Zinc biofortification of wheat through fertilizer applications in different locations of China

Field Crops Research 125 (2012) 1–7

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Zinc biofortification of wheat through fertilizer applications in different locations of China Yue-Qiang Zhang a , Yi-Xiang Sun b , You-Liang Ye c , Md. Rezaul Karim a , Yan-Fang Xue a , Peng Yan a , Qing-Feng Meng a , Zhen-Ling Cui a , Ismail Cakmak d , Fu-Suo Zhang a , Chun-Qin Zou a,∗ a

Key Laboratory of Plant-Soil Interaction, MOE; Key Laboratory of Plant Nutrition, MOA; Department of Plant Nutrition, China Agricultural University, Beijing 100193, PR China Soil and Fertilizer Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, PR China c Department of Plant Nutrition, Henan Agricultural University, Zhengzhou 450002, PR China d Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956, Turkey b

a r t i c l e

i n f o

Article history: Received 15 June 2011 Received in revised form 3 August 2011 Accepted 8 August 2011 Keywords: Flour Grain Phytic acid Wheat Zinc

a b s t r a c t Zinc (Zn) deficiency caused by inadequate dietary intake is a global nutritional problem in human populations, especially in developing countries. Biofortification of wheat and other staple foods with Zn is, therefore, an important challenge and a high-priority research task. In this study, one field experiment was conducted to examine the effects of soil and foliar Zn application with or without foliar urea application on Zn nutrition in whole grain and flour of wheat, and on flour processing traits. A second field experiment was conducted at four locations in China to evaluate the adaptability of foliar Zn and/or urea application on the enrichment of grain with Zn in wheat. The results showed that foliar Zn application was much more effective than soil Zn application in enrichment of wheat grain with Zn. Compared with no foliar Zn application, foliar 0.4% ZnSO4 ·7H2 O application resulted in best effect on grain Zn, with 58% increase in whole grain Zn, 76% increase in wheat flour Zn, and up to 50% decrease in the molar ratio of phytic acid to Zn in flour. Foliar Zn application had little effect on flour processing traits including protein concentration, peak viscosity, and dough development time. The second experiment showed that foliar Zn application had reliable adaptability in biofortification of wheat with Zn, while had no yield penalty in regional scale. The results suggest that foliar Zn application represents an effective approach to provide more dietary Zn from wheat-derived products to humans. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Zinc (Zn) is an important micronutrient in biological systems and is receiving growing attention worldwide because of increasing reports about Zn deficiencies in human populations and crop plants (Alloway, 2004; Cakmak, 2008; Hotz and Brown, 2004). Zinc deficiency is considered one of the top five micronutrient deficiencies in humans and is conservatively estimated to negatively affect nearly 1/3 of the world’s populations (Hotz and Brown, 2004; Stein, 2010). In China, approximately 100 million people with the majority living in rural areas suffer from Zn deficiency (Ma et al., 2008). Inadequate dietary intake of Zn has greatly contributed to the prevalence of Zn deficiency in humans. As one of main cereal crops worldwide, wheat (Triticum aestivum L.) is a major dietary source of calories, proteins, and bioavailable micronutrients. In many of

∗ Corresponding author at: Department of Plant Nutrition, China Agricultural University, Beijing 100193, PR China. Tel.: +86 10 6273 3539; fax: +86 10 6273 1016. E-mail address: [email protected] (C.-Q. Zou). 0378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2011.08.003

the developing countries, wheat is responsible for about 50% of the daily calorie intake (Cakmak, 2008). China produces more wheat than any other country, and the North China Plain (NCP) supplies nearly 70% of the total production in China (Liu et al., 2010). Wheat and its products make up more than 20% of Zn supplied in food in China, and this proportion is even higher in rural areas and in North China (Ma et al., 2008). An excessive and monotonous consumption of wheat-based products rapidly results in Zn malnutrition because wheat is inherently low in Zn and rich in compounds such as phytate that limit Zn bioavailability (Cakmak et al., 2010a; Welch and Graham, 2004). It is estimated that more than half of the wheat crop was cultivated on slightly to severely low Zn soils (Alloway, 2004; Cakmak, 2008; Liu, 1996), which further reduces the levels of Zn in wheat grain. The adoption of high-yield cultivars (also known as “green revolution crops”) seems to have aggravated this problem (Cakmak et al., 2010a; Stein, 2010; Zhao and McGrath, 2009). Furthermore, the processing of wheat after harvest substantially reduces the concentration of Zn and also other minerals, which further increases the Zn deficiency in humans (Cakmak, 2008; Kutman et al., 2011; Zhang et al., 2010b). Increasing the Zn

