ARTICLE

Elevated carbon dioxide and temperature effects on rice yield, leafgreenness, and phenological stages duration

Nuno Figueiredo • Corina Carranca • Henrique Trindade • Jose Pereira •

Piebiep Goufo • Joao Coutinho • Paula Marques • Rosa Maricato •

Amarilis de Varennes

Received: 30 January 2014 / Revised: 11 May 2014 / Accepted: 23 May 2014

� The International Society of Paddy and Water Environment Engineering and Springer Japan 2014

Abstract The present field experiment was conducted

during two consecutive cropping seasons in central Portugal

to study the effects of simultaneous elevation of carbon

dioxide concentration ([CO2]) (550 lmol mol-1) and air

temperature (?2–3 �C) on japonica rice (Oryza sativa L.

‘‘Ariete’’) yield, crop duration, and SPAD-values across the

seasons compared with the open-field condition. Open-top

chambers were used in the field to assess the effect of ele-

vated air temperature alone or the combined effect of ele-

vated air temperature and atmospheric [CO2]. Open-field

condition was assessed with randomized plots under

ambient air temperature and actual atmospheric [CO2]

(average 382 lmol mol-1). Results obtained showed that

the rice ‘‘Ariete’’ had a moderate high yielding under open-

field condition, but was susceptible to air temperature rise of

?2–3 �C under controlled conditions resulting in reduction

of grain yield. The combined increase of atmospheric [CO2]

with elevated air temperature compensated for the negative

effect of temperature rise alone and crop yield was higher

than in the open-field. SPAD-readings at reproductive stage

explained by more than 60 % variation the straw dry matter,

but this finding requires further studies for consolidation. It

can be concluded that potential increase in air temperature

may limit rice yield in the near future under Mediterranean

areas where climate change scenario poses a serious threat,

but long term field experiments are required.

Keywords Maturation duration � Modeling � Open-field �Open-top chamber � SPAD-reading

Introduction

Rice (Oryza sativa L.) is one of the most important food

crops in the world and a staple for more than half of the

N. Figueiredo � C. Carranca (&) � R. Maricato

Instituto Nacional de Investigacao Agraria e Veterinaria, Quinta

do Marques, Av. Republica, Nova Oeiras, 2784-505 Oeiras,

Portugal

e-mail: c.carranca.ean@clix.pt; corina.carranca@iniav.pt

C. Carranca � A. de Varennes

Biosystems Engineering Center (CEER) ISA/UL, Lisbon,

Portugal

C. Carranca

lnstituto de Ciencias Agrarias e Ambientais Mediterranicas

(ICAAM), Univ. Evora, Nucleo da Mitra, Apartado,

947002 Evora, Portugal

H. Trindade � J. Pereira � P. Goufo

Centre for the Research and Technology of Agro-Environmental

and Biological Sciences, CITAB, University of Tras-os-Montes

and Alto Douro, UTAD, Quinta de Prados, 5000-801 Vila Real,

Portugal

J. Pereira

Polytechnic Institute of Viseu, IPV, Agricultural Polytechnic

School of Viseu, ESAV, Quinta da Alagoa, 3500-606 Viseu,

Portugal

J. Coutinho

Chemistry Centre, University of Tras-os-Montes and Alto

Douro, UTAD, Quinta de Prados, 5000-801 Vila Real, Portugal

P. Marques

Centro Operativo e Tecnologico do Arroz, Salvaterra de Magos,

Portugal

123

Paddy Water Environ

DOI 10.1007/s10333-014-0447-x

global population. It has a wide physiological adaptability

and is grown successfully in tropical, subtropical, and

temperate regions. The optimum temperature for maximum

rice photosynthesis is 25–30 �C for daytime maxima and

20 �C for the nighttime maxima (Sreenivasan 1985; IRRI

(Int. Rice Res. Inst.) 1997). Higher yields are obtained in

temperate countries than in tropical areas; the average yield

of paddy rice in European countries is 5 t ha-1 and that of

Asia 4 t ha-1, but the maximum potential yield of modern

varieties is 13 t ha-1 in tropics and 15 t ha-1 in temperate

regions (Tran 1997; Biswas and Ntanos 2002). Rice pro-

ductivity does not only vary amongst countries but also

within the same country based on the different agro-eco-

logical zones and the production system used (Biswas and

Ntanos 2002). The European Union (EU) has a production

of rough rice of 3 million tons per year and ranks 17th

(0.5 %) among main world producers, whereas it ranks

only 19th in terms of consumption (3.5 million tons per

year). In Europe, more than 140 rice cultivars have been

produced in France, Spain, Italy, Greece, and Portugal

(Confalonieri and Bocchi 2005). In the EU, Portugal is the

first per capita consumer of rice and the fourth producer

(6 t ha-1, 28,000 ha) contributing to 5.3 % of the total

European production (Figueiredo et al. 2013). Rice varie-

ties mainly produced in Europe are japonica and indica.

The first generally refers to some traditional varieties

selected before the 2nd World War, but also to some

varieties selected between the 1970s and the 1990s

(semidwarf) and high yielding. These varieties require

lower temperature for ripening than indica varieties

(Krishnan et al. 2011).

