Pengxiang Chen

Summary Apple ( Malus × domestica ) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold‐hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors ( TF s), MYB 88 and the paralogous FLP ( MYB 124), in cold stress in apple and Arabidopsis . We found that MYB 88 and MYB 124 positively regulate freezing tolerance and cold‐responsive gene expression in both apple and Arabidopsis . Chromatin‐Immunoprecipitation‐ qPCR and electrophoretic mobility shift assays showed that Md MYB 88/Md MYB 124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 ( Md CSP 3 ) and CIRCADIAN CLOCK ASSOCIATED 1 ( Md CCA 1 ) genes. Dual luciferase reporter assay indicated that Md CCA 1 but not Md CSP 3 activated the expression of Md CBF 3 under cold stress. Moreover, Md MYB 88 and Md MYB 124 promoted anthocyanin accumulation and H 2 O 2 detoxification in response to cold. Taken together, our results suggest that Md MYB 88 and Md MYB 124 positively regulate cold hardiness and cold‐responsive gene expression under cold stress by C‐ REPEAT BINDING FACTOR ( CBF )‐dependent and CBF ‐independent pathways.

promoters and thus influence expression of their target genes under drought conditions. In addition, MdMYB88 and MdMYB124 were shown to regulate the deposition of cellulose and lignin root cell walls in response to drought. Taken together, our results provide novel insights into the importance of MdMYB88 and MdMYB124 in root architecture, root xylem development, and secondary cell wall deposition in response to drought in apple trees.

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.

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.