Biochemical Analyse To Function Of CIPK14 And EGY1 Of Arabidopsis In Stress Response And Light Reaction

Various abiotic stresses resulting from either high salinity or water deficit induce the expression of numerous stress-responsive genes and ultimate ABA synthesis in plants . Understanded the mechanisms that regulate the expression of stress-responsive genes is a fundamental issue in basic plant biology under abiotic stresses. To understand the mechanism of CIPK14( a Ser/Thr protein kinase that associates with a calcineurin B–like calcium sensor) and EGY1(the sterol regulatory element binding proteins) genes work in stress-response, the wild-type and some T-DNA insertion mutants of Arabidopsis were used in this study, and they all are in the Columbia background. The expression pattern of stress-responsive genes in response to stress- and ABA-induced calcium signal in cipk14 was also analysed. There is no report about SREBP function in plant, so the roles of EGY1 (SREBP2) regulating the key enzyme in MEP pathway was also studied in this research. We have first emphasized the light-regulation and clock-expression pattern of CIPK14 and EGY1 genes. Moreover, we also find that anthocyanin accumulation showed a positive correlation to EGY1 in light . The results of this study were listed as follows:( 1) High salt(300mM) and ABA( 100μM) strongly induced the expression of the CIPK14 gene. the induction level of all marker genes was significantly lower or retardation in the cipk14 mutant compared with wild-type plants on both stress treatment, which indicated the stress- and ABA-inducible expression pattern of the CIPK14 gene identified CIPK14 as a stress gene, implicating it in stress and ABA signal transduction. We tested whether cipk14 mutant plants were altered in their responses to ABA and abiotic stress conditions. And found that the germination of cipk14 mutant seeds was more sensitive to the stress of ABA, NaCl, Glucose and mannitol . To investigate whether the observed inhibition of seed germination by salt, glucose and mannitol was a consequence of ABA accumulation . We tested this possibility by including the ABA biosynthesis inhibitor norflurazon. We found that the germination rate of cipk14 mutant seeds with norflurazon on the salt-containing medium was rescued , which suggests that salt exerted their effect on cipk14 germination through ABA. We varied the ABA concentration to test root elongation of cipk14 mutant. The result is consistent with stress response as described previously. We assayed the drought tolerance of wild-type and cipk14 mutant plants by withdrawing watering, which indicates the function of CIPK14 in drought response.( 2 ) When cipk14 mutant was treated using different exogenous calcium under ABA and salt, stress related gene transcription level was invariable compare with wild type, illuminating that CIPK14 mediate the calcium-regulate stress gene expression. We proposed that CIPK14 is located upstream of transcription factors and downstream of the Ca2+ signal. This putative location for CIPK14 in the signaling pathways is further supported by our study. The transcripts of CIPK14 fluctuated with different exogenous calcium treatment while RD29A appeared the same diversification in wild type, but micro scale changes were found in the ABA and salt induction of RD29A in the cipk14 mutant compare with wild type by different exogenous calcium treatment. We also showed that the seeds germination rate was regulated by exogenous calcium under ABA and salt treatment in wild type but not CIPK14 insertion mutant. Externally supplied Ca2+ reduces the toxice Vects of NaCl, which was also confirmed in our study as previously report.( 3 ) Organisms ranging from bacteria to mammals possess an endogenous mechanism that temporally organizes biochemical, cellular, and behavioral activities . These daily rhythms are produced by the circadian biological clock and are manifested by the cyclic expression of genes and gene products controlled by one or more output pathways from the clock. In Arabidopsis thaliana,transcript abundance of 6% of the genes has been shown to be under circadian clock control, moreover , 68% of circadian clock control gene were involved in stress reponse. We have first proved that CIPK14 was up-regulated by far-red light and Circadian rhythms expression. On the other hand. The seedlings of the cipk14 seedlings grown in FR failed to green before 15h in Wc. while phyA and col-4 in FR greened efficiently in 30 min, 10h during subsequent Wc illumination respectively. the transcription of POR gene in the mutant seedlings compared with phyA and col-4 by RT-PCR showed that the transcription of POR gene in cipk14 mutant was suppressed significantly. It seemed that the disruption of CIPK14 reinforced the FR irradiation blocks greening of Arabidopsis. CIPK14 exerted a negative influence on phyA-dependent repression of POR genes in FR.( 4)High concentration of sal(t 300mM) and ABA( 100μM) strongly suppressed the expression of the EGY1 gene. The induction level of all marker genes in the EGYI mutant was similar to that of wild-type plants on both stress treatment. We tested whether EGY1 mutant plants were altered in their responses to ABA and abiotic stress conditions. We found that the germination of EGY1 mutant seeds was insensitive to the stress of ABA,NaCl. drought,salt and cold stress to a certain extent resulted in ultimate induction of ABA synthesis. So we think that EGY1 (SREBP2) might regulate the key enzyme in MEP pathway for ABA and other secondary metabolism synthesis under stress.( 5) We successfully isolated an Arabidopsis co-supression mutant, srebp2-35 , which has reduced chlorophyll accumulation . To further determined the synthesize of Chlorophyll in srebp2-35, the transcripts of DXR,GGPPS1,POR genes in col-4 and srebp2-35 which grown in natural conditions for 16 day then transferred to dark for figured times by RT-PCR assay. The result showed that all genes we monitored was significantly lower or retardation in the EGYI mutant compared with wild-type plants, which indicates that EGY1 involed in stimulating the key enzyme in the MEP pathway for chlorophyll synthesis.To further determine whether EGY1 acts as transcriptional modulators of sterol-responsive genes in MEP pathway. The expression of EGY1 was detected when the two isoprenoids pathway was blocked by using the two inhibitors—MEV for MVA pathway and FSM for MEP pathway. We found that the higher concentration of inhibitor was used the more transcripts of EGY1 was observed which accorded with the results above. While, we monitored the expression of EGY1 under exterior GA3, IAA and 2, 4-D which were the end products of MEP pathway by RT-PCR assay. the transcripts of EGY1 were markedly down regulated by exterior GA3 treatment. We concluded that EGY1 might involed in regulating the key enzyme in the isoprenoids pathway, which would be studied further.( 6)We extracted anthocyanins from rosette leaves of plants grown for 25 day on different concentrations of NaCl(0, 75, 100, 125 mM). The amount of anthocyanins in col-4 increased markedly with NaCl, and the induce level was significant higher than that of mutant. Interestingly, the transcription level of PAL,CHS gene in the EGYI mutant were similar to that of wild-type plants on salt stress treatment in dark. Previously study showed that, flavonoid genes are transiently expressed during germination in a light dependent manner in developing Arabidopsis seedlings. so we concluded that EGY1 is a positive regulator in light-induced accumulation of anthocyanins.( 7)We have first proved that EGY1 displays a diurnal rhythm of trans -cription level that peaks in the middle of the light phase. Likewise, the lower expression of PAL and CHS genes in blue light was fund because of EGY1 ineffective, which indicates that EGY1 might have positive influence to flavonoid genes in blue light induction.