9092 J. Agric. Food Chem.

2010, 58, 9092–9102

DOI:10.1021/jf101197h

Biofortification and Localization of Zinc in Wheat Grain

I. CAKMAK,*,† M. KALAYCI,‡ Y. KAYA,§ A. A. TORUN, N. AYDIN,^ Y. WANG,#

)
)
Z. ARISOY,§ H. ERDEM, A. YAZICI,† O. GOKMEN,† L. OZTURK,† AND W. J. HORST#
†
Faculty of Engineering and Natural Sciences, Sabanci University, 34956 Istanbul, Turkey, ‡Anatolian
Agricultural Research Institute, Eskisehir, Turkey, §BD International Agricultural Research Institute,

)
P.O. Box 125, Konya, Turkey, Department of Soil Science, Faculty of Agriculture, Cukurova
University, Adana, Turkey, ^Karamanoglu Mehmetbey University, 70100, Karaman, Turkey, and
#
Institute for Plant Nutrition, Leibniz University Hannover, 30419 Hannover, Germany

Zinc (Zn) deficiency associated with low dietary intake is a well-documented public health problem,
resulting in serious health and socioeconomic problems. Field experiments were conducted with
wheat to test the role of both soil and foliar application of ZnSO4 in Zn concentration of whole grain
and grain fractions (e.g., bran, embryo and endosperm) in 3 locations. Foliar application of ZnSO4 was
realized at different growth stages (e.g., stem elongation, boot, milk, dough stages) to study the effect
of timing of foliar Zn application on grain Zn concentration. The rate of foliar Zn application at each
growth stage was 4 kg of ZnSO4 3 7H2O ha-1. Laser ablation (LA)-ICP-MS was used to follow the
localization of Zn within grain. Soil Zn application at a rate of 50 kg of ZnSO4 3 7H2O ha-1 was effective
in increasing grain Zn concentration in the Zn-deficient location, but not in the locations without soil Zn
deficiency. In all locations, foliar application of Zn significantly increased Zn concentration in whole
grain and in each grain fraction, particularly in the case of high soil N fertilization. In Zn-deficient
location, grain Zn concentration increased from 11 mg kg-1 to 22 mg kg-1 with foliar Zn application
and to 27 mg kg-1 with a combined application of ZnSO4 to soil and foliar. In locations without soil Zn
deficiency, combination of high N application with two times foliar Zn application (e.g., at the booting
and milk stages) increased grain Zn concentration, on average, from 28 mg kg-1 to 58 mg kg-1. Both
ICP-OES and LA-ICP-MS data showed that the increase in Zn concentration of whole grain and grain
fractions was pronounced when Zn was sprayed at the late growth stage (e.g., milk and dough).
LA-ICP-MS data also indicated that Zn was transported into endosperm through the crease phloem.
To our knowledge, this is the first study to show that the timing of foliar Zn application is of great
importance in increasing grain Zn in wheat, especially in the endosperm part that is the predominant
grain fraction consumed in many countries. Providing a large pool of Zn in vegetative tissues during
the grain filling (e.g., via foliar Zn spray) is an important practice to increase grain Zn and contribute
to human nutrition.

KEYWORDS: Biofortification; crease phloem; wheat grain; zinc fertilization; zinc deficiency

INTRODUCTION return for humanity and global stability. This conclusion was
Zinc (Zn) deficiency is a well-documented public health pro- made in 2008 by a panel of eight economists including five Nobel
blem in the developing world, resulting in severe health and Laureates (www.copenhagenconsensus.com).
socioeconomic problems (1, 2). Impairments in brain function, Low dietary intake of Zn and very little dietary diversity appear
immune system and physical growth are the major consequences to be the major reasons for the widespread occurrence of Zn
of Zn deficiency in the human body. Based on diet and bioavail- deficiency in human populations (4-6). Diets consumed predo-
ability data, it is estimated that at least 1/3 of the world popula- minantly in the developing world are based on cereals which are
tion is affected by the Zn deficiency problem, particularly poor in the amount and bioavailability of Zn. Enrichment of
children (1, 3). Nearly 450 000 children under the age of five die cereal crops with Zn is, therefore, an important global challenge
annually because of Zn deficiency (4). Together with vitamin A and a high-priority research area (7). Plant breeding (e.g., genetic
deficiency, Zn deficiency has been identified as the top priority biofortification) and application of Zn fertilizers (e.g., agronomic
area to be addressed in order to achieve a very rapid and high biofortification) are two important agricultural tools to improve
grain concentration of Zn (8-10). The plant breeding approach
*To whom correspondence should be addressed. Phone: þ90-216- offers the most sustainable solution to enrichment of cereal grains
483-9524. Fax: þ90-216-483-9550. E-mail: cakmak@sabanciuniv. with Zn; it is, however, a long-term process. In addition, the
edu. genetic capacity of the newly developed high-Zn genotypes to