Crop duration is an important trait in rice and other

cereals, in particular because it correlates positively with

yield potential (De Raıssac et al. 2004). The growth

duration of a rice crop varies from 3 to 8 months

depending on the cultivar and environmental conditions.

Most japonica type varieties are medium and medium-

late cultivars (cycle longer than 150 days) with only few

early varieties (Krishnan et al. 2011). Akita (1989)

observed in IRRI rice varieties an increasing yield when

crop duration increased from 95 to 110 days, with a

maximum constant yield of 9 t ha-1 when the season

was longer than 110 days. In rice, most differences

among short-, medium- and late-term varieties are due to

the duration of the vegetative phase (De Raıssac et al.

2004). Variations in environmental factors can have

influence on the duration of this phase, even when

expressed in thermal time; sensitivity to photoperiod is

one classical example.

Atmospheric carbon dioxide (CO2) is a substrate for plant

photosynthesis. The effect of rising atmospheric CO2 con-

centration [CO2] on photosynthesis and productivity is

reported to be more pronounced in C3 plants such as rice

(Wassmann et al. 2009). High [CO2] reduces the stomata

conductance, resulting in reduced transpiration, and

increased net primary production (Haque et al. 2006; Ains-

worth 2008; Cheng et al. 2009). IPCC (2007) estimated that

atmospheric [CO2] has risen from approximately

280 lmol mol-1 in pre-industrial times to 380 lmol mol-1,

and will reach 550 lmol mol-1 by 2,050. In the absence of

strict control of emissions, the atmospheric [CO2] is likely to

reach 730–1,020 lmol mol-1 by 2,100. Since CO2 and

other greenhouse gases (GHGs) alter physical radiation

properties and the energy balance of the atmosphere, they

influence the global temperature regime. Therefore, simul-

taneously with the increase in the concentration of GHGs,

the global average air temperature is projected to increase

between 1.8 and 4.0 �C by the end of the present century

relative to the mean value for 1980–1999. The increase in

atmospheric [CO2] and projections of further increases in

global air temperature stimulated studies on the effects of

climatic variables on important food crops. These are par-

ticularly relevant for Mediterranean areas where climate

change has been projected with extreme events (Figueiredo

et al. 2013). To date, most studies have focused only on rice

responses to CO2 enrichment photosynthesis, water rela-

tions, phenology, organ formation, dry matter (DM) pro-

duction and distribution, carbon (C) and nitrogen

(N) metabolism, as well as grain yield and its components

(Wang et al. 2011).

The ability of rice plants to tolerate higher temperatures

depends on different thermo tolerance mechanisms at

biochemical and metabolic levels, membrane stability,

synthesis of heat shock proteins, and photosynthetic

activities (Krishnan et al. 2011). Mohammed and Tarpley

(2009) and Madan et al. (2012) reported that both high day

and high night temperatures have negative effects on rice

spikelet fertility and yields. High day temperatures beyond

a critical threshold during sensitive development stages

like gametogenesis and flowering lead to low seed-set.

Mohammed and Tarpley (2009) attributed the year-to-year

variation in rice grain yield and quality to the nighttime

temperature increase during the critical stages of devel-

opment. Although elevated [CO2] per se increases pro-

ductivity of C3 crops such as rice, the increasing frequency

and intensity of short-duration high temperature events

([33 �C) may pose a serious threat to agricultural pro-

duction (Madan et al. 2012). Most research on the indi-

vidual effects of CO2 and temperature alone effects have

been restricted to crop yield and phenological parameters

of plants grown in controlled environments. Data on

interactive effects of CO2 and temperature rise under field

conditions are rare.

The proper use of N fertilizers can also markedly

increase rice yield and improve quality. Crop N is closely

associated with leaf chlorophyll, since a great amount of

Paddy Water Environ

123

leaf N is contained in chlorophyll molecules. Leaf N is

related with grain yield in rice (Esfahani et al. 2008).

Nitrogen contributes to carbohydrate accumulation in cul-

ms and leaf sheaths during the pre-heading stage and in the

grain during the ripening stage (Swain and Sandip 2010;

Goufo et al. 2014a). Leaf N concentration is thus a sensi-

tive indicator for the dynamic changes in plant N, and N

monitoring during the growth period is essential to achieve

an efficient N fertilizer management and higher grain yield.

Readings of cereal leaves using a chlorophyll-meter read-

ing may provide information about the plant N status. The

Soil Plant Analysis Development (SPAD) meter is an

example of a simple, rapid, non-destructive, and portable

diagnostic tool to measure the greenness or relative chlo-

rophyll content of leaves (Gholizadeh et al. 2009; Neto

et al. 2011). For a particular plant species, a higher SPAD-

value usually indicates a healthier plant. SPAD-values are

thus appropriate to predict whether response to additional

topdress N is expected (Piekielek et al. 2008). Little

information on SPAD-values for rice across the season is

available, in particular in response to environmental

stresses.

The succession of rice development stages (phenology)

depends on air and floodwater temperature and on pho-

toperiod (day-length) (Krishnan et al. 2011). The different

phenological events differ in their sensitivity to high

temperature, depending on plant species and genotype.

When rice is exposed to high temperatures during the

vegetative stage, individual plant height, tiller number,

and DM may be considerably reduced (Krishnan et al.

