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Nectar is a very complex mixture of substances. Some components (sugars and amino acids) are considered primary alimentary rewards for animals and have been investigated and characterized in numerous species for many years. In contrast, nectar proteins have been the subject of few studies and little is known of their function. Only very recently have detailed studies and characterization of nectar proteins been undertaken, and then for only a very few species. This current work represents a first step in the identification of a protein profile for the floral nectar of Cucurbita pepo. In this regard, the species studied is of particular interest in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived from male and female flowers may differ.
Manually excised spots from two-dimensional (2-D) electrophoresis were subjected to in-gel protein digestion. The resulting peptides were sequenced using nanoscale LC–ESI/MS-MS (liquid chromatography–electrospray ionization/tandem mass spectrometry). An MS/MS ions search was carried out in Swiss-Prot and NCBInr databases using MASCOT software.
Two-dimensional electrophoresis revealed a total of 24 spots and a different protein profile for male and female flower nectar. Four main proteins recognized by 2-D electrophoresis most closely resemble β-d-xylosidases from Arabidopsis thaliana and have some homology to a β-d-xylosidase from Medicago varia. They were present in similar quantities in male and female flowers and had the same molecular weight, but with slightly different isoelectric points.
A putative function for xylosidases in floral nectar of C. pepo is proposed, namely that they may be involved in degrading the oligosaccharides released by the nectary cell walls in response to hydrolytic enzymes produced by invading micro-organisms. Several types of oligosaccharides have been reported to increase the pathogenic potential of micro-organisms. Thus, it is possible that such a mechanism may reduce the virulence of pathogens present in nectar.
Nectar is the most common reward produced by angiosperm flowers, and is gathered by a large variety of animal pollinators. For most flying pollinators, nectar is the main alimentary reward and can easily be digested and used as an energy source to fuel their flight (Nicolson, 2007). It is a complex solution containing mainly sugars, but also a plethora of other substances that are present at lower concentrations or in trace amounts. These include amino acids, organic acids, lipids, inorganic ions, vitamins, volatiles and alkaloids, and they are able to function as both attractants and repellents (Sangaravelan et al., 2005; Nicolson and Thornburg, 2007; Kessler et al., 2008). Since the 1930s, proteins have also been detected in nectar (Beutler, 1935), but it is only recently that nectar protein profiles have been better characterized (Carter and Thornburg, 2004; Carter et al., 2007; Kram et al., 2008; González-Teuber et al., 2009, 2010; Hillwig et al., 2010). However, proteins are seemingly not involved in attracting or repelling animals. Two general functional classes of proteins were found to occur in nectar: carbohydrate-metabolizing enzymes (invertase, transfructosidase and transglucosidase) and proteins that function in defence against micro-organisms (Nicolson and Thorburg, 2007). Sugary solutions, such as nectar, especially when exposed to the atmosphere, are excellent media in which fungi and bacteria can grow and multiply. Therefore, it is important that a plant defends its sugary secretions and protects them from being used by microbes as a source of carbon and energy to attack its reproductive system.
To date, very few species have been investigated for nectar proteins, and the few studies that have appeared in the literature demonstrate that floral nectar contains a large, heterogeneous assemblage of defence proteins (Carter and Thornburg, 2000, 2004; Naqvi et al., 2005; Carter et al., 2007; Kram et al., 2008; Hillwig et al., 2010, 2011). The floral nectar of an ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains five nectar proteins (nectarins) that function in a novel biochemical pathway – the nectar redox cycle (NRC), and this pathway produces high concentrations of hydrogen peroxide that protect against micro-organisms.
Although the NRC appears to be present in some other unrelated species (Carter and Thornburg, 2000), it was not found to occur in Petunia, even though this belongs to the same family (Solanaceae) as tobacco (Hillwig et al., 2010). The floral nectar of Petunia hybrida contains several RNases, a peroxidase and an endochitinase that have antimicrobial activity (Hillwig et al., 2010, 2011).
