TAO Zhi-qiang, WANG De-mei, CHANG Xu-hong, WANG Yan-jie, YANG Yu-shuang, ZHAO Guang-cai

Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/Key Laboratory of Crop Physiology and Ecology, Ministry of Agriculture, Beijing 100081, P.R.China

Abstract Content of wheat flour proteins affects the quality of wheat flour. Zinc nutrition in wheat can change the protein content of the flour. The inconsistency and instability of wheat grain quality during grainfilling while under high temperature stress(HTS) are major problems in the production of high quality wheat. At present, there is a lack of studies on zinc fertilizer and HTS effects on wheat flour protein and the content of its components. For this study, treatment combinations offour levels of zinc fertilizers and exposure to a short-term HTS, at 20 d after flowering (D20), were tested on two wheat cultivars with different gluten levels. Individuals of a strong gluten wheat, Gaoyou 2018 (GY2018), and a medium gluten wheat,Zhongmai 8 (ZM8), were grown in pots at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing in 2015-2017. We measured grain yield and weight and the activities of two enzymes (nitrate reductase and glutamine synthetase) from the flag leaves, collected at D10 and D20. Total protein content, protein yield, and content offour protein components (albumin, gliadin, glutenin, and globulin) were measured from flour produced from the pot-grown plants. HTS significantly increased the contents of total protein, albumin, gliadin, and glutenin in wheat grains, and reduced the grain yield, grain weight, protein yield, globulin content, and flag leaf nitrate reductase (NR) and glutamine synthetase (GS)activities. The results showed that HTS and zinc fertilizer had greater impacts on the strong gluten cultivar compared to the medium gluten cultivar. Under HTS, grain yield decreased by 13 and 8% in GY2018 and ZM8, respectively; protein yield decreased by 7 and 8% in GY2018 and ZM8, respectively. Zinc fertilizer increased: grain and protein yields; grain weight;total protein, albumin, gliadin, and glutenin contents; protein yield; and NR and GS activities. In contrast, zinc fertilizer reduced the content of globulin. The addition of 15 mg Zn kg-1 soil had the strongest effect on grain yield and quality as compared to the other three treatments (additions of 0, 30, and 45 mg Zn kg-1 soil). Zinc fertilizer also reduced the negative effects of HTS on protein yield, content, and components’ content. Therefore, wheat grown with additional zinc in the soil can improve the quality of the flour.

Keywords: Triticum aestivum L., zinc fertilizer, strong gluten, climate warming

1. lntroduction

Wheat is one of the world’s staple crops. In 2014, the total globaloutput of wheat was approximately 850 million tons(FAO 2017). Maintaining consistency in wheat flour quality is necessary to meet the demands of a growing population for more high quality wheat. Wheat quality is determined by gluten strength (ranging from weak to strong gluten wheats)and is affected by the proteins in wheat flour. Total protein content of wheat and its protein components determine flour processing quality and the commercial value offlour products (Goesaert et al. 2005). Genes, environmental factors, and cultivation methods control the content of protein and its components in wheat grain. During the grainfilling stage, high temperature stress (HTS) can not only reduce grain yield, but also affect flour protein components and its functional properties, and in turn, affect the rheological properties and baking quality of the flour (Blumenthal et al.1993; Asseng et al. 2015). However, Nuttall et al. (2017)reported that, due to climate change, the daily frequency of extreme high temperatures has been increasing, hence the frequency of HTS occurring in the late growth stages of wheat (e.g., seed production) will likely increase and detrimentally affect the future yield and quality of wheat.

