Functional And Mechanism Analysis Of A Gene Involved In Regulation Of Cold/drought Stress Responses In Arabidopsis

Drought and cold stresses profoundly affect crop growth and development, thus leading to decrease crop yield. Therefore, it is important for studying the mechanisms of the responses of plants to drought and cold stresses. In our preliminary study, it was found that the LWT1 gene encoding a function-unknown protein is induced by cold and drought stresses in Arabidopsis. The lwt1 mutant showed enhanced sensitivity to low temperature and drought stress. However, the action mechanisms is unclear. In this study, the function of the LWT1 gene and its regulation mechanism are further studied by using a gain-of-function method. The results are as follows:1. The full-length LWT1 coding sequence jwas amplified from cDNA of Arabidopsis thaliana (Col-0) and cloned into the pART27 vector. The resulting construct was transformed into Agrobacterium strain GV3101 for transformation into the Col-0 plants by the floral dip method. The transgenic lines were isolated by antibiotic screening and genetic identification.2. LWT1 over-expression lines showed enhanced tolerance to low temperature stress compared with the wild type, as indicated by higher survival rate and lower electrolyte leakage.3. LWT1 over-expression lines also displayed enhanced tolerance to drought stress compared with wild-type plants, as indicated by higher survival rate and lower water loss of detached leaves.4. There were no significant difference in fhe level of soluble sugar between the LWT1 over-expression plants and the wild type in response to low temperature and drought stresses. However, higher; proline accumulation was detected in the LWT1 over-expression plants than in the wild type, suggesting that LWT1 regulates the tolerance of plants to drought and cold stresses by altering the proline content.5. Under low temperature stress, the PDH1 gene encoding proline oxidase was induced in the wild type and the LWT1 over-expression plants, but the induced level was lower in the latter than the former. Under drought stress, the PDH2 gene endoding proline oxidase was induced in the wild type and the LWT1 over-expression plants, but a lower level was induced in the latter. However, there was no significant difference in expression of other proline degradation-related genes These results suggest that higher proline accumulation in LWT1 over-expression lines was associated with an increase in expression of proline degradation-related genes PDH1/PDH2.6. Analysis of the proline oxidase (PDH)showed that PDH activity was increased in both the LWT1 over-expression plants and the wild type under low temperature and drought stresses. However, a significantly lower increase was detected in LWT1 over-expression plants than in wide-type plants, suggesting that the PDH activity was regulated by LWT1.7. Analysis of yeast two-hybrid showed that LWT1 physically interact with PDH, suggesting that LWT1 suppressed its activity.Taken together, all these results suggest that low temperature and drought stress induces the LWT1 gene which leads to interaction bewteen LWT1 and PDH. LWT1 suppresses the PDH activity, thereby reducing the degradation of proline and increasing the accumulation of proline, which leads to the increased tolerance of plants to low temperature and drought stress.