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.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and co-expression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

MYB30-Interacting E3 Ligase1 (MdMIEL1) plays a negative role in apple (Malus × domestica) cold tolerance by mediating the degradation of its target proteins. Its protein stability decreases under cold stress; however, how MdMIEL1 is regulated under cold conditions remains unclear. Here, we report that the α-catalytic subunit of sucrose non-fermenting-1-related protein kinase, MdKIN10, a positive regulator of cold stress, interacts with MdMIEL1, leading to its phosphorylation and subsequent degradation. Specifically, cold-activated MdKIN10 phosphorylates MdMIEL1 at serine 198, which occurs near the AIM motif; this modification further enhances the interaction between MdMIEL1 and MdATG8i, thereby promoting MdMIEL1 degradation through the autophagy pathway. Furthermore, MdBBX7, which functions as a positive regulator in apple cold stress responses by positively regulating the expression of ω-3 fatty acid desaturase (MdFAD8) and C-repeat-binding factor 2 (MdCBF2), thus promoting fatty acid desaturation and cold-responsive gene expression, is a target of MdMIEL1. In addition, MdKIN10-mediated phosphorylation of MdMIEL1 at Ser198 attenuates the ability of MdMIEL1 to inhibit apple cold tolerance and decrease MdBBX7 protein abundance in response to cold stress. Our study reveals an intricate post-translational modification cascade of MdKIN10-MdMIEL1-MdBBX7 molecular module under cold stress in apple.