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concentration and Zn bioavailability in wheat grain and especially in wheat flour, which is the most frequently consumed product, is now an urgent challenge (Cakmak, 2008; Welch and Graham, 2004; Zhao and McGrath, 2009). In response to this problem, many approaches have been proposed and applied in developed countries (Bouis, 2003; Pfeiffer and McClafferty, 2007). In developing countries, biofortification of food crops with micronutrients is receiving increased attention (Bouis and Welch, 2010; Cakmak, 2008; Zhao and McGrath, 2009). The key tools of the biofortification are breeding, biotechnology, and fertilization. Although the progress made in the breeding of new genotypes with high Zn is promising (Bouis and Welch, 2010; Cakmak et al., 2010a; Pfeiffer and McClafferty, 2007), fertilization of food crops with Zn represents a short-term and complementary strategy, which is necessary to build the Zn pool for uptake or translocation (Cakmak, 2008). Because Zn has moderate mobility in phloem (Haslett et al., 2001), foliar Zn application alone or in combination with soil Zn application significantly increased the Zn concentration in wheat grain (Cakmak, 2008). Furthermore, a recent study indicated that the Zn concentration in wheat grain was greatly affected by the pool of physiologically available Zn in vegetative tissues treated with foliar application of Zn after flowering (Cakmak et al., 2010b). Because of the lack of plant available Zn in soil (Liu, 1996) and the limited uptake of Zn by roots during grain filling in the dry season of the NCP (Liu et al., 2010), we suspect that the rate of foliar Zn application may be the major factor which may determine the size of the Zn pool in vegetative organs of wheat and thereby to increase the Zn concentration in grain under field conditions with adequate macronutrient fertilization. Foliar Zn application was also affective in increasing Zn concentration in wheat flour (endosperm) (Cakmak et al., 2010b; Zhang et al., 2010a). Furthermore, Zn application potentially decreased the phosphorus (P) concentration in grain, the phytic acid (PA) concentration in grain, and consequently the PA to Zn molar ratio, which is widely used as an indicator of Zn bioavailability in diets (Cakmak et al., 2010a; Erdal et al., 2002). Similarly, increasing the rates of foliar Zn application around flowering stage is expected to increase the Zn concentration and bioavailability in flour to higher extent. A potential problem is that an increase in Zn concentration in flour may negatively affect its processing traits (Gomez-Becerra et al., 2010a; Peck et al., 2008), which could affect the acceptance of biofortified flour by consumers (Bouis, 2003; Welch and Graham, 2004). Increasing evidence has demonstrated the positive effects of nitrogen (N) on the Zn concentration in grain. Recent studies showed that adequate N supplied either by soil or foliar application maximized the Zn concentration in wheat grain when the Zn supply is not limited (Cakmak et al., 2010b; Kutman et al., 2010; Shi et al., 2010). It seems that N affects major steps in the route of Zn from vegetative organs to the grain, including root uptake, root-to-shoot transport and retranslocation of Zn by phloem, and finally accumulation in grain fractions (Cakmak et al., 2010a; Erenoglu et al., 2011). These results highlight importance of N nutritional status of plants in a successful biofortification of food crops with Zn (Cakmak et al., 2010a; Shi et al., 2010). Stability of a high Zn trait in newly developed (biofortified) genotypes is an important issue in the success of the breeding programs (Welch and Graham, 2004). Previous studies have, however, shown that the Zn concentrations in the wheat grain were largely affected by environment, genotype, and their interactions (Gomez-Becerra et al., 2010b; Joshi et al., 2010; Zhang et al., 2010b). Overall, very few attempts have been made under field conditions to explore the effects of fertilizer applications on concentrations and predicted bioavailability of Zn in wheat grain and mostly consumed flour, and to evaluate their adaptability in biofortification of wheat with Zn in regional scale. This study, therefore,