The nutritional status of a plant is one of the most important factors that affect the protein content of wheat grain. At present, many studies have reported the effects of macronutrients and micronutrients, such as nitrogen and sulfur, on wheat grain protein content (Tea et al. 2007;Klikocka et al. 2016) and effect of copper deficiency on dough extensibility (Flynn et al. 1987). The micronutrient zinc is known to play an important role in grain protein formation and nitrogen assimilation in winter wheat (Li et al. 2011). Zinc affects monomeric protein structure, the composition of high molecular weight glutenin subunits, and the proportions of glutenin and gliadin in total proteins, allof which affect the quality offlour (Liu et al. 2015). However,the mechanisms driving the effects are not clear (Starks and Johnson 1985; Peck et al. 2008). In China, Turkey,India, Pakistan, Australia, and other countries, most of the available zinc in the soil is low (Ozturk et al. 2006).Increasing the content of available zinc in soil could improve the protein content of wheat flour (Hemantaranjan and Garg 1988). Zhao et al. (2013) reported that the activities of nitrate reductase (NR) and glutamine synthetase (GS) in wheat flag leaves could significantly in fluence the content of protein in wheat flour. Zinc fertilizer can increase the activity of NR and GS in flag leaves, and thus, affect the content of protein components (Crawford 1995; Liu et al.2015). Furthermore, zinc can change the proportion of cysteine residues (Peck et al. 2008), and thus, affect the two disulfide bonds and the end-to-end formation of linear peptide molecules in the non-repetitive region of the peptide chain of high molecular weight glutenin subunits, and subsequently, affect the structure and rheological properties of gluten (Tamás et al. 2002).

At present, we lack research on how zinc regulates the protein content of wheat grains that have experienced high temperature stress. Therefore, this study examined the response characteristics of wheat grain protein and its components offour different glutens in strong gluten and medium gluten wheat cultivars that were grown with increasing levels of zinc fertilizer and exposed to a shortterm HTS after anthesis. The aim of this study was to provide empirical data to help elucidate the potential physiological mechanisms in response to additional zinc fertilizer and HTS effects, as wellas a theoretical basis and technical support to food producers and researchers for improving the grain yield and flour from wheat that may be growing under more stressful conditions due to climate change.

2. Materials and methods

2.1. Cultivars

Seeds of two varieties of winter wheat (Triticum aestivum L.) were obtained from wheatfields in northern North China. The strong gluten wheat, Gaoyou 2018 (GY2018),is considered a high quality wheat and the medium gluten wheat, Zhongmai 8 (ZM8) is considered of average quality.The optimal temperature range to grow these varieties is 15-32°C. In northern North China, the recorded average,maximum, and minimum daytime temperatures were 11.2,42, and -16°C, respectively, between 2000 and 2017.

2.2. Experimental design

The experiment was conducted at testfields in the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences(39°57′40′′N, 116°19′23′′E). Loam soil for pot experiments was collected from the top 30 cm of the soilat the testfield.The content of organic matter, total nitrogen, available nitrogen, available phosphorus, available potassium, and diethylenetriamene pentaacetate zinc (DTPA-Zn) in the soil was 16.5, 0.92, 55.2, 35.1, 189.6, and 0.85 mg kg-1,respectively. A totalof 10 kg of sieved (2-mm mesh), dry soilfilled each of 256 pots that were 25 cm in height and 25 cm in diameter. Seeds were sown on 15 October 2015 and 12 October 2016 and harvested on 5 June 2016 and 3 June 2017. We fertilized the potting soil with urea,superphosphate, and potassium chloride at respective concentrations of 90 mg N, 90 mg P, and 80 mg K per kg soil before sowing. When plants reached a three-leaf stage,seedlings were thinned to 10 per pot. Topdressing each pot with 1 g of urea dissolved in deionized water (DI) water occurred at the jointing stage of wheat growth. Zinc fertilizer was made from a solution of analytically pure chemical reagents and deionized water (ZnSO4·7H2O) and applied after fertilizing the potting soil with urea, superphosphate,and potassium chloride. Application of all fertilizers occurred once. There were four different concentrations of zinc fertilizer treatments: 0, 15, 30, and 45 mg Zn kg-1soil (named Zn0, Zn15, Zn30, and Zn45, respectively). To maintain soilfield capacity at 75%, plants were watered accordingly with the aid of a TZS-1K soil moisture analyzer(Zhejiang Top Cloud Agricultural Polytron Technologies Inc.,Zhejiang Province, China) which was used to measure soil moisture content every 3 d. The daytime temperature range was 15-38°C, the relative humidity reached an average of(50±15)% by the time wheat plants began to flower.

