Zhiqiang Tao,Xuhong Chang,Demei Wang,Yanjie Wang,Shaokang Ma,Yushuang Yang,Guangcai Zhao

Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Key Laboratory of Crop Physiology and Ecology,Ministry of Agriculture,Beijing 100081,China

Keywords:

A B S T R A C T The content of wheat flour proteins affects the quality of wheat flour.Sulfur nutrition in wheat can change the protein content of the flour.The inconsistency and instability of wheat grain quality during grain filling under high temperature stress(HTS)are a major challenge to the production of high-quality wheat.The effects of sulfur fertilization and HTS on wheat flour protein and its components are unknown.In this study,treatments varying two factors:sulfur fertilization and exposure to short-term HTS,at 20 days postanthesis,were applied to two wheat cultivars with differing gluten types.Plants of a stronggluten wheat(Gaoyou 2018)and a medium-gluten wheat(Zhongmai 8)were grown in pots in Beijing in 2015—2017.HTS significantly increased the contents of total protein,albumin,gliadin,glutenin,Cys,and Met in wheat kernels,but reduced grain yield,grain weight,protein yield,globulin content,and total starch accumulation.The HTS-induced increase in total protein amount was closely associated with nitrate reductase(NR)and glutamine synthetase(GS)activities in flag leaves.Sulfur fertilization increased grain and protein yields;grain weight;total protein,albumin,gliadin,glutenin,and globulin contents;protein yield;total starch;Cys,Met;and NR and GS activities.HTS and sulfur fertilization had larger effects on the strong-than on the medium-gluten cultivar.Sulfur fertilization also alleviated the negative effects of HTS on grain yield,protein yield,and starch content.Thus,growing wheat with additional soil sulfur can improve the quality of the flour.

1.Introduction

Wheat(Triticum aestivum L.)is one of the world's staple crops.In 2014,the total global output of wheat was approximately 850 Mt[1].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 and is affected by the proteins in wheat flour.Total protein content and protein components determine flour processing quality and the commercial value of flour products.Genes,environmental factors,and cultivation methods control the content of protein and its components in wheat grain.During the grain filling stage,high temperature stress(HTS)can not only reduce grain yield,but also affect flour protein components and its functional properties,and in turn,worsen the rheological properties and baking quality of the flour[2,3].Nuttall et al.[4]reported that owing to climate change,the daily frequency of extreme high temperatures is increasing;hence,the frequency of HTS occurring in the late growth stages of wheat will increase and will reduce the yield and quality of wheat.

The nutritional status of the wheat plant is one of the most important factors affecting the protein content of the grain.Studies have characterized the effects of macronutrients,such as nitrogen and sulfur,on wheat grain protein[5,6]and the effect of copper deficiency on dough extensibility[7].The macronutrient sulfur is known to play an important role in grain protein formation and nitrogen assimilation in winter wheat[8].Sulfur is an essential mineral nutrient for plant growth.It is the fourth major nutrient after nitrogen,phosphorus,and potassium.Ninety percent of the sulfur in the plant body is used to synthesize sulfur-containing amino acids,which are the major component of proteins[9].Sulfur is an important component of nitrogen metabolism enzymes such as nitrate reductase(NR)[10].A sulfur deficit will reduce the absorption of nitrogen,affecting the content of protein and the proportions of glutenin and gliadin in total protein,all of which affect the quality of flour.However,the mechanisms driving these effects in wheat flour are not clear[9,11].In the North China Plain,the humid Mediterranean region of northern Spain,and other regions,soil sulfur is low in some areas,owing mainly to a decrease in the use of S fertilization and of atmospheric deposition [9—12].Increasing sulfur fertilization application in S-limited soils could increase the content of protein and its components in wheat flour[8,13].Zhao et al.[14]reported that the activity of NR and glutamine synthetase(GS)in wheat flag leaves significantly influence the content of protein in wheat flour.Sulfur fertilization can increase the activity of NR and GS in flag leaves and thereby affect the content of protein components[15].Furthermore,sulfur fertilization can increase the plant content of sulfur amino acids,which favors synthesis of proteins rich in cysteine(Cys)and methionine(Met)[16],and the disulfide bond(-S-S-)in the sulfur-bearing protein Cys helps to improve the elasticity of gluten,which affects the processing quality of wheat[8].

To date,we lack research on how sulfur regulates the protein content of wheat grains that have experienced high temperature stress.Accordingly,this study examined the response of wheat grain protein and of four different gluten components in strong and medium-gluten wheat cultivars grown with sulfur fertilization and exposed to short-term HTS after anthesis.The aim was to provide empirical data to help elucidate the physiological mechanisms involved in response to additional sulfur fertilization and HTS effects,and also to provide a theoretical basis and technical support to food producers and researchers for improving grain yield and flour from wheat grown under more stressful conditions imposed by climate change.