pubs.acs.org/JAFC Published on Web 07/27/2010 © 2010 American Chemical Society

Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 9093
accumulate a sufficient amount of Zn in grain may depend on the Table 1. Concentration of DTPA-Extractable Zn, Fe, Mn and Cu, Mineral N
pool of plant-available Zn in soil solution. As reviewed by (Nmin), Organic Matter (OM), pH and Texture of Soils in the Experimental
Cakmak (7), application of Zn fertilizers represents a promising Sites in Samsun, Konya and Adana
solution to the problem in the short term and a complementary concn (mg kg-1)
approach to the plant breeding strategy.
location Zn Fe Mn Cu Nmin (kg ha-1) OM (%) pH texture
Convincing evidence is available about the effectiveness
of soil and foliarly applied Zn fertilizers in improving grain Samsun 1.59 18.6 3.48 6.96 57 2.7 7.75 clay
Zn concentrations in Zn-deficient wheat growing regions. Konya 0.18 8.3 5.13 1.97 110 1.5 8.31 clay
Strong increases in grain Zn were obtained in the case of Adana 0.49 5.8 6.64 1.24 22 1.0 7.51 clay
combined soil and foliar Zn applications of ZnSO4, raising
grain Zn by 3- or 4-fold (11). Application of Zn fertilizers not interesting research topic. According to Pearson et al (25), trans-
only improved nutritional quality but also contributed signifi- port of Zn into the endosperm increased as the grain matured,
cantly to grain production in Zn-deficient soils (7, 11-13). leading to a suggestion that late-season foliar application of Zn
Increases in grain Zn concentration by soil and/or foliar may maximize Zn accumulation in the endosperm. The crease
application of Zn fertilizers were also shown in pot trials phloem appears to be important in releasing Zn into endosperm.
under greenhouse conditions (14, 15). According to Kutman Destroying the crease phloem in wheat grain reduced substan-
et al. (15), the N nutritional status of plants has a critical role in tially Zn transport into the endosperm (25).
maximizing grain Zn concentration. When Zn nutrition of In the present study, field experiments were conducted in
plants was optimal, improving N nutrition of plants substan- 3 locations with wheat to study the role of timing of the foliar
tially enhanced grain Zn concentrations. There was a close Zn application on Zn concentrations of the whole grain and grain
positive correlation between tissue N and Zn concentrations in fractions (e.g., bran, embryo and endosperm) at different soil Zn
plants treated sufficiently with Zn and N fertilizers (15). These and N applications. The locations selected were different in levels
results indicate that a desirable biofortification of wheat grain of plant-available Zn concentration in soil. Laser ablation (LA)-
with Zn depends greatly on adequate supply of N. ICP-MS analytical technique has been used to monitor the
Remobilization of Zn from vegetative tissues into grain ap- localization of Zn in the grains of the plants treated by foliar
pears to be an important pathway for accumulation of Zn in Zn application at various growth stages. Currently, LA-ICP-MS
grain. More than 70% of the Zn reserves in the vegetative parts of technology is being increasingly used in localization of metals
wheat plants was remobilized (16), associated with the senescence such as Zn and Fe in plant samples (24, 26-28).
of leaf tissues and/or grain filling (17). There is convincing
molecular evidence for the role of leaf senescence in accumulation MATERIALS AND METHODS
of micronutrients in wheat grain. Expression of the NAM-B1 Field Experiments. Field experiments were conducted at 3 locations
gene was shown to be responsible for remobilization of Zn, Fe as follows: Bahri Dagdas International Agricultural Research Institute in
and also N from leaf tissue into grain during the leaf senescence, Konya (32 330 E, 37 510 N, 1009 m above sea level), Cukurova University
and delaying leaf senescence markedly reduced grain concentra- Research Farm in Adana (35190 E, 37020 N; 59 m above sea level) and
tions of Zn and Fe (18,19). The results of Pearson and Rengel (20) Black Sea Agricultural Research Institute in Samsun (36 200 E, 41170 N,
in wheat showed clearly that Zn reserves in vegetative parts are 4 m above sea level). The experiments in Konya were carried out in 2007
quickly depleted during the grain development period, indicating and 2008 using durum wheat (Triticum durum, cv. Selcuklu-97), while the
that grains are an important sink for Zn. Zinc has relatively high experiments in Samsun and Adana were conducted only in 2007 using
phloem mobility, and foliarly applied Zn is, therefore, easily bread wheat (Triticum aestivum). The bread wheat cultivars used were
Sagittario in Adana and O. zcan in Samsun. The experiments in the Konya
translocated into grain (14, 17). Based on these results it can be
location were not conducted on the same field so that there was not any
suggested that timing of foliar Zn application could be an concern with the residual Zn fertilizer effect. The total precipitation was
important issue in enrichment of wheat grain with Zn. Ozturk 242 mm in 2007 and 291 mm in Konya in 2008, 582 mm in Samsun in 2007
et al. (21) suggested that foliar application of Zn at an early stage and 354 mm in Adana in 2007.
of seed development could be a useful practice to produce a Soil properties at the experimental sites were different, especially
significant increase in grain Zn concentration in wheat because regarding the plant-available (DTPA-extractable) micronutrient concen-
the highest Zn accumulation in grain took place around early trations that were measured as described by Lindsay and Norvell (29). The
milk stage. Despite the existence of many studies related to foliar soils at the Konya location are known to be severely deficient in Zn (30)
application of Zn fertilizers, there is, however, no published and had lower levels of DTPA-extractable Zn than the Samsun and Adana
report about the effect of the timing of foliar Zn application on locations (Table 1). The soils of all three locations had adequate Fe, Mn
and Cu concentrations. Mineral N concentrations measured according to
grain Zn concentration. The importance of the late foliar applica-
Fabig et al. (31) were also different in all locations, being much higher at
tion of Zn fertilizers has been suggested as an effective way to the Konya location than the Samsun and Adana locations. The Adana
optimize grain Zn concentration (7). location had the lowest organic matter content. All soils were clay soils
Little is also known about how Zn is distributed within grain of with high pH values (Table 1).
plants treated with foliar Zn fertilizers at various vegetative and The experimental design consisted of split-plots in a randomized
reproductive growth stages. In most countries, wheat is eaten complete block design with five replications in each of 3 locations. Plot
after milling. The milling process removes the bran and embryo size was 6 m2 (1.2  5 m2). In Konya (e.g., Zn-deficient location), plants
parts of seeds that are highly rich in Zn, whereas the endosperm were grown with (50 kg of ZnSO4 3 7H2O/ha) and without soil Zn