Over the last 10 years in the winter wheat region of northern North China, daily maximum temperatures had reached approximately 38°C for 1.8 consecutive days during the grainfilling period in wheat growth. Thus, we exposed plants that reached 20 d after flowering (D20) to a 2-d HTS treatment at 38°C to simulate the potential growing trend of increased days of high temperatures in northern North China occurring during the wheat grain-filling period. We transferred plants to an artificial climate chamber where they were exposed to the heat stress for 5 h from 11:00 to 16:00 each day. The relative humidity of the indoor air was (45±5)%. After the 2-d heat treatment, all pots were returned to their previous locations in the field to continue growing until seed set. Control plants (NT) were grown at ambient temperatures in the field until harvest time. Pots were placed in a randomized block design. There were a totalof 16 replicates per zinc treatment. Eight of these replicates were used to determine NR and GS activities in flag leaves that were sampled at D10 and D22. Leaves were sampled during mornings with full sun. The other eight replicates were used to determine grain yield, weight, protein content and protein components. the field portion of the experiment wasfirst conducted on 15 October 2015 to 5 June 2016 and repeated 12 October 2016 to 3 June 2017.

2.3. Sampling methods

Uniform-sized ears were selected at the same flowering stage, and plants were repeatedly sampled at D10 and D20. Grain yield and mean weight per grain were calculated after seeds harvested from pots and dried. The seeds were air-dried for 30 d, and then crushed and ground into flour with a grinding machine, ZH10852 (Zhonghui Tiancheng Technology Co., Ltd., Beijing, China). The flour was used to determine protein content, protein yield, and content offour protein components (albumin, gliadin, glutenin, and globulin). The content of protein was determined by semimicro Kjeldahl nitrogen method, and then protein yield was determined by the protein content multiplied by the mean weight per grain (Zhao et al. 2013). NR and GS activities in flag leaves were determined by following the method described in Yu and Zhang (2012). The protein components of grains were determined by the sequential extraction method, i.e., albumin, globulin, gliadin, and glutenin were sequentially extracted with distilled water, 2% NaCl, 70%ethanol, and 0.5% KOH (Liu et al. 2015).

2.4. Data analysis

Using SPSS 21.0 software (SPSS Inc., Chicago, IL,USA), a variance analysis and interaction effect and factor contribution analysis (Eta2) were performed with a General Linear Model process, from “Analyze” to “General Linear Model” to “Univariate”. Eta2values range between 0 and 1 and represent the contributing proportion of effect from a factor in a model. The measured variables of grain weight,protein yield, total protein content, protein yield, and content offour protein components (albumin, gliadin, glutenin, and globulin), and flag leaf NR and GS activities were subjected to a post-hoc multiple comparisons test using Duncan’s method with a significance levelat P=0.05.

3. Results

3.1. Effects of zinc fertilizer and high temperature stress on grain yield

Exposure to HTS post-anthesis significantly reduced grain yield and weight (P＜0.05) below that of the NT group for plants of both cultivars. The in fluence of HTS on GY2018 was significantly higher than that on ZM8 (Figs. 1 and 2).

Zinc fertilizer significantly increased grain yield and weight of the two cultivars (P＜0.05). A consistent pattern was observed in yield and weight in both cultivars and both experimental years. The measures of grain yield and weight were consistently the highest in the Zn15 treatment and the lowest in the Zn0 treatment. Furthermore, yield and weight consistently decreased from Zn15 with greater additions of zinc. On average, yield and weight in the three zinc treatments of GY2018 of both years increased by a respective 34.3 and 30.7% (Zn15), 28.4 and 23.0% (Zn30),and 17.1 and 10.3% (Zn45) compared to the control (Zn0).In Zm8, for both years, average increases in yield and weight were approximately, 12.2 and 14.9% (Zn15), 6.1 and 7.5%(Zn30), and 3.5 and 4.0% (Zn45) respectively greater than Zn0 (Figs. 1 and 2).

Fig. 1 Effect of zinc fertilizer, high temperature stress (H), and no-stress (N) on grain yield from replicate experiments in 2016 and 2017 in Gaoyou 2018 (GY2018, A and B) and Zhongmai 8 (ZM8, C and D). Zn0, Zn15, Zn30, and Zn45 indicate the four levels of zinc fertilizer treatments, 0, 15, 30, and 45 mg Zn kg-1 soil, respectively. Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05.

