accomplished the role of chlorophyll in

enhancement of biomass accumulating attributes (Table 4.3.14-4.3.16 a, c). Increase of

selenium availability improved the peroxidase, phenolic content, chlorophyll content and

135

decreased lipid peroxidation (Sharma et al., 2014b). Application of selenium under heat

and UV light stress conditions enhanced the biosynthesis of antioxidants and chlorophyll

contents (Sieprawska et al., 2015).


(a) Results

Imposition of both heat treatments significantly decreased spike length, spikelets

per spike and grain filling duration compared to ‘no heat stress’. Whereas, grain filling

rate was significantly faster under heat compared to ambient conditions. Foliar

application of selenium significantly enhanced spike length, spikelets per spike, grain

filling rate and duration compared to control/water spray. Moreover, quite similar trends

were observed with different selenium concentrations in all main plots that resulted into

non-significant ‘heat × selenium’ effect over the years regarding growth of spike.

Significantly lesser and statistically similar spike length was measured under ‘heat

from spike to grain filling’ and ‘heat from flowering to grain filling’ compared to ‘no heat

stress’ over the years. While, significantly fewer number of spikelets per spike were

obtained under ‘heat from spike to grain filling’ compared to ‘no heat stress’ and ‘heat

from flowering to grain filling’ over the temporal inconsistencies. Whereas, grain filling

rate under ‘heat from flowering to grain filling’ was statistically alike to those under ‘no

heat stress’ and ‘heat from spike to grain filling’ during 2015-16. While, statistically alike

and significantly quicker grain filling rate was recorded under both heat stress treatments

compared to ‘no heat stress’ during 2016-17. Likewise, grain filling duration was

significantly shorter under heat stress treatments compared to ‘no heat stress’ during

2015-16. While, grain filling duration under ‘heat from flowering to grain filling’ was

statistically similar to those under ‘no heat stress’ and ‘heat from spike to grain filling’

during 2016-17.

Different concentrations of exogenous selenium varied significantly for spike

growth. Relatively more spike length and spikelets per spike were recorded with 75 and

100 mg L-1 exogenous selenium compared to control/water spray over the years. Whereas,

statistically higher grain filling rate and prolonged duration were evident with 50, 75 and

100 mg L-1 exogenous selenium over the control/water spray or 25 mg L-1 selenium over

the years (Table 4.3.5 and Table 4.3.6).

Additionally, with each unit application of selenium the spike length was

enhanced by 0.04 cm, 0.02-0.03 cm and 0.03 cm under ‘no heat stress’, ‘heat from spike

to grain filling’ and ‘heat from flowering to grain filling’, respectively over the years.

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While, spikelets per spike were improved by 0.03 under ‘no heat stress’, 0.02-0.03 under

‘heat from spike to grain filling’ and 0.02-0.03 under ‘heat from flowering to grain

filling’ with each 25 mg L-1 addition of foliar selenium over the years. Likewise, each 25

mg L-1 application of foliar selenium enhanced grain filling rate by 0.0002-0.0003 g per

day under ‘no heat stress’, 0.0003-0.0004 g per day under ‘heat from spike to grain

filling’ and 0.0001-0.0002 g per day under ‘heat from flowering to grain filling’ over the

two study years. Whereas, grain filling duration was enhanced by 0.02 days, 0.05-0.06

days and 0.02 days under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’ with each unit increment in foliar selenium over the temporal

variability. Moreover, dependence of spike length and spikelets per spike on exogenous

selenium was enhanced under the stress compared to ‘no heat stress’ over the years.

Foliar selenium triggered improvements in grain filling rate were higher under ‘heat from

spike to grain filling’ compared to ‘no heat stress in 2015-16. While, relatively more

improvements in grain filling rate were observed under ‘no heat stress/control compared

to both heat stress treatments in 2016-17. Likewise, improvements in grain filling

duration were more dependent on exogenous selenium under ‘heat from spike to grain

filling’ compared to control/no heat stress over the years. However, this dependence was

lesser under ‘heat from flowering to grain filling’ compared to ‘control/no heat stress’

over the temporal variations (Figure 4.3.5 and Figure 4.3.6).

(b) Discussion

Heat stress adversely impacted the biomass accumulation and ultimately reduced

the sources (leaves) for photosynthesis. Lesser biomass might have reduced the

partitioning of carbohydrates towards reproductive parts. Hence decreased spike length

and spikelets per spike might be consequence of decreased availability of carbohydrates.

Moreover, heat stress accelerated growth of spike in conjunction with impaired

availability of assimilates. Thus, decrease in spike length and spikelets per spike was an

adaptive response to poor synchronization of source supply and sink growth rate. While,

strong positive and pronouncing association of biological yield and plant height with

spike growth attributes was observed over the varying temperature stresses and years. It

further accomplished the heat mediated negative impacts on spike growth due to lesser

biological yield and plant height (Table 4.3.14-4.3.16 a, c). Higher temperature in rice

decreased above ground biomass, leaf area, spikelets per panicle and panicle length.

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Table 4.3.5: Effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat


Source of

variationDF

Spike length Spikelets per spike

2015-16 2016-17 2015-16 2016-17

Blocks 2 27.9 20.5 33.90 94.89

Heat (H) 2 150.2** 197.6** 196.07** 188.44**

Error I 4 2.2 4.0 2.54 3.58

Selenium (Se) 4 18.5** 18.8** 12.78** 14.30**

H × Se 8 1.8NS 2.5NS 0.38NS 1.06NS



TreatmentsSpike length (cm) Spikelets per spike

2015-16 2016-17 2015-16 2016-17

Heat stress (H)


Heat from spike to grain filling (H1) 14.5 B 12.6 B 11.9 C 11.0 C


Tukey’s HSD (p ≤ 0.05) 1.91 2.60 2.08 2.46


Control/ water spray (Se0) 15.0 D 13.4 C 14.3 B 13.4 C

25 mg L-1 selenium (Se25) 15.7 CD 14.9 BC 14.4 B 13.1 C

50 mg L-1 selenium (Se50) 17.0 BC 15.5 ABC 15.0 B 14.0 BC


100 mg L-1 selenium (Se100) 17.5 AB 16.1 AB 16.5 A 15.3 AB

Tukey’s HSD (p ≤ 0.05) 1.41 2.11 0.95 1.41

Year mean 16.8 15.42 15.4 14.4


Figure 4.3.5: Regression analysis for effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat

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Table 4.3.6: Effect of foliar applied selenium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat

139


Source of

variationDF

Grain filling rate Grain filling duration

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.00663 0.00172 243.9 66.7

Heat (H) 2 0.01103* 0.00795** 628.0** 503.1*

Error I 4 0.00073 0.00029 19.5 32.9

Selenium (Se) 4 0.00121** 0.00106** 19.6** 18.4**

H × Se 8 0.00010NS 0.00009NS 2.4NS 2.2NS



TreatmentsGFR (g per day) GFD (days)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)

No heat stress (H0) 0.12 B 0.11 B 34.74 A 32.43 A

Heat from spike to grain filling (H1) 0.17 A 0.16 A 21.87 B 20.96 B

Heat from flowering to grain filling (H2) 0.15 AB 0.13 A 27.11 B 25.27 AB

Tukey’s HSD (p ≤ 0.05) 0.035 0.022 5.750 7.467


Control/ water spray (Se0) 0.13 C 0.13 BC 26.18 B 24.68 B

25 mg L-1 selenium (Se25) 0.14 BC 0.12 C 26.83 B 25.18 B

50 mg L-1 selenium (Se50) 0.15 AB 0.14 ABC 27.92 AB 25.98 AB


100 mg L-1 selenium (Se100) 0.16 A 0.14 AB 28.72 AB 27.06 AB

Tukey’s HSD (p ≤ 0.05) 0.013 0.017 2.788 2.731

Year mean 0.15 0.13 27.91 26.22


Figure 4.3.6: Regression analysis for effect of foliar applied selenium on grain filling rate and grain filling duration of heat stressed wheat

140


Heat triggered decrease in photosynthesis and increase in photorespiration

reduced the availability of carbohydrates for development of reproductive organs (Laza et

141

al., 2015). Additionally, heat stress during post heading stages shortened the phenology of

spike and related attributes of wheat crop (Liu et al., 2016b).

Conversely, ever swelling demands of spike for carbohydrates due to more growth

rate and lesser duration could not be satiated and thus grain filling duration was reduced

under heat compared to ‘no heat stress’. While, negative feedback and reduced

availability of carbohydrates to reproductive parts was also accomplished from decrease

of 1000-grain weight and number of grains per spike under heat compared to those under

‘no heat stress’. Moreover, relative decrease of grain filling duration was also higher than

the increase of grain filling rate under heat compared to ‘no heat stress’ over the years.

Thus, increased grain filling duration could not compensate for reduced phenology and

resulted in reduced spikelets per spike, spike length, 1000-grain weight and number of

grains per spike. Moreover, strong positive and remarkable association of spike growth

attributes was observed with chlorophyll contents. Additionally, 1000-grains weight and

number of grain per spike also depicted strong positive and noticeable association with

spike growth attributes under varying conditions of heat and over the years (Table 4.3.14-

4.3.16 a, c). Increase of temperature accelerated senescence in wheat, adversely affected

spike attributes and yield components (Barlow et al., 2015). While, high temperature

stress reduced the biosynthesis of aminolaevulinic acid dehydratase and enhanced the

biosynthesis of protochlorophyllide oxidoreductase. Consequently, chlorophyll

degradation was more than the rate of biosynthesis, which resulted in accelerated

senescence and affected spike development (Iqbal et al., 2017).

Foliar application of selenium enhanced the biosynthesis of proline, glycine

betaine and soluble proteins. Consequently, the osmotic and water potential and the turgor

potential might have been improved. Retention of water in cells decreased the sensitivity

of wheat towards heat and thereby spike took more time for grain filling. While, more

turgor with the selenium availability might have activated hydrolases which ultimately

triggered the breakdown of cell wall cellulose and pectin. Simultaneously, the

biosynthesis of osmo-protectants enhanced the osmotic, water and turgor potential and

thus cell sustained to grow for longer duration which ultimately improved spike length,

spikelets per spike, grain filling rate and duration. While, strong positive and remarkable

association of osmo-protectants with attributes of spike growth and water relations was

recorded under varying temperature regimes over the years. It further confirmed the role

of osmo-protectants in growth of spike (Table 4.3.14-4.3.16 a, c). Application of selenium

enhanced the biosynthesis of proline, alleviated oxidative stress, and improved the

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membrane stability and turgor potential of cells under stress conditions (Bybordi, 2016).

Likewise, exogenous selenium improved the antioxidant defensive mechanism of plant,

enhanced membrane stability and relative leaf water contents under stress conditions

(Mozafariyan et al., 2016).

Likewise, improvements in antioxidants and phenolic contents under heat might

have prolonged the stay green by and carbohydrates partitioning to spike improved spike

growth. While, strong positive and pronounced correlation of antioxidants, phenolics and

chlorophyll contents with the spike attributes was recorded under ‘no heat stress’ (Table

4.3.14 a, c), ‘heat from spike to grain filling’ (Table 4.3.15 a, c) and ‘heat from flowering

to grain filling’ (Table 4.3.16 a, c) during 2015-16 and 2016-17. While, strong negative

and remarkable association of spike growth attributes was recorded with malondialdehyde

contents under the varying conditions of temperature and over the years. It accomplished

the importance of decreased lipid peroxidation for development of spike (Table 4.3.14 a,

c; Table 4.3.15 a, c and Table 4.3.16 a, c). Application of selenium enhanced the activities

of enzymatic and non-enzymatic antioxidants and improved bio-membrane stability

(Yildiztugay et al., 2017). While, antioxidant defense mechanism and quality was

improved with increasing selenium contents of plant (Mora et al., 2015).


