Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Université de Lille

Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species.

Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

The phenylpropanoid pathway in plants is responsible for the biosynthesis of a huge amount of secondary metabolites derived from phenylalanine and tyrosine. Both flavonoids and lignins are synthesized at the end of this very diverse metabolic pathway, as well as many intermediate molecules whose precise biological functions remain largely unknown. The diversity of these molecules can be further increased under the action of UDP-glycosyltransferases (UGTs) leading to the production of glycosylated hydroxycinnamates and related aldehydes, alcohols and esters. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. (De)-glycosylation therefore represents an extremely important regulation point in phenylpropanoid homeostasis. In this article we review recent knowledge on the enzymes involved in regulating phenylpropanoid glycosylation status and availability in different subcellular compartments. We also examine the potential link between monolignol glycosylation and lignification by exploring co-expression of lignin biosynthesis genes and phenolic (de)glycosylation genes. Of the different biological roles linked with their particular chemical properties, phenylpropanoids are often correlated with the plant's stress management strategies that are also regulated by glycosylation. UGTs can for instance influence the resistance of plants during infection by microorganisms and be involved in the mechanisms related to environmental changes. The impact of flavonoid glycosylation on the color of flowers, leaves, seeds and fruits will also be discussed. Altogether this paper underlies the fact that glycosylation and deglycosylation are powerful mechanisms allowing plants to regulate phenylpropanoid localisation, availability and biological activity.

Glycosylation Is a Major Regulator of Phenylpropanoid Availability and Biological Activity 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.

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

Interest in hemp (Cannabis sativa L.) is increasing due to the development of a new range of industrial applications based on bast fibers. However the variability of bast fiber yield and quality represents an important barrier to further exploitation. Primary and secondary fiber content was examined in two commercial hemp varieties (Fedora 17, Santhica 27) grown under contrasted sowing density and irrigation conditions. Both growing conditions and hemp varieties impact stem tissue architecture with a large effect on the proportion of secondary fibers but not primary fibers. Attenuated total reflectance infrared spectroscopy allowed the discrimination of manually-isolated native primary fibers and secondary fibers but did not reveal any clustering according to growing conditions and variety. Infrared data were confirmed by wet chemistry analyses that revealed slight but significant differences between primary and secondary fiber cell wall composition. Infrared spectroscopy of technical fibers obtained after mechanical defibering revealed differences with native primary, but not secondary fibers and also discriminated samples obtained from plants grown under different conditions. Altogether the results suggested that the observed variability of hemp technical fibers could be partially explained by i) differences in secondary fiber production and ii) differential behavior during mechanical defibering resulting in unequal separation of primary and secondary fibers.

Changes in hemp secondary fiber production related to technical fiber variability revealed by light microscopy and attenuated total reflectance Fourier transform infrared spectroscopy.

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.

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

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.

Simon Hawkins

UGSF – Unité de Glycobiologie Structurale et Fonctionnelle,
CNRS,
UMR 8576,
UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, CNRS, UMR 8576

Université de Lille

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Simon Hawkins

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Université de Lille

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

UGSF – Unité de Glycobiologie Structurale et Fonctionnelle,
CNRS,
UMR 8576,
UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, CNRS, UMR 8576