Molecular Mechanism Study Of BHLH122in Improving Plant Tolerance To Stresses

Drought and salt stresses are the major environmental factors limiting crop productivity worldwide.To reduce the adverse effects of abiotic stresses, plants have evolved multifaceted strategies, includingmorphological, physiological and biochemical adaptations. The cellular and molecular mechanisms ofplants adapt to environmental stresses have been investigated intensively and found the changes in geneexpression play an important role in this process. Basic helix-loop-helix proteins (bHLH) are foundthroughout the eukaryotic kingdom and constitute one of the largest families of transcription factors forexample; there are140members in Arabidopsis thalina and more than160members in Oryza sativa,what’s more, these members result in the redundancy of functions. bHLH involves in not only all kindsof biological processes but also plays important roles in plant stress responses to abiotic stresses. In ourstudy, we found that bHLH122could be introduced by salt, drought and osmotic stresses. Theexpression of bHLH122focused on stomas. In order to make clear the molecular mechanism ofbHLH122, we researched the bHLH122functions by virtues of molecular biology, plant physiology andbiochemistry methods.Basic on the former research, the main results are as follows:1. Compared with wild type,35S::bHLH122transgenic plants were more tolerant to drought stress.The stomatal aperture of35S::bHLH122after drought stress was40%~63%smaller than wild type.The average anthocyanin level in over-expression bHLH122was about75%less than the WT.These results suggested that more rapid appearance of wilting after water withholding in wild typecould be attributed at least in part to an inability of these plants to efficiently close their stomataand reduce transpiration.2. Addition of NaCl into MS plates inhibited cotyledon greening/expansion and biomasses of wildtype but not of35S::bHLH122transgenic plants. The35S::bHLH122plants were more resistant toNaCl but bhlh122mutant were more sensitive than wild type. Different from the responses to NaCltreatment, though the germination rates of transgenic plants growing on medium supplied withmannitol were similar to that of wild type but the average root length of the bHLH122transgeniclines was found3times more than the wild type. So we presumed that bHLH122were required toimprove plant resistance to salt and osmotic stresses.3. Through microarray analysis, we found approximately214genes showed statistically significantlychanged in expression in35S::bHLH122lines. Among them,87genes were up-regulated and127genes were down-regulated. Most of these genes have known or presumed functions associatedwith abiotic stress responses, such as protease inhibitor, WRKY/MYB transcriptional factors,protein kinase and so on.80%of these genes’ promoters contained5′-CACGTG-3′(G-box) or5′-CANNTG-3′(E-box) elements which were presumed as the binding domain of bHLH122. Atthe same time, we chose six genes to analysed using Real-Time PCR and the results wereaccordance with microarray results. These results indicated bHLH122could directly regulate the down-stream genes expressions which were necessary to resist abiotic stresses.4. The electrophoretic mobility shift assays further demonstrated that bHLH122could bind to bothE-box and G-box cis-elements but priority to E-box.5. The expression of bHLH122in aba2-1mutant and abi4-1mutant didn’t show any significantchange compared to wild type. CYP707A3participated in ABA metabolism and was the key genein ABA degradation. Abscisic acid is catabolized into inactive forms by oxidation pathway andCYP707A3can trigger this action. In microarray analysis and Real-Time PCR test, it wasdown-regulated harshly but revised in bhlh122mutants. In EMSA and ChIP assay, bHLH122could bind to E (G)-box in the promoter of CYP707A3gene and repressed gene translation. Inover-expression bHLH122transgenic plants, ABA content was high as two times as the content inwild type under drought stress. So we inferred bHLH122could improve the tolerance to abioticstress in Arabidopsis by accumulating more ABA.6. Through alignment to maize sequences, we found there were4proteins were similar toAtbHLH122, and CYP707A3had3proteins alike, these results implied maize may have similarmolecular mechanisms in the regulatory pathway of bHLH122.