Molecular Mechanism Of Trehalose Alleviating Osmotic Stress In Watermelon Suspension Cells

Watermelon(Citrullus lanatus)is one of the most important cash crops in the world.It is widely planted in China.Drought,salinity and high temperature are three common abiotic factors affecting the yield and quality of watermelon.Although a large number of studies have shown that trehalose is an important osmotic adjustment substance,which can effectively improve the resistance of various plants.However,is trehalose alleviating osmotic stress often encountered in watermelon production?What signal pathway does watermelon activate the protection mechanism of trehalose?Around the above scientific problems,the experimental material is the diploid-watermelon suspension cells,phenotypic characteristics of watermelon cells under different treatment conditions,bioinformatics analysis of trehalose synthesis and metabolism family genes,gene expression analysis and trehalose accumulation measurement were determined;which is uesed to clarify the relationship between trehalose and osmotic stress resistance of watermelon,to establish the correlation between trehalose signal substance,gene expression of trehalose biosynthesis pathway and trehalose content and to explore the signal transduction pathway of trehalose biosynthesis initiated by watermelon under osmotic stress.The main findings are as follows:(1)After pretreatment with exogenous trehalose,the cell growth,extracellular pH,relative content of intracellular ROS(reactive oxygen species)and microtubule skeleton of watermelon suspension culture were determined under osmotic stress.The results showed that osmotic stress could inhibit cell growth,induce extracellular alkalization,ROS bursting,and counteract the depolymerization of microtubular cytoskeleton.However,exogenous trehalose could alleviate the inhibitory effect on cell growth caused by osmotic stress,suppress the expression of extracellular alkalization and ROS,and maintain the integrity of the microtubular cytoskeleton.(2)Trehalose synthesis is catalyzed by two key enzymes:trehalose-6-phosphate synthase(TPS)and trehalose-6-phosphate phosphatase(TPP)in plants;furthermore,trehalose is reduced to two-molecule glucose by trehalase(TRE).The gene sequences of trehalose ClTPS,ClTPP and ClTRE in watermelon were obtained from the cucurbit genome database(CGD).The protein domains were identified by PFAM and SMART tools.The results showed that seven CITPS,seven ClTPP proteins and three ClTRE proteins,named ClTPS1-ClTPS7;ClTPP1-ClTPP7;ClTRE1-ClTRE3.(3)Bioinformatics analysis of trehalose catabolism family genes in watermelon showed that ClTPS family genes in watermelon were classified into two groups:Class Ⅰand Class Ⅱ.Class Ⅰ contained two genes(ClTPS2,ClTPS7),and the rest belonged to ClassⅡ.Chromosome localization analysis revealed that except for the ClTRE gene,the members of the ClTPS and ClTPP family genes were individually distributed on 11 chromosomes,and there was no tandem repeat.Signal peptide and phosphorylation sites analyzed that the 17 watermelon proteins in watermelon are non-secreted proteins and are not involved in the transport of proteins in cells,and the phosphorylation sites of ClTPS,ClTPP and ClTRE proteins are concentrated in the Serine site in watermelon;phylogenetic evolution shows that the TPS and TPP family genes are multiplied during evolution,while the TRE family genes are relatively conservative in evolution.(4)Prediction of cis-acting elements in promoter regions from trehalose anabolic pathway genes.The results showed that they contain not only some necessary cis-acting elements,but also many cis-acting elements response to stress and plant hormones.(5)Exogenous mannitol mimics osmotic stress to treat watermelon suspension cells.It is found that osmotic stress can up-regulate the expression of trehalose ClTPS,ClTPP and ClTRE family genes in different degrees and promote the increase of cell trehalose accumulation.(6)The cells were induced by exogenous hormones(ABA,MeJA,ethephon,SA).The results showed that the expression of ClTPS,ClTPP,ClTRE family gene and trehalose accumulation in watermelon suspension cells could respond to exogenous phytohormones(MeJA,ETH,ABA,SA)in varying degrees,among which the expression of ClTPS family gene was the highest induced by exogenous MeJA in watermelons cells;the expression of ClTPP and ClTRE genes was higher induced by exogenous MeJA and ETH in watermelons cells.However,trehalose accumulation in watermelon cells induced by different exogenous plant hormones was measured.The results that there was no trehalose in the watermelon cells of the control group,ABA,MeJA,ethephon and SA could induce the accumulation of trehalose in watermelon cells,among which MeJA induced the highest accumulation of trehalose in watermelon cells,followed by ABA and SA,and the lowest ethephon.(7)The expression levels of ClTPS,CITPP and ClTRE family genes in watermelon suspension cells responded to different degrees of jasmonic acid synthesis inhibitors(IBU).ethylene synthesis inhibitor(AVG)and abscisic acid synthesis inhibitor(FL).The expression of ClTPS family genes in watermelon is regulated by the synthesis of endogenous jasmonic acid and abscisic acid.The expression of most ClTPP family genes in watermelon is regulated by the synthesis of endogenous jasmonic acid and abscisic acid.The expression of ClTRE family genes in watermelon is regulated by the synthesis of abscisic acid.The synthesis of endogenous ethylene negatively regulates the expression of ClTPS,ClTPP and ClTRE family genes in watermelon.The trehalose content of watermelon cells induced by different endogenous signal molecular inhibitors was determined.Compared with mannitol treatment,different endogenous signal molecular inhibitors inhibited trehalose accumulation in watermelon cells.When jasmonic acid synthesis was inhibited,trehalose accumulation decreased significantly.(8)Watermelon cells were induced by exogenous hormones(ABA,MeJA,ethephon,SA).The results showed that different exogenous phytohormones also inhibited the growth of watermelon suspension cells to some extent.Among them,methyljasmonate had the least inhibitory effect on the growth of watermelon cells.ethylene(ETH)and abscisic acid(ABA)were the second;salicylic acid(SA)had the greatest inhibitory effect on the growth of watermelon suspension cells.(9)Exogenous addition of mannitol,jasmonic acid synthesis inhibitor(Ibuprofen,IBU),ethylene synthesis inhibition(AVG),and abscisic acid synthesis inhibitor(FL)induced watermelon cells.It was found that the growth of watermelon cells significantly decreased under mannitol stress;Compared with mannitol stress,After adding IBU and FL induction treatment,it had a significant inhibitory effect on the growth of watermelon cells under osmotic stress.However,after adding AVG pretreatment,although there was an inhibitory effect on the growth of watermelon under osmotic stress,there was no significant difference compared with mannitol stress.To sum up,exogenous addition of trehalose can improve the resistance of watermelon cells to osmotic stress,and the accumulation of endogenous trehalose in watermelon cells under osmotic stress is closely related to plant hormone signals,in which jasmonic acid signal may play a leading role in the signal pathway of watermelon trehalose synthesis.