The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

Abstract

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes

“…However, how a single protein kinase could act as a master regulator to incorporate various upstream signals to modulate a myriad of cellular activities remains largely unknown. In contrast to recent progress in discovering the downstream effectors of plant TOR signaling [e.g., S6K1, S6K2, E2Fa, E2Fb, RPS6, BZR1, BIN2 (19,22,27,44,45,47)], the upstream regulators of plant TOR signaling remain poorly defined. The small GTPases RHEB and RAG function as the key upstream regulators for mTOR activation in mammalians, but no orthologs of RHEB or RAG GTPases have been identified in plants (18,19).…”

Reciprocal regulation of photosynthesis and mitochondrial respiration by TOR kinase in Chlamydomonas reinhardtii

“…Numerous studies employing TOR kinase inhibitors (rapamycin and AZD8055) have shown leaf chlorosis and yellowing in Arabidopsis (Ren et al, ; Xiong et al, ) suggesting chloroplast damage during TOR kinase inhibition. Recent reports involving TOR inhibitor treatment have indicated that these effects could be due to transcriptional downregulation of genes encoding plastidic ribosomal proteins and photosynthetic proteins as well as those of the tetrapyrrole biosynthesis pathway in Arabidopsis (Dobrenel, Mancera‐Martínez, et al, ; Dong et al, ). Thus, the TORC1 complex has been shown to play a crucial role in biogenesis and maturation of chloroplast to promote leaf and cotyledon greening (Li, Gao, Xue, Wang, & Zhao, ; Li, Song, et al, ; Mohammed et al, ; Sun et al, ; Zhang et al, ).…”

Recent Discoveries on the Role of TOR (Target of Rapamycin) Signaling in Translation in Plants

“…Several other potential regulatory factors, including the RNA-binding protein LARP1 (Larelated protein), have been demonstrated to function under TOR control in TOP mRNA regulation (Tcherkezian et al, 2014). In plants, TOP-like motifs are abundant within mRNA 59-UTRs of ribosomal proteins (Dobrenel et al, 2016b), but whether these cis-elements are involved in translation regulation in plants requires further research. Interestingly, several recent publications revealed the existence of so-called R-motifs containing GA, G(A) 3 , G(A) 6 , etc., repeats (Dobrenel et al, 2016;Xu et al, 2017), and provided evidence that the R-motif regulates translation of its mRNA via interaction with poly(A)-binding proteins during immune regulation in plants .…”

“…Comparative studies of eS6 posttranslational modifications occurring in response to hormones and various abiotic signals documented phosphorylation of several sites within the C-terminal a-helix of eS6 (for review, see Browning and Bailey-Serres, 2015). Recently, phosphoproteomic analysis and western blot with an antibody specifically recognizing phosphorylated Ser-240 has suggested that phosphorylation of Ser-240 is TOR/S6K1 responsive, and, accordingly, TOR inactivation decreases eS6 phosphorylation at this site (Dobrenel et al, 2016b).…”