(e.g., the part consumed in human diet) is low in Zn (21, 22). application over 2 years. An aqueous solution of ZnSO4 was sprayed on
Therefore, biofortification of the endosperm with Zn is an the soil surface just before the planting and then incorporated into soil
(approximately 15-20 cm) by a disk-plowing. The basal fertilizer treat-
important topic in terms of human nutrition. Currently, most
ments in Konya were 70 kg of P2O5/ha applied as triple-superphosphate
molecular genetic approaches aimed at improving Zn and Fe and 80 kg N/ha applied as ammonium nitrate. In the Samsun and Adana
concentrations in seeds focus on endosperm expression of pro- locations, plants were grown with low (80 kg N/ha) and high (240 kg N/ha)
teins with high Fe- and/or Zn-binding capacity, like ferri- soil N applications which were applied as ammonium nitrate. The rate of
tin (9, 23, 24). Lack of information about the role of foliar Zn the P application was 80 kg of P2O5 ha-1 in each Adana and Samsun
fertilization in Zn enrichment of endosperm makes this issue an locations applied during the planting as triple superphosphate. Due to
9094 J. Agric. Food Chem., Vol. 58, No. 16, 2010 Cakmak et al.
existence of an adequate amount of plant-available Zn in soil, Zn was not Table 2. Grain Yield of Bread Wheat Grown under Field Conditions with Low
applied into soil in the Samsun and Adana locations. (N 80: 80 kg N ha-1) and High (N 240: 240 kg N ha-1) N Applications and
In Konya, foliar Zn applications were applied twice at different growth Treated by Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa at
stages as follows: (i) stem elongation þ booting, (ii) booting þ early milk the Adana and Samsun locations in 2007b
and (iii) early milk þ early dough, whereas in the Samsun and Adana grain yield (tons ha-1)
locations five different foliar Zn spraying times were applied as follows: (i)
stem elongation þ booting, (ii) booting þ early milk, (iii) early milk þ early Adana Samsun
dough, (iv) booting þ anthesis þ early milk, and (v) stem elongation þ
foliar Zn application stages N 80 N 240 N 80 N 240
booting þ early milk þ early dough. The control plots (no foliar Zn
application) were treated with a corresponding amount of water. At each control 3.33 3.44 6.50 6.48
foliar Zn application, approximately 4 kg of ZnSO4 3 7H2O ha-1 was stem þ booting 3.63 3.66 5.61 6.00
applied by spraying 0.5% (w/v) ZnSO4 3 7H2O solution. Foliar spray of booting þ milk 3.83 4.03 5.51 6.42
Zn was performed either on cloudy days or in the very late afternoon to milk þ dough 3.61 3.74 6.95 6.42
avoid possible leaf damage caused by salts on sunny day and at high day booting þ anthesis þ milk 3.95 3.65 6.01 5.56
temperature. Under given conditions, there was no visible leaf damage stem þ booting þ milk þ dough 4.01 3.66 5.20 5.89
associated with foliar applications of 0.5% (w/v) ZnSO4 3 7H2O. mean 3.70 3.72 5.96 6.17
Measurement of N, Zn and Fe in Whole Grain and Grain
Fractions. At full maturity, grains were harvested to determine grain LSD0.05 for Zn applications nsc ns ns ns
yield and the grain concentrations of N, Zn and Fe. Iron and Zn were LSD0.05 for N rates ns ns
measured by an inductively coupled plasma optical emission spectrometer a
(ICP-OES; Vista-Pro Axial; Varian Pty Ltd., Mulgrave, Australia) after Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The
digesting the grain samples in a closed microwave digestion system bread wheat cultivars used were Sagittario and Ozcan at Adana and Samsun
locations, respectively. The values shown are means of 5 independent replications.
(MarsExpress CEM Corp., Matthews, NC) in the presence of concen- c
Not significant.
trated HNO3 and H2O2. The grain N concentration was measured by a
Leco TruSpec CN analyzer (Leco Corp., St. Joseph, MI) using a 0.2 g ablation system UP193SS (New Wave Research, Fremont, CA) was used
ground grain sample. Grain samples were also analyzed for aluminum (Al) for determination of the Zn concentration and its distribution in wheat
concentrations to estimate possible contamination of grain samples grains. Samples and matrix-matched calibration standards were arranged
by soil, and the results showed that Al concentrations were low in grain in a laser ablation chamber (Supercell, New Wave Research, Fremont,
(<3 mg kg-1) indicating no contamination of grain samples with soil. CA) and ablated under identical experimental conditions, in order to allow
Analytical data were checked against certified values of a standard the calculation of Zn concentrations. The ablated material was trans-
reference material (SRM 8436 Durum Wheat Flour, National Institute ported by argon as a carrier gas to the plasma. The ICP-MS instrument
of Standards and Technology, Gaithersburg, MD). tuning was optimized with respect to the maximum ion intensity of low
By using the same methods as described above, the grain fractions masses. The carrier gas flow rate was adjusted to optimum output. 13C was
(e.g., embryo, bran and endosperm) were also analyzed for Zn, Fe and used as an internal standard to normalize for different quantities of seed
N. Fractionation of grain parts was performed by using the method of tissue ablated. The parameters for the LA-ICP-MS operation are sum-
Hidalgo and Brandolini (32) with some modifications. Initially, whole marized below:
embryos (i.e., the germ, including the scutellum) of about 100 seeds were
severed using a surgical blade. To separate the endosperm, de-embryo- ICP-MS: Agilent 7500 cx, Quadropol
nated seeds were milled with a vibrating agate cup mill (Pulverisette 9, Carrier gas flow: Ar 0.25 L min-1
Fritsch GmbH, Idar-Oberstein, Germany) for 20 s for bread wheat grains
Make up gas flow: Ar 1.25 L min-1
and 50 s for durum wheat grains at 700 rpm, and the resulting flour was
sieved with a 100 μm mesh plastic sieve. The sieved particles were treated as
Reaction mode: off
the endosperm, whereas the fraction remaining on the sieve (bran and RF power: 1300 W
shorts) was sieved again with a 1000 μm mesh plastic sieve in order to Laser: UP193SS, 193 nm
separate the bran from the shorts. Output energy: 2 J/cm2
Zinc Staining of Grains. Zinc staining of grains was conducted using Repetition rate: 10 Hz
dithizone reagent as described by Ozturk et al. (21), by incubating seeds Crater size: 25 μm
with 500 mg L-1 dithizone (1,5-diphenyl thiocarbazone) at room tempera- Scan speed: 10 μm s-1
64
ture for 30 min. The stained seeds were then rinsed with water and analyzed Measured elements: Zn, 13C
qualitatively by using a reflectance light microscope (Nikon SMZ1500,
Melville, NY) with a high-resolution digital camera (Diagnostic Instru- For calculation of grain Zn concentration, the Zn/C ratio (13C as
ments Inc., Sterling Heights, MI). internal standard) of the calibration standards and the grains was used.
Localization of Zn in Grains Using LA-ICP-MS. In preparation of Four cross sections from four grains of each treatment were analyzed. The
grain samples for LA-ICP-MS analyses, wheat grains were first soaked in images shown in this paper were representative of four independent
