Posttranslational regulation of photosynthetic activity via the TOR kinase in plants

Abstract

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

Fig. 1.. RSH enzymes interact with a subunit of the TOR complex and undergo TOR-dependent phosphorylation.

(A) Alignment of the Y2H LST8 interacting regions of RSH2/3, showing the minimal interaction zone (magenta dashed line). TargetP predicted CTP cleavage site (CS) shown. NTR, N-terminal region; SYN, synthetase domain; HYD, hydrolase domain. (B) A proximity labeling experiment showing selected regions from blots of protein extracts from N. benthamiana coexpressing TID LST8 fusions with RSH3-GFP or SSU-CFP. Leaves were infiltrated with biotin for 2 hours. NI, non-inoculated control; asterisk (*) represents endogenous biotinylated protein. (C) Liquid chromatography–mass spectrometry (LC-MS) identification of RSH3-GFP peptides in the biotinylated protein fraction from N. benthamiana coexpressing RSH3-GFP with TID-LST8 or TID-YFP. Blots of protein extracts from (D) N. benthamiana coexpressing TID-LST8 and RSH3_39–221-GFP or SSU_57–79-RSH_365–221-GFP, (E) Arabidopsis OX:RSH3-GFP seedlings, and (F) N. benthamiana expressing RSH3_39–221-GFP or a phosphodefective (P−) form and treated with AZD-8055 (AZD) or the mock control (CTRL). Arabidopsis seedlings were sampled 48 hours after treatment and N. benthamiana at 2 hours. Target protein phosphoforms are indicated by P+. (G) Map of RSH3_39–221-GFP phosphorylation sites identified by LC-MS following immunoprecipitation from N. benthamiana. Arrowheads indicate phosphorylation in putative TOR-dependent contexts (see also fig. S6). (H) Blots of protein extracts from N. benthamiana expressing RSH3_39–221 homologous regions from Solanum lycopersicum, Oryza sativa, and C. paradoxa treated with AZD-8055 (AZD) or the mock control (CTRL) for 2 hours. Filled arrowheads indicate target protein phosphoforms. SDS, SDS-PAGE separation; phos-tag, phos-tag SDS-PAGE separation. Full blots and source data are available in data S1. WT, wild type.

Fig. 2.. TOR activity regulates photosynthesis via RSH-dependent ppGpp synthesis.

Maximal efficiency of PSII (Fv/Fm) was measured in seedlings of the indicated Arabidopsis lines treated ±10 μM AZD for (A) 6 days (n = 17 to 35 plants) and (B) 48 hours (n = 17 to 20 plants). (C) Nucleotide quantification in adult plants treated ± 10 μM AZD for 48 hours (n = 3 biological replicates). (D) Images of 5-week-old Arabidopsis wild-type and mutant plants grown under short-day conditions (8-hour light/16-hour dark). (E) Fv/Fm measurements from plants of the same age treated ±10 μM AZD for 48 hours (n = 4 to 20). Wild type is qrt1-2 for (A) and Col-0 for the other panels. Graphs show mean (horizontal bar), median (column height), and 95% confidence interval (CI) (vertical line). Lowercase letters indicate statistical groups. FW, fresh weight. Source data are available in data S1.

Fig. 3.. TOR regulates RSH3 activity and cellular distribution via the RSH3 NTR.

(A) Maximal efficiency of PSII (Fv/Fm) was measured in Arabidopsis seedlings of the indicated lines treated ±10 μM AZD for 48 hours (n = 18 to 36 plants). RSH3, RSH3-GFP; SSU-RSH3, SSU-RSH3_65-END-GFP; WT, wild type (qrt1-2). (B) Fluorescence microscopy images of N. benthamiana leaves expressing RSH3_1–165-GFP P+ and RSH3_1–165-GFP P−. White arrows indicate nuclei surrounded by chloroplasts. Scale bars, 50 μm. (C) Quantification of GFP fluorescence in nuclei against GFP fluorescence in nuclei-proximal chloroplasts, n = 50 nuclei. (D) Immunoblots of protein extracts from N. benthamiana expressing native (N), phosphominus (P−), or phosphomimic (P+) RSH3_1–165-GFP and the non-inoculated control (NI). p, precursor protein; m, mature protein after chloroplast import. Graphs show mean (horizontal bar), median (column height), and 95% CI (vertical line). Lowercase letters indicate statistical groups. Source data available are in data S1.