A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax.

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes.

Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Targeted Metagenomics of Retting in Flax: The Beginning of the Quest to Harness the Secret Powers of the Microbiota.

The mechanical and chemical properties of natural plant fibers are determined by many different factors, both intrinsic and extrinsic to the plant, during growth but also after harvest. A better understanding of how all these factors exert their effect and how they interact is necessary to be able to optimize fiber quality for use in different industries. One important factor is the post-harvest process known as retting, representing the first step in the extraction of bast fibers from the stem of species such as flax and hemp. During this process microorganisms colonize the stem and produce hydrolytic enzymes that target cell wall polymers thereby facilitating the progressive destruction of the stem and fiber bundles. Recent advances in sequencing technology have allowed researchers to implement targeted metagenomics leading to a much better characterization of the microbial communities involved in retting, as well as an improved understanding of microbial dynamics. In this paper we review how our current knowledge of the microbiology of retting has been improved by targeted metagenomics and discuss how related '-omics' approaches might be used to fully characterize the functional capability of the retting microbiome.

The quest for tolerant varieties: the importance of integrating "omics" techniques to phenotyping.

The primary objective of crop breeding is to improve yield and/or harvest quality while minimizing inputs. Global climate change and the increase in world population are significant challenges for agriculture and call for further improvements to crops and the development of new tools for research. Significant progress has been made in the molecular and genetic analysis of model plants. However, is science generating false expectations? Are 'omic techniques generating valuable information that can be translated into the field? The exploration of crop biodiversity and the correlation of cellular responses to stress tolerance at the plant level is currently a challenge. This viewpoint reviews concisely the problems one encounters when working on a crop and provides an outline of possible workflows when initiating cellular phenotyping via "-omic" techniques (transcriptomics, proteomics, metabolomics).

Effects of a cold treatment of the root system on white clover (Trifolium repens L.) morphogenesis and nitrogen reserve accumulation.

The ability of white clover (Trifolium repens L.) to undergo cold acclimation is an important determinant of its persistence in mixed swards since growth rate at low temperatures sustains higher clover contents at the start of spring. During a re-growth period following defoliation, a gradual exposure of the root system (cv. Grasslands Huia) led to some physiological and morphological changes of cold-adaptive significance, similar to those developed by clover ecotypes originating in northern areas of Europe. Thus, cold exposure of the root system resulted in small-leaved prostrate forms of white clover after one month of re-growth. Similarly, cold exposure increased the ability of plants to store nitrogen since the application of low temperatures to the root system enhanced soluble protein accumulation in roots and in stolons. More specifically, cold exposure of the roots induced gene expression of a vegetative storage protein (17.3 kDa VSP) in both organs. These results demonstrate that the root system of clover plants should be a site of perception of the low-temperature stimulus, and gave rise to the question of the transduction of the cold signal from the roots to the aerial parts. On the basis of this study and taking into account molecular aspects concerning the clover VSP, it is suggested that this protein could participate in cold acclimation in addition to its role in nitrogen storage.

Exploring chloroplastic changes related to chilling and freezing tolerance during cold acclimation of pea (Pisum sativum L.).

Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Térèse, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DIGE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiometry of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover.

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.

The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature.

Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5 degrees C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.

Morphological pattern of development affects the contribution of nitrogen reserves to regrowth of defoliated white clover (Trifolium repens L.).

The contribution of nitrogen reserves to regrowth following defoliation was studied in white clover plants (Trifolium repens cv. Huia). This was found to be closely linked to the morphological pattern of development of the aerial parts during the same period. Low temperature (6 degrees C) and short day exposure (8 h photoperiod) were used to induce dwarf development, i.e. to increase branching rate and to enhance new sites of leaf production during a period of regrowth. Treated plants exhibited a large reduction in leaf area and a large increase in leaf pool size for the first 10 d of a subsequent regrowth under standard culture conditions (16 h daylight; 22/18 degrees C day/night). The contribution of nitrogen from storage compounds in organs remaining after defoliation (sources) to regrowing tissues (sinks) was assessed by 15N pulse-chase labelling during regrowth following shoot removal. The mobilization of nitrogen reserves from storage tissues of regrowing clover was closely linked to the pattern of differentiation of the newly developed organs. It appeared that regrowth was supported less by endogenous N for the first 10 d after defoliation in treated plants, compared with control plants grown continuously in standard conditions. It is assumed that dwarf plants exhibit a lower dependence upon the mobilization of soluble proteins previously accumulated in roots and uncut stolons. The relationship between leaf development rate and N-uptake recovery following defoliation is discussed.