Xiao-Fei Wang

The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation

The abundance of anthocyanins and proanthocyanins in apples is tightly regulated by three classes of regulatory factors, MYB, bHLH and WD40 proteins, only some of which have been previously identified. In this study, we identified an apple WD40 protein (MdTTG1) that promotes the accumulation of anthocyanins. The biosynthetic genes required downstream in the flavonoid pathway were up-regulated when MdTTG1 was over-expressed in Arabidopsis. Consistent with its role as a transcriptional regulator, an MdTTG1-GFP fusion protein was observed only in the nucleus. We assayed the expression patterns of this gene in different organs and found that they were positively correlated with anthocyanin accumulation in the apple. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that MdTTG1 interacted with bHLH transcription factors (TFs) but not MYB protein, whereas bHLH was known to interact with MYB in apples. However, based on a ChIP assay, MdTTG1 does not appear to bind to the promoter of the anthocyanin biosynthetic genes MdDFR and MdUFGT. Taken together, these results suggest that the apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.

MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple

MdSnRK1.1 regulates anthocyanin and proanthocyanidin accumulation in apple by phosphorylation and modulation of MdJAZ18 stability, explaining how jasmonic acid acts together with sucrose to regulate anthocyanin biosynthesis.

An apple NAC transcription factor negatively regulates cold tolerance via CBF-dependent pathway

Cold stress is an adverse stimulus that affects plant growth and development, and the C-repeat binding factor (CBF) cold-regulatory cascade has been regarded as a master regulator in the plant response to cold stress. Here, we showed that a NAC transcription factor modulated low-temperature tolerance. MdNAC029/MdNAP, an apple NAC gene was isolated and its role in regulating cold tolerance was investigated. MdNAC029 was responsive to low-temperature treatment, and over-expression of MdNAC029 reduced cold tolerance in apple calli and Arabidopsis. Furthermore, EMSA assays and transient expression assays demonstrated that MdNAC029 directly repressed the expression of MdCBF1 and MdCBF4 by binding to their promoters. Taken together, our data suggest that MdNAC029 functions as a negative regulator in regulating plant cold tolerance in a CBF-dependent manner, providing a deeper understanding of NAC transcription-factor-mediated cold tolerance.

MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple

Cold stress is a major external stimulator that affects crop quality and productivity. The CBF cold regulatory pathway has been regarded as a master regulator in the response to cold stress. In this study, we found that the apple bZIP transcription factor, MdHY5, was responsive to cold treatment both at the transcriptional and at the post-translational levels. Moreover, overexpression of MdHY5 enhanced cold tolerance in apple calli and Arabidopsis. Subsequently, EMSA assay and transient expression assay demonstrated that MdHY5 positively regulated the transcript of MdCBF1 by binding to G-Box motif of its promoter. Furthermore, MdHY5 also regulated the expression of CBF-independent cold-regulated genes. Taken together, our data suggest that MdHY5 positively modulates plant cold tolerance through CBF-dependent and CBF-independent pathways, providing a deeper understanding of MdHY5-regulated cold tolerance in apple.

An apple NAC transcription factor enhances salt stress tolerance by modulating the ethylene response

It is known that ethylene signaling is involved in the regulation of the salt stress response. However, the molecular mechanism of ethylene-regulated salt stress tolerance remains largely unclear. In this study, an apple NAM ATAF CUC transcription factor, MdNAC047, was isolated and functionally characterized to be involved in ethylene-modulated salt tolerance. MdNAC047 gene was significantly induced by salt treatment and its overexpression conferred increased tolerance to salt stress and facilitated the release of ethylene. Quantitative real-time-PCR analysis demonstrated that overexpression of MdNAC047 increased the expression of ethylene-responsive genes. Electrophoretic mobility shift assay, yeast one-hybrid and dual-luciferase assays suggested that MdNAC047 directly binds to the MdERF3 (ETHYLENE RESPONSE FACTOR) promoter and activates its transcription. In addition, genetic analysis assays indicated that MdNAC047 regulates ethylene production at least partially in an MdERF3-dependent pathway. Overall, we found a novel MdNAC047-MdERF3-ethylene-salt tolerance regulatory pathway, which provide new insight into the link between ethylene and salt stress.

Apple RING E3 ligase MdMIEL1 inhibits anthocyanin accumulation by ubiquitinating and degrading MdMYB1 protein

MdMYB1 is an important regulator for anthocyanin accumulation in apple (Malus × domestica). Here, an apple RING E3 ligase, MdMIEL1, was screened out as a partner of MdMYB1 with a yeast two-hybrid approach. Pull-down, bimolecular fluorescence complementation and coimmunoprecipitation assays further verified the interaction between MdMIEL1 and MdMYB1 proteins. Subsequently, in vitro and in vivo experiments indicated that MdMIEL1 functioned as a ubiquitin E3 ligase to ubiquitinate MdMYB1 protein, followed by degradation through a 26S proteasome pathway. Furthermore, transgenic studies in apple calli and Arabidopsis demonstrated that MdMIEL1 negatively regulated anthocyanin accumulation by modulating the degradation of MdMYB1 protein. Taken together, our findings provide a new insight into the molecular mechanism by which MdMIEL1 negatively regulates anthocyanin biosynthesis by ubiquitinating and degrading MdMYB1 protein.

Ectopic expression of an apple cytochrome P450 gene MdCYPM1 negatively regulates plant photomorphogenesis and stress response in Arabidopsis

Cytochrome P450s play an important role in plant growth and are involved in multiple stresses response. However, little is known about the functions of cytochrome P450s in apple. Here, a Malusxa0×xa0domestica cytochrome P450 monooxygenase 1 gene, MdCYPM1, was identified and subsequently cloned from apple Gala (Malusxa0×xa0domestica). To verify the functions of MdCYPM1, we generated transgenic Arabidopsis plants expressing the apple MdCYPM1 gene under the control of the Cauliflower mosaic virus 35S promoter. Four transgenic lines (#3, #5, #7 and #8) were selected for further study. The transgenic plants exhibited a series of skotomorphogenesis phenotypes relative to wild-type controls, such as reduction of the chlorophyll, anthocyanins content and hypocotyls elongation. In addition, overexpression of MdCYPM1 influenced auxin transport and flowering time in transgenic Arabidopsis. Furthermore, MdCYPM1 expression was induced by salt and mannitol treatments, and the transgenic plants were negatively regulated by salinity and osmotic stresses during germination. These results suggest that MdCYPM1 plays a vital role in plant growth and development.

Molecular cloning and functional characterization of MdPIN1 in apple

Abstract 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.

BTB protein MdBT2 inhibits anthocyanin and proanthocyanidin biosynthesis by triggering MdMYB9 degradation in apple

MdMYB9 is a positive regulator in the biosynthesis of anthocyanin and proanthocyanidin in apple. However, its posttranslational regulation is unclear. Here, we demonstrated that the BTB protein MdBT2 had a negative role in the biosynthesis of anthocyanin and proanthocyanidin. MdBT2 interacted with MdMYB9 and negatively regulated the abundance of MdMYB9 protein through the 26S proteasome system. The degradation of MdMYB9 by MdBT2 reduced the expression of MdMYB9-mediated anthocyanin and proanthocyanidin-related genes and reduced the accumulation of anthocyanin and proanthocyanidin, which functioned in an MdCUL3-independent pathway. Our results indicated that MdBT2 negatively regulated the stability of MdMYB9, which provides new insight into the homeostasis of anthocyanin and proanthocyanidin in apple.

Genome-wide analysis and identification of the SMXL gene family in apple ( Malus × domestica )

Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants.