Improved nitrogen nutrition enhances root uptake, root-to-shoot translocation and remobilization of zinc (65Zn) in wheat

Authors


Author for correspondence:
Ismail Cakmak
Tel: +90 216 4839524
Email: cakmak@sabanciuniv.edu

Summary

  • This study focussed on the effect of increasing nitrogen (N) supply on root uptake and root-to-shoot translocation of zinc (Zn) as well as retranslocation of foliar-applied Zn in durum wheat (Triticum durum).
  • Nutrient solution experiments were conducted to examine the root uptake and root-to-shoot translocation of 65Zn in seedlings precultured with different N supplies. In additional experiments, the effect of varied N nutrition on retranslocation of foliar-applied 65Zn was tested at both the vegetative and generative stages.
  • When N supply was increased, the 65Zn uptake by roots was enhanced by up to threefold and the 65Zn translocation from roots to shoots increased by up to eightfold, while plant growth was affected to a much smaller degree. Retranslocation of 65Zn from old into young leaves and from flag leaves to grains also showed marked positive responses to increasing N supply.
  • The results demonstrate that the N-nutritional status of wheat affects major steps in the route of Zn from the growth medium to the grain, including its uptake, xylem transport and remobilization via phloem. Thus, N is a critical player in the uptake and accumulation of Zn in plants, which deserves special attention in biofortification of food crops with Zn.

Introduction

Zinc (Zn) deficiency affects billions of people worldwide, especially in developing countries, where the diet is based on cereal grains with very low Zn concentrations (Welch & Graham, 2004; Cakmak et al., 2010b). As the fifth leading cause of disease in low-income countries according to the World Health Organization (WHO, 2002), Zn deficiency causes various health complications such as growth stunting, impaired mental development, high susceptibility to infectious diseases and poor birth outcomes (Kennedy et al., 2003; Hotz & Brown, 2004; Black et al., 2008).

Since the major cause of Zn deficiency in humans is insufficient dietary intake, and cereals such as wheat and rice are the most important sources of calories in many countries, increasing the Zn concentrations of cereal grains is currently a high-priority research area (Bouis, 2003; Cakmak, 2008; Stomph et al., 2009; White & Broadley, 2009). The recent literature indicates that the combination of agronomic biofortification with breeding is an applicable and sustainable approach to the Zn-deficiency problem in humans (Pfeiffer & McClafferty, 2007; Cakmak, 2008). Nitrogen (N) management appears to be a promising agronomic strategy for the biofortification of wheat with Zn. Under both glasshouse and field conditions, sufficiently high N application is effective in enhancing grain Zn concentration in wheat, especially if the plant-available Zn is sufficiently high in the soil or sufficient Zn is applied foliarly (Cakmak et al., 2010b; Kutman et al., 2010).

There are various physiological steps in the route taken by Zn from the rhizosphere to grains which are of great importance for accumulation of Zn in grain, such as root uptake, root-to-shoot translocation and remobilization (retranslocation) of Zn. The rate of root uptake of Zn may show high dependency on the activities of transporter proteins located in the plasma membrane root cells. Although it is not yet known which proteins are primarily responsible for root Zn uptake, it has been reported that zinc-regulated transporter/iron-regulated transporter-like proteins (ZIPs), including iron-regulated transporter 1 (IRT1), contribute to root Zn uptake (Ishimaru et al., 2005; Palmer & Guerinot, 2009). The root-to-shoot translocation of Zn might also be affected by the activity of transporter proteins contributing to xylem loading and unloading. Heavy metal ATPase (HMA) family proteins and yellow stripe-like (YSL) transporters have been implicated in xylem loading and unloading of Zn, respectively (Curie et al., 2009; Palmer & Guerinot, 2009). Phloem loading and unloading of Zn are important steps in determining the efficiency of Zn retranslocation from source to sink tissues. During the grain-filling stage, the sink activity of grains for Zn may be the driving force for Zn retranslocation. The nonproteinogenic amino acid nicotianamine (NA), which is the precursor of the mugineic acid (MA) family of phytosiderophores, is an important nitrogenous compound involved in phloem loading and mobility of Zn (Curie et al., 2009; Trampczynska et al., 2010). Phytosiderophores such as deoxymugineic acid also contribute to Zn translocation (Suzuki et al., 2008). Members of the YSL family, which is thought to be responsible for the phloem loading and unloading of NA-chelated Zn, are required for Zn remobilization from senescing leaves (Waters et al., 2006; Curie et al., 2009).