2011). Rice can grow with daytime temperatures as high

as 40 �C during the vegetative stage, whereas floral

development is very sensitive to high temperatures.

Therefore, temperature may affect the growth duration of

the rice crop to a great extent. Only one reference

(Bhattacharyya et al. 2013) was found to report the effects

of the interaction between temperature and CO2 elevation

on phenological stages, but no results were given for the

effects on crop duration.

In the present study, a japonica rice variety (Oryza

sativa L. cv. Ariete) was grown in open-field under nat-

ural sunlight, temperature and [CO2], but also in open-top

chambers (OTCs) to increase the temperature (by the

OTC effect) and the [CO2]. Rice management in the

OTCs was the same and simultaneous with the open-field

cultivation. Therefore, the overall objectives of this work

were to assess the simultaneous effects of elevated

[CO2] ? temperature and temperature per se in controlled

environments, in comparison with the natural ambient

open-field condition on i) rice yield and crop duration, ii)

leaf greenness across the season, and iii) SPAD-values at

specific phenological stage in order to predict the crop

yield.

Materials and methods

Field experiment

A field experiment with japonica rice variety (Oryza sativa

L. cv. Ariete) was conducted for two consecutive seasons

(2011 and 2012) at Salvaterra de Magos (Tagus Valley,

central Portugal; latitude: 39�2.20150N, longitude:

8�44.2570W, elevation: 18 m above sea level). This area is

the main region for rice production in Portugal. The

experimental design consisted of three treatments arranged

in a randomized complete block design and three repli-

cates, in a total of nine blocks (Fig. 1a). Each block was

4.0 m 9 4.0 m, 4.0 m apart from each other. Treatments

were as follows: elevated [CO2] ? temperature, elevated

temperature (OTC effect), and the unchambered (open-

field) control plots (around 375 lmol CO2 mol-1 air). To

change the climatic variables, six large open-top chambers

(OTC = 4 m wide 9 3 m height 9 2 m open-top diame-

ter, 30 tilt), covered with a polyethylene film (1-mm

thickness and 75 % light transmittance, provided by Es-

tufasMinho, S.A., Fao, Portugal) (Fig. 1b), were placed on

a previous prepared (chisel and laser) lowland for paddy

conditions: three OTCs were for elevated [CO2] ? tem-

perature and three for the temperature rise (OTC effect).

Details on the construction and operation of OTCs have

been provided by Pereira et al. (2013). In the three OTCs

for CO2 enrichment, a system using pure industrial CO2

injection was installed to fumigate CO2 during the day-

night time (24 h per day). It operated from May to October

2011 and 2012 in order to have a concentration of

550 lmol CO2 mol-1, which represented the expected

[CO2] range by the middle of the 21st century (IPCC 2007;

Goufo et al. 2014b). Several sensors connected to a data-

logger (DL2, Delta-T Devices, Cambridge, UK) were

installed inside each OTC (Fig. 1c) and outside the

chamber to monitor the climatic parameters: for [CO2], a

probe (GMP222, Vaisalia, Finland) was used; and for

temperature and humidity, a sensor (RHT2y, Delta-T

Devices, Cambridge, UK) was used. The air in each OTC

was circulated by two fans (EDM-100 �C 12 V, Soler &

Palau Ltd, Portugal). The CO2 fumigation system operated

in on–off mode control with a continuous sampling of CO2

level. When CO2 injection was necessary, the data-logger

acted over an electronic valve (7321B 12 V, Parker

Hannitin SpA, Gessate, Italy) linked to a pure industrial

CO2 tank through a high-density polyethylene tube. Inside

each elevated-[CO2] OTC, the CO2 distribution tube had

several emission holes and was located around the side-

walls and kept at the crop canopy level throughout the

season. Carbon dioxide concentration was continuously

monitored with an infrared gas transmitter (GMP111,

Vaisalia, Finland) linked to a real-time data acquisition and

Paddy Water Environ

123

a control system to maintain the concentration around the

target level. All data were collected and/or controlled with

a sampling interval of 30 s and storage time of 10 min

(Pereira et al. 2013; Goufo et al. 2014d).

Rice ‘Ariete’ was direct seeded in the open-field and

OTCs on 27 May, 2011 and 23 May, 2012 at a rate of

200 kg (dry seeds) ha-1 and cultivated under intermittent

water logging regime. In the OTC-treatments, rice was

cultivated as in the open-field, but was maintained at the

enriched [CO2] (552 ± 98.4 and 547 ± 73.0 lmol CO2

mol-1 air, respectively, in 2011 and 2012) and at the

ambient [CO2] (388 ± 27.2 and 375 ± 46.0 lmol CO2

mol-1 air, respectively, in 2011 and 2012). Temperature

was elevated in the six OTCs by the OTC effect (Fig. 2a, b).

The Anthropic soil (IUSS Working Group 2006) was

representative for rice production in Portugal. It had a clay

texture (17, 28 and 55 % of sand, silt and clay, respec-

tively) in the 0–60-cm layer. In the surface (0–20-cm

layer), the bulk density was 1.1 g cm-3, the pH(H2O)was

5.9, the cationic exchange capacity was 22.7 cmol(?) kg-1,

and the content of organic C and total N was 24 and 2.4 g1

kg-1, respectively. Methods used for the evaluation of the

physic-chemical characteristics of the soil are those rou-

tinely used in the Instituto Nacional de Investigacao Ag-

raria e Veterinaria.