2.Material and methods

2.1.Cultivars

Seeds of two cultivars of winter wheat were obtained from wheat fields 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 for growing these cultivars is 15—32 °C(Department of Plant Management,Ministry of Agriculture of the People's Republic of China 2016).In northern North China,recorded average,maximum,and minimum daytime temperatures were 11.2,42.0,and ?16.0 °C,respectively,between 2000 and 2017.

2.2.Experimental design

The experiment was conducted in test fields at the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences(39°57′40″N,116°19′23″E).The soil for pot experiments was silt loam(12.74%clay,9.03%sand,and 78.23%silt),collected from the top 30 cm of the soil in the test field.The contents of organic matter,total nitrogen,available nitrogen,available phosphorus,available potassium,and available sulfur in the soil were 16.51 and 0.92 g kg?1,and 55.22,35.13,189.62,and 26.05 mg kg?1,respectively.Ten kilograms of sieved(2-mm mesh)dry soil was placed in each of 128 pots that were 25 cm in height and 25 cm in diameter.Seeds were sown on October 15,2015 and October 12,2016,and harvested on June 5,2016 and June 3,2017,respectively.The pot soil was fertilized with urea,super phosphate,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 the three-leaf stage,seedlings were thinned to 10 per pot.Each pot was topdressed with1 g of urea dissolved in de-ionized water at the jointing stage of wheat growth.Sulfur fertilization consisting of S granules(98%S)was applied after fertilization with urea,superphosphate,and potassium chloride.All fertilizers were applied only once.Two sulfur fertilization levels were used:0(S0)and45(S45)kg ha?1.To maintain soil field capacity at 75%,plants were watered with the control of a TZS-1K soil moisture analyzer(Zhejiang Top Cloud Agricultural Polytron Technologies Inc.,Zhejiang province,China)used to measure soil moisture content every three days.The daytime temperature range was 15—38 °C,and the relative humidity reached an average of 50±15%by the time the wheat plants began to flower.

Over the last ten years in the winter wheat region of northern North China,daily maximum temperatures reached approximately 38°C for 1.8 consecutive days during the grain filling period in wheat growth.Thus,plants at 20 days after flowering were exposed to a two-day HTS treatment at 38°C to simulate the potential growing trend of increased days of high temperature in northern North China occurring during the wheat grain-filling period.Plants were transferred to an artificial climate chamber,where they were exposed to heat stress for 5 h from 11:00 to 16:00 each day at a relative humidity of 45%±5%.After the 2-day heat treatment,all pots were returned to their previous locations in the field to continue growing until seed set.Control(NT)plants were grown at ambient temperatures in the field until harvest.Pots were placed in a randomized block design.There were a total of 16 replicates per sulfur treatment.Eight of these replicates were used to determine nitrate reductase(NR)and glutamine synthetase(GS)activity in flag leaves that were sampled 10 and 22 days after flowering(DAA10 and DAA22,respectively).Leaves were sampled on mornings with full sun.The other eight replicates were used to determine grain yield,weight,protein content,and its components.

2.3.Sampling methods

Uniform-sized ears were selected at the same flowering stage,and plants were repeatedly sampled at 10 and 22 days after flowering and maturity.The seeds were air-dried for 30 days 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 of four protein components(albumin,gliadin,glutenin,and globulin).Protein content was determined by the semi-micro Kjeldahl nitrogen method[14].The content of the amino acids cysteine(Cys)and methionine(Met)in the grains was determined with a Hitachi L-8900 automatic amino acid analyzer(Hitachi High-Technologies Corporation,Beijing,China).NR and GS activity in flag leaves was determined following Yu and Zhang[17].The protein components of grains were determined by sequential extraction.Albumin,globulin,gliadin,and glutenin were sequentially extracted with distilled water,2%NaCl,70%ethanol,and 0.5%KOH[18].The content of total starch in grains was determined by the double-wavelength method[5].

2.4.Data analysis

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

3.Results

3.1.Effects of sulfur fertilization and HTS on grain yield

Post-anthesis exposure to HTS significantly reduced grain yield and grain weight(P＜0.05)compared with plants of the NT group in both cultivars.The influence of HTS on GY2018 was significantly greater than that on ZM8.

Fig.1–Effect of sulfur fertilization and high-temperature stress(HTS)on grain yield in two wheat cultivars(GY2018 and ZM8)from replicate experiments in 2016 and 2017.NT is the control.S0 and S45 indicate the level of sulfur fertilization treatments 0 and 45 kg sulfur ha?1.Bars represent standard deviations(SD).Letters signify significant differences.

