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.

Comparative Analysis of G-Layers in Bast Fiber and Xylem Cell Walls in Flax Using Raman Spectroscopy.

In a response to gravitropic stress, G-layers (gelatinous layers) were deposited in xylem cell walls of tilted flax plants. G-layers were produced in both tension wood (upper side) as expected but were also observed in opposite wood (lower side). Raman spectral profiles were acquired for xylem G-layers from the tension and opposite side as well as from the G-layer of bast fibers grown under non-tilted conditions. Statistical analysis by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) clearly distinguished bast fiber G-layers from xylem G-layers. Discriminating bands were observed for cellulose (380-1150-1376 cm-1), hemicelluloses (517-1094-1126-1452 cm-1) and aromatics (1270-1599-1658 cm-1). PCA did not allow separation of G-layers from tension/opposite-wood sides. In contrast, the two types of xylem G-layers could be incompletely discriminated through PLS-DA. Overall, the results suggested that while the architecture (polymer spatial distribution) of bast fibers G-layers and xylem G-layers are similar, they should be considered as belonging to a different cell wall layer category based upon ontogenetical and chemical composition parameters.

Raman spectroscopy mapping of changes in the organization and relative quantities of cell wall polymers in bast fiber cell walls of flax plants exposed to gravitropic stress.

Flax is an important fiber crop that is subject to lodging. In order to gain more information about the potential role of the bast fiber cell wall in the return to the vertical position, 6-week-old flax plants were subjected to a long-term (6 week) gravitropic stress by stem tilting in an experimental set-up that excluded autotropism. Stress induced significant morphometric changes (lumen surface, lumen diameter, and cell wall thickness and lumen surface/total fiber surface ratio) in pulling- and opposite-side fibers compared to control fibers. Changes in the relative amounts and spatial distribution of cell wall polymers in flax bast fibers were determined by Raman vibrational spectroscopy. Following spectra acquisition, datasets (control, pulling- and opposite sides) were analyzed by principal component analysis, PC score imaging, and Raman chemical cartography of significant chemical bonds. Our results show that gravitropic stress induces discrete but significant changes in the composition and/or spatial organization of cellulose, hemicelluloses and lignin within the cell walls of both pulling side and opposite side fibers.

A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.

BLISS: Shining a light on lignification in plants.

Lignin is a polyphenolic polymer of the plant cell wall formed by the oxidative polymerization of 3 main monomers called monolignols that give rise to the lignin H-, G- and S-units. Together with cellulose and hemicelluloses, lignin is a major component of plant biomass that is widely exploited by humans in numerous industrial processes. Despite recent advances in our understanding of monolignol biosynthesis, our current understanding of the spatio-temporal regulation of their transport and polymerization is more limited. In a recent publication, we have reported the development of an original Bioorthogonal Labeling Imaging Sequential Strategy (BLISS) that allows us to visualize the simultaneous incorporation dynamics of H and G monolignol reporters into lignifying cell walls of the flax stem. 11 Here, we extend the application of this strategy to other plant organs such as roots and rapidly discuss some of the contributions and perspectives of this new technique for improving our understanding of the lignification process in plants.

Spatial regulation of monolignol biosynthesis and laccase genes control developmental and stress-related lignin in flax.

BACKGROUND: Bast fibres are characterized by very thick secondary cell walls containing high amounts of cellulose and low lignin contents in contrast to the heavily lignified cell walls typically found in the xylem tissues. To improve the quality of the fiber-based products in the future, a thorough understanding of the main cell wall polymer biosynthetic pathways is required. In this study we have carried out a characterization of the genes involved in lignin biosynthesis in flax along with some of their regulation mechanisms. RESULTS: We have first identified the members of the phenylpropanoid gene families through a combination of in silico approaches. The more specific lignin genes were further characterized by high throughput transcriptomic approaches in different organs and physiological conditions and their cell/tissue expression was localized in the stems, roots and leaves. Laccases play an important role in the polymerization of monolignols. This multigenic family was determined and a miRNA was identified to play a role in the posttranscriptional regulation by cleaving the transcripts of some specific genes shown to be expressed in lignified tissues. In situ hybridization also showed that the miRNA precursor was expressed in the young xylem cells located near the vascular cambium. The results obtained in this work also allowed us to determine that most of the genes involved in lignin biosynthesis are included in a unique co-expression cluster and that MYB transcription factors are potentially good candidates for regulating these genes. CONCLUSIONS: Target engineering of cell walls to improve plant product quality requires good knowledge of the genes responsible for the production of the main polymers. For bast fiber plants such as flax, it is important to target the correct genes from the beginning since the difficulty to produce transgenic material does not make possible to test a large number of genes. Our work determined which of these genes could be potentially modified and showed that it was possible to target different regulatory pathways to modify lignification.

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.

BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants.

