Fig. 8: Coordinated regulation of TOE1 posttranscriptionally by the age pathway and BRs during vegetative phase change.

a, b The FLAG-rTOE1 protein insensitive to miR172 level in dwf5 (a), the FLAG-sTOE1protein sensitive to miR172 level in dwf5 (b). Ubi10::3×FLAG-TOE1 or Ubi10::3×FLAG-rTOE1 were transformed into the protoplasts from Col-0 and dwf5, respectively. Total protein was extracted from transformed protoplasts and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was first normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of Col-0. The band intensity was determined using image J. All experiments were repeated 3 times biologically with similar results. c A model for BR-mediated vegetative phase change in Arabidopsis. DWF5 is the rate-limiting enzyme in the BR biosynthetic pathway. In WT, the presence of normal levels of BRs suppresses the activity of BIN2 to phosphorylate SPL9 and TOE1, thereby maintaining a normal level of SPL9 and TOE1 for plants to proceed to vegetative phase change; in dwf5 with a reduced level of BRs, more phosphorylated BIN2 is accumulated and activated to destabilize more SPL9 and TOE1 by physical interaction. This leads to the downregulation of miR172 in dwf5. As the action of the SPL9-miR172-TOE1 mode predominates over that of BIN2-TOE1, TOE1 is upregulated in dwf5, thereby reducing the expression of GL1 to delay trichome production. Therefore, dwf5 exhibited a delayed vegetative phase change phenotype. During vegetative phase change, miR156 expression declines, while the expression of SPL genes increases; likewise, miR172 expression increases, while TOE1 expression declines. Due to the high expression of miR156 at the juvenile phase, very low expression of SPL9 alleviates the repression of TOE1 by miR172, leading to over-accumulation of TOE1. In this scenario, the BIN2-TOE1 mode is initiated to safeguard a homeostatic level of TOE1 required for normal juvenile development. Therefore, BIN2 functions in a dual manner to posttranscriptionally modify SPL9 and TOE1 with opposite outcomes for TOE1. The gradient change in the color of miR156, SPL9, miR172, and TOE1 represents the gradual change in the abundance of these genes during vegetative phase change. In addition to its function to regulate SPL9 and TOE1, BIN2 also regulates vegetative phase change through affecting the interaction between BZR1 and SPL9, or other unknown pathways. c was created with BioRender.com and Microsoft Powerpoint. P phosphorylation. Molecules in gray color represents the degraded proteins.