Sulfur fertilization significantly increased grain yield and weight of both 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 higher in the S45 treatment than in the S0 treatment.On average,yield and weight in the sulfur treatments of GY2018 of both years increased by 34.7%and 30.2%(S45),compared to the control(S0).In ZM8,for both years,average increases in yield and weight were approximately 11.2%and 14.2%greater under S45 than under S0(Figs.1 and 2).

The interaction effects of cultivar×temperature,cultivar×sulfur fertilization,temperature×sulfur fertilization,and cultivar×temperature×sulfur fertilization on grain yield and weight were significant at P＜0.01;however,the effect of year and the interaction effect of year,cultivar,temperature,and sulfur fertilization were not significant(Table 1).Sulfur fertilization had the largest contribution to total variation of grain yield and grain weight,followed by temperature,cultivar,cultivar×sulfur fertilization,temperature×sulfur fertilization,cultivar×temperature,and cultivar×temperature×sulfur fertilization (Table 2).Sulfur fertilization had a significant effect on grain yield and grain weight in wheat under HTS.Comparisons between the S0 treatment under HTS and the S0 treatment grown at ambient temperature in both experimental years revealed reductions in grain yield by 26.7%in GY2018 and 12.9%in ZM8.Grain weight was reduced by 12.9%in GY2018 and 14.3%in ZM8.In contrast,in the S45 treatments under HTS,grain yield was reduced by 6.5%and grain weight by 8.2%in GY2018 in 2016,compared to their respective sulfur treatment levels that were not applied under HTS.The same treatment comparisons were reduced 8.5%in yield and 6.9%in weight for GY2018 in 2017.For ZM8,the same treatment comparisons were reduced by 4.0%in yield and 5.9%in weight in 2016.In the 2017 treatment comparisons for Zm8,yield and weight were reduced by 4.9%and 5.6%,respectively(Figs.1 and 2).

3.2.Effects of sulfur fertilization and high temperature stress on nitrate reductase activity

The activity of NR,measured 10 days after flowering,was significantly affected by cultivar and sulfur fertilization(P＜0.05).However,the effect of year and the interaction effect of year,cultivar,and sulfur fertilization were not significant.The NR activity of flag leaves,22 days after flowering,was significantly affected by cultivar,temperature,sulfur fertilization,and temperature×sulfur fertilization(P＜0.01).However,the effect of year and the interaction effect of year,cultivar,temperature,and sulfur fertilization were not significant(Table 1).Sulfur fertilization had the greatest contribution to the total variation of NR activity,followed by temperature,temperature×sulfur fertilization,and cultivar(Table 2).Sulfur fertilization had a significant effect on the NR activity of flag leaves under HTS.Compared with the S0 treatment under HTS,the reduction in activity of NR was reduced in both cultivars in both years of S45 treatments under HTS(Fig.3).

Fig.2 – Effect of sulfur fertilization(S)(45 or 0 kg sulfur ha?1)and high-temperature stress(HTS)or no-stress(NT)on grain weight of each wheat cultivar(GY2018 and ZM8).Bars represent SD.Letters signify significant differences.

On the tenth day after flowering,NR activity was significantly greater in ZM8 than in GY2018(P＜0.05).There was no significant difference in NR activity between the two cultivars 22 days after flowering(Fig.3).

Both 10 or 22 days after flowering,NR activity of the two cultivars were significantly greater with sulfur fertilization than without(P＜0.05)(Fig.3).

3.3.Effects of sulfur fertilization and high temperature stress on glutamine synthetase activity

The activity of GS was significantly affected by cultivar,sulfur fertilization,and variety×sulfur fertilization at DAA10(P＜0.01).At DAA10,sulfur fertilization significantly increased the GS activity of ZM8 and GY2018,with activity of GS in ZM8 significantly higher than that in GY2018(Fig.4).GS activity was significantly affected by cultivar,temperature,sulfur fertilization,and sulfur fertilization×temperature at DAA22(P＜0.01)(Table 1).Heat stress significantly reduced(P＜0.05)the activity of GS in both wheat cultivars;there was a greater reduction of GS activity in GY2018 than in ZM8 in both 2016 and 2017(Fig.4).

Activity of GS was significantly higher(P＜0.05)in ZM8 than in GY2018.Sulfur fertilization had the greatest contribution to the total variation of GS activity,followed by temperature,sulfur fertilization×temperature,and cultivar(Table 2).The results showed that the activity of GS in both cultivars was sensitive to high-temperature stress and to sulfur fertilization.

