Yongli Luo,Yuhi Tng,Xin Zhng,Wenqin Li,Yongln Chng,Dngwei Png,b,Xu Xu,Yong Li,*,Zhenlin Wng,*

aState Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an 271018, Shandong, China

bCollege of Life Sciences, Shandong Agricultural University, Tai'an 271018, Shandong, China

cJiasixie Agronomy College of Weifang University of Science and Technology, Shouguang 262700, Shandong, China

Keywords:Triticum aestivum L.Interaction Cytokinin Nitrogen Staygreen wheat Flag leaf senescence

A B S T R A C T Premature senescence after anthesis reduces crop yields.Delaying leaf senescence could maintain photosynthetic activity for a longer period and lead to a higher photosynthetic rate.Recent studies have provided some insights into the interaction between cytokinin and nitrogen(N)in the regulation of plant development.In the present study,foliar application of exogenous 6-benzylaminopurine(6-BA)and lovastatin,an inhibitor of cytokinin synthesis,was combined with three N rates[0 kg ha?1(low nitrogen,LN),240 kg ha?1(normal nitrogen,NN),and 360 kg ha?1(high nitrogen,HN)]in two wheat cultivars,Wennong 6(with a staygreen phenotype)and Jimai 20(with a non-staygreen phenotype).Flag leaf senescence was assessed using a Gompertz growth curve.Grain mass,dry matter accumulation and distribution,total N of flag leaf,and concentrations of zeatin riboside(ZR)and abscisic acid(ABA)were also used to evaluate the functional characteristics of flag leaves.Grain mass was negatively correlated with initial senescence rate(r0)and duration of rapid chlorophyll loss(Chlloss),whereas it was positively correlated with maximum senescence rate(rmax),average senescence rate(raver),persistence phase(Chlper),total duration of flag leaf(Chltotal)and inflection point cumulative temperature(M).Compared to Jimai 20,Wennong 6 had larger raver,Chlper,and Chltotal.The concentration of ZR was highest under the 6-BA×NN treatment,followed by the 6-BA×HN and 6-BA×LN treatments.However,the concentration of ABA showed the opposite trend.It was concluded that the staygreen phenotype Wennong 6 was associated with greater grain mass and altered cytokinin metabolism and could be classified as a functional staygreen type.Foliar application of 6-BA interacting with N at the NN level(240 kg ha?1)may be a beneficial strategy for improving grain yield of wheat by regulating endogenous hormones and the flag leaf senescence process.Increasing endogenous cytokinin promoted the transport of dry matter to grain.

1.Introduction

Leaf senescence is a developmental process that involves degradation of macromolecules such as nucleic acids and proteins, accumulation of reactive oxygen species, and a transition from nutrient assimilation to nutrient remobilization[1–3].Premature senescence can lead to a reduction in canopy size,loss of light use efficiency,and decreased crop yield [4]. Delaying leaf senescence may enhance drought tolerance and improve the productivity of crop plants[4,5].Recent findings have indicated that flag leaves are major photosynthetic organs during the grain-filling period in cereal crops such as wheat(Triticum aestivum L.)and rice(Oryza sativa L.),making a major(41%–43%)contribution to crop yield[6,7].Accordingly,most studies of leaf senescence have focused on flag leaf senescence.

Plant hormones and related growth regulators play a vital role in the regulation of plant senescence.Besides hormones,nitrogen is another key factor in regulating leaf senescence of crops.Increasing evidence has highlighted the strong correlation between the concentration of cytokinins and nitrogen[8–10].Cytokinin synthesis is upregulated by nitrate()[10].Most studies [11–14] have shown thatpromotes the synthesis of highly active cytokinins such as zeatin,transzeatin riboside (tZR), and isopentenyl adenosine (IPR); in contrast,nutrition is a negative factor limiting the synthesis of cytokinins.Takei et al.[10]have provided direct evidence that AtIPT3 is a key determinant of nitratedependent cytokinin biosynthesis in Arabidopsis thaliana L.[15].AtIPT3 is partly regulated by a protein NRT1.1/CHL1 that functions as a nitrate transporter and nitrate sensor[16,17].However,other studies[18,19] have indicated that plants cultured in nutrient solution usingas nitrogen source accumulate higher levels of cytokinins thanfed plants.Recently[20],it was reported thatregulated stem cell dynamics in shoot meristems of Arabidopsis via changing cytokinin metabolism.The above studies have contributed to understanding of the close relationship between nitrogen()and cytokinins.

