In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are

Cellulose synthesis in plants requires beta-1,4-glucan chain initiation, elongation, and termination. The process of chain elongation is likely to be distinct from the process of chain initiation. We demonstrate that a CesA glucosyltransferase initiates glucan polymerization by using sitosterol-beta-glucoside (SG) as primer. Cotton fiber membranes synthesize sitosterol-cellodextrins (SCDs) from SG and uridine 5'-diphosphate-glucose (UDP-Glc) under conditions that also favor cellulose synthesis. The cellulase encoded by the Korrigan (Kor) gene, required for cellulose synthesis in plants, may function to cleave SG from the growing polymer chain.

Cellulose synthase (CesA) proteins are components of CesA complexes (rosettes) and are thought to catalyze the chain elongation step in glucan polymerization. Little is understood about rosette assembly, including how CesAs interact with each other or with other components within the complexes. The first conserved region at the N terminus of plant CesA proteins contains two putative zinc fingers that show high homology to the RING-finger motif. We show that this domain in GhCesA1 can bind two atoms of Zn 2؉ , as predicted by its structure. Analysis in the yeast two-hybrid system indicates that the N-terminal portions of cotton fiber GhCesA1 and GhCesA2 containing these domains can interact to form homo-or heterodimers. Although Zn 2؉ binding occurs only when the protein is in the reduced form, biochemical analyses show that under oxidative conditions, the GhCesA1 zinc-finger domain and also the full-length protein dimerize via intermolecular disulfide bonds, indicating CesA dimerization can be regulated by redox state. We also provide evidence that the herbicide CGA 325615 (Syngenta, Basel), which inhibits synthesis of crystalline cellulose and leads to a disruption of rosette architecture, may affect the oxidative state of the zinc-finger domain that is necessary for rosette stability. Taken together, these results support a model in which at least part of the process of rosette assembly and function may involve oxidative dimerization between CesA subunits.

The developing endosperm of rice (Oryza sativa, Os) synthesizes a large amount of storage proteins on the rough (r)ER. The major storage proteins, glutelins and prolamins, contain either intra or intermolecular disulfide bonds, and oxidative protein folding is necessary for the sorting of the proteins to the protein bodies. Here, we investigated an electron transfer pathway for the formation of protein disulfide bonds in the rER of the rice endosperm, focusing on the roles of the thiol-disulfide oxidoreductase, OsEro1. Confocal microscopic analysis revealed that N-glycosylated OsEro1 is localized to the rER membrane in the subaleurone cells, and that targeting of OsEro1 to the rER membrane depends on the N-terminal region from Met-1 to Ser-55. The RNAi knockdown of OsERO1 inhibited the formation of native disulfide bonds in the glutelin precursors (proglutelins) and promoted aggregation of the proglutelins through nonnative intermolecular disulfide bonds in the rER. Inhibition of the formation of native disulfide bonds was also observed in the seeds of the esp2 mutant, which lacks protein disulfide isomerase-like (PDIL)1;1, but shows enhanced OsEro1 expression. We detected the generation of H 2O2 in the rER of the WT subaleurone cells, whereas the rER-derived H2O2 levels decreased markedly in EM49 homozygous mutant seeds, which have fewer sulfhydryl groups than the WT seeds. Together, we propose that the formation of native disulfide bonds in proglutelins depends on an electron transfer pathway involving OsEro1 and OsPDIL.protein disulfide isomerase ͉ seed storage protein ͉ esp2 ͉ hydrogen peroxide ͉ protein body