(a) Results

Heat stress and selenium significantly affected the chlorophyll a, b contents, SOD,

POD, CAT and TPC. Different concentrations of selenium exhibited similar responses

under all heat treatments and resulted into non-significant interaction of heat and

selenium foliar spray for stay green trait. Whereas, selenium modulated improvements in

SOD, POD, CAT and TPC differed significantly under varying high temperature

environments and resulted into significant ‘heat × selenium’ effect over the years.

Significantly lesser chlorophyll a contents were recorded under ‘heat from spike

to grain filling’ compared to ‘no heat stress’ and ‘heat from flowering to grain filling’

over the years. While, significantly lesser and statistically alike chlorophyll b contents

were quantified under ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling’ compared to ‘no heat stress’ over the temporal variations. Moreover, ‘heat from

spike to grain filling’ and ‘heat from flowering to grain filling’ caused decrease in

chlorophyll a contents by 36-38% and 19-21%, respectively compared to ‘no heat stress’.

Whereas, chlorophyll b contents were diminished by 42-49% under ‘heat from spike to

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grain filling’ and 33-35% under ‘heat from flowering to grain filling’ compared to no heat

stress.

Varying concentrations of foliar applied selenium significantly improved

chlorophyll a and b contents compared to control/ water spray. Statistically similar and

significantly more chlorophyll a contents were recorded with 75 and 100 mg L-1 selenium

compared to other concentrations during 2015-16 and 2016-17. Likewise, relatively better

chlorophyll b contents were recorded with 75 and 100 mg L-1 exogenous selenium

compared to control/water spray over the years (Table 4.3.7).

Moreover, chlorophyll a contents were improved by 0.004, 0.003-0.004 and 0.003

mg g-1 FW under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’ with each unit foliar application of selenium over the period of

two years. While, each 25 mg L-1 exogenous selenium enhanced chlorophyll b contents by

0.002 mg g-1 FW under ‘no heat stress’, 0.0008-0.0010 mg g-1 FW under ‘heat from spike

to grain filling’ and 0.0008-0.0009 mg g-1 FW under ‘heat from flowering to grain filling’.

Besides, improvement in chlorophyll contents was generally more dependent on selenium

under stress compared to ambient conditions over the years (Figure 4.3.7).

Regards antioxidants, comparatively more activities of SOD, POD and CAT were

recorded under ‘no heat stress’ compared to both heat stress treatments. While, ‘heat from

spike to grain filling’ depicted more adverse impacts for SOD, POD, CAT and TPC

contents compared to ‘heat from flowering to grain filling’ over the years.

Under ‘no heat stress’, application of exogenous selenium at 75 mg L -1 either

exhibited significantly more or remained at par with 100 mg L-1 selenium regarding the

activities of SOD and POD over the years. While, application of 100 mg L -1 exogenous

selenium either depicted significantly more activities or it was statistically alike to 75 mg

L-1 foliar selenium concerning SOD and POD activities under both heat stress treatments

during 2015-16 and 2016-17. Likewise, significantly more activities of CAT and TPC

were recorded with 75 mg L-1 exogenous selenium during 2015-17 under ‘no heat stress’.

While, statistically similar and significantly more CAT and TPC were measured with 75

and 100 mg L-1 foliar selenium in 2016-17 under ‘no heat stress’. Whereas, significantly

higher activities of CAT and TPC were observed with 100 mg L -1 exogenous selenium

compared to other concentrations under heat over the years. While, relatively lesser and

statistically alike activities of SOD, POD, CAT and TPC were recorded with

control/water spray in all main plots over the two years study (Table 4.3.8 and Table

4.3.9).

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Moreover, each 25 mg L-1 selenium modulated increments in SOD were 0.53-0.57

U mg-1 protein under ‘no heat stress’, 0.37-0.41 U mg-1 protein under ‘heat from spike to

grain filling’ and 0.31 U mg-1 protein under ‘heat from flowering to grain filling’ over the

years. While, POD contents were enhanced by 0.03, 0.07 and 0.05-0.06 U mg-1 protein

under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain

filling, respectively with each unit application of foliar selenium over the years. While,

each unit of selenium mediated improvements in CAT were 0.23 U mg-1 protein under ‘no

heat stress’, 0.08-0.09 U mg-1 protein under ‘heat from spike to grain filling’ and 0.06-

0.08 U mg-1 protein under ‘heat from flowering to grain filling’ over the years. Each 25

mg L-1 addition of foliar selenium enhanced the TPC by 0.08-0.09, 0.06 and 0.03-0.05 mg

GAE g-1 under ‘no heat stress’, ‘heat from spike to grain filling’ and ‘heat from flowering

to grain filling’, respectively over the years. Moreover, SOD, POD, CAT and TPC

dependence on availability of foliar selenium was enhanced under stress conditions

compared to ambient conditions over the years (Figure 4.3.8 and Figure 4.3.9).

(b) Discussion

Decrease of chlorophyll, antioxidants and TPC under heat can be attributed to

degradation of chlorophyll. High temperature might have destabilized the reaction center

of photosystem-II (PS-II). It resulted in more energy transfer to reaction center and

accelerated photolysis of water. Whereas, transfer of electrons in electron transport chain

was slowed down under heat. Therefore, plethora of free electrons at reaction center of

PS-II reacted with lipids of bio-membranes on one side while activated the ground state

oxygen released from photolysis of water to singlet oxygen (1O2*) on the other hand.

Consequently, 1O2* triggered the breakdown of chlorophyll. While, impairment in flow of

electrons in electron transport chain of light reaction might have reduced reductants

(NADPH) to reduce carbon dioxide in photosynthesis. Poor partitioning of carbohydrates

was also confirmed from lesser 1000-grain weight and number of grains per spike under

heat compared to ‘no heat stress’. Moreover, strong positive and remarkable association

of chlorophyll contents with antioxidants and TPC confirmed the chlorophyll breakdown

damages to antioxidants activities (Table 4.3.14-4.3.16 a, c). Impairment in electron

generation at reaction center and transfer in electron transport chain triggered the

synthesis of ROS, which enhanced degradation of chlorophyll and restricted biosynthesis

of chlorophyll.

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Table 4.3.7: Effect of foliar applied selenium on chlorophyll a (Chl a) and on chlorophyll b (Chl b) contents of heat stressed wheat


Source of

variationDF

Chlorophyll a Chlorophyll b

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.499 0.530 0.0622 0.1086

Heat (H) 2 2.263** 2.425** 0.2502** 0.2970**

Error I 4 0.265 0.033 0.0022 0.0136

Selenium (Se) 4 0.194** 0.210** 0.0233** 0.0187**

H × Se 8 0.007NS 0.009NS 0.0011NS 0.0015NS



TreatmentsChl a (mg g-1 FW) Chl b (mg g-1 FW)

2015-16 2016-17 2015-16 2016-17

Heat stress (H)




Tukey’s HSD (p ≤ 0.05) 0.212 0.236 0.059 0.152


Control/ water spray (Se0) 1.57 B 1.50 B 0.39 C 0.36 D

25 mg L-1 selenium (Se25) 1.62 B 1.55 B 0.40 C 0.37 CD

50 mg L-1 selenium (Se50) 1.69 B 1.63 B 0.46 B 0.41 BC


100 mg L-1 selenium (Se100) 1.88 A 1.82 A 0.49 AB 0.45 AB

Tukey’s HSD (p ≤ 0.05) 0.143 0.153 0.039 0.044

Year mean 1.73 1.67 0.45 0.41


Figure 4.3.7: Regression analysis for effect of foliar applied selenium on chlorophyll a and on chlorophyll b contents of heat stressed wheat

146


147

Table 4.3.8: Effect of foliar applied selenium on superoxide dismutase (SOD) and peroxidase (POD) contents of heat stressed wheat


Source of variation

DFSuperoxide dismutase Peroxidase

2015-16 2016-17 2015-16 2016-17Blocks 2 1082.9 3985.1 30.5 26.0Heat (H) 2 27074.4** 24566.5** 933.4** 1131.4**Error I 4 45.3 361.4 6.2 2.9Selenium (Se) 4 2587.4** 2552.8** 39.2** 45.7**H × Se 8 329.6** 258.3** 3.9** 4.9**Error II 24 15.7 50.6 0.5 1.3



TreatmentsSOD

(Unit per mg protein)POD

(Unit per mg protein)2015-16 2016-17 2015-16 2016-17

No heat stress (H0)Control/ water spray (Se0) 128.3 c 125.0 c 30.7 d 29.0 d25 mg L-1 selenium (Se25) 131.7 c 126.7 c 30.9 cd 29.2 cd50 mg L-1 selenium (Se50) 161.7 b 156.7 b 32.9 b 31.0 ab75 mg L-1 selenium (Se75) 191.7 a 180.0 a 34.9 a 33.2 a100 mg L-1 selenium (Se100) 170.0 b 165.0 ab 32.6 bc 30.9 abHeat from spike to grain filling (H1)Control/ water spray (Se0) 50.8 d 51.2 c 14.6 d 11.3 c25 mg L-1 selenium (Se25) 65.2 c 60.2 bc 14.8 cd 11.5 c50 mg L-1 selenium (Se50) 74.2 bc 69.2 b 16.5 c 13.2 bc75 mg L-1 selenium (Se75) 79.6 b 74.6 b 18.8 b 15.4 b100 mg L-1 selenium (Se100) 89.7 a 94.7 a 21.0 a 18.4 aHeat from flowering to grain filling (H2)Control/ water spray (Se0) 95.7 d 94.0 c 19.3 b 16.0 c25 mg L-1 selenium (Se25) 100.4 cd 92.1 c 19.7 b 16.3 c50 mg L-1 selenium (Se50) 108.3 bc 103.3 bc 20.3 b 16.9 bc75 mg L-1 selenium (Se75) 114.8 b 111.5 ab 22.9 a 19.9 a100 mg L-1 selenium (Se100) 126.8 a 123.5 a 24.5 a 21.9 aTukey’s HSD (p ≤ 0.05) 9.52 17.08 1.70 2.74Year mean 112.6 108.5 23.6 20.9Tukey’s HSD (p ≤ 0.05) NS NS


significant

148

Figure 4.3.8: Regression analysis for effect of foliar applied selenium on superoxide dismutase and peroxidase contents of heat stressed wheat


149

Table 4.3.9: Effect of foliar applied selenium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat


Source of variation

DFCatalase Total phenolics

2015-16 2016-17 2015-16 2016-17Blocks 2 66.62 327.10 25.92 187.82Heat (H) 2 1255.42** 1395.74** 436.81** 505.44**Error I 4 10.04 8.29 1.61 9.09Selenium (Se) 4 228.14** 270.05** 60.53** 54.36**H × Se 8 59.80** 55.03** 4.28** 7.25**Error II 24 1.91 3.36 0.58 0.94



TreatmentsCAT (Unit per mg protein) TPC (mg GAE g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (Se0) 31.3 d 29.6 c 23.9 d 21.6 c25 mg L-1 selenium (Se25) 31.7 d 30.0 c 24.2 d 21.9 c50 mg L-1 selenium (Se50) 38.0 c 36.3 b 27.6 c 25.3 b75 mg L-1 selenium (Se75) 52.6 a 51.2 a 32.0 a 29.7 a100 mg L-1 selenium (Se100) 49.3 b 47.6 a 30.0 b 28.7 aHeat from spike to grain filling (H1)Control/ water spray (Se0) 20.9 c 18.2 c 14.8 d 11.5 c25 mg L-1 selenium (Se25) 21.1 c 18.3 c 15.2 cd 11.8 c50 mg L-1 selenium (Se50) 23.3 bc 19.6 bc 16.4 c 13.1 bc75 mg L-1 selenium (Se75) 24.6 b 22.9 ab 18.2 b 15.1 b100 mg L-1 selenium (Se100) 28.9 a 27.2 a 20.7 a 17.9 aHeat from flowering to grain filling (H2)Control/ water spray (Se0) 23.9 b 20.9 b 17.8 d 17.0 b25 mg L-1 selenium (Se25) 23.9 b 21.1 b 18.4 cd 17.9 b50 mg L-1 selenium (Se50) 25.3 b 21.9 b 20.0 bc 18.3 b75 mg L-1 selenium (Se75) 26.6 b 23.8 b 21.1 b 18.3 b100 mg L-1 selenium (Se100) 30.2 a 29.2 a 23.1 a 20.7 aTukey’s HSD (p ≤ 0.05) 3.32 4.40 1.83 2.33Year mean 30.1 27.9 21.6 19.2Tukey’s HSD (p ≤ 0.05) NS NS


significant

150

Figure 4.3.9: Regression analysis for effect of foliar applied selenium on catalase and total phenolic contents of heat stressed wheat


151

Escalated senescence under heat ultimately reduced carbohydrates availability for

reproductive parts (Mathur et al., 2014). While, heat stress decreased the soluble

carbohydrates by 10-25% in varying wheat genotypes depending on heat tolerance

(Talukder et al., 2014).