Cytokinins play a pivotal role in coordinating demand for and acquisition of nitrogen as well as nitrogen remobilization[21,22].Further studies[10]suggest that cytokinins function as a root-to-shoot long-distance signal;when the concentration of cytokinins is deficient,supplementary nitrogen will be transferred from root to shoot,and thus nitrogen supplementation causes an increase in cytokinin concentration in xylem sap.In a recent study[23],supplementation with exogenous cytokinin resulted in enhanced photosynthesis and increased biomass productivity under nitrogen stress.Cytokinins also function as a shoot-to-root long-distance signal.Cytokinins produced in roots or translocated from shoots suppress the expression of nitrogen uptake-associated genes such as AtNRT genes.As a result,nitrate uptake in the root is inhibited[22,24].In summary,cytokinins function as a root-to-shoot long-distance signal of nitrogen supplement and also act as a local signal or as a shoot-to-root long-distance signal.

Increasing numbers of studies have shed light on the interaction between cytokinin and nitrogen in the regulation of plant metabolism and development. Chang et al. [25]concluded that application of cytokinins combined with nitrogen was effective in improving the drought tolerance of creeping bentgrass(Agrostis stolonifera L.).Interaction between nitrogen availability and cytokinin promoted shoot branching in rice[26].Understanding how exogenous cytokinin signals interacting with nitrogen applied at varied rates affect the characteristics of flag leaf senescence in wheat cultivars that differ in staygreen performance would promote the objectives of increasing the yield and productivity of wheat by appropriate chemical application combined with nitrogen fertilization.Staygreen refers to the inherited delayed foliar senescence characteristic in crop plant species[27,28].The staygreen phenotype can be classified into five types,from type A to type E[29].

In the present study, the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20 were used to analyze the effects of cytokinin interaction with nitrogen rate on(i)senescence-associated endogenous hormones and(ii)the process of flag leaf senescence and related parameters.The focus was on increasing our understanding of the mechanisms of flag leaf senescence and elucidating the mechanisms of cytokinin interaction with nitrogen in the regulation of flag leaf senescence in wheat cultivars that differ in the staygreen property.

2.Materials and methods

2.1.Plant materials and growth conditions

The field experiments were conducted at a farm of Shandong Agricultural University,Tai'an,China(36°9′N,117°9′E;altitude 128 m)during the wheat growing season from October 2014 to June 2015 and from October 2015 to June 2016.The climate in this region is warm and semi-humid continental monsoon,with an average annual temperature of 13.7°C and an average annual precipitation of 631.5 mm.The soil was a sandy loam(pH 7.4)and maize(Zea mays L.)was the preceding crop.The 0–20 cm soil layer contained 17.81 g kg?1total organic matter,1.13 g kg?1total N,89.5 mg kg?1available N,16.87 mg kg?1available P2O5,and 109.60 mg kg?1available K2O.Two wheat genotypes,the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20,were grown in the experimental plots.The sowing dates were October 10,2014 and October 8,2015.The plant density was adjusted to 225 plants m?2at the three-leaf stage(GS13)[30].The harvesting dates were June 9,2015 and June 11,2016.Before planting,120 kg ha?1P2O5and 100 kg ha?1K2O were applied and incorporated into the soil as basal fertilizer.Pests,diseases,and weeds were controlled by appropriate chemical applications during the crop season.

2.2.Experimental design and treatments

The experiment was a 2×3×3[two cultivars,three N rates,and three exogenous hormone treatments(cytokinin,lovastatin,and water)] factorial design. Each treatment had three plots as repetitions in a randomized complete block design.The plot size was 3 m×3 m with 10 rows(0.25 m between rows).Nitrogen application treatments consisted of three N rates,0,240,and 360 kg ha?1,represented as low(LN),normal(NN),and high N(HN).Urea was applied as basal fertilizer at rates of 0,120,and 180 kg ha?1N before planting and another 0,120,and 180 kg ha?1N was applied at the jointing stage (GS31) as topdressing.Synthetic cytokinin[6-benzylaminopurine(6-BA)]and lovastatin,an inhibitor of cytokinin synthesis[31](both from Sigma,St Louis,MO, USA) were applied to leaves using an atomizer. The hormones were applied to all plant leaves for three days after anthesis(DAA)at 17:00 h.The effect of the application location of the hormones was not investigated. The foliar application method followed that used in previous studies[26,32,33].

