Xinchen Zhng,Honghong Wu,Linmu Chen,Linlin Liu,Xiochun Wn,*

aState Key Laboratory of Tea Plant Biology and Utilization,Anhui Agricultural University,Hefei 230036,Anhui,China

bDepartment of Botany and Plant Sciences,University of California,Riverside,CA 92527,USA

Keywords:Drought stress H+-ATPase Membrane potential Potassium Rehydration

ABSTRACT Drought stress is one of the main factors limiting yield in tea plants.The plant cell's ability to preserve K+homeostasis is an important strategy for coping with drought stress.Plasma membrane H+-ATPase in the mesophyll cell is important for maintaining membrane potential to regulate K+transmembrane transport.However,no research to date has investigated the possible relationship between plasma membrane H+-ATPase and mesophyll K+retention in tea plants under drought and subsequent rehydration conditions.In our experiment,drought stress inhibited plasma membrane H+-ATPase activities and induced net H+influx,leading to membrane potential depolarization and inducing a massive K+efflux in tea plant mesophyll cells.Subsequent rehydration increased plasma membrane H+-ATPase activity and induced net H+efflux,leading to membrane potential hyperpolarization and thus lowering K+loss.A first downregulated and then upregulated plasma membrane H+-ATPase protein expression level was also observed under drought and subsequent rehydration treatment,a finding in agreement with the change of measured plasma membrane H+-ATPase activities.Taken together,our results suggest that maintenance of mesophyll K+in tea plants under drought and rehydration is associated with regulation of plasma membrane H+-ATPase activity.

1.Introduction

Tea plant(Camellia sinensis(L.)O.Kuntze)is a woody perennial plant and economic crop[1].Its young leaves,especially the bud and youngest fully expanded leaf,are used for producing the popular nonalcoholic beverage,tea[2,3].Various abiotic stresses,including heavy-metal stress[4],fluoride stress[5–7],soil nutrient deficiency[8],and drought stress[9]exert a negative effect on tea plant growth and development.Among these stresses,drought stress is one of the most important limiting factors for agricultural growth of tea plants and quality of tea products[10,11].Water scarcity withers the bud and youngest fully expanded leaf,causing irreversible damage that is manifested in reduced quality of tea products and causing large economic losses[12].For example,severe drought stress caused an economic loss of about RMB 500 million in Hunan province,China,in 2011[13].Drought stress has also led to annual tea industry output losses of 4%–33%in other tea-producing countries,such as Kenya(4%–20%),Tanzania(33%),Sri Lanka(26%),and India(17%–31%)[14].

Although irrigation is the obvious answer to drought stress,management problems(such as insufficient labor or pumping capacity),or shortage of water sometimes prevent irrigation until after the onset of drought stress.However,rehydration alleviated drought-caused injury in four tea cultivars(TV-1,TV-20,TV-29,and TV-30)[15].Soluble sugars and proline contents as well as superoxide dismutase(SOD)and catalase(CAT)activities in two cultivars(Zhuyeqi and Ningzhou)increased under drought stress and then rapidly decreased following rehydration,indicating that tea plants can atleastpartlyrecoverfrom droughtstresswhen rehydrated[9].Understanding the mechanisms by which tea plants respond to drought stress and subsequent rehydration could aid in breeding a robust,drought stress-tolerant tea plant as well as in making irrigation management decisions.

To date,most studies of drought stress in tea plants have focusedon morphological,physiological,andmolecular factors.For example,lower numbers of new leaves and lower plant height were found in tea plants under drought stress than under control conditions[16].Reduced relative water content and biomass and increased proline content,CAT activity,and MDA(malondialdehyde)concentration were measured in four clones of tea roots(TV-1,TV-20,TV-29,and TV-30)exposed to drought stress in soil condition[17].Similarly,the activities of CAT and POD increased to cope with drought stress[18].Expression of HSP80 and SOD was upregulated in drought-tolerant relative to drought-sensitive varieties[19].A RNA-seq study[20]revealed differential expression of 762 protein kinase and 547 transcription factors in tea leaves under drought stress.However,the role of potassium in tea plant responses to drought and subsequent rehydration at the electrophysiologicallevel remains unknown.

