A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose.

Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.

The shape of plants depends on cellulose, a biopolymer that self-assembles into crystalline, inextensible microfibrils (CMFs) upon synthesis at the plasma membrane by multi-enzyme cellulose synthase complexes (CSCs). CSCs are displaced in directions predicted by underlying parallel arrays of cortical microtubules, but CMFs remain transverse in cells that have lost the ability to expand unidirectionally as a result of disrupted microtubules. These conflicting findings suggest that microtubules are important for some physico-chemical property of cellulose that maintains wall integrity.

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites.

WallGen, software to construct layered cellulose-hemicellulose networks and predict their small deformation mechanics.

We understand few details about how the arrangement and interactions of cell wall polymers produce the mechanical properties of primary cell walls. Consequently, we cannot quantitatively assess if proposed wall structures are mechanically reasonable or assess the effectiveness of proposed mechanisms to change mechanical properties. As a step to remedying this, we developed WallGen, a Fortran program (available on request) building virtual cellulose-hemicellulose networks by stochastic self-assembly whose mechanical properties can be predicted by finite element analysis.

Features of the primary wall CESA complex in wild type and cellulose-deficient mutants of Arabidopsis thaliana.

Evidence from genetics, co-precipitation and bimolecular fluorescence complementation suggest that three CESAs implicated in making primary wall cellulose in Arabidopsis thaliana form a complex. This study shows the complex has a M(r) of approximately 840 kDa in detergent extracts and that it has undergone distinctive changes when extracts are prepared from some cellulose-deficient mutants. The mobility of CESAs 1, 3, and 6 in a Triton-soluble microsomal fraction subject to blue native polyacrylamide gel electrophoresis was consistent with a M(r) of about 840 kDa.

Arabidopsis dynamin-like protein DRP1A: a null mutant with widespread defects in endocytosis, cellulose synthesis, cytokinesis, and cell expansion.

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology.

A mutation in an Arabidopsis ribose 5-phosphate isomerase reduces cellulose synthesis and is rescued by exogenous uridine.

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase.

Chimeric proteins suggest that the catalytic and/or C-terminal domains give CesA1 and CesA3 access to their specific sites in the cellulose synthase of primary walls.

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site.

Cortical microtubule arrays lose uniform alignment between cells and are oryzalin resistant in the Arabidopsis mutant, radially swollen 6.

The coordinated expansion of cells is essential to the formation of correctly shaped plant tissues and organs. Members of the radially swollen (rsw) class of temperature-sensitive arabidopsis mutants were isolated in a screen for reduced anisotropic expansion, by selecting plants with radially swollen root tips. Here we describe rsw6, in which cortical microtubules in the root epidermis are well organized in parallel arrays within cells, but neighboring cells frequently contain arrays differing in their mean orientation by up to 90 degrees.

Genes encoding ADP-ribosylation factors in Arabidopsis thaliana L. Heyn.; genome analysis and antisense suppression.

Vesicle trafficking delivers proteins to intracellular and extracellular compartments, cellulose synthase to the plasma membrane, and non-cellulosic polysaccharides to the cell wall. The Arabidopsis genome potentially encodes 19 proteins with sequence similarities to ARFs (ADP-ribosylation factors) and its relatives such as ARLs (ARF-like proteins). ARFs are essential for vesicle coating and uncoating in all eukaryotic cells, while ARLs play more diverse roles.

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization.

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils.

Mutation or drug-dependent microtubule disruption causes radial swelling without altering parallel cellulose microfibril deposition in Arabidopsis root cells.

As critical determinants of growth anisotropy in plants, cortical microtubules are thought to constrain the movement of cellulose synthase complexes and thus align newly deposited cellulose microfibrils. We tested this cellulose synthase constraint model using the temperature-sensitive mor1-1 mutant of Arabidopsis. Contrary to predictions, the disruption of cortical microtubules in mor1-1 root epidermal cells led to left-handed root twisting and radial swelling but did not alter the transverse orientation of cellulose microfibrils.

The cellulose-deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II, an enzyme processing N-glycans during ER quality control.

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 alpha-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.

Towards the mechanism of cellulose synthesis.

Recent research has provided insights into how plants make cellulose - the major structural material of their cell walls and the basis of the cotton and wood fibre industries. Arabidopsis thaliana mutants impaired in cellulose production are defective in genes encoding membrane-bound glycosyltransferases, an endo-1,4-beta-glucanase and several enzymes involved in the N-glycosylation and quality-control pathways of the endoplasmic reticulum. The glycosyltransferases form the rosette terminal complexes seen in plasma membranes making cellulose.

Mutant alleles of Arabidopsis RADIALLY SWOLLEN 4 and 7 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.

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis.

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.