Genes involved in xyloglucan biosynthesis and modification/degradation

1.1.7.1 Genes involved in xyloglucans biosynthesis

Several enzyme classes are involved in XG biosynthesis. The most important of these enzymes are β-glucan synthases taking part in backbone formation and α- xylosylransferases, β-galactosyltransferases as well as α-fucosyltransferases involved in side chains formation.

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As the xyloglucan backbone is structurally similar to those of cellulose, it was suggested that cellulose synthase-like (CSL) genes might be involved in XyG backbone biosynthesis. A transcriptional profiling approach applied on developing nasturtium seeds (Tropaeolum majus) led to the discovery of CSLC genes. A homologue, CSLC4, was identified in Arabidopsis. Both genes were heterologously expressed in Pichia where they were able to produce β1,4-D-glucan. Further analysis showed its localisation within the Golgi, high expression when xyloglucan is deposited in high quantities as well as co-expression of xyloglucan modifying enzymes, mainly the xylosylransferase AtXT1 in Arabidopsis. All these observations strongly suggest that A. thaliana CSLC4 encodes a xyloglucan specific β1,4-D-glucan synthase (Cocuron et al., 2007). Topological studies on CSLC4 showed that the enzyme is anchored in the Golgi membrane and that its catalytic domain is located into the cytosol, where it is suggested to use cytosolic UDP-glucose as a substrate. It is further supposed that the growing β-glucan chain is then translocated into the Golgi lumen. This process is similar to the proposed model of cellulose synthases which are located in the plasmamembrane and converting cytosolic substrate into extracellular β-glucan chains which are then assembled to cellulose (Davis et al., 2010).

Two α-xylosylransferases, AtXT1 and AtXT2, have been identified in A. thaliana. The first one was identified during investigation of seven potential galactosyltransferases, of which one showed xylosyltransferase activity. Together with pea xylosyltransferase, AtXT1 was expressed in Pichia. AtXT1 could use G5 substrate (GGGGG) as acceptor to give GXGGG, in addition it showed activity toward cellopentaose ((β-D-Glc-[1→4])4-D- Glc) and cellohexaose acceptor substrates (Faik et al., 2002). A second enzyme possessing xylosyltransferase activity, AtXT2, was described later on. Also this enzyme was able to catalyse the transfer of at least one D-xylose onto the forth position of the reducing end of cellopentaose or cellohexaose, to give GXGGG or GGXGGG (Cavalier and Keegstra, 2006). Both genes were knocked out in Arabidopsis and analysed in a double knockout plant. Plants were a little bit smaller than wildtype and roots growth was abnormal. In addition, there was no xyloglucan detectable in these double knockout plants (Cavalier et al., 2008). The fact that a mutant can survive without any detectable trace of xyloglucan is somehow questioning the physiological role of this hemicellulose in vivo.

A galactosyltransferase candidate acting on xyloglucan was identified by chemical mutagenesis of Arabidopsis, leading to mutant line mur3. Cell wall analysis of this line showed reduced L-fucose and D-galactose contents (Reiter et al., 1997). The

corresponding gene, MUR3, was identified later on as a xyloglucan specific galactosyltransferase. Further analysis of mur3 revealed absence of α-L-Fuc-(12)-β-

D-Gal-(12) side chain which is considered as being involved in xyloglucan binding to cellulose. Instead, an enhanced galactosylation of the second xylose residue was detected (to give XLXG). Apart from altered trichome papillae structure, mur3 plants did not show a visible phenotype compared to wildtype. Both XLXG and XXFG were described as forming straightened structures, indicating that galactosylation at the second D-xylose compensates absence of terminal L-fucose in side chain F (Madson et al., 2003).

A xyloglucan specific α1,2-fucosyltransferase, AtFUT1, has been identified in A.

thaliana using the amino acid sequence of a fucosyltransferase purified from pea microsomes. Enzymatic assays using non-fucosylated tamarind or nasturtium xyloglucan as a substrate and labelled GDP-L-fucose as a donor confirmed fucosyltransferase activity (Perrin et al., 1999). A. thaliana mutant mur2, defective in AtFuT1 gene, contained less than 2 % of fucosylated xyloglucan compared to wildtype.

