The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-β-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa β-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.
Plant cells are surrounded by a wall that defines their shape and size, provides mechanical strength and support, plays a role in communication, and is the cell’s primary defence against pathogens (Bacic et al., 1988). Walls and their components are also of great value to human society as sources of fuel, textiles, timber and paper. The wall is composed mostly of polysaccharide, generally with cellulosic microfibrils embedded in a matrix of non-cellulosic polysaccharides and pectic polysaccharides (Bacic et al., 1988; Carpita and Gibeaut, 1993). Some cells have more specialized walls characterized by the presence of particular polysaccharides. For example, callose, a 1,3-β-d-glucan, is abundant in the walls of pollen tubes (constituting 86% by weight of the carbohydrate in walls of Nicotiana alata pollen tubes; Li et al., 1999). Callose is also found at the cell plate, around the plasmodesmata and in sieve plates, and is deposited in response to abiotic stress, wounding or pathogen attack (Stone and Clarke, 1992).
Callose synthase (CalS; uridine-diphosphate glucose:1,3-β-d-glucosyl transferase; EC 188.8.131.52) enzymes maintain activity in vitro and can be partially purified, making them good candidates for further molecular and biochemical studies. The GSL genes have been proposed to encode the CalS enzymes in plants, based on their similarity to fungal FKS genes, which are believed to encode the fungal 1,3-β-d-glucan synthases (Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a; Østergaard et al., 2002; Saxena and Brown, 2000). Expression analysis, cellular location and functional analysis support a role for GSL proteins in callose synthesis. GSL proteins are large (200–220 kDa), integral membrane proteins predicted to contain 14–16 transmembrane domains, divided into two membrane regions separated by a large, central cytoplasmic region, and an N-terminal cytoplasmic extension (Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a; Østergaard et al., 2002; Verma and Hong, 2001). As each plant genome contains a number of GSL genes, Arabidopsis for example having 12, it has been proposed that different GSL proteins may be responsible for callose synthesis in different locations throughout the plant (Hong et al., 2001a). In some bacteria, e.g. Agrobacterium spp., the synthase that produces the 1,3-β-d-glucan curdlan is encoded by CrdS, a member of the CesA gene family (Stasinopoulos et al., 1999), so there remains a formal possibility, albeit remote, that members of either the CesA or related CSL families may produce callose in plants.
Some biochemical evidence also links GSL proteins with CalS activity. Detection by antibody labelling of GSL proteins has been demonstrated in CalS-enriched material from cotton fibres (Cui et al., 2001) and transgenic BY2 cells (Hong et al., 2001a), and a GSL protein, HvGSL1, has been directly identified from the callose-synthesizing region of a native gel containing membrane proteins from barley suspension culture cells (Li et al., 2003). The presence of GSL proteins in CalS-enriched material biochemically supports a role in callose synthesis, with enrichment of the GSL protein with CalS activity during purification predicted. It has also been suggested, based on the number of polypeptides present in CalS-enriched material, on the basis of yeast-two-hybrid analysis with AtCalS1 (AtGSL6), and on calmodulin-binding assays with CFL1 (GhGSL1), that GSL proteins may be part of a larger CalS complex (Cui et al., 2001; Hong et al., 2001a; Verma and Hong, 2001).
In pollen tubes, there is both molecular and biochemical evidence that suggests that the GSL gene products are involved in callose synthesis. Pollen tubes of N. alata contain a highly active, developmentally regulated CalS enzyme that does not require Ca2+ for activity and that can be activated in vitro by treatment with trypsin or certain zwitterionic detergents (Li et al., 1997, 1999; Schlüpmann et al., 1993). A GSL gene, NaGSL1, is abundantly and specifically expressed in N. alata pollen and pollen tubes, and is the main candidate for the pollen-tube CalS (Doblin et al., 2001). The orthologous gene in Arabidopsis is AtCalS5 (AtGSL2). The cals5-3 mutant has reduced AtCalS5 transcript levels in flowers and a severe callose deficiency in its pollen-tube walls and plugs, indicating that this gene is required for callose synthesis in Arabidopsis pollen tubes (Nishikawa et al., 2005). Another gene encoding a putative polysaccharide synthase, NaCSLD1, is also expressed in pollen and pollen tubes of N. alata (Doblin et al., 2001), and could also be considered a plausible candidate for the CalS based on analogy to the bacterial CrdS that is a member of the CesA gene family (see above). In this study, we used the N. alata pollen-tube CalS enzyme to establish a biochemical link between a GSL protein (NaGSL1) and callose synthesis. We enriched the CalS enzyme from pollen tubes using membrane fractionation followed by product entrapment, and used anti-GSL antibodies to show that NaGSL1 is enriched with CalS activity. We also used a proteomic approach to directly identify NaGSL1 and other proteins present in the CalS-enriched material. Combined, the results represent strong evidence for NaGSL1 being the CalS of N. alata pollen tubes, although the possibility that other proteins or co-factors are also required for callose synthesis in pollen-tube extracts could not be excluded.