6-BA was dissolved in 1 mol L?1HCl and then diluted so that stock solutions contained 1 mmol L?1HCl[34].Lovastatin was prepared in ethanolic NaOH(15%[v/v]ethanol,0.25%[w/v]NaOH),sonicated for 10 min,and heated at 50°C for 40 min[35].The spray concentration was 25 mg L?1(6-BA)and 300 mg L-?1(lovastatin)and the coverage was 100 mL m?2.All the solutions contained Tween-20 at a final concentration of 0.5%(v/v)as a surfactant.Control plants were sprayed with the same volume of deionized water containing the same concentration of Tween-20.

2.3.Flag leaf sampling

Sixty flag leaves from a plot were sampled every seven days from anthesis(GS61)to maturity.Half of the flag leaves were immediately frozen in liquid nitrogen for at least 30 min and stored at ?80°C for quantifying endogenous phytohormones.The other half were oven-dried at 70°C to constant weight for determination of total nitrogen concentration.

2.4.Determination of chlorophyll SPAD value

The relative chlorophyll content of flag leaves for each treatment was measured nondestructively with a handheld meter(SPAD 502,Minolta Camera Co.,Osaka,Japan).Ten healthy flag leaves in every treatment were tagged at anthesis(GS61)[30].Measurements were performed once a week from anthesis until 35 DAA.

2.5.Cumulative thermal time(degree days,°C d)

Spike flowering time was defined as that of first anther exsertion from the middle spikelets. Anthesis date was recorded when 50%of the spikes had begun flowering in the field.Daily thermal time was calculated as the average of maximum and minimum air temperature from anthesis to 35 DAA.Cumulative thermal time was then calculated as the summation of daily average temperature[(Tmax+Tmin)/2](as a base temperature of 0°C was assumed,Tmaxmeans daily maximum temperature and Tminmeans daily minimum temperature) [36]. Temperature data were obtained from Tai'an Meteorological Bureau,Shandong,China.

2.6.Flag leaf senescence

Chlorophyll SPAD values were fitted to cumulative thermal time after anthesis using the Gompertz growth equation(Eq.(1),Fig.1)describing leaf senescence kinetics according to Xie et al.[37]with modifications[38].here,G is chlorophyll SPAD value at cumulative thermal time t;t is cumulative thermal time after anthesis;a is the upper asymptote representing the maximum SPAD value predicted by the model during the period of measurement;b is an adjustable parameter that indirectly reflects the early senescence rate of the flag leaf.The initial senescence rate r0=b;the maximum senescence rate rmax=(a×r)/e;the average senescence rate raver=(r0+rmax)/2;inflection point cumulative temperature(M)is the cumulative thermal time when senescence rate is the maximum M=(ln b)/r;total duration of flag leaf(Chltotal)is defined as the period from anthesis to 90%senescence,Chltotal=[ln(ln 0.1)/b]/r;Chltotalconsists of two components:persistence phase(Chlper),from anthesis to tonset(the onset of senescence,10%senescence);and rapid loss phase,Chlloss=ttotal?tonset.

2.7.Determination of total nitrogen in flag leaf

Total nitrogen concentration in the flag leaf was determined by the Kjeldahl method[39]with slight modification.Powdered flag leaf samples(0.1 g)were digested in a Kjeldahl digestion flask by boiling with 8–10 mL of concentrated H2SO4,and 0.2 g of CuSO4and 3 g of K2SO4were added into the digestion flask as catalyst. When the mixture was clear,determination was performed with a FOSS Nitrogen Analyzer 2300(Foss Corporation,H?gan?s,Sweden)following Marcó et al.[40].Triplicate determinations were performed.