had two objectives. The first objective was to investigate the effect of the rates of foliar Zn application with or without soil Zn application and/or foliar N application at late growth stage on wheat grain yield, Zn and N nutrition in both grain and flour, and on the processing quality in flour of wheat grown under field conditions in China. The second objective was to evaluate the adaptability of foliar Zn application for Zn biofortification of wheat under different environments in China with local cultivars and optimal N input in fields. 2. Materials and methods 2.1. Field locations and materials Field experiments were conducted at five locations in three provinces of China (Table 1). The locations Feidong and Fengtai in Anhui province are in the southern NCP. The Wenxian location in Henan province is in the middle of the NCP, and the Quzhou location in Hebei Province is in the northern NCP. For analysis of basic soil properties, soil samples were collected at 0–30 cm depth at all locations before preplant fertilization. The properties of the soils are presented in Table 1. The most commonly cultivated wheat cultivars at each location were used in the experiments (Table 1). 2.2. Experimental design Two correlative field experiments were conducted during the 2008–2009 wheat cropping season. The first experiment was conducted at the Quzhou1 location. The experimental design was a split plot in randomized complete block design with four replicates. Main plot treatments consisted of two soil Zn application rates: 0 and 50 kg ha−1 of ZnSO4 ·7H2 O (23% Zn). Subplot treatments were foliar Zn applications at four ZnSO4 ·7H2 O rates: 0, 0.2%, 0.4%, and 0.5%. And sub-subplot treatments were foliar urea applications at two rates: 0 and 1% urea (46% N). Thus, there were 64 plots with each 9 m2 (3 × 3 m). Before sowing, 80 kg N ha−1 as urea, 35 kg P ha−1 as superphosphate, and 62 kg K ha−1 as potassium sulfate were applied and then mixed into soil by plow. Before the jointing stage, 120 kg N ha−1 as urea was topdressed. Field water condition was managed by flood irrigation with 3 times at prewintering, jointing and flowering stage with each 60 mm. Foliar applied solutions contained 0.01% (v/v) Tween 20 and were sprayed at the booting stage and 7 days after anthesis. The solution volume of 600 L ha−1 was used in all treatments. At maturity, 2 m2 of wheat plants in the center of each plot were harvested for determination of grain yield and yield components. The harvested grain was threshed and stored for milling and nutrient analysis. The second experiment was conducted at four locations (Quzhou2, Feidong, Fengtai, and Wenxian). These locations are main regions of winter wheat production in China and represent different ecological conditions (Liu et al., 2010). Three treatments were included at each location: control (recorded as−Zn); foliar application of Zn at the rate of 0.4% ZnSO4 ·7H2 O (recorded as +Zn); and foliar application of Zn at the rate of 0.4% ZnSO4 ·7H2 O plus N at the rate of 1% urea (recorded as +Zn+urea). The basal amounts of NPK fertilizers used in each location were based on the local optimum recommendation. The fertilization consisted of a preplanting application of 105 kg N ha−1 and 39 kg P ha−1 and 75 kg K ha−1 , and the first topdressing of 60 kg N ha−1 , and the second topdressing of 45 kg N ha−1 at Fengtai and Feidong locations. The fertilization consisted of a preplanting application of 120 kg N ha−1 and 26 kg P ha−1 and 50 kg K ha−1 , and the topdressing of 120 kg N ha−1 and 13 kg P and 25 kg K ha−1 at jointing stage at Wenxian location. The fertilization consisted of a preplanting application of 68 kg N ha−1 and 52 kg P ha−1 and 83 kg K ha−1 , and

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Table 1 The geographic coordinate, cultivars used and soil properties at each location. Location

