Interplay between brassinosteroids and TORC signaling in Arabidopsis revealed by integrated multi-dimensional analysis

Abstract

Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major processes coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we performed transcriptome, proteome, and phosphoproteome profiling of Arabidopsis mutants with altered levels of either BIN2 or RAPTOR1B, two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Additionally, we performed Multiplexed Assay for Kinase Specificity (MAKS), which provided a system-wide view of direct BIN2 substrates. Furthermore, phosphoproteomic data was used to reconstruct a kinase-signaling network and to identify novel proteins dependent on BR and/or TORC signaling pathways. Loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results provide genome-wide evidence for crosstalk between BR and TORC signaling and established a kinase signaling network that defines the molecular mechanisms of BR and TORC interactions in the regulation of plant growth/stress balance.

Interactions between autophagy and phytohormone signaling pathways in plants

“…The molecular connections between BR and TOR signaling provide hints as to how BR signaling regulates autophagy through regulation of TOR. Recently, a multi-omic analysis detailing transcriptome, proteome, and phosphoproteome changes in bin2 and raptor1b mutants was reported [83]. Integrated analysis revealed a significant overlap of gene products (i.e., transcript, protein, or phosphosites) that are regulated by both BIN2 and RAPTOR1B, suggesting that there is a shared regulatory core between BR and TOR signaling pathways.…”

“…Integrated analysis revealed a significant overlap of gene products (i.e., transcript, protein, or phosphosites) that are regulated by both BIN2 and RAPTOR1B, suggesting that there is a shared regulatory core between BR and TOR signaling pathways. Network analysis identified proteins that are required for both normal BR response and autophagy, which possibly participate in the balancing of plant growth and stress responses orchestrated by BIN2 and TOR [83]. From this proteome-wide dataset of BIN2, the phosphosite Serine 916 (Ser916) in RAPTOR1B was identified as potentially phosphorylated by BIN2.…”

Brassinosteroids modulate autophagy through phosphorylation of RAPTOR1B by the GSK3-like kinase BIN2 in Arabidopsis

“…We therefore hypothesized that BIN2 phosphorylates 13 proteins in the TOR pathway, thus regulating TOR activity. To determine whether BIN2 phosphorylates TOR complex components, we analyzed a previously reported proteome-wide dataset of BIN2 direct targets generated using the in vitro multiplexed assay for kinase specificity (MAKS) [66][67][68] . We also examined in vivo phosphoproteome data for sites differentially phosphorylation in bin2-D plants 66 .…”

“…To determine whether BIN2 phosphorylates TOR complex components, we analyzed a previously reported proteome-wide dataset of BIN2 direct targets generated using the in vitro multiplexed assay for kinase specificity (MAKS) [66][67][68] . We also examined in vivo phosphoproteome data for sites differentially phosphorylation in bin2-D plants 66 . In the MAKS assay, we identified the phosphopeptides TPPVpSPPR and TPPVpSPPRTNYLSGLR from RAPTOR1B, which contain the phosphosite Ser916, as significantly increased compared to the control samples with no BIN2 added (Figure 4A).…”