The link between senescence and enhanced remobilization of mineral nutrients is well established (Marschner, 1995). In recent genetic studies, the involvement of NAC (NAM, ATAF and CUC) family transcription factors in retranslocation of N, Zn and iron (Fe) during senescence has been documented. The activity of the NAM-B1 gene, associated with accelerated senescence, increases the efflux of nutrients including amino acids, Zn and Fe, and thereby results in higher grain partitioning of these nutrients in wheat (Uauy et al., 2006a,b; Distelfeld et al., 2007; Waters et al., 2009). Similarly, in rice, the OsNAC1 gene encodes a senescence-associated and abscisic acid (ABA)-dependent NAC transcription factor, which appears to be involved in the remobilization of amino acids, Zn and Fe from green tissues to seeds (Sperotto et al., 2009).

In a number of reports, radio-labeled Zn (65Zn) has been utilized for studying Zn uptake and root-to-shoot translocation (Cakmak et al., 1998; Erenoglu et al., 1999; Haslett et al., 2001) as well as remobilization (Erenoglu et al., 2001, 2002; Hajiboland et al., 2001; Haslett et al., 2001). These studies have contributed to our understanding of the physiological responses of cereals to Zn deficiency and the reasons behind the differential Zn-deficiency tolerance of genotypes. The roles of phloem and xylem transport in grain allocation of Zn have also been investigated in wheat (Pearson et al., 1995, 1996). The recent literature indicates that improved N-nutritional status of plants significantly enhances grain accumulation of Zn in wheat (Cakmak et al., 2010a,b; Kutman et al., 2010; Shi et al., 2010). However, to our knowledge, there is no experimental evidence regarding how root uptake, root-to-shoot transport and retranslocation from the source into sink organs of Zn are affected by N nutritional status of plants. This paper presents the results of several experiments dealing with the effects of varying N supply on root 65Zn uptake, root-to-shoot translocation of 65Zn, retranslocation of 65Zn from old leaves during vegetative growth, and retranslocation of 65Zn from flag leaves into developing grains in wheat.

Materials and Methods

Three solution culture experiments and one soil culture experiment were carried out in order to investigate the role of N supply in root uptake and root-to-shoot translocation of Zn. In addition, one solution experiment and one soil culture experiment were conducted for studying the effect of N nutrition on Zn retranslocation.

Growth chamber conditions

All solution culture experiments and the first soil culture experiment (uptake-related) were performed in a growth chamber under controlled climatic conditions (light : dark periods, 16 : 8 h; temperature (light : dark), 22 : 18°C; relative humidity (light : dark), 60 : 70%; photosynthetic flux density, 400 μmol m−2 s−1).

Solution culture

Seeds of durum wheat (Triticum durum Desf. cv Balcali2000) were imbibed in saturated CaSO4 solution for 0.5 h and germinated in perlite moisturized with saturated CaSO4 solution for 4–5 d at room temperature before being transferred to solution culture. Seedlings were grown in plastic pots containing 3 l of nutrient solution consisting of 0.9 mM K2SO4, 0.2 mM KH2PO4, 1 mM MgSO4.7H2O, 0.1 mM KCl, 100 μM Fe-EDTA, 1 μM H3BO3, 0.5 μM MnSO4.H2O, 0.2 μM CuSO4.5H2O, 0.2 μM NiCl2.6H2O and 0.14 μM (NH4)6Mo7O24.4H2O. Depending on the experiment and treatment group, different amounts of Zn were supplied in the form of ZnSO4.7H2O, and different concentrations of N were established by adding Ca(NO3)2.4H2O. Low N and medium N pots were supplemented with CaCl2.2H2O for complementing missing Ca. Nutrient solutions were continuously aerated and refreshed every 3–4 d. Depending on the nitrate supply, the pH of the nutrient solutions before refreshing ranged between 7.3 (low-N plants) and 7.6 (high-N plants).

Soil culture

Seeds of durum wheat (T. durum cv Balcali2000) were sown in plastic pots containing Zn-deficient soil, which was transported from a Zn-deficient location in central Anatolia (Cakmak et al., 1996). The soil is calcareous (18% CaCO3), has clay-loam texture, high pH (8.0 in dH2O) and low organic matter content (1.5%). As determined using the method described by Lindsay & Norvell (1978), the concentration of diethylenetriamine pentaacetic acid (DTPA)-extractable Zn is 0.1 mg kg−1. Before the seeds were sown, the following nutrients were homogeneously incorporated in the experimental soil as follows (kg–1 dry soil): 100 mg P in the form of KH2PO4, 25 mg S in the form of K2SO4 and 2.5 mg Fe in the form of Fe-EDTA. Depending on the experiment and treatment group, different amounts of Zn and N were supplied in the forms of ZnSO4.7H2O and Ca(NO3)2.4H2O, respectively. The pots were watered with deionized water, when required.