Enrichment of the pollen-tube CalS
We used a previously developed protocol (Turner et al., 1998) to enrich the CalS enzyme from N. alata pollen tubes grown in culture for 16 h. Briefly, this procedure involves the production of a pollen-tube homogenate (H) by sonication and then isolation of mixed membranes (MM) using a step gradient. Membranes are then fractionated by continuous density-gradient centrifugation, and fractions with the highest CalS activity, that are also enriched for plasma membrane (PM; Li et al., 1999; Turner et al., 1998), are pooled. CalS activity is then solubilized with the detergent digitonin, giving a digitonin supernatant (DS). The final step in the enrichment process is product entrapment, in which the solubilized CalS is incubated with its substrate, UDP-Glc, and the resulting insoluble product, callose, is pelleted along with any entrapped CalS enzyme (PE). At each stage of the enrichment, CalS activity is assayed in the presence of trypsin, which activates any inactive CalS (Li et al., 1997, 1999) ensuring that ‘total’ CalS activity is measured. This allows comparison between the total amount of enzymic activity and protein at each stage of the enrichment (see below) as the ratio of inactive/active CalS changes during the enrichment procedure (Li et al., 1999). The specific activity of CalS increased over 100-fold through the enrichment procedure (Figure 1c), a lower increase than reported previously (Turner et al., 1998) because in earlier studies trypsin was not routinely included in CalS assays, which would have lead to lower reported activities for H, MM and PM fractions.
Approximately equal amounts of protein from each stage of the enrichment were fractionated by SDS–PAGE and stained with Coomassie brilliant blue (Figure 1a). A polypeptide of the correct molecular weight to be NaGSL1 (220 kDa) was enriched with CalS activity and represented a major proportion of the protein in the PE. This 220 kDa polypeptide is identical to the 190 kDa polypeptide reported previously from N. alata pollen tubes (Turner et al., 1998), with the apparent difference in size resulting from the use of more accurate molecular weight markers in our study. The polypeptide at approximately 100 kDa, later identified by proteomics as an H+-ATPase, was not enriched during the latter phases of purification. No other polypeptides were detectably enriched in the product-entrapped material, as seen on the Coomassie brilliant blue-stained gel (Figure 1a). As the 220 kDa polypeptide was likely to be NaGSL1 based on its molecular weight, and because it was enriched with CalS activity, we investigated the enrichment and identity of this 220 kDa polypeptide further.
Enrichment of NaGSL1 with CalS activity
Affinity-purified anti-GSL antibodies specifically detected a 220 kDa polypeptide in pollen-tube preparations (Figure 1b) that was not detected by pre-immune serum (data not shown), and which was deduced to be NaGSL1. The abundance of NaGSL1 (as detected by the anti-GSL antibodies) increased substantially during the enrichment process (Figure 1b). NaGSL1 was barely detectable in H or MM, was more abundant in the PM and DS, and was present in high amounts in the PE. The increasing amount of NaGSL1 on the Western blot correlated with the increased abundance of the 220 kDa polypeptide on the Coomassie brilliant blue-stained gel and with increasing specific activity of CalS through the CalS enrichment procedure (Figure 1). Both NaGSL1 and CalS activity were readily detectable in the PM and both were greatly enriched by product entrapment.
