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Auxin response factors (ARFs) play key roles throughout the whole process of plant growth and development, and mediate auxin response gene transcription by directly binding with auxin response elements (AuxREs). However, their functions in abiotic stresses are largely limited, especially in apples. Here, the auxin response factor gene MdARF2(HF41569) was cloned from apple cultivar ‘Royal Gala’ (Malus×domesticaBorkh.). Phylogenetic analysis showed that ARF2 proteins are highly conserved among different species and MdARF2 is the closest relative to PpARF2 of Prunus persica, but they differ at the DNA level. MdARF2 contains three typical conserved domains including the B3 DNA-binding domain, Auxin_resp domain and AUX_IAA domain. The subcellular localization demonstrated that MdARF2 is localized in the nucleus. The three-dimensional structure prediction of the proteins showed that MdARF2 is highly similar with AtARF2, and they contain helices, folds, and random coils. The promoter of MdARF2contains cis-acting elements which respond to various stresses, as well as environmental and hormonal signals. Expression analysis showed that MdARF2is widely expressed in all tissues of apple, with the highest expression of MdARF2in root. Functional analysis with a series of MdARF2transgenic apple calli indicated that MdARF2can reduce the sensitivity to ABA signaling and enhance salt tolerance in apple. In summary, the results of this research provide a new basis for studying the regulation of abiotic stresses by ARFs.

ABSTRACTAuxin response factors (ARFs) mediate auxin response gene transcription by directly binding with AuxREs. However, whether ARF family genes involved in anthocyanin accumulation in apple is largely unexplored. In this study, thirty-three MdARF family members were identified in the apple (Malus × domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with ARF family members of Arabidopsis. Phylogenetic analysis revealed that thirty-three MdARF members were clustered into Class I, Class II and Class III. Functional domain analysis showed that MdARF proteins have some typical domains such as B3-like DNA-binding domain and Auxin_resp domain, as well as motif III and motif IV. Chromosome localisation analysis demonstrated that apple ARF members were widely distributed in the genome. Among these MdARFgenes, MdARF2was significantly correlated with anthocyanin variation according to extracting expression profiles from published RNA-seq data. Further functional identification in apple calli suggested that MdARF2 functions as a transcriptional repressor of anthocyanin biosynthesis. These findings provide evidence that MdARF family members play a role in the regulation of anthocyanin accumulation in apple.

MYB transcription factors (TFs) participate in many biological processes. However, the molecular mechanisms by which MYB TFs affect plant resistance to apple ring rot remain poorly understood. Here, the R2R3-MYB gene MdMYB73was cloned from “Royal Gala” apples and functionally characterized as a positive regulator of the defense response to Botryosphaeria dothidea. qRT-PCR and GUS staining demonstrated that MdMYB73was strongly induced in apple fruits and transgenic calli after inoculation with B. dothidea. MdMYB73overexpression improved resistance to B. dothideain apple calli and fruits, while MdMYB73suppression weakened. Increased resistance to B. dothideawas also observed in MdMYB73-expressing Arabidopsis thaliana. Interestingly, salicylic acid (SA) contents and the expression levels of genes related with SA synthesis and signaling were greater in MdMYB73-overexpressing plant materials compared to wild-type controls after inoculation, suggesting that MdMYB73 might enhance resistance to B. dothideaviathe SA pathway. Finally, we discovered that MdMYB73 interacts with MdWRKY31, a positive regulator of B. dothidea. Together, MdWRKY31 and MdMYB73 enhanced B. dothidearesistance in apples. Our results clarify the mechanisms by which MdMYB73 improves resistance to B. dothideaand suggest that resistance may be affected by regulating the SA pathway.