Localization of iron, zinc, and protein in seeds of spelt (Triticum aestivum sspseeds of spelt (Triticum aestivum ssp. spelta) genotypes with low and high

protein concentrationprotein concentration

Levent Ozturk1, Gamze Altintas1, Halil Erdem2, Ozay Ozgur Gokmen1, Atilla Yazici1, Ismail Cakmak*1

1Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey2Cukurova University, Faculty of Agriculture, Adana, Turkey

Fe and Zn deficiency exists in about half of the world

INTRO Fe and Zn deficiency exists in about half of the world

population (Welch and Graham, 2004) and inadequate nutrition with Fe and Zn can cause serious health problems (anemia, impaired immune system, poor cognitive function…)

P l d f F d Z d fi i Prevalence and extent of Fe and Zn deficiency among populations is associated with nutritional habits and dependence to cereal based staple foodsp p

To meet the daily requirement of humans, much higher amounts of Fe and Zn should be present in the grain, gexceeding the optimum level for the crop itself (Welch, 1999; Grusak and Cakmak, 2005)

Higher micronutrient concentration is also advantageous for better seedling vigor and field stand

INTRO

Enrichment of cereal grains with micronutrients either via breeding or application of micronutrient fertilizers is widely accepted as a feasible and sustainable solution to improve nutritional status and well-being of human beings (Cakmak, 2008)g ( , )

Existence of a large and useful genetic variation for the targeted micronutrients is of great importance for a g g psuccessful breeding program aiming at improving cereal grains with micronutrients

The genetic variation in micronutrient concentrations of modern cultivated wheat is, however, fairly limited…

INTRO

Spelt wheat (Triticum aestivum ssp. spelta) possess a higher genetic variation in protein and micronutrients (Campbell, 1997; Moudry and Dvoracek, 1999)

It is currently grown mainly for use in making traditional and regional specialty food products

S l i i ll h d illi i Spelt varieties generally have good milling properties and high baking quality with comparable levels of bioactive compounds similar to that of commercial pwheat (Zielijski et al., 2008)

INTRO

Using spelt wheat genotypes with contrasting grain protein…

localization of Fe, Zn and protein in seed

compare visual qualitative results vs. analytical data produced by instrumental p yanalysis

EXPERIMENTAL

10 spelt genotypes (USDA-ARS gene bank) of which five had low (i.e. ~12 %) and five had high (i.e. ~25 %) seed protein concentration were selected for stainingseed protein concentration were selected for staining experiments

Seed Fe and Zn were analyzed by ICP-OES and Seed Fe and Zn were analyzed by ICP OES and protein was determined by LECO N analyzer

Seeds were excised longitudinally along the crease by Seeds were excised longitudinally along the crease by a scalpel and incubated in staining solutions for Fe, Zn or protein

EXPERIMENTAL

Iron staining solution (Perls’ Prussian blue): 1 %potassium hexacyanoferrate (w/v) in 1% HCl (w/v)(Prom-u-thai et al., 2003)

Zinc staining solution: 500 mg L-1 1,5-diphenyl thi b (DTZ) i th l (O t k t lthiocarbazone (DTZ) in pure methanol (Ozturk et al., 2006)

P t i t i i l ti 2 1 ( / ) dil t d B df d Protein staining solution: 2:1 (v/v) diluted Bradford reagent (Bradford, 1976) by ethanol

Fe and Zn staining: 30 min at room temperature Fe and Zn staining: 30 min at room temperature

Protein staining: 15 min at 70oC

Concentration of protein, Fe, Zn and seed weight of selected low- and high-protein spelt genotypes

N Status Genotype Protein Fe Zn Seed wt.(%) (mg seed-1)(mg kg-1)

Sp 211 13 40 26 40Sp 912 12 25 33 45Sp 207 12 35 26 37Low-seed-N Sp 207 12 35 26 37Sp 89 11 36 26 40Sp 21 10 45 37 39

Mean 12 36 30 40

Low seed N

Mean 12 36 30 40

Sp 663 26 59 51 35Sp 244 25 79 66 41pSp 926 25 84 54 37Sp 818 25 70 53 37Sp 804 24 61 60 41

High-seed-N

pMean 25 71 57 38

Relationships between seed Fe, Zn and protein concentrationsconcentrations

75

100

g-1)

60

80

g-1)

60

80

g-1)

R2 = 0 788***25

50

Fe (m

g kg

R2 = 0 827***20

40Zn

(mg

kg

R2 0 775***

20

40

Zn (m

g kg

R2 = 0.788

00 10 20 30

Protein (%)

R2 = 0.827

00 10 20 30

Protein (%)

R2 = 0.775***

00 25 50 75 100

Fe (mg kg-1)Protein (%) Protein (%) Fe (mg kg )

Relationships between soil DTPA-Zn, seed Zn and d N t ti f fi ld i

50 50

seed N concentration of field-grown grain crops

30

40 40

tratio

n

tratio

n

20

30

20

30

Zn c

once

nt(m

g kg

-1)

Zn c

once

nt(m

g kg

-1)

R2 = 0.0630

10R2 = 0.515***

0

10

Seed

Seed

0.0 0.2 0.4 0.6 0.8 1.0 0 2 4 6 8

Extractable soil Zn concentration (mg kg-1)

Seed N concentration (%)

(Cakmak et al., 2009)

