Mechanism Analysis Of Dehydrin MtCAS31 In Affecting Aquaporion Autophagic Degradation And Nitrogenase Activity

As a major abiotic stress,drought negatively affects the plant growth,development and other biological process.To survival from the harmful environmental conditions,plants evolved many strategies to respond.Two kind of proteins function in drought response:the regulation proteins and functional proteins.Regulation proteins contain transcriptional factor,protein kinase et al..The functional proteins include the osmotic regulatory protein and the protein-protector such as late embryogenesis abundant proteins(LEA).Dehydrins are protein-protectors which belong to the LEA group and accumulate under drought stress.However,the study about the function of dehydrins was mainly focus on the protein protection in vitro.Their function in vivo is limited and required more study.MtCAS31 encodes a 31 kDa dehydrin.In our previous study,the drought tolerance of transgenic Arabidopsis expressing dehydrin MtCAS31 was highly improved.While in the present study,we further explored the function of MtCAS3 1 in Medicago truncatula.For the further study,we first generated the MtCAS31 overexpression lines(MtCAS31OE)and knockout mutant by Agrobacterium-mediated genetic transformation.Physiology evidence showed that MtCAS31 highly reduce the stomatal conductivity and transpiration rate,which in turns improved the drought tolerance of M.truncatula.To understand the function of MtCAS31 in drought response,MtCAS31 was used as bait to screen the abiotic stress induced Medicago cDNA library.Interestingly,an aquaporin MtPIP2;7 which belongs to PIP family was selected.Yeast two-hybrid,GST pull-down and BiFC assays further demonstrated the interaction between MtCAS31 and MtPIP2;7.The root conductivity(Lpr)of pip2:7 reduced but the drought tolerance was enhanced when compared to WT,suggesting that MtPIP2;7 was a negative regulator in drought response.Using immunoblotting analysis,MtPIP2;7 was degraded under dehydration(30%PEG 8000),which could be inhibited by 26S proteasome inhibitor MG 132 or autophagy inhibitor ConcA,suggesting that MtPIP2;7 was degraded via ubiquitin/26S proteasome degradation system and autophagic degradation pathway.To explore whether MtCAS31 affects the degradation of MtPIP2;7,WT and cas31 mutant were treated with dehydration.Protein inhibitors were applied and the immunoblotting analysis showed that MtCAS31 promoted autophagic degradation of MtPIP2;7.Yeast two-hybrid,BiFC assays and Co-immunoprecipitation(Co-IP)assay showed that MtCAS31 interacted with an autophagy initiation protein MtATG1.MtPIP2;7-MtCAS31-MtATG1 worked as a complex to affect the autophagy initiation of MtPIP2;7.Lpr measurement showed that the Lpr in cas31 was significantly higher than WT.Because the water transportation by aquaporin is passive,water tends to transport into soil from the roots under drought stress,which leads to the water loss.Bases on this reason,higher Lpr means higher water loss under drought stress.These results suggested that MtCSA31 promoted autophagic degradation of MtPIP2;7 thus reducing the water loss and in turns to enhance the drought tolerance.Beside the root,stem and leaf,the expression of MtCAS31 was also detected in nodules.So we further study the function of MtCAS31 in nodulation.According to acetylene reduction assay,we found that the nitrogenase activity was significantly lower in cas31 than in WT under drought stress.Besides,yeast two-hybrid,GST pull-down and BiFC assays showed that MtCAS31 interacted with leghemoglobin,MtLb120-1,an important protein in symbiotic nitrogen fixation(SNF).MtLb120-1 RNAi lines showed reduced nitrogenase activity,indicating that MtLb 120-1 affected the symbiotic nitrogen fixation efficiency.To study the molecular mechanism of the interaction between MtCAS31 and MtLb 120-1,thermal inactivation assay was conducted.The result showed MtCAS31 protected MtLb 120-1 through protein-protein interaction.According to previously study,the absence of leghemoglobin leads to not only the reduced nitrogenase activity but also the developmental defect of bacteroid.Transmission electron microscope and lugol staining showed that more starch granlue were accumulated in cas31 than in WT.The accumulation of starch granlue represents the nodule senescence.By RT-qPCR,two stress-induced senescence genes MtCP2 and MtCP3 were highly induced in cas31 than in WT under drought stress.These result suggested that MtCAS31 significantly delayed the nodule senescence under drought stress.In conclusion,our research identified the new functions of dehydrin in drought response.MtCAS31 mediated the MtPIP2;7 autophagic degradation thus to reduce the water loss under drought stress.Moreover,MtCAS31 reduced the negative effect of drought stress on SNF.Our study elucidated the molecular mechanism in drought response mediated by dehydrins,which help us understand the function of dehydrin profoundly.