Differential regulation of gene products in newly synthesized Brassica napus allotetraploids is not related to protein function nor subcellular localization

BACKGROUND: Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values) in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation) in synthetic B. napus. RESULTS: The complete set of non-additive proteins (335 in the stem and 205 in the root), as well as a subset of additive polypeptides (200 per organ), was identified by mass spectrometry. Several protein isoforms were found, and most of them (~55%) displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which ~50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under-represented among the proteins displaying non-additive values in the allopolyploids. CONCLUSION: Protein identification showed that functionally related polypeptides (isoforms and complex subunits) could be differentially regulated in synthetic B. napus in comparison to its diploid progenitors while such proteins are usually expected to display co-regulation. The genetic redundancy within an allopolyploid could explain why functionally related proteins could display imbalanced levels of expression. No functional category, no metabolic pathway and no subcellular localization was found to be over- or under-represented within non-additive polypeptides, suggesting that the differential regulation of gene products was not related to functional properties of the proteins. Thus, at the protein level, there is no evidence for the "genomic shock" expected in neo-polyploids and the overall topology of protein networks and metabolic pathways is conserved in synthetic allotetraploids of B. napus in comparison to its diploid progenitors B. rapa and B. oleracea

The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover

indicates that members of the Snf1-Related Kinases 2 family (SnRK2) are essential in mediating various stress-adaptive responses. Recent reports have indeed shown that one particular member, OPEN STOMATA (OST)1, whose kinase activity is stimulated by the stress hormone abscisic acid (ABA), is a direct target of negative regulation by the core ABA co-receptor complex composed of PYR/PYL/RCAR and clade A Protein Phosphatase 2C (PP2C) proteins. and that phospho-T451 is important for stabilization of ABF3. on T451 to create a 14-3-3 binding motif. In a wider physiological context, we propose that the long term responses to ABA that require sustained gene expression is, in part, mediated by the stabilization of ABFs driven by ABA-activated SnRK2s

Quantitative comparative phosphoproteomics to decipher signal transduction

Adaptation to changing environmental conditions takes place rapidly in all living organisms: cells must respond to a wide variety of signals. Through successive protein phosphorylation and de-phosphorylation steps, signal transduction cascades mediate between the perception of the extracellular conditions and the adaptation of intracellular processes. The first event after the perception of the external signal is the auto-phosphorylation of a receptor kinase, which, on its turn, phosphorylates down-stream targets, mainly kinase cascades. The downstream effectors, mitogen-activated protein kinases (MAPKs) and other kinases, regulate the activity of transcription factors, metabolic enzymes and other factors, thereby initiating the fungal adaptive response. Understanding these adaptive responses requires deciphering early phosphorylation events. While global proteomic analyses have been improved over decades, the global study of protein (de)phosphorylation events in living organisms only became possible recently. Significant advances in mass spectrometry based phosphoproteomics have taken place, including phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. Reports of fungal phosphoproteomic studies are increasing, but those dealing with filamentous ascomycetes are still rare. The first phosphoproteomic study of kinase dependent phosphorylation events has been reported for the model fungus Neurospora crassa. In Botrytis cinerea, phosphoproteomics have been used to study the global phosphoprotein content in axenic culture and the differential phosphorylation profiles between mycelia grown on glucose or tomato-cell wall as carbon-source. We have undertaken a comparative proteomics and phosphoproteomics study of the B. cinerea osmosensing pathway challenged by the fungicide fludioxonil. We compared mutants of the sensor histidine-kinase Bos1 and of the MAPK Sak1 to the parental wild-type. Strains were exposed (or not) to fludioxonil for 15 min. during exponential in vitro growth. Shotgun proteomics revealed considerable differences in protein content among the strains, but no treatment effect. These results indicate a strong transcriptional and/or translational regulation under Bos1 and Sak1 control, respectively, under standard conditions. Quantitative phosphoproteomics revealed a clear response to the fludioxonil treatment as well as (de)phosphorylation events controlled by Bos1, Sak1, or both