A lipase (JNP1) was reported from the floral nectar of Jacaranda mimosifolia, although its antimicrobial activity has not yet been demonstrated (Kram et al., 2008). Extrafloral nectar also has its own enzymatic defence against invasion by micro-organisms in that the predominant enzymes present are chitinase, β-1,3-glucanase and peroxidase (Gonzales-Teuber et al., 2009, 2010). Defence proteins, such as glucosidases, chitinases, hydrolases and thaumatine-like proteins, have also been detected in pollination drops (Wagner et al., 2007). These secretions are produced by gymnosperm ovules and have a similar chemical composition to angiosperm nectar (Nepi et al., 2009).
This paper describes the preliminary characterization of the nectar protein profile for Cucurbita pepo (Cucurbitaceae), a species that is particularly interesting since, being monoecious with unisexual flowers, it is a suitable subject for the detection of differences in the composition of nectar derived from male and female flowers. Moreover, clear sexual dimorphism exists in C. pepo in terms of nectary morphology, volume and concentration of nectar, as well as the dynamics of nectar production and re-absorption (Nepi and Pacini, 1993; Nepi et al., 2001). The first main proteins to be identified were β-xylosidases, and these are involved in xylo-oligosaccharide degradation. A putative functional role is proposed for these enzymes.
Plants were cultivated at The Botanical Gardens of the University of Siena during summer 2009. Male and female flowers of Cucurbita pepo L. were bagged with fine gauze on the day prior to opening, so as to prevent the removal of nectar and its contamination by visiting animals. Special care was taken not to contaminate nectar with pollen (in male flowers), and not to damage the nectary tissue (in both male and female flowers). Nectar for total protein determination was collected from ten male and six female flowers by means of a 20 µL pipette. Total protein determination was carried out using the Bradford assay (Bradford, 1976). For all the other analyses, nectar from 5–10 flowers was pooled in a vial. In this way, a total of ten vials for each of the two sexes were obtained, each one containing 100–200 µL of nectar (storage vials). All the vials were stored at –80 °C until immediately prior to analysis.
Samples for two-dimensional (2-D) electrophoresis were prepared by pipetting 20 µL of nectar from each storage vial and thereby obtaining a total of 200 µL of male and female nectar that then underwent protein separation. Nectar samples (200 µL) were supplemented with 4 vols of 20 % trichloroacetic acid (TCA) in acetone plus 0·07 % 2-mercaptoethanol. Proteins were precipitated for 45 min at –20 °C and then pelleted by centrifugation (15 min at 4 °C, 15 000 g). Pellets were washed with cold acetone containing 0·07 % 2-mercaptoethanol, and residual acetone was removed by air-drying. The protein pellet was suspended in about 250 µL of rehydration/solubilization buffer (8 m urea, 2 m thiourea, 2 % CHAPS, 40 mm Tris, traces of bromophenol blue) for 2-D electrophoresis.
The concentration of proteins in all samples was calculated using the commercial 2-D Quant Kit (GE HealthCare). The assay was carried out according to the manufacturer's instructions using bovine serum albumin (BSA) as standard and a Shimadzu UV-160 spectrophotometer set at 480 nm. Each sample and standards were analysed in replicate at least three times. Protein concentration was performed after re-suspension in the rehydration/solubilization buffer in order that equivalent amounts of female and male nectar proteins might be loaded in 2-D gels.
First dimension protein separation [isoelectric focusing (IEF)] was accomplished using Immobiline Dry-Strip (GE HealthCare), 11 cm long, with a 4–7 pH gradient. This pH gradient was used after a trial run with pH 3–10, where all the proteins were separated in the range pH 4–7. Each strip was hydrated in 200 µL of rehydration/solubilization buffer containing 18 mm dithiothreitol (DTT), 20 µL mL−1 IPG Buffer (pH 4–7) and protein samples (50 µg) of either male or female nectar. After hydration, strips were subjected to the first separation using the Multiphor II (GE HealthCare) at 300 V (1 min), from 300 to 3500 V (1·5 h), and at 3500 V (3·5 h). Prior to the second dimension separation, strips were equilibrated for 15 min in equilibration buffer (50 mm Tris–HCl pH 8·8, 6 m urea, 30 % glycerol, 2 % SDS, traces of bromophenol blue, 10 mg mL−1 DTT). Separations of proteins by SDS–PAGE were obtained by applying the strips to pre-cast gels (Criterion XT Bis-Tris Precast Gel, 10 %, 11 cm, Bio-Rad). Gels were run using the Criterion Cell (Bio-Rad) at 200 V, constantly for 1 h, and subsequently stained with Bio-Safe Coomassie (Bio-Rad).