2.8.Assays of endogenous ZR and ABA in flag leaf

For extraction and purification of endogenous ZR and ABA,the protocol described by Engelberth[41]was followed with some modifications.ZR and ABA were analyzed with a triplequadrupole mass spectrometer(ACQUITY UPLC I-Class/Xevo TQ-S, Waters, Milford, MA, USA) equipped with an electrospray ion source(ESI).The LC separation was performed on a reversed-phase C18 column(ACQUITY UPLC BEH,1.7 μm,2.1 mm×100.0 mm,Waters,Milford,MA,USA)using a binary solvent system composed of water with 0.1%acetonitrile,0.05%formic acid(mobile phase A)and MeOH(mobile phase B)at a flow rate of 0.4 mL min?1.Standard ZR and ABA were purchased from Sigma.Isotopically labeled internal standards including D6-ABA and D5-ZR were supplied by OlChemIm Ltd.(OlChemIm Ltd.,Olomouc,Czech Republic). Methanol, acetonitrile, and formic acid were purchased from CNW(CNW Technologies GmbH,Dusseldorf,Germany).

2.9.Kernel mass

For each plot,500 kernels were dried in an oven at 85°C for 48 h until constant weight to calculate thousand-kernel weight.Six replications were taken per treatment every year.Two-year data are shown in Fig.2.

2.10.Dry matter accumulation and distribution

At anthesis and maturity of 2015 and 2016,30 plants(ten for each replicate)from each treatment were harvested,divided into four parts(leaves,stems and sheath,spikelet rachis and glume,and kernels),dried at 70°C to constant weight and then weighed for dry matter (DM) determination. The following parameters were calculated following Yang et al.[42].

Fig.1–Gompertz growth curve fit for flag leaf senescence model.Total duration of flag leaf(ttotal)is defined as the period from anthesis to the time when 90%of chlorophyll SPAD value has been lost(90%senescence).ttotal is then divided into two phases:persistence and rapid loss.Duration of leaf persistence is from anthesis to 10%senescence(tonset);duration of rapid loss is from tonset to ttotal.tmsr indicates the cumulative thermal time when flag leaf senescence rate was maximum.

2.11.Statistical analysis

Analysis of variance was performed with DPS 7.05 software(Zhejiang University, Hangzhou, China). Data from each sampling date were analyzed separately.Means were tested by least significant difference at P ＜0.05(LSD0.05).DPS was used for fitting the Gompertz growth equation.Graphs and Gompertz growth curve were drawn with SigmaPlot 10.0(Systat Software,Inc.,San Jose,CA,USA).

3.Results

3.1.Kernel mass

Grain mass of Wennong 6 was higher than that of Jimai 20 under the same treatment(Fig.2).Grain mass was highest at the NN(240 kg ha?1)level,followed by HN(360 kg ha?1)and LN(0 kg ha?1)in the two different staygreen wheat cultivars.Exogenous 6-BA application significantly increased the grain mass of both cultivars.Compared to the control,cytokinin application enhanced the grain mass of Wennong 6 under LN,NN,and HN by respectively 3.00%,4.81%,and 4.72%in the 2016 growing season and by 3.67%,4.14%,and 3.96%in the 2015 growing season.For Jimai 20,grain mass was increased by 4.31%,6.59%,and 5.14%under the treatments 6-BA×LN,6-BA×NN,and 6-BA×HN,respectively,in the 2015 and 2016 growing seasons.The results from the 2015 growing season showed a similar trend.Grain mass was increased under LN,NN,and HN supplementated with exogenous 6-BA by 5.53%,7.64%,and 5.73%,respectively.The two-year results indicated that compared to the control treatment,the increase of grain mass showed the trend 6-BA×NN ＞6-BA×HN ＞6-BA×LN.In contrast, application of lovastatin markedly decreased grain mass when nitrogen rates were the same in the two growing seasons of 2015 and 2016.Thus,interactions between cytokinin and nitrogen promoted grain filling and ultimately increased grain mass,especially the interactions between 6-BA and the“optimum”nitrogen rate(240 kg ha?1).

