Annals of Botany 108: 521 527, 2011
doi:10.1093/aob/mcr165, available online at www.aob.oxfordjournals.org
Xylan-degrading enzymes in male and female flower nectar of Cucurbita pepo
M. Nepi1,*, L. Bini2, L. Bianchi2, M. Puglia2, M. Abate3 and G. Cai3
1
BIOCONNET, Biodiversity and Conservation Network, Department of Environmental Sciences  G. Sarfatti , University of
2
Siena, Via Mattioli 4, 53100 Siena, Italy, Department of Biotechnology, University of Siena, Via Torre Fiorentina 1, 53100
3
Siena, Italy and Department of Environmental Sciences  G. Sarfatti , University of Siena, Via Mattioli 4, 53100 Siena, Italy
* For correspondence. E-mail [e-mail]
Received: 24 February 2011 Returned for revision: 12 April 2011 Accepted: 4 May 2011 Published electronically: 3 August 2011
Background and Aims 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 inter-
est in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived
from male and female flowers may differ.
Methods 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.
Key Results 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 b-D-xylo-
sidases from Arabidopsis thaliana and have some homology to ab-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.
Conclusions 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.
Key words: Cucurbita pepo, nectar, proteins, defence, xylan-degrading enzymes.
INTRODUCTION defence against micro-organisms (Nicolson and Thorburg,
2007). Sugary solutions, such as nectar, especially when
Nectar is the most common reward produced by angiosperm exposed to the atmosphere, are excellent media in which fungi
flowers, and is gathered by a large variety of animal pollinators. and bacteria can grow and multiply. Therefore, it is important
For most flying pollinators, nectar is the main alimentary reward that a plant defends its sugary secretions and protects them
and can easily be digested and used as an energy source to fuel from being used by microbes as a source of carbon and energy
their flight (Nicolson, 2007). It is a complex solution containing to attack its reproductive system.
mainly sugars, but also a plethora of other substances that are To date, very few species have been investigated for nectar
present at lower concentrations or in trace amounts. These proteins, and the few studies that have appeared in the literature
include amino acids, organic acids, lipids, inorganic ions, vita- demonstrate that floral nectar contains a large, heterogeneous
mins, volatiles and alkaloids, and they are able to function as assemblage of defence proteins (Carter and Thornburg, 2000,
both attractants and repellents (Sangaravelan et al., 2005; 2004; Naqvi et al., 2005; Carter et al., 2007; Kram et al.,
Nicolson and Thornburg, 2007; Kessler et al., 2008). Since the 2008; Hillwig et al., 2010, 2011). The floral nectar of an orna-
1930s, proteins have also been detected in nectar (Beutler, mental tobacco (Nicotiana langsdorffii × Nicotiana sanderae)
1935), but it is only recently that nectar protein profiles have contains five nectar proteins (nectarins) that function in a
been better characterized (Carter and Thornburg, 2004; Carter novel biochemical pathway  the nectar redox cycle (NRC),
et al., 2007; Kram et al., 2008; González-Teuber et al., 2009, and this pathway produces high concentrations of hydrogen
2010; Hillwig et al., 2010). However, proteins are seemingly peroxide that protect against micro-organisms.
not involved in attracting or repelling animals. Two general Although the NRC appears to be present in some other unre-
functional classes of proteins were found to occur in nectar: lated species (Carter and Thornburg, 2000), it was not found to
carbohydrate-metabolizing enzymes (invertase, transfructosi- occur in Petunia, even though this belongs to the same family
dase and transglucosidase) and proteins that function in (Solanaceae) as tobacco (Hillwig et al., 2010). The floral
# The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [e-mail]
522 Nepi et al.  Proteins in floral nectar of Cucurbita pepo
nectar of Petunia hybrida contains several RNases, a peroxi- by air-drying. The protein pellet was suspended in about
dase and an endochitinase that have antimicrobial activity 250 mL of rehydration/solubilization buffer (8 M urea, 2 M
(Hillwig et al., 2010, 2011). thiourea, 2 % CHAPS, 40 mM Tris, traces of bromophenol
A lipase (JNP1) was reported from the floral nectar of blue) for 2-D electrophoresis.
Jacaranda mimosifolia, although its antimicrobial activity
has not yet been demonstrated (Kram et al., 2008).