Proteomic analysis of product-entrapped material
We used a proteomic approach to confirm the identity of the 220 kDa polypeptide as NaGSL1, and to determine the identity of other polypeptides in the PE. PE samples were therefore separated by SDS–PAGE and stained with silver, which is more sensitive than Coomassie brilliant blue. The 220 kDa polypeptide was the first polypeptide to appear with silver staining, indicating that it is the most abundant protein in the PE (Figure 2a, centre). However, as this type of staining is time-dependent and its intensity varies depending on the protein (i.e. staining is not quantitative), the PE was also subjected to RuBPs staining (Figure 2a, left). This staining is time-independent and suitable for quantitative comparison between polypeptide bands (Lamanda et al., 2004), and it only detected the 220 kDa polypeptide, confirming that this is the major protein in PE. Another sample of the PE fraction was over-stained with silver to detect all other proteins in addition to the 220 kDa polypeptide and thereby determine the regions to be excised for proteomic analysis (Figure 2a, right).
The 220 kDa polypeptide from two product-entrapped pellets (prepared separately from two batches of pollen tubes) was excised from a silver-stained gel and digested with trypsin. MALDI-TOF MS analyses of the resultant peptides are shown in Figure 2(b,c). The experimentally observed peptide masses were compared with a theoretical trypsin digest of the GenBank green plant sequences, and the 220 kDa polypeptide was identified as NaGSL1 (Table 1). All major peptide peaks in one digest (Figure 2b) and all but five in the other (Figure 2c) could be assigned to NaGSL1 or to peptides produced by the autolysis of trypsin. The five unassigned peptides may derive from NaGSL1 but contain post-translational modifications not included in the search criteria. Twenty-four peptides from one digest and 33 peptides from the other were derived from NaGSL1 (Figure 2b,c, respectively; Table S1), with 20 of these peptides being common to both digests. None of these peptides were phosphorylated. Overall 23% of the NaGSL1 sequence was covered, mainly regions predicted to be cytoplasmic (Figure 3). The high sequence coverage confirmed NaGSL1 as the major source of peptides in the 220 kDa polypeptide digest, and the low number of unassigned peptides further indicated that NaGSL1 is most likely the only 220 kDa polypeptide in PE. This also confirmed that the anti-GSL antibodies specifically detect NaGSL1 (Figure 1b).
Table 1. Identity of proteins in product-entrapped material from N. alata pollen tubes. Top hits in the GenBank green plant protein database as searched by MASCOT using MS spectra obtained from gel-slice samples shown in Figure 2(a)
Molecular weight (kDa)
Peptide mass matches
aThe 220 kDa polypeptide was analysed separately from two separate product-entrapped pellets.
bPercentage of peptide sequence coverage of the entire protein.
The 220 kDa polypeptide was also identified as NaGSL1 by LC-ESI-MS/MS peptide sequencing (Table 1). The experimentally observed masses and deduced amino acid sequences of six tryptic peptides matched those of peptides from NaGSL1 (Table 2, see Figure S1 for spectra), and no amino acid sequences were obtained from proteins other than NaGSL1. Each of these six peptides was also observed by MALDI-TOF MS, and their location within the NaGSL1 sequence is shown in Figure 3.
Table 2. Peptides from NaGSL1 sequenced by LC-ESI-MS/MS. Peptides identified in the GenBank green plant protein database (as searched by MASCOT) in the same 220 kDa gel slice as that analysed by MALDI-TOF MS in Figure 2(b)
Position (amino acid number)
Six peptides had experimental masses that matched those of peptides from a theoretical digest of NaGSL1. The amino acid sequence deduced from the MS/MS spectra (see Figure S1) of these six peptides matched the sequence of NaGSL1 peptides. Sequence could not be deduced from any other spectra.
Tryptic peptides from polypeptides in other selected regions of the PE lane in Figure 2(a) (right panel) were also analysed by MALDI-TOF MS. The 103 kDa polypeptide contained plasma membrane H+-ATPase (Table 1), as nine peptides had mass matches with peptides from a Daucus carota plasma membrane H+-ATPase. The 60 kDa polypeptide contained the β-subunit of mitochondrial ATPase (Table 1), based on seven peptides with mass matches with peptides from the N. sylvestris protein. Many peptides in the spectra from the 103 and 60 kDa polypeptides were unassigned (16 and 20 peptides, respectively; data not shown), probably because sequences for these proteins from N. alata pollen tubes are not available in GenBank. Tryptic peptides from other silver-stained protein bands (77, 61, 55 and 35 kDa) and from the 110–140 kDa region of the gel (where the 125 kDa NaCSLD1, if present, would be located) were also analysed, but there was insufficient material present in these gel slices to enable peptide masses to be obtained. Thus, any other polypeptides present in PE material were present in low amounts compared to NaGSL1, consistent with them not being clearly stained with Coomassie blue (Figure 1a) or RuBPs (Figure 2a, left panel).