Likewise, decrease of antioxidants, chlorophyll contents and TPC under heat can

also be attributed to aggravated lipid peroxidation. So, strong negative and remarkable

association of antioxidants, chlorophyll contents and TPC with malondialdehyde was

recorded under varying temperatures over the years (Table 4.3.14-4.3.16). It further

confirmed the adverse impacts of lipid peroxidation of antioxidants activities and TPC.

Plethora of ROS outcompeted the biosynthesis of antioxidants and disrupted the

membrane stability (Ashraf and Harris, 2013). Hydroxyl radical depicted the most

detrimental effects regarding the lipid peroxidation. While, singlet oxygen and superoxide

radicals exhibited relatively more damaging effects for chlorophyll degradation in wheat

under heat (Iqbal et al., 2017).

Impairment in biosynthesis of osmo-protectants under heat might be another

reason for inhibition of enzymatic activities and degradation of chlorophyll under heat.

Heat stress might have reduced the accumulation of proline, glycine betaine and soluble

proteins. Consequently, proline modulated detoxification of 1O2* was decreased and thus

it initiated cascade of reactions to synthesize ROS. Ultimately, chlorophyll contents and

carbohydrates partitioning towards grains was also reduced. Reduced biosynthesis of

proline also results in excessive synthesis of OH●-, which ultimately enhances the lipid

peroxidation of bio-membranes. Additionally, reduced accumulation of proline, glycine

betaine and soluble proteins depressed osmotic, water and turgor potential, which further

enhanced the sensitivity of cells for lipid peroxidation. Moreover, strong positive and

remarkable association of chlorophyll a and b contents with osmo-protectants was

recorded under ‘no heat stress’ (Table 4.3.14 a, d), ‘heat from spike to grain filling’

(Table 4.3.15 a, c) and ‘heat from flowering to grain filling’ (Table 4.3.16 a, c) during

2015-16 and 2-16-17. Likewise, strong positive and remarkable association of

antioxidants with osmo-protectants was observed under varying temperatures over the

years (Table 4.3.14-4.3.16 b, d). Thus, it was confirmed that enzyme activities were

diminished due to lesser accumulation of osmo-protectants. Excessive generation of ROS

decreased the activities of antioxidants, biosynthesis of proline, glycine betaine and

adversely affected chlorophyll fluorescence (Hemantaranjan et al., 2014). Imposition of

152

heat stress enhanced the synthesis of ROS, which ultimately depressed the water relations

(Kamal et al., 2017).

Selenium modulated improvements in chlorophyll contents, antioxidants and

phenolics, which can be ascribed to role of selenium in scavenging of ROS. Availability

of selenium might have escalated the spontaneous dismuatation (non-enzymatic) of 1O2●-

to H2O2. Together with non-enzymatic dismutation of 1O2●-, selenium improved activities

of SOD as well. Both enzymatic and non-enzymatic dismutation might have acted in

conjunction with each other to detoxify 1O2●-. Ultimately, SOD activities were not

restricted because of substrate (1O2●-) saturation. Afterwards, H2O2 might have enhanced

substrate availability for catalase and peroxidase. Concurrently, selenium might have

enhanced the activities of glutathione peroxidase, guaiacol peroxidase, ascorbate

peroxidase and catalase. Therefore, selenium modulated improvements in peroxidase and

catalase enzymes might have alleviated the saturation of substrate for POD and CAT.

Hence, boost in activities under selenium might have triggered the detoxification of H2O2

to H2O and oxygen and thus alleviated oxidative stress. Whereas, strong positive and

significant association of SOD, POD, CAT and TPC with each other was recorded under

‘no heat stress’ (Table 4.3.14 b, d), ‘heat from spike to grain filling’ (Table 4.3.15 b, d)

and ‘heat from flowering to grain filling’ (Table 4.3.16 b, d) over the years. It established

the enhancement in activities of POD, CAT and TPC with the enhancing SOD activities.

Exogenous application of selenium under the stress conditions improved antioxidant

defense mechanism, alleviated oxidative stress and improved yield components of wheat

under stress environment (Nawaz et al., 2015). Application of selenium under abiotic

stresses improved the detoxification of 1O2●- and H2O2 and ultimately enhanced membrane

integrity (Ahmad et al., 2016).

Selenium mediated improvements in biosynthesis of antioxidants and chlorophyll

might also be consequence seleno-methionine and seleno-cysteine synthesis. Availability

of selenium might have replaced the sulfur in sulpho-hydral groups of glutathione

reductase (GSH). Replacement of sulpho-hydral groups in fatty acids might have caused

the biosynthesis of seleno-methionine and seleno-cysteine (the heat stable amino acids).

Afterwards, GSH containing seleno-methionine and seleno-cysteine might have promoted

the quenching of H2O2. Hence, restrictions of substrate saturation of POD and CAT were

alleviated and thus relatively lesser H2O2 under selenium triggered the activities of POD

and CAT. Furthermore, strong positive and significant correlation of SOD with POD and

CAT established the rapid detoxification of H2O2 under selenium availability (Table

153

4.3.14-4.3.16 b, d). Application of selenium enhanced the stability of sulpho-hydral group

containing antioxidants such as glutathione reductase and glutathione s-transferase by

enhancing the synthesis of heat stable seleno-methionine and seleno-cysteine (Malik et

al., 2012). Application of selenium stabilized sulpho-hydral groups of fatty acids by

improving the detoxification of ROS through boost of antioxidants (Mroczek-Zdyrska

and Wójcik, 2011).

Detoxification of ROS, amplification of antioxidants and chlorophyll contents can

be attributed to selenium role in sustenance of reducing powers under heat. So, Feng et al.

(2013) concluded that application of selenium enhanced the availability of reductants for

antioxidants and improved output of photosynthesis.

Improvement in antioxidant activities under selenium availability can also be

consequence of decreased lipid peroxidation. Selenium availability might have replaced

thiol groups with the heat stable amino acids and thus conferred tolerance against heat.

Moreover, strong negative and significant association of antioxidants with

malondialdehyde confirmed lipid peroxidation caused damages for antioxidants activities

(Table 4.3.14-4.3.16 b, d). Application of selenium reorganized bio-membranes, regulated

unsaturation and galactolipids accumulation and ultimately enhanced membrane integrity

(Winkel et al., 2015). While, decrease of antioxidants and TPC with 100 mg L-1 foliar

selenium under no heat stress can be result of imbalance in selenium mediated ROS

quenching and synthesis of NADPH. Lesser ROS under normal conditions might inhibit

the assimilation of selenium as seleno-methionine and seleno-cysteine. Excessive free

selenium under ‘no heat stress’ might have caused outburst of ROS, which decreases

antioxidants with 100 mg L-1 foliar selenium. Excessive application of selenium under

ambient conditions caused an imbalance between thiols groups of chloroplast thylakoid,

NADPH synthesis and assimilation of selenium and enhanced ROS synthesis (Mroczek-

Zdyrska and Wójcik, 2011).


(a) Results

Imposition of heat stress either from ‘spike to grain filling’ or ‘flowering to grain

filling’ significantly decreased the biosynthesis of osmo-protectants (proline, glycine

betaine and soluble proteins) while enhanced lipid peroxidation compared to ‘no heat

stress’. Imposition of ‘heat from spike to grain filling’ proved more damaging than ‘heat

from flowering to grain filling’ for biosynthesis of osmo-protectants and lipid

peroxidation. While, exogenous selenium modulated improvements were significant and

154

varying responses were observed with different concentrations of selenium under

different high temperature environments. Hence interaction of heat stress and foliar

selenium differed significantly for biosynthesis of osmo-protectants and malondialdehyde

contents.

Statistically similar and comparatively more proline and glycine betaine were

quantified with 75 and 100 mg L-1 exogenous selenium under ‘no heat stress’ over the

years. Whereas, significantly more proline was observed with 100 mg L -1 foliar selenium

compared to other concentrations under ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’ over the two years of study. Likewise, significantly more

glycine betaine content was recorded with 100 mg L-1 compared to other concentrations

of foliar selenium under both heat stress treatments. However, some inconsistencies were

recorded over the years where 75 and 100 mg L-1 selenium produced statistically similar

glycine betaine under heat induced conditions. Statistically similar and relatively more

total soluble proteins and statistically alike and lesser malondialdehyde contents were

observed with 75 and 100 mg L-1 exogenous selenium under ‘no heat stress’ over the

years. While some discrepancies were recorded in soluble proteins under ambient

conditions in 2015-16 where 100 mg L-1 selenium synthesized significantly more soluble

proteins compared to other concentrations. Whereas, significantly more total soluble

proteins and significantly lesser malondialdehyde contents were recorded with 100 mg L -1

foliar selenium compared to other selenium concentrations under both heat stress

treatments over the years. Moreover, relatively higher malondialdehyde contents were

recorded for water spray under all three treatments of heat stress (Table 4.3.10 and Table

4.3.11).

Moreover, each 25 mg L-1 application of foliar selenium enhanced proline

synthesis by 0.009, 0.009 and 0.008 µmol g-1 under ‘no heat stress’, ‘heat from spike to

grain filling’ and ‘heat from flowering to grain filling’ respectively over the years. While,

each unit of foliar selenium modulated enhancements in glycine betaine was 0.59 µmol g -

1 under ‘no heat stress’, 0.42-0.49 µmol g-1 under ‘heat from spike to grain filling’ and

0.39-0.41 µmol g-1 under ‘heat from flowering to grain filling’ over the years. Similarly,

each 25 mg L-1 foliar selenium triggered enhancement in total soluble proteins was

0.0012-0.0013 mg g-1 under ‘no heat stress’, 0.0011-0.0015 mg g-1 under ‘heat from spike

to grain filling’ and 0.0012-0.0013 mg g-1 under ‘heat from flowering to grain filling’

over the years. Likewise, each unit application of selenium decreased the

malondialdehyde by 0.0016-0.0017, 0.004-0.006 and 0.003-0.005 µmol g-1 under ‘no heat

155

stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’ over the

years. Moreover, improvements in glycine betaine, soluble proteins and decrease in

malondialdehyde contents were more dependent on foliar selenium under the stress

environment compared to ambient conditions over the years. While, dependence of

proline on selenium was reduced slightly under ‘heat from flowering to grain filling’

compared to ‘no heat stress’ over the temporal variations. However, importance of

exogenous selenium for proline improvements was more under ‘heat from spike to grain

filling’ compared to ‘no heat stress’ over the years (Figure 4.3.10 and Figure 4.3.11).

(b) Discussion

Downregulation in biosynthesis of proline, glycine betaine and soluble proteins

under heat stress can be attributed to accelerated senescence under heat. Heat stress

triggered the enzymes that accelerate degradation of chlorophyll while inhibited the

chlorophyll synthesizing enzymes. Therefore, utilization of sunlight in photosynthesis

was decreased because of lesser chlorophyll contents. Henceforth, carbohydrates

availability for synthesis of carbon skeleton of amino acids (proline and glycine betaine)

and soluble proteins might have decreased. Hence, accumulation of osmo-protectants

under stress environment was lesser compared to ambient conditions. Whereas, lesser

grains per spike and 1000-grain weight under heat compared to control confirmed the

decreased availability of assimilates for synthesis of osmo-protectants. Likewise, decrease

of green leaf area and grain filling duration under heat also caused accelerated senescence

and thus reduced carbon chain availability for synthesis of amino acids and proteins.