Potassium is the most abundant macronutrient in plant cells.Maintenance of a high cytosolic K+/Na+ratio is critical for drought tolerance[21,22].For example,supplied potassium level in olive[23]and sunflower leaves[24]was directly associated with drought stress tolerance.An external supply of K2CO3significantly increased shoot potassium content and improved drought resistance in the drought-tolerant variety SN16 relative to the intolerant variety JM22[25].A barley cultivarwith thehighestleafpotassium contentalso displayed the strongest drought resistance[26].Membrane depolarization induced higherK+efflux in transgenic Arabidopsis(plasma membrane H+-ATPase mutants aha1–6 and aha1–7)than in the wild type,resulting in lower drought resistance[27].Thus,investigating the change of potassium levels in mesophyll cells may shed light on the response of tea plants to drought stress and subsequent rehydration.

Plasma membrane H+-ATPase,a key enzyme in maintenance of plasma membrane potential in plants,is involved in potassium homeostasis and drought tolerance[28,29].Decreased plasma membrane H+-ATPase activity in developing wheat embryos impaired resistance to short-term drought stress[30].Increased root hair cell plasma membrane H+-ATPase was observed in oat(Avena sativa L.)after the onset of drought stress[31].Similarly,tolerance of wheat seedlings to drought stress was associated with plasma membrane H+-ATPaseactivity[32].However,theroleofplasmamembraneH+-ATPase in maintaining mesophyll potassium homeostasis in tea plants under drought stress and rehydration treatment remains unknown.

In the present study,the role of plasma membrane H+-ATPase and mesophyll K+retention in the response of tea plants to drought and subsequent rehydration was investigated by monitoring plasma membrane H+-ATPase activity and expression level,potassium content,net K+and H+flux,and plasma membrane potential in tea leaves.

2.Materials and methods

2.1.Tea plant growth

Tea plants(Camellia sinensis cv.Shu-chazao)grown under identical conditions were obtained from the tea garden of Anhui Agricultural University,China.The plants were washed with deionized water and transferred to an artificial climate chamber for one month with a day length of 12 h day-1,temperature of 22 ± 1 °C,irradiance of 270 μmol m-2s-1,and relative humidity of 45%–50%.Plants were grown in nutrient solution for one week before treatment.The nutrient solution contained macronutrients(mmol L-1):N(1.427),P(0.1),K(0.513),Ca(0.392),and Mg(1.029),and micronutrients(μmol L-1):Zn(1.53),Cu(0.39),Mn(18.2),B(9.25),Mo(0.53),Al(0.77),and Fe(6.27)as EDTA salts for adaptation to hydroponic cultivation[33].

2.2.Drought and subsequent rehydration treatment

To mimic drought stress and subsequent rehydration,plants were placed into a 4-L pot filled with 10%PEG-6000(w/v)for two days,followed by draining of the PEG solution and rewatering with nutrient solution without PEG for three days.At the end of the drought and rehydration treatment,stomatal conductance,net photosynthesis rate,and transpiration rate during steadystateilluminationweremeasuredonthefirstfullyexpandedleaf from the top using a plant photosynthesis meter(LI-6400XT portable photosynthesis system,Li-COR Bioscience Inc.,U.S.A.).Thesettingswereasfollows:CO2concentration,370 μmol mol-1,lightintensity,500 μmol m-2s-1,and55%–60%relativehumidity,essentially following Ramegowda et al.[34].Photographs were takenbeforestress(day0),attheendofthedroughtstressperiod(day 2),and three days after rehydration(day 5)using a Canon EOS REBEL T4i digital camera(Canon,Japan).

2.3.Physiological measurements

The youngest and subsequent second fully expanded tea leaves were collected for measurement of physiological parameters.CAT activitywascalculatedbasedon the conversion of hydrogen peroxide per minute[35,36].MDA and sugar content were analyzed by thiobarbital acid chromogenic reaction[9].Plasma membrane protein concentration was quantified following Bradford[37].