Uptake and root-to-shoot translocation experiments

In the first experiment, 30 seedlings per pot were precultured in nutrient solution for 6 d with 0.5 mM (low), 1.0 mM (medium) or 4.0 mM (high) N and without or with 1 μM Zn supply. The roots of these 11-d-old seedlings were washed in 0.5 mM CaSO4 for 30 min and then single seedlings were transferred to micronutrient-free uptake solution containing only macronutrients and 1 μM ZnSO4 labeled with 123 kBq 65Zn. They were kept in the uptake solution for 2 h. The pH value of the uptake solution was between 5.5 (low-N plants) and 5.6 (high-N plants) during the 2 h uptake experiment. Thereafter, the roots were once again washed in 0.5 mM CaSO4 for 15 min, and the plants were transferred to 65Zn- and micronutrient-free nutrient solution containing only macronutrients, where they were kept for 24 h for the measurement of root-to-shoot translocation of 65Zn. The roots and shoots of the 12-d-old plants were harvested separately, oven-dried at 60°C and analyzed by PerkinElmer 2480 WIZARD2 Automatic Gamma Counter (PerkinElmer, Waltham, Massachusetts, USA).

In the second experiment, the seedlings were precultured in nutrient solution at the 3 N concentrations used in the first experiment for 17 d. All plants were supplied with marginal Zn (0.1 μM) during the preculture period. The 65Zn uptake and translocation experiment was started with 21-d-old marginal-Zn plants and conducted as already described.

The third experiment was a time-course depletion experiment in which seedlings were precultured in nutrient solution for 14 d with a 0.2 μM Zn supply. For the first 7 d of the preculture, all plants were first supplied with 1.0 mM N, and then, in order to study the effect of varied N supply on depletion of Zn from nutrient solution, plants were supplied with 0.1, 0.5  or 2.0 mM N (low, medium or high N, respectively) for the next 7 d. To start the depletion experiment, the roots of the seedlings were washed in 0.5 mM CaSO4 for 30 min and then transferred to micronutrient-free solution containing only macronutrients and 1 μM ZnSO4 labeled with 1 μCi 65Zn. The radioactivity in 5 ml of nutrient solution was measured by PerkinElmer 2480 WIZARD2 Automatic Gamma Counter at 12 different time points (0, 0.5, 1, 2, 3, 4, 6, 18, 24, 32, 48 and 72 h). Throughout the experiment, the volume of solution in the pots was kept constant by adding dH2O.

In the fourth experiment, plants were grown in soil culture under growth chamber conditions as already described. It was a four-replicate experiment with a factorial design. The soil was fertilized with 0.01 or 5.0 mg kg−1 Zn (low or high Zn, respectively); and 50, 100  or 200 mg kg−1 N (low, medium or high N, respectively). When the plants were 29 d old, the shoots were harvested and oven-dried at 60°C. The Zn and N concentrations were determined as described later (element analysis).

Retranslocation experiments

The first retranslocation experiment was conducted in a solution culture by testing the effect of N supply on Zn retranslocation in young wheat seedlings. Seedlings were precultured in nutrient solution for 6 d with 0.25, 1.0 or 4.0 mM N (low, medium or high N, respectively) and without or with 1 μM Zn supply. The tips (5 cm sections) of the oldest leaves of 11-d-old plants were dipped into 0.1% (w/v) ZnSO4 solution (pH 5.5) labeled with 1480 kBq 65Zn and containing 0.01% (w/v) Tween20 for 10 s. This foliar Zn application was repeated twice: once on the same day and once on the next day. When the leaf surfaces dried after the final foliar treatment, the tips (6 cm sections) of the oldest leaves in one-third of all plants were covered with aluminum foil to cause dark-induced senescence for 6 d. In another third of plants, dark treatment was started 3 d later and continued for 3 d. The remaining plants were kept uncovered for 6 d. Thereafter, plants were harvested by dividing into four parts as follows: the 65ZnSO4-applied (treated) part of the oldest leaf, the nontreated part of the oldest leaf, the remainder of the shoot and the roots. The 65ZnSO4-treated leaf parts were washed gently, first in 1 mM CaSO4 solution and then in deionized water. The 65Zn activity was measured in all samples as described earlier.

The effect of N nutrition on Zn retranslocation from source leaves to grains was studied in a soil culture experiment, which was conducted in a glasshouse equipped with an evaporative cooling system under natural daylight. In this eight-replicate experiment, the Zn-deficient potting soil was fertilized with 0.2 mg kg−1 Zn and 60,  180  or 540 mg kg−1 N (low, medium or high N, respectively). Tillers were removed just before flowering to obtain a single spike from each plant. When anthesis was completed (Zadoks stage: 69), the tips (7 cm sections) of flag leaves were dipped into 65Zn-labeled 0.1% (w/v) ZnSO4 solution containing 0.01% Tween20 (pH 5.5; 48 kBq 65ZnCl2 ml−1) for 10 s. The flag leaf treatment was repeated five times, once every 2 d. One day after the final treatment with 65Zn-labeled solution, the flag leaves were washed in 1 mM CaSO4 solution and deionized water. When the plants completely senesced, flag leaves and spikes were harvested. The 65Zn activity was determined in flag leaves, grains and husks by using the gamma counter mentioned earlier. Owing to the high risk of environmental contamination with 65Zn, the rest of the above-ground part of plants could not be ground and were not analyzed for 65Zn.