Moreover, strong positive and remarkable association of osmo-protectants with

chlorophyll a and b contents was recorded under varying temperatures over the years

(Table 4.3.14-4.316 a, c). High temperature stress inhibited the activities of antioxidants,

impaired the biosynthesis of osmo-protectants and osmolytes (Wang et al., 2014). High

temperature mediated boost in activities of chlorophyllide oxidoreductase escalated the

breakdown of chlorophyll. While, lesser activities of aminolaevulinic acid dehydratase

decreased the biosynthesis of chlorophyll under heat stress which reduced the synthesis of

carbohydrates (Hemantaranjan et al., 2014).

Decrease of osmo-protectants under high temperature stress can also be explained

in terms of impairment in activities of antioxidants and phenolics. Excessive heat caused

instability in energy absorption and electron transfer in electron transport chain might

have slowed down than the generation of free electrons at the reaction center from

photolysis of water. Hence, disruption of balance might have enhanced the availability of

156

free electrons which ultimately excited chlorophyll and led to synthesize 1O2*. Besides,

free electrons might have reacted with environmental oxygen at reaction center of

photosystem-II (PS-II) and generated 1O2●-.

Table 4.3.10: Effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat


Source of variation

DFProline Glycine betaine

2015-16 2016-17 2015-16 2016-17Blocks 2 2.941 0.337 1706.3 2200.5Heat (H) 2 15.298* 15.545** 31360.6** 40494.2**Error I 4 1.377 0.039 138.1 92.9Selenium (Se) 4 1.192** 1.105** 3640.5** 3144.9**H × Se 8 0.093** 0.082** 229.2* 355.2**Error II 24 0.017 0.016 70.7 69.6



TreatmentsProline (µmol g-1) Glycine betaine (µmol g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (Se0) 4.25 c 3.92 c 221.7 c 215.0 c25 mg L-1 selenium (Se25) 4.30 c 3.97 c 225.0 bc 218.0 bc50 mg L-1 selenium (Se50) 4.70 b 4.37 b 245.0 b 239.0 b75 mg L-1 selenium (Se75) 5.17 a 4.83 a 283.3 a 276.7 a100 mg L-1 selenium (Se100) 4.90 ab 4.57 ab 266.7 a 260.0 aHeat from spike to grain filling (H1)Control/ water spray (Se0) 2.40 c 2.07 c 130.7 c 122.7 b25 mg L-1 selenium (Se25) 2.47 c 2.13 c 148.3 bc 130.0 b50 mg L-1 selenium (Se50) 2.63 bc 2.30 bc 163.0 a 135.0 b75 mg L-1 selenium (Se75) 2.87 b 2.53 b 169.3 a 142.7 b100 mg L-1 selenium (Se100) 3.37 a 2.94 a 181.7 a 168.3 aHeat from flowering to grain filling (H2)Control/ water spray (Se0) 2.97 c 2.64 c 169.3 d 158.0 b25 mg L-1 selenium (Se25) 2.80 c 2.47 c 175.0 cd 163.3 b50 mg L-1 selenium (Se50) 2.90 c 2.57 c 189.7 bc 170.0 b75 mg L-1 selenium (Se75) 3.35 b 3.02 b 197.0 ab 176.3 b100 mg L-1 selenium (Se100) 3.75 a 3.38 a 210.0 a 200.0 aTukey’s HSD (p ≤ 0.05) 0.313 0.303 20.19 20.04Year mean 3.52 3.18 198.4 185.0Tukey’s HSD (p ≤ 0.05) NS NS

157


significant

Figure 4.3.10: Regression analysis for effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat


158

Table 4.3.11: Effect of foliar applied selenium on total soluble proteins (TSP) and malondialdehyde (MDA) contents of heat stressed wheat


Source of variation

DFTotal soluble proteins Malondialdehyde

2015-16 2016-17 2015-16 2016-17Blocks 2 0.0135 0.0128 0.0291 0.0402Heat (H) 2 0.5663** 0.6820** 0.6879** 1.6207**Error I 4 0.0005 0.0017 0.0067 0.0024Selenium (Se) 4 0.0267** 0.0201** 0.1048** 0.2658**H × Se 8 0.0024** 0.0012* 0.0107** 0.0312**Error II 24 0.0004 0.0005 0.0023 0.0015



TreatmentsTSP (mg g-1) MDA (µmol g-1)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (Se0) 0.75 d 0.70 c 0.70 a 0.86 a25 mg L-1 selenium (Se25) 0.79 cd 0.76 b 0.66 ab 0.81 ab50 mg L-1 selenium (Se50) 0.80 c 0.79 ab 0.61 abc 0.76 b75 mg L-1 selenium (Se75) 0.90 a 0.84 a 0.53 c 0.65 bc100 mg L-1 selenium (Se100) 0.85 b 0.81 ab 0.57 bc 0.73 cHeat from spike to grain filling (H1)Control/ water spray (Se0) 0.37 d 0.34 c 1.27 a 1.71 a25 mg L-1 selenium (Se25) 0.41 cd 0.38 bc 1.08 b 1.55 b50 mg L-1 selenium (Se50) 0.45 bc 0.40 b 0.99 b 1.46 b75 mg L-1 selenium (Se75) 0.46 b 0.41 b 0.97 b 1.26 c100 mg L-1 selenium (Se100) 0.53 a 0.47 a 0.83 c 1.07 dHeat from flowering to grain filling (H2)Control/ water spray (Se0) 0.49 c 0.37 c 1.05 a 1.46 a25 mg L-1 selenium (Se25) 0.51 bc 0.40 bc 0.96 ab 1.31 b50 mg L-1 selenium (Se50) 0.53 bc 0.42 bc 0.91 b 1.14 c75 mg L-1 selenium (Se75) 0.55 b 0.44 b 0.90 b 1.01 d100 mg L-1 selenium (Se100) 0.63 a 0.50 a 0.76 c 0.99 dTukey’s HSD (p ≤ 0.05) 0.046 0.051 0.115 0.093Year mean 0.60 0.53 0.86 B 1.12 ATukey’s HSD (p ≤ 0.05) NS 0.116


significant

159

Figure 4.3.11: Regression analysis for effect of foliar applied selenium on total soluble proteins and malondialdehyde contents of heat stressed wheat


160

Both 1O2* and 1O2

●- might have triggered degradation of chlorophyll, outcompeted

antioxidants and reacted with unsaturated fatty acids of membranes to synthesize OH●-.

Moreover, impairment in electron transfer in electron transport chain might have

decreased reduction of NADP to NADPH at ferredoxin of photosystem-I (PS-I). It might

have decreased the NAPDH and thereby restricted carbon dioxide fixation in

photosynthesis together with the generation of 1O2●- at the ferredoxin of photosystem-I.

All these cascades of reactions might have enhanced the synthesis of ROS and decreased

antioxidant activities by saturation of substrates. Consequently, membrane integrity was

decreased which aggravated the leakage of solutes and thus decreased osmo-protectants.

While, excessive ROS induced damages to chlorophyll might have reduced carbon chain

availability for amino acid synthesis concurrently. Furthermore, strong positive and

significant association of osmo-protectants with SOD, POD, CAT and TPC was recorded

under varying temperatures over the years. It established the relationship of osmo-

protectants with antioxidant enzymes (Table 4.3.14-4.3.16 b, d). Excessive synthesis of

ROS from chloroplast under heat stress decreased the reduction of carbon in

photosynthesis, depressed SOD, POD, CAT and phenolics and enhanced membrane

leakage (Mathur et al., 2014). Likewise, high light intensity and heat stress destabilized

photosystem-II, aggravated lipid peroxidation and impaired the activities of antioxidants

in wheat crop (Chen et al., 2017).

Foliar applied selenium might have enhanced efficacy of light reactions under

heat and thus improved chlorophyll contents also. Moreover, strong positive and

remarkable association of osmo-protectants with chlorophyll a and b contents was

recorded under varying temperature environments over the two years (Table 4.3.14-4.3.16

a, c). Application of selenium improved chlorophyll contents, enhanced energy

absorption by light harvesting complex, increased the energy of excitation, electron

transfer and eventually output quantum of light reaction in three wheat genotypes under

the stressed conditions. Moreover, application of higher concentrations of selenium under

ambient conditions often caused disruptions in photosynthetic machinery (Labanowska et

al., 2012). Exogenous application of selenium delayed the senescence, improved

antioxidant activities and ultimately increased biomass accumulation (Cheng et al., 2016).

Exogenous selenium modulated improvements in membrane stability might be

another reason for enhancement of osmo-protectants. Availability of selenium might have

activated transport proteins at bio-membranes that channelized various solutes across the

membranes under stress conditions. Moreover, selenium mediated biosynthesis of osmo-

161

protectants might have encapsulated thiol groups and points of unsaturation along the

carbon chain of fatty acids. Thus, selenium might have shielded the fatty acids from the

attack of OH●- and subsequent cascade of reactions of ROS generation. Besides, selenium

availability under heat stress might have regulated the degree of unsaturation in

galactolipids constituting ground structure of membranes of plastids and thereby

conferred heat tolerance. Moreover, strong negative and pronouncing association of

osmo-protectants with malondialdehyde under varying temperatures established the role

of osmo-protectants in the integration of bio-membranes (Table 4.3.14-4.3.16 b, d).

Availability of selenium enhanced the biosynthesis of antioxidants, osmo-protectants and

regulated solute transport across membranes under stress conditions (Kaur et al., 2014).

Exogenous application of selenium improved soluble sugars and free amino acids which

ultimately enhanced the water of wheat crop under stress conditions (Nawaz et al., 2015).

Selenium role in improvement of osmo-protectants and membrane integrity can

also be elucidated in terms of improved antioxidant defensive mechanism under heat.

Application of selenium might have enhanced non-enzymatic dismutation of 1O2●- to H2O2

and thus alleviated 1O2●- mediated substrate saturation of SOD. Subsequently,

comparatively lesser concentration of 1O2●- boosted the activities of SOD. Therefore, H2O2

concentration might have improved due to SOD mediated and non-enzymatic

dismutation. While increased availability of reductants enhanced the activities of DHAR,

MDHAR, CAT, ascorbate peroxidase and glutathione peroxidase which ultimately

detoxified ROS. Hence, oxidative stress was alleviated under selenium availability.

Alleviation of oxidative stress increased membrane stability and water relations of wheat

under stress. While, strong positive and remarkable association of antioxidants and

phenolics with osmo-protectants under varying conditions of temperature over the years

further accomplished the role of antioxidants in synthesis of osmo-protectants (Table

4.3.14 b, d; Table 4.3.15 b, d and Table 4.3.16 b, d). Foliar application of selenium

improved synthesis of osmo-protectants, uptake of osmolytes, chlorophyll contents and

antioxidant activities under stressed environment (Hajiboland et al., 2015). Likewise,

application of selenium enhanced the accumulation of osmolytes, photosynthetic

pigments and antioxidant defense system under the stress conditions (Abd-Allah et al.,

2016).

4.3.6. Water relations and quality attributes

(a) Results

162

Imposition of heat stress from ‘spike to grain filling’ and ‘flowering to grain

filling’ significantly depressed the water relations and degraded grain crude protein

contents compared to ‘no heat stress’. While, more deleterious impacts for these

parameters were recorded under ‘heat from spike to grain filling’ compared to ‘heat from

flowering to grain filling’ over the years. However, exogenous application of selenium

effectively alleviated the deleterious impacts and significantly enhanced the osmotic,

water and turgor potential and grain crude proteins. Varying responses of osmotic and

water potential were recorded with different foliar concentrations of selenium under ‘no

heat stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’.