Gel images were captured using the Fluor-S Multi-Imager and analysed by Quantity One (for 1-D gels) and PDQuest (for 2-D gels). Both types of software were obtained from Bio-Rad. Exposure times were 5–7 s for gels stained with Coomassie/silver. Analysis of protein spots in gels and blots was accomplished using the Spot Detection Wizard of PDQuest by selecting the faintest spot in the scan (for setting the sensitivity and minimum peak value parameters), the smallest spot in the scan (for setting the size scale parameter) and the largest spot (for setting the radius of the background subtraction rolling ball and the streak removal rolling disc). Further analysis of spots was achieved using the Spot and Matchset tools. Quantification of selected spots was automatically done by the PDQuest software after spot detection. To validate the quantification analysis, spots were also analysed using the ImageJ software and the Measure command (http://imagej.nih.gov/ij/index.html); in such a case, spots were measured as ‘integrated density’, i.e. the product of area and mean grey value. No critical differences in spot quantitation were found by the two methods.
Electrophoretic spots were visualized by a mass spectrometry (MS)-compatible silver staining procedure (Sinha et al., 2001). They were manually excised, destained and dehydrated with acetonitrile (ACN). They were then rehydrated in trypsin solution, and in-gel protein digestion was performed by overnight incubation at 37 °C. Tryptic digests were extracted from the gel using a 50 % (v/v) ACN and 0·1 % (v/v) triﬂuoroacetic acid solution. The resulting peptides were then subjected to peptide sequencing using nanoscale liquid chromatography–electrospray ionization/tandem mass spectrometry (LC–ESI/MS-MS), as described in detail by Meiring et al. (2002). Briefly, all the analyses were carried out on an LC–MS system consisting of a PHOENIX 40 (ThermoQuest Ltd, Hemel Hempstead, UK) and an LCQ DECA IonTrap mass spectrometer (Finnigan, SanJose, CA, USA). The peptides, after a manual injection (5 µL) in a six-port valve, were trapped in a C18 trapping column (20 mm × 100 µm ID × 360 µm OD; Nanoseparations, Nieuwkoop, The Netherlands) using 100 % solvent A (HPLC grade water + 0·1 % formic acid) under a flow rate of 5 µL min−1 for 10 min. A linear gradient to 60 % solvent B (ACN + 0·1 % formic acid) in 30 min was performed for analytical separation. A column flow rate of 100–125 nL min−1 on a C18 analytical column (30 cm × 50 µm ID × 360 µm OD; Nanoseparations) was obtained using a pre-column splitter restrictor. The LC pump, the mass spectrometer as well as the automatic mass spectra acquisitions were controlled using the Xcalibur™ 1·2 system software. The MS/MS ion search was carried out in Swiss-Prot and NCBInr databases using MASCOT (Matrix Science Ltd, London, UK, http://www-.matrixscience.com) software available online. The taxonomy was limited to Viridiplantae (green plants). The peptide precursor charge was set to 2+ or 3 + ; a mass tolerance of ±1·2 Da for precursor peptide and ±0·6 Da for fragment peptides was allowed, and the number of accepted missed cleavage sites was set to one. Alkylation of cysteine by carbamidomethylation was assumed as a ﬁxed modiﬁcation, while oxidation and phosphorylation? was considered as a possible modiﬁcation. Peptides with individual ions scores [–10 × log(P), where P is the probability that the observed match is a random event] > threshold score indicate identity or extensive homology (P <0·05).