3.2.Dry matter accumulation and distribution

Table 1 shows two-year means(from 2014 to 2015 and from 2015 to 2016)showing the effects of exogenous 6-BA on dry matter distribution in different organs of wheat at maturity under different nitrogen levels.The effect of exogenous 6-BA on total weight of either wheat cultivar was not significant.However, under the same nitrogen treatment, spraying exogenous 6-BA significantly increased the amount and distribution of dry matter in the grain at maturity.However,the amount and distribution of dry matter in sheath,spikelet rachis and glume and leaves were lower than those in the control treatment.Exogenous 6-BA increased the contribution of storage and assimilation before anthesis to grain in Jimai 20,but exogenous 6-BA increased the contribution of storage and assimilation after anthesis in Wennong 6.Under different nitrogen levels,the contributions of storage and assimilation before anthesis to grain in Jimai 20 were in the order NN ＞HN ＞LN and the contributions of storage and assimilation after anthesis to grain in Wennong 6 were in the order NN ＞HN ＞LN in both years(Table 2).

Fig.2–Grain mass of two different staygreen cultivars under various nitrogen and hormone treatments.Wennong 6 and Jimai 20 represent the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20.LN,NN,and HN represent 0,240,and 360 kg ha?1 of nitrogen application,respectively.CK,6-BA,and LOV denote spraying water,6-benzylaminopurine,and lovastatin with Tween-20.a,b,and c indicate significant difference among different hormone treatments(LSD,P ＜0.05).

3.3.Total N in flag leaf

Total nitrogen in flag leaf varied with wheat cultivar,N rate,hormone treatment,and measurement period.As senescence progressed,the N concentration in the flag leaf decreased.Nitrogen concentration of the flag leaf in the staygreen cultivar Wennong 6 was significantly higher than that in Jimai 20. Total nitrogen of the flag leaf increased with increasing N rate in all the treatments from anthesis to maturity(Fig.3).Cytokinin application had a positive effect on total nitrogen in the flag leaf.Total nitrogen in the flag leaf was highest in the exogenous 6-BA treatment,intermediate in the CK treatment,and lowest in the LOV treatment under the same N levels.

Compared to the control(CK),exogenous 6-BA increased total nitrogen of the flag leaf under LN,NN,and HN by 11.16%,13.46%,and 9.67%in Jimai 20 and by 7.93%,9.45%,and 9.25%in Wennong 6.However,exogenous LOV reduced total nitrogen of the flag leaf under LN,NN and HN by 12.75%,10.34%,and 11.20% in Jimai 20, and by 10.12%, 9.36%, and 7.11% in Wennong 6,implying that cytokinin×NN could increase N concentration of the flag leaf more effectively than cytokinin×LN and cytokinin×HN.

3.4.Flag leaf senescence

Table 2–Effects of exogenous 6-BA on dry matter translocation amount from vegetative organ to kernels after anthesis.

Compared with Jimai 20,Wennong 6 had shorter Chlloss,longer Chlper, and longer Chltotal(Table 3), implying that Wennong 6 maintained a longer effective duration of photosynthesis.These results further explain why Wennong 6 is defined as a staygreen wheat cultivar.The r0of Wennong 6 was lower than that of Jimai 20,but the rmax,raver,and M of Wennong 6 were higher than those of Jimai 20.

Senescence parameters of the flag leaf also varied with nitrogen and hormone treatment.In the staygreen wheat cultivar Wennong 6,r0was highest under NN,followed by LN,and lowest under HN.However,for Jimai 20,r0showed the trend HN ＞LN ＞NN. The rmaxof Wennong 6 decreased overall with N deficiency from 0.1519(HN)to 0.1397(LN)and that of Jimai 20 from 0.1208(HN)to 0.1010(LN).Generally,Chlper, Chltotal, and M increased with N rate. Chllossof Wennong 6 increase with N rate,but for Jimai 20,Chllossshowed the trend HN ＞LN ＞NN.The rmax,Chlper,Chltotal,and M were highest in the 6-BA treatment,followed by the control treatment,and lowest in the LOV treatment at the same N rate.In contrast,r0and Chllossdisplayed the opposite trend:exogenous LOV increased r0and Chlloss.The r0and Chllosswere lowest in the 6-BA×LN treatment,whereas rmax,Chlper,Chltotal,and M were highest in the 6-BA×HN treatment of exogenous level,followed by the 6-BA×NN and 6-BA×LN treatments(Table 3).

3.5.Concentration of ZR and ABA in flag leaf

The concentration of ZR and ABA first increased and then decreased as senescence progressed from 7 to 28 DAA.The concentration of ZR was highest at 14 DAA,whereas the flag leaf accumulated the most abundant ABA at 21 DAA(Figs.4 and 5). The concentration of ZR in the flag leaf of the staygreen wheat Wennong 6 was greater than that of Jimai 20;however,the concentration of ABA showed the opposite trend(Fig.5).