3.4.Effects of sulfur fertilization and high temperature stress on content of total grain protein and flour protein components

Total grain protein content and protein yield were significantly affected by cultivar,temperature,sulfur fertilization,cultivar×temperature,cultivar×sulfur fertilization,and temperature×sulfur fertilization(P＜0.01)(Table 1).Sulfur fertilization significantly increased grain protein content and protein yield in both wheat cultivars(P＜0.05).Compared to the S0 treatment,the S45 treatment protein content was greater by 16.6%in GY2018 in both years.In similar treatment comparisons,protein content was greater by 6.2%in ZM8 in both years(Fig.5).Similar results were observed for protein yield in both wheat cultivars and both replicate years.For GY2018,treatment comparisons of protein yield in S45 to that of S0 were greater by 51.3%.Similar comparisons in ZM8 produced greater differences by 21.5%(Fig.6).

The protein content of HTS-treated samples was significantly greater than that of the control samples(P＜0.05).However,protein yield was significantly lower(P＜0.05).In GY2018,compared to the control treatments(averaged over both sulfur treatment groups),protein content of the HTS treatment groups was 5.1%and 4.8%greater in 2016 and 2017,respectively.In similar treatment comparisons,for ZM8,protein content of controls was 2.6%and 2.8%greater than in the HTS treatments in 2016 and 2017,respectively(Fig.5).In contrast,in GY2018,the mean protein yields of the HTS treatment groups were 5.5%and 5.7%lower than those of the controls in 2016 and 2017,respectively.In ZM8,the mean protein yield was also lower in the same comparisons,by 7.7%in 2016 and 8.0%in 2017(Fig.6).

Fig.3 – Effect of sulfur fertilization(S)(45 or 0 kg sulfur ha?1)and high-temperature stress(HTS)or no-stress(NT)on nitrate reductase activity of flag leaves at 10(DAA10)and 22(DAA22)days after flowering in cultivars,GY2018 and ZM8.Bars represent SD.Letters signify significant differences.

The application of sulfur fertilization to plants that were heat-stressed caused increases in protein content and reductions in protein yield.Furthermore,additional sulfur had a greater effect on GY2018 than on ZM8.Grain protein content was significantly greater under the heat-stressed,sulfur added treatments than under the heat-stressed,S0 treatment in both replicates of both wheat cultivars.In contrast,for the same treatment comparison,grain protein yield was significantly lower in both replicates of both wheat cultivars.Under HTS,grain protein content was significantly greater in GY2018 than in ZM8.In both years,the reduction in grain protein content was significantly lower under the sulfur treatment in GY2018 than in ZM8.Moreover,there was significantly more protein in GY2018 than in ZM8(Figs.5 and 6).

The proportions of protein components in the two cultivars varied across treatment combinations(Fig.7).A ranking of the content of each component of grain protein in both cultivars is glutenin＞gliadin＞albumin＞globulin. The glutenin content and the ratio of glutenin to gliadin of the strong strong-gluten GY2018 was significantly higher than that of the medium-gluten ZM8.The HTS reduced the globulin content in both cultivars,whereas HTS increased the content of the other components.The ratio of glutenin to gliadin decreased in GY2018 under HTS,but no change was observed in ZM8.The ratio of glutenin to gliadin increased in GY2018 and ZM8 under the sulfur fertilization treatment.Lower concentrations of gliadin and glutenin were observed under the S0 treatment than under the S45 treatment in both GY2018 and ZM8.A similar trend was found for albumin and globulin content.There were increases of albumin and globulin content in GY2018 and in ZM8 with the addition of sulfur fertilization.The albumin and globulin contents were higher in ZM8 than in GY2018.

The contents of glutenin,gliadin,albumin,and globulin were significantly affected by cultivar,temperature,and sulfur fertilization(P＜0.05 or P＜0.01).The glutenin content was significantly affected by interaction effects of cultivar×sulfur fertilization(P＜0.01),temperature×sulfur fertilization(P＜0.05),and cultivar×temperature×sulfur fertilization(P＜0.01).The gliadin content was significantly affected by the interaction effect of cultivar×temperature×sulfur fertilization(P＜0.01)(Table 1).

The effect of cultivar had the greatest contribution to the total variation in gliadin,glutenin,albumin,and globulin content.This contribution was followed by that of sulfur fertilization and then by that of temperature(Table 2).In both years,comparison of sulfur-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 in ZM8.In contrast,in the S45 treatment under HTS compared to S0 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).

Fig.4 –Effect of sulfur fertilization(S)(45 or 0 kg sulfur ha?1)and high temperature stress(HTS)or no-stress(NT)on glutamine synthetase activity of flag leaves,10(DAA10)and 22(DAA22)days after flowering in each cultivar.Bars represent SD.Letters signify significant differences.

3.5.Effects of sulfur fertilization and high temperature stress on the content of total starch in grains

Post-anthesis exposure to HTS significantly reduced the content of total starch in grains(P＜0.05)compared with plants of the NT group for both cultivars.The influence of HTS was significantly higher on GY2018 than on ZM8.

