A Comparative Proteomic Analysis of Arabidopsis Mature pollen and

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Title: A Comparative Proteomic Analysis of Arabidopsis Mature pollen and Germinated Pollen
Running title: Proteomic Analysis During Arabidopsis Pollen Germination
Authors: Junjie Zou1#, Lianfen Song1#, Wenzheng Zhang1, Yi Wang1, Songlin Ruan1,2 and WeiHua Wu1*
1
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China
Agricultural University, Beijing 100193, China;
2
Institute of Biotechnology, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China
#
These authors contributed equally to this work.
*
Author for correspondence.
Tel: +86-(0)10-6273-1103;
Fax: +86-(0)10-6273-4640;
E-mail: <[email protected]>.
This work was supported by a competitive research grant (No. 30421002) for Creative Research
Groups sponsored by the National Science Foundation of China.
1
Abstract
Proteomic analysis was applied to generating the map of Arabidopsis mature pollen proteins and
analyzing the differentially expressed proteins that are potentially involving in the regulation of
Arabidopsis pollen germination. By applying two-dimensional electrophoresis and silver
staining, we resolved 499 and 494 protein spots from protein samples extracted from pollen
grains and pollen tubes, respectively. Using the matrix-assisted laser desorption ionization timeof-flight mass spectrometry method, we identified 189 distinct proteins from 213 protein spots
expressed in mature pollen or pollen tubes, and 75 new identified proteins were not reported
before in the researches of Arabidopsis pollen proteome. Comparative analysis revealed that 40
protein spots exhibit reproducible significant changes between mature pollen and pollen tubes.
And 21 proteins from 17 down-regulated and 6 up-regulated protein spots were identified.
Functional category analysis indicated that these differentially expressed proteins mainly
involved in signaling, cellular structure, transport, defense/stress responses, transcription,
metabolism, and energy production. The patterns of changes at protein level suggested the
important roles for energy metabolism-related proteins in pollen tube growth, accompanied by
the activation of the stress response pathway and modifications to the cell wall.
Keywords: Arabidopsis thaliana; 2D-PAGE; MALDI-TOF MS; pollen; pollen tube; proteome.
2
Sexual reproduction of flowering plants comprises several sequential steps from pollination
to fertilization. Pollen landing on a stigma is the first step, followed by pollen hydration,
germination, and pollen tube growth through intercellular spaces in the pistil. When the pollen
tube reaches the embryo sac of the ovary, it delivers sperm cells for double fertilization
(Franklin-Tong 1999a; McCormick 2004). The critical steps in this continuous process include
pollen germination on the stigma and pollen tube growth. The investigation of the regulatory
mechanisms for pollen germination and tube growth is important for fundamental studies of
fertility and reproduction in flowering plants. In addition, pollen germination is an ideal model
system for the investigation of important issues in cell biology, such as polarized tip growth,
cell–cell interactions, and signal transduction (Franklin-Tong 1999b).
Increasing efforts have been made to investigate the genetic and molecular mechanisms of
pollen germination and tube growth, and at least 150 genes involving in pollen development and
pollen tube growth have been studied (Twell 2002). Functional composition analysis of the
Arabidopsis pollen transcriptome has revealed that the mRNAs specifically or preferentially
presented in pollen mainly encode proteins potentially involved in cell wall metabolism, vesicle
transport, cytoskeleton, and signaling (Honys and Twell 2003; Honys and Twell 2004; Pina et al.
2005; Wang et al. 2008). These results may reflect the functional specialization of mature pollen
in the commitment of germination and tube growth. However, gene expression at mRNA
expression level lacks a direct correlation with protein level and activity (Greenbaum et al. 2003).
Although mature pollen grains may contain pre-synthesized mRNA for germination and other
mRNAs may be synthesized during pollen germination (Wang et al. 2008), newly transcribed
mRNAs are not necessarily translated into the corresponding proteins and post-translational
modifications may be also crucial to pollen function (Mascarenhas 1993; Taylor and Hepler
1997). Thus, detailed analysis at the protein level is an essential step toward the further
identification of regulatory components involving pollen germination and tube growth.