Fig.3–Nitrogen concentration in the flag leaf(%)taken from 7 days after anthesis(DAA)to 35 DAA.Wennong 6 and Jimai 20 represent the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20,respectively.LN,NN,and HN represent respectively 0,240,and 360 kg ha?1 of applied nitrogen application.CK,6-BA,and LOV denote spraying water,6-benzylaminopurine,and lovastatin with Tween-20.

The concentration of ZR in the flag leaf of Wennong 6 increased by 21.63%following spraying 6-BA treatment and that of Jimai 20 increased by 22.50%,whereas the concentration of ABA in flag leaf of Wennong 6 was reduced by 13.58%and that of Jimai 20 by 26.85%(Figs.4 and 5).In contrast,exogenous LOV significantly reduced the concentration of ZR and increased the concentration of ABA in all the treatments.ZR increased with N rate while endogenous ABA decreased,suggesting that N application could promote endogenous ZR synthesis and inhibit ABA synthesis.The concentrations of ZR and ABA were significantly affected by cytokinin×nitrogen treatment.The concentration of ZR increased most in the 6-BA×NN treatment,followed by the 6-BA×HN and 6-BA×LN treatments (Fig. 4). However, the concentration of ABA showed the opposite trend(Fig.5).

3.6.Relationships of senescence-related parameters of flag leaf and grain mass with concentrations of ZR and ABA

The results in Table 4 show that grain mass was negatively correlated with r0and Chlloss(r=?0.79**,?0.65**,P ＜0.01)but positively correlated with rmax,raver,Chlper,Chltotal,and M(r=0.91**,0.77**,0.83**,0.81**,0.83**,P ＜0.01),implying that the duration of chlorophyll persistence and senescence rate was tightly correlated with grain mass.The concentration of ZR from 14 to 28 DAA was negatively correlated with r0and Chllossbut positively correlated with rmax, raver, Chlper,Chltotal,and M.The concentration of ABA from 21 to 28 DAA was positively correlated with r0and Chlloss(except for the concentration of ABA at 21 DAA with Chlloss).In contrast,it was negatively correlated with rmax,raver,Chlper,Chltotaland M,indicating that ZR and ABA are involved in regulating the senescence process of flag leaves in two different staygreen cultivars.

4.Discussion

4.1.Interaction between flag leaf senescence and individual grain weight

In the present study,the Chlperof Wennong 6 was much larger than that of Jimai 20(Table 3)and the grain mass of Wennong 6 was significantly higher than that of Jimai 20 (Fig. 2).Accordingly,Wennong 6 could be classified as a functional staygreen phenotype,called type A,which is a combination of delayed onset and a normal rate of senescence phenotype.However, it remains debatable whether the functional staygreen trait contributes to higher yield. Kumudini [43]and Ismail et al.[44]reported that the functional staygreen trait could compromise yield in high-N species such as soybean(Glycine max(L.)Merr.)and cowpea(Vigna unguiculata(L.)Walp.).In contrast,functional staygreen crops such as rice(Oryza sativa L.),wheat,and sorghum(Sorghum bicolor(L.)Moench)are expected to achieve a higher grain yield than normal crops [45–47]. In the present study, Chltotalwas positively correlated with grain mass(r=0.81,P ＜0.01)and Chllosswas the opposite(r=?0.65,P ＜0.01),implying that the functional staygreen trait was beneficial for increasing grain yield.It can be concluded that the functional staygreen trait is favorable for high-C crops(yield and composition in highcarbon crops such as cereals)to achieve a higher grain yield but unfavorable for high-N crops(such as legumes).In a previous study[48],the maximal flag leaf senescence rate waspositively correlated with thousand-kernel weight(r=0.87,P ＜0.01), and the timing and rate of progression of leaf senescence could substantially affect the yield and quality of crops [49]. Consistently, our study also found that the maximal flag leaf senescence rate rmaxwas significantly and positively correlated with grain mass(r=0.91,P ＜0.01).There was also a positive correlation between the initial flag leaf senescence rate and grain mass(r=?0.79,P ＜0.01).

Table 3–Flag leaf senescence model and the corresponding parameters of wheat during post-anthesis under different treatments.