Sulfur fertilization significantly increased the content of total starch in grains of the two cultivars(P＜0.05).A consistent pattern was observed in the content of total starch in both cultivars and over both experimental years.Total starch content was consistently higher under the S45 treatment than under the S0 treatment.On average,the content of total starch under the sulfur treatments of GY2018 of both years increased by 14.2%(S45)compared to the control(S0).In ZM8,for both years,average increases in the content of total starch were approximately 12.3%(S45)greater than in S0(Table 3).

The interaction effects of cultivar×temperature,and of temperature×sulfur fertilization on the content of total starch were significant at P＜0.01;however,the effect of year and the interaction effect of year,cultivar,temperature,and sulfur fertilization were not significant(Table 1).Sulfur fertilization had the largest contribution to total variation of content of total starch,followed by temperature,cultivar,cultivar×temperature,and temperature×sulfur fertilization(Table 2).Sulfur fertilization had a significant effect on the content of total starch in wheat under HTS.Comparisons between the S0 treatment under HTS and the S0 treatment grown at ambient temperature for their respective cultivars in both experimental years revealed reductions in the content of total starch by about 16.5%in GY2018 and 9.7%in ZM8.In contrast,in the S45 treatments under HTS compared to their respective sulfur treatment levels that were not under HTS,the content of total starch was reduced by 10.4%and 11.6%in GY2018 in 2016 and 2017;and the content of total starch was reduced by 7.0%and 6.1%in ZM8 in 2016 and 2017(Table 3).

3.6.Effects of sulfur fertilization and high temperature stress on the content of cysteine and methionine in grains

The content of cysteine and methionine in kernels 22 days after flowering was significantly affected by cultivar,temperature,sulfur fertilization,and cultivar×sulfur fertilization(P＜0.05)(Table 1).However,the effect of year and the interaction effect of year,cultivar,temperature,and sulfur fertilization were not significant(Table 1).Cultivar had the greatest contribution to the total variation of the content of cysteine and methionine in grains,followed by sulfur fertilization,temperature,and cultivar×sulfur fertilization(Table 2).Compared to the S0 treatment,the content of cysteine and methionine in the S45 treatment was greater by 15.9%and 19.6%,respectively,in GY2018 in both years.In similar treatment comparisons,content of cysteine and methionine was greater by 9.0%and 11.5%in ZM8 in both years(Table 3).

The content of cysteine and methionine of HTS-treated samples was significantly greater than that of the control samples(P＜0.05).In GY2018,compared to the control treatments(averaged across both sulfur treatment groups),the content of cysteine and methionine of HTS treatment groups was 2.9%and 3.7%greater in both years,respectively.In similar treatment comparisons,for ZM8,the content of cysteine and methionine of HTS treatments was respectively 9.0%and 11.5%greater than those of controls in both years(Table 3).

4.Discussion

Nitrate reductase is important for the uptake and utilization of nitrogen because it affects crop yield and quality[19].Glutamine synthetase is a multifunctional enzyme involved in nitrogen metabolism and the regulation of many other metabolic processes.The activity of NR and GS were increased by sulfur fertilization up to 10—60 kg sulfur ha?1[20—22].In our study,45 kg sulfur ha?1treatment increased the activity of NR and GS,but the magnitude of the increase in the activity in the two cultivars varied.Thus,the amount of sulfur fertilization available in soil can affect the NR and GS activity of flag leaves,but the mechanism of these changes in activity needs further study.

Previous studies have yielded similar results:high temperature stress is known to reduce NR and GS activity in flag leaves[23].This observed reduction in activity of both enzymes caused by the HTS above 35°C may have accelerated the senescence of the plant in its later life stage of grain filling.Stress from high temperature may have been so severe that plants were unable to recover from or compensate for damage.Thus,the activity of NR in flag leaves decreased or possibly ceased.The likely consequence is thatis reduced to,which is subsequently reduced.Loss of availablewhich is a substrate of the GS reaction pathway,can cause a decrease in GS activity[23].This study also showed that the interaction effect of sulfur fertilization×temperature significantly affected NR and GS activities,showing that sulfur fertilization increased NR and GS activity in flag leaves of the two cultivars under high-temperature stress.

Fig.6 – Effect of sulfur fertilization(S)(0 and 45 kg sulfur ha?1)and high temperature stress(HTS)or no stress(NT)on grain protein yield of two wheat cultivars(GY2018 and ZM8).Bars represent SD.Letters signify significant differences.