NaGSL1 has been proposed to encode the CalS enzyme in pollen tubes of N. alata, based on its similarity to fungal 1,3-β-glucan synthase genes and its high expression in pollen tubes (Doblin et al., 2001). Here we have used a biochemical and proteomic approach to further link the NaGSL1 protein with pollen-tube CalS. A process of membrane fractionation and product entrapment was used to enrich CalS activity from pollen tubes, and a 220 kDa polypeptide in the CalS-enriched PE was shown by proteomics and Western analysis to be NaGSL1. The NaGSL1 polypeptide was abundant in CalS-enriched PE, and was specifically enriched with CalS activity, consistent with NaGSL1 being involved in callose synthesis. Taken together with the other biochemical and molecular studies of plant GSLs by ourselves and other laboratories (see below), we conclude that NaGSL1 is involved in the developmentally regulated deposition of callose in Nicotiana pollen tubes.
GSL proteins from somatic tissues have previously been implicated in wound-activated CalS activity, with HvGSL1 being identified in the callose-synthesizing region of a native gel containing membrane proteins from barley suspension culture cells (Li et al., 2003), and insertional mutations in AtGSL5 being associated with a loss of callose deposition in response to wounding or pathogen challenge in Arabidopsis (Jacobs et al., 2003; Nishimura et al., 2003). These systems contrast with the CalS encoded by NaGSL1 in N. alata pollen tubes, which is a developmentally regulated enzyme. Mutational studies of the Arabidopsis orthologue of NaGSL1, AtGSL2, indicated that this gene is required to produce the temporary callose walls that separate developing microspores as well as the callose in pollen-tube walls and plugs (Dong et al., 2005; Nishikawa et al., 2005). Another Arabidopsis GSL gene, AtGSL6, is important in the formation of callose in the cell plate (Hong et al., 2001a). Thus, GSL proteins appear to be involved in both developmentally regulated and wound-activated callose synthesis in plants, supporting the proposal that different GSL proteins are responsible for callose synthesis in different locations throughout the plant.
We conducted a thorough proteomic analysis of the other polypeptides in PE material, to examine their likely involvement, if any, in callose synthesis. The cellulose synthase-like (CSL) gene families have been implicated in polysaccharide synthesis, including CSLA (mannans; Dhugga et al., 2004; Liepman et al., 2005) and CSLF [(1,3;1,4)-β-glucan synthases] (Burton et al., 2006), but no peptides deriving from these gene products were detected in pollen tube samples enriched for CalS. Specifically, no polypeptides of the predicted molecular weight for NaCSLD1 (125 kDa), the other putative polysaccharide synthase highly expressed in pollen tubes (Doblin et al., 2001), were detected by SDS–PAGE using three staining methods, and no NaCSLD1 peptides were identified in the proteomic analysis, indicating that NaCSLD1 is either absent from the PE fraction or is present in such a low amount that it is below the level of detection. The other polypeptides that were identified at low levels in PE, a plasma membrane H+-ATPase and the β-subunit of a mitochondrial ATPase, are most likely contaminants. The 103 kDa plasma membrane H+-ATPase is abundant in the plasma membrane-enriched fraction and the digitonin supernatant, but is not enriched in PE (Figure 1a), suggesting that its presence in the callosic pellet is due to non-specific entrapment. The β-subunit of a mitochondrial ATPase was present at low levels that varied between CalS purifications and is from a subcellular compartment in which callose synthesis does not occur. ATPase polypeptides have also been detected by others characterizing PE material from somatic cells of plants (e.g. Bulone et al., 1995) and fungi (Schimoler-O’Rourke et al., 2003), and these workers have also concluded that these proteins are contaminants. The very low abundance of other polypeptides in the PE compared to the 220 kDa NaGSL1 polypeptide, which was always the predominant enriched protein in this fraction, precluded their identification by MS.