Quzhou1 Quzhou2 Wenxian Fengtai Feidong

Province

Hebei Hebei Henan Anhui Anhui

Geographic coordinate

36.8735◦ N, 115.0191◦ E 36.8724◦ N, 115.0201◦ E 34.9011◦ N, 112.9974◦ E 32.7157◦ N, 116.7095◦ E 31.9700◦ N, 117.5830◦ E

Cultivar

Han6172 Liangxing99 Ping’an8 Yannong19 Yangmai16

the first topdressing of 45 kg N ha−1 at regreening stage and the second topdressing of 150 kg N ha−1 at jointing stage at Quzhou2 location. Methods of foliar application and harvest were the same as described in the first experiment. 2.3. Sample preparation and analysis The grain samples were carefully and rapidly washed three times with deionized water with each 30 s, and were then dried at 60 ◦ C. Flour was obtained with a Quadrumat Junior mill (Brabender, Duisburg, Germany) according to the AACC approved method 26-21A (AACC, 2000); the flour extraction rate was 60–65%. Flour protein (dry weight basis) was measured with a nearinfrared transmittance analyzer Foss-Tecator 1241 (Foss, Hoganas, Sweden), which was calibrated by the Kjeldahl method (AACC approved method 46-12). The peak viscosity was measured with a rapid visco analyzer (RVA, Australia). Dough development time (DDT) was measured with a farinograph according to AACC approved method 54–21. Phytic acid (PA) concentrations in grain and flour samples without soil Zn application were determined by the anion-exchange method (Ma et al., 2005). A calibration curve for the colorimetric method was obtained with PA standards (P-3168, Sigma Co.). The PA/Zn molar ratio in samples was calculated by dividing millimoles of PA with millimoles of Zn. The samples were digested with HNO3 -H2 O2 by a microwave accelerated reaction system (CEM, Matthews, USA), and the Zn and P concentrations in the digested solution were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Shi et al., 2010). The N concentration in grain samples was analyzed by the micro-Kjeldahl procedure after digestion with H2 SO4 -H2 O2 . 2.4. Data analysis SAS software (SAS 8.0, USA) was used for statistical analysis. Data (except PA and PA/Zn in flour samples) in the first experiment were analyzed with a three-factor ANOVA procedure for split-plot design. Means were separated by Fisher’s protected least significance difference (LSD) at P < 0.05. The data for PA and PA/Zn in flour were analyzed with a two-factor ANOVA. The general linear models were used to evaluate the response of Zn concentration in grain and flour, and PA/Zn in flour to foliar Zn rates under different rates of soil Zn application and foliar urea application. Data in the second experiments were analyzed using a one-factor ANOVA, and means were separated by LSD at P < 0.05. 3. Results 3.1. Grain yield and grain nutrient concentrations in the first experiment Grain yield, harvest index, and thousand kernel weight of winter wheat were unaffected by applications of Zn or urea in the first experiment (Table 2). The concentration of Zn in grain was significantly affected by soil application of Zn, foliar application of Zn,

Soil property pH

Total N (g kg−1 )

DTPA-Zn (mg kg−1 )

7.3 7.3 8.0 6.2 5.7

0.62 0.65 1.21 0.53 0.94

0.40 0.38 0.72 0.74 1.59

and foliar application of urea but the interaction among the three factors was not significant (Table 2). The increase in the grain Zn concentration was less with soil application of Zn or foliar application of urea than with foliar application of Zn. Compared with the control, grain Zn concentrations were increased by 39%, 58%, and 73% by foliar application of ZnSO4 ·7H2 O at rates of 0.2%, 0.4%, and 0.5%, respectively (Table 2). Grain Zn concentration was positively correlated with foliar Zn rates (Fig. 1A). The increases in grain Zn concentrations caused by increasing the rate of foliar Zn application was not associated with any adverse effect on grain yield. Grain N and P concentrations were also not affected by Zn application or by foliar application of urea in the first experiment (Table 2).

3.2. Flour Zn, P, PA, and PA/Zn in the first experiment Both soil and foliar application of Zn significantly increased the Zn concentration in flour, but the increase was greater with foliar application than with soil application (Table 3). When compared with the control treatment, foliar application of ZnSO4 ·7H2 O at rates of 0.2%, 0.4%, and 0.5% increased the Zn concentration in flour by 60%, 76%, and 76%, respectively. According to the regression equation, the optimal Zn foliar application rate for achieving the highest Zn concentration in flour was 0.41% ZnSO4 ·7H2 O (Fig. 1B). P and PA concentrations in flour were not significantly affected by Zn application and/or foliar urea application except that foliar application of 0.4% ZnSO4 ·7H2 O reduced the PA concentration (Table 3; Fig. 1C). Consequently, the PA/Zn was mainly dependent on the Zn concentration in flour. The PA/Zn was continuously decreased with the increased rates of foliar ZnSO4 ·7H2 O application up to 0.4%; further increases in the application rate of foliar Zn increased the PA/Zn (Table 3; Fig. 1D). Foliar application of urea did not significantly affect the PA/Zn in wheat flour (Table 3).

