Developmental cues are encoded by the combinatorial phosphorylation of Arabidopsis RETINOBLASTOMA-RELATED protein RBR1

RETINOBLASTOMA-RELATED (RBR) proteins orchestrate cell division, differentiation, and survival in response to environmental and developmental cues through protein-protein interactions that are governed by multi-site phosphorylation. Here we explore, using a large collection of transgenic RBR phospho-variants to complement protein function in Arabidopsis thaliana, whether differences in the number and position of RBR phosphorylation events cause a diversification of the protein's function. While the number of point mutations influence phenotypic strength, phospho-sites contribute differentially to distinct phenotypes. RBR pocket domain mutations associate primarily with cell proliferation, while mutations in the C-region are linked to stem cell maintenance. Both phospho-mimetic and a phospho-defective variants promote cell death, suggesting that distinct mechanisms can lead to similar cell fates. We observed combinatorial effects between phosphorylated T406 and phospho-sites in different protein domains, suggesting that specific, additive, and combinatorial phosphorylation events fine-tune RBR function. Suppression of dominant phospho-defective RBR phenotypes with a mutation that inhibits RBR interacting with LXCXE motifs, and an exhaustive protein-protein interaction assay, not only revealed the importance of DREAM complex members in phosphorylation-regulated RBR function, but also pointed to phosphorylation-independent RBR roles in environmental responses. Thus, combinatorial phosphorylation defined and separated developmental, but not environmental, functions of RBR.