Yongli Luo ,Wenqian Li ,Cui Huang ,Junhao Yang,Min Jin,Jin Chen,Dangwei Pang,Yonglan Chang,Yong Li*,Zhenlin Wang*

National Key Laboratory of Crop Biology,College of Agronomy,Shandong Agricultural University,Tai’an 271018,Shandong,China

ABSTRACT Drought at the grain filling stage of wheat will cause premature leaf senescence,thus leading to considerable loss of wheat yield.Therefore,this paper aims to establish a cultivation technology for strong drought resistance,delayed senescence,and yield improvement based on the analysis of hormones homeostasis obtained by applying chemical control substances.Experiments were conducted with two genotypes of wheat.Four water irrigation treatments were applied to impose the water deficit,including well-watered control treatment(WW),mild water deficit(MiWD),moderate water deficit (MoWD),and severe water deficit(SWD).Exogenous abscisic acid(ABA)was sprayed on the plants at the anthesis stage of the wheat.As a result,exogenous ABA reduced initial senescence rate(r0),total duration of chlorophyll(Chltotal),rapid senescence phase(Chlloss),and the accumulated temperature at an inflection point(M)but improved the persistence phase (Chlper) of flag leaves under all of the four treatments.However,exogenous ABA produced inconsistent effects on photoassimilate relocation and grain weight under different treatments.It produced positive regulatory effects on grain weight under WW,MiWD,and MoWD treatments.On the one hand,spraying ABA during the persistence phase of flag leaves reduced the ratios of zeatin to gibberellin (Z/GA3),spermine to spermidine (Spm/Spd),and salicylic acid to ABA (SA/ABA),which prolonged active photosynthesis by stimulating high level of proline(Pro)and increased the activities of antioxidant enzymes,such as superoxide dismutase (SOD),peroxidase(POD),catalase(CAT),and ascorbate peroxidase(APX).Therefore,drought tolerance was enhanced,and more photosynthetic assimilates were accumulated.On the other hand,the rapid senescence phase and the transport rate of assimilates into grains were accelerated,resulting in higher grain weight,yield,and water use efficiency(WUE).However,under SWD treatment,exogenous ABA improved the ratio of SA/ABA,leading to low Pro content and low antioxidant enzyme activity of flag leaves in the rapid loss phase.Meanwhile,drought resistance declined and the transport duration of assimilates into grains was shortened,thus making photosynthetic assimilates redundant.Therefore,exogenous ABA can lead to the reduction in grain weight,yield,and WUE of wheat under SWD treatment.

Keywords:Wheat Water deficit stress Leaf senescence Hormones Exogenous ABA

1.Introduction

Wheat is one of the major crops in the world and enhancing wheat yield is of great significance for food security.With global climate change,drought occurs frequently at the grain filling stage of wheat,which influences the senescence of functional leaves and ultimately results in considerable yield loss and low stress tolerance of wheat[1,2].Therefore,drought has become the major environmental factor restricting wheat productivity[3–5].Plants show various physiological responses to drought stress conditions.Previous studies have shown that under drought stress,the stomatal conductance,chlorophyll synthesis,and electron transport of plants are inhibited,and as a result,photosynthetic activities are reduced[6,7].It has been also demonstrated that drought gives rise to oxidative stress [8].To cope with the damage caused by the oxidative stress,total antioxidant capacity is enhanced,including superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),and ascorbic acid,glutathione,flavonoids,phenols,and carotenoids[9,10].Moreover,as a compatible osmolyte,proline(Pro)possesses osmotic adjustment to maintain the structure of cell membranes and proteins under abiotic stress.Its accumulation drastically increases in response to water deficit stress [11].Furthermore,the synthesis,proportion,and transport of phytohormones are altered dramatically,especially abscisic acid (ABA),cytokinin,gibberellin (GA3),salicylic acid (SA),jasmonic acid (JA),and polyamines [12].

Leaves serve as the primary photosynthetic organs for energy harvesting and nutrient production at the grain filling stage of wheat[13].The senescence of functional leaves regulated by genotypes is also sensitive to environmental signals and cultivation technology [14,15].Drought at the grain filling stage leads to premature senescence of functional leaves,which shortens the photosynthetic period of the leaves.Moreover,the photosynthetic capacity of functional leaves is also reduced,which leads to decreased grain weight,poor grain quality,and serious economic loss [16,17].

Previous studies have documented that water deficit stress will significantly alter the homeostasis of endogenous hormones in plants [18,19],which in turn significantly influence the drought resistance of plants,either positively or negatively [20,21].It has long been considered that ABA plays a central role in the adaptation of plants to water deficit stress [22,23].The core response of plants to water deficit stress is reflected by the signal transduction pathway of ABA[24,25],wherein about 10%of signaling genes can be regulated by ABA [26].It has been reported that the synthesis and accumulation of ABA are the initial signals of plant in the response to drought stress [27] and that calcium-dependent protein kinase 6 can enhance drought tolerance by phosphorylating ABA-responsive element-binding factors (ABFs) [28].As indicated by previous research,ABA accumulates in plant roots under water deficit conditions[29,30].ABA-activated sucrose nonfermenting 1-related protein kinase 2s (SnRK2s) can phosphorylate NADPH oxidase Rboh F on the plasma membrane,producing O2-in the extraplastid space and subsequently forming H2O2,which serves as the signaling molecules used to regulate stomatal closure [31–33].Moreover,the activated SnRK2s protein kinases can also phosphorylate guard cell anion channel SLAC1,which subsequently regulates ABA-mediated stomatal closure under drought stress in order to decrease the water loss induced by transpiration [34].In addition,ABA-responsive transcription factor MdAREB2 directly activates the abundant expression of amylase and sugar transporter genes to increase the accumulation of soluble sugars,such as sucrose,which can enhance drought tolerance [35,36].