Protein is an important component of wheat grain,and the contents of albumin,globulin,and essential amino acids(Met)affect nutritional quality.Glutelin,gliadin,and non-essential amino acid(Cys)determine dough viscoelasticity,which has strong influence on the processing quality of flour[24].It is possible that the addition of sulfur fertilization increased NR and GS activities and consequently affected grain protein content and flour protein components.The contents of albumin,glutenin,gliadin,Cys,and Met increased with the application of sulfur fertilization(45 kg sulfur ha?1)and with HTS.Gluten is the main component of flour proteins.Gluten proteins include gliadin and glutenin,are the main storage proteins of wheat flour,and directly affect the processing quality of flour[25].In contrast,the non-gluten proteins,albumin and globulin,are mainly structural proteins with high nutritional value,because they are rich in Met and other important amino acids[26].Sulfur fertilization addition and HTS also increased the ratio of glutenin to gliadin in GY2018.The ratio of glutenin to gliadin is one of the important determinants for wheat processing quality traits.The increase in glutenin content and thus,gluten content,can cause sedimentation and stabilization time to increase,resulting in improved flour processing characteristics[14].The flour protein components examined in the present study may be affected by sulfur,HTS,Cys,and Met.Sulfur fertilization can increase content of sulfur-bearing protein Cy,then Cys promotes gluten formation and improve the elasticity of gluten and thereby increase the processing quality of wheat[8,16].Sulfur fertilization can increase content of sulfur bearing protein Met and thereby improve nutritional quality of wheat[6].In our study,the content of Cys and Met increased with the application of sulfur fertilization,and the content of Cys and Met was significantly affected by cultivar,temperature,sulfur fertilization,and cultivar×sulfur fertilization(Table 1).These results also showed that sulfur fertilization can help to improve the processing quality and nutritional quality of wheat under HTS.However,this study was limited to a pot experiment,and further research is needed to fully understand the underlying mechanisms of sulfur effects on flour protein components.

Exposure to high temperature stress caused a reduction in grain yield and grain weight,and an increase in grain protein content[27].Contents of grain protein,albumin,gliadin,and glutenin increased in plants of both cultivars that experienced short-term HTS 20 days after flowering.The likely reason is that the effect of HTS was greater on starch synthesis in grain than 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[2,28].On average,the content of total starch of GY2018 of both years decreased by 13.6%in HTS compared to the control(NT).In ZM8,for both years,average decreases in the content of total starch were approximately 8.1%(HTS)lower than those in NT,while the content of total grain protein of GY2018 of both years increased by 4.9%in HTS compared to the control(NT).In ZM8,for both years,average increases in the content of grain protein were approximately 2.7%(under HTS)higher than those in NT.Thus,the relative increase of the content of total grain protein under high temperature stress is the likely cause of the significant inhibition of starch synthesis.However,on average,the content of total starch of GY2018 in both years decreased by 11.0%in HTS compared to S45.In ZM8,for both years,average decreases in the content of total starch were approximately 6.5%lower under HTS than under S45,but the content of total grain protein of GY2018 of both years increased by 5.2%under HTS compared to S45.In ZM8,for both years,average increases in the content of total grain protein were approximately 3.6%(HTS)higher than S45.These results collectively demonstrate that the relative increase in the content of total grain protein under high temperature stress is the cause of the reduced inhibition of starch synthesis under sulfur fertilization treatment.

Fig.7 – Effect of sulfur fertilization(S)(0 and 45 kg sulfur ha?1)and high temperature stress(HTS)or no-stress(NT)on grain protein component of two wheat cultivars(GY2018 and ZM8).

Extreme heat can reduce the function of enzymes in plant chlorophyll,which uses light energy and water to produce sugars and energy that are important for seed growth[29].Thus,exposure to HTS 20 days after flowering in this study may have reduced photosynthetic rate,causing the subsequent reduction in grain weight and increase in protein content.Higher temperatures can also cause wheat to mature faster,which can shorten the grain filling period[28].Thus,grain weight is reduced,grain nitrogen content increases,and grain protein content increases correspondingly[23,30].HTS increased grain protein content in both cultivars,but reduced grain weight,the content of total starch,and protein yield.Additional sulfur fertilization reduced grain weight and total starch loss in HTS-treated plants of both cultivars(Fig.2,Table 3),and sulfur fertilization significantly(P＜0.05)increased grain protein content,protein yield,and total starch content in both wheat cultivars.Compared with ZM8 underHTS,grain protein content in GY2018 was significantly higher(Figs.5 and 6).

Table 3–Effects of sulfur fertilization and high temperature stress on the content of total starch,cysteine and methionine in grains in 2016 and 2017.