Total nitrogen in the flag leaf was highest in the 6-BA×HN treatment;however,grain mass was highest in the 6-BA×NN treatment,followed by 6-BA×HN and 6-BA×LN(Figs.2 and 3).High nitrogen may delay whole-plant senescence,slow remobilization of nonstructural carbohydrate,and reduce the transportation of carbohydrate to grain.Thus,there was a slight decrease in grain mass under the 6-BA×HN treatment compared to the 6-BA×NN treatment.In a previous study[50],the N remobilization rate(the rate of N remobilization from vegetative to reproductive organs)was correlated with leaf senescence and the N remobilization rate affected the N accumulation and distribution in different organs,indicating that N accumulation and distribution in different organs have an important role in regulating senescence.The finding that the concentration of cytokinin in the flag leaf of Wennong 6 was higher than that of Jimai 20 in all treatments was in accord with the findings that the staygreen phenotype can be the result of alterations in hormone metabolism and signaling, particularly cytokinins [42,47]. Although spraying exogenous 6-BA significantly increased the amount and distribution of dry matter in the grain at maturity,the amount and distribution of dry matter in the sheath,spike rachis,and glume and leaves were lower than those in the control treatment (Table 1). Under different nitrogen levels, the contributions of storage and assimilation before anthesis to grain in Jimai 20 were NN ＞HN ＞LN and the contributions of storage and assimilation after anthesis to grain in Wennong 6 were NN ＞HN ＞LN from 2015 to 2016 and from 2014 to 2015(Table 2).These results show that application of exogenous 6-BA increased the amount and distribution of dry matter in the grain at maturity,indicating that increasing endogenous cytokinin increased the transport of dry matter to grain.

Fig.4–Effects of exogenous hormone application and of nitrogen at varying rates on the concentration of zeatin riboside in flag leaves.Wennong 6 and Jimai 20 represent the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20.LN,NN and HN represent 0,240,and 360 kg ha?1 of nitrogen application,respectively.CK,6-BA,and LOV denote spraying water,6-benzylaminopurine,and lovastatin with Tween-20.

Fig.5–Effects of exogenous hormone application and nitrogen at varying rates on the concentration of ABA in flag leaves.Wennong 6 and Jimai 20 represent the staygreen wheat cultivar Wennong 6 and the non-staygreen wheat cultivar Jimai 20.LN,NN,and HN represent 0,240,and 360 kg ha?1 of nitrogen application,respectively.CK,6-BA,and LOV denote spraying water,6-benzylaminopurine,and lovastatin with Tween-20.

Table 4–Correlation coefficients of senescence parameters and grain mass with concentrations of ZRand ABA in two wheat cultivars.

4.2.Interactions between exogenous cytokinin and nitrogen at various rates in the regulation of flag leaf senescence

Nitrogen, the non-carbon mineral nutrient required in greatest abundance by plants,is a signal controlling many aspects of plant metabolism and development[51,52].Previous evidence[53]suggested that nutrient stress due to lack of nitrogen induces leaf premature senescence.In contrast,high nitrogen levels could delay leaf senescence[54].In the present study,the HN treatment significantly increased total nitrogen in the flag leaf. In addition, rmax, Chlper, Chltotal, and M increased with N rate.These findings suggest that high N level delay the initiation of flag leaf senescence,prolong the leaf functional period,and delay tmsr(the emergence period of the maximum senescence rate).However,the trends between r0and nitrogen level and between Chllossand nitrogen level in Wennong 6 were not consistent with those in Jimai 20.For example,r0was highest under the NN treatment in Wennong 6,but it was lowest in Jimai 20(Table 3).The trends between r0and nitrogen level and between Chllossand nitrogen level in Wennong 6 were not consistent with those in Jimai 20.The explanation for the above trend that the relationship between flag leaf senescence related parameters and nitrogen level shows different rules in the wheat cultivars is unclear.Evidently the staygreen cultivar Wennong 6 responds differently from Jimai 20 to different nitrogen rates.