Studies have found that ABA synthesized in roots is transported to leaves via xylem vessels under drought stress,which can promote the accumulation of ABA in leaves [37].Meanwhile,K+outchannels are activated while K+inchannels are suppressed on the guard cell membrane of the leaves under drought stress.As a result,the efflux of K+is improved and the influx of K+is reduced,which induces stomatal closure or inhibits stomatal aperture.Furthermore,the turgor pressure is regulated by activating the channels of K+,Ca2+,and anions,which can regulate the movement of ions in and out of cells.In this way,ABA-induced stomatal closure is mediated to reduce water loss[38–42].Additionally,endogenous ABA is highly correlated with the initiation of leaf senescence.High ABA content increases Ca2+concentration in guard cells,which results in leaf senescence [43,44].As shown by previous research,ABA receptor PYL9 and its downstream complex phosphatase type 2Cs (PP2Cs)as well as SnRK2s activate phosphorylation of ABF transcription factors.Meanwhile,they are related to ABA-insensitive 3/VP1RAV1(RAV1),which subsequently upregulates senescence-related genes and induces senescence of old leaves [20].Besides,it has been observed that the senescence of old leaves may improve drought resistance to extreme water deficit stress.

Studies have also shown that high ABA content can promote source nutrient remobilization and sink grain development in cereals [45–47].Compared to other cultivation measures,such as irrigation and nitrogen application,exogenous hormone regulation is time-bound and specific.It has been indicated that ABA holds a certain concentration threshold at which ABA initiates the onset of leaf senescence [43,44].Meanwhile,ABA can enhance the drought resistance of plants and promote the transport of nutrients to grains[48,49].Furthermore,it has been reported that exogenous ABA can improve the number of vascular bundles and the phloem area in the stalk of female inflorescences of corn and promote the transport of carbohydrates into grains [50].

Overall,the ABA-signaling pathway is critical for plants to cope with water deficit stress.On the one hand,high endogenous ABA content can reduce water loss of leaves,thus maintaining the integrity of the photosynthetic structure,improving plant resistance to drought stress,and ultimately maintaining the productivity of crop.On the other hand,leaf senescence is initiated when endogenous ABA concentration reaches a certain threshold.However,despite considerable progress made over the last few years,there is little knowledge regarding the mechanism of the ABAgoverned leaf senescence of wheat in response to drought.Therefore,this study aims to analyze how exogenous ABA coordinates the senescence of functional leaves and the transport of photoassimilates into grains by regulating homeostasis of endogenous hormones.This will ultimately provide a theoretical basis for the application of exogenous regulators in drought resistance and yield enhancement of wheat.

2.Materials and methods

2.1.Plant growth conditions

Experiments were conducted on the farm of Shandong Agricultural University,Tai’an,China (36°9′N,117°9′E) during two growing seasons of wheat (from October 2016 to June 2017 and from October 2017 to June 2018,respectively).Wheat was grown in deep cement tanks (300 cm long,250 cm wide,and 200 cm deep)in field condition under a canopy made of polycarbonate polyurethane to avoid the influence of shading and warming on wheat growth and development.The cement tanks were separated from each other by a 30 cm wide cement ridge.Each tank was filled with sandy loam,and a soil layer with the thickness of 20 cm,which contained 12.41 g kg-1of total organic matter,0.95 g kg-1of total N,80.23 mg kg-1of available N,20.23 mg kg-1of available P2O5,and 100.20 mg kg-1of available K2O.

2.2.Experimental design,soil drying treatments,and chemical applications

Experiments were performed using a split-split plot design.The whole plot factor was the wheat variety with different genotypes,namely the drought-tolerant wheat SN20 and the water-sensitive wheat F287.The wheat was sown on October 11,2016 and October 10,2017.Plant density was adjusted to 270 × 104plants ha-1at the three-leaf stage(GS13)[51].Then they were harvested on June 10,2017 and June 8,2018.The subplots were used for the water deficit treatments.Four irrigation treatments were set as described in previous research [52,53] and they were well-watered control treatment (WW),with a relative soil water content in the 30 cm thick soil layer (RSWC) of 75%–80%;mild water deficit (MiWD),with RSWC of 65%–70%;moderate water deficit (MoWD),with RSWC of 50%–60%;severe water deficit (SWD),with RSWC of 30%–40%.The sub-subplots were used for exogenous ABA treatment at anthesis stage.Starting from the 1st day after anthesis(May 3 in 2017 and May 1 in 2018),all plants had been sprayed for four days at 5:00 PM each day with 20 mg L-1of exogenous ABA (from Sigma-Aldrich,St Louis,MO,USA) at a dosage of 500 mL m-2using watering cans.To make the exogenous ABA be better attached to the plants,Tween-20 was added into the solution at a final concentration of 0.5%(v/v).Spraying water contained 0.5%(v/v) Tween-20 was taken as the control.The plot size was 3.0 m×2.5 m=7.50 m2.A total of 16 treatments[two genotypes of wheat,four water irrigation treatments,and two exogenous regulator treatments (exogenous ABA and the control water)] were arranged and each treatment was repeated thrice.N,P,and K fertilizers were used,which were urea (46% N),calcium superphosphate (16% P2O5),and potassium chloride (60% K2O),respectively.They were applied at a dosage of 240 kg ha-1,120 kg ha-1,and 150 kg ha-1,respectively.P and K fertilizers were applied once as the base fertilizer.In contrast,N fertilizer was applied twice,with one half being applied as a base fertilizer and the remaining half being top-dressed at the jointing stage (GS31) [51].Other management measures were the same as those followed in highyielding fields.

Irrigation was the same in all treatments before the anthesis stage.The water content in soil was monitored before the anthesis stage using ECH2O soil moisture monitoring systems (Decagon,Pullman,WA,USA),which were equipped with EM50 data collectors and EC-5 sensors.As a result,one datum was acquired every minute.The values of volumetric water content in soil (θ) were measured using the systems.When θ was about to reach the threshold of the water deficit treatment,supplemental irrigation(SI)was calculated as described in previous research[54].The supplementary irrigation was monitored using a water meter(Ningbo,Zhejiang,China) according to the following detailed steps.