Plant low‐K responses are partly due to Ca prevalence and the low‐K biomarker putrescine does not protect from Ca side effects but acts as a metabolic regulator

International audiencePotassium (K) deficiency is a rather common situation which impacts negatively on biomass, photosynthesis and N assimilation, making K fertilization often unavoidable. Effects of K deficiency have been investigated for several decades and recently, progress has been made in identifying metabolomics signatures thereby offering potential to monitor the K status of crops in the field. However, effects of low K conditions could also be due to the antagonism with other nutrients like calcium (Ca) and the well-known biomarker of K deficiency, putrescine, could be a response to Ca/K imbalance rather than K deficiency per se. To sort this out, we carried out experiments in sunflower grown at either low or high K, at high or low Ca, with or without putrescine added to the nutrient solution. Using metabolomics and proteomics analysis, we show that a significant part of the low-K response such as lower photosynthesis and N assimilation, is due to calcium and can be suppressed by low Ca conditions. Putrescine addition tends to restore photosynthesis and N assimilation but unlike low-Ca, does not suppress but aggravates the impact of low-K conditions on catabolism, including the typical fall-over in pyruvate kinase. We conclude that (i) the effects of K deficiency on key metabolic processes can be partly alleviated by the use of low Ca and not only by K fertilization, and (ii) in addition to its role as a metabolite, putrescine participates in acclimation to low K via the regulation of the content in enzymes involved in carbon primary metabolism

Comparative proteomics and phosphoproteomics to decipher signal transduction in Botrytis cinerea.

Adaptation to changing environmental conditions takes place rapidly in all living organisms: cells must respond to a wide variety of signals. Through successive protein phosphorylation and de-phosphorylation steps, signal transduction cascades mediate between the perception of the extracellular conditions and the adaptation of intracellular processes. Botrytis cinerea is a necrotrophic, polyphageous plant pathogen, that causes gray mold disease and can infect over 1000 plant species including several agronomically important crops (grapevine, strawberry, tomatoes …). Fungicides remain the most effective means to combat this disease. However B. cinerea rapidly adapts to fungicides. Presently, the phenylpyrrole fludioxonil is one of the most efficient fungicides against B. cinerea. Therefore deciphering the response to fludioxonil in B. cinerea is crucial. In Botrytis cinerea, the fungicide fludioxonil activates the Sak1 (Hog1-like) and Bmp3 (Slt2-like) MAPKs, which are respectively involved in osmoregulation, cell wall integrity, development and pathogenicity. In order to trace the transduction of fludioxonil to the MAPK pathways, we have undertaken a comparative proteomics and phosphoproteomics study of the B. cinerea osmosensing pathway challenged by the fungicide fludioxonil. We compared mutants of the sensor histidine-kinase Bos1 and of the MAPK Sak1 to the parental wild-type. Strains were exposed (or not) to fludioxonil for 15 min during exponential in vitro growth. Shotgun proteomics revealed considerable differences in protein content among the strains, but no treatment effect. These results indicate a strong transcriptional and/or translational regulation under Bos1 and Sak1 control, respectively, under standard conditions. One of the most relevant result is the abundance of 263 proteins controlled by Bos1 and Sak1 independently. Proteins of the oxidative stress response are under negative control of Bos1, while some proteases are positively regulated. In addition, proteins involved in translation are specifically regulated by Bos1. It appears that Bos1 is important in different biological processes. Sak1, on its turn, controls the abundance of proteins involved in oxidative stress response, early secretome, protein folding, primary metabolism and secondary metabolism. Quantitative phosphoproteomics is ongoing and it will permit to verify a clear response to the fludioxonil treatment as well as (de)phosphorylation events controlled independently by Bos1 or Sak1, or by both kinase

Metabolic responses to potassium availability and waterlogging reshape respiration and carbon use efficiency in oil palm

