Higher plants resist the forces of gravity and powerful lateral forces through the cumulative strength of the walls that surround individual cells. These walls consist mainly of cellulose, noncellulosic polysaccharides and lignin, in proportions that depend upon the specific functions of the cell and its stage of development. Spatially and temporally controlled heterogeneity in the physicochemical properties of wall polysaccharides is observed at the tissue and individual cell levels, and emerging in situ technologies are providing evidence that this heterogeneity also occurs across a single cell wall. We consider the origins of cell wall heterogeneity and identify contributing factors that are inherent in the molecular mechanisms of polysaccharide biosynthesis and are crucial for the changing biological functions of the wall during growth and development. We propose several key questions to be addressed in cell wall biology, together with an alternative two-phase model for the assembly of noncellulosic polysaccharides in plants.

Arabidopsis was transformed with double-stranded RNA interference (dsRNAi) constructs designed to silence three putative callose synthase genes: GLUCAN SYNTHASE-LIKE5 ( GSL5 ), GSL6 , and GSL11 . Both wound callose and papillary callose were absent in lines transformed with GSL5 dsRNAi and in a corresponding sequence-indexed GSL5 T-DNA insertion line but were unaffected in GSL6 and GSL11 dsRNAi lines. These data provide strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation. Deposition of callosic plugs, or papillae, at sites of fungal penetration is a widely recognized early response of host plants to microbial attack and has been implicated in impeding entry of the fungus. Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis. Paradoxically, the absence of callose in papillae or haustorial complexes correlated with the effective growth cessation of several normally virulent powdery mildew species and of Peronospora parasitica .

A characteristic feature of grasses and commercially important cereals is the presence of (1,3;1,4)-beta-d-glucans in their cell walls. We have used comparative genomics to link a major quantitative trait locus for (1,3;1,4)-beta-d-glucan content in barley grain to a cluster of cellulose synthase-like CslF genes in rice. After insertion of rice CslF genes into Arabidopsis, we detected (1,3;1,4)-beta-d-glucan in walls of transgenic plants using specific monoclonal antibodies and enzymatic analysis. Because wild-type Arabidopsis does not contain CslF genes or have (1,3;1,4)-beta-d-glucans in its walls, these experiments provide direct, gain-of-function evidence for the participation of rice CslF genes in (1,3;1,4)-beta-d-glucan biosynthesis.

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.

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The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca 2+ transporters, CAX1 (Ca 2+ /H + -antiporter), ACA4, and ACA11 (Ca 2+ -ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO 2 assimilation, and leaf growth rate; increased transcript abundance of other Ca 2+ transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca 2+ ], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca 2+ ] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

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.

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.