On the 7th day before the anthesis stage,undisturbed soil in the 30 cm thick soil layer was collected using 200 cm3rings.The soil layer in each plot was divided into three layers (0–10 cm,10–20 cm,and 20–30 cm),with each replicate being collected from each layer and being weighed and recorded as fresh weight (FW).Then each ring full of undisturbed soil was placed in an enamel dish filled with water.The water surface in the enamel dish was maintained 1–2 mm lower than the upper edge of the ring to prevent it from being flooded.When the soil was fully saturated to a constant weight,the water content in the soil was recorded as saturated soil water content (SSW).Subsequently,the soil was dried at 105–110 °C in an oven to a constant weight and recorded as dry weight (DW).The following equations were used to calculate the amount of supplementary irrigation required:

Where,SI(mm)is the amount of SI;γbd(g cm-3)is the soil bulk density;Dh(cm)is the depth of the soil layer(30 cm in this study);θt(%)is the weight-based target water content in soil after SI;θn(%)is the weight-based soil water content before SI,andV(cm3)is the volume of soil in the rings (200 cm3in this present study).

2.3.Sampling and measurements

2.3.1.Flag leaf senescence of wheat

Five complete and representative flag leaves were tagged.Their SPAD values were detected using the chlorophyll meter(SPAD 502,Minolta Camera CO.,Osaka,Japan) and recorded on the 5th,10th,15th,20th,25th,and 30th day after anthesis,with the top,middle,and basal parts of each leaf being measured.Fifteen readings were obtained for each treatment.Chlorophyll concentration index was expressed as the average of 15 readings.Accumulated temperature was initially set to zero and calculated as the sum of daily average temperature [(Tmax+Tmin)/2] from anthesis [55]:

Where,TmaxandTminrepresent the highest and the lowest temperature in one day,respectively.Meteorological data were acquired from the Tai’an meteorological station.The SPAD values were fitted to cumulative thermal time after anthesis using the Gompertz growth equation employed to describe leaf senescence kinetics (Eq.(4),Fig.S1) according to previous research [56,57].

whereGis chlorophyll SPAD value when accumulated thermal time was att;tis the accumulated thermal time after anthesis;ais the upper asymptotes that are the maximum SPAD value predicted by the model during the period of measurement;bis the adjustable parameters,which can indirectly reflect the early senescence rate of flag leaf;the initial senescence rater0=b;the maximum senescence ratermax=(a×r)/e;the average senescence rateraver=(r0+rmax)/2;the accumulated temperature at an inflection point(M) isM=lnb/r;total duration of flag leaf (Chltotalis defined as the duration from anthesis to the end of senescence,Chltotal=[ln(ln0.1/b)]/r;Chltotalconsisted of two components:persistence phase(Chlper) and rapid loss phase (Chlloss).Chlpermeans the duration from anthesis to the onset of senescence.and Chllossmeans the duration from the onset of senescence to the end of senescence,Chlloss=Chltotal-Chlper.

2.3.2.Determination of SOD,POD,CAT,and ascorbate peroxidase(APX) activities

SOD(EC 1.15.1.1.)activity was determined as described in previous research [58].The reaction mixture (3.9 mL) used contained enzyme extract,50 mmol L-1phosphate buffer(pH 7.8),0.1 mmol L-1EDTA,13.37 mmol L-1methionine,750 μmol L-1NBT,and 20 μmol L-1riboflavin.

POD(EC 1.11.1.7.)activity was measured as described by Zheng and Huystee [59].The reaction mixture (2.9 mL) used contained 10 mmol L-1phosphate buffer (pH 7.0),0.02 mol L-1guaiacol,and 0.04 mol L-1H2O2.

CAT (EC 1.11.1.6.) activity was determined according to the method described by Luna et al.[60].The reaction mixture(3.3 mL) used contained 100 mmol L-1phosphate buffer (pH 7.4),0.1 mmol L-1HEPES (pH=6.5),10 mmol L-1MgCl2,and 5 mmol L-1EDTA.

APX(EC 1.11.1.11.)activity was determined as described in previous research [61].The reaction mixture (1.7 mL) used contained 50 mmol L-1phosphate buffer (pH 7.8),0.1 mmol L-1EDTA,5 mmol L-1ascorbate,and 20 mmol L-1H2O2.

2.3.3.Determination of endogenous hormones and pro

Sampling was performed at 5-day intervals from anthesis.Thirty flag leaves were successively collected,immediately frozen in liquid nitrogen,and stored at -80 °C for the analysis of SA,JA,ABA,zeatin riboside (ZR),zeatin (Z),GA3,spermidine (Spd),spermine (Spm),putrescine (Put),and Pro.

Endogenous SA,JA,and ABA were quantified based on methods described in previous research [62].Ethyl ether was used to exact endogenous SA,JA,and ABA from the frozen leaves(100 mg),with extraction solvent comprising acetone/50 mmol L-1citric acid(70:30,v/v) and internal standards (20 ng;D6-SA,DHJA,and D6-ABA)being added.The ether phase was successively evaporated in vacuum,re-solubilized in methanol (100 μL),and filtered through a 0.22 μm membrane filter.Samples (5 μL) were injected for analysis using an Acquity UPLC system coupled to a Waters XEVO TQ-S triple-quadrupole mass spectrometer(Waters,Milford,MA,USA).

Endogenous ZR and Z were extracted according to the method described by Liu et al.[63],with precooled extraction buffer(methanol:water:acetic acid,80:19:1,v/v/v) being used as the extractant and the D5-ZR and D5-Z (20 ng) being used as internal standards.

Endogenous GA3was extracted according to the method described by Dave et al.[64],with the extraction buffer (isopropanol:chromatographic acetic acid,99:1) being used as the extractant.