All the experimental plots received the same rates and

types of fertilizers. A NP mineral fertilizer (20–20–0) was

mechanically broadcast at a depth of 20 cm in May as a basal

dressing preceding crop seeding at a rate of 60 kg NH4-

N ha-1, and a sulfamid (40 % N) was manually applied on

the floodwater at a rate of 60 kg N ha-1 as topdressing at

tillering, in July. No potassium was added to the soil in both

years as the soil was rich, whereas 60 kg ha-1 of phosphorus

(P) was incorporated into the soil as part of the basal dressing

in both seasons. After rice seeding, the water regime was

intermittent, i.e., flooding—midseason drainage (for plant

Fig. 1 A partial aspect of experimental site (a) installed in a rice field

at Salvaterra de Magos (central Portugal); an aspect of the octagonal

chamber with OTC (b); internal probes for CO2 (left, down) and air

temperature and humidity (right), a fan for air circulation (left, top),

and a closed PCV chamber for measurement of GHG emissions inside

the OTC (c)

Paddy Water Environ

123

rooting, about 1 week after rice germination, and twice for

weed control during a few days at the tillering)—refloo-

ding—drainage (3 weeks before crop harvest) (Fig. 3).

Floodwater height varied from 5 to 20 cm depending on

plant growth, in the open-field as well as the OTCs, as it

passed through holes located at the bottom of the polyeth-

ylene film surrounding the OTCs. Irrigation water had an

average pH 8.0, electric conductivity of 0.7 dS m-1, low

levels of mineral N, high level of chloride content (71 mg

Cl- l-1), 30–48 mg Ca2? l-1, 51–87 mg Na? l-1, and

7–10 mg K? l-1. ‘‘Ariete’’ is a cultivar moderately sensitive

to salinity and should not be negatively affected by salts

present in the water. The experiments were kept free from

weeds using herbicides. The cultural practices used in the

experiment for the two consecutive seasons were similar to

the typical agricultural management used by Portuguese rice

farmers for the last 14 years and have been thoroughly

described by Goufo et al. (2014c).

The climate of the region is Mediterranean-type. In the

open-field, daily meteorological data (rainfall, maximum,

minimum, and mean air temperature, solar radiation and

wind speed) were collected with an automatic weather

Fig. 2 Seasonal daily air temperature (maximum, average, minimum) in the open-field (O–F) and inside the chambers OTC during the rice

growth in 2011 (a) and 2012 (b); mean monthly temperature and accumulated rainfall during the growth seasons in 2011 and 2012 (c)

Paddy Water Environ

123

station placed near the site of the study. Mean ambient air

temperature during both growth cycles (2011 and 2012)

varied from 18 to 19 �C in May to 20–21 �C in August

(Fig. 2c). In both seasons in general, maximum air tem-

perature did not go over 34 �C (Fig. 2a, b). Minimum

rainfall occurred in June–July (\10 mm) and the maximum

in October (90–100 mm) for both years (Fig. 2c). The wind

speed in 2011 and 2012 ranged from 3.8 to 8.1 m s-1.

Global solar radiation did not vary in the two seasons and

averaged 5,787 W m-2. Inside the OTCs, the mean tem-

perature was ?2 ± 1.1 �C and ?3 ± 1.8 �C above the

open-field, respectively, in 2011 and 2012. In summer

2012, a higher number of days greater than 34 �C inside

the OTCs, including above 38 �C was registered compared

with the open-field this year and with the OTCs in

2011(Fig. 2a, b).

Across both seasons, SPAD-502 (Minolta, Japan) read-

ings were recorded in the afternoon (15–16 h) at different

phenological stages (4th leaf, tillering, internode elongation,

flowering, grain fill, and maturity) (Fig. 3) in each plot using

the youngest fully expanded Y-leaf of rice plants (Dober-

mann and Fairhurst 2000), also known as flag leaf in the

reproductive stage. One reading corresponded to the average

of ten measurements. At harvest (19 October 2011 and 10

October 2012), corresponding to 149 and 140 days after

sowing (DAS), all plants were removed from the center of

plots (corresponding to an average of 175 plants ha-1). Plant

material was separated into straw and grain, dried (65 �C for

48 h) and weighed to estimate yield (kg DM ha-1).

Statistical analysis

Analysis of variance (ANOVA) was performed by the

General Linear Model using the STATISTICA 6.0

software to evaluate effects of years, treatments [open-

field, and elevated [CO2] ? temperature and temperature

(OTC effect)], and growth phases (DAS) on DM yield

and SPAD-values. Means separation was determined for

significant differences by the Bonferroni’s test, at

P \ 0.05. Polynomial equations were established for

SPAD-values across seasons (P \ 0.05) depending on the

treatment. Linear regression equations to predict crop

yield were fitted for significant (P \ 0.05) overall SPAD-

values for both seasons at specific phenologic stage and

treatment.