Nectar xylosidase activity was detected according to the spectrophotometric method indicated by Kumar and Ramón (1996). The method is based on the enzymatic reaction between 4-nitrophenyl β-d-xylopyranoside (pNpX) and xylosidase. This reaction produces p-nitrophenol whose concentration is determined spectrophotometrically at 400 nm. A 10 µL aliquot of male nectar and 10 µL of female nectar were obtained by pipetting 1 µL of nectar from each one of the ten storage vials. The nectar sample was mixed with 240 µL of sodium acetate buffer (50 mm at pH 5·0) and 250 µL of 2 mm pNpX solution. The mixture was incubated at 50 °C for 30 min. The reaction was stopped by adding 1 mL of 2 m sodium carbonate solution and the release of p-nitrophenol was measured as indicated above. A reference blank was obtained by substituting the nectar with an equivalent volume of water. The assay was performed in triplicate.
To confirm that the xylosidase activity is present under natural conditions, 25 µL of female nectar (2·5 µL from each of the ten storage vials) was diluted 1:40 in a solution containing the oligosaccharides xylopentaose, xylotriose and xylobiose to give a final concentration of 0·55, 1·70 and 1·74 mg mL−1, respectively. The solution was incubated at 30 °C – a temperature comparable with that found during the summer flowering of C. pepo – and the chromatographic profile was detected at 24 and 48 h according to the methodology detailed below.
The presence of xylose and xylo-oligosaccharides (xylobiose, xylotreose and xylopentaose) was detected by high-performance liquid chromatography (HPLC). A 10 µL aliquot of male nectar and 10 µL of female nectar, obtained by pipetting 1 µL of nectar from each one of the ten storage vials, were diluted 1:40 with distilled water. The solution was analysed by isocratic HPLC operating with an LC1 Waters system. A 20 µL aliquot of sample and standard solution was injected. Water (MilliQ, pH 7) with a flow rate of 0·5 mL min−1 was used as the mobile phase. Sugars were separated in a Waters Sugar-Pack I column (6·5–300 mm), maintained at 90 °C and identified by a refractive index detector (Waters 2410). Determination was performed in triplicate.
The total protein content of male flower nectar was 583·06 ± 146·99 µg mL−1 (mean ± s.d., n = 10), while in female flowers it was 498 ± 178·08 µg mL−1 (mean ± s.d., n = 6). The difference was not statistically significant (Mann–Whitney U-test, Z = 0·976, P = 0·328). Several analyses by 2-D electrophoresis revealed a constant number of 24 spots for the female nectar (Fig. 1B, arrowheads), which apparently contains more polypeptides than the male nectar (Fig. 1A). While 15 spots were common to both male and female flower nectar (black arrowheads), nine were present in female flowers only (arrows) and two were present only in male flowers (Fig. 1A, white arrowheads). The different protein profile in male and female floral nectar may be related to the different ways in which nectar is presented (i.e. nectar exposure to external environment, see Nepi and Pacini, 1993) and/or to the different dynamics of nectar production and reabsorption that have been reported for the two sexes (Nepi et al., 2001; Nepi and Stpiczyńska, 2007). Due to the very different modes of presentation, female nectar is much more accessible to pollinators and much more exposed to the atmosphere than male nectar and thus more exposed to contamination by yeasts and bacteria. This may suggest a more complex defence arsenal against micro-organisms in female flowers, and this, in turn, may be related to the higher number of polypeptides. This supports the hypothesis that extrafloral nectar, being more exposed and less ephemeral, is characterized by the presence of a greater number of proteins than floral nectar (Heil, 2011).
Four of the 24 proteins recognized by 2-D electrophoresis were identified by MS and most closely resemble β-d-xylosidases from Arabidopsis thaliana, with some homology to a β-d-xylosidase from Medicago varia (Table 1). Results of the MS/MS analysis are summarized in Table 1, where the spot numbers match those reported in Fig. 1C and D. Accession number in the UniProtKB database, protein name, species, peptide sequence and Mascot Score/Mascot threshold score are also included. As the complete genome sequence of C. pepo has not yet been determined, the Mascot peptide sequence search was carried out setting a large taxonomy range to Viridiplantae. As a consequence, the majority of the peptide sequences found match the amino acid sequence of β-d-xylosidase in A. thaliana, one of the completely sequenced plant organisms. It is interesting to note that these peptides probably represent the most conserved of the amino acid sequences of β-d-xylosidase from A. thaliana and C. pepo. From the peptide sequence analysis, it is clear that the four spots can be assigned to β-d-xylosidase, as some common peptides have been found between them. The four spots had a molecular mass of approx. 70 kDa and a pI ranging from 5·6 to 6·3 (Fig. 1C). Having identical molecular weights, but slightly different isoelectric points (Fig. 1C, D), they are thought to be isoforms of the same protein, probably resulting from specific post-translational modifications. On the basis of spot quantification analyses, the four hypothetical isoforms were present in relatively similar quantities in both male and female flowers (Fig. 1E), with the exception of polypeptide 4, which appeared to be slightly more abundant in the female nectar.