Identified xyloglucan structures were XXLG and XLLG. Compared to mur1 which is blocked in de novo synthesis of L-fucose and exhibits a dwarfed phenotype, mur2 plants grew normally and showed no visible alterations (Vanzin et al., 2002). By searching for genes presenting sequence homologies to AtFuT1, nine other putative fucosyltransferases have been identified in Arabidopsis sharing up to 62 % amino acid sequence similarity to AtFuT1. Analysis of these genes by overexpression and in vitro enzymatic assays on AtFuT3, AtFuT4 and AtFuT5 indicated that they have other substrate specificity than xyloglucan (Sarria et al., 2001), as was shown recently for AtFuT4 and AtFuT6 involved in the fucosylation of AGPs (Wu et al., 2010).

1.1.7.2 Genes involved in xyloglucans modification/degradation

Degradation of Xyloglucans is more thoroughly understood than their biosynthesis.

There are four exoglycosidases involved in xyloglucan modification and/or degradation:

α1,2-fucosidases, β1,2-galactosidases, α1,6-xylosidases and β1,4-glucosidases which is disassembling the backbone. A comparative genomic approach showed that several essential enzymes involved in xyloglucan synthesis, modification or degradation appeared early during plant evolution (Del Bem and Vincentz, 2010).

An A. thaliana knockout plant defective in the xyloglucan-specific α-xylosidase AtXYL1 was investigated regarding enzymatic activity, quality of xyloglucan

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oligosaccharides and gene expression pattern. The mutated plant showed a decrease in the control of anisotropic growth of different plant organs with reduction in elongation but increasing the width of several organs, mostly siliques, leaves and sepals (Sampedro et al., 2010).

Removal of β1,2-linked D-galactose is catalysed by β-galactosidases (BGALs).

These enzymes are part of the GH family 35 hydrolases in the CAZy database and are suggested to play important roles in the modification of cell wall components, for example xyloglucan mobilisation in cotyledons (de Alcântara et al., 1999). Several candidates have been identified in A. thaliana and most of them are predicted to act extracellularly, potentially within the cell wall (Ahn et al., 2007).

After the removal of side chains, the xyloglucan backbone is degraded by the action of β-glucosidases. A β-glucosidase was studied in nasturtium (Tropaeolum) where it was active in the cotyledons during seed germination. This enzyme was not able to remove the β-D-glucose residue when side chains longer than substitution with D-xylose were present, indicating a steric hindrance (Crombie et al., 1998). Four genes sharing homology to the one of Tropaeolum have been identified in A. thaliana and each of their products was found in the apoplast (Iglesias et al., 2006).

A gene described as encoding an α1,2-fucosidase active on the xyloglucan oligosaccharide XXFG has been cloned in 1995 from pea epicotyls (Augur et al., 1995).

The first gene of A. thaliana described as encoding such an enzyme, AtFXG1, has been published in 2002 after its finding by sequence homologie with an α1,2-fucosidase purified from Brassica oleracea (de La Torre et al., 2002). A second gene, AtFUC1, was identified by the same authors based on homologie with animal enzymes but finally ended up to be an α1,3/4-fucosidase (Zeleny et al., 2006). AtFXG1 was heterologously produced and used with success for enzymatic assays against XXFG (de La Torre et al., 2002). AtFXG1 was described as being expressed in young leaves, apical region and inflorescence stems (Iglesias et al., 2006). Since AtFXG1 did not show strong homology to known α-L-fucosidases, new candidates have been searched in A.

thaliana. Based on sequence homology to an α1,2-fucosidase from Bifidobacterium a new candidate, called AtFUC95A, has been identified (At4g34260), grouped into CAZy family 95. Heterologous expression of the reading frame of At4g34260 in Nicotiana benthamiana leaves and Pichia pastoris and enzymatic assays using 2-fucosyllactose showed defucosylation, whereas other substrates having fucose in other linkages stayed untouched. The measured Km value for the used substrate has been found to be in accordance to that from the α1,2-fucosidase analysed form almond emulsion and

published by Kobata, 1982. In addition it could be shown that this enzyme was active against fucosylated xyloglucan XXFG fragment as well as against the xyloglucan polymer (Léonard et al., 2008).