deionized water overnight. Cross sections of the softened grains were measurements for each treatment.
obtained by using a microtome (MT.5530, Euromex Microscopen B.V., Statistical Analysis. JMP statistical software (SAS Institute, USA)
Arnhem, Netherlands). The cross sections were fixed on glass slides using was used in analysis of the experimental data. Comparison of means was
double face photostrip (Tesa AG, Hamburg, Germany) and subsequently performed by Student’s t test. Data was analyzed by ANOVA using the
air-dried for at least 24 h before analysis. general linear model (GLM). JMP Statistical software (SAS Institute) was
The matrix-matched calibration standards were prepared for the used in performing the statistical analyses. Student’s t test was used to
calibration of the grain Zn concentration. Wheat flour (10 g) was spiked compare means whenever ANOVA indicated significant differences
with 10 mL of Zn standard solution (ranging from 0.05 to 1000 Zn mg L-1) among treatments.
prepared from a 1000 mg L-1 ICP SPEX standard (Spex Industries Inc.,
Edison, NJ) and dried at 60 C for 48 h. The Zn-spiked flour was RESULTS
homogenized using a vibrating cup mill (Puverisette, Fritsch GmbH,
Idar-Oberstein, Germany) for 10 min. The flour was then pressed to pellets
As presented in Table 2, neither foliar Zn applications nor an
of 5 mm diameter. The Zn concentration of the flour pellets was increase in N rate from 80 to 240 kg N ha-1 affected grain
determined by digesting in concentrated nitric acid and measured by an yields at the Adana and Samsun locations. At the Konya
ICP-EOS (Spectroflame EOS, Spectro Analytical Instruments GmbH, location, with very low available soil Zn, applying Zn to the
Kleve, Germany) and ICP-MS (7500cx, Agilent Technology, (Santa soil increased the grain yield by 23% and 21% in the 2007 and
Clara, California, USA). A quadrupole ICP-MS coupled to a laser 2008 trials, respectively (Table 3). In the case of foliar Zn
Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 9095
application, wheat grain yield was not statistically affected in at Adana. Foliar Zn applications did not influence grain Fe
either of the two years. concentrations at Adana, but resulted in a significant increase at
An increase in the N application rate significantly increased Samsun (Table 4).
grain N concentration at Adana and Samsun, while foliar Zn At the Konya location, soil and foliar Zn applications did not
applications did not affect grain N (Table 4). All combinations of influence grain N concentrations in either year (Table 5). Soil Zn
foliar Zn treatments significantly increased grain Zn concentra- application increased grain Zn concentration nearly 2-fold in
tions at both locations, when compared to the control plots where 2007 and 2008. Relative increases in grain Zn concentrations
plants were not treated with foliar Zn. The increase in grain Zn by following foliar Zn application were much greater when soils
foliar Zn applications was more pronounced at Samsun (up to were not treated with Zn. Compared with soil Zn application,
2.4-fold increase) where grain Zn values were lower in the control enhancement in grain Zn concentrations was greater in foliar Zn
plots than at Adana. At both locations, the highest grain Zn treatments. A combined application of Zn to soil and foliage at
concentrations were obtained when Zn was applied 4 times. The the booting and milk stages increased grain Zn concentration
differences among the foliar Zn applications were relatively small almost 2.5-fold in 2007 and 2.6-fold in 2008. Iron concentrations
compared to the difference between any foliar Zn application and were not affected by the foliar Zn applications, but were
the control treatment. However, there was a clear trend for later decreased by soil Zn application.
applications (especially for the booting þ milk stages) to result in Among the grain fractions, the endosperm had the lowest Zn
higher grain Zn concentrations than the earlier applications, concentration (Table 6). In the case of the control plants (without
particularly at Samsun. Increasing the N rate from 80 to 240 kg foliar Zn application), the embryo had either greater (in Adana)
N ha-1 enhanced grain Zn concentrations at Samsun, but the or similar (in Samsun) Zn concentrations compared to the bran
magnitude of increase was not as high as in foliar Zn treatments. fraction. Foliar Zn treatments significantly increased Zn concen-
A similar trend was also observed in Adana, although not statis- trations in all three grain fractions. Although the endosperm had
tically significant, possibly resulting from relatively large field the lowest Zn concentrations, generally the greatest relative
variations. Nitrogen application also increased grain Fe con- increases in Zn concentrations were obtained in this fraction.
centrations, although the effect was not statistically significant Regarding the timing of foliar Zn applications, earlier application
(at stem and booting stages) had less effect on grain Zn than the
Table 3. Effect of Soil and Foliar-Applied ZnSO4 3 7H2O on Grain Yield of the later applications. A combination of booting and milk stage
Durum Wheat Cultivar Selcuklu-97 Grown in 2007 and 2008 on a Zn-Deficient applications resulted in the highest Zn concentration in all grain
Calcareous Soil at the Konya Locationa fractions at both locations. Foliar Zn treatment at booting þ milk
grain yield (ton ha-1) stages increased Zn concentrations in the endosperm from 11.5 to
foliar Zn
soil Zn (kg of ZnSO4 ha-1) application stages 2007 2008 18.5 mg kg-1 in Adana and from 6 to 13.5 mg kg-1 in Samsun as
an average of the two N rates. Embryos were the least affected,
0 control (no Zn application) 1.45 2.20 and the bran fractions were intermediate in terms of increase in
stem þ booting 1.49 2.42 Zn concentration as a result of foliar Zn application. Increases in
booting þ milk 1.38 2.38 grain Zn concentrations by the foliar Zn application has been also
milk þ dough 1.41 2.28 demonstrated by using the staining method for the grains from
50 control (no Zn application) 1.79 2.81
the Samsun location (Figure 1). Zinc was particularly concen-
stem þ booting 1.69 2.88
booting þ milk 1.77 2.68
trated in the aleurone and embryo fractions as shown by the
milk þ dough 1.75 2.88 intensity of red color (Figure 1).
Increased N rates showed a trend to increase grain Zn,
LSD0.05 for soil Zn application 0.14 0.20 although the effect was not as consistent as that of foliar Zn
LSD0.05 for foliar Zn application nsb ns treatment (Table 6). A combination of Zn and N treatments
a
Foliar Zn application was performed at different growth stages by applying 0.5% caused the greatest increase in endosperm Zn concentration,
ZnSO4 3 7H2O (approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth especially when foliar Zn was applied at booting þ milk stages
stage), while soil Zn application was prior to seeding. b Not significant. (e.g, from 11 to 20 mg of Zn kg-1 at Adana and from 5 to 15 mg