Thus, significant interaction of heat and foliar selenium for osmotic and water potential

was recorded over the years.

Under ‘no heat stress’, selenium at 75 mg L-1 depicted more osmotic and water

potential compared to other. While, under ‘heat from spike to grain filling’ and ‘heat from

flowering to grain filling’, 100 mg Se L-1 performed best in this regard (Table 4.3.12).

Moreover, each 25 mg L-1 increment in foliar selenium enhanced the osmotic

potential by 0.0021-0.0025, 0.0030-0.0031 and 0.0018-0.0034 MPa under ‘no heat

stress’, ‘heat from spike to grain filling’ and ‘heat from flowering to grain filling’,

respectively over the years. Whereas, each unit of foliar selenium modulated

improvements in water potential were 0.0026-0.0030 MPa under ‘no heat stress’, 0.0038-

0.0039 MPa under ‘heat from spike to grain filling’, 0.0025-0.0042 MPa under ‘heat from

flowering to grain filling’ over the two years. While, importance of selenium for

improvement of osmotic and water potential was enhanced under stress conditions

compared to ‘no heat stress’ over the years (Figure 4.3.12).

Exogenous selenium modulated improvements in turgor potential and grain crude

proteins were similar under varying conditions of temperature over the years. Thus, a

non-significant ‘heat × selenium’ effect for these parameters was recorded over the years.

Significantly lesser turgor potential was observed under ‘heat from spike to grain filling’

compared to ‘heat from flowering to grain filling’ and ‘no heat stress’ over the years.

Whereas, statistically alike and significantly more degradation of grain crude proteins was

recorded under both heat imposed conditions compared to ambient conditions over the

years. While, significantly more turgor potential and grain crude proteins were recorded

under ‘no heat stress’ compared to heat treatments over the temporal variations. So,

turgor potential was decreased by 51-59% and 34-46% under ‘heat from spike to grain

filling’ and ‘heat from flowering to grain filling’ compared to ‘no heat stress’ over the

163

years. While, a decrease of 20-22% in grain crude proteins was recorded under ‘heat from

spike to grain filling’ and 18-20% under ‘heat from flowering to grain filling’ compared

to ‘no heat stress’ over the years.

Varying concentrations of foliar selenium depicted significant improvements in

turgor potential and grain crude protein contents. Relatively more and statistically alike

turgor potential and grain crude proteins were recorded with 50, 75 and 100 mg L -1

selenium during 2015-16 and 75 and 100 mg L-1 selenium during 2016-17 compared to

other treatments. While, statistically similar and relatively lesser turgor potential and

grain crude proteins were recorded with control/water spray and 25 mg L-1 exogenous

selenium over the years (Table 4.3.13).

Besides, each unit application of foliar selenium enhanced turgor potential by

0.0004-0.0005 MPa under ‘no heat stress’, 0.0008 MPa under ‘heat from spike to grain

filling’ and 0.0006-0.0008 MPa under ‘heat from flowering to grain filling’ over the

years. While, each 25 mg L-1 modulated enhancements in grain crude proteins were

0.008-0.015, 0.007-0.011 and 0.009-0.013% under ‘no heat stress’, ‘heat from spike to

grain filling’ and ‘heat from flowering to grain filling’, respectively over the years.

Moreover, selenium mediated improvements in turgor potential and grain crude proteins

under stress environments was more compared to ‘no heat stress’ over the years (Figure

4.3.13).

(b) Discussion

Therefore, availability of carbon chain for synthesis of solutes and osmo-

protectants might have decreased and water relations were also decreased as an adaptive

response to lesser solutes. Decrease in availability of sucrose under heat was also

accomplished from lesser grains per spike and 1000-grain weight under heat compared to

ambient conditions. Moreover, decrease of chlorophyll contents might have decreased the

availability of carbon chain for synthesis of amino acids and subsequent grain crude

proteins. While, strong positive and pronouncing association of chlorophyll a and b

contents with water relations and grain crude proteins was observed under varying

temperatures over the years (Table 4.3.14-4.3.16 a, c). Heat stress disrupted the light

reactions, aggravated chlorophyll degradation, membrane leakage and decreased the

synthesis of soluble sugars, proteins and proline. Lesser synthesis of osmo-protectants

depressed water relations and enhanced the membrane leakage (Szymańska et al., 2017).

Imposition of heat stress from spike initiation to grain filling initiation reduced the

164

synthesis of proline, glycine betaine, soluble proteins and grain yield (Shahid et al.,

2017).

Depression of water relations and degradation of grain crude proteins under heat

stress can also be elucidated in context of aggravated lipid peroxidation under heat. Heat

stress might have triggered the synthesis of 1O2●-, 1O2

*, H2O2 and OH●- and plethora of

ROS might have overcome the antioxidants activities and thus aggravated lipid

peroxidation.

Table 4.3.12: Effect of foliar applied selenium on osmotic (ΨS) and water potential (ΨW) of heat stressed wheat


Source of variation

DFOsmotic potential Water potential

2015-16 2016-17 2015-16 2016-17Blocks 2 0.0195 0.0641 0.2233 0.1706Heat (H) 2 1.5048** 1.8871** 2.7087** 3.3382**Error I 4 0.0022 0.0027 0.0191 0.0047Selenium (Se) 4 0.1272** 0.0793** 0.1921** 0.1269**H × Se 8 0.0050* 0.0047** 0.0068* 0.0061*Error II 24 0.0016 0.0013 0.0022 0.0018



TreatmentsΨS (-MPa) ΨW (-MPa)

2015-16 2016-17 2015-16 2016-17No heat stress (H0)Control/ water spray (Se0) 1.11 c 1.15 e 0.73 b 0.79 d25 mg L-1 selenium (Se25) 1.08 c 1.11 d 0.68 b 0.74 cd50 mg L-1 selenium (Se50) 0.96 b 1.03 c 0.53 a 0.64 bc75 mg L-1 selenium (Se75) 0.85 a 0.92 a 0.42 a 0.51 a100 mg L-1 selenium (Se100) 0.91 ab 0.98 b 0.49 a 0.58 abHeat from spike to grain filling (H1)Control/ water spray (Se0) 1.73 d 1.81 e 1.58 d 1.69 d25 mg L-1 selenium (Se25) 1.67 cd 1.78 d 1.49 cd 1.63 cd50 mg L-1 selenium (Se50) 1.61 bc 1.73 c 1.40 bc 1.57 bc75 mg L-1 selenium (Se75) 1.53 b 1.58 b 1.31 b 1.40 a100 mg L-1 selenium (Se100) 1.41 a 1.53 a 1.18 a 1.33 aHeat from flowering to grain filling (H2)Control/ water spray (Se0) 1.61 d 1.70 e 1.38 c 1.51 c25 mg L-1 selenium (Se25) 1.52 cd 1.66 d 1.27 c 1.47 c50 mg L-1 selenium (Se50) 1.43 bc 1.61 c 1.15 b 1.41 bc

165

75 mg L-1 selenium (Se75) 1.38 b 1.58 b 1.09 b 1.35 ab100 mg L-1 selenium (Se100) 1.26 a 1.51 a 0.95 a 1.26 aTukey’s HSD (p ≤ 0.05) 0.096 0.021 0.113 0.102Year mean 1.34 1.44 1.04 1.19Tukey’s HSD (p ≤ 0.05) NS NS


significant

Figure 4.3.12: Regression analysis for effect of foliar applied selenium on osmotic and water potential of heat stressed wheat

166


Table 4.3.13: Effect of foliar applied selenium on turgor potential (ΨP) and grain crude protein contents of heat stressed wheat


Source of

variationDF

Turgor potential Grain crude proteins

2015-16 2016-17 2015-16 2016-17

Blocks 2 0.02393 0.01769 0.86 1.58

Heat (H) 2 0.17718** 0.20738** 32.84** 23.29**

Error I 4 0.00152 0.00063 0.70 0.92

Selenium (Se) 4 0.00712** 0.00560** 2.43** 1.00**

H × Se 8 0.00028NS 0.00031NS 0.09NS 0.02NS



TreatmentsΨP (MPa) Grain crude proteins (%)

2015-16 2016-17 2015-16 2016-17

167

Heat stress (H)




Tukey’s HSD (p ≤ 0.05) 0.051 0.033 0.985 1.250


Control/ water spray (Se0) 0.25 C 0.22 D 9.99 B 9.69 C

25 mg L-1 selenium (Se25) 0.28 B 0.24 CD 10.07 B 9.76 C

50 mg L-1 selenium (Se50) 0.31 A 0.25 BC 10.51 AB 9.92 BC

75 mg L-1 selenium (Se75) 0.31 A 0.27 AB 10.88 AB 10.32 AB


Tukey’s HSD (p ≤ 0.05) 0.022 0.025 0.985 0.459

Year mean 0.29 0.25 10.53 10.02


Figure 4.3.13: Regression analysis for effect of foliar applied selenium on turgor potential and grain crude protein contents of heat stressed wheat

168


Consequently, leakage of solutes from membrane might have enhanced and

thereby depressed osmotic, water and turgor potential. While, excessive ROS might have

also decreased the activities of enzymes involved in biosynthesis of grain crude proteins.

169

Furthermore, strong negative and conspicuous correlation of malondialdehyde with water

relations and grain proteins were observed under ‘no heat stress’ (Table 4.3.14 b, d), ‘heat

from spike to grain filling’ (Table 4.3.15 b, d) and ‘heat from flowering to grain filling’

(Table 4.3.16 b, d). It accomplished the negative impacts of lipid peroxidation on water

relations and grain quality of heat stressed wheat crop. High temperature stress enhanced

lipid peroxidation, disrupted photosystem-II, decreased the capability of membranes to

retain solutes and protein contents under high temperature stress conditions (Chen et al.,

2017). Post anthesis heat stress wheat inhibited the activities of sucrose

fructosyltransferase and fructan fructosyltransferase which declined carbo chain

availability for synthesis of amino acids (Wang et al., 2012). Lesser availability of

carbohydrates ultimately deteriorated grain quality (Rakszegi et al., 2014).

Improvement in water relations and grain proteins under foliar selenium might be

consequence of selenium-modulated improvement in biosynthesis of osmo-protectants.

Afterwards, osmo-protectants might have encapsulated ROS susceptible thiol groups and

unsaturated fatty acids along the carbon chain and thus imparted heat tolerance by

improving membrane stability. Osmo-protectants mediated alleviation of oxidative stress

might also have improved the partitioning of carbohydrates and amino acids to synthesize

grain proteins. While, solute retention inside cells might have enhanced osmotic, water

and turgor potential. Moreover, strong positive and remarkable association of water

relations and grain crude proteins with osmo-protectants under varying temperatures over

the years was recorded. It established the role of osmo-protectants in improving water

relations and grain crude protein contents (Table 4.3.14-4.3.16 b, d). Exogenous

application of selenium improved the synthesis of carbohydrates and dry matter

accumulation in wheat under drought stress conditions (Nawaz et al., 2014). Moreover,

foliar applied selenium improved membrane stability and thus conferred tolerance to heat

stressed wheat crop (Iqbal et al., 2015).

Another explanation is selenium-mediated improvement in grain filling rate.

Increased availability of carbohydrates for increased grain filling rate ultimately enhanced

the synthesis of amino acids which might have contributed for synthesis of amino acids

involved in membrane protection (proline and glycine betaine) and grain crude proteins.

While, strong positive and pronouncing association of grain filling rate with water

relations and grain proteins accomplished the role of accelerated grain filling rate to

improve water relations and quality of grain (Table 4.3.14-4.3.16 a, c). Availability of

170

selenium under heat stress enhanced the reductants output of light reactions which

enhanced carbon fixation ability under heat stress (Feng et al., 2013).

