Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.Cellulose, a polymer of ␤(1-4)-linked glucose, is the major component of most plant cell walls. In higher plants cellulose is synthesized at the plasma membrane by a very large complex that is believed to simultaneously synthesize many individual glucose chains that hydrogen bond together to form a microfibril (1-4). The cellulose synthase complex (CSC) 2 has been visualized in freeze fracture studies as a rosette structure resembling a hexamer with six distinct lobes (5-7). Most estimates suggest that each lobe of the rosette is itself a hexamer, such that the CSC is a 36-mer (4,8,9). Although the CSC is likely to be a major factor in determining microfibril structure, very little information is known about its organization. The only components of the CSC that have been identified to date are the cellulose synthase A (CESA) proteins that are presumed to be the catalytic subunits of the CSC (1, 4, 10 -12).The CESA family has been most intensively studied in Arabidopsis, and these studies have utilized the characterization of a wide range of mutants (4, 11). In primary cell walls a number of CESA proteins have been shown to be required for cellulose synthesis. Both CESA1 and CESA3 are essential (13-17). In contrast, CESA2, CESA5, and CESA9 appear to be partially redundant with CESA6 (18, 19). The situation is different in the secondary cell walls of developing xylem vessels where three nonredundant CESA proteins are required for cellulose synthesis: CESA4 (20), CESA7 (21), and CESA8 (22). Mutations compromising the function of any one of these t...

SUMMARYThere are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.

White campion (Silene latifolia) is one of the few examples of plants with separate sexes and with X and Y sex chromosomes. The presence or absence of the Y chromosome determines which type of reproductive organs--male or female--will develop. Recently, we characterized the first active gene located on a plant Y chromosome, SlY1, and its X-linked homolog, SlX1. These genes encode WD-repeat proteins likely to be involved in cell proliferation. Here, we report the characterization of a novel Y-linked gene, SlY4, which also has a homolog on the X chromosome, SlX4. Both SlY4 and SlX4 potentially encode fructose-2,6-bisphosphatases. A comparative molecular analysis of the two sex-linked loci (SlY1/SlX1 and SlY4/SlX4) suggests selective constraint on both X- and Y-linked genes and thus that both X- and Y-linked copies are functional. Divergence between SlY4 and SlX4 is much greater than that between the SlY1 and SlX1 genes. These results suggest that, as for human XY-linked genes, the sex-linked plant loci ceased recombining at different times and reveal distinct events in the evolutionary history of the sex chromosomes.

The cellulose synthase complex (CSC) exhibits a 6-fold symmetry and is known as a "rosette." Each CSC is believed to contain between 18 and 24 CESA proteins that each synthesize an individual glucan chain. These chains form the microfibrils that confer the remarkable structural properties of cellulose. At least three different classes of CESA proteins are essential to form the CSC. However, while organization of the CSC determines microfibril structure, how individual CESA proteins are organized within the CSC remains unclear. Parts of the plant CESA proteins map sufficiently well onto the bacterial CESA (BcsA) structure, indicating that they are likely to share a common catalytic mechanism. However, plant CESA proteins are much larger than the bacterial BcsA protein, prompting the suggestion that these plant-specific regions are important for interactions between CESA proteins and for conferring CESA class specificity. In this study, we have undertaken a comprehensive analysis of well-defined regions of secondary cell wall CESA proteins, with the aim of defining what distinguishes different CESA proteins and hence what determines the specificity of each CESA class. Our results demonstrate that CESA class specificity extends throughout the protein and not just in the highly variable regions. Furthermore, we find that different CESA isoforms vary greatly in their levels of site specificity and this is likely to be determined by the constraints imposed by their position within the CSC rather than their primary structure.Cellulose plays a central role in determining the mechanical properties of plant cell walls. It is important for both wall strength and rigidity and consequently plays an essential role in many aspects of plant growth (Delmer, 1999;Somerville, 2006). Cellulose is synthesized at the plasma membrane by a large protein complex, known as the cellulose synthase complex (CSC), which moves through the plane of the plasma membrane, simultaneously extruding many individual b-1,4-Glc chains. These chains are hydrogen bonded together to form the cellulose microfibril (Nishiyama, 2009). It is this microfibril that imparts the remarkable structural properties of cellulose that have led to it being so widely used by the plants. More recently, there has been increasing focus on using cellulose as an environmentally friendly and renewable source of biomass feedstock for biofuels (Carroll and Somerville, 2009) and other chemicals, which has stimulated interest in understanding how cellulose is synthesized.Several studies have visualized the CSC using electron microscopy of freeze fractured samples. The complex exhibits a 6-fold symmetry and is known as a "rosette" (Mueller and Brown, 1980;Haigler and Brown, 1986;Kimura et al., 1999). Despite the fact that cellulose synthase activity may be assayed in crude extracts (Lai-KeeHim et al., 2002), purification of an active CSC remains elusive. However, the crystal structure of the bacterial cellulose synthase, bacterial CESA (BcsA)/BcsB, together with the nascent glucan ...