Proteomic analyses of pollen development and germination can provide new insights on the
whole genome level into fascinating mechanisms of pollen development and tip-growth
regulation in higher plants (Chen et al. 2007; Dai et al. 2007). Proteomic analyses of rice anthers
provided comprehensive understanding of the proteins expression changes during microspore
and pollen development and in response to environment stresses (Imin et al. 2001,2004; Kerim et
al. 2003). By analyzing rice pollen proteins, Dai et al. (2006) identified several novel proteins
that may be involved in signal transduction, protein synthesis, assembly and degradation, and
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wall remodeling and metabolism. Proteomic analysis of tomato pollen showed that many of the
identified proteins have designated roles in defence mechanisms, energy conversion, pollen
germination, and pollen tube growth, and some possibly in sperm cell formation (Sheoran et al.
2007). By conducting a proteomic analysis of Arabidopsis pollen coat proteins, Mayfield et al.
(2001) reported that oleosins and lipases may play important roles in initiating pollination. More
recently, three independent proteomic analyses of Arabidopsis mature pollen were conducted
(Holmes-Davis et al. 2005; Noir et al. 2005; Sheoran et al. 2006), which provide a broad analysis
of the Arabidopsis pollen proteome and complement and extend the analysis of the pollen
transcriptome.
Proteomic analyses of the differentially expressed proteins during pollen tube growth in
Pinus strobus and Picea meyeri provide an important insight into the molecular basis of pollen
tube growth and effects of actin cytoskeleton disruption on global protein patterns in pollen tube
(Chen et al. 2006; Fernando 2005). Proteomic identification of differentially expressed proteins
associated with pollen germination and tube growth in rice revealed that these differentially
expressed proteins involve different cellular and metabolic processes with obvious functional
skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and
carbohydrate/energy metabolism (Dai et al. 2007). Obviously, a more comprehensive pollen
proteomic analysis of Arabidopsis, the model plant of dicots of angiosperms, particularly the
changes in protein expression profiles during the transitions from desiccated mature pollen to
germinating pollen and to growing pollen tubes, is needed to reveal the complex molecular
mechanisms of pollen germination and tube growth. Along with the development of methods for
collecting large quantities of Arabidopsis pollen (Johnson-Brousseau and McCormick 2004) and
high rates of pollen germination in vitro (Fan et al. 2001; Wang et al. 2008), it now becomes
possible to experimentally investigate changes in proteomic profiles during the process of pollen
germination and pollen tube growth. This study reports proteomic analysis of changes in protein
expression profiles between the mature pollen and growing pollen tubes. In addition, potential
roles of the differentially expressed proteins involved in the regulation of pollen germination and
tube growth are discussed.
Results and Discussion
4
Proteomic maps of Arabidopsis pollen and pollen tubes
After the 2-DE gels were aligned and matched, the protein spots shown on the gels of mature
pollen and pollen tubes were analyzed. There were totally 499 and 494 reproducible protein
spots were detected in the gels loaded with protein samples extracted from pollen grains and
pollen tubes, respectively. These proteins cover the pI (isoelectric point) range from 5 to 8, and
their MW (molecular weight) ranged from 10 to 110 kDa (Figures 1, 2 and 3). There were
totally 303 protein spots were excised from two sets of gels for MS analysis to generate PMFs
(peptide mass fingerprints). As shown in Table 1, 213 protein spots, representing 189 different
proteins, were identified after searching with the NCBI database. The identified proteins
corresponding to the spot numbers are shown in Figures 1, 2 and 3. The calculated MW of the
identified proteins ranged from 11.9 kDa to 108.9 kDa, and the calculated pI range was from
4.36 to 9.25, which is close to the experimental data as judged from the location of the spots on
the 2-DE gels (Table 1; Figures 1, 2 and 3).
It should be noted that the identified proteins did not always have a one-to-one correlation
with the spots on the gels according to other proteomic analyses, which may have resulted from
polypeptide variants that were present in different spots on the gel, but encoded by the same gene
(Kerim et al. 2003; Holmes-Davis et al. 2005; Noir et al. 2005). In our experiments, there were
24 proteins detected from different spots (see in Table 1). There may be several possibilities for
differential migration of the same protein. One possibility is a difference in post-translational
modification of the proteins in vivo, such as phosphorylation, glycosylation, or acetylation
(Krishna and Wold 1993; Jensen 2004). In most cases, these modifications do not significantly
affect the molecular weight of a protein, but may induce a pI shift on the gel (Holmes-Davis et al.
