Functional Analyses Of BZIP Transcription Factor HY5 In Ethylene Biosynthesis And Signaling Of Arabidopsis

Plants usually experience many biotic and abiotic stresses including cold, drought, salt and pathogen attack in the life cycles. Effcient mechanisms that plants respond to salt stress are gradually disclosed including ethylene and abscisic acid (ABA) signaling pathways. In the present investigation, three ethylene overproduction (eto) mutants and overexpressing ACS5 transgenic lines (OEACS5) exhibited increased seed germination and green cytoledon rate by enhanced ethylene biosynthesis under salt stress. The seed germination and green cytoledon rate were suppressed by ethylene biosynthesis inhibitor AVG and ethylene perception inhibitor AgNO3. The results of microarray and Real-time PCR showed that the expression level of seed germination regulatory gene ABI5 was decreased in eto mutants and OEACS5 transgenic lines, in contrast, the transcripts of ABI5 were increased in the downstream compounds of ethylene signal pathway mutants ein2 and ein3-1 compared to Col-0 did. HY5 was reported as the direct regulator of ABI5, and the inverstigation showed that hy5 increased salt tolerance in seed germination and early seedling growth. Compared to eto2, seed germination and the green cotyledon was reduced under salt stress in hybrid eto2×35S::HY5, but ein2 hy5 double mutant rescued the green cotyledon of ein2 to the level of Col-0 under salt stress, and the seed germination rate was higher in etr1-1 hy5 and ein3-1 hy5 double mutants than in Col-0 and single mutant etr1-1and ein3-1. These results showed that HY5 was affected by ACC via ethylene signal pathway, and the expression of ABI5 was reduced subsequently with the decreased HY5 content, and then enhanced the seed germination under salt stress.Further analyses showed that the ethylene emission of hy5 displayed 1.5-fold higher than Col-0; in contrast, there was no significant difference in ethylene production between Col-0 and 35S::HY5. The results of Real-time PCR showed that the expression of ACS2 and ACS5 was higher in hy5 mutant than this in Col-0 and the transcripts were little affected by exogenous ABA treatment, but the expression of these two genes were decreased in Col-0 after ABA treatment for 4 h. EMSA (Electrophoretic mobility shift assay) and ChIP (Chromatin immunoprecipitation) results showed that HY5 interacted with the G-box in the promoter of transcriptional repressor AtERF11 and regulated the expression. It was found that erf11 exhibited inhibited hypocotyl length response to 10μM ACC in ethylene"triple response"assay. Exogenous ABA caused a half reduction in ACS2 and ACS5 transcript levels in Col-0, but the levels in erf11 were little affected. EMSA and ChIP assay indicated that AtERF11 bound to the DRE in the promoters of ACS2 and ACS5 to repress its expression. Exogenous ABA slightly inhibited primary root growth in Col-0, however, primary root length in acs2-1, acs5-1and acs5-2 were decreased significantly, while acs2 was more sensitive in response to ABA in shoot growth. In addition ABA decreased shoot weight of Col-0 and acs5. Intresetingly, heptuple mutant, which corresponds to ~25% of the ethylene production compared to Col-0 plants, showed ABA tolerance in primary root length but ABA sensitivity in lateral root growth. Unexpectly, acs4-1, which increased ~40% ethylene emission than Col-0 did, showed ABA tolerance in primary root length. These inverstigation revealed that the interaction of ABA and ethylene might exist different integrations. In contrast, three eto mutants increased primary root length, whereas the data of latral root number and shoot weight after ABA treatment elaborated that exogenous ABA partly rescued the inhibition of ethylene in eto mutant seedling development and root elongation. Taken together we conclude that ABA represses ethylene biosynthesis at least in part through negative transcriptional regulation and HY5-AtERF11 regulon plays crucial role in this progress.