Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP⁺ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP⁺ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH–MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

Water deficit is one of the main challenges for apple (Malus × domestica) growth and productivity. Breeding drought-tolerant cultivars depends on a thorough understanding of the drought responses of apple trees. Here, we identified the zinc-finger protein B-BOX 7/CONSTANS-LIKE 9 (MdBBX7/MdCOL9), which plays a positive role in apple drought tolerance. The overexpression of MdBBX7 enhanced drought tolerance, whereas knocking down MdBBX7 expression reduced it. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified one cis-element of MdBBX7, CCTTG, as well as its known binding motif, the T/G box. ChIP-seq and RNA-seq identified 1,197 direct targets of MdBBX7, including ETHYLENE RESPONSE FACTOR (ERF1), EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), and GOLDEN2-LIKE 1 (GLK1) and these were further verified by ChIP-qPCR and electronic mobility shift assays. Yeast two-hybrid screen identified an interacting protein of MdBBX7, RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1). Further examination revealed that MdMIEL1 could mediate the ubiquitination and degradation of MdBBX7 by the 26S proteasome pathway. Genetic interaction analysis suggested that MdMIEL1 acts as an upstream factor of MdBBX7. In addition, MdMIEL1 was a negative regulator of the apple drought stress response. Taken together, our results illustrate the molecular mechanisms by which the MdMIEL1–MdBBX7 module influences the response of apple to drought stress.

Shotgun metagenomics has become a pivotal technology in microbiome research, enabling in‐depth analysis of microbial communities at both the high‐resolution taxonomic and functional levels. This approach provides valuable insights of microbial diversity, interactions, and their roles in health and disease. However, the complexity of data processing and the need for reproducibility pose significant challenges to researchers. To address these challenges, we developed EasyMetagenome, a user‐friendly pipeline that supports multiple analysis methods, including quality control and host removal, read‐based, assembly‐based, and binning, along with advanced genome analysis. The pipeline also features customizable settings, comprehensive data visualizations, and detailed parameter explanations, ensuring its adaptability across a wide range of data scenarios. Looking forward, we aim to refine the pipeline by addressing host contamination issues, optimizing workflows for third‐generation sequencing data, and integrating emerging technologies like deep learning and network analysis, to further enhance microbiome insights and data accuracy. EasyMetageonome is freely available at https://github.com/YongxinLiu/EasyMetagenome.

DNA-binding one zinc-finger (Dof) proteins constitute a family of transcription factors with a highly conserved Dof domain that contains a C2C2 zinc-finger motif. Although several studies have demonstrated that Dof proteins are involved in multiple plant processes, including development and stress resistance, the functions of these proteins in drought stress resistance are largely unknown. Here, we report the identification of the MdDof54 gene from apple and document its positive roles in apple drought resistance. After long-term drought stress, compared with nontransgenic plants, MdDof54 RNAi plants had significantly shorter heights and weaker root systems; the transgenic plants also had lower shoot and root hydraulic conductivity, as well as lower photosynthesis rates. By contrast, compared with nontransgenic plants, MdDof54-overexpressing plants had higher photosynthesis rates and shoot hydraulic conductivity under long-term drought stress. Moreover, compared with nontransgenic plants, MdDof54-overexpressing plants had higher survival percentages under short-term drought stress, whereas MdDof54 RNAi plants had lower survival percentages. MdDof54 RNAi plants showed significant downregulation of 99 genes and significant upregulation of 992 genes in response to drought, and 366 of these genes were responsive to drought. We used DAP-seq and ChIP-seq analyses to demonstrate that MdDof54 recognizes cis-elements that contain an AAAG motif. Taken together, our results provide new information on the functions of MdDof54 in plant drought stress resistance as well as resources for apple breeding aimed at the improvement of drought resistance.