ZmDnaJ-ZmNCED6 module positively regulates drought tolerance via modulating stomatal closure in maize.

Heat Shock Protein plays a vital role in maintaining protein homeostasis and protecting cells from stress stimulation. As one of the HSP40 proteins, DnaJ is a stress response protein widely existing in plant cells. The function and regulatory mechanism of ZmDnaJ, a novel chloroplast-localized type-III HSP40, in maize drought tolerance were characterized. Tissue-specific expression analysis showed that ZmDnaJ is highly expressed in the leaves, and is strongly drought-induced in maize seedlings. Overexpression of ZmDnaJ improved maize drought tolerance by enhancing stomatal closure and increasing ABA content to mediate photosynthesis. In contrast, the CRISPR-Cas9 knockout zmdnaj mutant showed lower relative water content and high sensitivity to drought stress. Moreover, Y2H, BiFC and Co-IP analyses revealed that ZmDnaJ interacts with an ABA synthesis-related protein ZmNCED6 to regulate drought tolerance. Similarly, ZmNCED6 overexpressed lines showed stronger oxidation resistance, enhanced photosynthetic rate, stomatal closure and ABA content, whilst the CRISPR-Cas9 knockout mutant showed sensitive to drought stress. More importantly, ZmDnaJ could regulate key drought tolerance genes (ZmPYL10, ZmPP2C44, ZmEREB65, ZmNCED4, ZmNCED6 and ZmABI5), involved in ABA signal transduction pathways. Taken together, our findings suggest that ZmDnaJ-ZmNCED6 module improves drought tolerance in maize.