Functional characterization of MdMYB73 reveals its involvement in cold stress response in apple calli and Arabidopsis

Recent studies have shown that the R2R3-MYB transcription factor MdMYB73 is involved in salt stress response in apple. However, no research was done whether MdMYB73 mediated cold tolerance in apple or not. In this study, we found that the expression of MdMYB73 was obviously induced by cold stress. Functional analysis showed that MdMYB73 significantly increased cold tolerance in transgenic apple calli and Arabidopsis. Quantitative real-time PCR （qRT-PCR） assay indicated that the expression levels of cold-responsive genes including MdCBF2, MdCBF3, MdCBF4, and MdCBF5 were obviously enhanced in MdMYB73 transgenic calli, suggesting that MdMYB73 increased cold tolerance via C-repeat binding factor （CBF） cold response pathway. Finally, we found that soluble sugar, which provides an osmoticum for cells, was increased in MdMYB73 transgenic calli compared to that in the wild type control. These findings provide a new insights into how MdMYB73 is involved in cold stress response.

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino acids.Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri.The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain.qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous NaCl.Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions.Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29.The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

Molecular cloning and functional identification of an apple flagellin receptor MdFLS2 gene

The leucine-rich repeat receptor kinase flagellin-sensing 2 gene(MdFLS2; Gene ID: MDP0000254112) was cloned from Royal Gala apple(Malus×domestica Borkh.). This gene contained a complete open reading frame of 3 474 bp that encoded 1 158 amino acids. The phylogenetic tree indicated that Prunus persica FLS2 exhibited the highest sequence similarity to MdFLS2. The PlantCare database suggests that the promoter sequence of MdFLS2 contains several typical cis-acting elements, including ethylene-, gibberellin-, salicylic acid-, and drought-responsive elements. Quantitative real-time PCR analysis showed that MdFLS2 was widely expressed in the different tissues of the apple and most highly expressed in the leaves. Furthermore, MdFLS2 was significantly induced by the flagellin elicitor peptide flg22. Treatment of the apple seedling leaves with flg22 resulted in an increase in leaf callose levels with increased treatment duration. An increase in the production of Oalong with the expression of disease-related genes was also observed. An oxidative burst was detected in the treated seedlings, but not in the control seedlings, indicating that flg22 had stimulated the expression of the MdFLS2 gene and its downstream target genes. Furthermore, the ectopic expression of MdFLS2 complemented the function of the Arabidopsis fls2 mutant and conferred enhanced flg22 tolerance to the transgenic Arabidopsis, suggesting that MdFLS2 acts as a positive regulator in the response to pathogens in apple.

Auxin response factor gene MdARF2 is involved in ABA signaling and salt stress response in apple

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×domestica Borkh.).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 DNAbinding 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 MdARF2 contains cis-acting elements which respond to various stresses,as well as environmental and hormonal signals.Expression analysis showed that MdARF2 is widely expressed in all tissues of apple,with the highest expression of MdARF2 in root.Functional analysis with a series of MdARF2 transgenic apple calli indicated that MdARF2 can 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.