Functional Study Of ABA Receptor GhPYL9-4D,GhPYL9-6A And GhPYL6-1A In Cotton

Cotton is one of the most important cash crops and fiber crops,which is planted in most countries in the world.China is one of the countries with the largest cotton cultivated area.Cotton fiber is the important raw material for textile industry.It can be used to make many kinds of fabrics,such as clothing,cotton cloth and so on.In recent years,many adverse environmental stimuli such as drought,high salt and cold seriously affect the growth of cotton in China,resulting in a decline in cotton yield and fiber quality.Therefore,how to enhance the tolerance of cotton to the stresses is the focus of scientists’ attention.Abscisic acid(ABA)is an important hormone in plants.It regulates stomatal closure,inhibits seed germination and primary root growth of the main root,and especially plays a key role in tolerance to the adverse environmental factors.Therefore,ABA is also known as stress hormone.ABA participates in the regulation of plant growth.The action of ABA requires a series of signal transduction components,including ABA receptor protein PYLs(Pyrabatin resistance 1-likes),A-lik PP2Cs(Protein phosphatase 2Cs),SnRK2s(SNF1-related protein kinases)and effect factors.It will help us to better undersand ABA signal transduction mechanism after cloning and functional analysis of PYLs genes in cotton.This will lay the foundation for further investigation on screening resistant genes and their tolerant mechanism in cotton to stress.In this study,40 ABA receptor genes were identified from the cotton database based on the amino acid sequence of Arabidopsis PYL proteins.According to the homology of Arabidopsis PYLs,three GhPYLs including GhPYL9-4D,GhPYL9-6A and GhPYL6-1,which are homologous to AtPYL8 and AtPYL5 were characterized.We analyzed the conservatism and evolutionary relationship between the three cotton PYLs and the two Arabidopsis PYLs by bioinformatic methods.The tissue localization and expression of the three cotton PYLs under stress were studied using the methods of RT-PCR and qRT-PCR.At the same time,interactions between each of the cotton PYLs with GhPP2CA were investigated by yeast two-hybrid method.Moreover,we constructed the three transgenic vectors of the cotton PYLs,and analyzed the effect of ABA,NaCl and mannitol on the seed germination and root growth of Arabidopsis thaliana overexpressing the individual gene.The main results are as follows:GhPYL9-4D,GhPYL9-6A and GhPYL6-lA have a conserved functional domain with PYL proteins of Arabidopsis thaliana.The tissue expression of GhPYL9-4D,GhPYL9-6A and GhPYL6-1A is specific.The gene expression patterns of GhPYL9-4D,GhPYL9-6A and GhPYL6-1A are different under ABA and drought stress.Results of qRT-PCR showed that the expression of GhPYL9-4D,GhPYL9-6A and GhPYL6-1A s decreased under ABA treatment but increased under drought stress.GhPYL9-4D,GhPYL9-6A and GhPYL6-lA can interact with GhPP2CA protein in vitro,and most of the interactions depended on ABA.Effects of ABA,NaCl or mannitol on seed germination and root growth of GhPYL9-4D GhPYL9-6A and GhPYL6-1A transgenic Arabidopsis plants were investigated.The results showed that the seed germination rate and green seedling rate of GhPYL9-4D overexpressors were markedly lower than those of the wild type(WT)in medium containing ABA.Moreover,the growth of primary roots was clearly inhibited by ABA medium.These results indicate that overexpression of GhPYL9-4D gene enhanced the sensitivity of seed germination and seedling growth to ABA.By contrast,there was no significant difference in seed germination rate,green seedling rate and main root length among GhPYL9-4D overexpressors and WT under salt stress.The seed germination rate of GhPYL9-4D overexpression plants did not significantly differ from that of WT in MS medium supplied with different concentrations of mannitol.However,the green seedling rate of GhPYL9-4D transgenic plants was remarkably lower than that of WT under osmotic stress.Furthermore,length of the primary roots from GhPYL9-4D overexpressors was significantly longer than that from WT in the presence of mannitol.These findings imply overexpresion of GhPYL9-4D enhanced tolerance of seedling growth to mannitol in Arabidopsis.Further investigations revealed that ABA inhibited the growth of primary root of GhPYL9-4D transgenic plants by inhibiting the length of the meristem zone and the number of cells in the meristem zone,and exogenic auxin can alleviate the differences in root growth between GhPYL9-4D overexpressors and WT after treatment with ABA.The levels of reactive oxygen species in roots from GhPYL9-4D overexpression plants were also evidently lower than those from WT.In addition,the expression of some drought tolerant related genes was pronouncedly increased in GhPYL9-4D overexpressors compared to WT.VIGS interference experiments showed that after silencing GhPYL9-4D,cotton showed a drought sensitive phenotype.We observed that ABA clearly inhibited the seed germination and main root growth of GhPYL9-6A overexpressors.In contrast,no significant difference in seed germination rate and primary root growth was found between GhPYL9-6A overexpressing plants and WT under salt stress.After mannitol treatment,the seed germination rate of GhPYL9-6A overexpressors was not significantly different from that of WT,however the length of primary roots of the transgenic plants was prominently longer than that of WT.These data suggest that overexpression of GhPYL9-6A increases the sensitivity of Arabidopsis plants to ABA and their tolerance to osmotic stress.While overexpression of GhPYL6-1A not only enhanced the sensitivity of seed germination and primary root growth to ABA,but also increased the their sensitivity to NaCl and mannitol treatments.Subcellular localization analysis showed that GhPYL9-4D and GhPYL9-6A were localized in plasma membrane,and GhPYL6-1A was in the plasma membrane and nucleus.