thanatos is a nonconditional semi-dominant mutant allele of AtCesA3
Cellulose is synthesized at the plasma membrane by hexameric rosette terminal complexes to form crystalline microfibrils that act as a framework for the deposition of the noncellulosic wall polymers. Genetic mutant analyses and physical interaction experiments show that a functional complex requires at least three different CesA isoforms (Taylor et al., 2000, 2003; Desprez et al., 2007; Persson et al., 2007). All CesA proteins share a domain structure of two transmembrane domains (TMDs) close to the amino-terminus, six TMDs towards the carboxy-terminus and a large hydrophilic domain that faces the cytosol (Fig. 2c). This domain contains the conserved DXD motif, two other single aspartate residues and the Q/RXXRW consensus that are collectively referred to as the D,D,D,Q/RXXRW motif (Pear et al., 1996; Saxena & Brown, 1997). This motif is characteristic of the class of processive β-glycosyltransferases (GTs) that belong to the GT2 family (http://www.cazy.org). The cytoplasmic catalytic domain bridging TMD2 and TMD3 contains a class-specific region (CSR; Vergara & Carpita, 2001) that may contribute to subfunctionalization within the gene family. The relatively short hydrophilic N-terminus contains the first hypervariable region (HVR1) and the cysteine-rich zinc-finger domains (ZFD) that are common among CesA proteins. The ZFDs act as redox-regulated multimerization domains and may be involved in the formation of homo- or heterodimers of CesA monomers for assembly of the rosette complex (Kurek et al., 2002).
thanatos is a novel mutant allele of CesA3 located downstream of the conserved DXD motif in the globular region that modulates substrate catalysis and binding of processive glycosyltransferases (Saxena et al., 2001). Map-based cloning and sequencing of the than locus revealed a single-nucleotide transition from G to A causing the amino acid substitution of proline578 to serine. Ab initio analysis predicted a radical change in the three-dimensional configuration of the mutated AtCesA3 domain flanking the proline residue. Curiously, the same proline has been mutated in the fra5 allele of AtCesA7, a gene involved in secondary cell wall formation (Zhong et al., 2003). Noticeable growth phenotypes are only observed when the fra5 mutant cDNA is overexpressed in wild-type plants. Nevertheless, the fra5 mutation causes a dominant negative effect in terms of fiber cell wall thickness and cellulose content. By contrast, than has a dramatic nonconditional growth phenotype even in heterozygous plants, probably because primary walled rather than secondary walled cells are affected. Both than and fra5 mutants reveal the critical role of the proline residue in the structure and function of cellulose synthase complexes. The incorporation of a than defective CesA3 subunit into the rosette complex could impair the production of normal (1→4)-β-D-glucan chains. Our results indicate that the predicted drastic conformational change caused by proline to serine substitution even in one of the three subunits may impair the construction of an active rosette or prevent its assembly. Nevertheless, further experimental evidence is required to show whether the defective than polypeptide associates into the complex and how it affects the activity of the whole rosette complex.
To date, six mutant alleles of CesA3 have been characterized. The constitutive expression of the vegetative storage protein1(cev1; Ellis & Turner, 2001; Ellis et al., 2002), ectopic lignification1-1 (eli1-1) and eli1-2 (Cano-Delgado et al., 2003) mutations occur in the central catalytic domain. These mutants show constitutive expression of defense-related genes or ectopic lignin deposition. The rsw5 mutant is a temperature-sensitive allele of CesA3 in the carboxy-terminal domain following TMD8 (Wang et al., 2006). Whereas these CesA3 missense mutations are recessive, the conditional semi-dominant alleles ixr1-1 and ixr1-2 that lie between TMDs 7 and 8 confer resistance to the potent cellulose synthesis inhibitor isoxaben (Scheible et al., 2001). However, than is sensitive to isoxaben application. It appears that isoxaben resistance is coupled with missense mutations located in close proximity to the carboxy-terminus of cellulose synthases which is quite distant from the catalytic and substrate binding domain (Fig. 2c). Consistent with this observation, ixr2-1, the isoxaben-resistant allele of CesA6, also carries a missense mutation close to the carboxy-terminus (Desprez et al., 2002). The ixr1-1, ixr1-2 and ixr2-1 semi-dominant isoxaben-resistant alleles suggest that isoxaben affects the sensitivity of the CesA3- and CesA6-containing complex (Desprez et al., 2002, 2007).