3.3. Flour processing traits in the first experiment Zinc application had no effect on flour traits including protein concentration, peak viscosity, and DDT (Table 4). Foliar application of urea remained without effect on peak viscosity or DDT but slightly increased the protein concentration in wheat flour (Table 4).

3.4. Adaptability of foliar applications of Zn at different locations in the second experiment Foliar application of Zn with or without urea did not significantly affect grain yield at any location, but significantly increased the Zn concentration in grain at all four locations (Table 5). Compared with control, the increase in grain Zn concentration caused by foliar application of Zn was 38, 26, 44, and 69% at Quzhou2, Wenxian, Fengtai and Feidong locations, respectively. Foliar application of Zn with or without urea did not significantly affect the concentrations of N, P, or PA in grain, but significantly decreased the PA/Zn in grain at all locations (Table 5).

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Table 2 The grain yield and grain nutrient concentrations of winter wheat as affected by application of Zn and urea in the first experiment at the Quzhou1 location. Treatment

Grain yield (t ha−1 )a

Soil application rate of ZnSO4 ·7H2 O (kg ha−1 ) 5.4 0.0 5.5 50.0 nsb LSD0.05 Foliar application rate of ZnSO4 ·7H2 O (%) 0.0 5.4 5.6 0.2 5.5 0.4 5.4 0.5 ns LSD0.05 Foliar application rate of urea (%) 5.5 0.0 1.0 5.4 ns LSD0.05 Significance of variation ns Soil Zn (S) ns Foliar Zn (F) ns Urea (U) ns S×F S×U ns ns F×U S×F×U ns

Harvest index (%)

TKW (g)

Grain nutrient concentration Zn (mg kg−1 )

N (g kg−1 )

P (g kg−1 )

45.3 45.3 ns

37.1 37.4 ns

37.1 38.7 1.0

22.7 22.1 0.5

3.1 3.1 ns

45.3 45.3 45.7 45.0 ns

37.9 37.4 36.9 36.7 ns

26.6 37.1 42.0 46.0 1.5

22.4 22.1 22.6 22.4 ns

3.1 3.0 3.1 3.1 ns

45.8 44.8 ns

37.4 37.1 ns

36.9 38.9 1.0

22.6 22.1 ns

3.1 3.0 ns

ns ns ns ns ns ns ns

ns ns ns ns ns ns ns

**

*

***

ns ns ns ns ns ns

ns ns ns ns ns ns ns

***

ns ns ns ns

TKW: thousand kernel weight. a The yield was calculated on the basis of 13% moisture. b Not significant. * Significant at P < 0.05. ** Significant at P < 0.01. *** Significant at P < 0.001.

Fig. 1. The relationship between the rates of foliar Zn application and Zn concentrations in wheat grain (A) and flour (B) and the PA concentration (C) and PA/Zn molar ratio (D) in wheat flour in the first experiment at the Quzhou1 location. PA: phytic acid.

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Table 3 Nutritional traits of wheat flour as affected by application of Zn and urea in the first experiment at the Quzhou1 location. Treatment

Nutrient traits in wheat flour Zn (mg kg−1 )

Soil application rate of ZnSO4 ·7H2 O (kg ha−1 ) 9.5 0 10.1 50 0.5 LSD0.05 Foliar application rate of ZnSO4 ·7H2 O (%) 0 6.4 10.3 0.2 11.3 0.4 11.3 0.5 0.8 LSD0.05 Foliar application rate of urea (%) 9.7 0 1.0 10.0 ns LSD0.05 Significance of variation * Soil Zn (S) *** Foliar Zn (F) Urea (U) ns ns S×F ns S×U * F×U S×F×U ns

Total P (g kg−1 )

PA (g kg−1 )

PA/Zn

0.81 0.80 nsb

0.84 –a

9.3 –

0.82 0.81 0.78 0.81 ns

0.86 0.83 0.77 0.88 0.09

14.2 8.2 7.0 8.0 1.0

0.81 0.80 ns

0.84 0.83 ns

9.3 9.4 ns

ns ns ns ns ns ns ns

– ns ns – – ns –

– ***

ns – – **

–

PA: phytic acid. a Not measured. b Not significant. * Significant at P < 0.05. ** Significant at P < 0.01. *** Significance at P < 0.001.

