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Problem tissue prints on XET test papers were used, as described by Fry (1997). The activity thus found in vitro is merely indicative for its presence in vivo, as xyloglucans are absent in the Charophyta and Chlorophyta (Popper and Fry, 2003). However this technique allows the localization of enzymes that are capable of incorporating xyloglucan oligosaccharides into a xyloglucan matrix and thus displaying XET activity. These enzymes can include XTHs or XTH-like enzymes. In accordance, searches were made for the presence of an XTH or XTH-like gene in one of the closest relatives of the vascular plants, Chara vulgaris. Combining these data with recent findings on the phylogenetic relationship of XTHs and family GH16 hydrolases, the possible origin and evolution of the XTH–xyloglucan interacting mechanism itself as well as the evolution of PCW elongation in general are discussed.Physcomitrella patens (Hedw.) Bruch & Schimp. was grown in Petri dishes on minimal medium (Schaefer, 2001) covered with cellophane discs. Plants were grown in a growth chamber (TCPS, Werchter, Belgium) at 26 °C, 16-h light per day (Sylvania, cool white), with a photosynthetic photon flux of 50–80 µmol m-2 s-1. Seven-day-old gametophytes were used to prepare RNA and tissue homogenates.Most bryophytes used for tissue printing were collected in the wild in Hechtel-Eksel (Belgium), Phaeoceros carolinianus (Michx.) Prosk. was collected in Boom (Belgium), Anthoceros agrestis Paton nom. cons. prop. near Ghent (Belgium), Chara vulgaris L. in the National Botanical Garden (Meise, Belgium) and marine algae were collected in Wimereux (France). All bryophytes and algae were directly processed in the lab upon arrival to minimize stress and changes in growth conditions.The Selaginella kraussiana XTH amino acid sequence, Sk-XHT1 (accession no. AY580314), was used in a protein blast against the draft Physcomitrella patens database (http://www.cosmoss.org; Lang et al., 2005). The retrieved sequences were analysed in expasy (http://www.expasy.org) and aligned using ClustalW (http://www.ebi.ac.uk/clustalw/).RNA from P. patens was prepared using a slightly modified Plant Concert RNA protocol (Invitrogen). The RNA was kept in 50 % isopropanol overnight, at -20 °C, to achieve optimal precipitation and a maximal extraction yield. Five micrograms of total RNA was reverse-transcribed using Superscript II RNase H-Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. The 3'-end of the CAC43709 cDNA (http://www.cosmoss.org) was subsequently obtained by 3' RACE using a gene-specific internal primer and an oligo-dT26. The full-length cDNA sequence of CAC43709 was constructed in silico and subsequently amplified from total cDNA using a specific primer set and a High Fidelity (Roche) proofreading enzyme mix.The XET and control test papers were made as described by Fry (1997). A filter paper (Whatman no. 1) was dipped into a solution of 0·5 % 1-1-1 trichloro-2-methylpropan-2-ol and 1 % 200-kDa tamarind xyloglucan, and dried overnight. The dried papers were subsequently dipped into a 5 µm XLLGol-sulforhodamine (XGO-SRs) in 70 % acetone solution to become XET test papers, or into a 5 µm trisaccharide-SR solution (=non-XET substrate) to serve as control papers. They were all left to dry in the dark before use. The trisaccharide-SR preparation contained two main fluorescent compounds (one major and one minor), both of which ran on a silica-gel thin-layer chromatography plate (in BuOH/HOAc/H2O 2 : 1 : 1) between maltose-SR and maltotriose-SR, suggesting that they are trisaccharides with at least one residue smaller than Glc. The material used for preparation of these SR derivatives was a minor fraction of small oligosaccharides obtained by cellulase digestion of tamarind xyloglucan. Their exact structures are unknown, but one may possibly be Xyl-Glc-Glc. They do not act as XET acceptor substrates for a crude preparation of cauliflower enzymes (S. Fry, University of Edinburgh, pers. comm.).Physcomitrella patens gametophytic tissue (7 g) was homogenized with several 15-s pulses (IKA Labortechnik, Staufen, Germany) in 300 mm sodium acetate, 20 mm calcium chloride, 1 mm dithiothreitol at pH 5·5 adjusted with sodium hydroxide (Iannetta and Fry, 1999). XET activity was tested by spotting 5 µL of the homogenate onto both a control and an XET test paper, which were then incubated in a sealed acetate