Determination of protein concentration
Extrafloral nectar also has its own enzymatic defence against
invasion by micro-organisms in that the predominant The concentration of proteins in all samples was calculated
enzymes present are chitinase, b-1,3-glucanase and peroxidase using the commercial 2-D Quant Kit (GE HealthCare). The
(Gonzales-Teuber et al., 2009, 2010). Defence proteins, such assay was carried out according to the manufacturer s instruc-
as glucosidases, chitinases, hydrolases and thaumatine-like tions using bovine serum albumin (BSA) as standard and a
proteins, have also been detected in pollination drops Shimadzu UV-160 spectrophotometer set at 480 nm. Each
(Wagner et al., 2007). These secretions are produced by gym- sample and standards were analysed in replicate at least three
nosperm ovules and have a similar chemical composition to times. Protein concentration was performed after re-suspension
angiosperm nectar (Nepi et al., 2009). in the rehydration/solubilization buffer in order that equivalent
This paper describes the preliminary characterization of the amounts of female and male nectar proteins might be loaded in
nectar protein profile for Cucurbita pepo (Cucurbitaceae), a 2-D gels.
species that is particularly interesting since, being monoecious
with unisexual flowers, it is a suitable subject for the detection
Two-dimensional electrophoresis
of differences in the composition of nectar derived from male
and female flowers. Moreover, clear sexual dimorphism exists First dimension protein separation [isoelectric focusing
in C. pepo in terms of nectary morphology, volume and con- (IEF)] was accomplished using Immobiline Dry-Strip (GE
centration of nectar, as well as the dynamics of nectar pro- HealthCare), 11 cm long, with a 4 7 pH gradient. This pH
duction and re-absorption (Nepi and Pacini, 1993; Nepi gradient was used after a trial run with pH 3 10, where all
et al., 2001). The first main proteins to be identified were the proteins were separated in the range pH 4 7. Each strip
b-xylosidases, and these are involved in xylo-oligosaccharide was hydrated in 200 mL of rehydration/solubilization buffer
degradation. A putative functional role is proposed for these containing 18 mM dithiothreitol (DTT), 20 mL mL21 IPG
enzymes. Buffer (pH 4 7) and protein samples (50 mg) of either male
or female nectar. After hydration, strips were subjected to
the first separation using the Multiphor II (GE HealthCare)
MATERIALS AND METHODS
at 300 V (1 min), from 300 to 3500 V (1.5 h), and at 3500
Plants were cultivated at The Botanical Gardens of the V (3.5 h). Prior to the second dimension separation, strips
University of Siena during summer 2009. Male and female were equilibrated for 15 min in equilibration buffer (50 mM
flowers of Cucurbita pepo L. were bagged with fine gauze Tris HCl pH 8.8, 6 M urea, 30 % glycerol, 2 % SDS, traces
on the day prior to opening, so as to prevent the removal of of bromophenol blue, 10 mg mL21 DTT). Separations of pro-
nectar and its contamination by visiting animals. Special teins by SDS PAGE were obtained by applying the strips to
care was taken not to contaminate nectar with pollen (in pre-cast gels (Criterion XT Bis-Tris Precast Gel, 10 %,
male flowers), and not to damage the nectary tissue (in both 11 cm, Bio-Rad). Gels were run using the Criterion Cell
male and female flowers). Nectar for total protein determi- (Bio-Rad) at 200 V, constantly for 1 h, and subsequently
nation was collected from ten male and six female flowers stained with Bio-Safe Coomassie (Bio-Rad).
by means of a 20 mL pipette. Total protein determination
was carried out using the Bradford assay (Bradford, 1976).
Analysis of gels
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 Gel images were captured using the Fluor-S Multi-Imager
the two sexes were obtained, each one containing 100 and analysed by Quantity One (for 1-D gels) and PDQuest
200 mL of nectar (storage vials). All the vials were stored at (for 2-D gels). Both types of software were obtained from
 80 8C until immediately prior to analysis. 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
Precipitation of proteins with TCA/acetone
PDQuest by selecting the faintest spot in the scan (for
Samples for two-dimensional (2-D) electrophoresis were setting the sensitivity and minimum peak value parameters),
prepared by pipetting 20 mL of nectar from each storage vial the smallest spot in the scan (for setting the size scale par-
and thereby obtaining a total of 200 mL of male and female ameter) and the largest spot (for setting the radius of the back-
nectar that then underwent protein separation. Nectar ground subtraction rolling ball and the streak removal rolling
samples (200 mL) were supplemented with 4 vols of 20 % disc). Further analysis of spots was achieved using the Spot
trichloroacetic acid (TCA) in acetone plus 0.07 % and Matchset tools. Quantification of selected spots was auto-
2-mercaptoethanol. Proteins were precipitated for 45 min at matically done by the PDQuest software after spot detection.
