Identification of the functions of other GT2 enzymes from plants

Following the identification of the CesA gene family as the source of cellulose synthase enzymes, bioinformatic analyses of the CesA genes, EST databases and in the genome sequences of Arabidopsis and rice revealed that the CesA genes were in fact a subgroup within a larger, cellulose synthase gene super-family (Richmond & Somerville 2000; Fig. 5.4). The other eight subgroups were referred to as cellulose synthase-like (Csl) genes and were assumed to encode polysaccharide synthases that mediated the synthesis of P-linked wall polysaccharides. All subgroups of the cellulose synthase gene super-family encoded GT2 enzymes.

Defining the functions of the Csl genes has proved to be somewhat more difficult than expected. It took several years before Dhugga et al. (2004) reported that the CslA subgroup encoded enzymes that synthesized (1,4)-P-d- mannans. Dhugga et al. (2004) detected a relatively small number of CslA transcripts (15 from 15 000 ESTs) in developing guar seeds, at a stage when mannan synthesis in the seed was peaking. When the CslAs were transformed into soybean, the transgenic soybean plants showed greatly enhanced mannan synthase activity. The role of CslAs in mannan synthesis was confirmed in Arabidopsis, rice and the moss Physcomitrella, through heterologous expression of CslAs from these species in insect cells (Liepman et al. 2005; Liepman et al. 2007). The recombinant CslA proteins synthesized (1,4)-P-d-mannans when supplied with GDP-mannose, or (1,4)-p-d-glucomannans when supplied both GDP-mannose and GDP-glucose; one of the CslA proteins synthesized (1,4)-p-d-glucan when supplied with GDP-glucose alone (Liepman et al. 2005). Thus, plant CslA genes are members of an extended multigene family and while many are involved in (1,4)-P-d-glucomannan synthesis, it is not yet known whether all CslA proteins are (1,4)-P-d-glucomannan synthases (Liepman et al. 2007).

The CslF, CslH and CslJ groups of genes are the three so-called Poaceae-specific subfamilies of the large GT2 cellulose synthase-like gene family (Fig. 5.4). Members of the CslF and CslH gene families have been shown to be involved in the synthesis of (1,3;1,4)-P-d-glucans (Burton et al. 2006; Burton

CsIC

of^t

CslA

CesA

OsCslF7 830 OsCslF3 868

CsIC

CesA

of^t

CslA

OsCslF7 830 OsCslF3 868

CslF

0sCsiF4 889 (cereals)

OsCslFI 860 0sCslF2 889

OsCslHI 750 CslH OsCslH2 762 (cereals)

CsIB (dicots)

CslG (dicots)

CslE

Figure 5.4 Phylogenetic tree for the family GT2 cellulose synthase gene superfamily from plants. The cellulose synthase (CesA) and cellulose synthase- like (Csl) groups of genes are shown. Modified from Richmond & Somerville (2000).

CslF

0sCsiF4 889 (cereals)

OsCslFI 860 0sCslF2 889

OsCslHI 750 CslH OsCslH2 762 (cereals)

CsIB (dicots)

CslG (dicots)

CslE

Figure 5.4 Phylogenetic tree for the family GT2 cellulose synthase gene superfamily from plants. The cellulose synthase (CesA) and cellulose synthase- like (Csl) groups of genes are shown. Modified from Richmond & Somerville (2000).

et al. 2008; Doblin et al. 2009), as outlined in detail in the following section, but it is not yet confirmed that all CslF or CslH genes encode (1,3;1,4)-P -d-glucan synthases.

I t has recently been reported that the CslC gene family in Arabidopsis includes genes that encode polysaccharide synthases that are responsible for the synthesis of the (1,4)-P-d-glucan backbone of cell wall xyloglucans (Cocuron et al. 2007). These authors searched for genes that were expressed at relatively high levels during the last stages of nasturtium (Tropaeolum majus) seed development, when large amounts of xyloglucan are deposited as a storage polysaccharide. High levels of transcripts for a single member of the CslC subfamily detected and it was subsequently shown that the CslC gene was coexpressed with a xylosyl transferase gene. Heterologous expression of the CslC gene in Pichia pastoris resulted in the production of short-chain (1,4)-P-d-oligoglucosides, but not xyloglucan. On the basis of these results the authors concluded that the CslC gene family encodes proteins that synthesize the (1,4)-P-d-glucan backbone of xyloglucans.

There have been a number of studies in which various mutants that carry lesions in other Csl genes have been examined. For example, there is evidence that members of the Arabidopsis and tobacco CslD gene subfamily are required for pollen tube formation and for normal root hair formation (Doblin et al. 2001; Bernal et al. 2007; Kim et al. 2007; Bernal et al. 2008 communication). This suggest that at least some members of the AtCslD subgroup are involved in the synthesis of a polymer that is required for tip growth (Bernal et al. 2008), but the nature of the polysaccharide is not yet known.

In summary, there is some information available on the CslA, CslC, CslD and CslFgene subfamilies that encode GT2 enzymes, but direct evidence for involvement of the genes in the biosynthesis of clearly identified wall polysaccharides is not always strong. Little or no information has been published on the CslB, CslE, CslG or CslJ subgroups. Thus, it is probably too early to say with any confidence that the Csl and CesA clades of the cellulose synthase gene superfamily (Fig. 5.4) are 'well-formed' in the sense that all genes within a clade, or subfamily, are involved in the biosynthesis of a single class of wall polysaccharide. On the other hand, there is no evidence to date that this is not so.

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