Mixed-Linkage Glucan Is the Main Carbohydrate Source and Starch Is an Alternative Source during Brachypodium Grain Germination.

Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

Advances in Cell Wall Matrix Research with a Focus on Mixed-Linkage Glucan.

Mixed ß(1,3;1,4)-linkage glucan (MLG) is commonly found in the monocot lineage, at particularly high levels in the Poaceae family, but also in the evolutionally distant genus, Equisetum. MLG has several properties that make it unique from other plant cell wall polysaccharides. It consists of ß1,4-linked polymers of glucose interspersed with ß1,3-linkages, but the presence of ß1,3-linkages provides quite different physical properties compared to its closest form of the cell wall component, cellulose. The mechanisms of MLG biosynthesis have been investigated to understand whether single or multiple enzymes are required to build mixed linkages in the glucan chain. Currently, MLG synthesis by a single enzyme is supported by mutagenesis analyses of cellulose synthase-like F6, the major MLG synthase, but further investigation is needed to gather mechanistic insights. Because of transient accumulation of MLG in elongating cells and vegetative tissues, several hypotheses have been proposed to explain the role of MLG in the plant cell wall. Studies have been carried out to identify gene expression regulators during development and light cycles as well as enzymes involved in MLG organization in the cell wall. A role of MLG as a storage molecule in grains is evident, but the role of MLG in vegetative tissues is still not well understood. Characterization of a cell wall component is difficult due to the complex heterogeneity of the plant cell wall. However, as detailed in this review, recent exciting research has made significant impacts in the understanding of MLG biology in plants.

Activity and Action of Cell-Wall Transglycanases.

Transglycanases (endotransglycosylases) are enzymes that "cut and paste" polysaccharide chains. Several transglycanase activities have been discovered which can cut (i.e., use as donor substrate) each of the major hemicelluloses [xyloglucan, mannans, xylans, and mixed-linkage ß-glucan (MLG)], and, as a recent addition, cellulose. These enzymes may play interesting roles in adjusting the wall's physical properties, influencing cell expansion, stem strengthening, and fruit softening.Activities discussed include the homotransglycanases XET (xyloglucan endotransglucosylase, i.e., xyloglucan-xyloglucan endotransglycosylase), trans-ß-mannanase (mannan -mannan endotransglycosylase), and trans-ß-xylanase (xylan -xylan endotransglucosylase), plus the heterotransglycanases MXE (MLG -xyloglucan endotransglucosylase) and CXE (cellulose -xyloglucan endotransglucosylase).Transglycanases acting on polysaccharide donor substrates can utilize small, labeled oligosaccharides as acceptor substrates, generating easily recognizable polymeric labeled products. We present methods for extracting transglycanases from plant tissues and assaying them in vitro, either quantitatively in solution assays or by high-throughput dot-blot screens. Both radioactively and fluorescently labeled substrates are mentioned. A general procedure (glass-fiber blotting) is illustrated by which proposed novel transglycanase activities can be tested for.In addition, we describe strategies for detecting transglycanase action in vivo. These methods enable the quantification of, separately, XET and MXE action in Equisetum stems. Related methods enable the tissue distribution of transglycanase action to be visualized cytologically.

Comprehensive cross-genome survey and phylogeny of glycoside hydrolase family 16 members reveals the evolutionary origin of EG16 and XTH proteins in plant lineages.

Carbohydrate-active enzymes (CAZymes) are central to the biosynthesis and modification of the plant cell wall. An ancient clade of bifunctional plant endo-glucanases (EG16 members) was recently revealed and proposed to represent a transitional group uniting plant xyloglucan endo-transglycosylase/hydrolase (XTH) gene products and bacterial mixed-linkage endo-glucanases in the phylogeny of glycoside hydrolase family 16 (GH16). To gain broader insights into the distribution and frequency of EG16 and other GH16 members in plants, the PHYTOZOME, PLAZA, NCBI and 1000 PLANTS databases were mined to build a comprehensive census among 1289 species, spanning the broad phylogenetic diversity of multiple algae through recent plant lineages. EG16, newly identified EG16-2 and XTH members appeared first in the green algae. Extant EG16 members represent the early adoption of the ß-jellyroll protein scaffold from a bacterial or early-lineage eukaryotic GH16 gene, which is characterized by loop deletion and extension of the N terminus (in EG16-2 members) or C terminus (in XTH members). Maximum-likelihood phylogenetic analysis of EG16 and EG16-2 sequences are directly concordant with contemporary estimates of plant evolution. The lack of expansion of EG16 members into multi-gene families across green plants may point to a core metabolic role under tight control, in contrast to XTH genes that have undergone the extensive duplications typical of cell-wall CAZymes. The present census will underpin future studies to elucidate the physiological role of EG16 members across plant species, and serve as roadmap for delineating the closely related EG16 and XTH gene products in bioinformatic analyses of emerging genomes and transcriptomes.