Results

Crop productivity

Years (2011 and 2012) and treatments (open-field, tem-

perature and [CO2] ? temperature elevation) significantly

affected yield (Table 1). A higher grain yield (11 t ha-1)

was observed in 2011 (Fig. 4) due to increased [CO2]

(552 ± 98 lmol mol-1 air), higher than the yield

(7 t ha-1) obtained in plants grown in the open-field this

year (388 ± 27 lmol CO2 mol-1 air). The lowest yield

(5 t ha-1) was measured in 2012 in the OTCs for

[CO2] ? temperature elevation (Fig. 4). This year, the

maximum temperature in summer (August and September)

was above 34 �C (Fig. 2b) and caused a 29 % decrease in

weight compared with the average grain yield in the open-

field for both seasons (7 t ha-1), where the maximum daily

temperature was always below 34 �C (Fig. 2a, b). As to

straw DM yield, no significant differences were observed

between treatments in 2011 and in the open-field in 2012

(6 t ha-1), but straw DM was reduced 50 % due to the

Fig. 3 Cultural practices and

date of sampling in seasonal rice

‘Ariete’ growths (2011 and

2012), at Salvaterra de Magos

(central Portugal) (Pereira et al.

2013)

Paddy Water Environ

123

increase in temperature by the OTC effect (Fig. 2b) in 2012

(3 t ha-1) (Fig. 4).

Leaf greenness and duration of phenological stages

Years (2011 and 2012), treatments (open-field, temperature

and [CO2] ? temperature elevation) and dates of sampling

(DAS) significantly affected leaf greenness given by

SPAD-values taken in the youngest fully expanded Y-leaf

across both seasons (Table 2). In general, the interaction of

tested factors also affected the SPAD-readings. The overall

SPAD-value in 2011 (41) was significantly higher than in

2012 (SPAD = 40). Considering the mean effect of years

and sampling dates, a significantly lower SPAD-value was

measured in the open-field (SPAD = 37) compared with

the OTCs (SPAD = 42), where SPAD-values did not differ

from each other.

Changes in the relative amount of chlorophyll over time,

measured indirectly by the SPAD technique were also

observed (Table 2; Fig. 5A). Greater SPAD-values were

observed at the flowering phase (68-77 DAS) for all

treatments in response to topdressing at tillering and

the rise of temperature and [CO2] ? temperature

(SPAD = 49). Under the open-field, SPAD-values differed

in both seasons, with lower values in 2012. Lower SPAD-

values in 2012 in the open-field were probably a conse-

quence of a lower ambient [CO2] (\350 lmol mol-1 of

air) from middle July till middle September and/or simul-

taneous higher maximum daily temperatures ([34 �C)

during this period. In 2011, the ambient [CO2] was

Year 2011 Year 2012

Straw

0

2000

4000

6000

8000

10000

12000

14000

16000

Dry

mat

ter

(kg

DM

ha-1

)

GrainOpen-field CO2+Temperature Temperature Open-field CO2+Temperature Temperature

Fig. 4 DM yield by rice ‘Ariete’ cultivated in clay loam soil at Salvaterra de Magos (central Portugal) in response to the interaction effect of

years and treatments (open-field, and temperature and CO2 ? temperature elevation). (n = 36; vertical bars denote 0.95 confidence intervals)

Table 2 ANOVA for SPAD-values in leaves of japonica rice ‘Ari-

ete’ cultivated in clay loam soil at Salvaterra de Magos (central

Portugal)

ANOVA F value P

Year (Y) 3.9 *

Treatment (T) 184.1 ***

Time of sampling (S) 202.1 ***

Y 9 T 43.3 ***

Y 9 S 15.9 ***

T 9 S 10.8 ***

Y 9 T 9 S 4.1 ***

Year = 2011, 2012; Treatments = open-field (control), and temper-

ature and CO2 ? temperature elevation; time of sampling = days

after sowing (DAS); P Probability value, *, *** F-values significant

for P \ 0.05 and P \ 0.001, respectively according to the Bonfer-

roni’s test

Table 1 ANOVA results for DM yield of japonica rice ‘Ariete’

cultivated in clay loam soil at Salvaterra de Magos (central Portugal)

for two continuous seasons (2001 and 2012)

ANOVA F value P

Year (Y) 86.4 ***

Treatment (T) 11.6 ***

Plant organ (P) 41.8 ***

Y 9 T 31.6 ***

Y 9 P – ns

T 9 P 5.0 *

Y 9 T 9 P 6.0 **

Year = 2011, 2012; treatment = open-field (control), and tempera-

ture and CO2 ? temperature elevation; plant organ = straw, grain;

P Probability value, ns Data not shown, *, **, *** F-values non-

significant (P C 0.05), and significant for P \ 0.05, P \ 0.01, and

P \ 0.001, respectively according to the Bonferroni’s test

Paddy Water Environ

123

maintained in the range of 361–415 lmol CO2 mol-1 air

along the season, and maximum daily temperatures were

lower than 34 �C (Fig. 2a). For both seasons, the lowest

SPAD-values were measured at the 4th leaf stage (39–42

DAS) and at maturity (103–112 DAS).

