Histone Methyltransferase MdSUVH1 Binds to the DNA-Methylated Promoters of Drought-Responsive Genes and Activates their Expression to Enhance Drought Resistance in Apple

Abstract

Apple is one of the most widely cultivated fruit crops worldwide, yet its orchards are frequently plagued by drought exacerbated by global warming and water scarcity. Therefore, understanding how plants regulate their responses to drought at the molecular level is crucial for genetic improvement of drought resistance. Previous studies have implicated suppressor of variegation homolog (SUVH) proteins in DNA methylation recognition and chromatin-associated regulation of plant development and stress responses. However, the specific mechanisms by which SUVH proteins modulate drought tolerance by binding to methylated regions of drought-responsive genes warrant further investigation. In this study, we first characterized an apple SUVH1 protein that positively regulated drought tolerance in apple plants as demonstrated by the phenotypes of MdSUVH1 transgenic lines under drought stress. Expression analysis showed that MdSUVH1 can modulate the expression levels of drought-responsive genes in apple. Given that SUVH proteins possess DNA-binding capabilities, we hypothesize that MdSUVH1 may regulate the expression of drought-responsive genes by binding to methylated regions in their promoters. Notably, we found that two ERF transcription factor genes, MdERF1B and MdERF4, were regulated by MdSUVH1 under drought stress and that DNA methylation was present in their promoters. Electrophoretic mobility shift assays (EMSAs) showed binding of MdSUVH1 to tested methylated promoter probes, whereas Dual-LUC assays showed activation of the corresponding promoter reporters. Moreover, we found that MdERF4 could inhibit the expression of MdNAC029 and MdPP2-10B, two genes implicated in the negative regulation of drought tolerance, by suppressing the transcriptional activities of their promoters. MdERF1B can activate the expression of drought-responsive genes MdRD29A and MdRD29B. Collectively, these findings suggest that MdSUVH1 contributes to the regulation of MdERF1B and MdERF4 through methylated-promoter recognition, while these two transcription factors regulate distinct downstream drought-responsive genes that contribute to drought tolerance in apple. Our findings support a proposed regulatory model linking MdSUVH1-associated methylated-promoter recognition with ERF-mediated drought responses, providing valuable theoretical insights and genetic resources for the molecular breeding of drought-tolerant apple cultivars.