Endogenous hormones affect the plant growth and development of crops[55,56]and play an important role in the regulation of leaf senescence[57].He et al.[58]have proposed that the more rapidly cytokinin was transported from roots into leaves,the more delayed was leaf senescence initiation.In contrast,the translocation of ABA from roots to shoots was perhaps retarded in the staygreen cultivar,also resulting in delaying leaf senescence.The present results showed that exogenous 6-BA significantly enhanced endogenous ZR and decreased ABA.Although rmax,Chlper,Chltotal,and M were significantly increased by exogenous 6-BA,r0and Chllosswere decreased by 6-BA(Table 3).The concentration of ZR from 14 to 28 DAA was negatively correlated with r0and Chllossbut positively correlated with rmax,raver,Chlper,Chltotal,and M.The concentration of ABA from 21 to 28 DAA was positively correlated with r0and Chllossbut negatively correlated with rmax,raver,Chlper,Chltotal,and M(Table 4).These findings indicate that ZR and ABA are involved in regulating the senescence process in flag leaves in two different staygreen cultivars.

Nitrogen regulates plant metabolism and development by regulating the concentration of endogenous hormones[26].Most studies have shown that root-derived NO3?promotes the synthesis of high active cytokinins(display high activity in promoting plant growth and development,such as zeatin,tZR,and IPR)via enhanced expression of IPT that catalyze the limiting step of cytokinin biosynthesis[15].However,NH4+nutrition is a negative factor limiting the synthesis of cytokinins[11–14].Recently[20],it was reported that nitrate regulated stem cell dynamics in Arabidopsis shoot meristems via cytokinin metabolism.The above results contribute to understanding the tight relationship between nitrogen and cytokinin.The results of the present study suggest that the concentration of ZR increased with nitrogen rate, while endogenous ABA decreased(Figs.4 and 5), in agreement with the results of Singh et al.[8].That ZR increased with nitrogen rate showed that nitrogen could regulate cytokinin biosynthesis and degradation. Cytokinin, in turn, is an important factor that mediates the absorption, transport,assimilation,and metabolism of nitrogen[59].Recently,it has been reported that low nitrogen application with exogenous cytokinin results in higher photosynthetic performance and enhanced biomass productivity [23]. Cytokinins also function as a mobile signal controlling the efficiency of N uptake and assimilation [60] and can regulate nitrogen acquisition and assimilation in response to the changing plant C/N ratio[61].For this reason,we focused on the effect of the cytokinin interaction with nitrogen rate on flag leaf senescence and grain yield.Prior to this study,little information was available to elucidate the mechanisms by which the cytokinin×nitrogen rate interaction affected the flag leaf senescence process and the grain mass of cereal crops.Chang et al.[25]demonstrated that foliar application of cytokinin at 10 or 100 μmol L?1plus 7.5 kg ha?1N may be beneficial for improving drought stress resistance of creeping bentgrass(Agrostis stolonifera L.)by improving antioxidant metabolism.Our study showed that r0and Chllosswere lowest under the treatment with exogenous 6-BA interacting with LN level,but that rmax, Chlper, Chltotal, and M were highest under the treatment of exogenous 6-BA interacting with HN level,followed by the 6-BA×NN and 6-BA×LN treatments(Table 3).Grain mass,total nitrogen of flag leaf,and ZR concentration of ZR were increased most under the 6-BA×NN interaction treatment.

5.Conclusion

The flag leaf senescence process was studied using a Gompertz growth curve. The total duration of flag leaf senescence was divided into two phases:persistence and rapid loss.Longer Chltotal,longer Chlper,larger M,and shorter Chllosswere positively associated with larger grain mass.The flag leaf senescence process and related parameters differed significantly between Wennong 6 and Jimai 20.The functional staygreen trait can be favored for high-C crops such as winter wheat to achieve a higher grain yield,whereas it is unfavorable for high-N crops to achieve a higher grain yield.ZR and ABA were involved in regulating the senescence process of flag leaves in two different staygreen cultivars.The staygreen phenotype Wennong 6 was associated with greater grain mass and altered cytokinin metabolism.Foliar application of 6-BA interacting with nitrogen at the NN level(240 kg ha?1)can be beneficial for improving grain yield of wheat by regulating the flag leaf senescence process,which is mediated by endogenous hormones.

Acknowledgments

This study was supported by the National Key Research and Development Program of China (2017YFD0301001 and 2016YFD0300403),the National Basic Research Program of China(2015CB150404),and the Shandong Province Mount Tai Industrial Talents Program.