Likewise, selenium might have regulated the antioxidant activities by triggering

the enzymatic (SOD) and non-enzymatic dismutation of 1O2●-. While selenium might have

alleviated H2O2 mediated oxidative stress through enhancement of reductants that

ultimately boosted the recuing potential of CAT, POD, DHAR and MDHAR. Resultantly,

decrease of ROS produced suitable environment for chlorophyll to synthesize

carbohydrates and partition these for the synthesis of amino acids and soluble sugars.

While, amino acids thus contributed both towards osmotic adjustments as well as in grain

protein synthesis. Besides, strong positive and significant association of antioxidants and

phenolics with water relations and grain crude protein contents was recorded under

varying heat stress treatments over the years (Table 4.3.14-4.3.16 a, c). Application of

selenium improved the activities of antioxidants, alleviated oxidative stress, enhanced

membrane integrity and ultimately increased pollen fertility (Tedeschini et al., 2015).

Likewise, application of selenium decreased malondialdehyde contents and H2O2 while

boosted the activities of glutathione reductase and redox potential of POD under UV

stress (Mostafa and Hassan, 2015).

171

Table 4.3.14 (a): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2015-16

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.94*GY 0.99** 0.93*BY 0.98** 0.90* 0.99**HI 0.99** 0.95* 0.99** 0.98**SY 0.92* 0.81NS 0.95* 0.98** 0.92*PH 0.70NS 0.79NS 0.75NS 0.77NS 0.71NS 0.79NS

SL 0.90* 0.91* 0.90* 0.88* 0.91* 0.80NS 0.85NS

SPS 0.87NS 0.79NS 0.92* 0.94* 0.88* 0.96* 0.90* 0.88*GFR 0.99** 0.93* 0.98** 0.96** 0.99** 0.88* 0.65NS 0.90* 0.84NS

GFD 0.98** 0.95* 0.98** 0.97** 0.99** 0.91* 0.79NS 0.96** 0.91* 0.98**Chl a 0.90* 0.80NS 0.94* 0.97** 0.90* 0.99** 0.83NS 0.81NS 0.98** 0.86NS 0.90*Chl b 0.99** 0.92* 0.99** 0.99** 0.99** 0.96* 0.78NS 0.91NS 0.94* 0.97** 0.99** 0.95*SOD 0.98** 0.93* 0.98** 0.97** 0.97** 0.91* 0.80NS 0.96** 0.93* 0.97** 0.99** 0.91* 0.99**POD 0.92* 0.92* 0.92* 0.90* 0.92* 0.82NS 0.85NS 0.99** 0.89* 0.92* 0.97** 0.84NS 0.93*CAT 0.95* 0.84NS 0.98** 0.99** 0.95* 0.99** 0.80NS 0.85NS 0.97** 0.92* 0.94* 0.99** 0.98**TPC 0.98** 0.90* 0.99** 0.99** 0.98** 0.96* 0.80NS 0.92* 0.95* 0.96** 0.99** 0.96* 0.99**LP 0.98** 0.92* 0.99** 0.98** 0.98** 0.94* 0.80NS 0.94* 0.94* 0.97** 0.99** 0.94* 0.99**GB 0.96** 0.89* 0.98** 0.99** 0.96** 0.97** 0.83NS 0.92* 0.97** 0.94* 0.98** 0.97** 0.97**TSP 0.93* 0.92* 0.95* 0.96** 0.93* 0.96* 0.90* 0.90* 0.96** 0.89* 0.95* 0.96** 0.96**

MDA - 0.99** - 0.96** - 0.99** - 0.99** - 0.85NS - 0.74NS - 0.80NS - 0.92* - 0.92* - 0.96** - 0.99** - 0.93* - 0.99**ΨS 0.99** 0.94* 0.99** 0.98** 0.99** 0.93* 0.76NS 0.93* 0.91* 0.99** 0.99** 0.92* 0.99**ΨW 0.99** 0.95* 0.99** 0.97** 0.99** 0.90* 0.74NS 0.93* 0.88* 0.99** 0.99** 0.89* 0.99**ΨP 0.92* 0.95* 0.89* 0.83NS 0.92* 0.70NS 0.54NS 0.85NS 0.95* 0.94* 0.90* 0.66NS 0.86NS

GCPC 0.96* 0.84NS 0.98** 0.99** 0.95* 0.99** 0.74NS 0.82NS 0.94* 0.94* 0.94* 0.98** 0.98*** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

172

Table 4.3.14 (b): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2015-16

Parameters SOD POD CAT TPC LP GB TSP MDA ΨS ΨW ΨP

POD 0.97**CAT 0.95* 0.88*TPC 0.99** 0.94* 0.98**LP 0.99** 0.96** 0.97** 0.99**GB 0.98** 0.93* 0.99** 0.99** 0.99**TSP 0.95* 0.92* 0.96** 0.96** 0.96* 0.98**

MDA - 0.98** - 0.94* - 0.96** - 0.98** - 0.99** - 0.98** - 0.97**ΨS 0.99** 0.95* 0.96** 0.99** 0.99** 0.98** 0.95* - 0.99**ΨW 0.99** 0.95* 0.94* 0.98** 0.99** 0.97** 0.93* - 0.99** 0.99**ΨP 0.87NS 0.86NS 0.75NS 0.84NS 0.86NS 0.80NS 0.78NS - 0.89* 0.90* 0.93*

GCPC 0.94* 0.85NS 0.99** 0.98** 0.96** 0.98** 0.95* - 0.96** 0.96** 0.94* 0.77NS

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

173

Table 4.3.14 (c): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2016-17

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.98**GY 0.99** 0.96**BY 0.99** 0.97** 0.99**HI 0.99** 0.95* 0.99** 0.99**SY 0.83NS 0.91* 0.83NS 0.87NS 0.80NS

PH 0.92* 0.97** 0.93* 0.95* 0.91* 0.98**SL 0.92* 0.96** 0.92* 0.94* 0.90* 0.97** 0.99**

SPS 0.91* 0.89* 0.95* 0.96* 0.93* 0.87NS 0.93* 0.92*GFR 0.98** 0.98** 0.98** 0.99** 0.97** 0.88* 0.96* 0.93* 0.93*GFD 0.88* 0.83NS 0.92* 0.93* 0.91* 0.80NS 0.87NS 0.88* 0.98** 0.87NS

Chl a 0.87NS 0.80NS 0.92* 0.91* 0.91* 0.72NS 0.83NS 0.81NS 0.97** 0.88** 0.98**Chl b 0.98** 0.95* 0.99** 0.99** 0.99** 0.85NS 0.94* 0.93* 0.97** 0.97** 0.95* 0.93*SOD 0.98** 0.98** 0.99** 0.99** 0.98** 0.88* 0.96* 0.94* 0.93* 0.99** 0.88* 0.88* 0.98**POD 0.91* 0.96* 0.92* 0.95* 0.90* 0.97** 0.99** 0.98** 0.94* 0.96** 0.87NS 0.84NS 0.93*CAT 0.94* 0.89* 0.97** 0.96** 0.97** 0.79NS 0.89* 0.86NS 0.97** 0.95* 0.96* 0.98** 0.97**TPC 0.98** 0.93* 0.99** 0.99** 0.99** 0.80NS 0.90* 0.88* 0.95* 0.98** 0.93* 0.95* 0.99**LP 0.98** 0.97** 0.99** 0.99** 0.98** 0.88* 0.96** 0.94* 0.96** 0.99** 0.92* 0.92* 0.99**GB 0.96** 0.94* 0.99** 0.99** 0.95* 0.86NS 0.94* 0.92* 0.98** 0.98** 0.95* 0.95* 0.99**TSP 0.96** 0.95* 0.95* 0.95* 0.95* 0.84NS 0.91* 0.93* 0.88* 0.91* 0.88* 0.82NS 0.96*

MDA - 0.95* - 0.96** - 0.96** - 0.98** - 0.95* - 0.93* - 0.98** - 0.98** - 0.97** - 0.96* - 0.94* - 0.90* - 0.98**ΨS 0.99** 0.97** 0.99** 0.99** 0.99** 0.88* 0.96** 0.95* 0.96** 0.98** 0.93* 0.91* 0.99**ΨW 0.99** 0.97** 0.99** 0.99** 0.99** 0.88* 0.96** 0.95* 0.96** 0.98** 0.93* 0.91* 0.99**ΨP 0.99** 0.98** 0.99** 0.99** 0.99** 0.86NS 0.95* 0.88* 0.94* 0.98** 0.91* 0.89* 0.99**

GCPC 0.91* 0.84NS 0.95* 0.94* 0.95* 0.73NS 0.84NS 0.83NS 0.97** 0.90* 0.98** 0.99** 0.96*** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

174

Table 4.3.14 (d): Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2016-17


POD 0.96**CAT 0.95* 0.90*TPC 0.98** 0.91* 0.99**LP 0.99** 0.96** 0.97** 0.99**GB 0.98** 0.95* 0.99** 0.99** 0.99**TSP 0.92* 0.87NS 0.87NS 0.90* 0.92* 0.90*

MDA - 0.96** - 0.97** - 0.94* 0.90* - 0.97** - 0.97** - 0.95*ΨS 0.99** 0.95* 0.97** 0.98** 0.99** 0.99** 0.96* - 0.99**ΨW 0.99** 0.95* 0.96** 0.98** 0.99** 0.99** 0.96* - 0.99** 0.99**ΨP 0.99** 0.94* 0.95* 0.98** 0.99** 0.98* 0.97** - 0.98** 0.99** 1.00*

GCPC 0.90* 0.84NS 0.99** 0.97** 0.93* 0.96** 0.87NS - 0.92* 0.94* 0.94* 0.94** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

175

Table 4.3.15 (a): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2015-16

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.91*GY 0.91* 0.92*BY 0.94* 0.93* 0.99**HI 0.90* 0.95* 0.99** 0.99**SY 0.98** 0.94* 0.98** 0.99** 0.96**PH 0.86NS 0.98** 0.84NS 0.86NS 0.87NS 0.87NS

SL 0.93* 0.90* 0.79NS 0.82NS 0.78NS 0.86NS 0.88*SPS 0.85NS 0.97** 0.95* 0.94* 0.97** 0.91* 0.92* 0.81 NS

GFR 0.94* 0.95* 0.99** 0.99** 0.99** 0.98** 0.87 NS 0.85 NS 0.96*GFD 0.93* 0.99** 0.93* 0.94* 0.94* 0.95* 0.98** 0.89* 0.95* 0.94*Chl a 0.98** 0.94* 0.97** 0.98** 0.95* 0.99** 0.86 NS 0.91* 0.90* 0.99** 0.94*Chl b 0.98** 0.96** 0.92* 0.94* 0.92* 0.97** 0.94* 0.92* 0.90* 0.94* 0.98** 0.96**SOD 0.98** 0.90* 0.93* 0.95* 0.90* 0.96** 0.82 NS 0.92* 0.87 NS 0.97** 0.90* 0.99** 0.94*POD 0.89* 0.91* 0.99** 0.99** 0.98** 0.96** 0.82 NS 0.76 NS 0.95* 0.98** 0.98** 0.96* 0.90*CAT 0.88* 0.82NS 0.98** 0.96** 0.95* 0.938 0.70 NS 0.69 NS 0.87 NS 0.96* 0.82 NS 0.93* 0.83 NS

TPC 0.89* 0.88* 0.99** 0.98** 0.99** 0.95* 0.77 NS 0.74 NS 0.93* 0.98** 0.87 NS 0.95* 0.86 NS

LP 0.85NS 0.83NS 0.97** 0.96** 0.96* 0.92* 0.71 NS 0.70 NS 0.89* 0.96** 0.82 NS 0.92* 0.82 NS

GB 0.99** 0.91* 0.93* 0.95* 0.91* 0.97** 0.83 NS 0.92* 0.87 NS 0.97** 0.91* 0.99** 0.95*TSP 0.93* 0.84NS 0.95* 0.96** 0.93* 0.95* 0.73 NS 0.81 NS 0.86 NS 0.97** 0.84 NS 0.97** 0.86 NS