Although no proteins other than NaGSL1 likely to be involved in callose synthesis were therefore detected in PE material, studies in other plant systems have found proteins associated with GSL proteins. For instance, the GhGSL1 protein from cotton fibres binds calmodulin in the presence of Ca2+in vitro, and contains a putative calmodulin-binding domain in the N-terminal cytoplasmic region (Cui et al., 2001). Also, yeast two-hybrid analyses indicate that AtGSL6, the putative cell-plate CalS from Arabidopsis, interacts with phragmoplastin, a cell plate-associated, dynamin-like GTP-binding protein, and with UGT1, a novel UDP-Glc transferase (Hong et al., 2001a,b). Further, previous biochemical studies on enrichment of the wound-induced Ca2+-dependent callose synthase have identified co-purifying polypeptides with molecular weights of approximately 30–80 kDa. For example, polypeptides at 55 and 70 kDa in pea tissue (Dhugga and Ray, 1994) and at 38 and 78 kDa in mung bean (Kudlicka and Brown, 1997) co-purify with CalS activity, but none of these polypeptides have been linked to specific gene products related to polysaccharide synthesis. However, association of a GSL with other proteins and the identity of these proteins may relate to differences in the cellular environment of different GSLs, and the way they are regulated during growth and development. GhGSL1 may be regulated by the Ca2+-binding calmodulin protein, but pollen-tube CalS is Ca2+-independent (Schlüpmann et al., 1993), consistent with NaGSL1 not containing the putative calmodulin-binding domain found on GhGSL1 (data not shown). Similarly, proteins that associate with AtGSL6 may locate it to the cell plate (e.g. phragmoplastin) or allow it to channel UDP-Glc to CalS (e.g. UGT1), but the high cytoplasmic UDP-Glc concentrations in the pollen tube (3.5 mm; Schlüpmann et al., 1994) mean that a specific protein to feed UDP-Glc into CalS is not be required in this cell type. As our analysis focussed on higher molecular weight polypeptides, it is possible that proteins of less than 30 kDa could be involved in callose synthesis in pollen tubes and have escaped identification in this study.
The data presented here add to the accumulating evidence that the GSL proteins are involved in callose synthesis. However, the molecular mechanisms underlying such activity and the amino acid residues important in binding the substrate, UDP-Glc, are yet to be determined. Indeed, such molecular analysis has not been performed for any members of glycosyltransferase family 48 (GT48), the GT family containing the GSL and FKS proteins. None of these proteins contain a recognized UDP-Glc binding motif, such as the D,D,D,QXXRW motif found in the GT2 family containing the CesA and CSL proteins (Saxena and Brown, 2000; Saxena et al., 1995). An FKS protein from Neurospora crassa binds UDP-Glc in vitro (Schimoler-O’Rourke et al., 2003) and polypeptides of approximately 200 kDa, presumably GSL proteins, in CalS preparations from cotton fibres (Delmer et al., 1991) and red beet (Mason et al., 1990) were labelled with radioactive UGP-Glc, showing that GT48 proteins can bind UDP-Glc. Several conserved motifs in the GSL proteins have been proposed as candidates for this function (Li et al., 2003), but none have been examined experimentally.
Whether GSL proteins interact with each other is also unknown, although it seems likely that CalS complexes throughout the plant, including the pollen tube, contain more than one GSL polypeptide. This conclusion is based on analyses of plasma membrane proteins from spinach leaves (Kjell et al., 2004) and barley suspension culture cells (Li et al., 2003) fractionated under non-denaturing conditions, where GSL proteins migrate as high molecular weight complexes that are well in excess of the size of a single GSL polypeptide. Furthermore, the products of CalS activity, 1,3-β-glucans, form helices composed of three parallel chains (Kjell et al., 2004; Stone and Clarke, 1992), consistent with the presence of three catalytic subunits in a CalS complex. This situation is analogous to that observed in cellulose synthesis: cellulose microfibrils contain 36 or more chains of cellulose (Herth, 1983), and the size of the cellulose synthase complex visualized in electron microscopy studies is consistent with the presence of 36 CesA proteins, possibly arranged in six groups of six proteins (Doblin et al., 2002). If several GSL proteins are associated in a CalS complex, these complexes may contain only one type of GSL (a homo-oligomer), or may contain a number of different GSL proteins (a hetero-oligomer), as occurs with cellulose synthesis in which different CesA proteins associate to form an active complex (Gardiner et al., 2003; Taylor et al., 2003). CalS complexes from different plant tissues may thus differ in the presence and identity of associated proteins and also in the type and number of GSL proteins involved.
