To isolate sequences containing the 1,3-beta glucosidase domain (GH17) from charophycean algae, Physcomitrella patens and selected embryophytes (Arabidopsis thaliana, Populus trichocarpa and Oryza sativa) BLAST (Altschul et al., 1990) searches were performed using as query five representative GHL17 sequences from Arabidopsis thaliana (At3g13560, At3g57260, At4g14080, At4g31140, At5g42100). For charophycean algae we searched the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) non-redundant (NR), high-throughput genome sequence (HTGS), whole genome shotgun (WGS), genome survey sequence (GSS) and expressed sequence tag (EST) databases. We obtained partial ESTs that were translated to amino acid sequences using Expasy translate tool. Presence of GH17 domain was confirmed in these sequences using the Conserved Domain (Marchler-Bauer et al., 2007) and SMART (http://smart.embl-heidelberg.de; Letunic et al., 2012) search engines. To isolate GH17 proteins from embryophytes sequenced genomes (Physcomitrella patens, Populus trichocarpa and Oryza sativa) a BLAST search against the Refseq protein database for each specific organism was performed using as query the same five Arabidopsis representative listed above and the GHL17 consensus domain sequence (cl18348). Similarly, to isolate beta-1,3-glucanases from fungi representatives (Candida albicans, Aspergillus clavatus, Aspergillus fumigatus, Aspergillus niger, Candida glabrata, Debaryomyces hansenii, Ashbya gossypii, Fusarium graminearum, Kluyveromyces lactis, Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Yarrowia lipolytica) the consensus domain sequence (ci18819) was used to search the reference genome databases. Only protein sequences containing GH17 domain (confirmed in SMART) and predicted to be complete were considered. Aramemnon (http://aramemnon.uni-koeln.de/request.ep) was also used to search and/or confirm the identity of the proteins isolated in the Rice annotation project database or in Phytozome.

To eliminate redundancies, and/or to identify overlapping regions in isolated ESTs, sequences obtained for each organism were aligned using Muscle (Edgar, 2004). The resulting sequences are summarized in Table 1. These were screened for characteristic features of this family, the presence of a secretory signal peptide (SP), glycosyl phosphatidylinositol anchor (GPI) and carbohydrate-binding module (X8), using the prediction programs SMART, SignalP 4.1 Serve, Phobius, GPI-SOM, FragAnchor, PredGPI and BIG-PI respectively (Eisenhaber et al., 2003; Fankhauser and Maser, 2005; Poisson et al., 2007; Pierleoni et al., 2008; Petersen et al., 2011; Letunic et al., 2012). According to the results obtained full length sequences were classified in the following types: type 0 showed no obvious SP (non-secreted proteins); type 1 contains SP and might (or might not) contain one or more X8 domains (predicted secreted proteins); type 2 contains SP, one or more X8 domains and GPI anchor and type 3 contains SP and GPI anchor but not X8 domain. The presence of GPI anchor in type 2 and 3 proteins was used to predict their membrane localization. The classification of the sequences analyzed is provided in Table 2.

All sequences isolated from representatives of charophycean algae and fungi, P. patens, Oryza sativa and Arabidopsis thaliana (Table 1) were aligned using Muscle (Edgar, 2004). Sequences from algae were incomplete which generate large gaps. These gaps were mostly avoided when only the domain was used. Therefore we constructed trees with both, full sequences and domain only. These alignments are provided in Supplementary data 1. To calculate the best fitting model of amino acid evolution MEGA5 was used (Tamura et al., 2013). This suggests WAG+G+F as the best model under the Akaike Information Criterion. Dendograms were obtained using three different methods of tree reconstruction [maximum likelihood (ML), neighbor-joining (NJ) and Bayesian inference (Bayesian)]. A majority-rule consensus tree was built by Bayesian inference using Mr. Bayes (Huelsenbeck and Ronquist, 2001). Convergence was reached after 960000 generations (3720000 when using domain only) and posterior probabilities were calculated for each clade. Using the same model a ML analysis was performed with MEGA5 (Tamura et al., 2013) and bootstrap values were determined from a population of 100 replicates. A NJ tree was also generated using Phylip (Felsenstein, 1997) as well as bootstrap values, which were determined from a population of 100 replicates. The tree was visualized using Figtree (http://tree.bio.ed.ac.uk/software/figtree/). A similar protocol was followed for phylogenetic comparison of Arabidopsis thaliana and Populus trichocarpa sequences (alignments provided in Supplementary data 2). In this case convergence was reached after 45000 generations.