Pooling SPAD-data recorded for both seasons in the open-

field, a significant polynomial regression equation was fitted

(SPAD = 16 ? 0.629x-0.004x2, x = DAS, n = 8,

R2 = 0.75, P = 0.03). In the fitted model (Fig. 5Ba), SPAD-

readings increased from 35 at the 4th leaf stage (40 DAS) to

about 40 at 75 DAS (flowering), and decreased thereafter to

30 at maturity (123 DAS). A second polynomial regression

equation was adjusted for pooled SPAD-measurements in

‘Ariete’ cultivated for both years in the OTCs ([CO2] ?

temperature and temperature elevation): SPAD =

-0.37 ? 1.178x-0.007x2, x = DAS, n = 30, R2 = 0.64,

P \ 0.001. From Fig. 5B(b), SPAD-values increased from

about 35 at the 4th leaf stage (39 DAS) to 46 at flowering (75

DAS), and decreased sharply to 33 at maturity (123 DAS).

The two curves were similar, with the main difference being

the greater SPAD-value estimated at flowering in plants

cultivated in the OTCs (SPAD = 46) compared with the

lower value obtained in the open-field (SPAD = 40). From

these curves, the maturity onset did not differ significantly

between treatments (open-field, and temperature and

[CO2] ? temperature elevation).

A

26

29

32

35

38

41

44

47

50

39 51 63 75 87 99 111 123

SP

AD

-val

ue

Days after sowing

1st topdressing

tilleringelongation

flowering grain fill maturity

SPAD =-0.37 + 1.178x -0.007x2, x=DAS n=30; R²= 0.64; P<0.001

262932353841444750

39 51 63 75 87 99 111 123

SP

AD

- val

ue

Days after sowing

temperature carbon dioxide

1st topdressing

tillering flowering grain fill maturityelongation

a b

B

Open-field CO2+Temperature Temperature

Year 2011

53 58 66 73 87 108 121

DAS

20

22

24

26

28

30

32

34

36

38

40

42

44

46

48

50

52

54SP

AD

-val

ue

Year 2012

53 58 66 73 87 108 121

DAS

SPAD = 16 + 0.629x-0.0042x2, x=DAS n=8; R²= 0.75; P<0.05

Fig. 5 A SPAD-values in Y-leaf of rice ‘Ariete’ cultivated in clay

loam soil at Salvaterra de Magos (central Portugal) in response to the

interaction effect of years (2011 and 2012), treatments (open-field,

and temperature and CO2? temperature elevation) and sampling date

(DAS). (n = 126; vertical bars denote 0.95 confidence intervals);

B Polynomial regression equations for SPAD-values obtained in the

rice under the open-field (a) and open-top chambers for CO2 ? tem-

perature and temperature elevation (b)

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123

Since N is closely associated with leaf chlorophyll it

was expected to be related to DM yield. Then, linear

relationships between SPAD-values and rice yield were

established for the overall seasons (Table 3). SPAD-data

determined at three phenological stages (elongation, grain

fill and maturity) were closely related (P \ 0.05) to straw

DM and grain yield at harvest (Table 3). Significant

relationships were found for SPAD-values measured in

plants cultivated in the OTCs at elongation, grain fill, and

maturity which predicted by 60 % variation the straw DM

yield. Under the open-field conditions, a significant model

was only fitted for SPAD-values measured at maturity

which explained with reasonable accuracy (67 % varia-

tion) the straw DM. As to grain yield, predicts were not

so confident.

Discussion

Climate change effects on crop yield and duration

Plant DM accumulation is driven by the interception of

solar radiation and subsequent conversion of solar energy

into biomass (C assimilation), a process that also depends

on water and N supply (Weerakoon et al. 2005). In the

present study, the open-field condition (high solar radia-

tion, long day-length: 14 h/10 h day/night light regime,

moderate temperature) at Salvaterra de Magos (central

Portugal) contributed to a high grain yield (7 t ha-1) of

japonica Ariete cultivar (Fig. 4), higher than the average

productivity in the country (6 t ha-1) but within the range

reported for temperate climates (Tran 1997; Biswas and

Ntanos 2002). Nevertheless, rice ‘Ariete’ responded dif-

ferently to rises in temperature (OTC effect) and [CO2].

Although the genotype was bred for a Mediterranean area

(Italy) and was largely adopted in south European countries

accounting for 40 % of the total cultivated area in Portugal,

it was susceptible to an average temperature increase of

?2–3 �C throughout the season (Fig. 2c) which resulted in

a bulk reduction of 29 % in grain yield in 2012 (Fig. 4).

This was particularly relevant inside the OTCs in 2012

when the maximum daily temperature was frequently

above 34 �C in the summer, during the reproductive phase,

higher than that observed for the other treatments. This

finding confirmed that reported by Krishnan et al. (2011)

and Madan et al. (2012) who stated that daily daytime

temperatures above the critical threshold of 33 �C during

the gametogenesis and flowering may affect the spikelet

fertility, causing a low seed-set and yield reduction.

Ainsworth (2008) and Madan et al. (2012) referred

increases of 24–30 % in rice grain yield with an elevation

of [CO2] in a growth chamber, whereas De Costa et al.