4. Discussion In the current study, application of Zn without or with urea did not affected grain yield or yield components at all locations. This might be due to relatively high DTPA-extractable Zn concentrations in the experimental soils and thus the high Zn nutritional status of plants in the absence of Zn application and adequate soil water

content by irrigation. On low Zn soils, Zn concentrations in cereal grain are generally below 15 mg kg−1 (Cakmak et al., 2010a, 2010b; Erdal et al., 2002). In the present study, grain Zn concentrations were higher than 20 mg kg−1 which indicated that the experimental plants were not under Zn-deficiency stress. In contrast to grain yield, Zn concentrations in grain were significantly increased by Zn application at all locations. The Zn

Table 4 The processing quality traits of wheat flour as affected by application of Zn and urea in the first experiment at the Quzhou1 location. Treatment

Flour processing traits Protein concentration (%)

Soil application rate of ZnSO4 ·7H2 O (kg ha−1 ) 13.3 0.0 13.3 50.0 nsa LSD0.05 Foliar application rate of ZnSO4 ·7H2 O (%) 13.2 0.0 13.4 0.2 13.4 0.4 0.5 13.1 LSD0.05 ns Foliar application rate of urea (%) 13.0 0 13.6 1.0 0.3 LSD0.05 Significance of variation ns Soil Zn (S) ns Foliar Zn (F) *** Urea (U) ** S×F ns S×U ns F×U S×F×U ns DDT: dough development time. a Not significant. * Significant at P < 0.05. ** Significant at P < 0.01. *** Significance at P < 0.001.

Peak viscosity (RVU)

DDT (min)

206 203 ns

4.5 4.5 ns

205 202 207 204 ns

4.5 4.4 4.6 4.7 ns

205 204 ns

4.6 4.5 ns

ns ns ns ns ns ns ns

ns ns ns ns ns *

ns

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Table 5 Grain yield, Zn concentration, PA concentration and PA/Zn molar ratio of wheat as affected by application of Zn and urea at different locations in NCP (the second field experiment). Treatment

Location Quzhou2

Wenxian

Fengtai

Feidong

6.2a 6.1a 6.1a

6.7a 6.0a 6.2a

6.3a 6.4a 6.4a

35.0b 44.1a 43.3a

20.8b 29.8a 31.6a

24.8b 42.0a 48.5a

21.8a 21.7a 22.0a

20.9a 21.0a 21.3a

19.5a 19.0a 19.8a

3.0a 2.8a 3.2a

2.5a 2.5a 2.4a

2.7a 2.8a 2.7a

6.9a 7.0a 7.0a

6.8a 7.1a 7.0a

6.4a 6.5a 6.2a

19.6a 15.1b 16.0b

32.1a 23.5b 22.0b

25.5a 15.4b 12.1b

−1

Grain yield (t ha ) −Zn 6.2a a +Zn 6.0a 5.9a +Zn+urea Grain Zn concentration (mg kg−1 ) 25.3b −Zn +Zn 34.9a +Zn+urea 35.2a Grain N concentration (g kg−1 ) 21.7a −Zn 21.3a +Zn 21.9a +Zn+urea Grain P concentration (g kg−1 ) 3.0a −Zn 3.0a +Zn 3.0a +Zn+urea Grain PA concentration (g kg−1 ) 7.4a −Zn +Zn 7.3a +Zn+urea 7.0a Grain PA/Zn molar ratio 32.3a −Zn 21.4b +Zn +Zn+urea 19.6b

a In each column, means for a parameter with the same letters are not significantly different at P < 0.05.

