Mutant Alleles of Arabidopsis RADIALLY SWOLLEN 4 and RSW7 Reduce Growth Anisotropy Without Altering the Transverse Orientation of Cortical Microtubules or Cellulose Microfibrils

The anisotropic growth of plant cells depends on cell walls having anisotropic mechanical properties, which are hypothesized to arise from aligned cellulose microfibrils. To test this hypothesis and to identify genes involved in controlling plant shape, we isolated mutants in Arabidopsis thaliana in which the degree of anisotropic expansion of the root is reduced. We report here the characterization of mutants at two new loci, RADIALLY SWOLLEN 4 (RSW4) and RSW7. The radial swelling phenotype is temperature sensitive, being moderate (rsw7) or negligible (rsw4) at the permissive temperature, 19°C, and pronounced at the restrictive temperature, 30°C. After transfer to 30°C, the primary root’s elongation rate decreases and diameter increases, with all tissues swelling radially. Swelling is accompanied by ectopic cell production but swelling is not reduced when the extra cell production is eliminated chemically. A double mutant was generated, whose roots swell constitutively and more than either parent. Based on analytical determination of acid-insoluble glucose, the amount of cellulose was normal in rsw4 and slightly elevated in rsw7. The orientation of cortical microtubules was examined with immunofluorescence in whole mounts and in semi-thin plastic sections, and the orientation of microfibrils was examined with field-emission scanning electron microscopy and quantitative polarized-light microscopy. In the swollen regions of both mutants, cortical microtubules and cellulose microfibrils are neither depleted nor disoriented. Thus, oriented microtubules and microfibrils themselves are insufficient to limit radial expansion; to build a wall with high mechanical anisotropy, additional factors are required, supplied in part by RSW4 and RSW7

Temperature-Sensitive Alleles of Radially Swollen2 Link the KORRIGAN Endo-1,4-fl-Glucanase to Cellulose Synthesis and Cytokinesis

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