Table 4. Nitrogen, Zn and Fe Concentrations in Whole Grain of Bread Wheat Grown under Field Conditions with Low (N 80: 80 kg N ha-1) and high (N 240: 240 kg N
ha-1) N Applications and Treated by Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa at the Adana and Samsun Locations in 2007b
concn

grain N (%) grain Zn (mg kg-1) grain Fe (mg kg-1)

ADANA SAMSUN ADANA SAMSUN ADANA SAMSUN

foliar Zn treatment stages N 80 N 240 N 80 N 240 N 80 N 240 N 80 N 240 N 80 N 240 N 80 N 240

control 1.97 2.32 1.89 2.27 32 37 23 29 36 41 29 32

stem þ booting 2.07 2.29 1.95 2.20 51 58 42 42 39 41 35 36
booting þ milk 2.12 2.25 1.83 2.21 56 56 49 55 38 40 34 36
milk þ dough 2.07 2.39 1.92 2.31 57 64 44 51 35 41 33 34
booting þ anthesis þ milk 1.97 2.45 2.02 2.37 58 65 53 60 38 41 36 38
stem þ booting þ milk þ dough 2.07 2.37 1.96 2.40 65 70 56 63 36 43 36 39
mean 2.05 2.34 1.93 2.29 53 58 45 50 37 41 34 37

LSD0.05 for foliar Zn applications nsc ns ns ns 7.4 7.4 5.1 5.1 ns ns 3.2 3.2
LSD0.05 for soil N applications 0.12 0.27 ns 2.3 ns 0.9
a
Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The bread wheat cultivars used were Sagittario and Ozcan at Adana and Samsun locations,
respectively. The values shown are means of 5 independent replications. c Not significant.
9096 J. Agric. Food Chem., Vol. 58, No. 16, 2010 Cakmak et al.
Table 5. Nitrogen, Zn and Fe Concentrations in Whole Grain of Durum Wheat Cultivar Selcuklu-97 Grown in 2007 and 2008 on a Zn-Deficient Calcareous Soil with
(50 kg of ZnSO4 3 7H2O) and without Soil Zn Application and Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa at the Konya locationb
grain concn

2007 2008
-1 -1 -1
soil Zn application (kg of ZnSO4 ha ) foliar Zn application stages N (%) Zn (mg kg ) Fe (mg kg ) N (%) Zn (mg kg-1) Fe (mg kg-1)

0 control (no Zn application) 2.98 11.7 42 3.20 10.4 58

stem þ booting 3.13 18.8 40 3.18 12.7 54
booting þ milk 3.09 26.9 42 3.20 17.9 58
milk þ dough 3.09 25.4 47 3.24 18.8 59
50 control (no Zn application) 3.06 21.7 35 3.32 21.5 46
stem þ booting 2.92 25.5 38 3.34 26.1 46
booting þ milk 3.04 29.0 38 3.32 27.0 45
milk þ dough 2.96 29.3 38 3.32 25.2 44

LSD0.05 for soil Zn application nsc 1.8 2.0 ns 2.7 4.0

LSD0.05 for foliar Zn application ns 2.6 3.0 ns 2.8 ns
a
Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The values shown are means of 5 independent replications. c Not significant.

Table 6. Zinc Concentrations of the Bran, Embryo and Endosperm Fractions in the Grain of Bread Wheat Grown under Field Conditions with Low (N 80:
80 kg N ha-1) and High (N 240: 240 kg N ha-1) N Applications and Treated by Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa at the Adana and
Samsun locations in 2007b
Zn concn (mg kg-1)

Adana Samsun
-1
N application rate (kg ha ) foliar Zn treatment stages bran embryo endosperm bran embryo endosperm

80 control (no Zn) 42 70 11 64 68 5

stem þ booting 72 96 15 115 102 11
booting þ milk 88 106 17 143 120 12
milk þ dough 88 98 16 121 105 11
240 control (no Zn) 75 75 12 72 74 7
stem þ booting 105 105 18 121 106 12
booting þ milk 104 112 20 156 131 15
milk þ dough 110 112 20 144 109 14

LSD0.05 for foliar Zn applications 12 4 1 8 9 2

LSD0.05 for N applications 12 n.s 3 11 nsc ns
a
Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The bread wheat cultivars used were Sagittario and Ozcan at Adana and Samsun locations,
respectively. The values shown are means of 5 independent replications. c Not significant.

