Most recent achievements in plant cell wall biosynthesis research resulted from the anal­ysis of Arabidopsis thaliana cell wall phenotype mutants and the availability of genome sequence data (see detailed review in Chapter 5) (3, 4). The cellulose synthase complex (CelS), known as rosettes in higher plants, was first observed using electron microscopy and the freeze-fracture sample preparation technique. Rosettes appear in hexagonal geometry with a honeycomb pattern arrayed in the plasma membrane (5,6). Rosettes are believed to be responsible for the synthesis ofelementary fibrils in most current plant cell wall biosynthesis models (7). More recently, mutant analyses [reviewed by Doblin and coworkers (4)] and immunolabeling (8) have confirmed that these rosettes are composed of cellulose synthase (CesA) proteins, and that at least three types of CesA isoforms (a1, a2, and p) are required

Cellulose elementary fibril

6 core chains

Figure 3.4 Model of plant cell wall cellulose elementary fibril and its synthesis. In this model, at least three types of cellulose synthases (CesA subunits, a1, a2, and p) are needed to spontaneously assemble the rosettes that composed of 6 x 6 CesA enzymes synthesizing 36-chain cellulose elementary fibril. The rosettes may also form arrays in the cell membrane, in this case, a number of rosettes synthesize a bundle of elementary fibril, the macrofibril. The estimated dimensions of elementary fibril are 3 x 5.5nm that agrees with direct measurement using atomic force microscopy (see also Figure 3.9). The depiction of the glucan chains is based generally on an X-ray structure of cellulose Ip. It has been proposed that the cellulose elementary fibril may contain three groups of glucan chains: in group C1 (red) there are 6 crystalline chains;in group C2 (green) there are 12 sub-crystalline chains with a small degree of disorder; and in group C3 (blue) there are 18 surface chains that are sub-crystalline with a large degree of disorder. (Modified from Ding and Himmel, 2006;Himmel etal., 2007) (Reproduced in color as Plate 1.) for the spontaneous assembly of single rosettes. The next question is — how many CesA proteins are assembled into single rosettes?

A model of rosettes has been recently proposed (9) (see Figure 3.4) in which three types of interactions are needed for the spontaneous assembly of rosettes in the plasma membrane, these are p-p, at-p, and a2-p. The p-p interaction facilitates the assembly of six subunits into rosettes, as well as the array of rosettes in plasma membrane. The six subunits of rosettes are identical and each rosette is composed of one molecule of at, two molecules of a2, and three molecules of p. This simplified rosette model seems probable because of the proposed at positioning and the fewer types of interactions required for the rosette assembly in plasma membrane. There is no direct biochemical evidence yet to confirm how the rosettes are assembled, probably the zinc finger domains of CesA proteins, plasma

membrane, and microtubules are all involved. During rosette assembly, the a1 molecule from each asymmetric subunit is always indexed to the center of the rosettes to ensure the correct positioning of each of the three types of CesA proteins. The rosette assembly probably starts with the dimerization (p-p) of the N-terminal zinc finger domain of CesAs. The next step is an a-p interaction, with each a isoform interacting with two p isoforms. Here as well, there is no direct evidence yet to prove how cells control these interactions among the catalytic units. One possibility is that different protein-protein interactions (i. e., p-p and a-p) occur under different physiological conditions or in different locations in the cell. Experiments of in vitro assembly or in vivo labeling of co-expressed CesA proteins might confirm this hypothesis. Furthermore, having multiple CesA proteins co-expressed in the same cell does not necessarily mean that they are all assembled into the same rosettes (10). Each position (i. e., at, a2, and p) in different rosettes may have different CesA proteins or different CesA proteins may fit in the same type of position in one rosette. For example, there are three p positions in one subunit and 18 positions in one rosette (the p position might be occupied by different CesA enzymes).

Most researchers agree that cellulose is synthesized in the plasma membrane, whereas hemicelluloses are assembled and secreted from the Golgi vesicles (11). Figure 3.5 is a schematic diagram showing cellulose being synthesized by the rosettes that comprise 36 CesA

enzymes. This enzyme complex thus produces 36 (3 -1,4-glucan chains, which simultaneously coalesce to form the cellulose elementary fibril. Hemicelluloses are synthesized in Golgi apparatus and secreted to cell wall. The hemicellulose particle interacts with the surface of newly synthesized cellulose elementary fibrils that usually form a ribbon-like bundle (the macrofibril) (9). During cell wall expansion, the macrofibril is thought to split into single elementary fibrils, the hemicellulose particles unfold and layer upon the microfibrils where some fraction of these polysaccharides start to coat the cellulose surface though numerous hydrogen bonds.