2005; Noir et al. 2005). Another possibility is alternative splicing of mRNAs during translation
(Smith et al. 1989; Brett et al. 2002;) or chemical modification of the proteins during sample
preparation.
Among the 303 protein spots analyzed by MALDI-TOF MS (matrix-assisted laser
desorption ionization time-of-flight mass spectrometry), 213 protein spots representing 189
proteins were identified. This is the greatest number of identified proteins in Arabidopsis pollen
and pollen tubes so far. One previous study (Holmes-Davis et al. 2005) identified totally 135
proteins from the Arabidopsis mature pollen proteome, and 60 of them were identified in the
present study. Another study identified 121 proteins from the Arabidopsis mature pollen
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proteome (Noir et al. 2005), and 76 of them were detected in our study. Compared with the
proteomic analysis of Arabidopsis Landsberg ecotype pollen (Sheoran et al. 2006), 39 out of 95
identified proteins in the previous study were common in the present study. These differences
may be attributed to the methods of protein extraction, choices of IPG strips with different pH
ranges, spots selection for analysis and the ecotype used (Sheoran et al. 2006). In our study, 75
new proteins were identified which were not reported in the previous studies (see in Table 1).
Most of the newly identified proteins were classified into metabolism, energy and protein fate.
Especially, we found that spots 42, 50, 65, 184, 191, 212, 288 were proteasome-related proteins.
The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in
eukaryotes and has been proposed as being involved in pollen tube growth (Sheng et al. 2006).
Inhibition of proteasome activity can significantly prevent pollen tube development and
markedly alter tube morphology (Sheng et al. 2006). The pre-synthesized proteome-related
proteins in mature pollen indicate their important roles for rapid pollen germination and tube
growth. Some other newly identified proteins (spots 3, 6, 7, 9, 20, 31, 32, 33, 73, 214, 269) are
differently expressed between mature pollen and pollen tubes.
According to the gene sequences and a homological comparison with other known proteins,
the identified proteins were classified into 15 different functional categories (Table 1; Figure 4A),
including energy (31.2%), metabolism (24.9%), protein fate (13.2%), protein synthesis (1.1%),
cell structure (6.3%), signal transduction (5.8%), defense/stress responses (4.8%), transport
(3.7%), cell fate (2.6%), storage protein (1.1%), subcellular localization (0.5%), cell type
differentiation (1.1%), development (0.5%), transcription (1.1%), and unclassified proteins
(2.1%) . Among the 189 proteins identified, there were approximate 70% of them classified into
3 categories, including energy (31.2%), metabolism (24.9%) and protein fate (13.2%). This
result may indicate the special requirement of these categories of proteins for pollen development
or pollen germination. There were only four proteins (spots 31, 43, 183, 207) out of the 189 that
could not be functionally classified as they were not observed to contain any known conserved
domains.
Changes in protein expression profiles during the transition from the mature pollen grains
to the germinated pollen
Based on the analysis of the reproducible protein spots of mature pollen grains and pollen tubes,
40 protein spots in total exhibited significant and reproducible changes during the transition
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from the mature pollen grains to germinated pollen materials (Figures 2, 3 and 5). There were 23
protein spots representing 21 proteins identified by MALDI-TOF MS (Table 2). Database
analysis revealed that most of these proteins have either an unknown function, or are not yet
characterized for their potential function in pollen development or germination. Further detailed
functional characterization of these proteins may expand our understanding of complex
mechanisms in regulation of pollen germination and pollen tube growth.
Analysis of the
subcellular locations (www.arabidopsis.org) of these proteins showed that most of these proteins
are targeted to, or associated with, the cytoplasm and endomembranes. Further functional
characterization of these proteins may reveal their potential roles in the regulation of pollen
germination.