2.4.Plasma membrane potential measurements

The first tea leaf was cut with a～30 degree angel into small pieces(5 mm×8 mm)to expose mesophyll cells and then incubated with BSM (basic saltmedium)containing 0.1 mmol L-1CaCl2and0.5 mmol L-1KCl(pH 5.7,nonbuffered)overnight(refer to Wu et al.[38]for details).A noninvasive micro-test technique(NMT;Nanjing Forestry University,Nanjing,China)was used to measure plasma membrane potential.Microelectrodes were prepared by the Nanjing Forestry University NMT Service station.Microelectrodes were prepared freshly and tested to ensure the accuracy of membrane potential measurements.First,a glass microelectrode was backfilled with 3 mol L-1KCl.Then,to make electrical contact with the electrolyte solution,an Ag/AgCl wire was slowly inserted into the back of this microelectrode.Reference microelectrodes(YG003-Y05)were obtained from Nanjing Forestry University NMT Service station.After penetration of tea plant leaf mesophyll cells,plasma membrane potential was recorded for 10 min and outputted to a monitoring computer as Excel(Microsoft,U.S.A.)files for further analysis.At least eight individual samples(n=8)were used for each treatment[39].

2.5.Flux measurement of H+and K+

Leaf samples were prepared as described above.Tea plants were subjected to drought for two days and subsequently to three days of rehydration.Net fluxes of H+and K+were measured noninvasively using the NMT technique[40].Net flux of H+and K+was measured by moving microelectrodes repeatedly between two positions(5 and 35 μm)adjacent to the exposed tea leaf mesophyll cells.The youngest fully expanded leafwasused.BSM(0.1 mmol L-1CaCl2and 0.5 mmol L-1KCl,pH 5.7,non-buffered)solution was used.Net H+and K+flux was calculated by Fick's law of diffusion.

2.6.Determination of K+content

K+content in tea plants and leaves(the youngest and subsequent second fully expanded leaves)was measured using Inductively coupled plasma(ICP)optical emission spectrometry(Optima 2100DV;Perkin Elmer Inc.,Shelton,CT,USA)following Mekawy et al.[41].Samples were dried in an oven at 70°C for 48 h and gently agitated in 5%H2SO4overnight.K+contentwasthenmeasuredbyICPand calculated from K+standard curves.

2.7.Plasma membrane H+-ATPase isolation and hydrolytic activities

To isolate plasma membrane H+-ATPase,3.0–3.5 g of leaves(the youngest and subsequent second fully expanded tea leaves)from tea plants after each treatment(control,drought,and rehydration)were sampled.Leaf samples were then washed with distilled water and ground in a mortar with ice in 18 mL of buffer solution(25 mmol L-1Hepes-Tris(pH 7.6),50 mmol L-1mannitol,3 mmol L-1EGTA,3 mmol L-1EDTA,250 mmol L-1KCl,2 mmol L-1PMSF,1%PVP,0.1%BSA and 2 mmol L-1DTT).The homogenate was filtered through four layers of cheesecloth,collected in a 13-mL Beckman tube,and centrifugedat10,000 ×gfor10 minat4 °C.Thesupernatantwas placed in a new 13-mL Beckman tube and centrifuged at 50,000 ×g for 45 min at 4 °C to obtain the microsomal fraction.An aqueous polymer two-phase system method[42]was used to obtain plasma membranes from the microsomal fraction by partitioning with 6.2%Dextran T-500 and 6.2%PEG-3350.The purity of the isolated plasma membrane was tested by addition of 1 mmol L-1NaN3(mitochondrial membrane H+-ATPase inhibitor)and 50 mmol L-1KNO3(tonoplast H+-ATPase inhibitor).Activities of the isolated plasma membrane H+-ATPase were calculated from the release of phosphate[33].

2.8.Gel electrophoresis and immunodetection of plasma membrane H+-ATPase

Isolation of plasma membrane protein was essentially as described in Zhang et al.[33].Concentrations of plasma membrane protein were determined by BCA protein assay kit(Beyotime Biotechnology,Shanghai,China).Membrane vesicles containing 20 μg proteins were solubilized in SDS loading buffer(250 mmol L-1Tris-HCl(pH 6.8),10%(w/v)SDS,0.5%(w/v)BPB(Bromophenol blue),50%(v/v)glycerine.After 8 min protein denaturation,samples were loaded onto SDSPAGE.For Western blot analysis,after SDS-PAGE separation,gel was transferred to a PVDF(Polyvinylidene Fluioride)membrane filter.For identification and quantification of plasma membrane H+-ATPase,the membrane was then incubated with a polyclonal antibody to visualize plant H+-ATPase(Agrisera Antibodies product AS07 260,rabbit polyclonal serum lyophilized,Agrisera company,Sweden).The first antibody was diluted 1:1000 in TBST buffer(1%TBS(Tris-HCl)(w/v)and 20%Tween(v/v))and incubated overnight at 4°C.After rinsing in TBST,the membrane filters were incubated with 1:2500(v/v)diluted secondaryantibody(affinity purified goat anti-rabbit IgG antibody,Cell Signaling Technology,Inc.Boston,MA,USA).After rinsing in TBST,the PVDF membrane was subjected to detection(ChemiDoc MP imaging system,Bio-Rad company,USA)[43].

