The Dynamics of the Cell Wall Proteome of Developing Alfalfa Stems.

In this study, the cell-wall-enriched subproteomes at three different heights of alfalfa stems were compared. Since these three heights correspond to different states in stem development, a view on the dynamics of the cell wall proteome during cell maturation is obtained. This study of cell wall protein-enriched fractions forms the basis for a description of the development process of the cell wall and the linking cell wall localized proteins with the evolution of cell wall composition and structure. The sequential extraction of cell wall proteins with CaCl2, EGTA, and LiCl-complemented buffers was combined with a gel-based proteome approach and multivariate analysis. Although the highest similarities were observed between the apical and intermediate stem regions, the proteome patterns are characteristic for each region. Proteins that bind carbohydrates and have proteolytic activity, as well as enzymes involved in glycan remobilization, accumulate in the basal stem region. Beta-amylase and ferritin likewise accumulate more in the basal stem segment. Therefore, remobilization of nutrients appears to be an important process in the oldest stem segment. The intermediate and apical regions are sites of cell wall polymer remodeling, as suggested by the high abundance of proteins involved in the remodeling of the cell wall, such as xyloglucan endoglucosylase, beta-galactosidase, or the BURP-domain containing polygalacturonase non-catalytic subunit. However, the most striking change between the different stem parts is the strong accumulation of a DUF642-conserved domain containing protein in the apical region of the stem, which suggests a particular role of this protein during the early development of stem tissues.

Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases.

The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,ß-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given.

Copper Trafficking in Plants and Its Implication on Cell Wall Dynamics.

In plants, copper (Cu) acts as essential cofactor of numerous proteins. While the definitive number of these so-called cuproproteins is unknown, they perform central functions in plant cells. As micronutrient, a minimal amount of Cu is needed to ensure cellular functions. However, Cu excess may exert in contrast detrimental effects on plant primary production and even survival. Therefore it is essential for a plant to have a strictly controlled Cu homeostasis, an equilibrium that is both tissue and developmentally influenced. In the current review an overview is presented on the different stages of Cu transport from the soil into the plant and throughout the different plant tissues. Special emphasis is on the Cu-dependent responses mediated by the SPL7 transcription factor, and the crosstalk between this transcriptional regulation and microRNA-mediated suppression of translation of seemingly non-essential cuproproteins. Since Cu is an essential player in electron transport, we also review the recent insights into the molecular mechanisms controlling chloroplastic and mitochondrial Cu transport and homeostasis. We finally highlight the involvement of numerous Cu-proteins and Cu-dependent activities in the properties of one of the major Cu-accumulation sites in plants: the cell wall.

Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism.

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

Ups and downs in alfalfa: Proteomic and metabolic changes occurring in the growing stem.

The expanding interest for using lignocellulosic biomass in industry spurred the study of the mechanisms underlying plant cell-wall synthesis. Efforts using genetic approaches allowed the disentanglement of major steps governing stem fibre synthesis. Nonetheless, little is known about the relations between the stem maturation and the evolution of its proteome. During Medicago sativa L. maturation, the different internodes grow asynchronously allowing the discrimination of various developmental stages on a same stem. In this study, the proteome of three selected regions of the stem of alfalfa (apical, intermediate and basal) was analyzed and combined with a compositional analysis of the different stem parts. Interestingly, the apical and the median regions share many similarities: high abundance of chloroplast- and mitochondrial-related proteins together with the accumulation of proteins acting in the early steps of fibre production. In the mature basal region, forisomes and stress-related proteins accumulate. The RT-qPCR assessment of the expression of genes coding for members of the cellulose synthase family likewise indicates that fibres and the machinery responsible for the deposition of secondary cell walls are predominantly formed in the apical section. Altogether, this study reflects the metabolic change from the fibre production in the upper stem regions to the acquisition of defence-related functions in the fibrous basal part.

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.

Physiological and proteomic responses of different willow clones (Salix fragilis x alba) exposed to dredged sediment contaminated by heavy metals.

High biomass producing species are considered as tools for remediation of contaminated soils. Willows (Salix spp.) are prominent study subjects in this regard. In this study, different willow clones (Salix fragilis x alba) were planted on heavy-metal polluted dredging sludge. A first objective was assessment of the biomass production for these clones. Using a Gupta statistic, four clones were identified as high biomass producers (HBP). For comparison, a group of four clones with lowest biomass production were selected (LBP). A second objective was to compare metal uptake as well as the physiological and proteomic responses of these two groups. All these complementary data's allow us to have a better picture of the health of the clones that would be used in phytoremediation programs. Cd, Zn, and Ni total uptake was higher in the HBPs but Pb total uptake was higher in LBPs. Our proteomic and physiological results showed that the LBPs were able to maintain cellular activity as much as the HBPs although the oxidative stress response was more pronounced in the LBPs. This could be due to the high Pb content found in this group although a combined effect of the other metals cannot be excluded.