of Zn kg-1 at Samsun). There was no consistent effect of concentration in the embryo also increased with foliar Zn applica-
the N treatments on Fe concentrations in grain fractions, with tion in the case of soil Zn application, although not as much as with
exception of increased Fe concentration in the bran fraction at the the nil soil Zn treatment. Neither soil nor foliar Zn applications
Adana location (data not shown). Increasing the N rate was affected N concentrations in any grain fraction (data not shown).
effective in improving N concentrations in almost all grain frac- Localization and distribution of Zn within grains were also
tions at both locations, mainly in the endosperm fraction studied by using LA-ICP-MS (Figures 2 and 3) using grain from
(Table 7). Foliar applied Zn did not influence grain N concentra- plants grown with or without foliar application of Zn at Adana
tions in any grain fraction. and Konya. In all grain samples tested by LA-ICP-MS, Zn
Under severe Zn deficiency at the Konya location, soil and foliar was particularly concentrated in the embryo, aleurone layer
Zn applications significantly enhanced Zn concentrations in all and crease tissue, whereas the endosperm fraction contained very
grain fractions (Table 8). The embryo fraction had more Zn than low Zn (Figures 2 and 3). LA-ICP-MS analysis also showed that
the bran and endosperm fractions. Endosperm Zn was less affected Zn concentrations greatly increased in the aleurone layer, embryo
by soil Zn application than the other grain fractions. However, part and crease tissue after Zn foliar application (Figures 2 and 3).
foliar application of Zn at later growth stages resulted in greater In the grain samples without foliar Zn application from both
increases (nearly 1.9-fold) in endosperm Zn concentrations than experimental locations, endosperm Zn was uniformly distributed.
the soil Zn application (0.38-fold). A combination of soil and foliar Foliar application of Zn markedly increased endosperm Zn
Zn applied at late growth stages caused more than 2-fold increase concentration of the grains. This increase was very distinct at
in endosperm Zn concentrations (e.g., from 8 mg kg-1 to 17 mg both experimental locations and more pronounced when Zn was
kg-1). Similar increases in Zn concentration after foliar application sprayed at later growth stages (Figure 2). The application of Zn by
of Zn at late growth stages were also found in the bran and foliar sprays (particularly at later stages) enhanced endosperm Zn
especially in the embryo fractions (Table 8). Soil Zn applications concentrations primarily near the crease, leading to an increas-
resulted in a decrease in Fe concentrations in the all grain fractions, ingly steep gradient from the crease tissue to the external border
mainly in the bran and embryo fractions (Table 8). Iron con- of the endosperm. The greater role of late-season foliar appli-
centrations in bran and embryo parts tended to increase with foliar cation of Zn in increasing Zn concentrations of the embryo
Zn applications only when Zn was not applied to the soil. Iron part compared to the foliar Zn application at earlier growth
Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 9097
stage (Table 8) was also demonstrated and confirmed by using the stages (33, 34). It is, therefore, not surprising that seeds with high
LA-ICP-MS analysis (Figure 3). Zn concentrations contribute greatly to high seedling vigor, good
field establishment and large yield under Zn-deficient soil
DISCUSSION conditions (35-37).
Soil and foliar applications of ZnSO4 were highly effective in Foliar Zn applications were more effective at increasing grain
increasing grain Zn concentration in wheat, confirming earlier Zn concentrations than soil applied Zn. Whereas two foliar appli-
results (11, 13, 14). At the Konya location with low plant- cations were effective at achieving high grain Zn concentrations
available Zn, soil Zn application also resulted in significant (Tables 4 and 5), four foliar applications produced the highest Zn
increases in grain yield. Foliar Zn applications were, however, concentrations, but this practice may not be feasible in commer-
not as effective as the soil Zn application in improving yields, cial production. To our knowledge, this is the first study to show
indicating the importance of adequate available soil Zn during the that the timing of foliar Zn application is a critical factor in
early growth stages. It is known that Zn is particularly important increasing grain Zn concentration. Compared to the early appli-
for ensuring good root growth and improving tolerance to cations (e.g., stem elongation þ booting stages), foliar applica-
various environmental stress factors during the early growth tion of Zn later in the growing season (e.g., at booting þ milk or
milk þ dough stages) resulted in greater increases in grain Zn
concentration. A combination of late foliar Zn applications with
either high soil N or soil Zn application caused further increases in
grain Zn (e.g., from 23 to 55 mg kg-1 with high N application at
Samsun and from 12 to 29 mg kg-1 with soil Zn application at the
Zn-deficient Konya locations). In order to achieve a measurable
biological impact on human health, grain Zn concentrations
should be increased by at least 10 mg kg-1 in a given region (7, 8).
The increases in grain Zn concentrations achieved through foliar
Zn applications were around 17 mg kg-1 at Konya and more than
30 mg kg-1 at the other locations.
High N supply positively affected grain concentration of both
Zn and Fe (Table 4). Similar results were recently reported by
Kutman et al. (15) in a greenhouse study with durum wheat. The
reasons for the positive impact of N nutrition on Zn and Fe
concentrations of plants might be the possible contributions of
adequate N nutrition to (i) pool of transporter proteins mediating
absorption and transport of Zn and Fe, (ii) release of Zn- and Fe-
solubilizing phytosiderophores from roots into the rhizosphere,
Figure 1. Staining and localization of Zn in a bread wheat grain (iii) facilitation of Zn and Fe translocation from vegetative tissues
€
(cv. Ozcan) without (left) and with (right) foliar spray of 0.5% ZnSO4 3 7H20 into grains by nitrogenous compounds such as peptides or nico-
at booting and milk stages (approximately 4 kg of ZnSO4 3 7H2O ha-1 tianamine (10). Understanding the mechanisms contributing to an
applied at each growth stage) at the Samsun location in 2007. Zinc increase in grain Zn by N fertilization deserves further research.
concentrations of the grains were 25 mg kg-1 (left) and 52 mg kg-1 (right). Increases in the concentration of whole-grain Zn through soil
The longitudinally cut seed surface was stained for Zn by dithizone reagent and/or foliar Zn applications were reflected proportionally in all
(500 mg L-1 1,5-diphenyl thiocarbazone dissolved in absolute methanol; grain fractions analyzed (Tables 6 and 8). In the case of the late-
incubation at room temperature for 30 min; see Materials and Methods for season foliar Zn application, increases in Zn concentrations of the
more detail). Red color formation indicates Zn localization, especially in the grain fractions were more distinct as shown both by the ICP-OES
embryo and aleuron layer of the grains. (EMB: embryo. ALE: Aleuron. data (Tables 6 and 8) and by the LA-ICP-MS data for endosperm
END: Endosperm.) and embryo (Figures 2 and 3). Here, particular attention should