Our current knowledge of the biochemistry of the pollen-tube CalS forms an ideal platform from which to conduct further studies into the mechanism(s) involved in catalytic activity and regulation of callose synthesis in this cell type. However, as discussed above, the regulation of CalS complexes is likely to be very different in different locations in the plant, and hence regulation of CalS in other cell types will need to be studied separately.
Plants of N. alata Link & Otto (self-incompatibility genotype S2S3) were grown as previously described (Schlüpmann et al., 1993), and pollen was harvested and pollen tubes grown in growth medium using established conditions (Li et al., 1997).
Pollen-tube homogenate and mixed membranes were prepared from pollen tubes grown in culture for 16 h (Schlüpmann et al., 1993). Continuous density-gradient centrifugation (without a flotation step), solubilization of CalS with digitonin (using a 10:1 detergent:protein weight ratio) and product entrapment of CalS (using a 30 min incubation of solubilized membranes with UDP-Glc, followed by centrifugation at 1500 g for 5 min at room temperature) were all conducted as described previously (Turner et al., 1998) with the noted modifications.
Standard assay of CalS activity
Incorporation of radioactivity from UDP-[U-14C]Glc into product insoluble in 66% v/v ethanol was used to assay CalS activity from pollen tubes as described previously (Turner et al., 1998). Each assay contained between 3 and 30 μg protein and 10 μg trypsin (TPCK-treated, Sigma; http://www.sigmaaldrich.com/).
Protein concentration determination and SDS–PAGE
Protein concentrations were determined using a Coomassie brilliant blue dye-binding assay (Bio-Rad; http://www.bio-rad.com/) with BSA as the standard. Polyacrylamide gels were prepared using standard methods (Sambrook et al., 1989). Protein samples were mixed with SDS–PAGE loading buffer and incubated at 37°C for 1 h before electrophoresis. Gels were stained with either Coomassie brilliant blue using the GelCode® Blue Stain Reagent (Pierce; http://www.piercenet.com), silver (Rabilloud, 1992) or Ruthenium II Tris (barthophenanthroline disulphonate, RuBPs; Lamanda et al., 2004) with fluorescence detected on a Typhoon 8600 imager (Amersham Biosciences; http://www5.amershambiosciences.com/) with 532 nm excitation and 610BP30 emission filter settings.
The sequence corresponding to a 46 kDa peptide (amino acids 1018–1418 from the N-terminus of NaGSL1) was amplified by PCR from N. alata pollen tube cDNA and cloned into the pProEX HTa vector (Invitrogen; http://www.invitrogen.com). The resultant plasmid was transformed into BL21(DE3) cells along with the RIG plasmid (Baca and Hol, 2000). Expression was induced in a 500 ml culture by addition of IPTG to 0.6 mm, and the peptide was enriched using Ni-NTA (Qiagen; http://www.qiagen.com/) affinity chromatography as described by the manufacturer. Ni-NTA-eluted peptides were fractionated by SDS–PAGE and detected by staining with 1 m KCl. Peptides were excised from the gel, and the gel slices washed with water, lyophilized, ground to a powder and finally resuspended in half the original gel-slice volume of water. Antibodies were raised by intramuscular injection of the resuspended gel powder (100–200 μg protein) with Freund’s complete adjuvant (Sigma) into New Zealand white rabbits (Monash University, Melbourne, Australia). Booster injections were given 21, 49 and 77 days after the initial injection, and the rabbits were exsanguinated 94 days after immunization.
Total IgG was purified from the immune serum using Protein A–Sepharose CL-4B (Amersham Biosciences) according to the manufacturer’s instructions. Anti-GSL antibodies were affinity purified by incubating the IgG fraction with the bacterially expressed peptide immobilized on nitrocellulose membrane strips (approximately 5 μg protein 0.5 cm−2 membrane). Membrane strips carrying immobilized peptide were incubated at 4°C overnight in 3% w/v milk powder in PBS, washed, incubated overnight with 1.5 ml of the IgG-purified immune serum diluted with 3 ml PBS, and washed again. The bound antibodies were then eluted in 600 μl 0.2 m glycine–HCl (pH 2.5) and neutralized immediately with 300 μl 1 m KPO4 (pH 9.0), 5% w/v BSA.
Western blotting was performed using standard methods (Sambrook et al., 1989), with an OSMONIC™ Nitropure 22 μm nitrocellulose membrane (GE Osmonics Labstore; http://www.osmolabstore.com). After blocking, the membrane was incubated in a 1:300 dilution of affinity-purified anti-GSL antibodies in PBS, followed by incubation in a 1:10 000 dilution of a goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Pierce), and detection was performed using SuperSignal® West Pico chemiluminescent substrate (Pierce).
