Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF)

Abstract

Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in shortand long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast.phosphoproteomics | Arabidopsis thaliana I n the field of chloroplast biogenesis, interest in protein phosphorylation historically focused on photosynthesis-related proteins, with the initial discovery of thylakoid membrane protein phosphorylation dating back to the late 1970s (1-4). Almost a decade later, AtpB, RNA-binding proteins, and transcription factors were recognized as phosphoproteins in thylakoid membranes and stroma fractions (5-7). Because of recent large-scale functional genomics and phosphoproteomics approaches, ∼200 chloroplast phosphoproteins are known today, and several kinases have been identified that are most likely involved in their phosphorylation (8). However, the exact kinase/substrate relationships are not known for most of the proteins, and efforts are underway to identify in vivo substrates of known kinases. Phosphoproteomics data suggest that chloroplast functions are regulated by a highly complex phosphoprotein network in which one kinase phosphorylates several substrates and one substrate is probably phosphorylated by several kinases at different sites (9, 10).Although we currently do not understand all nodes in the chloroplast phosphoprotein network, candidate proteins and experimental tools are available to address the above questions. Two of the best-characterized chloroplast kinases are STN7 and STN8. STN7 is the ortholog of the Stt7 kinase from Chlamydomonas reinhardtii that was identified in screens for strains with a defect in state transitions (11). This process ba...

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

“…This kinase is phosphorylated under state 2 conditions, suggesting the existence of a kinase cascade. However, no phosphorylation could be detected in STN8 [44].…”

“…A proteome-wide screen for STN8 substrates by comparative quantitative phosphoproteomics with WT and STN8-deficient plants confirmed the presence of D2, CAS and PsbH among the substrates of this kinase [44]. However, in this screen, no differences were detected in the phosphorylation state of D1 and CP43 between wild-type and stn8.…”

“…This protein is involved in cyclic electron flow [51] and is part of a large supercomplex comprising PSI, cyt b 6 f and LHCII in C. reinhardtii [52]. Spectroscopic analysis with stn7, stn8 and the stn7/stn8 double mutant revealed a Stn8-specific effect on the transition between linear and cyclic electron flow [44]. In the absence of STN8, the stability of cyclic electron flow is reduced.…”

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…By genome annotation, protein kinases were found to make up about 5.5% of the Arabidopsis genome [4], which is nearly twice of that in human [5], indicating the high specificity and a complex network of phosphorylation events in plants [4]. In recent years, benefitting from the new advances in proteomics technologies including phosphopeptide enrichment, high-accuracy mass spectrometry (MS), and associated bioinformatics, large-scale analyses of protein phosphorylation have been carried out in variety of plant species including Medicago Truncatula [6], Oryza sativa [7], Glycine max [8], Brassica napus [8], and Arabidopsis thaliana [8][9][10][11][12][13][14][15][16][17][18]. However, our understanding of plant phosphoproteomes remains very limited with respect to their complexity and functions.…”

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana : is it caused by genetic variation in the STN kinases?

“…The STN7 kinase is involved in phosphorylation of LHCII, CP29, CP26 and TSP9 proteins, whereas the STN8 kinase phosphorylates PSII core D1, D2, PsbH and to some extent CP43 proteins (for reviews, see [10,11]). Besides the PSII core proteins, STN8 has additional targets, including the chloroplast calcium-sensing protein CAS [12], and a protein involved in cyclic electron transport (PGRL1) [13], which is a part of the PSI-LHCI-LHCII supercomplex [14]. Analysis of the protein phosphorylation profiles of the stn7 and stn8 mutants of Arabidopsis showed residual phosphorylation of the LHCII and PSII core proteins, respectively.…”