The presence of xylosidases was supported by the enzymatic assay. This revealed xylosidase activity of 0·23 ± 0·04 and 0·29 ± 0·06 µm min−1 mL−1 in female and male nectar, respectively. The occurrence of such activity under conditions similar to those that occur in nature (temperature = 30 °C) was confirmed by the increase in xylose concentration and a corresponding decrease in the concentration of the xylo-oligosaccharides – and especially xylobiose – in the mixture nectar + oligosaccharides after incubation at 24 and 48 h (Fig. 2).
Neither xylose nor the xylo-oligosaccharides tested was detected in male and female nectar, indicating that lack of xylosidase activity is simply due to the absence of the appropriate substrate.
Endoxylanases and xylosidases are key enzymes in the degradation of xylans, the major hemicelluloses found in the secondary walls of most higher plants. Xylans have a relatively complex structure based on a β-1,4-linked d-xylose backbone, substituted to varying degrees (Minic et al., 2004). Endo-β-1,4-xylanases hydrolyse the insoluble xylan backbone into shorter, soluble xylo-oligosaccharides, while β-d-xylosidases hydrolyse xylo-oligosaccharides and xylobiose from their non-reducing ends to liberate d-xylose (Minic et al., 2004, and references therein). Side chain-cleaving enzymes, such as α-l-arabinofuranosidase, are also important, and they are recognized as a limiting step in achieving efficient hydrolysis of the polysaccharide polymer (Tuncer and Ball, 2003).
Plants use these enzymes for dynamic regulation of cell wall morphology, structure and composition during their development (Minic et al., 2004). These same classes of enzymes represent important components in the offensive arsenal of phyto-pathogens, both fungi and bacteria, and they are used to degrade cell wall polymers when invading plant tissue (Beliën et al., 2006).
The interaction between plants and pathogens induces a diverse array of responses from both sides. Plant defence responses, including cell wall strengthening, production of antimicrobial compounds, ethylene biosynthesis and rapid, localized cell death (Aro et al., 2005; Beliën et al., 2006, and references therein), are frequently triggered by pathogen- or plant-derived molecules that have been termed ‘elicitors’ (Bucheli et al., 1990; Esquerré-Tugayé et al., 2000). Plant responses can be stimulated by the direct interaction of a specific pathogen peptide with the plant cell and do not involve intermediate compounds. Specific fungal xylanases are reported to be able to stimulate plant responses directly (Sharon et al., 1993; Noda et al., 2010).
Xylo-oligosaccharides are recognized as important signals for defence responses in plants, and are most probably involved in the elicitation of phytoalexins, ethylene synthesis, PR (pathogenesis-related) proteins (Ryan and Farmer, 1991) and xylanase inhibitor proteins (Beliën et al., 2006) following plant tissue invasion by fungi. At the same time, these wall-derived molecules increase the pathogenic potential of micro-organisms. It was demonstrated that the production of plant cell wall-degrading enzymes in micro-organisms (cellulase, hemicellulase, pectinase and ligninases) can be induced by the presence of wall polymers, or molecules derived from these polymers. For example, the presence of xylobiose and various other oligosaccharides in cultures of the fungus Trichoderma reesei is known to induce cellulase and xylanase expression (Aro et al., 2005).