(2003) observed a larger range of increase of rice grain

yield by [CO2] elevation, varying from ?4 % to ?175 %,

depending on plant genotype and season duration. In

addition, Cheng et al. (2009) and Shoor et al. (2012)

Table 3 Linear regressions between SPAD-values and dry matter yield (kg DM ha-1) of japonica rice ‘Ariete’ cultivated in Salvaterra de

Magos (central Portugal)

Treatment Plant fraction Date of SPAD reading Linear regression R2 P n

�C Straw 61–70 DAS (elongation) Y2 years = 28,208–518.83x 0.74 0.028 6

�C Straw ? Grain 61–70 DAS (elongation) Y2 years = 20,391–340.16x 0.51 0.009 12

CO2 Straw 89–112 DAS (grain fill) Y2 years = -13,699 ? 407.68x 0.50 0.033 9

CO2 Grain 89–112 DAS (grain fill) Y2 years = -28,640 ? 774.90x 0.65 0.009 9

CO2 Straw ? Grain 89–112 DAS (grain fill) Y2 years = -21,169 ? 591.29x 0.46 0.002 18

�C Straw 89–112 DAS (grain fill) Y2 years = -18,573 ? 491.92x 0.67 0.007 9

�C Straw ? Grain 89–112 DAS (grain fill) Y2 years = -10,323 ? 323.39x 0.42 0.003 18

CO2 ? �C Straw 89–112 DAS (grain fill) Y2 years = -15,719 ? 440.81x 0.51 0.001 18

CO2 ? �C Grain 89–112 DAS (grain fill) Y2 years = -19,129 ? 546.22x 0.38 0.007 18

CO2 ? �C Straw ? Grain 89–112 DAS (grain fill) Y2 years = -17,424 ? 493.51x 0.37 0.000 36

Open-field Straw 118–123 DAS (maturity) Y2 years = 418 ? 169.27x 0.67 0.047 6

CO2 Straw 118–123 DAS (maturity) Y2 years = 16,626–362.27x 0.78 0.020 6

CO2 Straw ? Grain 118–123 DAS (maturity) Y2 years = 19,733–423.25x 0.47 0.013 12

�C Straw ? Grain 118–123 DAS (maturity) Y2 years = 12,778–240.45x 0.52 0.008 12

CO2 ? �C Straw 118–123 DAS (maturity) Y2 years = 16,407–358.11x 0.74 0.000 12

CO2 ? �C Grain 118–123 DAS (maturity) Y2 years = 18,992–393.71x 0.39 0.030 12

CO2 ? �C Straw ? Grain 118–123 DAS (maturity) Y2 years = 17,700–375.91x 0.45 0.000 24

All Straw 118–123 DAS (maturity) Y2 years = 11,845–221.54x 0.40 0.005 18

DAS Days after sowing, R2 Determination coefficient, P Probability level, n Number of observations

Paddy Water Environ

123

verified that rice yield increased with CO2 enrichment, but

this was reduced when N availability was low, which was

not the present situation. The simultaneous effect of [CO2]

and temperature elevation has not been extensively studied

(Wassmann et al. 2009), in particular under field condi-

tions. In the present experiment, the [CO2] elevation in

2012 did not compensate for the negative effect of high

temperature. In contrast, in 2011, the [CO2] elevation lar-

gely compensated for the negative effect of temperature on

grain yield (11 t ha-1). This year, maximum daily tem-

peratures were lower than 34 �C, except during the late

season (maturity). In agreement with the present observa-

tion, Madan et al. (2012) in a pot experiment observed that

elevated [CO2] did not compensate for the negative effect

of high temperature (38 �C) on grain yield in a highly

susceptible rice genotype. The present field observations

suggested that the interaction effect of [CO2] and temper-

ature elevation on rice yield was not consistent and

strongly depended on the degree of temperature elevation,

in particular, the daily maximal temperature during the

reproductive phase. Our results are in agreement with

previous studies carried out in other environments such as

in India and Philippines (Matsui et al. 1997; Satapathy

et al. 2014) where the same [CO2] and temperature inter-

action effects were observed. Although ‘Ariete’ is a

semidwarf high yielding japonica rice variety, the results

showed that it is sensitive to high temperature during the

reproductive stage and perhaps it will not be appropriate in

the near future climate change scenario for Mediterranean

areas where more extreme variations in weather will result

in adverse effects on the genotype. This insight includes a

selection for a better adapted variety to high temperatures.

Climate change effects on leaf greenness

and phenological stages duration

It is widely accepted that chlorophyll plays a pivotal role in

regulating photosynthesis, including the capture of sunlight

and the conversion of luminous energy. SPAD-value rep-

resents the relative content of chlorophyll, which is con-

venient and effective for the research of chlorophyll level

without damaging rice organs. As to leaf greenness, in the

present study the elevated temperature and the simulta-

neous rise of [CO2] ? temperature led to increased SPAD-

values (?14 %) in comparison with the average values in

the open-field (Fig. 5A) confirming that the negative

impact of high temperature on grain yield of the present

rice genotype was not due to effects on chlorophyll levels.