envelope and kept overnight at room temperature. XET activity is defined as the transfer of the non-labelled xyloglucans to fluorescent acceptor-substrates (XGO-SRs). Non-reacted fluorescent xyloglucan oligosaccharides and trisaccharides were washed away after the assay in 90 % formic acid/ethanol/water (1 : 1 : 1) for 1 h followed by a 5-min wash in water. XET activity is seen as the remaining fluorescence on the XET test paper upon UV illumination. Images were taken using an Olympus C-5050 ZOOM digital camera with identical settings (1/10 shutter time, F 2.0 diaphragm) to allow comparison of the resulting fluorescence.Both XET and control test papers used for the tissue prints were initially dipped into 25 mm MES buffer adjusted to pH 5·5. Fresh tissue of bryophytes and algae was subsequently pressed onto the test papers (see above) and sealed into an acetate envelope. This ‘sandwich’ was incubated in the dark at room temperature for 3 d under a constant pressure of 3·2 g cm-2 to ensure that the tissue was kept in contact with the test papers. Pictures of the print were taken before and after it was washed (as described above), using daylight and UV illumination. In some tissue prints, chlorophyll was transferred to the blot, especially at the sites were the tissue was cut. This contaminant, however, was largely washed away in formic acid/ethanol/water. The remaining autofluorescence was extremely weak and of a different colour than that of the SR substrates. It thus did not interfere with the observations. The pigments of brown and red algae were not as easily removed by washing and hence caused some problems in analysing the presence of XET activity. An overlay of the tissue and fluorescence images allowed sites with (high) leachable XET activity to be located exactly.RNA of Chara vulgaris was prepared and reverse transcribed as described for Physcomitrella above. The resulting cDNA served as template in a PCR reaction using a degenerate primer based upon variations present in the catalytic domain of angiosperm XTHs and an oligo-dT26 primer [annealing temperature of 50 °C during 45 s]. A 480-bp cDNA sequence was amplified, sequenced and analysed in silico (http://www.expasy.org/tools/dna.html, http://elm.eu.org/). Its homology to XTHs and 1,3-,1,4-ß-d-endoglucanases was analysed using ClustalW (http://www.ebi.ac.uk/clustalw/).Blast analyses of the Physcomitrella genome with the amino acid sequence of the most primitive XTH thus far characterized, i.e. the lycopodiophyte XTH, Sk-XTH1 (Van Sandt et al., 2006), identified one putative full-length XTH (CAC43710) and 17 fragmentary putative XTH ESTs. An identical experiment using the different Arabidopsis XTH amino acid sequences gave an identical set of hits. Some of the 18 hits differed in only a few amino acids and probably originated from sequencing errors. Seven ESTs were suggested to be part of Arabidopsis thaliana XTH precursor genes (Rensing et al., 2005; http://www.cosmoss.org). Yet only one EST (CAC43709; cDNA: AX172659) and one full-length sequence (CAC43710; cDNA: AX172661) included a variant of the catalytic domain, which is one of the criteria for a gene to become annotated as ‘putative XTH’. The cDNA sequence of AX172659 was completed using 3' RACE PCR and the deduced amino acid sequences were analysed in silico.Both moss amino acid sequences showed high homology with known XTH amino acid sequences of numerous vascular plants. Most domains and some well-positioned amino acids, characteristic of vascular plant XTHs (Campbell and Braam, 1998; Johansson et al., 2003, 2004; Henriksson et al., 2003; Van Sandt et al., 2006), were found in both moss cDNAs. Yet some notable differences were present in the amino acid composition of the catalytic domain. For clarity, in Fig. A both Physcomitrella amino acid sequences were aligned with one representative vascular plant XTH, i.e. Sk-XTH1 from the lycopodiophyte Selaginella kraussiana (Van Sandt et al., 2006). A secretion signal at the N-terminus allowed both enzymes to be secreted in the apoplast (Fig. A, lower-case letters). The catalytic domain of both putative Physcomitrella XTHs (Fig. A, bold) differed in two (CAC43709, CEFDFEFLG) and three (CAC43710, YELDMEFLG) amino acids, respectively, compared with the ‘average’ functional site DEIDFEFLG, conserved among most XTHs of seed plants (Nishitani, 1997) and present in family 16 glycoside hydrolases (Henrissat et al., 