 20 8C and then pelleted by centrifugation (15 min at 4 8C, To validate the quantification analysis, spots were also ana-
15 000 g). Pellets were washed with cold acetone containing lysed using the ImageJ software and the Measure command
0.07 % 2-mercaptoethanol, and residual acetone was removed (http://imagej.nih.gov/ij/index.html); in such a case, spots
Nepi et al.  Proteins in floral nectar of Cucurbita pepo 523
were measured as  integrated density , i.e. the product of area the ten storage vials. The nectar sample was mixed with
and mean grey value. No critical differences in spot quanti- 240 mL of sodium acetate buffer (50 mM at pH 5.0) and
tation were found by the two methods. 250 mL of 2 mM pNpX solution. The mixture was incubated
at 50 8C for 30 min. The reaction was stopped by adding
1mL of 2 M sodium carbonate solution and the release of
Protein identification by LC ESI/MS-MS
p-nitrophenol was measured as indicated above. A reference
Electrophoretic spots were visualized by a mass spec- blank was obtained by substituting the nectar with an equival-
trometry (MS)-compatible silver staining procedure (Sinha ent volume of water. The assay was performed in triplicate.
et al., 2001). They were manually excised, destained and dehy- To confirm that the xylosidase activity is present under
drated with acetonitrile (ACN). They were then rehydrated in natural conditions, 25 mL of female nectar (2.5 mL from
trypsin solution, and in-gel protein digestion was performed each of the ten storage vials) was diluted 1:40 in a solution
by overnight incubation at 37 8C. Tryptic digests were containing the oligosaccharides xylopentaose, xylotriose and
extracted from the gel using a 50 % (v/v) ACN and 0.1% xylobiose to give a final concentration of 0.55, 1.70 and
(v/v) trifluoroacetic acid solution. The resulting peptides 1.74 mg mL21, respectively. The solution was incubated at
were then subjected to peptide sequencing using nanoscale 30 8C  a temperature comparable with that found during
liquid chromatography electrospray ionization/tandem mass the summer flowering of C. pepo  and the chromatographic
spectrometry (LC ESI/MS-MS), as described in detail by profile was detected at 24 and 48 h according to the method-
Meiring et al. (2002). Briefly, all the analyses were carried ology detailed below.
out on an LC MS system consisting of a PHOENIX 40
(ThermoQuest Ltd, Hemel Hempstead, UK) and an LCQ
Determination of xylose and xylo-oligosaccharides
DECA IonTrap mass spectrometer (Finnigan, SanJose, CA,
USA). The peptides, after a manual injection (5 mL) in a The presence of xylose and xylo-oligosaccharides (xylo-
six-port valve, were trapped in a C18 trapping column biose, xylotreose and xylopentaose) was detected by high-
(20 mm × 100 mm ID × 360 mm OD; Nanoseparations, performance liquid chromatography (HPLC). A 10mL
Nieuwkoop, The Netherlands) using 100 % solvent A (HPLC aliquot of male nectar and 10 mL of female nectar, obtained
grade water + 0.1 % formic acid) under a flow rate of 5 mL by pipetting 1 mL of nectar from each one of the ten storage
min21 for 10 min. A linear gradient to 60 % solvent B vials, were diluted 1:40 with distilled water. The solution
(ACN + 0.1 % formic acid) in 30 min was performed for was analysed by isocratic HPLC operating with an LC1
analytical separation. A column flow rate of 100 125 nL Waters system. A 20 mL aliquot of sample and standard
min21 on a C18 analytical column (30 cm × 50 mm ID × solution was injected. Water (MilliQ, pH 7) with a flow rate
360 mm OD; Nanoseparations) was obtained using a pre- of 0.5mL min21 was used as the mobile phase. Sugars were
column splitter restrictor. The LC pump, the mass spec- separated in a Waters Sugar-Pack I column (6.5 300 mm),
trometer as well as the automatic mass spectra acquisitions maintained at 90 8C and identified by a refractive index detec-
were controlled using the XcaliburTM 1.2 system software. tor (Waters 2410). Determination was performed in triplicate.