3.Results and discussion

3.1.The effect of drought and subsequent rehydration on photosynthesis and antioxidant activities in tea plants

Phenotypic changes of tea leaves in response to drought stress were recorded after two days of treatment with 10%(w/v)PEG solution.Phenotypic changes in response to subsequent rehydration were recorded on the fifth day after commencement of drought stress.The leaves of tea plants started to wilt after two days of drought stress;however,after subsequent rehydration,the wilting of the leaves was clearly reversed(Fig.1-A).Tea plants showed a significant reduction of stomatal conductance(52.1%)(Fig.1-B)under drought stress compared with control plants.Net photosynthesis(Fig.1-C)and transpiration rate(Fig.1-D)were also reduced by 46.9%and 38.5%,respectively.After three days of rehydration,leaf stomatal conductance was significantly higher than in drought-stressed leaves,although it was still lower than in the non-stressed control(Fig.1-B),showing an alleviation effect of rehydration on drought stressed tea plants.Net photosynthesis rate(Fig.1-C)and transpiration rate(Fig.1-D)showed similar changes,further confirming that rehydration alleviated drought-induced impairment of photosynthesis in tea plants.This is in agreement with previous finding that water deficit impaired plant photosynthesis and growth can be alleviated by re-watering[44].

Fig.1–Effects of drought and subsequent rehydration on the wilting phenotype and photosynthetic parameters of the youngest and subsequent second fully expanded tea leaves.A,Wilting phenotype of youngest and subsequent second fully expanded tea leaves in tea plants after two days of drought stress and three days of subsequent rehydration.The white broken lines indicate the wilting phenotypic changes of leaves under drought and subsequent rehydration.Stomatal conductance(B),net photosynthesis rate(C),and transpiration rate(D)were measured on the youngest fully expanded leaves.

MDA is a major cytotoxic product of lipid peroxidation,and changes in MDA level correspond to the degree of lipidmembrane oxidation[45].After two days of drought stress,MDA concentrations were increased by 14.4%compared with the control.With a subsequent three days of rehydration,MDA content was decreased and recovered to the control level(Fig.2-A).Similar results were also reported in tobacco[46]and rice[47].In the present study,catalase activity and sugar content were higher by 219.4%and 35.1%in drought-stressed tea leaves than in control leaves(Fig.2-B,C).After three days of rehydration,they fell to levels similar to those of the control group(Fig.2-B,C).Our results support the notion that antioxidant system[48]and osmotic adjustment[49]play an important role in drought stress resistance.Furthermore,plasma membrane protein concentration in droughtstressed tea leaves was increased by 51.9%[15],and the protein concentration was nearly equal to that of the control after three days of rehydration(Fig.2-D).Coomassie Brilliant Blue staining revealed a similar result(Fig.S1).

3.2.Effects of drought and rehydration on potassium content and net K+flux

Potassium concentration was reduced by 7.5%(Fig.3-A)in tea leaves(the youngest and subsequent second fully expanded leaves)and also by 14.4%in tea plants(Fig.3-B)after drought stress,and recovered to the control level after three days of rehydration.To further estimate the dynamic change of K+,NMT ion flux measurement was used to study net K+flux in mesophyll cells in the first tea leaf.K+efflux was relatively stable in the control.Upon exposure to PEG(mimicking drought stress),a massive mesophyll K+efflux was detected,ranging from 2376.73 to 5577.70 pmol cm-2s-1(Fig.3-C).The total K+efflux from drought-stressed tea leaves reached 469,185.80 pmol cm-2s-1,which was about 3-fold higher than that in the control treatment(Fig.3-D).However,after subsequent rehydration,net K+loss was significantly lowered to value similar to that in the control group(Fig.3-C,D).This is in agreement with the notion that potassium homeostasis is crucial in mediating response to drought stress[50].

Fig.2–Physiological changes of tea plants under drought and subsequent rehydration.MDA content(A),CAT activity(B),sugar content(C),and plasma membrane protein concentration(D)in youngest and second fully expanded tea leaves after drought and subsequent rehydration.