The quantification of endogenous polyamines,including permidine (Spd),spermine (Spm),and putrescine (Put),as well as Pro was performed as described in previous research [65] with some simplification being adopted to avoid derivation,which greatly simplifies the extraction process.Formic acid water(5 mL,0.1%) was added to the samples,which were subsequently transferred into a microwave digester (60 °C,10 min,600 W).The supernatant was successively evaporated under vacuum at 25 °C,resolubilized,and filtered through a 0.22 μm membrane filter.Sample solution (5 μL) was injected for analysis.

2.3.4.Dry matter accumulation and distribution of wheat

At the mature stage,thirty plants were randomly sampled from each plot,with 10 plants being considered as repetitions.The plant samples were divided into stems+sheaths,flag leaves,the second leaves from the top,other leaves,grains,spike axis,and kernel husks.All the organs were dried at 70 °C to a constant weight and weighed.The distribution of dry matter of different organs was calculated for different treatments.

2.3.5.Grain yield components and water use efficiency (WUE)

At the mature stage,20 uniform and representative wheat spikes were chosen to calculate grain number.A subplot of 1 m2was harvested and the number of spikes per area was counted.Then,the grain was dried.1000-kernel weight was calculated,and the yield is reported at 13% grain moisture.

WUE was calculated as follows:

WUE (kg ha-1mm-1)=(grain yield per hectare)/(water consumption during the whole growth period).Water consumption during whole growth period (ET) was calculated as described in previous research as follows [66]:ET=P+SI +ΔW.Where,P represents precipitation,SI represents supplementary irrigation(mm),and ΔWrepresents soil water storage consumption (mm).In this study,the soil water content in the 200 cm thick tanks at sowing time was subtracted from that in the tanks at harvest time.Meanwhile,the influence of groundwater and surface leakage was ignored since groundwater was below 5 m.

2.4.Statistical analysis and plotting

Statistical analysis was performed using DPS7.05 software (developed by Zhejiang University,Hangzhou,China),and multiple comparisons were performed using the least significant difference tests.Meanwhile,graphs were drawn using Microsoft Excel 2007 and SigmaPlot 10.0 (Systat Software,Inc.,San Jose,CA,USA).

3.Results

3.1.Exogenous ABA affecting wheat yield and its components in response to water deficit treatments

The effects of exogenous ABA on grain yield and its components under different water deficit treatments are shown in Table 1.It can be seen from the table that 1000-grain weight and grain yield of SN20 are both higher than those of F287.Water deficit during the grain filling stage had no significant effect on spike number(P>0.05).However,it dramatically reduced the 1000-grain weight,superior grain weight,and inferior grain weight,and ultimately reduced grain yield (P<0.01).In addition,exogenous ABA significantly influenced 1000-grain weight,superior grain weight,and grain yield (P<0.01).However,the effects of exogenous ABA on 1000-grain weight,superior grain weight,and grain yield varied with different water deficit treatments.During the growing season of wheat in 2016–2017,spraying ABA enhanced the 1000-grain weight of F287 by 3.11%,5.20%,and 6.83%,respectively under WW,MiWD,and MoWD treatments,and improved the 1000-grain weight of SN20 by 4.35%,4.52%,and 3.24%,respectively under WW,MiWD,and MoWD treatments.Similarly,the superior grain weight of exogenous ABA-treated F287 was enhanced by 3.83%,4.90%,and 3.03%,respectively under WW,MiWD,and MoWD treatments,while the superior grain weight of exogenous ABA-treated SN20 was improved by 1.02%,1.86%,and 0.24%,respectively under WW,MiWD,and MoWD treatments compared to the control.Moreover,the grain yield of exogenous ABA-treated F287 was increased by 10.43%,10.04%,and 10.35%,respectively under WW,MiWD,and MoWD treatments,while the grain yield of exogenous ABA-treated SN20 was improved by 7.16%,9.41%,and 12.49%,respectively under WW,MiWD,and MoWD treatments compared to the control.In contrast,the 1000-grain weight,superior grain weight,and yield trended to decrease under SWD treatment.In detail,the 1000-grain weight of F287 and SN20 was reduced by 3.28% and 2.82%,respectively;the superior grain weight of F287 and SN20 was reduced by 1.80%and 1.90%,respectively;the grain yield of F287 and SN20 was decreased by 9.08%and 13.16%,respectively.Meanwhile,the genotype,water deficit,hormone treatment,and two-way interactions (water deficit × hormone treatment) significantly affected the 1000-grain weight and yield of wheat(P<0.01).Furthermore,the water deficit,hormone treatment,and two-way interactions (water deficit × hormone treatment) influenced superior grain weight of wheat noticeably (P<0.01).In addition,the grain number,1000-grain weight,superior grain weight,inferior grain weight,and yield were significantly different in the two wheat growing seasons,which indicated that climatic conditions had a remarkable influence on yield components (P<0.01).

3.2.Effects of exogenous ABA on WUE in two genotypes of wheat

The WUE of SN20 was higher than that of F287 in response to all the treatments (Fig.S2).Besides,the WUE of both genotypes of wheat under MiWD treatment was higher than that under WW treatment.During the growing season of wheat in 2016–2017,the WUE of F287 and SN20 was improved by 1.21% and 0.52%,respectively under MiWD treatment compared to WW treatment,while it was reduced under MoWD and SWD treatments compared to WW treatment.

The effects of ABA on WUE varied under different water deficit treatments (Fig.S2).Under WW,MiWD,and MoWD treatments,exogenous ABA enhanced the WUE of F287 by 3.42%,3.89%,and 4.51%,respectively,and it enhanced the WUE of SN20 by 1.66%,4.15%,and 2.67%,respectively.However,under SWD treatment,exogenous ABA decreased WUE of F287 and SN20 by 5.36% and 4.17%,respectively.Similar trends were observed in grain yield(Table 1).

Table 1 Effects of exogenous ABA on grain yield and its components under different water deficit stress treatments.