Mineral analysis

For Zn analysis, dry samples were ground and subjected to acid digestion in a closed-vessel microwave system (MarsExpress; CEM Corp., Matthews, NC, USA) using 30% H2O2 and 65% HNO3. Zinc concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES; Vista-Pro Axial; Varian Pty Ltd, Mulgrave, Australia). Nitrogen concentrations of dried and ground samples were determined using a LECO TruSpec C/N Analyzer (Leco Corp., St Joseph, MI, USA). Measurements were checked using certified standard reference materials obtained from the National Institute of Standards and Technology (Gaithersburg, MD, USA).

Calculations and statistical analysis

The Zn translocation index (%) was calculated by dividing the radioactivity in the shoot by the total radioactivity in the shoot and root and multiplying the quotient by 100.

Similarly, the Zn retranslocation index (%) was calculated by dividing the radioactivity in nontreated plant parts by the total radioactivity in treated and nontreated parts and multiplying the quotient by 100.

The significance of the effects of the treatments and their interactions on the reported traits was evaluated by one-way, two-way or three-way ANOVA, depending on the experimental design. Then, significant differences between means were determined using Fisher’s protected least significant difference (LSD) test at the 5% level ( 0.05).

Results

Role of N supply in Zn uptake and root-to-shoot translocation

In the first solution culture experiment, where the plants were precultured with or without Zn supply at three different N concentrations, two-way ANOVA revealed significant effects of N and Zn supply on the shoot and root DWs and the root-to-shoot ratio (Supporting Information, Table S1). The shoot DW increased significantly, when the N concentration was increased from low to medium or high, whereas the root DW decreased significantly when the N concentration was increased from medium to high (Table 1). Consequently, the root-to-shoot ratio showed a clear negative response to increasing N supply. Zinc supply also had a significant positive effect on the shoot DW, but its effects on the root DW and root-to-shoot ratio were generally not significant. According to the results of two-way ANOVA, not only the N and Zn supplies but also their interaction had significant effects (< 0.001) on the N concentrations of the shoot and root (Table S1). Both in the absence and in the presence of Zn supply, increasing the N supply resulted in marked increases in the N concentrations of the shoot and root (Table 2). At low or medium N, high Zn led to lower N concentrations, whereas at high N, high Zn resulted in higher N concentrations.

Table 1.   Shoot and root DWs and root-to-shoot ratios of 12-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.5 mM), medium (1.0 mM) or high (4.0 mM) nitrogen (N) in a nutrient solution with (1 μM) and without zinc (Zn) supply
N supplyShoot DW (mg per plant)Root DW (mg per plant)Root : shoot (%)
−Zn+Zn−Zn+Zn−Zn+Zn
  1. Values are means of five independent replicates. For each trait, means in columns followed by different upper-case letters and means in rows followed by different lower-case letters are significantly different from each other according to two-way ANOVA followed by least significant difference test (< 0.05).

Low177Aa202Ab165Ba176Ba94Cb87Ca
Medium222Ba263Bb163Ba174Ba73Bb66Ba
High205Ba260Bb127Aa149Ab62Ab57Aa
Table 2.   Shoot and root nitrogen (N) concentrations of 12-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.5 mM), medium (1.0 mM) or high (4.0 mM) N in a nutrient solution with (+Zn, 1 μM) and without (−Zn) zinc (Zn) supply
N supplyShoot N concentation (%)Root N concentration (%)
−Zn+Zn−Zn+Zn
  1. Values are means of three independent replicates. For each trait, means followed by different letters are significantly different from each other according two-way ANOVA followed by least significant difference test (< 0.05).

Low2.94b2.75a1.58b1.38a
Medium4.38d3.77c2.65d2.13c
High5.49e6.18f4.60e4.99f

Both the main effects of N and Zn supplies and the N by Zn interaction affected the 65Zn uptake and translocation rates significantly (< 0.001; Table S1). Plants precultured without Zn supply exhibited markedly higher 65Zn uptake and translocation rates than the corresponding plants grown with sufficient Zn supply (Fig. 1). Both the Zn uptake and root-to-shoot translocation rates showed a striking positive response to increasing N application. In terms of Zn uptake and translocation rate, Zn-deficient plants were even more responsive to increasing N supply than the Zn-sufficient plants. Increasing the N supply from low to high doubled the Zn uptake rate in Zn-sufficient plants, while it almost tripled the Zn uptake rate in Zn-deficient plants. Moreover, the effect of N on Zn uptake and translocation rates was gradual in Zn-deficient plants, while in Zn-sufficient plants, increasing the N supply from medium to high did not affect these rates (Fig. 1).

Figure 1.

