Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall–specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function

In a screen to identify novel cellulose deficient mutants, three lines were shown to be allelic and define a novel complementation group, irregular xylem5 (irx5). IRX5 was cloned and encodes a member of the CesA family of cellulose synthase catalytic subunits (AtCesA4). irx5 plants have an identical phenotype to previously described mutations in two other members of this gene family (IRX1 and IRX3). IRX5, IRX3, and IRX1 are coexpressed in exactly the same cells, and all three proteins interact in detergent solubilized extracts, suggesting that three members of this gene family are required for cellulose synthesis in secondary cell walls. The association of IRX1 and IRX3 was reduced to undetectable levels in the absence of IRX5. Consequently, these data suggest that IRX5, IRX3, and IRX1 are all essential components of the cellulose synthesizing complex and the presence of all three subunits is required for the correct assembly of this complex.

SummaryPrevious studies using co-expression analysis have identified a large number of genes likely to be involved in secondary cell-wall formation. However, the function of very few of these genes is known. We have studied the cell-wall phenotype of irx7, irx8 and irx9, three previously described irregular xylem (irx) mutants, and irx14 and parvus-3, which we now show also to be secondary cell-wall mutants. All five mutants, which have mutations in genes encoding putative glycosyltransferases, exhibited large decreases in xylan. In addition, all five mutants were found to have the same specific defect in xylan structure, retaining MeGlcUA but lacking GlcUA side branches. Polysaccharide analysis by carbohydrate gel electrophoresis (PACE) was used to determine the xylan structure in Arabidopsis, and revealed that side branches are added to approximately one in every eight xylose residues. Interestingly, this ratio is constant in all the lines analysed despite the wide variation in xylan content and the absence of GlcUA branches. Xylanase digestion of xylan from wild-type plants released a short oligosaccharide sequence at the reducing end of the xylan chain. MALDI-TOF MS analysis indicated that this sequence of sugars was absent in xylan from irx7, irx8 and parvus-3 mutants, but was present in irx9 and irx14. This is consistent with previous NMR analysis of xylan from irx7, irx8 and irx9, and suggests that PARVUS may be involved in the synthesis of a xylan primer whereas IRX14 may be required to synthesize the xylan backbone. This hypothesis is supported by assays showing that irx9 and irx14 are both defective in incorporation of radiolabel from UDP 14 C-xylose. This study has important implications for both our understanding of xylan biosynthesis and the functional analysis of cell-wall biosynthesis genes.

Recessive mutations at three loci cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis.These irregular xylem (irx) mutations were identified by screening plants from a mutagenized population by microscopic examination of stem sections. The xylem cell defect was associated with an up to eightfold reduction in the total amount of cellulose in mature inflorescence stems. The amounts of cell wall-associated phenolics and polysaccharides were unaffected by the mutations. Examination of the cell walls by using electron microscopy demonstrated that the decreases in cellulose content of irx lines resulted in an alteration of the spatial organization of cell wall material.This suggests that a normal pattern of cellulose deposition may be required for assembly of lignin or polysaccharides. The reduced cellulose content of the stems also resulted in a decrease in stiffness of the stem material. This is consistent with the irregular xylem phenotype and suggests that the walls of irx plants are not resistant to compressive forces. Because lignin was implicated previously as a major factor in resistance to compressive forces, these results suggest either that cellulose has a direct role in providing resistance to compressive forces or that it is required for the development of normal lignin structure. The irx plants had a slight reduction in growth rate and stature but were otherwise normal in appearance. The mutations should be useful in facilitating the identification of factors that control the synthesis and deposition of cellulose and other cell wall components.

All plant tissue is ultimately derived from the meristems, and the molecular mechanisms that control growth of apical meristems have been widely studied (reviewed in). In contrast, much less attention has been paid to vascular meristems, such as the cambium and procambium, even though these meristems are the source of woody tissue and therefore generate the majority of plant biomass. Although biomass may represent a novel source of renewable energy, little is known about the molecular regulation of vascular-meristem activity. The vascular meristems participate in a highly ordered developmental process with a very prominent polarity. This polarity results in precisely orientated divisions of meristematic initials that generate files of cells, which differentiate into highly specialized and spatially separated xylem and phloem cells. The factors that are necessary to establish and maintain this polarity remain unknown. This manuscript describes the identification of the PXY mutant in which the spatial organization of vascular development is lost and the xylem and phloem are partially interspersed. The PXY gene encodes for a receptor-like kinase (RLK) that defines a novel role for RLKs in the meristem where it functions to maintain the cell polarity required for the orientation of cell division during vascular development.