These differentially expressed proteins between mature pollen grains and pollen tubes were
grouped into nine functional categories, including metabolism, energy, defense/stress responses,
signal transduction, transport, and cell structure (Figure 4B). It is known that many processes,
such as cell signaling, vesicle transport and fusion, expansion of the cell wall and plasma
membrane, and actin dynamics, play essential roles in pollen germination and tube growth (Brett
et al. 2002; Franklin-Tong 1999; Krishna and Wolf 1993; Mascarenhas 1993). Previous pollen
transcriptome analyses have shown that a high proportion of genes in signal transduction, cell
wall biosynthesis, and cytoskeletal dynamics are preferentially and selectively expressed in
pollen (Pina et al. 2005). The present proteomic analysis demonstrated that approximately 29%
of the proteins that were differentially expressed during the transition from mature pollen to
germinated pollen belong to these functional categories. The proteins associated with energy and
metabolism accounted for 38%, and the stress/defense responses proteins accounted for 19% of
the differentially expressed proteins during the transition from mature pollen to germinated
pollen.
Proteins involved in cell structure
Pollen tube growth is a typical tip growth process. Thus, the apical cell wall must be plastic for
stretching and for the incorporation of newly synthesized wall materials, and must be also rigid
to withstand high internal turgor pressures (Bosch and Hepler 2005). The apical cell wall of a
pollen tube is a pectic network, and pectin methylesterases (PMEs) act as key regulators of
pollen tube growth (Bosch et al. 2005; Bosch and Hepler 2005). In the present proteomic
7
analysis, two proteins (At1g48020, spot 20 in Figure 5B and At4g24640, spot 23 in Figure 5B)
that match with pectin methylesterase inhibitor (PMEI) were identified. The At1g48020 was
disappeared and At4g24640 was dramatically decreased in pollen tubes, as compared to mature
pollen grains. Wolf et al. (2005) reported that At1g48020 (named as AtPMEI1) inhibited PME
activity in flowers and siliques, and AtPMEI1 was preferentially expressed in pollen. Our
results, together with the previous reports, suggest that PMEI may play an important role in
loosening the cell wall during pollen tube growth. Further functional characterization of the
PMEI may increase our understanding of cell wall construction during pollen tube growth.
Signaling proteins
It is well known that Ca2+ is an essential messenger and that Ca2+-regulated proteins serve as key
signal transducers in the regulation of pollen tube growth. In plants, members of the annexin
family are considered to be a group of calcium sensors involved in a number of signaling
processes, such as interactions with the actin cytoskeleton and Golgi-mediated vesicle secretion
(Clark et al. 2005). These annexin-associated processes also occur in a growing pollen tube
(Franklin-Tong 1999; Hepler et al. 2001; Robinson and Messerli 2002). In the present study, 2DE gel analyses revealed that the expression of annexin1 (AnnAt1, At1g35720, spot 4 in Figure
5B) was significantly decreased during the transition from mature pollen grains to germinating
pollen. AnnAt1 is a 317-amino acid residue protein with a molecular weight of 37 kDa (pI 5.0).
This protein mainly exists in a soluble form in the cytosol, but is also associated with the
membranes (Lee et al. 2004). AnnAt1 protein has been reported to be involved in plant
responses to NaCl, ABA, osmotic, and oxidative stress (Lee et al. 2004; Gorecka et al. 2005). It
is known that these stress responses are closely related to Ca2+-signaling events (Xiong et al.
2002). Our proteome analysis showed that AnnAt1 was significantly down-regulated, with a
2.7-fold change, from mature pollen grains to pollen tubes, suggesting its potential function in
the regulation of pollen tube growth. Another protein (At1g30580, spot 270 in Figure 5B)
identified in mature pollen grains matched a predicated GTP-binding protein with an unknown
function. GTP-binding proteins have been reported to play important roles in pollen germination
and pollen tube growth (Ma et al. 1999; Zheng and Yang 2000). The novel GTP-binding protein
(At1g30580) identified in this study was significantly down-regulated during pollen germination.
8
It is worthwhile to further characterize this protein for its potential function in the regulation of
pollen tube growth.
The ADK1 (At5g63400, spot 18 in Figure 5B) protein was only detected in the mature
pollen samples. ADK is an enzyme involved in the adenylate metabolic network, by which
adenosine (Ado) is converted into AMP using one molecule of ATP (Moffatt et al. 2000). In
animal cells, ADK has been reported to modulate methyltransferase activity, the production of
polyamines and secondary compounds, and cell signaling (Young et al. 2006). In plants, an
analysis of a mutation in ADK1 has suggested that the function of ADK was to modulate root
cap morphogenesis and gravitropism (Young et al. 2006). It is also known that the expression
level of ADK1 is low in leaves, but very high in flowers, stems, and roots (Moffatt et al. 2000).
