Differential Changes Of Mitogen-activated Protein Kinase4 Under Cold Stress In Arabidopsis Thaliana

The growth of land plants is greatly affected by avariety of environmental stresses, such as dehydration, low temperature, heat, and mechanical perturbation(touch), wounding and pathogen infection. Environmental stresses induce various biochemicals, physiological and molecular responses, including gene expression. It is thought that the seresponses are promoted as a result of the perception of environmental stimuli and signal transduction in plant cells, but them echanisms by which plants sense and transducer signals in response to environmental stresses are largely unknown.The mitogen-activated protein kinase(MAPK) cascade is a universal module of signal transduction from the cell surface to the nucleus. It plays a crucial role in the regulation of biochemical and physiological changes as sociated with extracellular stimuli. MAPK cascades have been reported to function in various signal transduction pathways in eukaryotes.A possible MAPK cascade in Arabidopsis comprising ATMEKK1, MEK1 and ATMKK2 and ATMPK4 has been identified based on analyses of the yeast two-hybrid system and complementation of yeast mutants related to the MAPK cascade. MEK1 can phosphorylate and activate ATMPK4 in vitro. The ATMEKK1 mRNA level increases markedly in response to low-temperature, high-salinity and mechanical stresses. Although this result suggests that the possible MAPK cascade can be activated by environmental stresses, it was unclear whether the secomponents are activated enzymatically inresponse to environmental stresses.We have already identified a possible Arabidopsis MAPK cascade, ATMEKK1→MEK1/ATMKK2→ATMPK4, by yeast two-hybrid analysis and complementation analysis of yeast mutants. Transcription of the ATMEKK1 gene was rapidly increased by low-temperature, high-salinity and mechanical stresses. This finding suggests that ATMEKK1 and its downstream factors function in a signal transduction pathway for environmental stresses in Arabidopsis. In this study, we show that cold stress induce the changes of ATMPK4 with its transcript and protein levels.To determine if ATMAPK4 can respond to cold stress in vitro, we expressed ATMPK4 in E.coli. Then to analyze the activation of ATMPK4 under environmental stress conditions, we raised polyclonal antibodies against the entire amino acids of ATMPK4. A proteomic analysis was carried out aiming at understanding of the molecular adaptation mechanisms of cold stress in Arabidiopsis (Arabidopsisthanliana, ecotypeCol-0). The seedlings were exposed to 4℃for 1, 2, 4, 8 and 24h respectively, together with the controls which were grown at normal temperature. Using specific antibodies, we examined the protein expression of ATMPK4 under stress conditions. Expression of ATMP4 was analyzed by western-blot with antibodies to ATMPK4. At the same time we quantitated mRNA of Arabidiopsis MAPK4 by realtime PCR. The results showed that 5 cold-treated groups are all higher then the untreated group at RNA level. Expression of the ATMPK4 genes are correlated with their Expression of the ATMPK4 proteins. The physiological implications, which were derived from the experimental data, are discussed, suggesting that there is a complex metabolicnet work in plants responsive to cold stress.