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Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3'-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends > or = 1230 bp from the 3' end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-(14)C-Glc into cellulose and into mixed-linkage (1 --> 3),(1 --> 4)-beta-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis.

Cell walls of the Arabidopsis mutant mur2 contain less than 2% of the wild-type amount of fucosylated xyloglucan because of a point mutation in the fucosyltransferase AtFUT1. The mur2 mutation eliminates xyloglucan fucosylation in all major plant organs, indicating that Arabidopsis thaliana fucosyltransferase 1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase activity in Arabidopsis. Despite this alteration in structure, mur2 plants show a normal growth habit and wall strength. In contrast, Arabidopsis mur1 mutants that are defective in the de novo synthesis of l-fucose exhibit a dwarfed growth habit and decreased wall strength [Reiter, W. D., Chapple, C. & Somerville, C. R. (1993) Science 261, 1032-1035]. Because the mur1 mutation affects several cell wall polysaccharides, whereas the mur2 mutation is specific to xyloglucan, the phenotypes of mur1 plants appear to be caused by structural changes in fucosylated pectic components such as rhamnogalacturonan-II. The normal growth habit and wall strength of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan fucosylation in facilitating xyloglucan-cellulose interactions or in modulating growth regulator activity.

Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1-->3), (1-->4)-beta-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa and was separated from a much larger (1-->3)-beta-D-glucan (callose) by gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were separated by HPLC and GLC. The trisaccharide beta-D-Glcp-(1-->4)-beta-D-Glcp-(1-->3)-D-Glc, the tetrasaccharide [beta-D-Glcp-(1-->4)]2-beta-D-Glcp-(1-->3)-D-Glc, and longer cellodextrin-(1-->3)-D-Glc oligosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhanced synthesis of MG, presumably by removing inhibitory compounds. The Golgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was restored by addition of either MgCl2 or, to a lesser extent, MnCl2. Synthesis continued for well over 1 h; addition of KOH to raise the pH from 7.2 to 8.0 during the reaction increased the rate of synthesis, which indicates that a transmembrane pH gradient may facilitate synthesis of MG.