MDA 0.97** - 0.84NS - 0.89* - 0.92* - 0.86NS - 0.93* -0.75 NS -0.91* -0.81 NS -0.93* -0.85 NS -0.97** -0.90*ΨS 0.92* 0.90* 0.98** 0.98** 0.97** 0.96** 0.79 NS 0.81 NS 0.92* 0.99** 0.89* 0.98** 0.90*ΨW 0.95* 0.91* 0.98** 0.99** 0.97** 0.98** 0.82 NS 0.85 NS 0.92* 0.99** 0.91* 0.99** 0.92*ΨP 0.99** 0.92* 0.90* 0.93* 0.88* 0.96** 0.87 NS 0.95* 0.85 NS 0.94* 0.93* 0.98** 0.97**

GCPC 0.96** 0.90* 0.98** 0.99** 0.96** 0.99** 0.83 NS 0.82 NS 0.89* 0.97** 0.93* 0.98** 0.95* * = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

176

Table 4.3.15 (b): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2015-16


POD 0.92*CAT 0.91* 0.98**TPC 0.92* 0.99** 0.99**LP 0.91* 0.98** 0.99** 0.99**GB 0.99** 0.92* 0.90* 0.92* 0.90*TSP 0.97** 0.96* 0.97** 0.97** 0.86 NS 0.97**

MDA -0.99** -0.89* -0.88* -0.89* -0.88* -0.99** -0.97**ΨS 0.96** 0.99** 0.98** 0.99** 0.98** 0.96** 0.99** -0.94*ΨW 0.98** 0.99** 0.96** 0.98** 0.97** 0.98** 0.99** -0.96** 0.99**ΨP 0.98** 0.88* 0.85 NS 0.87 NS 0.84 NS 0.99** 0.93* -0.97** 0.92* 0.95*

GCPC 0.95* 0.97** 0.95* 0.95* 0.94* 0.96** 0.96** -0.93* 0.97** 0.97** 0.95** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

177

Table 4.3.15 (c): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2016-17

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.97**GY 0.92* 0.94*BY 0.97** 0.98** 0.98**HI 0.86NS 0.89* 0.99** 0.94*SY 0.94* 0.92* 0.78NS 0.88* 0.68NS

PH 0.93* 0.97** 0.99** 0.98** 0.97** 0.81NS

SL 0.83NS 0.84NS 0.73NS 0.83NS 0.64NS 0.93* 0.74NS

SPS 0.64NS 0.73NS 0.88* 0.78NS 0.93* 0.41NS 0.86NS 0.39NS

GFR 0.89* 0.95** 0.88* 0.90* 0.85NS 0.81NS 0.94* 0.39NS 0.75NS

GFD 0.93* 0.96** 0.98** 0.97** 0.96** 0.80NS 0.99** 0.67NS 0.86NS 0.95*Chl a 0.98** 0.94* 0.95* 0.97** 0.91* 0.87NS 0.94* 0.76NS 0.71NS 0.85NS 0.94*Chl b 0.99** 0.98** 0.95* 0.99** 0.92* 0.90* 0.97** 0.79NS 0.73NS 0.91* 0.96** 0.99**SOD 0.92* 0.83NS 0.89* 0.90* 0.86NS 0.78NS 0.85NS 0.71NS 0.65NS 0.68NS 0.97** 0.96** 0.91*POD 0.88* 0.82NS 0.93* 0.90* 0.93* 0.69NS 0.89* 0.61NS 0.65NS 0.72NS 0.88* 0.95* 0.91*CAT 0.83NS 0.76NS 0.91* 0.86NS 0.92* 0.62NS 0.85NS 0.56NS 0.79NS 0.65NS 0.84NS 0.91* 0.91*TPC 0.87NS 0.81NS 0.93* 0.90* 0.93* 0.69NS 0.88* 0.62NS 0.79NS 0.70NS 0.87NS 0.95* 0.90*LP 0.87NS 0.80NS 0.91* 0.89* 0.91* 0.69NS 0.86NS 0.62NS 0.78NS 0.68NS 0.85NS 0.94* 0.90*GB 0.84NS 0.74NS 0.85NS 0.89* 0.84NS 0.68NS 0.97** 0.62NS 0.65NS 0.58NS 0.77NS 0.91* 0.85NS

TSP 0.93* 0.84NS 0.88* 0.84NS 0.84NS 0.82NS 0.84NS 0.76NS 0.61NS 0.68NS 0.83NS 0.96** 0.92*MDA - 0.93* - 0.89* - 0.95* - 0.96* - 0.93* - 0.81NS - 0.92NS - 0.77NS - 0.75NS - 0.76NS - 0.90* - 0.97** - 0.95*

ΨS 0.90* 0.90* 0.99** 0.96** 0.98** 0.74NS 0.96* 0.69NS 0.87NS 0.81NS 0.95* 0.95* 0.94*ΨW 0.92* 0.91* 0.99** 0.97** 0.97** 0.77NS 0.96* 0.73NS 0.84NS 0.81NS 0.94* 0.96** 0.95*ΨP 0.95* 0.91* 0.93* 0.96** 0.89* 0.88* 0.91* 0.85NS 0.67NS 0.76NS 0.89* 0.97** 0.96*

GCPC 0.90* 0.85NS 0.95* 0.93* 0.95* 0.72NS 0.91* 0.64NS 0.80NS 0.76NS 0.91* 0.96** 0.93** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

178

Table 4.3.15 (d): Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2016-17


POD 0.97**CAT 0.96** 0.99**TPC 0.98** 0.95* 0.99**LP 0.98** 0.99** 0.99** 0.99**GB 0.99** 0.97** 0.98** 0.98** 0.99**TSP 0.99** 0.95* 0.93* 0.96* - 0.98** 0.97*

MDA - 0.98** - 0.98** - 0.96** - 0.98** - 0.98** - 0.96** 0.98**ΨS 0.93* 0.97** 0.96** 0.97** 0.96** 0.92* 0.92* - 0.98**ΨW 0.95* 0.97** 0.96* 0.97** 0.96** 0.93* 0.94* - 0.99** 0.99**ΨP 0.97** 0.93* 0.91* 0.94* 0.94* 0.93* 0.98** - 0.99** 0.95* 0.96**

GCPC 0.97** 0.99** 0.99** 0.99** 0.99** 0.96** 0.95* - 0.98** 0.98** 0.98** 0.94** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

179

Table 4.3.16 (a): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2015-16

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.94*GY 0.94* 0.91*BY 0.92* 0.87NS 0.99**HI 0.94* 0.93* 0.95* 0.90*SY 0.88* 0.82NS 0.96** 0.99** 0.84NS

PH 0.89* 0.82NS 0.84NS 0.88* 0.70NS 0.90*SL 0.94* 0.98** 0.94* 0.93* 0.92* 0.90* 0.89*SPS 0.97** 0.98** 0.96** 0.94* 0.94* 0.91* 0.89* 0.99**GFR 0.89* 0.83NS 0.90* 0.94* 0.76NS 0.96* 0.99** 0.90* 0.91NS

GFD 0.73NS 0.76NS 0.58NS 0.60NS 0.72NS 0.60NS 0.87NS 0.78NS 0.76NS 0.79NS

Chl a 0.95* 0.99** 0.93* 0.90* 0.92* 0.87NS 0.88* 0.99** 0.99* 0.88* 0.80NS

Chl b 0.95* 0.96** 0.94* 0.94* 0.89* 0.92* 0.94* 0.99** 0.99* 0.94* 0.81NS 0.99**SOD 0.95* 0.92* 0.98** 0.94* 0.90* 0.88* 0.76NS 0.91* 0.95* 0.81NS 0.53NS 0.92* 0.90*

POD 0.92* 0.97** 0.93* 0.87NS 0.96** 0.80NS 0.72NS 0.94* 0.95* 0.75NS 0.59NS 0.95* 0.91*CAT 0.86NS 0.87NS 0.92* 0.86NS 0.98** 0.79NS 0.60NS 0.85NS 0.88* 0.67NS 0.36NS 0.84NS 0.81NS

TPC 0.95* 0.92* 0.99** 0.95* 0.99** 0.90* 0.78NS 0.93* 0.96* 0.83NS 0.54NS 0.92* 0.91*LP 0.79NS 0.90* 0.85NS 0.78NS 0.99** 0.70NS 0.53NS 0.85NS 0.86NS 0.59NS 0.41NS 0.86NS 0.79NS

GB 0.96** 0.92* 0.99** 0.97** 0.94* 0.93* 0.82NS 0.93* 0.96** 0.87NS 0.57NS 0.93* 0.93*TSP 0.87NS 0.83NS 0.90* 0.83NS 0.97** 0.75NS 0.58NS 0.80NS 0.85NS 0.59NS 0.35NS 0.81NS 0.77NS

MDA -0.92* -0.82NS -0.93* - 0.88* - 0.96** - 0.83NS -0.70NS -0.80NS -0.87NS -0.75NS -0.43NS -0.82NS -0.81NS

ΨS 0.97** 0.90* 0.98** 0.95* 0.97** 0.90* 0.81NS 0.90* 0.94* 0.85NS 0.56NS 0.90* 0.90*ΨW 0.97** 0.90* 0.98** 0.95* 0.97** 0.91* 0.82NS 0.91* 0.95* 0.82NS 0.57NS 0.91* 0.91*ΨP 0.97** 0.89* 0.99** 0.97** 0.94* 0.95* 0.87NS 0.92* 0.95* 0.91* 0.62NS 0.91* 0.93*

GCPC 0.90* 0.88* 0.96** 0.92* 0.99** 0.86NS 0.68NS 0.87 NS 0.91* 0.75NS 0.42NS 0.87NS 0.85NS

* = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

180

Table 4.3.16 (b): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2015-16


POD 0.97**CAT 0.97** 0.96**TPC 0.99** 0.97** 0.97**LP 0.90* 0.96** 0.96* 0.90*GB 0.99** 0.95* 0.95* 0.99** 0.87NS

TSP 0.97** 0.94* 0.99** 0.96* 0.91* 0.93*MDA -0.97** -0.90* -0.94* -0.96** -0.81NS -0.96* -0.97**

ΨS 0.99** 0.94* 0.94* 0.99** 0.84NS 0.99** 0.95* -0.98**ΨW 0.99** 0.93* 0.93* 0.99* 0.83NS 0.99** 0.94* -0.98** 0.99**ΨP 0.97** 0.91* 0.89* 0.97** 0.79NS 0.99** 0.89* -0.95* 0.99** 0.99**

GCPC 0.99** 0.95* 0.99** 0.99** 0.92* 0.98** 0.98** -0.96** 0.97** 0.97** 0.94** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

181

Table 4.3.16 (c): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2016-17

Parameters GPS TGW GY BY HI SY PH SL SPS GFR GFD Chl a Chl bTGW 0.83NS 1.00GY 0.96* 0.82NS

BY 0.96* 0.82NS 0.99**HI 0.95* 0.82NS 0.99** 0.99**SY 0.96** 0.82NS 0.99** 0.99** 0.99**PH 0.96** 0.72NS 0.97** 0.98** 0.97** 0.98**SL 0.94* 0.88* 0.87NS 0.87NS 0.88* 0.88* 0.84NS

SPS 0.83NS 0.87NS 0.94* 0.94* 0.94** 0.93* 0.84NS 0.81NS

GFR 0.80NS 0.74NS 0.94* 0.94* 0.94** 0.93* 0.87NS 0.69NS 0.97**GFD 0.65NS 0.48NS 0.68NS 0.67NS 0.78NS 0.67NS 0.65NS 0.77NS 0.65NS 0.59NS

Chl a 0.91* 0.85NS 0.92* 0.91* 0.92* 0.91* 0.86NS 0.97** 0.89* 0.81NS 0.86NS

Chl b 0.84NS 0.88* 0.89* 0.89* 0.89* 0.88* 0.79NS 0.91* 0.94* 0.84NS 0.82NS 0.98**SOD 0.91* 0.96** 0.91* 0.91* 0.90* 0.91* 0.85NS 0.87NS 0.90* 0.83NS 0.45NS 0.85NS 0.85NS