Oil palm is by far the major oil‐producing crop on the global scale, with c. 62 Mt oil produced each year. This species is a strong potassium (K)‐demanding species cultivated in regions where soil K availability is generally low and waterlogging due to tropical heavy rains can limit further nutrient absorption. However, the metabolic effects of K and waterlogging have never been assessed precisely. Here, we examined the metabolic response of oil palm saplings in the glasshouse under controlled conditions (nutrient composition with low or high K availability, with or without waterlogging), using gas exchange, metabolomics and proteomics analyses. Our results showed that both low K and waterlogging have a detrimental effect on photosynthesis but stimulate leaf respiration, with differential accumulation of typical metabolic intermediates and enzymes of Krebs cycle and alternative catabolic pathways. In addition, we found a strong relationship between metabolic composition, the rate of leaf dark respiration, and cumulated respiratory loss. Advert environmental conditions (here, low K and waterlogging) therefore have an enormous effect on respiration in oil palm. Leaf metabolome and proteome appear to be good predictors of carbon balance, and open avenues for cultivation biomonitoring using functional genomics technologies

Label-Free Quantitative Proteomics Reveal the Involvement of PRT6 in <i>Arabidopsis thaliana</i> Seed Responsiveness to Ethylene

In Arabidopsis thaliana, the breaking of seed dormancy in wild type (Col-0) by ethylene at 100 μL L−1 required at least 30 h application. A mutant of the proteolytic N-degron pathway, lacking the E3 ligase PROTEOLYSIS 6 (PRT6), was investigated for its role in ethylene-triggered changes in proteomes during seed germination. Label-free quantitative proteomics was carried out on dormant wild type Col-0 and prt6 seeds treated with (+) or without (−) ethylene. After 16 h, 1737 proteins were identified, but none was significantly different in protein levels in response to ethylene. After longer ethylene treatment (30 h), 2552 proteins were identified, and 619 Differentially Expressed Proteins (DEPs) had significant differences in protein abundances between ethylene treatments and genotypes. In Col, 587 DEPs were enriched for those involved in signal perception and transduction, reserve mobilization and new material generation, which potentially contributed to seed germination. DEPs up-regulated by ethylene in Col included S-adenosylmethionine synthase 1, methionine adenosyltransferase 3 and ACC oxidase involved in ethylene synthesis and of Pyrabactin Resistance1 acting as an ABA receptor, while DEPs down-regulated by ethylene in Col included aldehyde oxidase 4 involved in ABA synthesis. In contrast, in prt6 seeds, ethylene did not result in strong proteomic changes with only 30 DEPs. Taken together, the present work demonstrates that the proteolytic N-degron pathway is essential for ethylene-mediated reprogramming of seed proteomes during germination

Comparative quantitative proteomics of osmotic signal transduction mutants in Botrytis cinerea explain mutant phenotypes and highlight interaction with cAMP and Ca2+ signalling pathways

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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

Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiaeSaccharomyces\ cerevisiae

International audienceThe yeast $Saccharomyces\ cerevisiae$ is a facultative aerobe able to adapt its metabolism according to the carbon substrate. The mechanisms of these adaptations involve at least partly the mitochondria but are not yet well understood. To address the possible role of protein phosphorylation event in their regulation, it is necessary in a first instance to determine precisely the phosphorylation sites that show changes depending on the carbon source. In this aim we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media. Label free quantitative analysis of protein accumulation revealed significant variation of 176 mitochondrial proteins including 108 proteins less accumulated in glucose medium than in lactate and galactose media. We also showed that the responses to galactose and glucose are not similar. Stable isotope dimethyl labeling allowed the quantitative comparison of phosphorylation levels between the different growth conditions. This study enlarges significantly the map of yeast mitochondrial phosphosites as 670 phosphorylation sites were identified, of which 214 were new and quantified. Above all, we showed that 90 phosphosites displayed a significant variation according to the medium and that variation of phosphorylation level is site-dependent