In-gel trypsin digestion
Regions excised from a silver-stained gel were diced into approximately 1 mm3 pieces, destained with 5 min washes in 50 mm NH4HCO3 with 0.5% hydrogen peroxide, and then dried under vacuum. Polypeptides were reduced with 10 mm DTT in 50 mm NH4HCO3 for 1 h at 56°C, and alkylated with 55 mm iodoacetamide in 50 mm NH4HCO3 for 45 min at room temperature. The gel pieces were washed with 50 mm NH4HCO3 then 100% acetonitrile for 5 min each, and dehydrated under vacuum. The gel pieces were covered with 12.5 ng μl−1 sequencing grade modified trypsin (Promega; http://www.promega.com/) in 50 mm NH4HCO3 and incubated on ice for 45 min, then overnight at room temperature. Peptides were eluted by washing for 5 min with agitation once with 20 μl 50 mm NH4HCO3 and twice with 20 μl 50% v/v acetonitrile, and then once with 20 μl 5% v/v formic acid and twice with 40 μl 100% acetonitrile. Gel pieces were rinsed with 15 μl 50 mm NH4HCO3 between each wash. All washes and rinses were combined and the volume reduced to near dryness, and the semi-dried tryptic peptides then reconstituted with 60 μl of 0.1% v/v formic acid and filtered through a 0.20 μm Minisart RC4 single-use syringe filter (Sartorius; http://www.sartorius.com).
For MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry), 2 μl of filtered tryptic peptides were mixed with 10 μl 0.1% v/v formic acid, and salts removed using a ZipTip® pipette tip (Millipore; http://www.millipore.com). Peptides were eluted from the ZipTip with 1 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% v/v acetonitrile, 0.1% v/v formic acid directly onto a MALDI-TOF MS target plate. MALDI-TOF MS was conducted on a Voyager-DE STR instrument (Applied Biosystems; http://www.appliedbiosystems.com/), with 50 random shots averaged per spectrum using an accelerating voltage of 20 kV, 69% grid voltage, and a delay time of 166 ns. The spectra were baseline-corrected, noise-filtered and the peaks de-isotoped and then internally calibrated using the autolyzed trypsin peaks of 842.500 and 2211.105 Da. Spectra were then searched using MASCOT software (Matrix Science) run on an in-house server. MASCOT parameters were: database, NCBI NR protein database; taxonomy, green plants (Viridiplantae); enzyme, trypsin; fixed modifications, carbamidomethyl; variable modifications, oxidized methionine and phosphorylation; peptide tolerance, 0.1 Da; number of missed cleavage sites, up to 1. Matches were considered significant if the MASCOT ion score was greater than that required to show identity or extensive homology at a confidence level of <0.05.
For LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry), 40 μl of tryptic peptide solution was injected into a LC-Packings UltiMate capillary LC system (LC-Packings; http://www.lcpackings.com), and pre-concentrated and desalted on a 300 μm × 5 mm Vydac C18 pre-column (Grace VyDac; http://www.vydac.com). The pre-column was placed in line with a VyDac C18 column (75 μm internal diameter × 15 cm, 3 μm 100 Å−1). Peptides were eluted from the C18 column onto the MS via an ESI source using a gradient of 0–70% v/v acetonitrile in 0.1% v/v formic acid over 60 min at a flow rate of 0.25 μl min−1. MS and MS/MS data were acquired on a QSTAR XL™ hybrid quadrupole TOF instrument (Applied Biosystems/MDS Sciex; http://www.mdssciex.com) using analystQS software (Applied Biosystems/MDS Sciex) in data-dependent acquisition mode. Spectra were also searched using the MASCOT software. Parameters were as above, except that peptide tolerance for MS was increased to 0.25 Da and the MS/MS tolerance was 0.125 Da. Identifications were considered positive when the MASCOT ion score was over 20 and the e-value was <1. MS/MS spectra were also validated manually.
This work was supported by the Grains Research and Development Corporation through funding to the Cereal Functional Genomics Group (L.B., M.D., E.N. and A.B.) and by a University of Melbourne Research Development Grant to S.M.R. L.B. acknowledges an Australian Post-Graduate Award.