Expression of a Mutant Form of Cellulose Synthase AtCesA7 Causes Dominant Negative Effect on Cellulose Biosynthesis

Abstract

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.Cellulose is the most abundant biopolymer produced by plants. It is the major component of plant cell walls and, in particular, is synthesized in large quantities during secondary wall formation of tracheary elements and fibers in wood. Cellulose synthase, the enzyme responsible for cellulose biosynthesis, is located in the plasma membrane. It is imaged by transmission electron microscopy as a rosette consisting of six particles, which is termed the terminal rosette complex (Brown and Montezinos, 1976). The identification of the rosette complexes as the sites of cellulose synthesis was confirmed by the immunolocation of putative cellulose synthase catalytic subunits (CesAs) in the rosette subunits (Kimura et al., 1999). It is proposed that as many as 36 CesA protein molecules together with other putative associated proteins form one rosette complex, which produces 36 (1,4)-␤-glucan chains that polymerize and crystallize into a microfibril with an estimated diameter of 8 to10 nm (Ha et al., 1998). The monosaccharide donor substrate used by CesAs, UDP-Glc, has been postulated to be provided by membrane-associated form(s) of Suc synthase (Amor et al., 1995). Recent in vitro studies suggest that the initiation of ␤-1,4-glucan synthesis starts with the addition of Glc units to a primer, sitosterol lipid, to form lipid-linked oligosaccharides called sitosterol cellodextrin (Peng et al., 2002). Cellodextrins are then thought to be cleaved from the sitosterol primer probably by a KOR cellulase (Nicol et al., 1998) and further elongated by addition of more Glc molecules to form a long chain of ␤-1,4-glucan (Peng et a...

Global expression analysis of CESA and CSL genes in Arabidopsis

“…The CESA4 (irx5), CESA7 (irx3), and CESA8 (irx1) genes are strongly expressed in stems and rather weakly expressed in other tissues. This is compatible with genetic evidence indicating that these genes encode components of cellulose synthase complexes that mediate secondary cell wall synthesis in xylem cells (Turner and Somerville 1997;Taylor et al 2003;Zhong et al 2003). The high level of expression of the CESA1 (rsw1), CESA3 (Turner and Somerville 1997); prc, procuste (Fagard et al 2000); STG4425 (Goubet et al 2003); rat, resistance to Agrobacterium tumefaciens.…”

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

“…Although genetic approaches have been used successfully to analyze the functions of many AtCESA genes (Arioli et al 1998;Taylor et al 1999Taylor et al , 2000Fagard et al 2000;Scheible et al 2001;Burn et al 2002;Desprez et al 2002;Zhong et al 2003) and some genes involved in the biosynthesis of matrix polysaccharides (Bouton et al 2002;Iwai et al 2002;Vanzin et al 2002;Madson et al 2003), genetic studies for CSL genes have not been as successful in determining their biochemical functions. So far, mutations in only a few CSL genes have yielded speciWc phenotypes, including AtCSLA7 (Goubet et al 2003), AtCSLA9/rat4 (Zhu et al 2003), AtCSLD3/kojak (kjk) (Favery et al 2001; Fig.…”

Cellulose Synthesis and Its Regulation

“…irx3 W859stop irregular, collapsed xylem, cellulose deficiency Ler (Taylor et al, 1999) fra5 P557T semi-dominant allele, reduced fiber cell wall thickness, cellulose content deficiency, dwarf plants, short roots and etiolated hypocotyls Col (Zhong et al, 2003) mur10-1 W444stop collapsed xylem, dwarf aerial organs, reduced female fertility, reduced tensile strength in hypocotyls, altered primary cell wall composition Col (Bosca et al, 2006) mur10-2 H734Y Col (Bosca et al, 2006) exi5 W954stop small rosette leaves, flowers, siliques, reduced cell elongation, dwarf plants, altered vasculature and cell morphology, cellulose content deficiency, impaired water transport (Taylor et al, 2000) irx1-2 S679L irregular, collapsed xylem, cellulose deficiency, dwarf plants Ler (Taylor et al, 2000) lew2-1 W217stop increased tolerance to drought and osmotic stress, increased proline, soluble sugars, and abscisic acid content, collapsed xylem, cellulose content deficiency C24 (Chen et al, 2005) lew2-2 L792F Col (Chen et al, 2005) fra6 R362K reduced fiber cell wall thickness, reduced cellulose content Col (Zhong et al, 2003) exi1-1 splicing variant small rosette leaves, flowers, siliques, reduced cell elongation, dwarf plants, altered vasculature and cell morphology, cellulose content deficiency, impaired water transport…”