The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation

Abstract

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana , CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca 2+ -dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

TheArabidopsis thalianaPeptideAtlas; harnessing world-wide proteomics data for a comprehensive community proteomics resource

“…Together this strongly suggests that CAS is phosphorylated at T380 and very unlikely at S378 (although some spectra are ambiguous). A recent paper suggested T376, S378, and T380 as the major p-sites based on phosphoproteomics and in vitro phosphorylation assays on CAS variants (Cutolo et al, 2019), with an earlier study suggesting T380 as the main p-site (Vainonen et al, 2008). PeptideAtlas showed no sequence coverage for T376 despite the very high sequence coverage of the protein (possible peptides with one missed cleavage are SFGT 376 RSGTK or IIPAASRSFGT 376 R or SFGT 376 RSGTK but these were not observed) (Figure 13B,C).…”

“…Darker red rectangles indicate higher numbers of PSMs for each peptide. The predicted thylakoid transmembrane domain is indicated as a blue rectangle (we note that the N- and C-termini as facing the chloroplast stromal site based on (Cutolo et al, 2019)).…”

Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis

“…In contrast, the relative abundance of TAP38 decreased to a similar extent in both LL and HL plants compared to ML (Figure 3e). The calcium sensor kinase protein CAS, which also plays a role in regulating photo-acclimation in high light by promoting dephosphorylation of LHCII (Cutolo et al, 2019), also increased in abundance in HL by approximately 70% and decreased in LL by approximately 30% (Figure 3e).…”

Chloroplast Calcium Signaling in the Spotlight

“…Ca 2+ sensors are indeed present in plastids, although their activity in deciphering organellar Ca 2+ signatures has not been fully demonstrated so far. Nevertheless, a recent work points to CAS as mediator of light response and photoacclimation (Cutolo et al, 2019).…”