Fig. 2 Effect of zinc fertilizer additions (Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively) and high temperature stress (H)or no-stress (N) on grain weight from replicate experiments in 2016 and 2017 in Gaoyou 2018 (GY2018, A and B) and Zhongmai 8(ZM8, C and D). Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05.

The results indicated that HTS reduced grain yield and grain weight in both cultivars; whereas, zinc fertilizer increased grain yield and grain weight compared to the control (Zn0). to gether, zinc fertilizer and HTS also caused increases in grain yield and grain weight in both cultivars. The interaction effects of cultivar×temperature,cultivar×zinc fertilizer, temperature×zinc fertilizer, and cultivar×temperature×zinc fertilizer on grain yield and weight were significant at P＜0.01 or P＜0.05; however, the effect of year and the interaction effect of year, cultivar, temperature,and zinc fertilizer were not significant (Table 1). Zinc fertilizer had the largest contribution to total variation of grain yield and grain weight, followed by temperature, cultivar, cultivar×zinc fertilizer, temperature×zinc fertilizer, cultivar×temperature,and cultivar×temperature×zinc fertilizer (Table 2). Zinc fertilizer had a significant effect on grain yield and grain weight in wheat under HTS. Comparisons between the Zn0 treatment under HTS and the Zn0 treatment grown at ambient temperature of their respective cultivars in both experimental years revealed reductions in grain yield by about 26% in GY2018 and 13% in ZM8. Grain weight was reduced by about 14% in GY2018 and 15% in ZM8. In contrast, in Zn15, Zn30, and Zn45 treatments under HTS compared to their respective zinc treatment levels that were not under HTS, grain yield was reduced by 6.7-12.1% and grain weight by 8.0-13.9% in GY2018 in both years. For ZM8, the same treatment comparisons were reduced by 3.8-9.6% in yield and 5.4-11.8% in weight in both years(Figs. 1 and 2).

3.2. Effects of zinc fertilizer and high temperature stress on NR activity

HTS reduced NR activity in both cultivars; whereas, zincfertilizer increased NR activity compared to the control(Zn0). to gether, zinc fertilizer also caused increases in NR activity under HTS in both cultivars. The activity of NR,measured at D10, was significantly affected by cultivar and zinc fertilizer (P＜0.05). However, the effect of year and the interaction effect of year, cultivar, and zinc fertilizer were not significant. The NR activity offlag leaves, at D22, was significantly affected by cultivar, temperature,zinc fertilizer, cultivar×temperature, cultivar×zinc fertilizer,temperature×zinc fertilizer, and cultivar×temperature×zinc fertilizer (P＜0.01). However, the effect of year and the interaction effect of year, cultivar, temperature, and zinc fertilizer were not significant (Table 1). Zinc fertilizer had the greatest contribution to the total variation of NR activity,followed by temperature, temperature×zinc fertilizer, cultivar,cultivar×temperature×zinc fertilizer, cultivar×zinc fertilizer,and cultivar×temperature (Table 2). Zinc fertilizer had a significant effect on the NR activity offlag leaves under HTS.Compared with the Zn0 treatment under HTS, the reduction in activity of NR was less in both cultivars in both years of Zn15, Zn30, and Zn45 treatments under HTS (Fig. 3).

Table 1 Results of the ANOVA of effects of cultivar (C), temperature (T), zinc fertilizer (Zn), and year (Y) on wheat grain yield, grain weight, nitrate reductase activity (NR), glutamine synthetase activity (GS), grain protein content, grain protein yield, and contents of albumin, globulin, gliadin, and glutenin in 2016 and 2017

Table 2 Results offactor contribution analysis (Eta2) of cultivar (C), temperature (T), zinc fertilizer (Zn), and year (Y) on wheat grain yield, grain weight, nitrate reductase activity (NR), glutamine synthetase activity (GS), grain protein content, grain protein yield, and contents of albumin, globulin, gliadin, and glutenin in 2016 and 2017

On the 10th d after flowering, NR activity in ZM8 was significantly greater than that in GY2018 (P＜0.05). There was no significant difference in NR activity between the two cultivars at D22 (Fig. 3).