3.3.Effects of exogenous ABA on dry matter distribution

It can be seen from Fig.1 that there was no significant difference in total plant weight between SN20 and F287.However,the dry matter weights of stems+sheaths,flag leaves,the second leaves from the top,and spike axis+kernel husks of F287 and their distribution ratios were higher than those of SN20,respectively.Incontrast,the dry matter weight of the kernel of F287 and its distribution ratio were lower than that of SN20 and its distribution ratio,respectively (Fig.S3).The above results suggest that more photosynthetic assimilates were transported into the grains of SN20 at a higher translocation rate compared to F287.As the water deficit stress increased,dry matter weights of the total plant weight,flag leaves,the second leaves from the top,spikes,and grain yield and their distribution ratios were all reduced,whereas those of stems+sheaths were enhanced.As also shown in Fig.1,exogenous ABA enhanced the total plant weight as well as the grain weight and its proportion under WW,MiWD,and MoWD treatments,while it reduced the dry matter weights of stems+sheaths,flag leaves,and the second leaves from the top and their proportions under WW,MiWD,and MoWD treatments.In contrast,exogenous ABA reduced the dry matter weight of kernels and its proportion under SWD treatment.The above results demonstrate that under WW,MiWD,and MoWD treatments,exogenous ABA plays a positive regulatory role in the distribution of dry matter and is conducive to the transport of photosynthates from wheat stems+sheaths and functional leaves to grains,but it decreased kernel weight and its distribution ratio under SWD treatment.

3.4.Role of exogenous ABA in regulating flag leaf senescence of wheat under water deficit stress

As can be observed in Fig.2,exogenous ABA applied in the wheat reducedr0,Chltotal,Chlloss,andM,but improved Chlperof flag leaves.The exogenous ABA has inconsistent effects onraverof the two genotypes of wheat.It enhanced theraverof flag leaves of F287 but reduced that of SN20 under WW,MiWD,and MoWD treatments.

3.5.Variance analysis of flag leaf senescence-related parameters of wheat

Fig.1.Effects of exogenous ABA on dry matter accumulation of kernels,spike axis+kernel husks,leaves,and stems+sheaths in two genotypes of wheat under different water deficit stress treatments during the growing seasons in 2016–2017 and 2017–2018.

The effects of genotypes,water deficit treatments,and exogenous ABA on flag leaf senescence-related parameters of wheat were analyzed.As shown in Table S1,the genotypes and water deficit treatments had significant effects onr0,rmax,raver,Chltotal,Chlper,Chlloss,andM,withP<0.01 andP<0.05,respectively.In addition,exogenous ABA significantly affectedrmax,Chltotal,Chlper,Chlloss,andM(P<0.05).The interaction effects of genotypes and exogenous ABA were also remarkable on Chltotal.

3.6.Effects of exogenous ABA on concentration and proportions of endogenous hormones

3.6.1.Effects of exogenous ABA on concentrations of endogenous hormones in flag leaves of wheat under water deficit stress

As shown in Fig.3A–I,spraying ABA enhanced the concentrations of SA,ABA,ZR,GA3,and Spd,but reduced the concentrations of JA,Z,Spm,and Put.Among these hormones,the concentration of ZR showed the strongest change trend,while the changes in Spm were the minimal.Exogenous ABA improved the concentrations of ZR by 42.91%,57.33%,58.14%,and 64.73%,respectively under WW,MiWD,MoWD,and SWD treatments,but reduced the concentrations of Spm by 16.00%,15.95%,16.00%,and 19.73%,respectively.The results indicate that exogenous ABA significantly changed the homeostasis of endogenous hormones in flag leaves of wheat.Among them,ZR showed the strongest response,while Spm responded less sensitively.

3.6.2.Effects of ABA on the ratios between endogenous hormones in flag leaves of wheat under water deficit stress

The dynamic balance of endogenous hormones and the interaction between hormones regulate leaf senescence under abiotic stress.Meanwhile,optimizing hormone homeostasis is significant for the coordination of the relationship between leaf senescence and yield formation.To a certain extent,the ratios between different endogenous hormones can reflect the response of interaction between hormones to water deficit stress.Fig.4 shows that Z/GA3and Spm/Spd were reduced by exogenous ABA.Moreover,Spm/Put was improved and SA/ABA was reduced by ABA under WW,MiWD,and MoWD treatments.However,exogenous ABA had opposite effects on Spm/Put and SA/ABA under SWD treatment.The results of correlation analysis in Table 2 and Fig.5 suggest that the increase in Z/GA3,SA/ABA,Spm/Spd,and Spm/Put is not conducive to the increase in grain weight.The above results indicate that exogenous ABA can regulate the flag leaf senescence by decreasing Z/GA3and Spm/Spd under WW,MiWD,and MoWD treatments,resulting in higher grain weight.However,spraying ABA reduced grain weight under SWD treatment owing to the increase in SA/ABA.

3.7.Relationship between flag leaf senescence and endogenous hormones of wheat

3.7.1.Relationship between flag leaf senescence-related parameters and endogenous hormones

Fig.2.Effects of exogenous ABA on flag leaf senescence-related parameters of wheat under different water deficit stress treatments. r0 and raver represent the initial senescence rate and average senescence rate,respectively.Chltotal and Chlper refer to the total duration and the persistent duration of flag leaf,respectively.M denotes the cumulative temperature at an inflection point when senescence rate is the maximum.Chlloss represents the rapid loss phase.CK and ABA denote exogenous spraying water and abscisic acid,respectively.

