Richard E. Williamson

Summary: rsw3 is a temperature‐sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map‐based strategy, and shows high similarity to the catalytic α‐subunits of glucosidase II from mouse, yeast and potato. These enzymes process N‐linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative β‐subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER‐retention signal) with β‐subunits in mammals and yeast. The genes encoding the putative α‐ and β‐subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose‐deficient mutants, but some – notably, production of multiple rosettes and a lack of secreted seed mucilage – are not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2(AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1⁻) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3antisense plants. In keeping with the mild phenotype compared with Rsw1⁻, reductions in total cellulose levels in antisenseCesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, likeCesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 inrsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31°C.

Cellulose microfibrils containing crystalline β-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-β-glucanases. The mutants also accumulate a readily extractable β-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2show single amino acid mutations in the putative endo-1,4-β-glucanase encoded by KOR. The F₁ from crosses betweenkor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots ofrsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported forrsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2⁻ phenotype generally resembles the Kor⁻and cellulose-deficient Rsw1⁻ phenotypes, but anther dehiscence is impaired in Rsw2-1⁻. The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where thecelA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-β-glucanase related to RSW1 and KOR, respectively.

Therswl mutant ofArabidopsis thaliana is mutated in a gene encoding a cellulose synthase catalytic subunit. Mutant seedlings produce almost as much cellulose as the wild type at 21 °C but only about half as much as the wild type at 31 °C. We used this conditional phenotype to investigate how reduced cellulose production affects growth and morphogenesis in various parts of the plant. Roots swell in all tissues at 31 °C, and temperature changes can repeatedly switch them between swollen and slender growth patterns. Dark-grown hypocotyls also swell, whereas cotyledons and rosette leaf blades are smaller, their surfaces are more irregular and their petioles shorter. Leaf trichomes swell and branch abnormally. Plants readily initiate inflorescences at 31 °C which have shorter but not fatter bolts and stomata which bulge above the uneven surface of internodes. Bolts carry the normal number of flowers, but their stigmas protrude beyond the shortened sepals and petals. Anthers dehisce normally, but self-fertilisation is reduced because the stigma is well above the anthers. Anther filaments are short and show a crumpled surface. Viable pollen develops, but female reproductive competence and postpollination development are severely impaired. We conclude that theRSW1 gene is important for cellulose synthesis in many parts of the plant and that reduced cellulose synthesis suppresses organ expansion rather than organ initiation, causes radial swelling only in the root and hypocotyl, but makes the surfaces of many organs uneven. We discuss some possible reasons to explain why different organs vary in their responses. The morphological changes suggest that RSW1 contributes cellulose to primary walls but do not yet exclude a role during secondary-wall deposition.