HTS increased glutenin and gliadin content in both cultivars,and the ratio of glutenin to gliadin increased in GY2018,but no change was observed in ZM8(Fig. 7). The glutenin content and the ratio of glutenin to gliadin of the strong-gluten GY2018 were significantly higher than that of the medium-gluten ZM8.Compared with the S0 treatment under HTS,sulfur treatments increased glutenin content,gliadin content,and the ratio of glutenin to gliadin.These changes suggest that the content of main storage proteins of wheat flour also increased.Higher amounts of storage protein increases gluten strength,which indicates better flour processing quality,and GY2018 is a prime example of a strong-gluten wheat. Application of 45 kg sulfur ha-?1to plants under HTS improved the processing quality of flour in the strong-gluten wheat GY2018 and the medium-gluten wheat ZM8.Sulfur addition was especially beneficial for GY2018 because it alleviated some of the negative effects of HTS on grain weight and grain yield.

5.Conclusions

The addition of sulfur fertilization increased grain yield and weight,increased NR and GS activity within days but not weeks after anthesis,and increased grain protein yield,content,and components(glutenin,gliadin,albumin,and globulin),and contents of total starch,cysteine,and methionine.High-temperature stress reduced grain yield and weight,total starch content,and NR and GS activities,but increased grain protein content and content of cysteine and methionine.HTS also reduced grain protein yield and globulin content,and reduced glutenin,gliadin,albumin.Sulfur fertilization and high-temperature stress had different effects on different wheat cultivars.Sulfur fertilization 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 methods to increase grain yield while maintaining grain quality,in view of the potential threats to plant growth from climate change.

Acknowledgments

This work was supported by the National Key Research and Development Program of China(2016YFD0300407),and China Agriculture Research System(CARS-03).

R E F E R E N C E S

[1]FAOSTAT,Food and Agriculture Organization of the United Nations,Rome,Italy,http://faostat.fao.org/2017(Agricultural Data).

[2]C.S.Blumenthal,F.Bekes,I.L.Batey,C.W.Wringely,H.J.Mass,D.J.Mares,E.W.R.Barlow,Interpretation of grain quality results from wheat variety trials with reference to higher temperature stress,Aust.J.Agric.Res.42(1991)325—334.

[3]S.Asseng,F.Ewert,P.Martre,R.P.Rotter,D.B.Lobell,D.Cammarano,B.A.Kimball,M.J.Ottman,G.W.Wall,J.W.White,M.P.Reynolds,P.D.Alderman,P.V.V.Prasad,P.K.Aggarwal,J.Anothai,B.Basso,C.Biernath,A.J.Challinor,G.D.Sanctis,J.Doltra,E.Fereres,M.Garcia-Vila,S.Gayler,G.Hoogenboom,L.A.Hunt,R.C.Izaurralde,M.Jabloun,C.D.Jones,K.C.Kersebaum,A.K.Koehler,C.Müller,K.S.Naresh,C.Nendel,G.O'Leary,J.E.Olesen,T.Palosuo,E.Priesack,E.E.Rezaei,A.C.Ruane,M.A.Semenov,I.Shcherbak,C.St?ckle,P.Stratonovitch,T.Streck,I.Supit,F.Tao,P.J.Thorburn,K.Waha,E.Wang,D.Wallach,J.Wolf,Z.Zhao,Y.Zhu,Rising temperatures reduce global wheat production,Nat.Clim.Chang.5(2015)143—147.

[4]J.G.Nuttall,G.J.O'Leary,J.F.Panozzo,C.K.Walker,K.M.Barlow,G.J.Fitzgerald,Models of grain quality in wheat-a review,Field Crops Res.202(2017)136—145.

[5]I.Tea,T.Genter,N.Naulet,M.Lummerzheim,D.Kleiber,Interaction between nitrogen and sulfur by foliar application and its effects on flour bread-making quality,J.Sci.Food Agric.87(2007)2853—2859.

[6]H.Klikocka,M.Cybulska,B.Barczak,B.Narolskil,B.Szostak,A.Kobia?ka,A.Nowak,E.Wójcik,The effect of sulphur and nitrogen fertilization on grain yield and technological quality of spring wheat,Plant Soil Environ.62(2016)230—236.

[7]A.G.Flynn,J.F.Panozzo,W.K.Gardner,The effect of copper deficiency on the baking quality and dough properties of wheat flour,J.Cereal Sci.6(1987)91—98.

[8]Q.S.Sun,J.Huang,X.J.Wu,H.D.Jiang,Q.Zhou,Effect of different acidities of acid rain on nitrogen and sulfur metabolism and grain protein levels in wheat after anthesis,Acta Ecol.Sin.36(2016)190—199(in Chinese with English abstract).

[9]G.M.Yang,G.C.Zhao,L.H.Liu,Y.S.Yang,Sulphur effect on protein components and grain yield of wheat,Chin.J.Soil Sci.38(2007)89—92(in Chinese with English abstract).

[10]U.Swamy,M.Wang,J.N.Tripathy,S.K.Kim,M.Hirasawa,D.B.Knaff,J.P.Allen,Structure of spinach nitrite reductase:implications for multi-electron reactions by the iron-sulfur:siroheme cofactor,Biochemistry 44(2005)16054—16063.