The MS/MS ion search was carried out in Swiss-Prot and
NCBInr databases using MASCOT (Matrix Science Ltd,
RESULTS AND DISCUSSION
London, UK, http://www-.matrixscience.com) software avail-
able online. The taxonomy was limited to Viridiplantae The total protein content of male flower nectar was 583.06 +
(green plants). The peptide precursor charge was set to 2+ 146.99 mg mL21 (mean + s.d., nź10), while in female
or 3 + ; a mass tolerance of +1.2 Da for precursor peptide flowers it was 498 + 178.08mg mL21 (mean + s.d., nź6).
and +0.6 Da for fragment peptides was allowed, and the The difference was not statistically significant (Mann
number of accepted missed cleavage sites was set to one. Whitney U-test, Zź0.976, Pź0.328). Several analyses by
Alkylation of cysteine by carbamidomethylation was 2-D electrophoresis revealed a constant number of 24 spots
assumed as a fixed modification, while oxidation and phos- for the female nectar (Fig. 1B, arrowheads), which apparently
phorylation? was considered as a possible modification. contains more polypeptides than the male nectar (Fig. 1A).
Peptides with individual ions scores [ 10 × log(P), where P While 15 spots were common to both male and female
is the probability that the observed match is a random flower nectar (black arrowheads), nine were present in
event] . threshold score indicate identity or extensive female flowers only (arrows) and two were present only in
homology (P ,0.05). 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
Enzyme assays for xylosidase activity
(i.e. nectar exposure to external environment, see Nepi and
Nectar xylosidase activity was detected according to the Pacini, 1993) and/or to the different dynamics of nectar pro-
spectrophotometric method indicated by Kumar and Ramón duction and reabsorption that have been reported for the two
(1996). The method is based on the enzymatic reaction sexes (Nepi et al., 2001; Nepi and Stpiczyńska, 2007). Due
between 4-nitrophenyl b-D-xylopyranoside (pNpX) and xylo- to the very different modes of presentation, female nectar is
sidase. This reaction produces p-nitrophenol whose concen- much more accessible to pollinators and much more exposed
tration is determined spectrophotometrically at 400 nm. A to the atmosphere than male nectar and thus more exposed
10 mL aliquot of male nectar and 10mL of female nectar to contamination by yeasts and bacteria. This may suggest a
were obtained by pipetting 1 mL of nectar from each one of more complex defence arsenal against micro-organisms in
524 Nepi et al.  Proteins in floral nectar of Cucurbita pepo
AC
IEF
Spot 1 2 3 4
Male
Mr 71 70 70 69
250 -
pl 5·6 5·8 6·1 6·3
150 -
100 -
75 - D
1 2 3 4
50 -
Male
37 -
25 - Female
pH 4 pH 7
B
Female
8000
E
250 -
Male
150 -
6000
Female
100 -
75 -
4000
1 2 3 4
50 -
2000
37 -
0
1 2 3 4
25 -
b-xylosidase isoforms
FIG. 1. Two-dimensional electrophoresis of proteins extracted from nectar of male and female flowers. (A) A representative gel of proteins from a male flower.
Proteins were separated by isoelectrofocusing (IEF; pH 4 7) in the first dimension and with standard SDS PAGE in the second dimension. The molecular
masses of standards are shown on the left. (B) Proteins of female flowers separated under identical conditions. Gels were stained with Bio-Safe Coomassie.
(C) The spots enclosed in the boxes highlighted in (A) and (B) are the proteins with xylosidase-like sequences and have been numbered (1 4); the calculated
molecular mass (Mr) and isoelectric point (pI) are also shown. (D) Magnification of the highlighted box of both the male and female protein pattern. (E) Relative
quantitation of spots 1 4 (x-axis); the intensity of spots is shown as the relative integrated density (y-axis).
female flowers, and this, in turn, may be related to the higher they are thought to be isoforms of the same protein, probably
number of polypeptides. This supports the hypothesis that resulting from specific post-translational modifications. On the
extrafloral nectar, being more exposed and less ephemeral, is basis of spot quantification analyses, the four hypothetical iso-
characterized by the presence of a greater number of proteins forms were present in relatively similar quantities in both male
than floral nectar (Heil, 2011). and female flowers (Fig. 1E), with the exception of polypep-
Four of the 24 proteins recognized by 2-D electrophoresis tide 4, which appeared to be slightly more abundant in the
were identified by MS and most closely resemble female nectar.