In the present study, the authors propose a functional role for β-xylosidase in C. pepo nectar that takes into account all the above observations. The invasion of the nectar by micro-organisms is followed by damage to the nectary cell walls due to the action of cellulases and xylanases produced by the pathogens. This action may induce the release of several oligosaccharides from the cell walls. After the invasion of the nectar by micro-organisms, it is likely that the relative abundance of the plant cell wall-derived oligosaccharides displaying differing degrees of polymerization (DP) is important to the plant. For example, their presence at very low concentrations may be useful in ‘alerting’ the plant's defence mechanism against micro-organisms (Shibuya and Minami, 2001), but increased levels may be detected promptly by the latter, thus resulting in their increased ability to damage the plant cell wall via xylanase activity. Therefore, the degradation of surplus xylo-oligosaccharides may help keep pathogens present in nectar at a reduced state of activity. It is important to point out that the DP of xylo-oligosaccharides capable of inducing the xylanase activity of micro-organisms varies widely: xylobiose (DP 2) is reported to be the elicitor of xylanases in the fungi Aspergillus and Trichoderma (Aro et al., 2005), while the same enzyme activity is stimulated by xylo-oligosaccharides with a DP of 6–30 in the bacterium Prevotella bryantii (Miyazaki et al., 2005). Thus, it is very likely that the nectar defence system, which must be effective against a wide range of micro-organisms, is equipped with a complete set of enzymes involved in xylan degradation (endo-β-1,4-xylanases, β-d-xylosidases and α-l-arabinofuranosidases), although only two of them were identified in the present study (Table 1). Another strategy involving the direct inhibition of microbial xylanase activity has been reported for Nicotiana (Harper et al., 2010). Here, the nectarin NEC4 functions as a defence agent that inhibits a xyloglucan-specific endoglucanase produced by fungi during pathogenesis. Thus, it appears that protection of nectar from invasion by micro-organisms may be direct (by inhibiting pathogen enzyme activity involved in cell wall degradation, as in Nicotiana) or indirect (by regulating the concentration of oligosaccharides released by cell walls and that elicit the cell wall-degrading activity of the micro-organism).
An alternative explanation for the function of xylosidases found in the floral nectar of C. pepo takes into account the possibility that these enzymes are derived from nectary cell walls, from where they are mobilized by nectar flow. Although the more common method for nectar exudation is via the modified stomata of the nectary epidermis, nectar exudation through the epidermal cell wall cannot be ruled out (Nepi et al., 1996; Nepi, 2007). Xylosidases and endo-xylanases may be involved in the reorganization of cell walls during the development of nectary parenchyma and the changes in cell shape and volume that accompany this process (Nepi et al., 1996). Reorganization of cell walls may also increase wall permeability to nectar. These enzymes, once released into the nectar, help defend the latter from fungal or bacterial invasion, as described above.
It is interesting to note that a xylosidase was also found in the pollination drop of Juniperus communis (Wagner et al., 2007). Both pollination drop and nectar are secretions that have a very similar chemical composition, even though their functions are entirely different (Nepi et al., 2009). Whereas the former is very common amongst gymnosperms, and is the landing site for pollen, the latter is widely distributed amongst angiosperms and is the most common reward for pollinators. Since both are sugary solutions and are more or less exposed to the environment, they are equally subject to contamination and invasion by micro-organisms. The presence of xylosidases in both these secretions may account for a certain degree of conservatism in the defence proteins of these two groups of plants.
Although this is the first attempt at determining the proteins present in C. pepo nectar, many of which still remain to be identified, it is clear that most of the main proteins (i.e. those present at high concentrations) found in this species are involved in protecting the plant against attack by micro-organisms. This defence strategy appears to fulfil an important requirement of floral nectar, as revealed by the probable presence of four isoforms of the same enzyme. However, the proteins identified here do not have a direct lethal effect, but rather an inhibitory effect on the pathogenic potential of micro-organisms. Furthermore, they cannot be responsible for the recently demonstrated inhibition of growth and metabolic activity of Escherichia coli and Erwinia tracheiphila by nectar of C. pepo ssp. texana (Sasu et al., 2010), and it is likely that other proteins or other substances are responsible for this. In short, it appears that the nectar of C. pepo possesses a complex chemical defence ‘arsenal’ that we are only just beginning to discover.
The authors are grateful to the personnel of the Botanical Gardens of the University of Siena for growing the plants used in this study.