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.

The irregular xylem 1 ( irx1 ) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.

Cellulose synthesis in the developing xylem vessels of Arabidopsis requires three members of the cellulose synthase (CesA) gene family. In young vessels, these three proteins localize within the cell, whereas in older vessels, all three CesA proteins colocalize with bands of cortical microtubules that mark the sites of secondary cell wall deposition. In the absence of one subunit, however, the remaining two subunits are retained in the cell, demonstrating that all three CesA proteins are required to assemble a functional complex. CesA proteins with altered catalytic activity localize normally, suggesting that cellulose synthase activity is not required for this localization. Cortical microtubule arrays are required continually to maintain normal CesA protein localization. By contrast, actin microfilaments do not colocalize with the CesA proteins and are unlikely to play a direct role in their localization. Green fluorescent protein-tagged CesA reveals a novel process in which the structure and/or local environment of the cellulose synthase complex is altered rapidly.

The irregular xylem3 ( irx3 ) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3 , and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 ( rsw1) plants, irx3 plants show no increase in the accumulation of ␤ -1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases. INTRODUCTIONFor many plant cells, the cell wall is synthesized in two distinct stages. During the initial phase of cellular growth, a primary cell wall is laid down and continuously expanded by processes that include relaxation of interchain linkages and addition of new polymers and matrix materials. Cellulose usually comprises ‫ف‬ 20 to 30% of the dry weight of the primary wall (Fry, 1988). After the cessation of expansion and division, a secondary cell wall is synthesized within the bounds of the primary wall. This wall is usually much thicker than the primary wall and comprises the bulk of terrestrial biomass. Cellulose accounts for ‫ف‬ 40 to 90% of the secondary cell wall, depending upon the cell type. In some heavily thickened cells, such as xylem cells, the secondary wall also may contain a high proportion of lignin that contributes to the mechanical strength.The mechanisms involved in the synthesis of secondary cell walls are not understood in detail (Emons and Mulder, 1998). It generally is accepted that for both primary and secondary walls, the cellulose component is synthesized by enzyme complexes situated at the plasma membrane. Many freeze-fracture studies have identified plasma membrane particles known as rosettes that appear to be associated with the ends of microfibrils (Brown, 1996). The spacing of these rosettes also correlates with the distribution of the microfibrils (Giddings et al., 1980). It has been suggested that each rosette consists of a hexameric complex, which results in the synthesis of 36 ␤ -glucan chains that are thought to be present in a primary microfibril (Delmer and Amor, 1995). The differences in physical properties of primary and secondary plant cell walls are partly due to differences in the number of individual cellulose chains in the microfibril unit. In contrast to the ‫ف‬ 36 individual chains in primary microfibrils (Delmer and Amor, 1995), the secondary cell walls of some algae contain fibrils containing up to 12,000 individual...