A multiple-level study of metal tolerance in Salix fragilis and Salix aurita clones.

The response of two willow clones (Salix fragilis (Sf) and Salix aurita (Sa)) to the presence of metals (Zn, Cu, Cd, Ni) was studied. Rooted cuttings were planted in control and contaminated soil. After 100days, different parameters (biomass, chlorophyll fluorescence (Fv/Fm), pigment and sugar concentrations, electrolyte leakage and proteome-level changes) were analyzed. The growth of Sa was not influenced by metals whereas Sf produced significantly less biomass when exposed to the pollutants. Furthermore, although Sa did not show a growth reduction in the presence of metals, the overall view of the physiological results among others the changes in the accumulation of sugars and pigments indicated that metals had a more severe impact on this clone. The response at the proteome level confirmed these observations. The growth reduction and the proteomic changes in Sf indicate that this clone adjusts its metabolism to maintain cellular homeostasis. Sa on the contrary maintains growth but the physiological and proteomics data suggests that this can only be done at the cost of cellular deregulation. Therefore high biomass is not linked with a good tolerance strategy. In a long-term study the survival of Sa might be compromised making it a poorer candidate for phytoremediation efforts. BIOLOGICAL SIGNIFICANCE: In the last centuries human activity has resulted in the dispersal of heavy metals with potential phytotoxic effects over large areas. The increased knowledge of the responses of Salix-species, a group of trees with potential as biomass producer but also as phytoremediation agent, when growing on metal-polluted substrate provided by this study has the potential to help in the improved selection of clones with more or less potential for these aims. Contrary to most studies the trees in the current study were exposed to a mixture of metals, thereby facing a closer resemblance to the situation on soils polluted by human activity. Whereas many papers focused on the two main phenotypic characteristics (biomass and accumulation), fewer papers studied proteomic and physiological parameters which allow to have a global view of the tolerance of probable willow candidates for phytoremediation purposes. Our data demonstrates that higher biomass production in presence of metals is not necessarily linked with higher tolerance whereas growth reduction might indicate longer long-term tolerance. In the long term and in the purpose of a future use in phytoremediation, the survival of this high producer clone could be compromised.

Callose and cellulose synthase gene expression analysis from the tight cluster to the full bloom stage and during early fruit development in Malus × domestica.

Apple (Malus × domestica) is an economically important temperate fruit-bearing crop which belongs to the family of Rosaceae and its pomaceous fruit is one of the most commonly cultivated. Several studies have demonstrated that the cell wall plays a pivotal role during flower and fruit development. It takes active part in pollen tube growth and contributes to determine the fruit firmness trait through the action of cell wall-related enzymes (i.e. polygalacturonase and pectinmethylesterase). We have investigated the expression of callose and cellulose synthase genes during flowering from tight cluster to anthesis and during early fruit development in domesticated apple. We also link the changes observed in gene expression to the profile of soluble non-structural carbohydrates at different developmental stages of flowers/fruitlets and to the qualitative results linked to wall polysaccharides' composition obtained through near-infrared spectroscopy. This work represents an important addition to the study of tree physiology with respect to the analysis of the expression of callose and cellulose synthase genes during flower and early fruit development in domesticated apple.

From tolerance to acute metabolic deregulation: contribution of proteomics to dig into the molecular response of alder species under a polymetallic exposure.

Alnus spp. are actinorhizal trees commonly found in wet habitats and able to grow effectively in soil slightly contaminated with metal trace- elements. Two clones belonging to two Alnus species, namely, A. incana and A. glutinosa, were grown in hydroponics and exposed for 9 weeks to a Cd + Ni + Zn polymetallic constraint. Although responding by a similar decrease in total biomass production, the proteomic analysis associated with the study of various biochemical parameters including carbohydrate and mineral analyses revealed that the two clones have a distinct stress-responsive behavior. All parameters indicated that the roots, the organ in direct contact with the media, are more affected than the leaves. In fact, in A. glutinosa the response was almost completely confined to the roots, whereas many proteins change significantly in the roots and in the leaves of the treated A. incana. In both clones, the changes affected a broad range of metabolic processes such as redox regulation and energy metabolism and induced the production of pathogenesis-related proteins. In particular, changes in the accumulation of bacterial proteins that were not identified as coming from the known symbionts of Alnus were reported. Further investigation should be performed to identify their origin and exact role in the plant response to the polymetallic exposure tested here.

Physiological and proteome study of sunflowers exposed to a polymetallic constraint.

The new energy requirements of the growing world population together with the actual ecological trend of phytoremediation have made challenging the cultivation of energetic crops on nonagricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterization and biochemical analyses were combined to emphasize the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni, and Zn concentrations. Leaves and roots of sunflowers reaching the stage "2-extended leaves" and exposed to different trace metal concentrations were harvested and analyzed by 2D-DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reactions and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression, and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as a remediative tool of trace element polluted soils is limited.

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry.

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus × Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.