65Zn uptake (a) and root-to-shoot translocation rates (b) of 12-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.5 mM), medium (1.0 mM) or high (4.0 mM) N and without (closed bars) or with (open bars) standard (1 μM) Zn supply. Values are means of five independent replicates. Error bars represent 1 SD. For each trait, means followed by different letters are significantly different from each other according to two-way ANOVA followed by least significant difference test (< 0.05).

In the second solution culture experiment, where the plants were precultured with low Zn supply at the same three N concentrations, the effect of N supply on the shoot and root DWs as well as the root-to-shoot ratio was generally very similar to the effect of N in the first experiment (Fig. S1, Table S2). In the second experiment, the root-to-shoot ratio at the highest N supply was much lower than in the first experiment (Table 1, Fig. S1). One-way ANOVA demonstrated highly significant effects (< 0.001) of N on the Zn uptake and translocation rates as well as the Zn translocation index (Table S2). The Zn uptake and translocation rates responded dramatically to N supply (Fig. 2). While the Zn uptake rate was almost quadrupled when the N supply was increased from low to high, the Zn translocation rate increased by 800%. Consequently, the 24 h Zn translocation index was significantly improved by N supply (Fig. 2).

Figure 2.

65Zn uptake rates (a), root-to-shoot translocation rates (b) and 24 h translocation indices (c) of 22-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with marginal (0.1 μM) Zn and low (0.5 mM; N1), medium (1.0 mM; N2) or high (4.0 mM; N3) N supply. Values are means of seven independent replicates. Error bars represent 1 SD. For each trait, means followed by different letters are significantly different from each other according to one-way ANOVA followed by least significant difference test (< 0.05).

The third uptake-related solution experiment was a time-course experiment, where the depletion of 65Zn by plants was followed for 72 h at three N concentrations in solution. As revealed by two-way ANOVA (Table S3), the shoot DW was significantly affected by the N supply, the plant age (d after sowing (DAS)) and the N × DAS interaction, while the root DW was affected only by DAS, and the root-to-shoot ratio only by the N supply (< 0.001). At the beginning of the depletion experiment, the shoot DW tended to increase when the N supply increased (Fig. 3). The positive response of the shoot DW to N application became much more pronounced at the end of the depletion experiment. At both time points, the root-to-shoot ratio was reduced by high N supply. The root and shoot N concentrations were significantly (< 0.001) affected by the N supply, DAS and N × DAS interaction (Table S3). As expected, there was a very close positive relationship between N supply and tissue N concentrations (Fig. 4).

Figure 3.

 Shoot and root DW (a) and root-to-shoot ratios (b) of 18- and 21-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with 0.2 μM Zn and low (0.1 mM; black bars), medium (0.5 mM; white bars) or high (2.0 mM; grey bars) N supply for 7 d before the depletion experiment. Values are means of four independent replicates. Error bars represent 1 SD. DAS,days after sowing.

Figure 4.

 Root (a) and shoot (b) N concentrations of 18- and 21-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with 0.2 μM Zn and low (0.1 mM; black bars), medium (0.5 mM; white bars) or high (2.0 mM; grey bars) N supply for 7 d before the depletion experiment. Values are means of four independent replicates. Error bars represent 1 SD. For each trait, means followed by different letters are significantly different from each other according to two-way ANOVA followed by least significant difference test (< 0.05). DAS, days after sowing.

The Zn depletion rate, that is, the rate of decrease in the activity of the labeled solution, was strongly dependent (< 0.001) on the N supply in the preculture solution (Table S3). High-N plants exhausted the Zn in the solution in 6 h, whereas medium-N plants needed 32 h and low-N plants 72 h to deplete all the Zn (Fig. 5). In the first 6 h, the average Zn depletion rate of high-N plants was more than twice as high as that of medium-N plants, which was more than twice as high as that of low-N plants.

Figure 5.

 (a) 65Zn depletion (0 → 72 h) in 65Zn-labeled solutions containing ten 18–21-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with 0.2 μM Zn and low (0.1 mM; closed squares), medium (0.5 mM; open squares) or high (2.0 mM; diamonds) N supply for 7 d before the depletion experiment. (b) Average 65Zn depletion rates (0 → 6 h) of 10 18-d-old seedlings per root DW (N1, low N supply; N2, medium N supply; N3, high N supply). Values are means of three independent replicates. Error bars represent 1 SD. Means followed by different letters are significantly different from each other according to one-way ANOVA followed by least significant difference test (< 0.05).

In the soil experiment, the shoot growth was positively affected by both N and Zn treatments (Tables 3, S4). When the soil N application was increased, the shoot N concentration was enhanced significantly under both low- and high-Zn conditions. High-Zn supply improved the shoot Zn markedly. In the case of the shoot Zn concentration, both the main effects of the N and Zn supplies and the effect of the N × Zn interaction were highly significant (< 0.001). For the low-Zn treatment, increasing the N supply tended to enhance the shoot Zn concentration, but the effect was not significant. By contrast, the shoot Zn concentration showed a dramatic positive response to increasing N application in the case of high-Zn treatment.

