Interfering small ubiquitin modifiers (SUMO) improves the thermotolerance of apple by facilitating the activity of MdDREB2A.

Heat stress, which is caused by global warming, threatens crops yield and quality across the world. As a kind of post-translation modification, SUMOylation involves in plants heat stress response with a rapid and wide pattern. Here, we identified small ubiquitin modifiers (SUMO), which affect drought tolerance in apple, also participated in thermotolerance. Six isoforms of SUMOs located on six chromosomes in apple genome, and all the SUMOs were up-regulated in response to heat stress condition. The MdSUMO2 RNAi transgenic apple plants exhibited higher survival rate, lower ion leakage, higher catalase (CAT) activity, and Malondialdehyde (MDA) content under heat stress. MdDREB2A, the substrate of MdSUMO2 in apple, was accumulated in MdSUMO2 RNAi transgenic plants than the wild type GL-3 at the protein level in response to heat stress treatment. Further, the inhibited SUMOylation level of MdDREB2A in MdSUMO2 RNAi plants might repress its ubiquitination, too. The accumulated MdDREB2A in MdSUMO2 RNAi plants further induced heat-responsive genes expression to strengthen plants thermotolerance, including MdHSFA3, MdHSP26.5, MdHSP18.2, MdHSP70, MdCYP18-1 and MdTLP1. In summary, these findings illustrate that interfering small ubiquitin modifiers (SUMO) in apple improves plants thermotolerance, partly by facilitating the stability and activity of MdDREB2A.

The NAC transcription factor MdNAC29 negatively regulates drought tolerance in apple.

Drought stress is an adverse stimulus that affects agricultural production worldwide. NAC transcription factors are involved in plant development and growth but also play different roles in the abiotic stress response. Here, we isolated the apple MdNAC29 gene and investigated its role in regulating drought tolerance. Subcellular localization experiments showed that MdNAC29 was localized to the nucleus and transcription was induced by the PEG treatment. Over-expression of MdNAC29 reduced drought tolerance in apple plants, calli, and tobacco, and exhibited higher relative conductivity, malondialdehyde (MDA) content, and lower chlorophyll content under drought stress. The transcriptomic analyses revealed that MdNAC29 reduced drought resistance by modulating the expression of photosynthesis and leaf senescence-related genes. The qRT-PCR results showed that overexpression of MdNAC29 repressed the expression of drought-resistance genes. Yeast one-hybrid and dual-luciferase assays demonstrated that MdNAC29 directly repressed MdDREB2A expression. Moreover, the yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that MdNAC29 interacted with the MdPP2-B10 (F-box protein), which responded to drought stress, and MdPP2-B10 enhanced the repressive effect of MdNAC29 on the transcriptional activity of the MdDREB2A. Taken together, our results indicate that MdNAC29 is a negative regulator of drought resistance, and provide a theoretical basis for further molecular mechanism research.

Fine-tuning of SUMOylation modulates drought tolerance of apple.

SUMOylation is involved in various aspects of plant biology, including drought stress. However, the relationship between SUMOylation and drought stress tolerance is complex; whether SUMOylation has a crosstalk with ubiquitination in response to drought stress remains largely unclear. In this study, we found that both increased and decreased SUMOylation led to increased survival of apple (Malus × domestica) under drought stress: both transgenic MdSUMO2A overexpressing (OE) plants and MdSUMO2 RNAi plants exhibited enhanced drought tolerance. We further confirmed that MdDREB2A is one of the MdSUMO2 targets. Both transgenic MdDREB2A OE and MdDREB2AK192R OE plants (which lacked the key site of SUMOylation by MdSUMO2A) were more drought tolerant than wild-type plants. However, MdDREB2AK192R OE plants had a much higher survival rate than MdDREB2A OE plants. We further showed SUMOylated MdDREB2A was conjugated with ubiquitin by MdRNF4 under drought stress, thereby triggering its protein degradation. In addition, MdRNF4 RNAi plants were more tolerant to drought stress. These results revealed the molecular mechanisms that underlie the relationship of SUMOylation with drought tolerance and provided evidence for the tight control of MdDREB2A accumulation under drought stress mediated by SUMOylation and ubiquitination.

The apple columnar gene candidate MdCoL and the AP2/ERF factor MdDREB2 positively regulate ABA biosynthesis by activating the expression of MdNCED6/9.

MdCoL, which encodes a putative 2OG-Fe(II) oxygenase, is a strong candidate gene for control of the columnar growth phenotype in apple. However, the mechanism by which MdCoL produces the columnar trait is unclear. Here, we show that MdCoL influences abscisic acid (ABA) biosynthesis through its interactions with the MdDREB2 transcription factor. Expression analyses and transgenic tobacco studies have confirmed that MdCoL is likely a candidate for control of the columnar phenotype. Furthermore, the ABA level in columnar apple trees is significantly higher than that in standard apple trees. A protein interaction experiment has showed that MdCoL interacts with MdDREB2. Transient expression and electrophoretic mobility shift assays have demonstrated that MdDREB2 binds directly to the DRE motif in the MdNCED6 and MdNCED9 (MdNCED6/9) gene promoters, thereby activating the transcription of these ABA biosynthesis genes. In addition, a higher ABA content has been detected following co-overexpression of MdCoL-MdDREB2 when compared with the overexpression of MdCoL or MdDREB2 alone. Taken together, our results indicate that an interaction between MdCoL and MdDREB2 promotes the expression of MdNCED6/9 and increases ABA levels, a phenomenon that may underlie the columnar growth phenotype in apple.