Whether at D10 or D22, NR activity of the two cultivars were significantly greater with the addition of any of the three concentrations of Zn fertilizer than compared to no zinc addition (P＜0.05). The greatest difference was observed between Zn15 and Zn0. However, activity rates gradually decreased with the increase of levels of Zn fertilizer (P＜0.05)(Fig. 3).

3.3. Effects of zinc fertilizer and high temperature stress on GS activity

The activity of GS was significantly affected by cultivar, zinc fertilizer, and variety×zinc fertilizer at D10 (P＜0.01). At D10,zinc fertilizer significantly increased the GS activities of ZM8 and GY2018 where activity of GS in ZM8 was significantly higher than that in GY2018. GS activity was generally lower with the addition of 0 and 45 mg Zn kg-1soil than in the other two Zn fertilizer treatments (Fig. 4). The GS activity was significantly affected by cultivar, temperature,zinc fertilizer, and cultivar×temperature at D22 (P＜0.01 or P＜0.05) (Table 1). At D22, zinc fertilizer had no effect on GS activity in ZM8 and GY2018; however, the activity of GS in ZM8 was significantly higher than that in GY2018.Heat stress significantly decreased (P＜0.05) the activity of GS in both wheat varieties; there was a greater reduction of GS activity in GY2018 than in ZM8 in both 2016 and 2017 (Fig. 4).

Fig. 3 Effect of zinc fertilizer, high temperature stress (H), and no-stress (N) on nitrate reductase activity offlag leaves at 10 (D10)and 22 (D22) days after flowering in cultivars, Gaoyou 2018 (GY2018) in 2016 (A) and 2017 (B) and Zhongmai 8 (ZM8) in 2016(C) and 2017 (D). Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively. Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05.

Temperature had the greatest contribution to the total variation of GS activity, followed by cultivar,cultivar×temperature, and zinc fertilizer (Table 2). The results showed that the activity of GS in both cultivars was sensitive to high temperature stress.

3.4. Effects of zinc fertilizer and high temperature stress on content of total grain protein and flour protein components

The content of total grain protein and protein yield were significantly affected by cultivar, temperature, zinc fertilizer,cultivar×zinc fertilizer, temperature×zinc fertilizer, and cultivar×temperature×zinc fertilizer (P＜0.01) (Table 1). The results of interaction effects indicated that HTS generally reduced protein yield in both cultivars, while zinc fertilizer generally increased protein content and yield. Zinc fertilizer to gether with HTS generally increased protein content and yield in both cultivars. Zinc fertilizer significantly increased grain protein content and yield in both wheat cultivars (P＜0.05). Compared with allother Zn treatments,the highest grain protein content and yield achieved was observed in the Zn15 treatment in both wheat cultivars.Compared to Zn0, Zn30, and Zn45 treatments, Zn15 treatment protein content was greater by 16.6, 9.2, and 12.1%, respectively, in GY2018 in both years. In similar treatment comparisons, protein content was greater by 6.1,1.7, and 3.2%, respectively, in ZM8 in both years (Fig. 5).Similar results were observed for protein yield in both wheat varieties and both replicate years. For GY2018, treatment comparisons of yield in Zn15 to that in Zn0, Zn30, and Zn45 were greater by 52.4, 15.6, and 32.7%, respectively. Similar comparisons in Zm8 produced greater differences by 21.9,8.6, and 13.8%, respectively (Fig. 6).

Fig. 4 Effect of zinc fertilizer additions (Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively) and high temperature stress(H) or no-stress (N) on glutamine synthetase activity offlag leaves, 10 (D10) and 22 (D22) days after flowering in each cultivars,Gaoyou 2018 (GY2018) in 2016 (A) and 2017 (B) and Zhongmai 8 (ZM8) in 2016 (C) and 2017 (D). Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05. Abs indicates absorbance.

The protein content of HTS-treated samples was significantly greater than that of the control samples(P＜0.05). In contrast, protein yield was significantly lower(P＜0.05). In GY2018, protein content of HTS treatment groups averaged across all four Zn treatment groups was 4.6% greater in both years than the control treatment. In similar treatment comparisons, for ZM8, protein content of the HTS treatments in both years were 3.4% greater than that of the controls (Fig. 5). Similarly, in GY2018, mean protein yield of HTS treatment groups was 7.4% lower than the controls in both years. In ZM8, mean protein yield was also lower by 7.7% in both years when comparing the same groups (Fig. 6).

Fig. 5 Effect of zinc fertilizer additions (Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively) and high temperature stress(H) or no-stress (N) on grain protein content of each wheat cultivar, Gaoyou 2018 (GY2018) in 2016 (A) and 2017 (B) and Zhongmai 8 (ZM8) in 2016 (C) and 2017 (D). Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05.

Fig. 6 Effect of zinc fertilizer additions (Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively) and high temperature stress(H) or no-stress (N) on grain protein yield of each wheat cultivar, Gaoyou 2018 (GY2018) in 2016 (A) and 2017 (B) and Zhongmai 8(ZM8) in 2016 (C) and 2017 (D). Bars represent standard deviations (SD). Letters mean significant differences at P＜0.05.

The application of zinc fertilizer to plants that were heatstressed caused increases in protein content and reductions in protein yield. Furthermore, additional zinc had a greater effect on GY2018 than that on ZM8. Grain protein content was significantly greater in the heat-stressed, zinc-added treatments than in the heat-stressed, Zn0 treatment in both replicates of both wheat varieties. Conversely, of the same treatment comparison, grain protein yield was significantly lower in both replicates of both wheat varieties. Compared with ZM8 under HTS, grain protein content was significantly greater in GY2018 under HTS. In both years, the reduction in grain protein content was significantly less in the Zn treatments in GY2018 than that in ZM8. Moreover, there was significantly more proteins in GY2018 than those in ZM8 (Figs. 5 and 6).

Fig. 7 illustrates the proportions of protein components in the two cultivars across all treatment combinations. A ranking of the content of each component of grain protein in both cultivars is glutenin＞gliadin＞albumin＞globulin. The high temperature treatment reduced the globulin content in both cultivars whereas, HTS increased the contents of the other components. The ratio of glutenin to gliadin increased in GY2018, but no change was observed in ZM8.The glutenin content and the ratio of glutenin to gliadin of strong gluten GY2018 was significantly higher than that of medium gluten ZM8. Lower concentrations of gliadin and glutenin were observed in both the Zn0 and Zn45 treatments than in the mid-level Zn treatments. Of all zinc fertilizer treatments, the highest gliadin content was measured in Zn15 in GY2018 and in Zn30 in ZM8. The highest glutenin content was measured in Zn15 in both GY2018 and ZM8.

A similar trend was also found in albumin content and the opposite trend in globulin content. There was an increase of albumin in GY2018 and ZM8 with the addition of zinc fertilizer from 0-15 mg Zn kg-1soil, followed by a decrease of albumin when zinc fertilizer additions increased from 15-45 mg Zn kg-1soil. The albumin content was higher in ZM8 than that in GY2018. A different trend was observed in globulin content where there was a decrease of globulin content in GY2018 and ZM8 with the increase of zinc fertilizer.

The contents of glutenin, albumin, and globulin were significantly affected by main and interaction effects of cultivar, temperature, zinc fertilizer, cultivar×temperature,cultivar×zinc fertilizer, temperature×zinc fertilizer, and cultivar×temperature×zinc fertilizer (P＜0.01). The content of gliadin content was significantly affected by main and interaction effects of cultivar, temperature, zinc fertilizer, and cultivar×zinc fertilizer (P＜0.01 or P＜0.05) (Table 1). The effect of cultivar had the greatest contribution to the total variation in glutenin, albumin, and globulin contents. This was followed by the contributions of zinc fertilizer, temperature,cultivar×zinc fertilizer, cultivar×temperature×zinc fertilizer,zinc fertilizer×temperature, and cultivar×temperature.The effect of cultivar had the greatest contribution to the total variation of gliadin content, followed by zinc fertilizer,cultivar×zinc fertilizer, and temperature (Table 2). In both years, comparison of zinc-added treatments in combination with HTS between the two cultivars indicated that glutenin,gliadin, albumin, and globulin contents were more strongly affected in GY2018 than those in ZM8. In contrast, in Zn15,Zn30, and Zn45 treatments under HTS compared to their respective zinc treatment levels that were not under HTS,glutenin, gliadin, albumin, and globulin contents were greater in both GY2018 and ZM8 in both years (Fig. 7).

