DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLA acts as a transcriptional regulator and is recruited to target promoters by binding to transcription factors (TFs) and histone H2A through its GRAS domain. Recent studies have shown that DELLA stability is post-translationally regulated through two mechanisms: polyubiquitination induced by the phytohormone gibberellin, which leads to its rapid degradation, and conjugation of small ubiquitin-like modifiers (SUMO) to increase its accumulation. In addition, DELLA activity is dynamically regulated by two different glycosylations: the DELLA-TF interaction is enhanced by O-fucosylation but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear, as previous studies have shown conflicting results, ranging from those showing that phosphorylation promotes or reduces DELLA degradation to others showing that phosphorylation does not affect its stability. Here, we identify phosphorylation sites in REPRESSOR ga1-3 (RGA, AtDELLA) purified from Arabidopsis thaliana by mass spectrometry analysis and show that phosphorylation of two RGA peptides at the PolyS and PolyS/T regions promotes H2A binding and enhanced RGA activity. Association of RGA with target promoters. Notably, phosphorylation does not affect RGA-TF interactions or RGA stability. Our study reveals the molecular mechanism by which phosphorylation induces DELLA activity.
To elucidate the role of phosphorylation in regulating DELLA function, it is critical to identify DELLA phosphorylation sites in vivo and perform functional analyzes in plants. By affinity purification of plant extracts followed by MS/MS analysis, we identified several phosphosites in RGA. Under conditions of GA deficiency, RHA phosphorylation increases, but phosphorylation does not affect its stability. Importantly, co-IP and ChIP-qPCR assays revealed that phosphorylation at the PolyS/T region of RGA promotes its interaction with H2A and its association with target promoters, revealing the mechanism by which phosphorylation induces RGA function.
RGA is recruited to target chromatin through the interaction of the LHR1 subdomain with TF and then binds to H2A through its PolyS/T region and PFYRE subdomain, forming the H2A-RGA-TF complex to stabilize RGA. Phosphorylation of Pep 2 in the PolyS/T region between the DELLA domain and the GRAS domain by an unidentified kinase enhances RGA-H2A binding. The rgam2A mutant protein abolishes RGA phosphorylation and adopts a different protein conformation to interfere with H2A binding. This results in destabilization of transient TF-rgam2A interactions and dissociation of rgam2A from target chromatin. This figure depicts only RGA-mediated transcriptional repression. A similar pattern could be described for RGA-mediated transcriptional activation, except that the H2A-RGA-TF complex would promote target gene transcription and dephosphorylation of rgam2A would decrease transcription. Figure modified from Huang et al.21.
All quantitative data were statistically analyzed using Excel, and significant differences were determined using Student’s t test. No statistical methods were used to preliminarily determine the sample size. No data were excluded from the analysis; the experiment was not randomized; the researchers were not blind to the distribution of data during the experiment and the evaluation of the results. The sample size is indicated in the figure legend and source data file.
For more information about the study design, see the Natural Portfolio Report Abstract associated with this article.
Mass spectrometry proteomics data have been contributed to the ProteomeXchange consortium through the PRIDE66 partner repository with dataset identifier PXD046004. All other data obtained during this study are presented in the Supplementary Information, Supplementary Data Files, and Raw Data Files. Source data is provided for this article.
Post time: Nov-08-2024