Measurements of cellulose content in segregating populations showed a gene dose-dependent effect of the than mutation on cellulose synthesis, with cellulose content reduced by 19% in heterozygotes and by 42% in homozygotes. This gene dosage effect was also apparent when genomic DNA containing the promoter and coding sequence of the AtCesA3 (P578S) mutant gene was introduced into the wild-type A. thaliana background. The than-like phenotypic variation in transgenic lines appears to be correlated with the copy-number-dependent expression of the integrated transgene.
FTIR spectra showed changes in ester content, which appear to be characteristic of cellulose-deficient mutants (Fagard et al., 2000; Schindelman et al., 2001). As in the eli1-1 and eli1-2 mutant alleles of CesA3, the reduction of cellulose synthesis in the than mutant induces ectopic lignification. A regulatory mechanism may monitor cell wall integrity by inducing lignification as a mechanical barrier when the tensile strength of the wall is compromised (Cano-Delgado et al., 2003). In contrast to all AtCesA mutant alleles isolated to date, only than has a nonconditional dominant-negative effect on growth under normal conditions as a result of defective cellulose deposition during primary cell wall formation, while fra5 exhibits a dominant growth phenotype only upon overexpression of the mutant allele (Zhong et al., 2003).
thanatos perturbs cell morphology and plant growth
Microscopic analysis of than embryos revealed that the mutation caused defects in cell expansion and elongation. The embryos had a swollen phenotype and impaired development, and the formation of the shoot apical hook in than etiolated seedlings was dramatically affected. The swollen phenotype persisted during the elongation of the primary root and resulted in abnormal organization of the root cell files. The cellular abnormalities in than genetic segregants probably originate from the reduction of anisotropic cell expansion (Wasteneys, 2004).
Transverse sections through the vascular bundles of inflorescent stems showed abnormal cell shapes and organization both in than heterozygous plants and in wild-type plants expressing the AtCesA3 (P578S) transgene. Similar phenotypes have been previously described for either the hypocotyls of wild-type dark-grown seedlings treated with herbicides that inhibit cellulose biosynthesis (Desprez et al., 2002) or mutant alleles of CesA proteins involved in primary wall cellulose synthesis, including rsw1-1/cesa1 (Arioli et al., 1998) and prc1/cesa6 (Fagard et al., 2000). The phenotype of than heterozygous seedlings is similar to that produced by the prc1-1/cesa6 mutant allele, whereas the than homozygous lethal phenotype resembles that of rsw1-2, the strong nonconditional mutant allele of CesA1. These data are consistent with observations that both CesA3 and CesA1 are unique components of the primary wall cellulose synthase complex, while CesA6 may be functionally redundant with other CesA6-related CesAs (Persson et al., 2007).
Cellulose deficiency may also induce feedback mechanisms affecting the synthesis of other wall components. Recent fluorescent live-cell imaging of CesA6 (Paredez et al., 2006) and CesA3 (Desprez et al., 2007) identified significant intracellular reservoirs of CesA proteins in the Golgi that do not exclusively coincide with cellulose synthase complex assembly. The intracellular trafficking of CesAs could play an important role in the developmental and environmental regulation of cell wall composition. The semi-dominant thanatos mutation provides a further tool for use in dissecting the interactions of cellulose synthase subunits in assembly of a functional rosette and contributes to better understanding of plant growth mechanisms.