For all treatments, the SPAD-measurements were gen-

erally maintained above 35 across the seasons, in particular

during panicle formation, panicle differentiation, and grain

fill, indicating an adequate nutritional N status (Balasubr-

amanian et al. 1999; Dobermann and Fairhurst 2000; Liu

et al. 2013). No relevant differences were observed for the

duration of each phenological stage (49–62 DAS for til-

lering, 61–79 DAS for elongation, 68–77 DAS for panicle

formation, 89–112 DAS for grain fill, and 118–123 DAS

for maturing), but the higher temperature in 2012 advanced

the harvest day by more than one week (140 DAS) com-

pared with 149 DAS in 2011. Bhattacharyya et al. (2013)

tested in India a photoperiod non-sensitive Naveen cultivar

and similar to the present experiment, they observed no

response of rice phenological stages duration to treatments

([CO2] and temperature enrichment per se) and observed

results identical to the present—maximum tillering: 60

DAS, panicle differentiation: 86 DAS, grain fill: 93 DAS,

and maturity: 123 DAS. According to Krishnan et al.

(2011), the different phenological stages differ in their

sensitivity to high temperature, depending on plant geno-

type. The variety Ariete is a medium-late cultivar (140–149

DAS) compared with tropical varieties, and was in agree-

ment with observations in Greece and Italy (harvest at

149–150 DAS) where flowering occurred 55 days earlier

(94 DAS) than harvest (Biswas and Ntanos 2002; Confa-

lonieri and Bocchi 2005). In the present study, 63 days

were needed to mature ‘Ariete’ in OTCs in 2012 by the

elevated maximum daily temperature during the repro-

ductive phase ([34 �C), whereas in 2011 and in both

seasons in the open-field 81 days were required, when

maximum daily temperatures were lower than 34 �C. The

large season duration improved the japonica genotype

productivity, as indicated by De Costa et al. (2003) and

Krishnan et al. (2011).

Two identical polynomial curves (Fig. 5Ba,b) were fitted

for the relative chlorophyll content in plants grown in the

open-field and OTCs. An initial exponential phase of

SPAD-values was observed in both curves with a maximum

value at panicle differentiation. The only difference

observed between curves was the maximum SPAD-value

which was greater in the OTCs. Overall, the decline in

relative leaf chlorophyll (leaf greenness) since flowering

until the maturity was highest in the OTC-treatments

(28 %) compared with the open-field (25 %), suggesting

that [CO2] and temperature elevation enhanced senescence.

Oh-e et al. (2007) reported that high temperatures can

reduce the photosynthetic rate by 40–60 % at mid-ripening,

leading to more rapid senescence. Models fitting the present

SPAD-values agreed with those determined by Liu et al.

(2013) who found that the relationships between SPAD-

values and the days after heading stage were well described

by quadratic curve equations, with days as independent

variable, but contradicts as to treatments response. These

authors observed the greatest SPAD-values for natural

ambient conditions (SPAD = 34.58 ? 0.644x-0.014x2,

x = days after heading, n = 6, R2 = 0.93, P = \0.01),

and the smaller readings for high air temperature

Paddy Water Environ

123

(SPAD = 34.86 ? 0.530x-0.013x2, x = days after head-

ing, n = 6, R2 = 0.83, P = 0.03). Present models partially

agree with curves determined by Haque et al. (2006) for

changes in leaf chlorophyll during post-heading period of

rice. Present results can be used to understand and exploit

the beneficial effects that a rise in temperature and [CO2]

can have on an earlier maturity of rice ‘Ariete’, which can

be favorable in Mediterranean areas where water shortage is

a reality even though a reduction on grain yield is expected.

Regression analysis indicated that there were significant

relationships between SPAD-values at elongation, grain

fill, and maturity stages especially with straw DM for both

years under environmental stress. SPAD-readings deter-

mined during the reproductive phase explained reasonably

the variation of rice yield, but preferably the straw DM.

The present relationships agree with Esfahani et al. (2008)

who observed significant regression equations between N

and SPAD-readings within each growth stage of rice.

Nevertheless, further research is needed to obtain a robust

model relating SPAD-values and crop yield.

Conclusion

In a 2 year field experiment, the rice ‘Ariete’ had a moderate

high yielding under the open-field condition, but was sus-

ceptible to temperature elevation of ?2–3 �C under con-

trolled conditions resulting in reduction of grain yield. The

combined increase of atmospheric [CO2] with elevated air

temperature compensated for the negative effect of temper-

ature elevation alone, and crop yield was higher than in the

open-field. Hence, it can be concluded that potential increase

in air temperature may limit rice yields in the near future

under Mediterranean areas where climate change scenario

poses a serious threat but long term field experiments are

recommended for consolidation of present findings.

Relative chlorophyll content in ‘Ariete’ increased sig-

nificantly in consequence of temperature and [CO2] ?

temperature elevation, but the duration of phenological

stages did not depend on climatic conditions. SPAD-read-

ings at the reproductive stage explained by more than 60 %

of the variation in crop yield, in particular the straw DM,

but this finding requires further studies for consolidation.

Acknowledgement Authors acknowledge COTArroz and its staff

for facilities, climatic data, and help for the field work, as well as the

reviewers for their constructive suggestions. Authors also acknowl-

edge the Portuguese Foundation for Science and Technology (FCT,

Portugal) for the financial support through the project PTDC/AGR-

AAM/102529/2008. This work was also supported by European

Union Funds (FEDER/COMPETE-Operational Competitiveness

Programme) and by national funds (FCT) under the project FCOMP-

01-0124-FEDER-022692.

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