Functional Characterization Of MsDREB6.2 Transcription Factor From (Malus Sieversii Roem.) In Response To Drought Stress

Plants are continually exposed to ever-changing environmental stresses, such as drought, high salinity, and extreme temperatures, which limit their growth and productivity throughout their life cycle. Dehydration-responsive element binding factors (DREBs) play important roles in plant growth, development, and stress signaling pathways in model plants. Most studies of DREB to date have focused on DREB Is, DREB2s, and DREB5s, while knowledge of the DREBA-6 subgroup is limited. We isolated a DREB gene from Malus sieversii (Ledeb.) Roem., one of the most drought tolerant apple rootstocks in China, a MsDREB6.2, and investigated its functions in response to drought stress. Some main results have been achieved as follows:1. We searched for DREBA6 subgroup members in the M. sieversii genome by conducting a BLAST search against the apple sequence database, and identified 10 putative DREB6 genes from M. sieversii. One of the most drought-responsive genes, MsDREB6.2, was selected for further analysis. The expression of MsDREB6.2 was highest in young roots, followed by mature roots, and was strongly reduced by high sality. Subcellular localization analyses revealed that MsDREB6.2 was targeted to the nucleus and MsDREB6.2 protein showed transcriptional activation ability in the (3-galactosidase assay.2. To investigate the function of MsDREB6.2 in apple plants, we overexpressed this gene in a rootstock variety (M26) of apple. We also generated transgenic M26 plants by taking advantage of chimeric repressor gene-silencing technology (CRES-T). Overexpression of MsDREB6.2 results in cytokinin-deficient developmental phenotypes in transgenic M26 plants. On the contrary, suppression of MsDREB6.2 partly eliminated the reduction in plant height, but the root growth of CRES-T line plants was retarded. In this study, overexpression and suppression of MsDREB6.2 resulted in a significant decrease and increase, respectively, in the four kinds of CK metabolites analyzed in the roots of 35S:MsDREB6.2 and 35S:MsDREB6.2SRDX plants. No significant differences were found in the expression of IPT genes between transgenic and control plants. On the other hand, compared with the control plants, MdCKX4a expression was markedly increased and decreased, respectively, in the root of 35S:MsDREB6.2 and 35S:MsDREB6.2SRDX plants. EMSA and ChIP experiments showed that MsDREB6.2 specifically binds to the DRE present in the promoter region of MdCKX4a, in vitro and in vivo, respectively. Our results suggest that overexpression of MsDREB6.2 increased the expression of a key cytokinin (CK) catabolism gene, MdCKX4a, which led to a significant reduction in endogenous CK levels, and promoted root formation in transgenic M26 plants.3. Overexpression of MsDREB6.2 in M26 plants increases tolerance to drought stress. However, the 35S:MsDREB6.2SRDX plants were more sensitive to drought stress compared with control plants. Under drought stress,35S:MsDREB6.2 and 35S:MsDREB6.2SRDX plants exhibited a significant decrease and increase in the stomatal apertures compared with control plants. Overexpression and suppression of MsDREB6.2 altered the expression of some CK-signalling genes in transgenic M26 plants. Furthermore, benzylaminopurine (BA) sprayed on leaves increased stomatal apertures of transgenic and control plants. In addition, the expression of MdHK2, MdHK3a, and MdHK4a were significantly upregulated in all plants after BA treatment, suggestting that the CK-signalling genes might play important roles in the MsDREB6.2-mediated decrease in stomatal apertures.4. Compared with control plants,35S:MsDREB6.2 and 35S:MsDREB6.2SRDX plants exhibited a distinct increase and decrease, respectively, in Lo under drought stress conditions. The qRT-PCR results indicate that MsDREB6.2 is a positive regulator of MdPIPl;3 and Mdy-TIP expression. EMSA showed that MsDREB6.2 bound specifically to the DRE in the promoter of both of MdPIPl;3 and Mdy-TIP in vitro. The effect of the two aquaporin (AQP) genes on Lo was further characterized using the AQP inhibitor HgCl2. These results suggest that up-regulation of MdPIP1;3 and Mdy-TIP participates in the MsDREB6.2-mediated increase in Lo under drought stress.In summary, MsDREB6.2 plays a positive role in response to drought strss in apple plant. On the one hand, MsDREB6.2 increased the expression of a key cytokinin (CK) catabolism gene, MdCKX4a, which led to a significant reduction in endogenous CK levels, and promoted root formation in tapple plants, and enhances the expression of two AQP genes, MdPIP1;3 and Mdy-TIP. The enlarged root systems and the up-regulation of AQP genes might together maintain the Lo at a high value under drought stress, thereby facilitating optimal water uptake and rapid recovery at the rewatering stage. On the other hand, MsDREB6.2 reduces stomatal aperture and density, which results in lower water loss rates in apple plants under drought stress, the CK-signalling genes might play important roles in the MsDREB6.2-mediated decrease in stomatal apertures.