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In order to get an idea of the potential activity of AtFXG1, the amino acid sequences of both AtFXG1 and AtFUC95A were analysed by BLAST search. Whereas AtFUC95A shows homologies to other known α1,2-fucosidases, as already shown by Léonard et al. (2008), the result of AtFXG1 revealed that this protein shares homologies to several esterases from other organism. In order to investigate this aspect, AtFXG1 was analysed regarding a putative esterase activity. AtFXG1 was seemingly able to catalyse the hydrolysis of the artificial substrate PNP-acetate more efficient than AtFUC95A or the control, as its activity was the double of those of AtFUC95A and the control.
AtFUC95A is expressed throughout the plant and is localised to the apoplast
Several enzymes are necessary for xyloglucan modification and degradation.
(Iglesias et al., 2006) analysed the expression of the four main enzyme classes involved, but included AtFXG1 as the α1,2-fucosidase acting on xyloglucans. Its expression pattern was found to correlate with what one can expect when modification of xyloglucans in growing tissue takes place. In A. thaliana roots it was shown that xyloglucan modifications take place in muro. One antibody, CCRC-M1, recognizing α1,2-linked terminal L-fucose residue, was used to study the pattern of distribution of cell wall polysaccharides. The results showed that the distribution depends on cell type and development. It was suggested that these different pattern are related to cell wall modifications, for example as an adaptation during root growth. It was also shown that the pattern of epitope localisation within the apoplast was different according to cell types. In some cells, α1,2-linked terminal L-fucose residue containing epitopes were situated close to the plasmamembrane, in others they were present in the outer parts of the wall (Freshour et al., 2003). We could show that AtFUC95A is present in the apoplast, even though our detection method did not allow a close distinction between the different layers of the cell wall. CCRC-M1 was later on used to investigate the distribution pattern in the fucose-deficient mutant mur1. Expression was detected in roots but no labelling was observed in the aboveground tissue, whereas hypocotyl, leaves and stipules were labelled in the wildtype (Freshour et al., 2003). Our data about the expression of AtFUC95A obtained by GUS-staining indicates expression in the same or similar developmentally active tissue. AtFUC95A was strongly expressed in the hypocotyl as well as vessels of young seedlings, in the root differentiation, elongation and/or maturation zone but not in the root tip, where fucosylated xyloglucan were also detected. Strong expression in zones with cell division indicates involvement of
AtFUC95A in developmental processes. The strong difference that we find between the xyloglucan fucosylation pattern of wild type plants and of Atfuc95A speaks for a high implication of in muro modifications in the final xyloglucan side chain pattern.
Considerations on the reason of mur1 phenotype
After obtaining GMD single and double knockouts, we analysed at first the sugar- nucleotide level. GDP-L-fucose was reduced in the gmd1 mutant and not detectable in both gmd2 and gmd1/gmd2 double knockout mutants. Both gmd2 and gmd1/gmd2 mutants were dwarfed as initially described by Reiter et al. (1993), and the wild type phenotype could by restored by external supplementation with L-fucose. Analysis of RG-II side chains revealed in addition to the replacement of L-fucose by L-galactose on the chain A, the presence of very truncated structures. The mutant mur1 has been very thoroughly studied (O'Neill et al., 2001; Glushka et al., 2003; Reuhs et al., 2004).
Comparison with mutants affected for specific xyloglucan fucosylation could rule out a consequence of the fucosylation of this kind of polymer in the dwarf phenotype of mur1 (Ryden et al., 2003). The conclusion of the works published until now is that the dwarfed phenotype and the cell wall alteration observed in mur1 results from the exchange of L- fucose by L-galactose, supposed to alter the affinity of RG-II for boron and therefore decrease its dimerisation so crucial for cell wall integrity (O'Neill et al., 2001; Glushka et al., 2003; Reuhs et al., 2004). According to this theory, the structure of RG-II cannot afford the slightest change without dramatic consequences. This theory is not in accordance with our observations. First of all, RG-II is not a unique structure but possesses diversity, far from being negligible. Among the observation that we made on RG-II, the study of tomato RG-II revealed the presence, in this organism, of about 20 percent of chain A naturally containing L-galactose instead of L-fucose. Nevertheless, in wild type tomato, a dimerisation rate of RG-II of about 97% is described (Voxeur et al., 2011). This is not compatible with L-fucose replacement by L-galactose being the cause of the mur1 phenotype. We think that it is therefore crucial to re-examine the RG-II structure present in dimeric and monomeric fraction of RG-II and postulate that in tomato GME-RNAi line as well as in mur1, the monomeric RG-II will contain very truncated chains A stopping were the fucose is described as being inserted.
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