Our results indicate that ADK1 is only expressed in mature pollen grains and not in pollen tubes,
which may indicate a possible involvement of ADK1 in pollen maturation development.
Proteins associated with transport events
It is well known that the transport of various ions, such as Ca2+ , K+ and Cl-, is extremely
important for pollen germination and tube growth (Hepler et al. 2001; Robinson and Messerli
2002), and many transporter proteins have been reported to regulate pollen germination and tube
growth (Bock et al. 2006). Among the differentially expressed proteins between the mature
pollen grains and the pollen tubes, one protein (At4g16160, spots 36 and 37 in Figure 5A) was
categorized for potential functions in membrane transport. At4g16160 was detected in different
locations on the gels (spots 36 and 37 in Figure 5A), and its expression was up-regulated during
the transition from mature pollen grains to growing pollen tubes. This protein has been
previously suggested to be a transporter protein (named as AtOEP16-S) because it shares a 32%
identity with pea OEP16 that was the first chloroplast outer membrane channel protein to be
isolated (Pohlmeyer et al. 1997; Drea et al. 2006). The GUS expression analysis of AtOEP16-S
demonstrated that pollen grains and seeds contain a high level of GUS activity, which is
consistent with the results of in situ hybridization, and that the AtOEP16-S promoter appeared to
be most active during the maturation phase in both pollen and seed (Pohlmeyer et al. 1997; Drea
et al. 2006). In addition, the expression of AtOEP16-S in siliques is related to the ABA
signaling pathway because it requires the transcription factors ABI3 and ABI5 (Drea et al. 2006).
The increased expression of AtOEP16-S in pollen tubes in the present study (spots 36 and 37 in
9
Figure 5A) may indicate that this protein functions as a potential transporter for facilitating ion
and/or metabolite fluxes during pollen tube growth.
Proteins related to stress responses
Pollen germination begins with pollen hydration, and pollen tube growth requires a relatively
high turgor pressure at the pollen tube tip (Bosch et al. 2005). During this continuous growth
process, pollen and pollen tubes appear to utilize a series of strategies against various stresses
(Zonia and Munnik 2004). In the present study, four proteins (At1g60740, At1g65970,
At2g30870 and At4g08390) were identified and they may be related to pollen responses to stress.
The proteins At1g60740 and At1g65970 both matched with spot 3 (Figure 5A) and only
expressed in pollen tube samples. These two proteins are predicted to be putative type 2
peroxiredoxin (PRXII) and are named as AtPRXII-D and AtPRXII-C. They each have an ORF
encoding 162 amino acids and have a high degree of similarity. It has been reported that the
GUS activity in AtPRXII-D::GUS plants was not only detected in mature pollen (similar to
AtPRXII-C), but also in germinating pollen, pollen tubes as well as fertilized ovules (Bréhélin et
al. 2003). However, the corresponding spot on the gel for these two proteins was exclusively and
remarkably detected in pollen tube samples in this study, which indicates that these two proteins
were either synthesized during pollen germination or present in the mature pollen grains in an
undetectably low abundance. The peroxiredoxin (PRX) family includes a group of alkyl
hydroperoxide reductases consisting of antioxidant enzymes that protect macromolecules from
damage caused by ROS, and the donor molecules of these reducers are all thio-containing
substances such as thioredoxin (TRX) and glutaredoxin (GRX) (Bréhélin et al. 2003). The
significant expression of AtPRXII-D and AtPRXII-C in growing pollen tubes may protect some
required components for pollen tube growth, such as the TRX protein (spot 98 in Figure 1) that
was detected without changes in expression in different materials in this study.
The expressions of two other stress-related proteins (At2g30870, At4g08390) were downregulated in pollen tubes (spot 6 and spot 7 in Figure 5B). At2g30870 is a dehydration earlyresponse gene (ERD13) that has been reported as a stress-induced gene (Bianchi et al. 2002).