Drought is known as a limiting factor for tea yield and quality[14].Drought stress limits the movement of soluble ions in the rhizosphere and restricts K+diffusion in the soil towards plant roots,limiting K+availability to plants.Low K concentrations can further impair plant resistance to drought stress[21].Adequate plant potassium facilitatesosmoticadjustment,which maintains high turgor pressure and low osmotic potential,thusimprovingtheabilityofplantstotoleratedrought stress[51].In our study,drought stress induced a massive mesophyll K+efflux(Fig.3-C)and caused a significantly lower potassium concentration in drought-stressed than in nonstressed plants(Fig.3-A,B).This finding is in agreement with a report that drought stress induced a massive K+efflux in soybean leaf mesophyll[52].Not surprisingly,the subsequent rehydrationsignificantlyreducednetmesophyllK+efflux(Fig.3-C),in accord with the finding that leaf potassium content in rehydrated tea leaves was similar to that in the non-stressed control(Fig.3-A).It is also in agreement with previous reports thatmesophyllK+retentionisanimportantmechanisminplant resistance to abiotic stress,such as salt stress[53,54].These results suggest that the tea plant's response to drought stress and subsequent rehydration is tightly linked with its ability to maintain mesophyll K+.Upadhyaya et al.[17]reported that drought stress decreased relative water content and biomass and that applying potassium alleviated drought-induced damage and promoted plant growth.Adequate potassium fertilizer effectively protected the winter wheat cultivar SN16 from drought stress[25].Our results indicate that maintaining mesophyll potassium homeostasis is important for tea plants'response to drought stress and subsequent rehydration.

3.3.The effect of drought and subsequent rehydration on plasmamembranepotential,netH+flux,andplasma membrane H+-ATPase activity in mesophyll cells

Potassium homeostasis played a role in tea plants'response to drought stress and subsequent rehydration.K+transmembrane transport is known to be associated with plasma membrane potential[55].Accordingly,membrane potential in tea mesophyll cells was measured.Drought stress induced membrane potential depolarization,revealing lower(P＜0.05)membrane potential(about-80 mV)in drought-stressed tea leaves than in the non-stressed control(about-120 mV)(Fig.4-A).Subsequent rehydration hyperpolarized plasma membrane potential(about-152 mV)of mesophyll cells,and the value was higher,by 27.3%,than in the control(Fig.4-B).

Plasma membrane H+-ATPase accumulated in mesophyll cells[28]is the key component in maintenance of plasma membrane potential in plants[56].To further investigate the underlying interrelationship among plasma membrane H+-ATPase,net H+flux,and plasma membrane potential in tea mesophyll cells,the effects of drought stress and subsequent rehydration on net H+flux and plasma membrane H+-ATPase activity were studied.Drought stress caused a rapid H+influx,ranging from 21.73 to 33.73 pmol cm-2s-1(Fig.4-C),and the total net H+influx value reached 2771.4 pmol cm-2s-1(Fig.4-D),further confirming that drought stress induced plasma membrane potential depolarization in tea mesophyll cells(Fig.4-A).Subsequent rehydration induced net H+efflux,and the total amount of net H+efflux was similar to that in the non-stressed control(Fig.4-C,D).

Fig.3–Effects of drought and subsequent rehydration on K+efflux and K+content in tea plants.Effects of drought andsubsequent rehydration on K+content in tea leaves(youngest and subsequent second fully expanded leaves)(A)and tea plants(B).Dynamic change(C)and the total amount(D)of K+flux of mesophyll cells of youngest fully expanded leaves under drought and subsequent rehydration.

To establish a possible relationship between plasma membrane H+-ATPase and net H+flux,we firstly checked the purity of plasma membranes isolated from the microsomal fraction.Enzyme activity of isolated plasma membrane was decreased about 80%by Na3VO4and was inhibited by＜10%by KNO3and NaN3(Fig.5-A).The purity of plasma membranes isolated from the microsomal fraction of tea mesophyll cells was high enough for use in further experiments[33].Plasma membrane H+-ATPase activity isolated from mesophyll cells was reduced by 28.4%after drought stress.With subsequent rehydration,this value recovered to levels equal to those in the control group(Fig.5-B).To investigate whether the changesofplasmamembraneH+-ATPaseactivitywere associated with H+-ATPase protein expression level,the expression patterns of plasma membrane H+-ATPase protein in tea mesophyll cells grown under drought stress and rehydration treatment were analyzed by Western blotting.The band intensity of plasma membrane H+-ATPase in drought-stressed tea plants was significantly lower than that in the non-stressed control.After subsequent rehydration,the band intensity of plasma membrane H+-ATPase was significantly increased in comparison with that in drought-stressed plants(Fig.5-C).