concentrations in grain increased gradually with increased application rates of foliar Zn, which was similar with previous results (Cakmak et al., 2010b; Modaihsh, 1997). The relationship between the Zn concentrations in grain and the foliar application rates of Zn was well described by a linear regression equation (Fig. 1A). These results are consistent with the hypothesis that a high pool of available Zn within plant tissues after flowering is the primary limitation for the retranslocation of Zn to grain when adequate NPK fertilizers are supplied in fields of the NCP. In well agreement with these observations, Cakmak et al. (2010b) showed in field tests that increasing pool of Zn in the vegetative tissue during the reproductive growth stages (for example by spraying foliar Zn fertilizers) represents an important field practice in maximizing accumulation of Zn in grain. To have a measurable effect on human health, biofortification should increase the Zn concentration in wheat grain to about 40–45 mg kg−1 (Ortiz-Monasterio et al., 2007). This Zn concentration in grain was achieved by foliar Zn application at three of five locations in the current study. The targeted levels of Zn in grain for better human nutrition can be easily reached by optimizing rate and timing of foliar Zn application (Cakmak et al., 2010b). Fertilizer strategy (e.g., agronomic biofortification) appears as a highly effective short-term solution to micronutrient malnutrition problem and should be implemented in the target countries, at least until currently on-going breeding programs develop promising wheat varieties with high grain Zn concentrations (Cakmak, 2008; Cakmak et al., 2010b; Zhao and McGrath, 2009). Foliar application of Zn was much superior to soil application of Zn for increasing Zn concentrations in grain and flour, even though much less Zn is applied in the foliar than in the soil application (Cakmak et al., 2010b; Erdal et al., 2002). Adding Zn to soil is relatively inefficient because of the poor mobility of Zn in soil and because of rapid adsorption of Zn in calcareous and/or clayey soils with neutral or higher pH (Alloway, 2004). Another problem with soil application of Zn is that the wheat roots and applied Zn may have different distributions in the soil profile, which

would decrease the uptake by roots (Holloway et al., 2010). In addition, in most cases, under field conditions topsoil with high density of roots is often dry during the reproductive growth stage; and meanwhile the root activity is generally declined due to less photo-assimilate allocation. Thus root uptake from soil or fertilizer Zn is severely limited and therefore accumulation of Zn in grain is largely dependent on retranslocation of Zn from the vegetative tissues (Cakmak, 2008; Cakmak et al., 2010b). Therefore, foliar application of Zn in such environments (e.g., NCP) represents an efficient practice to maintain high Zn concentrations in the vegetative tissue during the retranslocation period and to contribute significantly to Zn biofortification of wheat grain with Zn under field conditions. Foliar application of Zn to winter wheat increased not only the Zn concentration in the whole grain but also the Zn concentration in the flour. Irrespective of Zn application, Zn concentrations were much lower in the flour than in the whole grain, which is consistent with published results (Cakmak et al., 2010b; Kutman et al., 2011; Zhang et al., 2010a). As the case with Zn concentrations in whole grain, Zn concentrations in flour were not increased by soil application of Zn or foliar application of urea. The proportional increase in Zn concentration caused by foliar application of 0.4% ZnSO4 ·7H2 O was greater in flour than in whole grain, but further increases in the foliar application rate caused minimal increases in the flour Zn concentrations but substantial increases in the whole grain Zn concentration (Fig. 1A and B). The failure of Zn concentration in flour to increase with increased application rate may reflect the negative feedback of the crease phloem, which may play a key role in Zn compartmentation among the roadmap of Zn transport from nucellar projection to target endosperm (Pearson et al., 1998). Recent evidence demonstrates that crease phloem is the key path for delivery of Zn to the endosperm (Cakmak et al., 2010b). Based on the results presented here and those of Cakmak et al. (2010b), foliar application of 0.4% ZnSO4 ·7H2 O can be recommended for the biofortification of wheat with Zn without causing any foliar damage and without reducing yield. Phosphorus and PA concentrations in whole grain or flour were generally not affected by Zn and/or urea application at any location. The PA/Zn molar ratio, however, was substantially decreased with the increase of Zn concentration in grain or flour. The relationship between PA/Zn in flour and the foliar application rate of ZnSO4 ·7H2 O was well described by a quadratic equation (Fig. 1D). This finding indicated that the effect of foliar Zn application on predicted Zn bioavailability was dose-dependent and that foliar Zn application is useful to increase Zn bioavailability not only in whole grain but also in wheat flour (Cakmak et al., 2010a; Kutman et al., 2011). Foliar applications of urea at the late growth stage, however, had only minor effects on Zn and N concentrations in grain at all five locations in this study. The likely explanation is that the basal N application was adequate for wheat production (Cui et al., 2010) and that the dose of foliar urea application was too low to increase the N concentration in grain. Kutman et al. (2010) documented synergistic effects between Zn and N when the concentration of both elements in grain varied in a given experiment. Depending on the results from all locations in the NCP, we conclude that the N nutrition of the experimental plants was not the limiting factor for the retranslocation of Zn from vegetative tissue to grain and Zn accumulation in grain. Flour processing traits such as peak viscosity and DDT are important parameters for cooking and taste. In the current study, foliar application of Zn with or without urea had little effect on flour processing traits. This suggested that biofortified flour with Zn may not affect the acceptance by consumers, likely due to the small quantities of Zn in flour (Bouis, 2003).