4. Discussion

4.1. Response of NR and GS to zinc fertilizer and high temperature stress

Nitrate reductase is important to the uptake and utilization of nitrogen because it affects crop yield and quality (Raun and Johnson 1999). GS is a multifunctional enzyme involved in nitrogen metabolism and the regulation of many other metabolic processes. In higher plants, more than 95%of NH4

+is assimilated by glutamine synthetase/glutamate synthase (GS), GS is central to nitrogen metabolism where it participates in the regulation of key enzymes. A lack of GS activity can negatively impact a variety of nitrogen metabolizing enzymes as wellas some glucose metabolizing enzymes (Foyer et al. 2003). Zinc fertilizer has been shown to enhance nitrogen assimilation (Shi et al. 2011).Additionally, studies showed that the activities of NR and GS increased with Zn fertilization of up to 10 mg Zn kg-1soil(Ghosh and Srivastava 1994; Liu et al. 2015). In our study,15-45 mg Zn kg-1soil treatments increased the activities of NR and GS, but the magnitude of the increases in the two cultivars varied. Thus, the amount of zinc fertilizer available in soil can affect the NR and GS activities offlag leaves,but the mechanism of these changes in activity need to be further studied.

Similar to our observations, other researchers have also observed that high temperature stress causes NR and GS activities in flag leaves to decrease (Liu et al. 2007). The observed reduction in both enzyme’s activity caused by HTS above 35°C may have accelerated senescence of the plant while in its later life stage of grainfilling. The stress from the high temperature may have been so severe that plants were unable to recover from or compensate for the damage. Thus, the NR activity in the flag leaves decreased or possibly ceased completely. The likely consequence is that NO3

-is deoxidized to NH4+and the NH4

+is subsequently reduced. As a substrate of the GS reaction pathway, the loss of available NH4+can cause a decrease in GS activity (Liu et al. 2007). The results of this study also showed that the interaction effect of zinc fertilizer×temperature×cultivar had a significant effect on NR activities, and temperature×cultivar had a significant effect on GS activities. Overall, these results indicate that zinc fertilizer could significantly improve NR activity in flag leaves of the two cultivars that have been exposed to a high temperature stress.

Fig. 7 Effect of zinc fertilizer additions (Zn0-45 mean 0, 15, 30, and 45 mg Zn kg-1 soil, respectively) and high temperature stress(H) or no-stress (N) on different grain protein components of each wheat cultivar, Gaoyou 2018 (GY2018) in 2016 (A) and 2017(B) and Zhongmai 8 (ZM8) in 2016 (C) and 2017 (D).

4.2. Response of the content offlour protein components to zinc fertilizer and high temperature stress

Gluten is the main component offlour proteins. Gluten proteins include gliadin and glutenin, and are the main storage proteins of wheat flour that directly affect the processing quality offlour (Goesaert et al. 2005). In contrast, the non-gluten proteins, albumin, and globulin,are mainly structural proteins with high nutritional value because they are rich in lysine and other important amino acids (Wieser and Seilmier 1998). Availability of nutrients,such as N, S, Cu, and Zn, affect flour protein components(Zhao et al. 2013). It is possible that the addition of zinc fertilizer significantly enhanced NR and GS activities and subsequently affected grain protein content and flour protein components (Liu et al. 2015). The content of albumin,glutenin, and gliadin increased and then decreased with the increase of zinc fertilizer (0-45 mg Zn kg-1soil), where contents peaked in the 15 mg Zn kg-1soil treatment. Zinc fertilizer addition also increased the ratio of glutenin to gliadin in GY2018. The ratio of glutenin to gliadin is correlated with wheat quality traits. The increase in glutenin content and thus, gluten content, can cause sedimentation and stabilization time to increase which results in improved flour processing characteristics (Zhao et al. 2013). The flour protein components that we examined may be affected by zinc and cysteine. Cysteine is a deoxidizer that alters the two sulfur bonds between and within protein molecules that promotes gluten formation. Cysteine-rich binding proteins with low relative molecular mass are able to bind to zinc with high affinity. Thus, Zn combined with cysteine rich binding proteins can affect flour protein composition (Zhang et al.2012; Liu et al. 2015). Although this greenhouse experiment provided valuable evidence,field studies are required to fully understand the underlying mechanisms and consequences of zinc fertilizer on flour protein components.