The correlations between the grain weight and flag leaf senescence-related parameters in 2016–2017 are shown in Table 2.It can be seen that there are significant negative correlations between the grain weight andr0andraverof the two genotypes of wheat (r=-0.72*and -0.68**,respectively).In contrast,there are positive correlations between the grain weight andrmax,Chltotal,Chlper,and M(r=0.68*,0.80**,0.72*,and 0.86**,respectively).Besides,negative correlations exist between the superior grain weight andr0andraver(r=-0.83**,-0.81**,respectively),while positive correlations exist between the superior grain weight andrmax,Chltotal,Chlper,and M (r=0.75*,0.76*,0.84**,and 0.85**,respectively).Moreover,there is a significant negative correlation between the inferior grain weight andr0(r=-0.68*),while there are positive correlations between the inferior grain weight and Chltotal,Chlper,and M (r=0.87**,0.72*,and 0.91**,respectively).All these indicate that long total duration,long persistence phase of flag leaf senescence,and high maximum senescence rate are favorable for the increase in superior and inferior grain weights,and for the increase in 1000-grain weight in wheat ultimately.

The correlations between endogenous hormones and flag leaf senescence-related parameters were analyzed and the results are also shown in Table 2.It can be seen that there are significant positive correlations between endogenous SA concentration andr0andraver(r=0.90**and 0.87**,respectively).Conversely,there are negative correlations between SA content andrmax,Chltotal,Chlper,andM(r=-0.83**,-0.68*,-0.78*,and -0.79*,respectively).Similar to SA,JA content and ABA content are positively correlated withr0andraversignificantly but are negatively correlated tormax,Chltotal,Chlper,andM.In addition,significant negative correlations can be found between endogenous Z concentration andr0andraver(r=-0.69*and -0.68*,respectively),while significant positive correlations exist between Z content and Chltotal,Chlper,andM(r=0.88**,0.72*,and 0.93**,respectively).Endogenous ZR and GA3show similar trends.There are significant positive correlations between endogenous Spm andr0andraver(r=0.98**and 0.94**,respectively),while significant negative correlations exist between endogenous Spm andrmaxand Chlper(r=-0.89**and -0.92**,respectively).There are significant positive correlations between endogenous Spd and ChltotalandM(r=0.93**and 0.94**,respectively),and there is a significant negative correlation between endogenous Put and Chlloss(r=-0.83**).The correlations between endogenous hormones and flag leaf senescence-related parameters in 2017–2018 are shown in Table S2.They are similar to the correlations observed between the grain weight and flag leaf senescence-related parameters.Overall,these results suggest that endogenous hormones in flag leaves of wheat influence grain weight by regulating the flag leaf senescence.

3.7.2.Correlations between flag leaf senescence-related parameters and proportions of endogenous hormones of wheat

To study the effects of the interaction between hormones on the flag leaf senescence of wheat,the correlations between the propositions of different endogenous hormones and flag leaf senescencerelated parameters were analyzed.The ratios between endogenous hormones that were significantly correlated with senescencerelated parameters were selected(Fig.5)and the results are as follows.Z/GA3is significantly negatively correlated withMand Chltotal(r=-0.91**and -0.80**,respectively).Spm/Spd is significantly negatively correlated withrmax(r=-0.66**).SA/ABA is significantly positively correlated withr0andraver(r=0.79**and 0.81**,respectively),while it is significantly negatively correlated with Chlper(r=-0.70**).Furthermore,Spm/Put is significantly positively correlated withr0andraver(r=0.66**and 0.65**,respectively),while it is significantly negatively correlated with Chlper(r=-0.74**).The results shown in Table 2 and Table S2 jointly suggest that the increase in Z/GA3,SA/ABA,Spm/Spd,and Spm/Put is not conducive to the increase in grain weight of wheat.

Table 2 Correlations between flag leaf senescence-related parameters and grain weight,concentration of hormones in 2016–2017.

Fig.3.Endogenous hormones content in flag leaves of two genotypes of wheat under different water deficit treatments.(A) Content of endogenous salicylic acid (SA).(B)Content of jasmonic acid(JA).(C)Content of abscisic acid(ABA).(D)Content of zeatin(Z).(E)Content of zeatin riboside(ZR).(F)Content of gibberellin(GA3).(G)Content of spermine (Spm).(H) Content of spermidine (Spd).(I) Content of putrescine (Put).CK and ABA denote exogenous spraying water and abscisic acid.Box boundaries indicate upper and lower quartiles,and circles indicate outliers.

3.8.Effects of ABA on Pro content under water deficit stress

Fig.6 shows that the content of free Pro increased,decreased,and increased successively with grain filling,and the lowest content of free Pro occurred on the 15th day after anthesis (DAA).Compared to the sensitive wheat F287,the content of free Pro in wheat SN20 was much lower at early and middle grain filling stages(from 5 to 20 DAA),while it was higher at the later grain filling stage(from 20 to 30 DAA),suggesting that the osmotic regulatory mechanism of wheat varied with different drought resistance.Meanwhile,water deficit stress improved the content of free Pro.In 2017–2018,the content of free Pro in F287 increased by 3.95%,14.55%,and 32.43%,respectively and the content of free Pro in SN20 increased by 8.59%,15.15%,and 39.42%,respectively,under MiWD,MoWD,and SWD treatments compared to WW treatment.2016–2017 witnessed the same trends.In addition,the effects of exogenous ABA on the content of free Pro varied under different water deficit treatments.Spraying ABA enhanced the content of Pro under WW,MiWD,and MoWD treatments,while it reduced the content of Pro under SWD treatment.

3.9.Effects of exogenous ABA on the activity of antioxidant enzymes in flag leaves of wheat

As shown in Figs.7,S4,S5,and S6,the activity of antioxidant enzymes(SOD,POD,CAT,and APX)responded differently to exogenous ABA at various stages of flag leaf senescence.In 5–15 DAA(persistence phase of flag leaves),spraying ABA enhanced the activity of SOD,POD,CAT,and APX.In contrast,in 20–30 DAA 9(rapid loss phase of flag leaves),exogenous ABA sprayed reduced the activity of SOD,POD,CAT,and APX.Moreover,the activity of antioxidant enzymes was reduced to a higher extent under MiWD,MoWD,and SWD treatments compared to WW treatment.The results indicate that exogenous ABA enhanced the ability of reactive oxygen species(ROS)scavenging in the persistence phase,thus prolonging the effective duration of chlorophyll photosynthesis in flag leaves under water deficit stress.The results also indicate that spraying ABA decreased the activity of antioxidant enzymes in the rapid senescence phase.As a result,the rapid senescence phase was shortened,thus accelerating the senescence of flag leaves at the late stage of wheat.

4.Discussion

4.1.Role of exogenous ABA in coordinating flag leaf senescence and transport of assimilates into grain of wheat under water deficit stress

It was reported that treatment with ABA increased the expression of sugar-induced starch biosynthesis genes,which modulated early seed development [67].Researchers also reported that ABA was involved in regulating the biosynthesis and degradation of starch in developing grains [68].Higher ABA content in leaves was positively associated with grain filling by enhancing remobilization [69].Yang et al.[45] found that high level of endogenous ABA promoted the transfer of nutrients from senescent leaves to grains.Moreover,under MoWD treatment,exogenous ABA promoted the transport of carbohydrates to grains,ultimately enhancing grain yield of crops [50,70].It was also reported that exogenous ABA increased WUE under WW and MoWD treatments,which,however,did not benefit grain yield of soybean in responses to MoWD and SWD conditions [71].

Fig.4.Ratios between endogenous hormones in flag leaves of two genotypes wheat under different water deficit treatments.(A)Ratio of zeatin(Z)to gibberellin (GA3).(B)Ratio of spermine (Spm) to putrescine (Put).(C) Ratio of spermine (Spm) to spermidine (Spd).(D) Ratio of salicylic acid (SA) to abscisic acid (ABA).CK and ABA denote exogenous spraying water and abscisic acid,respectively.Box boundaries indicate upper and lower quartiles,and circles indicate outliers.

This study indicated that exogenous ABA reducedr0,Chltotal,Chlloss,and M,but improved Chlper.Furthermore,hormone treatment had highly significant effects onrmax,Chltotal,Chlper,Chlloss,andM.Under WW,MiWD,and MoWD treatments,exogenous ABA increased the dry matter weights of the total plant and kernel weight and their distribution ratios,but reduced the dry weights of stems,flag leaves,and the second leaves from the top and their proportions.The results of this study revealed that under WW,MiWD,and MoWD treatments,on the one hand,exogenous ABA prolonged the highly active photosynthetic period of flag leaves,thus increasing the accumulation of photosynthetic assimilates.Meanwhile,it accelerated the rapid senescence process and the transport of assimilates to grains,thus promoting the transport of photosynthetic assimilates from stems+sheath,flag leaves,and the second leaves from the top to the grains.Finally,both grain weight and yield were enhanced,which was consistent with previous studies [45,50].Moreover,spraying ABA increased grain weight by 4.03%,5.43%,and 5.39%,respectively,significantly enhanced grain yield by 4.30%,5.90%,and 6.16%,respectively,significantly enhanced WUE by 2.54%,4.02%,and 3.59%,respectively under WW,MiWD,and MoWD treatments,which was consistent with previous research [20].

Fig.5.Correlation between the ratios between endogenous hormones and senescence-related parameters.(A)Correlation between the ratio of zeatin to gibberellin(Z/GA3)and cumulative thermal time(M).(B)Correlation between Z/GA3 and total duration of flag leaf(Chltotal).(C)Correlation between spermine to spermidine(Spm/Spd)and the max senescence rate(rmax).(D)Correlation between the ratio of salicylic acid to abscisic acid(SA/ABA)and the initial senescence rate(r0).(E)Correlation between SA/ABA and the average senescence rate(raver).(F)Correlation between SA/ABA and Chlper.(G)Correlation between the ratio of spermine to putrescine(Spm/Put)and r0.(H)Correlation between Spm/Put and raver.(I) Correlation between Spm/Put and duration of chlorophyll persistence (Chlper).

However,exogenous ABA decreased grain weight,yield,and WUE by 3.27%,3.34%,and 4.77%,respectively under SWD treatment.The possible reason is that SWD treatment led to an imbalance of starch biosynthesis and degradation in developing grains,which significantly impaired starch content and grain weight[67].Furthermore,exogenous ABA reducedr0,Chltotal,Chlloss,andMbut improved Chlper.These results are similar to those of previous studies wherein ABA played a positive role in coordinating most responses of roots to MiWD and MoWD while the responses to strong SWD were ABA-independent[72].The above results suggest that under SWD treatment,spraying ABA shortened the transport time of photosynthetic assimilates to grains,making photosynthetic assimilates redundant.Therefore,this is not conducive to grain filling,thus reducing the grain weight of wheat.

4.2.Exogenous ABA altering the balance of endogenous hormones in flag leaves of wheat under water deficit stress

Fig.6.Effects of exogenous ABA on free proline of two genotypes of wheat under water deficit stress treatments during the growing seasons in 2016–2017 and 2017–2018.The different letters mean the values are significantly different P=0.05.