b-D-xylosidases from Arabidopsis thaliana, with some hom- The presence of xylosidases was supported by the enzy-
ology to a b-D-xylosidase from Medicago varia (Table 1). matic assay. This revealed xylosidase activity of 0.23 + 0.04
Results of the MS/MS analysis are summarized in Table 1, and 0.29 + 0.06mM min21 mL21 in female and male nectar,
where the spot numbers match those reported in Fig. 1C and respectively. The occurrence of such activity under conditions
D. Accession number in the UniProtKB database, protein similar to those that occur in nature (temperatureź30 8C)
name, species, peptide sequence and Mascot Score/Mascot was confirmed by the increase in xylose concentration and a
threshold score are also included. As the complete genome corresponding decrease in the concentration of the xylo-oligo-
sequence of C. pepo has not yet been determined, the saccharides  and especially xylobiose  in the mixture
Mascot peptide sequence search was carried out setting a nectar + oligosaccharides after incubation at 24 and 48 h
large taxonomy range to Viridiplantae. As a consequence, (Fig. 2).
the majority of the peptide sequences found match the Neither xylose nor the xylo-oligosaccharides tested was
amino acid sequence of b-D-xylosidase in A. thaliana, one detected in male and female nectar, indicating that lack of
of the completely sequenced plant organisms. It is interesting xylosidase activity is simply due to the absence of the appro-
to note that these peptides probably represent the most con- priate substrate.
served of the amino acid sequences of b-D-xylosidase from Endoxylanases and xylosidases are key enzymes in the
A. thaliana and C. pepo. From the peptide sequence analysis, degradation of xylans, the major hemicelluloses found in the
it is clear that the four spots can be assigned tob-D-xylosidase, secondary walls of most higher plants. Xylans have a relatively
as some common peptides have been found between them. The complex structure based on a b-1,4-linked D-xylose backbone,
four spots had a molecular mass of approx. 70 kDa and a pI substituted to varying degrees (Minic et al., 2004). Endo-
ranging from 5.6 to 6.3 (Fig. 1C). Having identical molecular b-1,4-xylanases hydrolyse the insoluble xylan backbone into
weights, but slightly different isoelectric points (Fig. 1C, D), shorter, soluble xylo-oligosaccharides, while b-D-xylosidases
SDS
PAGE
Relative integrated density
Nepi et al.  Proteins in floral nectar of Cucurbita pepo 525
Xylopentose Xylotriose
2·0 Xylobiose Xylose
1·8
1·6
1·4
1·2
1·0
0·8
0·6
0·4
0·2
0
02448
Time (h)
FIG. 2. Concentration of xylose and xylo-oligosaccharides in the mixture
nectar + xylo-oligosacharides at 0, 24 and 48 h of incubation at 30 8C. The
more evident and steady decrease is that of xylobiose.
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
a-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 develop-
ment (Minic et al., 2004). These same classes of enzymes rep-
resent 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
ż
67/30
54/32
45/32
34/30
27/27
31/31
51/30
47/30
39/34
67/31
76/33
Score
 1
!
Concentration (mg mL
)
Peptide
K.HYTAYDLDNWK.G
R.GQETPGEDPLLSSK.Y
K.LPMTWYPQSYVEK.V
+
Ox (M)
K.LPMTWYPQSYVEK.V
K.LPMTWYPQSYVEK.V
+
Ox (M)
K.LPMTWYPQSYVEK.V
K.HYTAYDLDNWK.G
K.LPMTWYPQSYVEK.V
K.LPMTWYPQSYVEK.V
+
Ox (M) (HW)
K.HYTAYDLDNWK.G
GQETPGEDPLLSSK
Species
Arabidopsis thaliana
Medicago varia
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Medicago varia
T
ABLE
1. Proteins identified using LC ESI/MS-MS
Protein name
Probable b-
D
-xylosidase 5
b-Xylosidase/a-
L
-arabinofuranosidase1
b-
D
-Xylosidase 4
b-
D
-Xylosidase 4
b-
D
-Xylosidase 4
b-
D
-Xylosidase 4
Probable b-
D
-xylosidase 5
b-
D
-Xylosidase 4
b-
D
-Xylosidase 4
Probable b-
D
-xylosidase 5
b-Xylosidase/a-
L
-arabinofuranosidase1

Accession
Q9LJN4
A5JTQ2
Q9FLG1
Q9FLG1
Q9FLG1
Q9FLG1
Q9LJN4
Q9FLG1
Q9FLG1
Q9LJN4
A5JTQ2
UniProtKB accession number of matched protein.