At4g08390 encodes an ascorbate peroxidase that should localize to the chloroplast stroma and
the mitochondria, according to database analysis (www.arabidopsis.org). As one of the ROSscavenging enzymes, it plays a major role in the removal of H2O2 in living cells. Although both
10
proteins were detected in the mature pollen samples, as shown in this study, the transcription of
these two genes was very low in mature pollen (Honys and Twell 2004; Pina et al. 2005). These
results may suggest that these two proteins may accumulate in mature pollen grains for
protecting pollen from dehydration and oxidation damage during the pollen late maturation
phase.
Proteins involved in energy and metabolism
It is known that pollen tube development and growth has large energetic and biosynthetic
requirements (Hepler et al. 2001). In order to meet these demands, mature pollen grains contain
abundant carbohydrates as energy sources, and pollen germination and tube growth are
accompanied by high metabolic activity (Tagede and Kuhlemeier 1997). In the present study, 8
differentially expressed proteins (At5g65690, At1g08480, At3g22850, At3g52300, At1g01050,
At1g23730, At5g26667, At1g75270) were identified and categorized as energy and metabolism
proteins, and account for 38% of all differentially expressed proteins. According to a database
analysis, these proteins are closely related to many processes in metabolism and energy
production, such as oxidative phosphorylation, carbohydrate metabolism, and sugar metabolism.
However, no detailed information is available for their specific function so far. Further detailed
functional characterization of these proteins is required to reveal their potential functions in
pollen germination and tube growth.
Materials and Methods
Pollen grain collection and in vitro pollen germination
Arabidopsis plants (ecotype, Columbia) were grown under the same condition described by
Wang et al. (2008). Mature pollen grains were collected from freshly anther-dehisced flowers
using a vacuum cleaner as described previously (Johnson-Brousseau and McCormick 2004) and
were immediately frozen in liquid nitrogen for protein extraction. The “thin liquid layer”
germination methods were used for collection of large quantity of pollen tubes (Wang et al.
2008). After incubation at 25°C for 3.5 h, the averaged pollen tube length was about 140 µm.
The pollen tube samples were collected and centrifuged at 1500 × g at 4°C for 3 min. The
supernatants were discarded and the pelleted pollen tubes were resuspended in double-distilled
11
water supplemented with 1 mM PMSF and quickly frozen in liquid nitrogen for protein
extraction.
Protein extraction
The proteins were extracted following the methods described previously with slight
modifications (Damerval et al. 1986; Natera et al. 2000). The frozen samples of pollen grains or
pollen tubes were ground in liquid nitrogen into fine powder and collected in a sterilized 2 mL
centrifuge tube. The homogenate was precipitated with cold protein extraction buffer containing
10% TCA (w/v) and 0.07% β-mercaptoethanol (v/v) in acetone for 1 h at -20°C, and then was
centrifuged at 15 000 × g for 25 min at 4°C. The precipitate was washed three times with the
same cold protein extraction buffer without 10% TCA (w/v), followed by a 1 h incubation at 20°C and subsequent centrifugation at 15 000 × g for 25 min at 4°C for each wash. The pellets
were vacuum-dried, weighed, and dissolved in a lysis buffer containing 7 M urea, 2 M thiourea,
4% CHAPS (w/v), 50 mM DTT, and 0.5% carrier ampholytes (pH 3-10). After 1 h of lysis at
room temperature with vortexing every 10 min, the homogenate was centrifuged at 15 000 × g
for 20 min. The supernatant was transferred to a 1.5 mL centrifuge tube and stored in aliquots at
-80°C. Protein samples extracted from four collections of pollen or pollen tubes were combined
together for further analysis. The protein concentration of the supernatant was determined by
Bradford assay, with a range of known concentrations of BSA as standard (Bradford 1976).
