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 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 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.

Plant peptide hormones play important roles in plant growth and development.  Among these hormones, the C-TERMINALLYENCODED PEPTIDE (CEP) belongs to a newly found peptide family that regulates root development in Arabidopsis as well as in other species.  However, nothing is known about the CEP genes in apple (Malus×domestica, MdCEP).  In this study, a total of 27 apple CEP genes were identified through a genome-wide analysis and were phylogenetically divided into three classes (I, II and III).  The predicted MdCEP genes were distributed across 10 of 17 chromosomes with different densities.  Next, the gene structures and motif compositions of the MdCEP genes were analyzed.  Subsequently, the expression analysis suggested that the MdCEP genes were highly activated in roots and that MdCEP23 may play an important role in regulating the growth and development of roots.  Moreover, all of the MdCEP genes were responsive to multiple abiotic stresses, indicating that MdCEP genes may be involved with various aspects of physiological processes in apple.  Nearly one-third of MdCEP genes had a significant response to low nitrogen treatment.  Most of the MdCEP genes were up-regulated under stress, including mannitol, polyethylene glycol (PEG) and abscisic acid (ABA), suggesting that MdCEP genes may be involved in the drought stress response.  This study provides insight into the putative functions of the MdCEP genes using a genome-wide analysis of the CEP gene family. 

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.

Auxin has been identified to play critical roles in regulating plant growth and development.  The polar transport of auxin is regulated by auxin transporters.  In the present study, an auxin efflux carrier gene MdPIN1 was cloned from Malus×domestic, Royal Gala, and introduced into wild-type Arabidopsis thaliana (Col-0).  The transgenic plants exhibited the phenotype of inhibition of primary root (PR) elongation and increased lateral root (LR) number in compared with Col-0.  Overexpression of MdPIN1 affected auxin transport, and enhanced phototropic responses and geotropism reaction, whereas had no significant difference in the auxin biosynthesis.  These findings suggest that the MdPIN1 gene plays a vital role in auxin transport and root development.