Concerted changes in the phosphoproteome and metabolome under different CO2/O-2 gaseous conditions in arabidopsis rosettes

Considerable efforts are currently devoted to understanding the regulation of primary carbon metabolism in plant leaves, which is known to change dramatically with environmental conditions, e.g. during light/dark transitions. Protein phosphorylation is believed to be a key factor in such a metabolic control. In fact, some studies have suggested modifications in the phosphorylation status of key enzymes in the dark compared with the light, or when photosynthesis varies. However, a general view of the phosphoproteome and reciprocal alterations in both the phosphoproteome and metabolome under a wide spectrum of CO2 and O-2 conditions so as to vary both gross photosynthesis and photorespiration is currently lacking. Here, we used an instant sampling system and strictly controlled gaseous conditions to examine short-term metabolome and phosphoproteome changes in Arabidopsis rosettes. We show that light/dark, CO2 and O-2 mole fraction have differential effects on enzyme phosphorylation. Phosphorylation events that appear to be the most important to regulate metabolite contents when photosynthesis varies are those associated with sugar and pyruvate metabolism: sucrose and starch synthesis are major phosphorylation- controlled steps but pyruvate utilization (by phosphoenolpyruvate carboxylase and pyruvate dehydrogenase) and pyruvate reformation (by pyruvate orthophosphate dikinase) are also subjected to phosphorylation control. Our results thus show that the phosphoproteome response to light/dark transition and gaseous conditions (CO2, O-2) contributes to the rapid adjustment of major pathways of primary C metabolism

Differential protein phosphorylation regulates chloroplast movement in response to strong light and darkness in Arabidopsis thaliana

International audiencePhototropin-dependent chloroplast movement is essential to the photosynthetic acclimation of mesophyll cells to incident light. Chloroplast movement involves many cellular actors, such as chloroplast-associated actin filaments and proteins that mediate signalling between phototropins and chloroplast motion. In the past few years, genetic approaches have identified several key proteins but the intrinsic mechanisms of the signalling cascade, such as phosphorylation events, remain undefined. Here, we took advantage of phosphoproteomics to examine the involvement of protein phosphorylation in chloroplast movement in darkness or under high light, at different CO2 mole fractions (100, 380 or 1,000 ppm) to vary photosynthetic activity. Amongst the 100 relevant identified phosphopeptides, 19 (corresponding to 8 proteins) were differentially phosphorylated in darkness vs. high light. There was no significant CO2 effect on the observed phosphorylation patterns. We further characterized the phosphorylation sites in THRUMIN1, which is believed to be crucial for the attachment of chloroplast-associated actin filaments to the plasma membrane and thus for chloroplast movements. The mutant thrumin1 was complemented with a mutated protein in which phospho-sites were substituted to a phosphomimetic (Asp) or a non-phosphorylatable (Ala) residue. While the phosphomimetic substitution altered the chloroplast response in the light only, both light and dark responses were altered with the non-phosphorylatable substitution. Our data suggest a key role of protein phosphorylation, including that of THRUMIN1, in the light/dark control of chloroplast movements

Arabidopsis thaliana 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase 2 activity requires serine 82 phosphorylation

International audiencePhosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3-phosphoglycerate and 2-phosphoglycerate, a step of glycolysis. PGAMs can be sub-divided into 2,3-biphosphoglycerate dependent (dPGAM) and independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post-translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified a iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark-adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D-structural modelling of AtiPGAM2 based on non-plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82-phosphopeptide reflects iPGAM2 steady-state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed

Metabolic leaf responses to potassium availability in oil palm (Elaeis guineensis Jacq.) trees grown in the field

Oil palm growth and production is highly dependent on potassium (K) fertilization. Presently, monitoring K fertilization is difficult since it depends on soil properties, crosses and other nutrients. To adjust K fertilization precisely during cultivation, leaf biomarkers that can indicate changes in tree K status before the appearance of symptoms on fruit production and yield, are required. However, the metabolic response of oil palm leaves to K availability is poorly documented. Here, we investigated the response of oil palm leaf metabolome and proteome to K availability in two crosses (Deli x La Mé, and Deli x Yangambi) grown in the field. Our result show that one to two years only after the onset of K fertilization treatments, there were changes in N metabolism, photosynthesis and mitochondrial metabolism, with a differential effect in the two crosses. In particular, there were changes in sugars, amino and organic acids pointing to modifications in photosynthetic and catabolic (Krebs cycle) capacity and this agreed with the effect seen on enzyme content. Therefore, K availability led to rapid changes in leaf primary metabolism, opening avenues for the utilization of leaf metabolic signature as a marker of K nutrition in oil palm.C. Mirande-Ney is grateful to CIRAD for the PhD Fellowship and financial support for travel, experiments, and analyses. The authors also thank IDEEV for its financial support, and the company SOCFINDO for access to oil palm field, experimental material and technical support. The authors also acknowledge the support of Bertrand Gakiere from the metabolomics facility Metabolism-Metabolome (IPS2) and Jean Ollivier (CIRAD) for providing agronomical data.http://pappso.inra.f

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

<div><p>Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to <i>Arabidopsis thaliana</i> rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.</p></div

Phosphoregulation of the photorespiratory enzyme, glycolate oxidase, in response to light and CO2 content in Arabidopsis thaliana

International audienc

Photosynthetic parameters.

<p>leaf net photosynthesis (<b>A</b>), leaf-to-air water vapour drawdown (<b>B</b>), leaf glucose content (<b>C</b>) and Gly-to-Ser ratio (<b>D</b>), under the different photosynthetic contexts used (LC, NC and HC: 100, 380 and 1000 µmol mol⁻¹ CO₂; D: darkness).</p

Eukaryotic ribosomal protein and initiation factor phosphopeptides identified by nanoLC-MS/MS.

<p>a- Phosphopeptides sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070692#pone.0070692.s002" target="_blank">Table S2</a>.</p><p>b- A phosphorylation site is considered as new when absent from refs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070692#pone.0070692-Turkina1" target="_blank">[8]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070692#pone.0070692-Muench1" target="_blank">[11]</a> and PhosPhAt 4.0 database(<a href="http://phosphat.mpimp-golm.mpg.de/" target="_blank">http://phosphat.mpimp-golm.mpg.de/</a>).</p><p>c- Phosphorylation patterns are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070692#pone-0070692-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070692#pone-0070692-g004" target="_blank">4</a>.</p><p>d- By ‘punctually’, we mean occasional detection of phosphorylation, with no clear pattern.</p

Phosphorylation pattern of ribosomal proteins.

<p><b>A</b>, Heat map representation of the phosphorylation level of significant phosphopeptides (phosphorylated peptides that showed statistically significant changes with conditions). A hierarchical clustering analysis is shown on the left. All significant phosphopeptides had a very similar pattern, except for RPL13D, which was minimally phosphorylated under ordinary conditions (NC). Unavailable data (non-detected peptides) are indicated with a grey cell. <b>B</b> and <b>C</b>, Detailed phosphorylation pattern of RPS6A/B and RPS14A. LC, NC, HC and D: low, normal and high CO₂ and darkness.</p