The sequence of matched peptides.
Mascot score/Mascot threshold score for each identified peptide.
* The spot number as given in Fig. 1C and D.

!
ż
2
3
4
Spot no.*
1
526 Nepi et al.  Proteins in floral nectar of Cucurbita pepo
these polymers. For example, the presence of xylobiose and It is interesting to note that a xylosidase was also found in
various other oligosaccharides in cultures of the fungus the pollination drop of Juniperus communis (Wagner et al.,
Trichoderma reesei is known to induce cellulase and xylanase 2007). Both pollination drop and nectar are secretions that
expression (Aro et al., 2005). have a very similar chemical composition, even though their
In the present study, the authors propose a functional role for functions are entirely different (Nepi et al., 2009). Whereas
b-xylosidase in C. pepo nectar that takes into account all the the former is very common amongst gymnosperms, and is
above observations. The invasion of the nectar by micro- the landing site for pollen, the latter is widely distributed
organisms is followed by damage to the nectary cell walls amongst angiosperms and is the most common reward for pol-
due to the action of cellulases and xylanases produced by the linators. Since both are sugary solutions and are more or less
pathogens. This action may induce the release of several oligo- exposed to the environment, they are equally subject to con-
saccharides from the cell walls. After the invasion of the nectar tamination and invasion by micro-organisms. The presence
by micro-organisms, it is likely that the relative abundance of of xylosidases in both these secretions may account for a
the plant cell wall-derived oligosaccharides displaying differing certain degree of conservatism in the defence proteins of
degrees of polymerization (DP) is important to the plant. For these two groups of plants.
example, their presence at very low concentrations may be Although this is the first attempt at determining the proteins
useful in  alerting the plant s defence mechanism against present in C. pepo nectar, many of which still remain to be
micro-organisms (Shibuya and Minami, 2001), but increased identified, it is clear that most of the main proteins (i.e.
levels may be detected promptly by the latter, thus resulting those present at high concentrations) found in this species
in their increased ability to damage the plant cell wall via are involved in protecting the plant against attack by micro-
xylanase activity. Therefore, the degradation of surplus organisms. This defence strategy appears to fulfil an important
xylo-oligosaccharides may help keep pathogens present in requirement of floral nectar, as revealed by the probable pres-
nectar at a reduced state of activity. It is important to point ence of four isoforms of the same enzyme. However, the pro-
out that the DP of xylo-oligosaccharides capable of inducing teins identified here do not have a direct lethal effect, but
the xylanase activity of micro-organisms varies widely: rather an inhibitory effect on the pathogenic potential of
xylobiose (DP 2) is reported to be the elicitor of xylanases in micro-organisms. Furthermore, they cannot be responsible
the fungi Aspergillus and Trichoderma (Aro et al., 2005), for the recently demonstrated inhibition of growth and meta-
while the same enzyme activity is stimulated by bolic activity of Escherichia coli and Erwinia tracheiphila
xylo-oligosaccharides with a DP of 6 30 in the bacterium by nectar of C. pepo ssp. texana (Sasu et al., 2010), and it is
Prevotella bryantii (Miyazaki et al., 2005). Thus, it is likely that other proteins or other substances are responsible
very likely that the nectar defence system, which must be for this. In short, it appears that the nectar of C. pepo possesses
effective against a wide range of micro-organisms, is a complex chemical defence  arsenal that we are only just
equipped with a complete set of enzymes involved in xylan beginning to discover.
degradation (endo-b-1,4-xylanases, b-D-xylosidases and
a-L-arabinofuranosidases), although only two of them were
identified in the present study (Table 1). Another strategy invol- ACKNOWLEDGEMENTS
ving the direct inhibition of microbial xylanase activity has
The authors are grateful to the personnel of the Botanical
been reported for Nicotiana (Harper et al., 2010). Here, the nec-
Gardens of the University of Siena for growing the plants
tarin NEC4 functions as a defence agent that inhibits a
used in this study.
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
LITERATURE CITED
pathogen enzyme activity involved in cell wall degradation,
Aro N, Pakula T, Penttila M. 2005. Transcriptional regulation of plant cell
as in Nicotiana) or indirect (by regulating the concentration
wall degradation by filamentous fungi. FEMS Microbiology Reviews
of oligosaccharides released by cell walls and that elicit the
29: 719 739.
cell wall-degrading activity of the micro-organism).