Table 3.   Shoot DW and nitrogen (N) and zinc (Zn) concentrations of 29-d-old wheat plants (Triticum durum cv Balcali2000) grown on a Zn-deficient soil with low (0.01 mg Zn kg−1 soil) or high (5.0 mg Zn kg−1 soil) Zn and low (50 mg N kg−1 soil), medium (100 mg N kg−1 soil) or high (200 mg N kg−1 soil) N supply
Zn supplyN supply
LowMediumHigh
  1. Values are means of four independent replicates. For each trait, means followed by different letters are significantly different from each other according to two-way ANOVA followed by least significant difference test (< 0.05).

Shoot DW (g per plant)
 Low0.85a0.85a0.97b
 High0.86a1.04b1.19c
Shoot N concentration (%)
 Low1.18a2.24b4.31d
 High1.14a1.92b3.71c
Shoot Zn concentration (mg kg−1)
 Low5.0a6.0a7.1a
 High29.8b37.9c56.7d

Role of N supply in Zn retranslocation during vegetative and generative phases

The effect of N supply on retranslocation of Zn from old leaves was studied during the vegetative growth of plants with and without Zn supplies by treating the tips of the oldest leaves in 65Zn-labeled ZnSO4 solution. In this experiment, the root DW tended to increase under Zn-deficient conditions, but did not clearly respond to different N applications (Tables 4, S5). By contrast, the shoot DW was highly responsive to increasing N supply and the root-to-shoot ratio was reduced by high N in both the absence and presence of Zn supply. When the 65Zn-applied sections of the oldest leaves were covered and kept in the dark, their DWs were significantly reduced (Tables 5, S5). The most marked biomass losses were observed in the 65Zn-treated sections kept in dark for 6 d.

Table 4.   Shoot and root DWs and root-to-shoot ratios of 17-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.25 mM), medium (1.0 mM) or high (4.0 mM) nitrogen (N) in a nutrient solution with (+Zn, 1 μM) and without (−Zn) zinc (Zn) supply
N supplyShoot DW (mg per plant)Root DW (mg per plant)Root : shoot ratio (%)
−Zn+Zn−Zn+Zn−Zn+Zn
  1. Values are means of six independent replicates. For each trait, means in columns followed by different upper-case letters and means in rows followed by different lower-case letters are significantly different from each other according to two-way ANOVA followed by least significant difference test (< 0.05).

Low80Aa77Aa104Aa95Aa130Cb123Ca
Medium133Ba122Ba129Bb112Ba98Bb91Ba
High147Ba154Ca120Bb109Ba82Ab71Aa
Table 5.   Dry weights of 65Zn-applied and dark-treated leaf sections of 17-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.25 mM), medium (1.0 mM) or high (4.0 mM) nitrogen (N) in a nutrient solution with (+Zn, 1 μM) and without (−Zn) zinc (Zn) supply
N supplyDry weight of 65Zn-applied leaf section (mg per section)
No dark treatment3 d dark treatment6 d dark treatment
−Zn+Zn−Zn+Zn−Zn+Zn
  1. Values are means of six independent replicates. Means followed by different letters are significantly different from each other according to three-way ANOVA followed by least significant difference test (< 0.05).

Low3.67ab5.22c3.82b3.40ab1.98a1.75a
Medium4.80bc5.07c5.02c4.77bc1.90a1.87a
High6.73d6.10cd4.98c5.05c1.85a2.65a

Three-way ANOVA demonstrated that the main effects of the N supply, Zn supply and dark treatment on the Zn retranslocation index, that is, the ratio of the activity in nontreated plant parts to the total activity, including the activity in the treated section, were highly significant (< 0.001), whereas none of the interactions had significant effects (Table S5). The Zn retranslocation index was higher in Zn-deficient than in Zn-sufficient plants (Fig. 6). Keeping the 65Zn-treated sections in the dark for 3 d did not have any significant effect on Zn retranslocation, but keeping the treated section covered for 6 d resulted in statistically significant increases. Only Zn-sufficient low-N plants did not respond to dark treatment. The Zn retranslocation index increased in both Zn-deficient and Zn-sufficient plants, when the N concentration in the preculture solution was increased from low to medium. Increasing the N concentration from medium to high did not lead to further enhancement of Zn retranslocation in Zn-deficient plants.

Figure 6.

 Percentage of 65Zn retranslocated in 6 d out of 65Zn-applied and dark-treated oldest leaves to other parts of 17-d-old wheat (Triticum durum cv Balcali2000) seedlings precultured with low (0.25 mM), medium (1.0 mM) or high (4.0 mM) N and without (closed bars) or with (open bars) standard (1 μM) Zn supply. Values are means of six independent replicates. Error bars represent 1 SD.

