A barley cellulose synthase-like CSLH gene mediates (1,3;1,4)-β- d -glucan synthesis in transgenic Arabidopsis

Abstract

The walls of grasses and related members of the Poales are characterized by the presence of the polysaccharide (1,3, 1,4)-␤-Dglucan (␤-glucan). To date, only members of the grass-specific cellulose synthase-like F (CSLF) gene family have been implicated in its synthesis. Assuming that other grass-specific CSL genes also might encode synthases for this polysaccharide, we cloned HvC-SLH1, a CSLH gene from barley (Hordeum vulgare L.), and expressed an epitope-tagged version of the cDNA in Arabidopsis, a species with no CSLH genes and no ␤-glucan in its walls. Transgenic Arabidopsis lines that had detectable amounts of the epitopetagged HvCSLH1 protein accumulated ␤-glucan in their walls. The presence of ␤-glucan was confirmed by immunoelectron microscopy (immuno-EM) of sectioned tissues and chemical analysis of wall extracts. In the chemical analysis, characteristic tri-and tetra-saccharides were identified by high-performance anion-exchange chromatography and MALDI-TOF MS following their release from transgenic Arabidopsis walls by a specific ␤-glucan hydrolase. Immuno-EM also was used to show that the epitopetagged HvCSLH1 protein was in the endoplasmic reticulum and Golgi-associated vesicles, but not in the plasma membrane. In barley, HvCSLH1 was expressed at very low levels in leaf, floral tissues, and the developing grain. In leaf, expression was highest in xylem and interfascicular fiber cells that have walls with secondary thickenings containing ␤-glucan. Thus both the CSLH and CSLF families contribute to ␤-glucan synthesis in grasses and probably do so independently of each other, because there is no significant transcriptional correlation between these genes in the barley tissues surveyed.

Pattern of Deposition of Cell Wall Polysaccharides and Transcript Abundance of Related Cell Wall Synthesis Genes during Differentiation in Barley Endosperm

“…However, CSLH1 transcript levels are generally much lower than CSLF transcript levels in endosperm and in vegetative tissues. At their peak, CSLH1 transcript levels are considerably lower than both CSLF6 (,200 normalized transcripts levels; data not shown) that is steady in the early stages of endosperm development and CSLF9 that is highest at 8 DAP, declining to baseline levels by 12 DAP Doblin et al, 2009). After this time, only CSLF6 has significant levels of expression throughout the developing endosperm, suggesting that this gene is responsible for the accumulation of (1→3, 1→4)-b-D-glucan during differentiation.…”

“…Transcript profiles of members of the CslF family, that encode (1→3, 1→4)-b-D-glucan synthases Doblin et al, 2009), are shown in Figure 11C. Two genes dominate the expression profile: Transcripts of CslF9 peak early in grain development at 8 DAP and decrease to negligible levels by 12 DAP whereas the transcript levels of CslF6 shows a concurrent increase to become the dominant transcript present throughout the majority of grain development (12-38 DAP).…”

The Maize Mixed-Linkage (1→3),(1→4)-β-d-Glucan Polysaccharide Is Synthesized at the Golgi Membrane

“…This monoclonal antibody has been used to show dramatic changes in epitope abundance of (1/3),(1/4)-b-D-glucan in the cell walls of developing tissues (Meikle et al, 1994;Trethewey et al, 2005;McCann et al, 2007) and its appearance in the cell walls of Arabidopsis (Arabidopsis thaliana) following heterologous expression of genes thought to encode its synthases Doblin et al, 2009). The failure to detect (1/3),(1/4)-b-D-glucan in Golgi membranes and only in the cell wall prompted Fincher (2009) to conclude that cellodextrin oligomers of the (1/3),(1/4)-b-D-glucan may be initiated in the Golgi membrane, but the actual polymerization of the polysaccharide occurs at the plasma membrane.…”

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos

“…The family GT2 members (CslF and CslH) catalyze the biosynthesis of (1,3)(1,4)-β-D-glucan in barley (Burton et al , 2008, Burton, Wilson, 2006, Doblin, Pettolino, 2009). The mRNA levels of CslF6 (MLOC_57200) were very high (over 200 RPKM) and showed a substantial decrease at 48 h in four barley varieties (Fig.…”

“…The glucuronosyl substitution of the backbone is catalyzed by members of the glycosyltransferase family 8 (GT8) (Mortimer et al , 2010), while the -(1,3)-linked arabinosyl substitution for the HX backbone is catalyzed by members of the glycosyltransferase family 61 (GT61) (Anders et al , 2012). Members of the cellulose synthase-like family F (CslF) and cellulose synthase-like family H (CslH) (both GT2) are responsible for the biosynthesis of (1,3)(1,4)-β-D-glucans (Burton et al , 2006, Doblin et al , 2009). Furthermore, a suite of hydrolytic enzymes may regulate polysaccharide levels in germinating barley grains.…”