Jenny Renaut

An improved protocol to study the plant cell wall proteome

Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.

Plant proteome changes under abiotic stress--contribution of proteomics studies to understanding plant stress response.

Plant acclimation to stress is associated with profound changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. In this review, proteomics studies dealing with plant response to a broad range of abiotic stress factors--cold, heat, drought, waterlogging, salinity, ozone treatment, hypoxia and anoxia, herbicide treatments, inadequate or excessive light conditions, disbalances in mineral nutrition, enhanced concentrations of heavy metals, radioactivity and mechanical wounding are discussed. Most studies have been carried out on model plants Arabidopsis thaliana and rice due to large protein sequence databases available; however, the variety of plant species used for proteomics analyses is rapidly increasing. Protein response pathways shared by different plant species under various stress conditions (glycolytic pathway, enzymes of ascorbate-glutathione cycle, accumulation of LEA proteins) as well as pathways unique to a given stress are discussed. Results from proteomics studies are interpreted with respect to physiological factors determining plant stress response. In conclusion, examples of application of proteomics studies in search for protein markers underlying phenotypic variation in physiological parameters associated with plant stress tolerance are given.

Gradual Soil Water Depletion Results in Reversible Changes of Gene Expression, Protein Profiles, Ecophysiology, and Growth Performance in Populus euphratica , a Poplar Growing in Arid Regions

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.

Quantitative changes in protein expression of cadmium-exposed poplar plants

Cadmium (Cd) pollution is a worldwide major concern having, among others, deleterious effects on plants. In the present work, the effects of a 20 μM Cd exposure in hydroponics culture during 14 days were evaluated in young poplar leaves. Proteins were analysed by 2‐D DIGE, followed by MALDI‐TOF‐TOF identification. Additionally, growth and other physiological parameters were monitored during the experiment. Treated plants exhibited an inhibition of growth and visual symptoms appeared after 7 days. A significant accumulation of Cd in all organs was recorded by ICP‐MS analysis. A number of changes in the expression of proteins with various functions were identified; in particular a decreased abundance of oxidative stress regulating proteins, whereas pathogenesis‐related proteins showed a drastic increase in abundance. Furthermore, a large number of proteins involved in carbon metabolism showed a decrease in abundance, while proteins involved in remobilizing carbon from other energy sources were upregulated. In conclusion, the negative effect of Cd could be explained by a deleterious effect on protein expression from the primary carbon metabolism and from the oxidative stress response mechanism. Accumulation of Cd in stems of poplar, coupled with a low impact of Cd on physiological parameters, promotes the use of poplar trees for phytoremediation purposes.

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress‐elicited changes in plants at the proteome level. Hydroponically grown 2‐wk‐old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H2O2 contents in roots. A total of 23 As‐regulated proteins including predicted and novel ones were identified using 2‐DE coupled with MS analyses. The expression levels of S‐adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST‐tau, and tyrosine‐specific protein phosphatase proteins (TSPP) were markedly up‐regulated in response to arsenate, whereas treatment by H2O2 also regulated the levels of CS suggesting that its expression was certainly regulated by As or As‐induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose‐dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

PROTEOME ANALYSIS OF NON-MODEL PLANTS: A CHALLENGING BUT POWERFUL APPROACH

Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives.

Recent developments in the application of proteomics to the analysis of plant responses to heavy metals

Pollution of soils by heavy metals is an ever‐growing problem throughout the world, and is the result of human activities as well as geochemical weathering of rocks and other environmental causes such as volcanic eruptions, acid rain and continental dusts. Plants everywhere are continuously exposed to metal‐contaminated soils. The uptake of heavy metals not only constrains crop yields, but can also be a major hazard to the health of humans and to the entire ecosystem. Although analysis of gene expression at the mRNA level has enhanced our understanding of the response of plants to heavy metals, many questions regarding the functional translated portions of plant genomes under metal stress remain unanswered. Proteomics offers a new platform for studying complex biological functions involving large numbers and networks of proteins, and can serve as a key tool for revealing the molecular mechanisms that are involved in interactions between toxic metals and plant species. This review focuses on recent developments in the applications of proteomics to the analysis of the responses of plants to heavy metals; such studies provide a deeper understanding of protein responses and the interactions among the possible pathways that are involved in detoxification of toxic metals in plant cells. In addition, the challenges faced by proteomics in understanding the responses of plants to toxic metal are discussed, and some possible future strategies for meeting these challenges are proposed.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.

Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica L. Batsch)

In the temperate climate of the northern hemisphere, winter survival of woody plants is determined by the ability to acclimate to freezing temperatures and to undergo a period of dormancy. Cold acclimation in many woody plants is initially induced by short photoperiod and low, non-freezing temperatures. These two factors (5°C and short photoperiod) were used to study changes in the proteome of bark tissues of 1-year-old peach trees. Difference in-gel electrophoresis technology, a gel-based approach involving the labeling of proteins with different fluorescent dyes, was used to conduct a quantitative assessment of changes in the peach bark proteome during cold acclimation. Using this approach, we were able to identify differentially expressed proteins and to assign them to a class of either ‘temperature-responsive’ or ‘photoperiod-responsive’ proteins. The most significant factor affecting the proteome appeared to be low temperature, while the combination of low temperature and short photoperiod was shown to act either synergistically or additively on the expression of some proteins. Fifty-seven protein spots on gels were identified by mass spectrometry. They included proteins involved in carbohydrate metabolism (e.g., enolase, malate dehydrogenase, etc), defense or protective mechanisms (e.g., dehydrin, HSPs, and PR-proteins), energy production and electron transport (e.g., adenosine triphosphate synthases and lyases), and cytoskeleton organization (e.g., tubulins and actins). The information derived from the analysis of the proteome is discussed as a function of the two treatment factors: low temperature and short photoperiod.