A barley cellulose synthase-like CSLH gene mediates (1,3;1,4)-beta-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)-beta-D-glucan (beta-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 HvCSLH1, 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 beta-glucan in its walls. Transgenic Arabidopsis lines that had detectable amounts of the epitope-tagged HvCSLH1 protein accumulated beta-glucan in their walls. The presence of beta-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 beta-glucan hydrolase. Immuno-EM also was used to show that the epitope-tagged 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 beta-glucan. Thus both the CSLH and CSLF families contribute to beta-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.

Fig. 1.

(A) Schematic of the transfer DNA of the HvCSLH1::pGBW15 construct used in gain-of-function experiments in Arabidopsis. Arrows indicate the direction of gene transcription. (B) Northern blot showing transcript levels in mature leaves of HvCSLH1 transgenic T₁ plants. (Upper) X-ray film exposure. (Lower) Corresponding ethidium bromide-stained gel. The observed 2.5-kb transcript corresponds to the expected size of the tagged HvCSLH1 mRNA. (C) Western blot showing 3×HA-tagged HvCSLH1 protein levels in 3-week-old pooled seedlings of HvCSLH1 transgenic T₂ lines. Mixed microsomal membrane protein (30 μg/lane) was loaded, and blots were probed with the anti-HA antibody. (B and C) Numbers refer to transgenic lines. Col-0 is the wild-type untransformed line. Lines 8 and 14 are from the same blot; all other lines are from different blots.

Fig. 2.

Transmission electron micrographs showing detection of β-glucan in walls of HvCSLH1-expressing lines with a β-glucan antibody (20). (A–C) Lines 8, 16, and 11, respectively; (D) wild-type Col-0 control; (E) line 6. A and D show cells of the vascular bundle; B and C show mesophyll cells; E shows epidermal cells. Scale bar represents 0.5 μm in A–C and E and 1 μm in D.

Fig. 3.

HPAEC profiles of oligosaccharides released upon β-glucan hydrolase digestion of total walls (AIR) prepared from HvCSLH1 transgenic Arabidopsis lines 16–1 and 16–2 (A) and174 and 208 (B). Samples were taken from plants at 145 d (16–1, leaf; 16–2: leaf and stem), 21 d (174, entire seedlings) and 56 d (208, leaf), respectively. AIR positive control: barley mature leaf (entire sheath); AIR negative control: wild-type Arabidopsis Col-0 (mature leaves). AIR samples were loaded at similar concentrations. Laminaribiose (G3G_R) and a cellodextrin series (degree of polymerization [DP] 3–6) were used as standards. Arabidopsis samples are plotted on the left y-axis; standards and positive control are plotted on the right y-axis. G3G_R (DP2), G4G3G_R (3-O-β-cellobiosyl-Glc, DP3), and G4G4G3G_R (3-O-β-cellotriosyl-Glc, DP4) peaks are indicated, as are the cellodextrins.

Fig. 4.

Transmission electron micrographs showing the detection of the 3×HA-tagged HvCSLH1 protein by a gold-labeled anti-HA antibody in sections of high-pressure-frozen leaves of Arabidopsis transgenic line 11. (A and B) Mesophyll cells. cw, cell wall; er, endoplasmic reticulum; G, Golgi body; pm, plasma membrane; v, vacuole. Scale bar represents 0.5 μm in A and 0.2 μm in B. Black arrows indicate Golgi-associated vesicle labeling; white arrow indicates plasma membrane.

Fig. 5.

HvCSLH1 expression in barley as determined by qPCR and in situ PCR analyses. (A) Normalized levels of HvCSLH1 transcript in a range of tissues. Control genes for normalization were GAPDH, cyclophilin, and α-tubulin. (B) Normalized levels of HvCSLH1 transcript in developing endosperm 0–24 days after pollination. Control genes were GAPDH, α-tubulin, and EF1a. (C) Normalized levels of HvCSLH1 transcript in 10-day-old first leaf. Control genes were GAPDH, cyclophilin, and HSP70. Error bars on qPCR plots indicate SD. (D–F) In situ PCR images of the maturing zone of a 7-day-old first leaf using probes for 18S RNA (positive control, D), HvCSLH1 (F), and a negative control (E). Scale bar represents 100 μm.