A graphical representation of the GH17 domain alignment was performed using weblogo3 (Crooks et al., 2004). In the logo the overall height of the stack indicates the sequence conservation at that position.

Construction of p35S-mCitrine-PdBG1 (At3g13560) was described elsewhere (Benitez-Alfonso et al., 2013). N-terminal and GPI-anchor domains were predicted for At4g31140 and At5g58090 using SignalP 4.1 Serve and GPI-SOM (Fankhauser and Maser, 2005; Petersen et al., 2011). mCitrine protein fusions were obtained by overlapping PCR (Tian et al., 2004) and expressed in the binary vector pB7WG2.0 using Gateway technology. The mCitrine was fused in frame between amino acids 454–455 in the case of At4g31140 and between amino acids 445–446 in the case of At5g58090.

Transient expression was verified by agroinfiltration in Nicotiana benthamiana leaves. Stable transgenic lines were generated using the floral dip method, followed by selection with BASTA. T2 seeds were sterilized and germinated in long day conditions on plates containing MS medium supplemented with BASTA (25 μg/ml).

Callose deposition at PD was detected in plant samples vacuum infiltrated with 0,1% (w/v) aniline blue in 0,1M sodium phosphate (pH 9.0) and incubated in the dark for 1–2 h before imaging.

Confocal analysis was performed on a Zeiss LSM700 Inverted microscope using a 488 nm excitation laser for mCitrine, the 405 nm laser for aniline blue fluorochrome and 585 nm laser to detect chloroplast autofluorescence. Emission was collected using the filters: BP 505–530 for mCitrine, the DAPI filter for aniline blue (463 nm) and LP 615 filter for chloroplasts (581 nm). The images corresponded to stacks of z- optical sections. Sequential scanning was used to image tissues expressing mCitrine and stained with aniline blue.

The presence of intercellular connections (phragmoplast and/or less evolved PD) has been described in some species belonging to the Charophytes (Figure 1) but so far, in this lineage, regulation of PD by callose metabolism has only been demonstrated in embryophytes (Scherp et al., 2001; Schuette et al., 2009). The presence of β-1,3 glucans in the cell wall of unicellular organisms indicate an ancient origin for this metabolic pathway but how and when it evolved to control PD transport is unknown (Sorensen et al., 2011). In an attempt to answer this question, we isolated sequences encoding GH17 domains from charophytes, bryophytes, and vascular plants. Based on the availability of sequence information, we selected representative species from the charophycean orders: Klebsormidiales (Klebsormidium flaccidum), Zignematales (Penium margaritaceum), Coleochatales (Chaetosphaeridium globosum) and Charales (Nitella mirabilis). 14 partial transcripts were isolated but only 7 (2 from Klebsormidium, 1 from Penium, 1 from C. globosum and 3 from Nitella) contained key aminoacids forming the active site of GHL17 (Table 1).

Full-length GHL17 sequences were isolated from moss (Physcomitrella patents) and from monocots (Oryza sativa) and dicots (Arabidopsis thaliana and Populus trichocarpa) model plants using genome information and protein annotation databases. In total we were able to identify 18 sequences in Physcomitrella, 24 sequences in Oryza sativa, 50 sequences in Arabidopsis thaliana and 54 in Populus trichocarpa (Table 1). The increasing number of sequences isolated in land plants with respect to those isolated in algae and moss suggests that an expansion in this gene family have occurred during or immediately after land colonization.

We used prediction tools to determine the structure and localization of the proteins encoded by the sequences identified. This was not possible for algae representatives because only partial transcripts were isolated. For moss, rice, Arabidopsis and Populus sequences, secretory signal peptides (SP) and the presence of C-terminal GPI anchoring domains were predicted using several bioinformatics websites (see Material and Methods). GHL17 sequences were also classified according to the presence of one or more carbohydrate binding domains (named X8 or CBM43). We classified sequences in 4 types according to the presence of one or more of these features (see Material and Methods and Table 2). Type 2 and 3 displayed a SP and GPI-anchor signature that predicts their localization at the PM or at membranous subdomains (such as PD). From the 18 sequences isolated in Physcomitrella only 4 were classified as type 2. Arabidopsis genome contained 21 membrane predicted sequences (42% of the total), which were experimentally verified in a proteomic analysis (Borner et al., 2003). The number of membrane predicted GHL17 was very similar in rice and Populus trichocarpa (22 in rice, 21 in poplar). When comparing moss and vascular plants a major increase in the number of predicted membrane-targeted proteins is detected consistent with the hypothesis that GHL17 evolved and expanded to support or adopt specialized functions at membraneous domains in terrestrial environments.