A better in vivo understanding of lignin formation within plant cell walls will contribute to improving the valorization of plant-derived biomass. Although bioorthogonal chemistry provides a promising platform to study the lignification process, methodologies that simultaneously detect multiple chemical reporters in living organisms are still scarce. Here, we have developed an original bioorthogonal labeling imaging sequential strategy (BLISS) to visualize and analyze the incorporation of both p-hydroxyphenyl (H) and guaiacyl (G) units into lignin in vivo with a combination of strain-promoted and copper-catalyzed azide-alkyne cycloadditions. On our path to BLISS, we designed a new azide-tagged monolignol reporter for H units in metabolic lignin engineering and used it in conjunction with an alkyne-tagged G unit surrogate to study lignification dynamics in flax. Here, we show that BLISS provides precise spatial information on the zones of active lignification and reveals polarization in single-cell lignification dynamics.

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.

Pretreatments, conditioned medium and co-culture increase the incidence of somatic embryogenesis of different Cichorium species.

Somatic embryogenesis (SE) in Cichorium involves dedifferentiation and redifferentiation of single cells and can be induced by specific in vitro culture conditions. We have tested the effect of various treatments on the incidence of SE (ISE) of an interspecific embryogenic hybrid (C. endivia x C. intybus) and of different commercial chicories (C. endivia and C. intybus) that are typically recalcitrant to SE in standard culture conditions. We found that the ISE of the hybrid is significantly increased by pretreatment of tissues by submersion in solutions of glycerol, abscisic acid, spermine, putrescine or of combinations of these compounds. Interestingly, the most efficient of these pretreatments also had an unexpectedly high effect on the ISE of the C. intybus cultivars. The ISE of the hybrid and of the commercial chicories were increased when explants were co-cultured with highly embryogenic chicory explants or when they were cultured in conditioned medium. These observations established that unidentified SE-promoting factors are released in the culture medium. HPLC analyses of secreted Arabino-Galactan Proteins (AGPs), which are known to stimulate SE, did not allow identifying a fraction containing differentially abundant AGP candidates. However, pointing to their role in promoting SE, we found that the hybrid had a drastically higher ISE when amino sugars and L-Proline, the putative precursors of secreted AGPs, were both added to the medium.

EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar.

• The eucalyptus R2R3 transcription factor, EgMYB1 contains an active repressor motif in the regulatory domain of the predicted protein. It is preferentially expressed in differentiating xylem and is capable of repressing the transcription of two key lignin genes in vivo. • In order to investigate in planta the role of this putative transcriptional repressor of the lignin biosynthetic pathway, we overexpressed the EgMYB1 gene in Arabidopsis and poplar. • Expression of EgMYB1 produced similar phenotypes in both species, with stronger effects in transgenic Arabidopsis plants than in poplar. Vascular development was altered in overexpressors showing fewer lignified fibres (in phloem and interfascicular zones in poplar and Arabidopsis, respectively) and reduced secondary wall thickening. Klason lignin content was moderately but significantly reduced in both species. Decreased transcript accumulation was observed for genes involved in the biosynthesis of lignins, cellulose and xylan, the three main polymers of secondary cell walls. Transcriptomic profiles of transgenic poplars were reminiscent of those reported when lignin biosynthetic genes are disrupted. • Together, these results strongly suggest that EgMYB1 is a repressor of secondary wall formation and provide new opportunities to dissect the transcriptional regulation of secondary wall biosynthesis.

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.).

BACKGROUND: In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies 1 showed that the use of the beta-D-glucosyl Yariv reagent (beta-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that beta-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation 2. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used beta-GlcY to block SE in order to identify genes potentially involved in this process. RESULTS: Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. beta-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by beta-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by beta-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. CONCLUSION: The use of two different chicory genotypes differing in their responsiveness to SE induction, together with beta-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

Immunolocalization of non-symbiotic hemoglobins during somatic embryogenesis in chicory.

Hemoglobins are ancient O(2)-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum x C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.

Glutathione-S-Transferase is Detected During Somatic Embryogenesis in Chicory.

Glutathione S-tranferases (GSTs) are a heterogeneous family of proteins, which perform diverse pivotal catalytic and non-enzymatic functions during plant development and in plant stress responses. Previous studies have shown that a GST activity (EC 2.5.1.18) is closely linked with the precocious phases of somatic embryogenesis in leaf tissues of an interspecific chicory hybrid (Cichorium intybus L. var. sativa x C. endivia L. var. latifolia). In order to learn more about the involvement of this enzyme in this process, in situ-hybridization as well as immunolocalization were performed in parallel. GST-mRNAs and proteins were colocalized in small veins, particularly in young protoxylem cell walls. During cell reactivation, the in situ and protein signals became less intense and were associated with chloroplasts. The GST-mRNAs and corresponding proteins were not always colocalized in the same tissues. While high amounts of transcripts could be detected in multicellular embryos, the proteins were not well labeled. Our results indicated that GSTs belong to a complex anti-oxidant mechanism within the cell, and also at the cell wall level. GSTs presence in reactivated cell and multicellular embryos is discussed in relation to redox cell status.

The novel gene CpEdi-9 from the resurrection plant C. plantagineum encodes a hydrophilic protein and is expressed in mature seeds as well as in response to dehydration in leaf phloem tissues.

The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants. CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.