The E3 ubiquitin ligase MIEL1 promotes thermo-responsive growth by blocking auto-ubiquitination and self-degradation of XBAT31

Global warming poses a serious threat to plant growth, development, morphology, and yield quality. Among those effects, mild warm temperature acts as a signal that promotes morphological changes in plants, a phenomenon known as thermomorphogenesis. In the model plant Arabidopsis thaliana, hypocotyl elongation serves as a primary research indicator, establishing a regulatory network centered on the transcription factor PIF4. Thermosensors (such as ELF3, phyB, and PIF7) convey warm temperature signals to PIF4, which in turn activates the expression of auxin-regulated genes, leading to hypocotyl elongation. However, the upstream regulatory mechanisms of plants precisely perceive and transmit mild warm signals to achieve adaptive growth in harmony with nature, and remain poorly understood.

Previous studies found that the E3 ligase XBAT31 is a positive regulator through ubiquitination and further degradation of ELF3, thereby relieving ELF3’s inhibitory effect on the transcriptional activity of PIF4 in thermomorphogenesis. In this study, the researchers identified another E3 ligase, MIEL1, by yeast two-hybrid screen. The study confirmed that MIEL1 interacts with both the full-length and the N-terminal domain of XBAT31 in vitro and in vivo. Phenotypic analysis revealed that MIEL1 positively mediates thermoresponsive hypocotyl elongation. Both MIEL1 and XBAT31 function upstream of the ELF3 signaling pathway.

To elucidate the regulatory relationship between these two E3 ligases, the research team found that mutation of MIEL1 leads to obviously lower XBAT31 protein levels, whereas mutation of XBAT31 has no effect on MIEL1 protein levels. In vitro ubiquitination assays confirmed that XBAT31 and MIEL1 cannot ubiquitinate each other, suggesting that MIEL1 likely stabilizes XBAT31 through an alternative mechanism. Further research demonstrated that XBAT31 is capable of self-interaction in vitro and in vivo, and that this interaction mediates XBAT31N’s auto-ubiquitination and subsequent degradation. The study identified eight key auto-ubiquitination sites. Mutation of these sites inhibited the degradation of the XBAT31N protein in the MIEL1 mutant background. Additionally, ubiquitination levels of XBAT31N increased in the MIEL1 mutant. How does MIEL1 participate in the XBAT31 auto-ubiquitination process to influence protein abundance? 3D structural modeling predictions and yeast three-hybrid results indicated that MIEL1 can effectively inhibit the interaction between XBAT31 molecules, thereby suppressing XBAT31’s auto-ubiquitination and subsequent degradation, ultimately maintaining XBAT31 protein stability.

In summary, two E3 ligases cooperatively and hierarchically regulate thermoresponsive growth to adapt to ambient high temperature. This study reveals the molecular mechanism by which the E3 ligase MIEL1 inhibits XBAT31 auto-ubiquitination in thermomorphogenesis, expanding our understanding of the regulatory roles of two E3 ligases and the construction of upstream signaling networks. Research on the E3 ligases MIEL1 and XBAT31 contributes to the development of crops better adapted to global warming, providing genetic targets for crop biotechnology breeding and aiding in the cultivation of new heat-tolerant varieties.