In order to study the effect of N nutrition on Zn retranslocation during the generative period of wheat development, a second retranslocation experiment was conducted, where the plants were grown in soil culture at low or high Zn and three different N concentrations. The tips of the flag leaves were treated with 65Zn-labeled ZnSO4 solution when flowering was complete. The N supply had significant effects on the grain yield and grain N concentration (Table S6). Both values increased markedly when the N concentration was increased from low to medium, but increasing the N concentration from medium to high could not further increase these values (Table 6). The ratio of the radioactivity in the grains to the activity in the flag leaves, which is a measure of retranslocation, was also significantly affected by the N concentration (= 0.018; Table S6). It was almost doubled when the N concentration was increased from low to medium, but then remained the same (Fig. 7). There was no measurable 65Zn activity in the husks.

Table 6.   Grain yield and grain nitrogen (N) concentrations of mature wheat plants (Triticum durum cv Balcali2000) grown on zinc (Zn)-deficient soil with a Zn supply of 0.2 mg Zn kg−1 soil and with low (60 mg N kg−1 soil), medium (180 mg N kg−1 soil) or high (540 mg N kg−1 soil) N supply
N supplyGrain yield (mg per plant)Grain N concentration (%)
  1. Values are means of eight independent replicates. For each trait, means followed by different letters are significantly different from each other according to one-way ANOVA followed by least significant difference test (< 0.05).

Low901a2.22a
Medium1423b3.48b
High1263b3.38b
Figure 7.

 Grain 65Zn : flag leaf 65Zn ratio in mature wheat plants (Triticum durum cv Balcali2000) grown on Zn-deficient soil with marginal (0.2 mg Zn kg−1 soil) Zn and low (60 mg N kg−1 soil), medium (180 mg N kg−1 soil) or high (540 mg N kg−1 soil) N supply. Flag leaves were treated with 65Zn six times after anthesis, once every 2 d. Values are means of eight independent replicates. Error bars represent 1 SD. Means followed by different letters are significantly different from each other according to one-way ANOVA followed by least significant difference test (< 0.05).

Discussion

In all nutrient solution experiments reported here, the N concentrations of both shoots and roots were enhanced (Fig. 4; Table 2), while the root-to-shoot biomass ratio was reduced by increasing N supply (Tables 1, 4), especially in the second experiment (Fig. S1). This negative response of the root-to-shoot ratio to increasing N concentration is well documented in the literature (Levin et al., 1989; Marschner, 1995).

The first step in the route of Zn from root to shoot and finally to grain is the Zn uptake by the root system. According to the results of the 65Zn uptake experiments, the rate of Zn uptake increases significantly when the N status of the plant is improved (Figs 1, 2, 5). The effect of increasing N supply on the rate of Zn uptake was much stronger in the second experiment (Fig. 2) than in the first experiment (Fig. 1), probably because of a much lower root-to-shoot ratio and thus a better N nutritional status of plants in the second experiment (Table 1, Fig. S1). Most probably, the abundance of root Zn uptake transporters, including ZIPs such as IRT1 and other unknown proteins (Ishimaru et al., 2005; Palmer & Guerinot, 2009; Pedas et al., 2009), is enhanced by increasing tissue concentrations of N. The striking positive effect of N-nutritional status on root-to-shoot translocation of 65Zn is shown in Figs 1 and 2. The stimulation of root-to-shoot translocation of Zn by N might be related to enhanced xylem-transport and xylem-loading of Zn, possibly as a result of N-stimulated activities of transporter proteins involved in xylem loading and enhanced production of nitrogenous compounds facilitating Zn transport in plants such as nicotianamine and deoxymugineic acid (Suzuki et al., 2008; Curie et al., 2009; Palmer & Guerinot, 2009). Since the reported rates of Zn uptake and translocation are normalized with respect to root biomass, the effects cannot be related to the effects of N nutrition on root growth.

It could be claimed that the root-to-shoot translocation of Zn is in fact not limited by N status, but by Zn uptake. Then the measured enhancement of Zn translocation rate by high N could be a result of increased Zn uptake, but Fig. 2 demonstrates that this is not the case. Improved N-nutritional status increases not only the 65Zn translocation rate, but also the 65Zn translocation index, that is, the ratio of activity in the shoot to the total activity (65Zn uptake) in the plant. The improvement of 65Zn translocation index, which is a value normalized with respect to uptake, indicates that N-nutritional status is an important factor for root-to-shoot translocation of Zn.

In the depletion experiment, the average rate of depletion was calculated based on the results of the first 6 h of the 72 h period (Fig. 5) for two reasons. First, high-N plants depleted all 65Zn in the solution in the first 6 h. Secondly, when the plants were 18 d old at the beginning of the depletion experiment, the differences between the shoot dry matters of plants precultured at different N concentrations were too small to explain the huge differences between the depletion rates (Fig. 3). This indicates that the effect of N on Zn depletion rate is not related to the dry matter production of the experimental plants. Owing to the nature of the depletion experiment, apoplastic 65Zn in roots was not removed and measured. However, the substantial differences in the depletion rate caused by varied N supply (Fig. 5) cannot be explained by the concentrations of apoplastic 65Zn.