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5. Conclusion Foliar Zn application significantly increased the Zn concentration and the predicted bioavailability in both whole grain and flour of wheat. The extent of increasing concentration and predicted bioavailability of Zn depended mostly on the rate of foliar Zn application rather than soil Zn application or foliar urea application. Foliar application of Zn at the rate of 0.4% ZnSO4 ·7H2 O provided the best benefit to increase the Zn concentration in wheat flour and to decrease the molar ratio of phytic acid to Zn in flour. Up to regional scale, foliar Zn application showed a reliable adaptability in biofortification of various wheat cultivars with Zn without causing any yield penalty. A supplementation of foliar urea seems small importance in Chinese wheat production because of a general optimal or excessive N supply. Foliar Zn application, therefore, represents a preferential agronomic practice to deliver more Zn from wheat-derived products to people in China and other countries. Acknowledgements This research was supported by the 973 project (No. 2009CB118605), the National Natural Science Foundation of China (30871592), an Innovative Group Grant of NSFC (300821003), Special Fund for Agro-scientific Research in the Public Interest (201103003) and the HarvestPlus China program (#8231). The authors would like to thank Dr. Yong Zhang in Chinese Academy of Agricultural Sciences for analyzing processing traits of flour and Dr. Jianfen Liang in China Agricultural University for her assistance in PA analysis. References AACC, 2000. Approved Methods of the American Association of Cereal Chemists, 10th ed. AACC International, St. Paul, USA. Alloway, B.J., 2004. Zinc in soils and crop nutrition. IZA Publications, Brussels. Bouis, H.E., Welch, R.M., 2010. Biofortification - a sustainable agricultural strategy for reducing micronutrient malnutrition in the global south. Crop Sci. 50, S20–S32. Bouis, H.E., 2003. Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proc. Nutr. Soc. 62, 403–411. Cakmak, I., 2008. Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant Soil 302, 1–17. Cakmak, I., Kalayci, M., Kaya, Y., Torun, A.A., Aydin, N., Wang, Y., Arisoy, Z., Erdem, H., Yazici, A., Gokmen, O., Ozturk, L., Horst, W.J., 2010b. Biofortification and localization of zinc in wheat grain. J. Agric. Food Chem. 58, 9092–9102. Cakmak, I., Pfeiffer, W.H., McClafferty, B., 2010a. Biofortification of durum wheat with zinc and iron. Cereal Chem. 87, 10–20. Cui, Z., Chen, X., Zhang, F., 2010. Current N management status and measures to improve the intensive wheat–maize system in China. Ambio 39, 376–384. Erdal, I., Yilmaz, A., Taban, S., Eker, S., Torun, B., Cakmak, I., 2002. Phytic acid and phosphorus concentrations in seeds of wheat cultivars grown with and without zinc fertilization. J. Plant Nutr. 25, 113–127. Erenoglu, E.B., Kutman, U.B., Ceylan, Y., Yildiz, B., Cakmak, I., 2011. Improved N nutrition enhances root uptake, root-to-shoot translocation and remobilization of zinc (65 Zn) in wheat. New Phytol. 189, 438–448.

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