Localization of iron, zinc, and protein in seeds of...
Transcript of Localization of iron, zinc, and protein in seeds of...
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