Exposure to the high temperature stress caused a reduction in grain yield and grain weight, and an increase in grain protein content (Wang et al. 2008). Results also showed that the amounts of grain protein components,albumin, gliadin, and glutenin, increased in plants of both cultivars that experienced the short-term HTS at D20. The likely reason is that the effect of HTS on starch synthesis in grain was greater than that on protein synthesis. A large decrease in starch can result in a decrease in grain weight and a relatively large increase in grain protein content(Blumenthal et al. 1991; Zhang et al. 2012).

Furthermore, extreme heat can reduce the function of chlorophyll enzymes, which can limit availability of sugar and energy for seed growth (Wahid et al. 2007).Therefore, exposure to HTS at D20 may have reduced the photosynthetic rate, and subsequently, caused the reduction in grain weight and increase in protein content. Higher temperatures can also cause wheat to mature faster, which can shorten the grainfilling period (Akter and Islam 2017).Thus, grain weight was reduced, the grain nitrogen content was increased, and the grain protein content was increased correspondingly (Jenner et al. 1991; Liu et al. 2007).

4.3. Response of wheat grain production and flour quality to zinc fertilizer and high temperature stress

The type of wheat (strong gluten or medium gluten wheat)is dependent upon grain quality and its end use. Wheat with high protein content for bread is considered strong gluten and wheat with a lower protein content for slender and steamed bread is considered a medium gluten wheat.Certain varieties of wheat are better for particular food uses depending on the flour quality. Bread wheat has high protein content and strong gluten strength, noodle wheat has a moderate protein content and gluten strength, and biscuit wheat has a low protein content and gluten strength(Shewry et al. 1997). HTS increased grain protein content in both cultivars, but reduced grain weight and protein yield(Liu et al. 2007). The results of this study also showed that additional zinc fertilizer reduced the extent of grain weight loss in HTS-treated plants of both cultivars (Fig. 2),and zinc fertilizer significantly (P＜0.05) increased grain protein content and protein yield of both wheat cultivars.Furthermore, compared with ZM8 under HTS, grain protein content of GY2018 was significantly higher (Figs. 5 and 6).The high protein content of GY2018 makes it a high quality wheat that is suitable for bread-making; whereas, the lower protein content of ZM8 makes it a lower quality wheat that is more suitable for noodle-making.

The glutenin content and the ratio of glutenin to gliadin of strong gluten GY2018 was significantly higher than that of medium gluten ZM8. Compared with the Zn0 treatment under HTS, Zn treatments increased glutenin content,gliadin content, and the ratio of glutenin to gliadin. These changes suggest that the content of the main storage proteins of wheat flour also increased. Higher amounts of storage protein increases gluten strength where greater strength indicates a better processing quality offlour, and GY2018 is a prime example of a strong gluten wheat. These results showed that application of 15 mg Zn kg-1soil zinc fertilizer to plants under HTS is beneficial to improving the processing quality offlour in strong gluten wheat GY2018 and medium gluten wheat ZM8. Zinc addition is especially beneficial to GY2018 because it alleviated some of the negative effects of HTS on grain weight and grain yield.

5. Conclusion

The addition of zinc fertilizer increased grain yield and weight, positively affected NR and GS activities within days but not weeks after anthesis, and affected grain protein yield, content and components (glutenin, gliadin, abumin,and globulin). High temperature stress reduced grain yield and weight and NR and GS activities but increased grain protein content. HTS also decreased grain protein yield and likely affected the synthesis of protein components.Zinc fertilizer and high temperature stress had different effects on different wheat cultivars. Zinc fertilizer appears to alleviate the negative effects of high temperature stress on wheat yield and flour quality. The results of this study may be helpful to farmers seeking ways to increase their grain yield while maintaining grain quality in light of the potential threats to plant growth from climate change.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2016YFD0300407)and the earmarked fund for the China Agriculture Research System (CARS-03).