Benoît Valot

Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase.

MINT‐7260208: OST1 (uniprotkb:Q940H6) and ATRBOHF (uniprotkb:O48538) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)

Extent of N‐terminal modifications in cytosolic proteins from eukaryotes

Most proteins in all organisms undergo crucial N‐terminal modifications involving N‐terminal methionine excision, N‐α‐acetylation or N‐myristoylation (N‐Myr), or S‐palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N‐terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N‐Myr experiments were performed with both plant and animal N‐myristoyltransferases, for accurate prediction of the modification. N‐terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N‐termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N‐terminal methionine (Met) cleavage and N‐acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.

The use of metabolomics integrated with transcriptomic and proteomic studies for identifying key steps involved in the control of nitrogen metabolism in crops such as maize

Linking plant phenotype to gene and protein expression and also to metabolite synthesis and accumulation is one of the main challenges for improving agricultural production worldwide. Such a challenge is particularly relevant to crop nitrogen use efficiency (NUE). Here, the differences in leaf gene transcript, protein, and metabolite accumulation in maize subjected to long-term nitrogen (N)-deficient growth conditions at two important stages of plant development have been studied. The impact of N deficiency was examined at the transcriptomic, proteomic, and metabolomic levels. It was found that a number of key plant biological functions were either up- or down-regulated when N was limiting, including major alterations to photosynthesis, carbon (C) metabolism, and, to a lesser extent, downstream metabolic pathways. It was also found that the impact of the N deficiency stress resembled the response of plants to a number of other biotic and abiotic stresses, in terms of transcript, protein, and metabolite accumulation. The genetic and metabolic alterations were different during the N assimilation and the grain-filling period, indicating that plant development is an important component for identifying the key elements involved in the control of plant NUE. It was also found that integration of the three omics studies is not straightforward, since different levels of regulation seem to occur in a stepwise manner from gene expression to metabolite accumulation. The potential use of these omics studies is discussed with a view to improve our understanding of whole plant nitrogen economics, which should have applications in breeding and agronomy.

Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate–regulated by interaction of PII with the biotin carboxyl carrier subunit

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the Vmax of the ACCase reaction without altering the Km for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

On the mechanisms of cadmium stress alleviation in Medicago truncatula by arbuscular mycorrhizal symbiosis: A root proteomic study

The arbuscular mycorrhizal (AM) symbiosis belongs to the strategies plants have developed to cope with adverse environmental conditions including contamination by heavy metals such as cadmium (Cd). In the present work, we report on the protective effect conferred by AM symbiosis to the model legume Medicago truncatula grown in presence of Cd, and on the 2‐D‐based proteomic approach further used to compare the proteomes of M. truncatula roots either colonised or not with the AM fungus Glomus intraradices in Cd‐free and Cd‐contaminated substrates. The results indicated that at the proteome level, 9 out of the 15 cadmium‐induced changes in nonmycorrhizal roots were absent or inverse in those Cd‐treated and colonized by G. intraradices, including the G. intraradices‐dependent down‐accumulation of Cd stress‐responsive proteins. Out of the twenty‐six mycorrhiza‐related proteins that were identified, only six displayed changes in abundance upon Cd exposure, suggesting that part of the symbiotic program, which displays low sensitivity to Cd, may be recruited to counteract Cd toxicity through the mycorrhiza‐dependent synthesis of proteins having functions putatively involved in alleviating oxidative damages, including a cyclophilin, a guanine nucleotide‐binding protein, an ubiquitin carboxyl‐terminal hydrolase, a thiazole biosynthetic enzyme, an annexin, a glutathione S‐transferase (GST)‐like protein, and a S‐adenosylmethionine (SAM) synthase.

Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves

Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in plant water status, and their possible involvement in plant growth-related processes.

A proteomic approach to decipher chilling response from cold acclimation in pea (Pisum sativum L.).

Two pea lines (Pisum sativum L.) with contrasted behaviours towards chilling and subsequent frost were studied by a proteomic approach to better understand cold acclimation. Following a chilling period, the Champagne line becomes tolerant to frost whereas Terese remains sensitive. Variance analysis allowed to select 260 statistically variable spots with 68 identified proteins (35 in leaves, 18 in stems, and 15 in roots). These proteins were shared out in proteins related to chilling response or cold acclimation. The better adaptation of Champagne to chilling might be related to a higher content in proteins involved in photosynthesis and in defence mechanisms. Moreover Champagne might prevent freezing damage particularly thanks to a higher constitutive expression of housekeeping proteins related to Terese. After three days of subsequent frost, proteomes of previously chilled plants also showed significant differences compared to unchilled plants. Out of 112 statistically variable spots (44 in leaves, 38 in stems, and 30 in roots), 32 proteins were identified. These proteins were related to frost response or frost resistance. It seems that Champagne could resist to frost with the reorientation of the energy metabolism.

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N‐glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC‐MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty‐one proteins were identified in the N‐glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido‐reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.