Genotype GenotypeGrain protein status

Fe staining of spelt seeds

Low-grain-protein (LGP) High-grain-protein (HGP)Genotype Genotype

Sp 211 Sp 663

Sp 912 Sp 244Sp 912 Sp 244

Sp 207 Sp 926

Sp 89 Sp 818

Sp 21 Sp 804

Genotype GenotypeGrain protein status

Zn staining of spelt seeds

Low-grain-protein (LGP) High-grain-protein (HGP)Genotype Genotype

Sp 211 Sp 663

Sp 912 Sp 244Sp 912 Sp 244

Sp 207 Sp 926

Sp 89 Sp 818

Sp 21 Sp 804

Genotype GenotypeGrain protein status

Protein staining of spelt seeds

Low-grain-protein (LGP) High-grain-protein (HGP)Genotype Genotype

Sp 211 Sp 663

Sp 912 Sp 244Sp 912 Sp 244

Sp 207 Sp 926

Sp 89 Sp 818

Sp 21 Sp 804

Cakmak et al., 2009

X-Ray Fluorescence Imaging of Zn and Fe in Excised Wheat Seeds

Localization of Fei l

ZINCin aleurone

IRON

Localization of Zn in aleurone

(in collaboration with Dr. A. Zoubir, HORIBA Jobin Yvon S.A.S. - France)

Localization of Zn in bread wheat

Seed Zn: 26 mg kg-1 Seed Zn: 47 mg kg-1

oca at o o b ead eat

g g(after foliar Zn-application)

Staining Zn (Zn: mg kg–1)

Ozturk et al., 2005

Zinc Mobilization in T. dicoccoidesDuring Germination

Seed Zn: 17 ppm

g

Seed Zn: 17 ppm

Seed Zn: 61 ppm

Ozturk et al., 2005

CONCLUSIONS

Seed Fe and Zn correlates well with seed protein in spelt wheat

Positive correlations found in protein and Fe or Zn t b ifi (i K M C h dseem to be specific (i.e. K, Mn, Ca showed no

correlation with protein)

S d t i ld b l l i k f F d Z i Seed proteins could be a local sink for Fe and Zn in wheat grain

Variation in spelt genotypes constitutes a valuable Variation in spelt genotypes constitutes a valuable breeding tool for enrichment of cultivated wheat with protein, Fe and Zn

CONCLUSIONS

Staining of spelt seeds by using specific dyes for Fe, Zn and protein revealed that…Zn and protein revealed that…

Fe is preferentially localized in the Fe is preferentially localized in the scutellum and aleuron

whereas Zn and protein is localized whereas Zn and protein is localized in the whole germ (i.e. including scutellum, radicle and plumule) and th l llthe aleuron as well

CONCLUSIONS

The natural brownish color of spelt seed can mask DTZ staining, nevertheless it was clearly shown that embryo as a whole and aleuron are the sites of Zn localizationlocalization

Embryo and aleuron are the most protein dense parts of spelt seedp

As the seed protein increased, Coomasie dye binding and thus blue color formation also increased virtually in yall tissues of the seeds including the endosperm

CONCLUSIONS

First report on protein staining of seed tissue in spelt wheat showing a substantial visual difference inwheat showing a substantial visual difference inrelation to seed protein level

The proposed method for protein staining could be The proposed method for protein staining could be helpful for pre-screening of large germplasms for high grain protein content

The mechanisms affecting the metal deposition into different components of seeds and its dependency on N nutrition remains to be a challenging scientificN nutrition remains to be a challenging scientific research topic

Perls’ Prussian blue staining of excised seeds for Fe resulted in staining of scutellum and aleuron in a ggreenish-blue color particularly in high-grain-proteingenotypes

Fe staining was also evident in the endosperm of high-grain-protein genotypes

Incubating the excised seeds in dithizone for Zn localization resulted in staining of the whole embryo in red color, including the scutellum

The intensity of the red color formation in the embryo was similar for low- or high-grain-protein genotypes

Th l t diff i dithi t i i f The only apparent difference in dithizone staining of low and high-grain-protein spelt genotypes was the darker staining of endosperm in seeds of high-grain-g p g gprotein genotypes

RESULTS

Average Fe and Zn concentrations were 36 Average Fe and Zn concentrations were 36and 30 for low-grain-protein and 71 and 57mg kg-1 for high-grain-protein genotypes

High-grain-protein genotypes contained nearly 2-fold more Zn and Fe than the low-

i t i tgrain-protein genotypes

Average seed weights were similar for both low and high grain protein genotypeslow- and high-grain-protein genotypes

RESULTS

Fe Zn or protein concentration did not Fe, Zn or protein concentration did not correlate with seed weight

Without any exception high-grain-protein Without any exception, high grain protein genotypes had higher seed Fe and Zn concentration

Significant and positive correlations were found between seed Fe, Zn and protein concentrationsconcentrations

Mazzolini et al (1985) reported analytical data for the Mazzolini et al. (1985) reported analytical data for the accumulation of Fe and Zn in the embryonic parts such as the radicle, leaf primordium and scutellum and showed that Fe concentration was almost four timesshowed that Fe concentration was almost four times higher in the scutellum than the leaf primordium

The consequence of low Fe concentration in the leaf The consequence of low Fe concentration in the leaf primordium is not well understood

Reduced level of Fe in germinating shoot tissue can be Reduced level of Fe in germinating shoot tissue can be an adaptive mechanism to cope with Fe induced photo-oxidative stress

Fe could be retained in the scutellum via phytic acid where it is mostly abundant in the grain (Lin et al., 2005 J t l 2005)2005; Joyce et al., 2005)

Staining of seeds with the modified Bradford reagent resulted in formation of the typical blue color of Coomassie dye binding on the protein-rich seed parts

In all spelt genotypes tested, embryo and partly aleuron were the main targets of Coomassie dye binding, whereas endosperm stained less compared to embryo or aleurony

Similar to dithizone staining, the major distinction in seeds stained with Bradford reagent was the darkergstaining of the endosperm in the high-grain-protein genotypes