Nutrients can be retranslocated from source to sink tissues via phloem during both vegetative and generative stages of development (Marschner, 1995). Senescence in source tissues is known to stimulate nutrient remobilization (Marschner, 1995; Gregersen et al., 2008), but it is not a prerequisite for remobilization. In wheat and rice, Zn retranslocation out of nonsenescent leaves has been demonstrated (Hajiboland et al., 2001; Erenoglu et al., 2002). Darkness is a signal for the initiation of senescence (Krupinska et al., 2003). Induction of senescence in selected leaves by absolute shading is an experimental method which can be used to study the effects of senescence on nutrient remobilization (Zhang et al., 1996). Retranslocation of 65Zn out of both nonsenescent and senescent leaves was clearly enhanced when the N supply was increased from low to medium, but high N did not make consistent and significant contributions, probably because medium N was already high enough under these experimental conditions (Fig. 6). Here, senescence was induced in the 65Zn-treated parts of the oldest leaves by dark treatment, and two sets of data provided evidence for senescence. First, the DW of the treated leaf sections decreased (Table 5); and secondly, their soil plant analysis development (SPAD) values decreased very significantly, which is an indicator of chlorophyll degradation (data not shown). Three-day-long dark treatment initiated senescence but was not sufficiently long to allow for marked remobilization enhancement. By contrast, after 6-d-long dark treatment, both the biomass loss in treated sections and the enhancement of Zn retranslocation index were highly significant (Fig. 6; Table 5). The improvement of retranslocation by improved N-nutritional status might be related to increased abundance of NA and phytosiderophores involved in the chelation of Zn in the phloem (Suzuki et al., 2008; Curie et al., 2009; Trampczynska et al., 2010) and increased activity of YSL proteins responsible for the phloem loading and unloading of NA-chelated Zn (Waters et al., 2006; Curie et al., 2009).

The positive effect of improved N nutrition on grain Zn in wheat was clearly documented in recent reports (Cakmak et al., 2010a,b; Kutman et al., 2010; Shi et al., 2010). Grain protein is considered as a sink for Zn, and under Zn-sufficient conditions, there is a strong positive correlation between grain Zn and grain N (Cakmak et al., 2010b; Kutman et al., 2010). The retranslocation of the flag leaf-applied 65Zn to grains and the grain N concentration were improved in medium-N plants as compared with low-N plants (Fig. 7), which shows that improved retranslocation of Zn under improved N-nutritional status is at least partially responsible for the positive effects of N on grain Zn. When the N supply was further increased from medium to high, neither the grain N concentration nor the 65Zn retranslocation from the flag leaf was affected. Because the tillers were removed, the medium N concentration was already high enough under the given experimental conditions.

The data presented here demonstrate that the N-nutritional status of wheat is critical in root uptake and root-to-shoot translocation of Zn as well as Zn remobilization during both vegetative and generative stages of development. This study sheds light on the physiological components underlying the previously reported positive effects of N application on grain Zn concentration. Up to a certain limit, increasing the N supply improves grain Zn concentration, probably by affecting major steps including uptake, xylem transport and phloem transport of Zn. The positive effects of N on uptake, translocation and remobilization of Zn can be explained by increased abundance of transporter proteins and nitrogenous chelators involved in these processes. It is well documented that Zn significantly affects biosynthesis and structural and functional integrity of proteins (Cakmak, 2000; Broadley et al., 2007). Substantial amounts of proteins in biological systems are Zn-dependent. For example, in eukaryotic cells, Zn-binding proteins make up nearly 10% of the proteomes (Andreini et al., 2006). It might therefore be possible that abundance and activity of the transporter proteins contributing to root uptake and loading of Zn into xylem and phloem tissues such as yellow stripe 1-like transporter proteins (Curie et al., 2009) are affected by the Zn nutritional status. In future, molecular physiological studies are needed to explore and demonstrate how the abundance or activities of those transporter proteins depend on the N- and Zn-nutritional status of plants.

The results of this paper have important nutritional implications for humans. Zinc deficiency is a growing public health and socioeconomic issue caused by low dietary intake of Zn in the developing world (Hotz & Brown, 2004; Black et al., 2008). Biofortification of cereal crops with Zn is therefore a growing global challenge. The results presented here indicate that N-nutritional status of plants is an important critical player in root uptake and accumulation of Zn in plants and deserves special attention in the biofortification of food crops with Zn.

Acknowledgements

This study was financially supported by the HarvestPlus Biofortification Challenge Program (http://www.harvestplus.org).

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