2001). However, both glutamic acid residues and the second aspartic acid residue of the motif (xExDxExxx), which play a crucial role in the cleavage of the donor substrate in family GH16 enzymes, were maintained. As in most vascular plant XTHs, the catalytic domain of both moss amino acid sequences was immediately followed by a potential N-glycosylation site (N-X-T/S, Shakin-Eshleman et al., 1996) (Fig. A, dashed line). There was a notable variation in the C-terminal part of both putative Physcomitrella XTHs and in vascular plant XTHs. However, important residues involved in the maintenance of three-dimensional stability (Fig. A, underlined) and the recognition of the acceptor substrates in higher plant XTHs were also encoded by both moss cDNAs (Fig. A, superimposed). The conservation of most of the XTH identity motifs made both Physcomitrella amino acid sequences putative XTH enzymes. Thus, the in silico data suggested the presence of at least two potential XTH-encoding cDNAs in the bryophyte P. patens.A dot-spot of homogenized Physcomitrella tissue on XET test paper revealed a bright fluorescent spot in Fig. B, resulting from the specific incorporation of fluorescently labelled XyG oligosaccharides (X), whereas no fluorescence was seen on control test paper (C). This confirmed the presence of at least one functional XTH in Physcomitrella.The presence of XET activity in other bryophytes was assayed by tissue printing the gametophyte and/or sporophyte of 15 species, representing members of the three major groups of bryophytes, i.e. the Bryophyta, the Anthocerophyta and the Marchantiophyta (Table ). To localize specifically the sites of the tissue in which enzymes, capable of xyloglucan transglucosylation, are expressed, an image showing the tissue (left) and the tissue print (right) is shown in Figs . Here the xyloglucan transglucosylation, i.e. XET activity, was visible as an orange fluorescent spot under UV illumination.The early diversification of the Bryophyta or mosses is linked to spore dispersion and this is reflected in the taxonomy of this group (Goffinet and Buck, 2004). Based on the position of the gametangia and on the branching of the stem, however, two major groups can be distinguished, acrocarps and pleurocarps (Mägdefrau, 1982; Buck and Goffinet, 2000). As growth patterns differ in the two groups, XET activity was assayed in different acrocarp and pleurocarp species (Table , pleurocarpous mosses are designated with an asterisk). The expression of XET-displaying enzymes in the pleurocarp mosses corresponded to the site just beneath the apical meristem of the moss (Fig. A, see circle), whereas the other internal parts of the gametophyte displayed no visible XET activity on the print. No incorporation of fluorescence was visible in the control assay (Fig. B), indicating that the fluorescence in Fig. A was indicative of the XET activity of XTHs or XTH-like enzymes. In acrocarp mosses the same pattern of XET activity was visible when a young moss was assayed (Fig. C, see circle). However, the XET signal near the acrocarp apex was clearly weaker than that observed in pleurocarp mosses. This was probably caused by the high density of leaves covering the apex of most acrocarps, making it more difficult for the growing part to come into contact with the test paper. Remarkably, no XET activity was visible near the apex when older acrocarp gametophytes, bearing sporophytes, were pressed onto the test paper (Fig. E). The remainder of both the young and the old acrocarp gametophytes did not display XET activity (Fig. E, see circle, example of an acrocarp moss). By contrast, young capsules showed no or only very little XET activity (Fig. E, left), and were therefore in direct contact with the XET assay paper leaving a bright XET signal. No signal was detected at the site where the seta was pressed onto the paper. In the control assay, the test paper revealed no incorporation of fluorescence (Fig. The Sphagnales, or peat mosses, do not belong within the acrocarps or pleurocarps and are completely isolated within the Bryophyta. The gametophyte of a peat moss displayed a more widespread XET signal, from the capitulum down to the youngest branches that are still stretching out to their full length (Fig. Similar results were obtained in the Anthocerophyta. Pressing the gametophyte onto the test paper, a faint, but clear incorporation of XGO-SRs was visible at the edges of the thallus (Fig. I), but