Table 7. Nitrogen Concentrations of the Bran, Embryo and Endosperm Fractions in the Grain of Bread Wheat Grown under Field Conditions with Low (N 80:
80 kg N ha-1) and High (N 240: 240 kg N ha-1) N Applications and Treated by Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa in the Adana and
Samsun locations in 2007b
N concn (%)

Adana Samsun
-1
N application rate (kg ha ) foliar Zn treatment bran embryo endosperm bran embryo endosperm

80 control (no Zn) 2.12 2.85 2.15 2.56 2.47 1.57

stem þ booting 2.28 2.80 2.23 2.54 2.54 1.53
booting þ milk 2.30 2.85 2.17 2.50 2.43 1.36
milk þ dough 2.16 2.80 2.09 2.55 2.52 1.46
240 control (no Zn) 2.60 3.11 2.50 2.87 2.68 1.74
stem þ booting 2.50 3.08 2.44 2.77 2.64 1.61
booting þ milk 2.49 3.04 2.46 2.76 2.58 1.71
milk þ dough 2.52 3.02 2.49 2.82 2.64 1.75

LSD0.05 for foliar Zn applications nsc ns ns ns ns ns

LSD0.05 for N applications 0.17 0.15 0.25 0.28 ns 0.17
a
Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The bread wheat cultivars used were Sagittario and Ozcan in Adana and Samsun locations,
respectively. The values show the mean of 5 independent replications. c Not significant.
9098 J. Agric. Food Chem., Vol. 58, No. 16, 2010 Cakmak et al.
Table 8. Zinc and Fe Concentrations of the Bran, Embryo and Endosperm Fractions in the Grain of Durum Wheat Cultivar Selcuklu-97 Grown in 2007 under Field
Conditions with (50 kg of ZnSO4 3 7H2O) and without Soil Zn Application and Foliar Spray of 0.5% ZnSO4 3 7H2O at Different Growth Stagesa in the Konya Locationb
concn (mg kg-1)

Zn Fe
-1
soil Zn application (kg of ZnSO4 ha ) foliar Zn application stages bran embryo endosperm bran embryo endosperm

0 control (no Zn) 20 38 8 58 89 23

stem þ booting 28 47 10 57 89 24
booting þ milk 35 62 15 52 90 27
milk þ dough 41 63 15 79 100 27
50 control (no Zn) 33 52 11 54 85 20
stem þ booting 34 58 13 49 85 23
booting þ milk 44 68 17 52 87 22
milk þ dough 45 69 16 52 90 20

LSD0.05 for soil Zn application 3.0 3.4 1.0 5.3 3.8 1.9
LSD0.05 for foliar Zn application 4.8 4.2 4.8 5.4 4.2 5.4
a
Approximately 4 kg of ZnSO4 3 7H2O ha-1 applied at each growth stage. b The values show the mean of 5 independent replications.

