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. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

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. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis.

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. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene.

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. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3(WT) protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3(rsw5)) does not.

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. Putative beta-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with beta-subunits in mammals and yeast. The genes encoding the putative alpha- and beta-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.

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. 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 CesA3 were 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 CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 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 degrees C.