[11]Y.X.Xie,H.Zhang,Y.J.Zhu,L.Zhao,J.H.Yang,F.N.Cha,C.Liu,C.Y.Wang,T.C.Guo,Grain yield and water use of winter wheat as affected by water and sulfur supply in the North China Plain,J.Integr.Agric.16(2017)614—625.

[12]P.Gallejones,A.Castellon,A.Del Prado,O.Unamunzaga,A.Aizpurua,Nitrogen and sulphur fertilization effect on leaching losses,nutrient balance and plant quality in a wheat-rapeseed rotation under a humid Mediterranean climate,Nutr.Cycl.Agroecosyst.93(2012)337—355.

[13]C.Z?rb,D.Steinfurth,V.G?dde,K.Niehaus,K.H.Niehaus,Metabolite profiling of wheat flag leaf and grains during grain filling phase as affected by sulfur fertilization,Funct.Plant Biol.39(2012)156—166.

[14]P.Zhao,F.Yang,F.Q.Sui,Q.Y.Wang,The effects of zinc and nitrogen on wheat nitrogen using,yield and grain protein content,J.China Agric.Univ.18(2013)28—33(in Chinese with English abstract).

[15]J.B.Geng,Q.Ma,J.Q.Chen,M.Zhang,C.L.Li,Y.C.Yang,X.Y.Yang,W.T.Zhang,Z.G.Liu,Effects of polymer coated urea and sulfur fertilization on yield,nitrogen use efficiency and leaf senescence of cotton,Field Crops Res.187(2016)87—95.

[16]L.M.Tabe,M.Droux,Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein,Plant Physiol.128(2002)1137—1148.

[17]X.Z.Yu,F.Z.Zhang,Activities of nitrate reductase and glutamine synthetase in rice seedlings during cyanide metabolism,J.Hazard.Mater.225—226(2012)190—194.

[18]H.E.Liu,Q.Y.Wang,Z.Renge,P.Zhao,Zinc fertilization alters flour protein composition of winter wheat genotypes varying in gluten content,Plant Soil Environ.61(2015)195—200.

[19]W.R.Raun,G.V.Johnson,Improving nitrogen use efficiency for cereal production,Agron.J.91(1999)357—363.

[20]C.H.Foyer,M.Parry,G.Noctor,Markers and signals associated with nitrogen assimilation in higher plants,J.Exp.Bot.54(2003)585—593.

[21]A.F.Arata,S.E.Lerner,G.E.Tranquilli,A.C.Arrigoni,D.P.Rondanini,Nitrogen×sulfur interaction on fertiliser-use efficiency in bread wheat genotypes from the Argentine Pampas,Crop Pasture Sci.68(2017)202—212.

[22]M.M.A.Blake-Kalff,M.J.Hawkesford,F.J.Zhao,S.P.McGrath,Diagnosing sulfur deficiency in field-grown oilseed rape(Brassica napus L.)and wheat(Triticum aestivum L.),Plant Soil 225(2000)95—107.

[23]P.Liu,W.S.Guo,H.C.Pu,C.N.Feng,X.K.Zhu,Y.X.Peng,Effects of transient high temperature after anthesis on grain protein content and physiological mechanism in wheat(Triticum aestivum L.),Acta Agron.Sin.33(2007)1516—1522(in Chinese with English abstract).

[24]W.Y.Li,S.H.Yan,Z.L.Wang,Comparison of grain protein components and processing quality in responses to dim light during grain filling between strong and weak gluten wheat cultivars,Acta Ecol.Sin.32(2012)265—273(in Chinese with English abstract).

[25]H.Goesaert,K.Brijs,W.S.Veraverbeke,C.M.Courtin,K.Gebruers,J.A.Delcour,Wheat flour constituents:how they impact bread quality,and how to impact their functionality,Trends Food Sci.Technol.16(2005)12—30.

[26]H.Wieser,W.Seilmeier,The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour,J.Sci.Food Agric.76(1998)49—55.

[27]J.Wang,C.N.Feng,W.S.Guo,X.K.Zhu,C.Y.Li,Y.X.Peng,Effects of high temperature after anthesis on starch traits of grain in wheat,J.Triticeae Crops 28(2008)260—265(in Chinese with English abstract).

[28]N.Akter,M.R.Islam,Heat stress effects and management in wheat.A review,Agron.Sustain.Dev.37(2017)37.

[29]A.Wahid,S.Gelani,M.Ashraf,M.R.Foolad,Heat tolerance in plants:an overview,Environ.Exp.Bot.61(2007)199—223.

[30]C.F.Jenner,The physiology of starch and protein deposition in the endosperm of wheat,Aust.J.Plant Physiol.18(1991)211—226.