Two-dimensional gel electrophoresis and silver staining
Protein samples (300 µg) were diluted in a rehydration buffer containing 8 M urea, 2% CHAPS
(w/v), 50 mM DTT, 0.5% pH 3-10 carrier ampholytes (v/v), and 0.01% bromophenol blue (w/v)
for gel electrophoresis using a 17 cm IPG strip (pH 5-8, Bio-Rad). After active rehydration
(20°C, 50 V) for 12 h in a Bio-Rad PROTEAN® IEF Cell, the IEF (isoelectric focusing) was
preformed at 20°C following the manufacturer’s protocols. After IEF, the IPG strips were treated
in a equilibration buffer containing 6 M urea, 0.375 M Tris-HCl (pH 8.8), 2% SDS (w/v), 20%
glycerol (v/v), and 2% DTT (w/v) for 15 min, and subsequently, in the same buffer containing
2.5 % iodoacetamide (w/v) without DTT for another 15 min. The second-dimension separations
were carried out on 12.5% SDS-PAGE gels with a running buffer in a PROTEAN® Plus Dodeca
cell (Bio-Rad) electrophoresis tank at 16°C. The gels were run with a constant voltage at 100 V
12
for one hour, and then at 200 V until the tracking dye reached the bottom of the gels
(approximate 6 hours).
After electrophoresis, a silver-staining procedure compatible with mass spectrometric
analysis was performed with a slight modification (Yan et al. 2000). The gels were fixed in 40%
ethanol (v/v) and 10% acetic acid (v/v) for one hour, washed twice for ten minutes in Milli-Q
water, and then sensitized with a 30% ethanol (v/v), 6.8% sodium acetate (w/v), and 0.2%
sodium thiosulfate (w/v) solution for 30 min. The gels were then rinsed with Milli-Q water three
times for ten minutes each. The gels were incubated in 0.25% (w/v) silver nitrate for 20 min,
rinsed twice with Milli-Q water for one minute, and then developed in a solution of 2.5%
sodium carbonate (w/v) with formaldehyde (37% ,w/v) added (400 µL/L) before use. When the
desired intensity of staining was achieved, development was stopped with 1.46%
EDTA·Na2·2H2O (w/v) for 10 min. The gels were stored in 1% acetic acid (v/v) at 4°C until
further analysis. Three representative gels per sample were used for analysis.
Gel scanning and image analysis
The gels were scanned by ScanMaker 6000 (Microtek). Image analysis was carried out with
PDQuest 6.2 software (Bio-Rad), including the quantitative analysis. After background
subtraction and spot detection, the volume of each spot from three replicate gels was normalized
against the total valid spots. Protein spots with reproducible and statistically significant changes
in intensity (greater than 2-fold, paired t-test, p<0.05) were considered to be differentially
expressed proteins. Except for the differentially expressed protein spots, the best focused 263
spots present in both pollen and pollen tube samples were picked and subjected to detailed
proteomic analysis.
In-gel digestion and mass spectrometry
In-gel digestion of proteins for MALDI-TOF MS was performed according the method described
by Gharahdaghi et al. (1999) with some modifications. Before digestion, the protein spots were
cut from the gels and rinsed twice with Milli-Q water, and then destained twice for 15 min using
200 µL of a freshly prepared 1:1 solution of 100 mM Na2S2O3 and 30 mM K3Fe(CN)6 by slow
vortexing. After being rinsed twice for 5 min with Milli-Q water, the samples were dehydrated
twice for 10 min in 200 µL of dehydration solution containing 25 mM NH4HCO3 and 50% ACN
13
(v/v), shrunk for 10 min in 100 µ L of ACN, and then completely dried under vacuum. For
digestion, the samples were incubated in 10 µ g/mL Roche sequencing-grade modified trypsin
buffer (containing 1 mM CaCl2, 25 mM NH4HCO3, pH 8.3) for 45 min on ice. And then the
remaining solution was removed and replaced with 10 µL of 25 mM NH4HCO3 for digestion at
37°C for 12 h. The digest solution was transferred to a 0.5 mL centrifuge tube where the
digested peptides were extracted using 40 µL of 0.1% TFA (v/v) by vortexing 20 min at room
temperature, followed by 30 µL of a 70% ACN (v/v) and 5% TFA (v/v) solution twice by
vortexing for 20 min. The digested samples from different tubes of the same original material
were combined and vacuum-dried. A piece of the gel was cut from a protein-free region and
processed in parallel with the samples as a control.
