| Drought is one of the abiotic stresses that seriously threatens the growth and development of plants and the crop yield,it causes increasing huge economic losses in the world every year,and especially with the global warming is getting more serious.Therefore,it is of great theoretical significance and application value for the development of modern agriculture to screen and investigate the plant drought responsive genes by means of molecular biology and genetics.The plant stomata are surrounded by pairs of guard cells,and are micropores for water and gas exchange between plants and the outside.The opening and closing of stomatal are fine regulated by changes in turgidity and volume of guard cells,and play essential roles in plant drought response,and the phytohormone abscisic acid(ABA),is the key regulator during stomatal movements.ABA and drought could induce solute ions efflux of guard cells and in turn shrink stomatal aperture to save water and improves the plant’s ability of drought-resistance.The LRR-RKs represent the largest group of RKs in the Arabidopsis thaliana genome,comprising 216 members,and have been found to be involved in plant invorionment interactions.In our previous research,we analysed lots of candidate genes which were specifically expressed in guard cells by proteomics analysis and screened two unknown gnens At3g08680(KIN8)and At2g26730(KIN9).KIN8 and KIN9 belong to LRR-RKs family,and are both highly expressed in guard cells,so their function in stomatal movents is worthy of investigation.In this study,we further performed a series of experiments to investigate the function of the two interested LRR-RKs,KIN8 and KIN9 in ABA-mediated stomatal movement and drought stress response by using molecular biology,cell biology,genetics techniques.In the study of KIN9,GFP fusion protein assay and GUS staining results indicated that,KIN9 was subcellularly localized in cytoplasm and cell membrane,and highly expressed in guard cells.The transcription of KIN9 was decreased in response to drought and ABA treatments.The stomatal movement,leaf water loss and whole plant drought treatment bioassays indicated that,the kin9 mutant was hypersensitive to ABA than Col-0,and with lower water loss rate and enhanced drought-resistant ability.The phenotypic analyses with KIN9 over-expression lines and complementation lines showed that,the ABA hyposensitivity of kin9 mutants could be restored in KIN9-C-1 and KIN9-C-2 complemented lines.On the contrary,the KIN9 overexpression lines KIN9-OE-1 and KIN9-OE-2 showed the opposite phenotypes compared with the loss-of-function mutants.These results indicated that KIN9 negatively regulates ABA-mediated stomatal movement and plant drought response.An Arabidopsis transcription factor NAP was identified as a KIN9-interacting protein,and the interaction was further confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays(BiFC).Bused on the collected data,we proposed a hypothesis that,in response to drought,KIN9 is a negative regulator,which could phosphorylate and activate NAP,and thus to regulate downstream target gene transcription.As for the research of KIN8,KIN8 was found be to subcellulally localized in cytoplasm and plasma membrane.The transcription of KIN8 could be induced by drought and ABA treatments.In the ABA-mediated stomatal movement experiment,the mutant kin8 was more sensitive to ABA than Col-O.Besides,the rate of water loss from detached leaves were lower in the mutant than Col-0.The phenotypic analyses with KIN8 over-expression lines showed that,KIN8-OE-1 and KIN8-OE-2 had the opposite phenotypes to the loss-of-function mutants.So,we conclude that KIN8 also serves as a negative regulator in ABA-mediated stomatal movement and drought response.In conclusion,our research reveals the roles of KIN8 and KIN9 in stomatal movement and plant drought resistance.It will contribute to perfect the guard cell signal transduction network and the potential application in the development of water-use-efficiency modern agriculture. |
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