Apple E3 ligase MdPUB23 mediates ubiquitin-dependent degradation of MdABI5 to delay ABA-triggered leaf senescence.

ABSCISIC ACID-INSENSITIVE5 (ABI5) is a core regulatory factor that mediates the ABA signaling response and leaf senescence. However, the molecular mechanism underlying the synergistic regulation of leaf senescence by ABI5 with interacting partners and the homeostasis of ABI5 in the ABA signaling response remain to be further investigated. In this study, we found that the accelerated effect of MdABI5 on leaf senescence is partly dependent on MdbHLH93, an activator of leaf senescence in apple. MdABI5 directly interacted with MdbHLH93 and improved the transcriptional activation of the senescence-associated gene MdSAG18 by MdbHLH93. MdPUB23, a U-box E3 ubiquitin ligase, physically interacted with MdABI5 and delayed ABA-triggered leaf senescence. Genetic and biochemical analyses suggest that MdPUB23 inhibited MdABI5-promoted leaf premature senescence by targeting MdABI5 for ubiquitin-dependent degradation. In conclusion, our results verify that MdABI5 accelerates leaf senescence through the MdABI5-MdbHLH93-MdSAG18 regulatory module, and MdPUB23 is responsible for the dynamic regulation of ABA-triggered leaf senescence by modulating the homeostasis of MdABI5.

MdMYB10 affects nitrogen uptake and reallocation by regulating the nitrate transporter MdNRT2.4-1 in the red flesh apple.

Nitrate is the major nitrogen sources for higher plants. In addition to serving not only as a nutrient, it is also a signaling molecule that regulates plant growth and development. Although membrane-bound nitrate transporter/peptide transporters (NRT/PTR) have been extensively studied and shown to regulate nitrate uptake and movement, little is known about how these factors are regulated by the external nitrogen environment. Red flesh apple, the coloration of which is determined by the transcription factor MdMYB10, had higher nitrate uptake efficiency than non-red flesh apple. Nitrate assimilation and utilization were increased in red flesh apple cultivar, and comparative transcriptome analysis showed that the expression of genes encoding the NRT2s was increased in red flesh apple. In vitro and in vivo experiments showed that MdMYB10 directly bound to the MdNRT2.4-1 promoter to transcriptionally activate its expression, resulting in enhanced nitrate uptake. MdMYB10 also controlled nitrate reallocation from old leaves to new leaves through MdNRT2.4-1. Overall, our findings provide novel insights into the mechanism by which MdMYB10 controls nitrate uptake and reallocation in apple, which facilitates adaptation to low nitrogen environment.

Functional identification of apple MdMYB2 gene in phosphate-starvation response.

Inorganic phosphate (Pi) starvation severely affects the normal growth and development of plants. Here, a Pi-responsive gene, named MdMYB2 (MDP0000823458), was cloned and functionally identified in apple. Overexpression of MdMYB2 regulated the expression of Pi starvation-induced (PSI) genes and then promoted phosphate assimilation and utilization. The ectopic expression of MdMYB2 in Arabidopsis influenced plant growth and flowering, which was partially rescued by application of exogenous gibberellin (GA). These results indicated that MdMYB2 may be an essential regulator in phosphate utilization and GA-regulated plant growth and development.

EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 Act in a Regulatory Loop That Synergistically Modulates Ethylene Biosynthesis and Anthocyanin Accumulation.

Ethylene regulates climacteric fruit ripening, and EIN3-LIKE1 (EIL1) plays an important role in this process. In apple (Malus domestica), fruit coloration is accompanied by ethylene release during fruit ripening, but the molecular mechanism that underlies these two physiological processes is unknown. In this study, we found that ethylene treatment markedly induced fruit coloration as well as the expression of MdMYB1, a positive regulator of anthocyanin biosynthesis and fruit coloration. In addition, we found that MdEIL1 directly bound to the promoter of MdMYB1 and transcriptionally activated its expression, which resulted in anthocyanin biosynthesis and fruit coloration. Furthermore, MdMYB1 interacted with the promoter of ETHYLENE RESPONSE FACTOR3, a key regulator of ethylene biosynthesis, thereby providing a positive feedback for ethylene biosynthesis regulation. Overall, our findings provide insight into a mechanism involving the synergistic interaction of the ethylene signal with the MdMYB1 transcription factor to regulate ethylene biosynthesis and fruit coloration in apple.

The molecular cloning and functional characterization of MdMYC2, a bHLH transcription factor in apple.

The basic helix-loop-helix (bHLH) Leu zipper transcription factor MYC2 is an important regulator in the Jasmonic acid (JA) signaling pathway. In this study, the apple MdMYC2 gene was isolated and cloned on the basis of its homology with Arabidopsis thaliana MYC2. Quantitative real time PCR (qRT-PCR) analysis demonstrated that MdMYC2 transcripts were induced by Methyl Jasmonate (MeJA) treatment and wounding. The MdMYC2 protein interacted with itself and bound the G-Box motif of the AtJAZ3 gene. MdMYC2 interacted with the MdJAZ2 protein, which is a repressor protein in the JA signaling pathway. Furthermore, we obtained transgenic apple calli that either overexpressed or suppressed the MdMYC2 gene. Expression analysis with qRT-PCR demonstrated that the transcript levels of JA-regulated anthocyanin biosynthetic genes, such as MdDFR, MdUF3GT, MdF3H and MdCHS, were markedly up-regulated in the MdMYC2 overexpressing calli and down-regulated in the suppressing calli compared with the WT control. As a result, the overexpressing calli produced more anthocyanin, and the suppressing calli produced less. Finally, the MdMYC2 gene was ectopically expressed in Arabidopsis. Both phenotypic investigation and expression analysis demonstrated that the MdMYC2 transgenic Arabidopsis lines were more sensitive to MeJA than the WT control. Together, these results indicate that the apple MdMYC2 gene plays a vital role in the JA response.