PEG and soil drought stress caused the acidification of apoplastic pH and K+uptake of three barley genotypes,XZ5,Tadmor and ZJU9,in root epidermis[57],indicating that plasma membrane H+-ATPase and K+flux were associated with drought stress in barley.In our study,drought stress inhibited plasma membrane H+-ATPase activity(Fig.5-B),induced a significant mesophyll H+influx(Fig.4-C),resulting in a depolarized plasma membrane potential(Fig.4-A).Thus,the significant K+efflux observed in drought-stressed tea plants(Fig.3-C)could be at least partially attributed to the membrane depolarization induced activation of voltage gated KOR(K+outward rectifying channel)channel[58].Plasma membrane depolarization-induced K+efflux through the voltage-gated KOR/GORK channel has been shown in other plant species.For example,NaCl-induced massive K+efflux via stimulation of plasma membrane potential depolarization was found in Arabidopsis[59]and barley[60].Similarly,plasma membrane potential depolarization activated the GORK channel in Arabidopsis mesophyll cells,accelerating K+efflux[61].

Fig.4–Changes in plasma membrane potential and net H+flux in tea mesophyll cells subjected to drought and subsequent rehydration.Changes(A)and mean value(B)of membrane potential in mesophyll cells of youngest fully expanded leaves subjected to drought and subsequent rehydration.Dynamic changes of net H+flux(C)and total H+flux(D)in mesophyll cells of youngest fully expanded leaves subjected to drought and subsequent rehydration.

Fig.5–Effects of drought and subsequent rehydration on plasma membrane H+-ATPase in youngest and subsequent second fully expanded tea leaves.A.Test of the purity of the isolated plasma membrane H+-ATPase;B.Comparison of plasma membrane H+-ATPase activity in tea leaves subjected to drought and subsequent rehydration;C.Protein level(by Western blotting)of plasma membrane H+-ATPase isolated from youngest and subsequent second fully expanded tea leaves under control,drought,and rehydration treatment.Actin protein was loaded as control.

Compared with the decreased activity under drought stress,subsequent rehydration increased plasma membrane H+-ATPase activity(Fig.5-B)and induced a significant H+efflux than the one under drought stress(Fig.4-C),stimulating membrane potential hyperpolarization(Fig.4-A).Plasma membrane-located AKT/KAT K+uptake channels are known to be activated by plasma membrane hyperpolarization[55].Stimulation of plasma membrane H+-ATPase hyperpolarized membrane potential in Vicia faba guard cell and regulated K+influx[62].Li et al.reported that Os-AKT1,which requires a more negative membrane potential(hyperpolarization)to be activated,is involved in mediating K+uptake[63].Membrane potential hyperpolarization-activated AtAKT1 is known to mediate K+uptake under low-K+stress[64].Our results suggested that rehydration-inducedK+influxinteaplants(Fig.3-C)isassociated withincreasedactivityofplasmamembraneH+-ATPaseandthus plasma membrane potential.To our knowledge,ours is the first finding that plasma membrane H+-ATPase may be associated with potassium homeostasis in tea plant response to drought stress and subsequent rehydration.

4.Conclusions

Maintaining mesophyll potassium homeostasis is important for tea plants'response to drought stress and subsequent rehydration treatment and is associated with the regulation of plasma membrane H+-ATPase activity.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.06.001.

Acknowledgments

This work was supported mainly by the Science Foundation for Anhui Province(KJ2017A126)to Xianchen Zhang and the Opening Fund of State Key Lab of Tea Plants Biology and Utilization at Anhui Agricultural University(SKLTOF20170112)to Honghong Wu.It was also supported by the National Natural Science Foundation of China(11008389)and the National Basic Research Program of China(11000206)to Xiaochun Wan.We thank Mr.Joseph Hartley from University of Tasmania,Australia for hishelpinreading thismanuscript.WethankProf.James C.Nelson from Kansas State University for his proofreading of the manuscript.