Beliën T, Van Campenhout S, Robben J, Volckaert G. 2006. Microbial
An alternative explanation for the function of xylosidases
endoxylanases: effective weapons to breach the plant cell-wall barrier
or, rather, triggers of plant defence systems? Molecular Plant-Microbe
found in the floral nectar of C. pepo takes into account the
Interaction 19: 1072 1081.
possibility that these enzymes are derived from nectary cell
Beutler R. 1935. Nectar. Bee World 24: 106 162.
walls, from where they are mobilized by nectar flow.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of
Although the more common method for nectar exudation is
microgram quantities of protein utilizing the principle of protein dye
via the modified stomata of the nectary epidermis, nectar binding. Analytical Biochemistry 72: 248 254.
Bucheli P, Doares SH, Albersheim P, Darvill A. 1990. Host pathogen inter-
exudation through the epidermal cell wall cannot be ruled
actions. XXVI. Partial purification and characterization of heat-labile
out (Nepi et al., 1996; Nepi, 2007). Xylosidases and
molecules secreted by the rice blast pathogen that solubilize plant cell
endo-xylanases may be involved in the reorganization of cell
wall fragments that kill plant cells. Physiological and Molecular Plant
walls during the development of nectary parenchyma and the
Pathology 36: 159 173.
Carter C, Thornburg RW. 2000. Tobacco Nectarin I: purification and
changes in cell shape and volume that accompany this
characterization as a germine-like, manganese superoxide dismutase
process (Nepi et al., 1996). Reorganization of cell walls may
implicated in the defence of floral reproductive tissues. Journal of
also increase wall permeability to nectar. These enzymes,
Biological Chemistry 275: 35726 36733.
once released into the nectar, help defend the latter from
Carter C, Thornburg RW. 2004. Is the nectar redox cycle a floral defence
fungal or bacterial invasion, as described above. against microbial attack? Trends in Plant Science 9: 320 324.
Nepi et al.  Proteins in floral nectar of Cucurbita pepo 527
Carter C, Healy R, O Tool NM, et al. 2007. Tobacco nectaries express a Nepi M. 2007. Nectary structure and ultrastructure. In: Nicolson S, Nepi M,
novel NADPH oxidase implicated in the defence of floral reproductive Pacini E. eds. Nectaries and nectar. Dordrecht: Springer, 129 166.
tissues against microorganisms. Plant Physiology 143: 389 399. Nepi M, Pacini E. 1993. Pollination, pollen viability and pistil receptivity in
Esquerré-Tugayé M, Boudart G, Dumas B. 2000. Cell wall degrading
Cucurbita pepo. Annals of Botany 72: 527 536.
enzymes, inhibitory proteins, and oligosaccharides participate in the mol- Nepi M, Stpiczyńska M. 2007. Nectar resorption and translocation in
ecular dialogue between plants and pathogens. Plant Physiology and
Cucurbita pepo L. and Platanthera chlorantha Custer (Rchb.). Plant
Biochemistry 38: 157 163.
Biology 9: 93 100.
González-Teuber M, Eilmus S, Muck A, Svatos A, Heil M. 2009.
Nepi M, Ciampolini F, Pacini E. 1996. Development and ultrastructure of
Pathogenesis-related proteins protect extrafloral nectar from microbial
Cucurbita pepo nectaries of male flowers. Annals of Botany 81: 251 262.
infestation. The Plant Journal 58: 464 473.
Nepi M, Guarnieri M, Pacini E. 2001. Nectar secretion, reabsorption, and
González-Teuber M, Pozo MJ, Muck A, Svatos A, Adame-Álvarez RM,
sugar composition in male and female flowers of Cucurbita pepo.
Heil M. 2010. Glucanases and chitinases as causal agents in the protec-
International Journal of Plant Sciences 162: 353 358.
tion of Acacia extrafloral nectar from infestation by phytopathogens.
Nepi M, von Aderkas P, Wagner R, Mugnaini S, Coulter A, Pacini E. 2009.
Plant Physiology 152: 1705 1715.
Nectar and pollination drops: how different are they? Annals of Botany
Harper AD, Stalnaker SH, Wells L, Darvill A, Thornburg R, York WS.
104: 205 219.
2010. Interaction of Nectarin 4 with a fungal protein triggers a microbial
Nicolson SW. 2007. Nectar consumers. In: Nicolson S, Nepi M, Pacini E. eds.
surveillance and defense mechanism in nectar. Phytochemistry 71:
Nectaries and nectar. Dordrecht: Springer, 289 342.