be given to the changes in Zn concentration in the endosperm vegetative tissue into developing grain via the phloem may be
fraction, as this is the predominant fraction consumed in many the major pathway for Zn accumulation in grain. Accordingly,
countries. In addition to the ICP-OES data on Zn concentra- Waters et al. (52) showed that withholding supply of Zn in
tions in grain fractions, the LA-ICP-MS data (Figure 2) also nutrient solution during postanthesis stimulated Zn remobiliza-
showed that the application of foliar Zn late in the growing season tion from the leaf tissue, while in case of the continuous supply
produced a large increase in the concentration of endosperm and of Zn in the nutrient solution there was no net remobilization
whole-grain Zn. Similarly, the application of foliar N fertilizers from leaf tissue. Based on these results from controlled growth
after flowering enhanced grain protein concentration more than conditions (15,52) and field trials described in this study, it can be
the N application at earlier growth stages, such as booting (38,39). suggested that increasing the pool of Zn in vegetative tissue during
During the early stage of seed development, both protein syn- the reproductive growth stage (for example through foliar spray-
thesis (40, 41) and Zn accumulation (21) are maximized in wheat ing of Zn) is of great importance for maximizing grain Zn
grain, suggesting that an increase in protein synthesis possibly accumulation under field conditions with limited soil-Zn supply.
creates a sink for Zn. Zinc is known to be primarily required In agreement with this suggestion, Pearson and Rengel (20)
for biosynthesis of proteins (17, 42) and affect protein composi- emphasized the importance of the flag leaf and stem as Zn
tion of wheat grain (13). Up to 10% of proteins in biological reservoirs in wheat and showed that these plant parts rapidly
systems require Zn for their function and structural integrity (43). depleted Zn during the grain filling period. Increasing molecular
Accordingly, Zn concentrations are extremely high (e.g., 600 mg evidence is also available showing that remobilization of Zn from
kg-1) in the protein bodies of wheat grain (44) and correlate senescing leaf tissue through the action of the NAM-B1 gene plays
positively with the protein concentrations in different wheat a critical role in accumulation of Zn in wheat grains (18, 19, 52).
genotypes (45-47). These observations suggest that foliar appli- Although it was not examined in this study, foliarly applied
cation of Zn during early stage of seed development may maxi- Zn in the late growth stages might be important for alleviation
mize accumulation of Zn in grain due to a possible high sink of subsoil Zn deficiency problems. Previous studies with
activity for Zn at this stage of seed development. radiolabeled Zn showed that an important part of foliarly
The distinct increases in grain Zn concentrations following absorbed Zn is translocated to plant roots (48, 49). Under field
foliar application (especially at the late growth stage) indicate that conditions, transport of Zn into subsoil roots may improve
Zn is easily translocated via the phloem into grain. Similar plant growth and yield capacity (53). In most cases, concentra-
conclusions were also reported earlier by Haslett et al. (48) and tions of plant-available Zn in the subsoil is low when compared
Erenoglu et al. (49) in the experiments conducted with wheat to the top soil due to the low mobility of Zn in the soil and the
under controlled environmental conditions. Based on these impossibility of applying Zn to the subsoil through common
results it can be speculated that a major part of the Zn loaded fertilizer application methods. In semiarid regions, roots may
into grains during grain development under field conditions, as not be able to absorb sufficient Zn from the topsoil because this
described in this study, was most probably due to retranslocation part of soil is often dry. Therefore, roots rely on the available
from the vegetative tissues via the phloem. Contrary to this Zn in the subsoil (which contains moisture long after the
suggestion, some reports for rice and Arabidopsis plants show topsoil had dried), especially during the late growth stages.
that continued root uptake and translocation into seeds during Having adequate amounts of Zn available to subsoil roots is of
the seed-filling period appears to be the major way for accumula- great importance for the protection of roots from various stress
tion of Zn into seed (50, 51). It seems likely that in the case factors such as salinity, B toxicity, and soil-borne diseases,
of high availability of Zn in the growth medium, such as in and also for the maintenance of the structural integrity of root
growth chamber or greenhouse pot experiments, continuous root cell-membranes (34, 54).
uptake and translocation into grain during the grain-filling period It remains unclear how Zn is transported into the developing
contributes significantly to grain accumulation of Zn (15,50-52). grain. The LA-ICP-MS data (Figure 2) suggest that Zn is
However, when the availability of Zn in the growth medium is transported into the endosperm through the crease phloem in a
limited such as under field conditions in calcareous soils with similar way to sucrose. It is widely believed that sucrose is
limited soil moisture, the role of root uptake and translocation transported into the endosperm cavity and then into the endo-
into grains during the grain-filling period is possibly minimal. sperm via the crease phloem (55). As demonstrated by the LA-
Under field conditions, the remobilization of Zn from the ICP-MS measurements, there was a gradient in the endosperm
Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 9099

Figure 2. Localization of Zn in cross sections of wheat grains harvested at the Zn-deficient Konya location without soil Zn application (A) and Zn-adequate
Adana (B) locations in 2007. Grains subjected to LA-ICP-MS analysis were from plants which were either not treated (no foliar Zn application) or treated with
foliar spray of ZnSO4 3 7H2O at the stem elongation and booting or at the milk and dough stages. The laser ablation scanning of the seed cross section started
at the seed coat and moved through the cross section passing through the crease (cr) in the direction as shown by the white arrow in the pictures on the left.
The diagrams in the middle show the Zn concentrations along the entire cross section (white arrow). The diagrams on the right show the Zn concentrations in
the endosperm between the aleurone layers of the seed surface and the crease (black arrow). Four cross sections from four grains of each treatment were
analyzed. Thus, the images shown are representative of four independent measurements of each treatment. See Materials and Methods for more details.
9100 J. Agric. Food Chem., Vol. 58, No. 16, 2010 Cakmak et al.

Figure 3. Localization of Zn in cross sections including the embryo (emb) of wheat grains harvested at the Zn-deficient Konya location in 2007. Grains
subjected to LA-ICP-MS analysis were from plants which were either not treated (no foliar Zn application) or treated with foliar spray of ZnSO4 3 7H2O at the
stem elongation and booting or at the milk and dough stages. The laser ablation scanning of the seed cross section started at the seed coat and moved through
the cross section passing through the endosperm and the embryo, as shown by the white arrow in the pictures on the left. The diagrams on the right show the
Zn concentrations along the entire cross section (white arrow). Four cross sections from four grains of each treatment were analyzed. Thus, the images shown
are representative of four independent measurements of each treatment. See Materials and Methods for more details.

Zn concentration from the crease tissue to the external border foliar. In locations without soil Zn deficiency, combination
of the endosperm. This gradient became steeper with the late of high N application with two foliar Zn applications (e.g., at
application of Zn, indicating an important role of phloem the booting and milk stages) increased grain Zn concentration, on
and crease tissues in the delivery of Zn into the endosperm. average, from 28 mg kg-1 to 58 mg kg-1. Timing of the foliar Zn
A similar conclusion was drawn by Pearson et al. (25) in an application was found to be an important factor in increasing
experiment with wheat by using radiolabeled Zn (65Zn) under grain Zn concentration. An increase in grain Zn concentration by
controlled environmental conditions. Their data indicated that application of foliar Zn fertilizers was more pronounced when Zn
most of the 65Zn was transported into the endosperm via the was applied in the late compared with the early growth stages.
crease phloem. This suggests that providing a large pool of Zn in the vegetative
In conclusion, application of Zn to soils and/or foliage repre- tissue during the reproductive growth stages (e.g., by foliar
sents a very useful and rapid approach in enrichment with Zn of spraying of Zn fertilizers) is an important field practice for maxi-
wheat grains (particularly the endosperm and thus white wheat mizing grain Zn accumulation. There is, now, an urgent need for
flour). In Zn-deficient location, grain Zn concentration increased research on bioavailability of grain Zn derived from late foliar
from 11 mg kg-1 to 22 mg kg-1 with foliar Zn application and to Zn applications. High bioavailability of grain Zn is important for
27 mg kg-1 with combined application of ZnSO4 to soil and human nutrition, and this would make the fertilization strategy
Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 9101
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