POD 0.92* 0.97** 0.85NS 0.98** 0.85NS 0.86NS 0.80NS 0.93* 0.82NS 0.71NS 0.51NS 0.87NS 0.85NS

CAT 0.83NS 0.92* 0.76NS 0.76NS 0.74NS 0.76NS 0.71NS 0.79NS 0.71NS 0.62NS 0.23NS 0.69NS 0.67NS

TPC 0,85NS 0.80NS 0.76NS 0.76NS 0.74NS 0.76NS 0.77NS 0.74NS 0.63NS 0.59NS 0.94* 0.62NS 0.55NS

LP 0.80NS 0.99** 0.76NS 0.75NS 0.75NS 0.75NS 0.66NS 0.85NS 0.79NS 0.65NS 0.38NS 0.78NS 0.80NS

GB 0.90* 0.90* 0.83NS 0.83NS 0.82NS 0.83NS 0.80NS 0.82NS 0.75NS 0.69NS 0.30NS 0.73NS 0.69NS

TSP 0.93* 0.88* 0.86NS 0.86NS 0.85NS 0.87NS 0.86NS 0.84NS 0.76NS 0.72NS 0.99** 0.76NS 0.70NS

MDA -0.99** -0.80NS -0.98** - 0.98** - 0.98** - 0.98** -0.98** -0.92* -0.87NS -0.86NS -0.71NS -0.93* -0.87NS

ΨS 0.97** 0.88* 0.93* 0.93* 0.93* 0.94** 0.93* 0.88* 0.83NS 0.80NS 0.49NS 0.84NS 0.78NS

ΨW 0.97** 0.92* 0.93* 0.93* 0.92* 0.93** 0.90* 0.91* 0.85NS 0.79NS 0.49NS 0.86NS 0.81 NS

ΨP 0.91* 0.98** 0.87NS 0.87NS 0.86NS 0.87NS 0.80NS 0.93* 0.85NS 0.74NS 0.51NS 0.88* 0.86NS

GCPC 0.96* 0.96* 0.94* 0.93* 0.93* 0.94* 0.88* 0.95* 0.90* 0.82NS 0.61NS 0.93* 0.91** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); GPS = Grains per spike; TGW = 1000-grain weight; GY = Grain yield; BY = Biological yield; HI = Harvest index; SY = Straw yield; PH= Plant height; SL = Spike length; SPS = Spikelets per spike; GFR = Grain filling rate; GFD = Grain filling duration; Chl a = Chlorophyll a; Chl b = Chlorophyll b; SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

182

Table 4.3.16 (d): Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2016-17


POD 0.97**CAT 0.95* 0.95*TPC 0.89* 0.88* 0.95*LP 0.95* 0.97** 0.95* 0.83NS

GB 0.96** 0.95* 0.99** 0.98** 0.92*TSP 0.96* 0.94* 0.96** 0.98** 0.88* 0.99**

MDA - 0.89* - 0.87NS - 0.76NS - 0.78NS - 0.74NS - 0.84NS - 0.88*ΨS 0.96** 0.94* 0.92* 0.94* 0.86NS 0.97** 0.99** - 0.95*ΨW 0.98** 0.97** 0.94* 0.93* 0.90* 0.97** 0.98** - 0.94* 0.99**ΨP 0.98** 0.99** 0.95* 0.87NS 0.97** 0.95* 0.94* - 0.87NS 0.99** 0.97**

GCPC 0.98** 0.98** 0.90* 0.84NS 0.93* 0.93* 0.93* - 0.94* 0.96** 0.98** 0.98*** = Significant (p ≤ 0.05); ** = Significant (p ≤ 0.01); NS = Non-significant (p ≤ 0.05); SOD = Superoxide dismutase; POD = Peroxidase; CAT = Catalase; TPC = Total phenolic contents; LP = Leaf proline; GB = Glycine betaine; TSP = Total soluble proteins; MDA = Malondialdehyde; Ψ S = Osmotic potential; ΨW = Water potential; ΨP = Pressure potential; GCPC = Grain crude protein contentsn (number of pairs of observations) = 45

183

SUMMARY CHAPTER-5

The present research work was conducted to determine the thermo-sensitivity of

Pakistani wheat genotypes and alleviation of negative implications of heat on varying

terminal phenological stages through exogenous application of potassium and selenium. The

research work was comprised of two phases. In the first phase wheat genotypes were

screened for terminal thermo-sensitivity and tolerance under field conditions during 2014-15.

Heat was imposed in main plots viz. H0 = no heat stress (control) and H1 = Heat imposition

from complete emergence of spike to grain filling initiation (early milk stage) (Feekes scale =

10.50 to 11.0). Different genotypes were randomized in split plots. A medium heat tolerant

genotype was selected on the basis of recorded response variables and used in further

experimentation.

Imposition of heat stress deleteriously impacted the metabolism of all genotypes.

Antioxidants (superoxide dismutase, peroxidase, catalase and total phenolics) and osmo-

protectants (proline, glycine betaine and soluble proteins) were enhanced in genotypes ‘AAS-

2011, Chakwal-50 and Mairaj-2008’ under high temperature environment compared to ‘no

heat stress’. While, in all other genotypes biosynthesis of antioxidants and osmo-protectants

was depressed under heat compared to control. Likewise, adverse impacts of heat on spike

growth, the stay green trait, grain yield and yield components were relatively lesser in

genotypes ‘AAS-2011, Chakwal-50 and Mairaj-2008’ than other genotypes. On the basis of

recorded attributes, genotypes ‘AAS-2011, Chakwal-50 and Mairaj-2008’ depicted

terminal heat tolerance while genotypes ‘Fareed-2006’ and ‘Punjab-2011’ exhibited

medium tolerance. Genotypes AARI-2011, Galaxy-2013, Millat-2011, Pakistan-2013,

NIBGE-NIAB-1 and Kohistan-97 could not produce remarkable responses under heat

and were characterized as terminal heat susceptible on the basis of recorded

parameters.

During the 2nd year of study (2015-16), more detailed studies of heat were performed

to explicate relative damages of terminal heat on different terminal stages under field

environment. Moreover, negative implications of heat stress were alleviated through

exogenous application of varying concentrations of potassium and selenium. The second and

third experiment were comprised of heat stress in main plots viz. H0 = no heat imposition

(control); H1 = Heat imposition from complete emergence of spike to grain filling initiation

184

(early milk stage) (Feekes scale = 10.50 to 11.0)’ and H2 = Heat imposition from flowering

initiation to grain filling initiation (early milk stage) (Feekes scale = 10.5.1 to 11.0)’. In the

second experiment, subplot treatments were comprised of varying concentrations of foliar

potassium viz. control/water spray, 15, 30, 45 and 60 g L-1. While, split plot treatments in the

third experiment were different concentrations of foliar selenium viz. control/water spray, 25,

50, 75 and 100 mg L-1. Second and third experiments were repeated during 2016-17.

Heat stress either from ‘spike to grain filling’ or ‘flowering to grain filling’ adversely

affected yield and related components, biomass accumulation attributes, spike growth, stay

green, antioxidants, osmo-protectants, lipid peroxidation and quality compared to ‘no heat

stress’. While negative implications of heat were more pronouncing under ‘heat from

spike to grain filling’ compared to ‘heat from flowering to grain filling’. Varying

concentrations of foliar potassium differed significantly. Relatively more and statistically

alike grain yield, yield components, biomass accumulating attributes, spike growth attributes,

chlorophyll contents and grain crude protein contents were recorded with 45 and 60 g L -1

exogenous potassium generally. Whereas, statistically similar and relatively more

antioxidants, osmo-protectants, water relation attributes and statistically similar and relatively

lesser lipid peroxidation were recorded with 45 and 60 g L-1 under ‘no heat stress’. While,

application of 60 g L-1 exogenous potassium depicted significantly more antioxidants,

osmo-protectants, water relation attributes and significantly lesser lipid peroxidation

compared to other concentrations. Whereas, relatively poor and statistically similar

responses were observed with control/water spray and 15 g L-1 exogenous potassium for most

of instances.

Likewise, statistically alike and relatively more yield, yield components, spike

attributes, biomass accumulation, turgor potential and grain crude protein contents were

observed with 75 and 100 mg L-1 exogenous selenium compared to other concentrations.

While, application of selenium at 100 mg L-1 proved toxic for antioxidants, osmo-protectants,

lipid peroxidation, water and osmotic potential under ‘no heat stress’. Hence, application of

100 mg L-1 exogenous selenium either depicted significantly lesser or 100 mg L-1

selenium was at par with 75 mg L-1 foliar selenium under ambient conditions.

Contrarily, application of 100 mg L-1 selenium proved advantageous under both

treatments of heat stress and depicted significantly more antioxidants, osmo-

protectants, lipid peroxidation, water and osmotic potential compared to other

concentrations. While, comparatively poor and statistically similar responses were observed

185

with control/water spray and 25 mg L-1 exogenous selenium. Moreover, importance of

availability of potassium and selenium was enhanced for almost all the studied

attributes under heat stress conditions compared to ‘no heat stress’. Thus, significant

associations of biochemical attributes were observed with growth, yield and other

morphological attributes.

CONCLUSION

Not surprisingly, the imposition of heat stress adversely affected biochemical and

morphological attributes of all genotypes tested. While, genotypes ‘AAS-2011, Chakwal-50

and Mairaj-2008’ depicted heat tolerance; ‘Fareed-2006’ and ‘Punjab-2011’ depicted

medium tolerance and genotypes ‘AARI-2011, Galaxy-2013, Millat-2011, Pakistan-2013,

NIBGE-NIAB-1 and Kohistan-97’ were susceptible to terminal heat. Imposition of ‘heat

from spike to grain filling’ and ‘heat from flowering to grain filling’ significantly affected

yield components, grain yield, spike growth attributes, antioxidants, osmo-protectants, water

relations and quality attributes compared to ‘no heat stress’. Whereas, imposition of ‘heat

stress from spike to grain filling’ was more damaging compared to ‘heat from flowering to

grain filling’. Under ‘no heat stress’ application of exogenous potassium at 45 g L-1 or

selenium at 75 mg L-1 depicted more promising results. Whereas, application of potassium at

60 g L-1 or selenium at 100 mg L-1 depicted more promising morphological and biochemical

responses under ‘heat from spike to grain filling and ‘heat from flowering to grain filling’.

Moreover, foliar application of potassium or selenium proved more advantageous under heat

imposition compared to no heat stress. In addition, biochemical attributes (such as superoxide

dismutase, catalase, peroxidase, chlorophyll contents, proline, glycine betaine, phenolics and

malondialdehyde) significantly modulated changes in growth, yield components and grain

yield under varying temperatures. This, significant association of biochemical attributes with

morphological attributes can be used as futuristic roadmap to improve heat resistance in

wheat. Likewise, improving the capability of wheat to uptake potassium and selenium can

also prove potent tool as stratagem to improve terminal heat tolerance.

FUTURE PERSPECTIVES

1. Potassium and selenium modulated regulations in stomatal conductance, glutathione

peroxidase, ascorbate peroxidase and reducing powers (NADPH) should be explored in

future.

2. Heat stress and exogenous potassium and selenium triggered alterations in fertility of

pollens and ovule development should constitute the bases for futuristic roadmaps.

186

3. It is suggested to conduct experiments on thermo-sensitivity of genotypes over years and

locations.

4. Experiments on potassium and selenium modulated alleviations in adversities of heat stress

should be conducted using multiple genotypes and on multiple locations in future to

determine heat alleviation potential of these nutrients.

5. Heat stress is often accompanied by physiological drought under field conditions. Hence, it

is suggested to conduct experiments on combined imposition of drought and heat stress in

wheat crop in future.

187

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