Research on GHL17 protein structure revealed two strictly conserved glutamate residues that act as the proton donor and the nucleophile in all reactions catalyzed by glycosyl hydrolases (Jenkins et al., 1995; Wojtkowiak et al., 2013). A number of aromatic and hydrophilic residues located near the catalytic cleft, presumably involved in substrate specificity and enzyme activity, are also conserved among all plant GHL17 proteins (Wojtkowiak et al., 2013).

To study the molecular evolution of the GH17 domain in green algae, moss and plants, we translated and aligned the domain region of the retrieved sequences using MEGA5 (Supplementary data 1). We also included sequences isolated from fungi representatives to analyze domain conservation in a different lineage. The results revealed that the glutamate catalytic residues (E) are highly conserved among all charophycean representatives, fungi and embryophytes (highlighted in red in the alignment shown in Supplementary data 1 and in Figure 2). Similarly, the residues surrounding the catalytic site are mostly conserved in all selected representatives (Supplementary data 1, Figure 2). Moreover a region contained the aromatic residues Tyr200 and Phe203 (location refer to At2g05790 sequence), which is involved in substrate interaction (Wojtkowiak et al., 2013), is also conserved in all streptophytes (Figure 2).

The high degree of similarity between the catalytic sites of GHL17 proteins in green algae, fungi and land plants supports the ancestral origins of this metabolic pathway.

The phylogenetic distribution of Arabidopsis GHL17 sequences has been studied before (Doxey et al., 2007). Based on tree topology, these proteins were grouped into three distinct clades: α, β, and γ. Predicted membrane GHL17 were evenly distributed in clade α and β. We investigated the evolutionary origin of these clades by comparing the phylogenetic distribution of GHL17 sequences isolated from charophycean green algae, fungi Physcomitrella patens, Oryza sativa and Arabidopsis thaliana. Although plants and fungi evolved in a different lineage, they share a common eukaryotic origin, which is reflected in the conservation of key aminoacids in the GH17 domain (Supplementary data 1).

Unrooted phylogenetic trees were generated using three search algorithms: Bayesian inference (Bayesian), Maximum Likelihood (ML) and Neighbor Joining (NJ) (Figure 3A and supplementary data 3). The tree topology was generally well supported by all 3 methods, with the exception of several higher order branches in ML and NJ bootstrap values. The three phylogenetic clades (α, β, and γ) described by Doxey et al. (2007) are color coded in Figure 3A. Fungi selected sequences branch off at the same point as some algae representatives and near the point of connection of plant sequences forming the clade beta. This suggests a more ancestral origin for this clade (Figure 3B). Clade alpha and gamma contained embryophytes only and, for the purpose of this paper, they could be considered as a single clade (Figure 3C).

Only partial transcripts were isolated for algae representatives hence gaps were introduced in the alignments that could affect the accuracy and reliability of the trees. To confirm the tree topology, we manually eliminate these gaps to generate trees containing the sequence region encoding the domain only (marked in yellow in Supplementary data 1). As shown in supplementary data 3, the distribution of sequences in the different clades and the relationship between the different branches was conserved in these “domain only” trees.

As in Arabidopsis, even distribution of predicted membrane sequences between the alpha and the beta clade was observed in rice (Figures 3B,C). Interestingly, type 3 proteins were almost exclusively found in the alpha clade. In summary our phylogenetic analysis suggest that GHL17 membrane proteins contained in clade alpha appeared in early embryophytes presumably to adopt new functions at the cell periphery.

Since cell wall composition and PD complexity evolved during land plant colonization, it seems logical to assume that callose, and specialized callose metabolic enzymes, were adopted at some stage during this evolutionary process to regulate PD aperture. The presence of charophytic sequences and the proximity to a fungi branch suggests a more ancestral origin for membrane proteins included in the beta clade (Figure 3B). We hypothesize that PD-targeted GHL17 proteins evolved with the appearance of early embryophytes, hence likely be contained within the alpha clade (Figure 3C).