when removed from the gametophyte, the sporophyte foot generated a clear spot of XET activity on the test paper (Fig. In the Marchantiophyta both leafy and thalloid liverworts were assayed. In both groups the gametophytes showed clear XET activity. In thalloid liverworts an intense XET signal was visible at the edges of the gametophyte thallus, as seen in a Riccia species (Fig. The presence of extractable XET activity was also studied in the Charophyta. In simple charophytic algae, such as the Zygnematales, cell division is often not localized into meristematic regions. Also, the anatomy of these organisms is not particularly suitable for tissue printing. Therefore, Chara vulgaris was analysed. This species has a highly organized thallus with clear spatial segregation of meristematic activity and cell expansion. In Chara clear XET activity was visible at the site where the apex was pressed onto the XET test paper, but also at sites corresponding to other parts of the gametophyte (Fig. A, see circles). A clear fluorescent signal was present at the site of the node and at the sites where the branchlets were pressed onto the test paper (Fig. B, see circle). The control assays confirmed the specificity of the XET-generated signal (Fig. Studying the other major lineage of green plants, the Chlorophyta, the presence of enzymes displaying XET activity was found in the ulvophycean algae Ulva linza, previously known as Enteromorpha linza. At the base, where the foot is attached to substrate, fluorescently labelled xyloglucan was clearly incorporated in the test paper (Fig. F) confirmed the specificity of the XET activity in Chlorophyta. Other chlorophytes, however, displayed no XET activity (Fig. Different representatives of the Rhodophyta (Fig. I) were also tested for XET activity. A substantial amount of autofluorescence was visible, both in experiments and in controls, hampering the detection of XET activity. However, none of the species assayed appeared to show a signal of XET activity on the prints (Fig. The fluorescence found on the XET tissue print of Chara vulgaris indicated that there are enzymes expressed in the charophycean cell wall that are able to transglucosylate xyloglucans in vitro. Based upon these findings RNA was prepared and searches were made for the presence of XTH or XTH-like transcripts in Chara. 3' RACE using a degenerate primer, based upon the variations present in the catalytic domain of vascular plant XTHs, resulted in the amplification of a 480-bp fragment, named Chara2. To identify its homology with angiosperm XTHs, the deduced amino acid sequence was analysed in silico. As the evolutionary connection between XTHs and (1,3-1,4)-ß-d-glucan endohydrolases was recently demonstrated by Strohmeier et al. (2004), the relationship of Chara2 with both enzyme groups was studied in an alignment (ClustalW) including two monocot XTHs of barley (HvXTH3, HvXTH4), two dicot XTHs of Arabidopsis (AtXTH4, AtXTH22) and two family 16 (1,3-1,4)-ß-d-glucan endohydrolases (Fig. ). Both groups of enzymes shared a homologous catalytic site (shown in italics), which in most XTHs was immediately followed by an N-linked glycosylation site (underlined). Strohmeier et al. (2004) stated that one of the key differences between XTHs and (1,3-1,4)-ß-d-glucan endohydrolases is the insertion of three amino acids in XTHs, i.e. the PYX motif (boxed in Fig. ). This motif is absent in the Chara2 sequence and in the two (1,3-1,4)-ß-d-glucan endohydrolases; another key difference is the substitution of a methionine in (1,3-1,4)-ß-d-glucan endohydrolases by an aromatic amino acid residue, which is generally a tyrosine in XTHs. Interestingly, this substitution is present in Chara2 (Fig. , arrow). In addition, the sequence of Chara2 shaded in grey shows more homology with that of the corresponding acceptor binding loop sequence of XTHs than do the endoglucanase sequences (Fig. 4, acceptor binding loop is shaded in grey, homologous amino acids are marked in white).Thus far, XET action has been shown to be present in all vascular plants from the very ‘primitive’ lycopodiophyte Selaginella kraussiana up to ‘more evolved’ angiosperms (Vissenberg et al., 2003). Little is known about the presence of cell-wall-modifying enzymes in even ‘earlier’ land plants and algae. Thus far, extractable XET activity was detected in the liverwort Marchantia and the.
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