Molecular Mechanism Of Betaine Alleviating Osmotic Stress In Watermelon Suspension Cells

Watermelon(Citrullus lanatus)is one of the important economic crop in the world.It is widely cultivated in most regions of China.During the process of produce,in order to make full use of the land,the watermelons are often planted in the sandy soil of drought and less rain or with the high salinity of the seaside sand because its drought tolerance and the roots prefer sandy soil with good ventilation.Drought,salt stress and so on will first produce osmotic stress to watermelon,and then affect its yield and quality.The studies showed that betaine as an osmotic regulator can effectively improve the resistance of plants under stress stress.However,is betaine alleviating osmotic stress often encountered in watermelon production?What signal pathway does watermelon activate the protection mechanism of betaine?Around the above scientific problems,the experimental material is the diploid-watermelon suspension cells and the osmotic stress was simulated by mannitol in this study,meanwhile combined the exogenous application of betaine.The correlation of the osmotic stress tolerance and betine was determined in watermelon by measuring the cell mitosis index,intracellular reactive oxygen species(ROS)and extracellular pH.Through the determination of the gene expression of CICMO and ClBADH and the accumulation of betaine to study the protection mechanism of endogenous betaine was initiated by osmotic stress.The sequence of key genes was analyzed by bioinformatics and the possible signaling material to start the protection mechanism of watermelon betaine was predicted.By using exogenous signal substances and corresponding inhibitors,we explored the possible signal pathways of watermelon response to osmotic stress to initiate betaine protection mechanism.The main results are as follows:(1)Different concentrations of mannitol treated watermelon suspension cells,the cell mitotic index decreased,while the intracellular reactive oxygen species(ROS)and the extracellular pH were significantly increased.The results showed that osmotic stress inhibited the growth of watermelon cells obviously,and the higher the concentration of the treatment,the more obvious the inhibition effect.However,exogenous betaine can effectively alleviate the inhibitory effect of osmotic stress on cell growth.(2)Screening watermelon CICMO gene sequence(Cla001775)and CIBADH gene sequence(Cla019160)from the watermelon database(CGD),and using bioinformatics analysis tools to analyze.The results showed that CICMO and ClBADH gene encoding 417 and 503 amino acids respectively,their molecular weight of the protein are 47.2kDa and 54.5kD,and their isoelectric point are 6.01 and 5.29.They both belong to hydrophilic stable proteins.The protein alignment analysis showed that ClCMO has the Rieske[2Fe-2S]and Fe-binding motif that are two typical conserved domains.ClBADH conserved domains have ten peptide sequences and Csy binding sites of typical aldehyde dehydrogenase.Phylogenetic tree analysis showed that the ClCMO and ClBADH of watermelon were closely related to Cucumis sativus and Cucumis melo from Cucurbit,but were not related to CDH and BADH from Escherichia coli.(3)Prediction of cis acting elements in promoter regions from ClCMO and CIBADH gene.The results showed that they contain not only some necessary cis-acting elements,but also many cis-acting elements that response to stress and plant hormones.(4)Exogenous mannitol simulated osmotic stress could induce the expression of ClCMO and CIBADH genes and increase the accumulation of betaine in watermelon,and gene expression was positively correlated with betaine accumulation.(5)After adding exogenous hormones(abscisic acid,methyl jasmonate,ethephon and salicylic acid)induced watermelon cells the relative expression of ClCMO and CIBADH genes increased after induction,and the relative expression of genes increased more significantly by exogenous MeJa induced.After determining the content of betaine,it was found that abscisic acid,methyl jasmonate and ethephon could induce betaine accumulation,but MeJa induced the highest content,which was basically consistent with the expression level after challenged by osmotic stress.(6)By adding jasmonic acid synthesis inhibitors(ibuprofen,IBU)and the ethylene synthesis inhibitor(AVG)the relative expression of ClCMO and CIBADH under osmotic stress and the accumulation of betaine showed increasing trend,while IBU has obvious inhibitory effects on osmotic stress and the relative expression of and the accumulation of betaine.In conclusion,exogenous betaine has a significant effect on alleviating osmotic stress induced inhibition of cell growth in watermelon.Osmotic stress can induce the synthesis and accumulation of endogenous betaine in watermelon.The accumulation of endogenous betaine in watermelon cells is closely related to plant hormone signals,and jasmonate signals may play a leading role in the initiation of the molecular protection mechanism of watermelon betaine.