The Bayesian tree shows (with high support values) 10 predicted membrane proteins (type 2 and 3) from Arabidopsis contained in the alpha clade whereas 10 type 2 sequences appeared in a compact clade within the beta subgroup surrounded by sequences isolated from green algae (Figures 3B,C). Data from several publications reported the intracellular localization of several GHL17 proteins in Arabidopsis. The root developmental regulators At3g13560, At2g01630, and At1g66250 (Benitez-Alfonso et al., 2013) and the virus-induced protein At5g42100 (Levy et al., 2007) were PD-localized whereas At3g57260 was preferentially expressed in the apoplast (Zavaliev et al., 2013). Confirming our hypothesis, all PD localized proteins were grouped in the alpha clade (Figure 3C).

The localization of few GHL17 proteins from Populus has been recently reported (Pechanova et al., 2010; Rinne et al., 2011). To test the relationship between the appearance of the alpha clade and protein localization, we constructed a Bayesian tree with GHL17 sequences isolated from Arabidopsis and from Populus trichocarpa. BLAST searches against the Populus genome identified a total of 54 non-redundant sequences containing the GH17 domain (Table 1). Classification of these sequences according to bioinformatic predictions identified 21 putative membrane proteins (Table 2). A multiple sequence alignment was conducted and unrooted phylogenetic trees were generated using the Bayesian, ML and NJ algorithms (Figure 4 and Supplementary data 2 and 4). According to tree topology, Populus GHL17 proteins also appeared grouped in 3 clades α, β, and γ, each well supported by high probability values in each tree (Figure 4 and Supplementary data 4). As before, type 3 proteins were contained within the α clade whereas type 2 proteins were distributed between the α and β clades.

Orthologs of PtGHL17_18 and PtGHL17_26 were both found to target PD whereas PtGHL17_48 and PtGHL17_49 orthologs were mainly localized at the PM and lipid bodies (Rinne et al., 2011). As expected, PtGHL17_18 and PtGHL17_26 are membrane predicted proteins contained in the alpha clade (Figure 4). The results confirmed a potential link between the phylogenetic distribution of GHL17 proteins and their intracellular localization.

To identify novel PD components the proteomic composition of PD-enriched cell walls has been analyzed (Bayer et al., 2006; Fernandez-Calvino et al., 2011). Several GHL17 proteins were isolated through these screens, including the predicted membrane localized proteins At3g13560, At5g42100, At4g31140, and At5g58090. Different from At3g13560 and At5g42100 (included in the alpha clade), At4g31140 and At5g58090 were found in clade beta. Successful separation of PD membranous section from the desmotubule and the PM is quite challenging (if not impossible) therefore a number of false positives was expected. The results presented above suggest that proteins excluded from the alpha clade are not likely targeted to PD sites. Therefore, we tested the intracellular localization of At4g31140 and At5g58090 using as control At3g13560-mCitrine (a previously PD-localized GHL17 protein). m-Citrine fluorescent fusions were obtained and expressed transiently in tobacco leaves. The results are shown in Figure 5. Transient expression of either At4g31140-mCit or At5g58090-mCit led to protein accumulation in the apoplast (Figures 5A–C). At5g58090-mCit also appears to be associated with the endoplasmic reticulum (data not shown).

Transient assays can be misleading. Therefore we obtained stable transgenic lines expressing p35s-At5g58090-mCit to confirm the subcellular localization of this protein. Leaves isolated from 10 days-old seedlings expressing p35s-At5g58090-mCit and leaves isolated from seedlings overexpressing At3g13560-mCit (grown in the same plate) were stained with aniline blue to reveal callose deposits at PD sites. The intracellular localization of these proteins in stable lines reproduced the results obtained in transient assays (Figures 5D,E): At5g58090-mCit was found at the cell periphery and in the apoplast whereas At3g13560-mCit was found in a punctuated pattern along the cell wall (presumably PD sites). Co-localization with callose deposits at PD was found for At3g13560 but not for At5g58090 (white arrows in Figures 5D,E). This result suggests that PD localization of GHL17 proteins could be related to their evolutionary origin, hence with the appearance of the alpha clade.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.