As revealed by the results of this study,exogenous ABA enhanced the content of endogenous SA,ABA,ZR,GA3,and Spd but reduced the content of JA,Z,Spm,and Put.Plants adapt to drought by regulating the homeostasis of endogenous hormones,which do not work independently but produce synergistic or antagonistic effects on plants development [73].Previous studies have shown that hormones modulate each other’s biosynthesis or responses.Pretreatment with SA can lead to low expression of the ABA biosynthesis geneNCED3and the signal transduction genePDF1.2.Hence,the ratios of ABA/SA and JA/SA are decreased.SA and ABA produce antagonistic effects on sucrose accumulation,which can enhance drought tolerance inBrassica napus[74].Cytokinin is an antagonist to ABA while there is a synergistic relationship between GA and SA.Exogenous GA3induced high expression of isochorismate synthase1 and nonexpressor of pathogenesis related genes 1 (NPR1),which are involved in SA biosynthesis and SA action,respectively[75].As indicated by the findings in this study,exogenous ABA reduced Z/GA3,Spm/Spd and SA/ABA,but improved Spm/Put under WW,MiWD,and MoWD treatments,while it reduced Spm/Put but improved SA/ABA under SWD treatment.Interactions between different endogenous hormones on flag leaf senescence process of wheat response to drought stress were shown in Fig.8.According to the results that the increase in SA/ABA,Z/GA3,Spm/Put,and Spm/Spd is not conducive to the increase in grain weight and that exogenous ABA reduces the ratios of SA/ABA,Z/GA3,and Spm/Spd under WW,MiWD,and MoWD treatments,exogenous ABA regulates the flag leaf senescence by decreasing SA/ABA,Z/GA3,and Spm/Spd.Actually,exogenous ABA regulated the flag leaf senescence by decreasing Z/GA3and Spm/Spd under WW,MiWD,and MoWD treatments,resulting in higher grain weight.However,spraying ABA decreased grain weight under SWD treatment owing to the enhancement of SA/ABA.

4.3.Exogenous ABA influencing Pro accumulation and the activity of antioxidant enzymes in flag leaves of wheat by regulating endogenous hormones homeostasis

Pro accumulation serves as an important indicator of plant response to abiotic stress[76,77].Free Pro level in wheat increased under water deficit stress [78].The high level of Pro can maintain the productivity of crops under stress condition[78].Previous findings also indicate that hormones influence the accumulation level of Pro as signals[79,80].Exogenous ABA signaling can significantly improve the levels of free Pro and soluble sugar as well as other osmoregulatory substances,resulting in improved osmotic adjustment ability in plants [81].Sripinyowanich et al.[82] also found that exogenous ABA can upregulate the expression of the genes(OsP5CS1,OsP5CR) related to Pro biosynthesis under stress conditions [83].The findings in this study show that spraying ABA improved the level of Pro under WW,MiWD,and MoWD treatments but reduced it under SWD treatment.Free Pro is known to contribute to the osmotic adjustment,thus high level of free Pro is beneficial for enhanced drought tolerance of the wheat under water deficit stress treatments.

Studies have shown that exogenous ABA can enhance the activity of CAT,APX,glutathione peroxidases (GPX),and glutathione reductase (GR),thus enhancing the ability of ROS scavenging[84,85].This can effectively alleviate the reduction in the maximum light energy conversion efficiency and the light quantum efficiency of photosystem II (PSII) under water deficit stress [86].In this study,in 5–15 DAA(persistence phase of flag leaves),spraying ABA enhanced the activity of SOD,POD,CAT,and APX.In contrast,in 20–30 DAA (rapid loss phase of flag leaves),spraying ABA decreased the activity of SOD,POD,CAT,and APX.These results had not been reported previously as known by the authors of this paper.

Fig.7.Role of exogenous ABA in regulating the activity of superoxide dismutase(SOD)in two genotypes of wheat under different water deficit stress treatments during the growing seasons of wheat in 2016–2017 and 2017–2018.The different letters mean the values are significantly different at P=0.05.DAA means the days after anthesis.

Fig.8.Interactions between different endogenous hormones on flag leaf senescence of wheat in response to drought stress.ABA,abscisic acid;Z,zeatin;GA3,gibberellin;SA,salicylic acid;Spm,spermine;Spd,spermidine;Put,putrescine;r0,initial senescence rate;raver,the average senescence rate;rmax,the maximum senescence rate;Chlper,the persistence phase;Chltotal,total duration of chlorophyll; M,accumulated temperature at an inflection point.The red solid arrow means stimulatory effect.The red dotted arrow means possible stimulatory effect.The blue solid arrow means inhibitory effect.The blue dotted arrow means possible inhibitory effect.The up orange arrows and down orange arrows mean drought has positive or negative effects on endogenous hormones,respectively.The up green arrows and down green arrows mean exogenous ABA has positive or negative effects on endogenous hormones,respectively.

5.Conclusions

The homeostasis of endogenous hormones in flag leaves of wheat changed considerably under water deficit stress.Compared to WW treatment,the ratios of Z/GA3,Spm/Spd,and Spm/Put were enhanced under water deficit treatments.As a result,the flag leaf senescence process changed as follows:r0andraverwere improved by 0.02/°C (40.49%) and 0.009/°C (40.49%),respectively,while Chltotal,Chlper,andMwere reduced by 79.51/°C (9.06%),69.87/°C(31.08%),and 79.43/°C(11.58%),respectively.Meanwhile,the rapid loss phase was initiated earlier and the senescence process was accelerated.This can shorten the total duration and prolong photosynthetic period of flag leaves,eventually leading to premature senescence of the leaves.Under WW,MiWD,and MoWD treatments,ABA could tailor internal physiological changes to adapt drought stress.Exogenous ABA improved Pro accumulation and enhanced the activity of antioxidant enzymes (SOD,POD,CAT,and APX) in the persistence phase.However,the decrease in the ratios of SA/ABA,Z/GA3,and Spm/Spd prolonged the persistence phase of flag leaves.Consequently,more photosynthates were accumulated and the transport of assimilates into grains was accelerated,ultimately increasing both grain yield and WUE of the wheat.However,when subjected to SWD treatment,spraying ABA enhanced the ratio of SA/ABA but reduced Pro content and the activity of antioxidant enzymes.Thus the transport time of assimilates to grains was shortened,leading to the redundancy of photoassimilates and the decrease in both grain yield and WUE.

CRediT authorship contribution statement

Zhenlin Wang and Yong Li designed the research.Yongli Luo wrote the paper and analyzed the data.Wenqian Li and Cui Huang did some experiments.All the authors discussed the data and made comments on the paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the National Key Research and Development Program of China (grant Nos.:2017YFD0301001 and 2016YFD0300403),the Shandong Province Mount Tai Industrial Talents Program,and the National Natural Science Foundation of China (grant No.:31801295).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.08.012.