1963 1969.
Nicolson SW, Thornburg RW. 2007. Nectar chemistry. In: Nicolson S, Nepi
Heil M. 2011. Nectar: generation, regulation and ecological functions. Trends
M, Pacini E. eds. Nectaries and nectar. Dordrecht: Springer, 215 264.
in Plant Science 16: 191 200.
Noda J, Brito N, González C. 2010. The Botrytis cinerea xylanase Xyn11A
Hillwig MS, Liu X, Liu G, Thornburg RW, MacIntosh GC. 2010. Petunia
contributes to virulence with its necrotizing activity, not with its catalytic
nectar proteins have ribonuclease activity. Journal of Experimental
activity. BMC Plant Biology 10: 38. doi:10.1186/1471-2229-10-38.
Botany 61: 2951 2965.
Ryan CA, Farmer EE. 1991. Oligosaccharide signals in plants: a current
Hillwig MS, Kanobe C, Thornburg RW, MacIntosh GC. 2011.
assessment. Annual Review of Plant Physiology 42: 651 674.
Identification of S-RNase and peroxidase in petunia nectar. Journal of
Sangaravelan N, Nee man G, Inbar M, Izhakiet I. 2005. Feeding responses
Plant Physiology 168: 734 738.
of free-flying honeybees, to secondary compounds mimicking floral
Kessler D, Gasse K, Baldwin IT. 2008. Field experiments with transformed
nectars. Journal of Chemical Ecology 31: 2791 2804.
plants reveal the sense of floral scents. Science 321: 1200 1202.
Sasu MA, Wall KL, Stephenson AG. 2010. Antimicrobial nectar inhibits a
Kram B, Bainbridge E, Perera M, Carter C. 2008. Identification, cloning
florally transmitted pathogen of a wild Cucurbita pepo (Cucurbitaceae).
and characterization of a GDSL lipase secreted into the nectar of
American Journal of Botany 97: 1025 1030.
Jacaranda mimosifolia. Plant Molecular Biology 68: 173 183.
Sharon A, Fuchs Y, Anderson JD. 1993. The elicitation of ethylene biosyn-
Kumar S, Ramón D. 1996. Purification and regulation of the synthesis of a
thesis by a Trichoderma xylanase is not related to the cell wall degra-
b-xylosidase from Aspergillus nidulans. FEMS Microbiology Letters
dation activity of the enzyme. Plant Physiology 102: 1325 1329.
135: 287 293.
Shibuya S, Minami E. 2001. Oligosaccharides signalling for defence
Meiring HD, van der Heeft E, ten Hove GJ, de Jong APJM. 2002.
responses in plants. Physiological and Molecular Plant Pathology 59:
Nanoscale LC MS(n) technical design and applications to peptide and
223 233.
protein analysis. Journal of Separation Science 25: 557 568.
Sinha P, Poland J, Schnölzer M, Rabilloud T. 2001. A new silver staining
Minic Z, Rihouey C, Trung Do C, Lerouge P, Jouanin L. 2004. Purification
apparatus and procedure for matrix-assisted laser desorption/ionization-
and characterization of enzymes exhibiting D-xylosidase activities in stem
time of flight analysis of proteins after two-dimensional electrophoresis.
tissues of Arabidopsis. Plant Physiology 135: 867 878.
Proteomics 1: 835 840.
Miyazaki K, Hirase T, Kojima Y, Flint HJ. 2005. Medium-to large-sized
Tuncer M, Ball AS. 2003. Co-operative actions and degradation analysis of
xylo-oligosaccharides are responsible for xylanase induction in
purified xylose-degrading enzymes from Thermonospora fusca BD25
Prevotella bryantii B14. Microbiology 151: 4121 4125.
on spelt xylan. Journal of Applied Microbiology 94: 1030 1105.
Naqvi SM, Harper A, Carter C, Ren G, Guirgis A, York WS, Thornburg
RW. 2005. Tobacco Nectarin IV is a specific inhibitor of fungal xylosi- Wagner R, Mugnaini S, Sniezko R, et al. 2007. Proteomic evaluation of
dases secreted into the nectar of ornamental tobacco plants. Plant gymnosperm pollination drop proteins indicates highly conserved and
Physiology 139: 1389 1400. complex biological functions. Sexual Plant Reproduction 20: 181 189.
Copyright of Annals of Botany is the property of Oxford University Press / UK and its content may not be
copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.