To acquire the PMF data, the vacuum-dried samples were dissolved in 10 µL of a 70%
ACN (v/v) and 0.1% TFA (v/v) solution. Then the samples were spotted onto the Anchorchip
target plate (600 μm, Bruker Daltonics, Germany) (1.0 µL) twice and 0.3 µL of 4 mg/mL CHCA
matrix solution dissolved in 70% ACN (v/v), and 0.1% TFA (v/v) once. The sample spot was
desalted with 1 µL of 0.1% TFA (v/v) twice, and completed dried. The tryptic peptide masses
were generated using a MALDI-TOF/TOF mass spectrometer (AUTOFLEX II TOF-TOF; Bruker
Daltonics, Germany). The peptide calibration standard mono (Bruker Daltonics) was used as an
external calibration to ensure the accuracy of protein identification.
Data analysis
The PMF data searches were performed using MASCOT search tools
(http://www.matrixscience.com) in the NCBI non-redundant public protein database.
Arabidopsis thaliana (thale cress) was selected for the taxonomic category. All of the peptide
masses were assumed to be monoisotopic and [M+H]+. Carbamidomethylcysteine was
considered to be a fixed modification, while oxidation of methionine was considered to be a
variable modification. The mass accuracy was set to ±100 ppm, and the maximum number of
missed cleavages was set at one. The identified proteins should have more than 4 matched
peptides, and the percentage of sequence coverage should be greater than 10%. All of the
positive protein identification scores were significant (p<0.05, mascot score>60). Functional
categories were assigned according to the data from MIPS (http://mips.gsf.de/projects/funcat),
BBC (http://bbc.botany.utoronto.ca /welcome.htm), plant energy biology
14
(http://www.plantenergy.uwa.edu.au/applications/suba/flatfile.php), and protein information
resource (PIR, http://pir.georgetown.edu/). The lowest p value was selected when multiple
functional categories were allotted to one protein. The subcellular location and preferentially
expression pattern of the proteins were deduced by using the TAIR database
(http://www.arabidopsis.org) and Gene Atlas tool of Genevestigator
(https://www.genevestigator.ethz.ch/).
Acknowledgments
We thank Dr. Heven Sze (Department of Cell Biology & Molecular Genetics, University of
Maryland) and Dr. Tai Wang (Institute of Botany, Chinese Academy of Sciences, China) for
critical reading of this article. We also thank Mr. Jidong Feng (State Key laboratories of
AgroBiotechnology, China Agricultural University) for his assistance in mass spectrometry
analysis.
15
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Figure legends
Figure 1. A representative proteome map of mature pollen. All labeled spots are proteins without
changes in intensity between pollen and the pollen tube two-dimensional gels (see Table 1 for
details).
Figure 2. Present and up-regulated protein spots during the transition from mature pollen to
pollen germination and pollen tube growth. The spots marked by rectangles are the identified
proteins (listed in Table1, 2).
Figure 3. Disappeared and down-regulated protein spots during the transition from mature pollen
to pollen germination and pollen tube growth. The spots marked by rectangles are the identified
proteins (listed in Table 1, 2).
Figure 4. Functional categories of the total and differentially expressed proteins identified in
Arabidopsis pollen grains and pollen tubes. Each different color represents the percentage of
proteins in the corresponding functional category. The putative functions of these proteins are
listed in Table 1.
(A) Functional categories of total identified proteins.
(B) Functional categories of differentially expressed proteins.
Figure 5. An enlarged view of the differentially expressed protein spots on the 2-DE gels between
pollen and pollen tubes.
(A) Up-regulated protein spots.
(B) Down-regulated protein spots.
21
Tables
Table 1. Proteins identified in mature pollen grains and pollen tubes of Arabidopsis.
Spot No.a
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
Eukaryotic translation initiation factor
5A, putative
Expressed protein
5.55/17.4
5.81/17.1
42%
79
5.73/33.9
5.78/33.6
31%
114
6.0/50.3
7.03/53.9
18%
107
AGI name
Protein identity
Cell fate (5)b
28c
At1g26630
56c
At1g63000
137
At3g17240
236c
At1g56340
2-oxoglutarate dehydrogenase, E3
subunit
Calreticulin 1 (CRT1)
4.50/47.7
4.20/48.5
26%
99
237c
At1g09210
Calreticulin 2 (CRT2)
4.36/48.4
4.12/48.1
16%
71
297
c
At1g63000
Expressed protein
5.73/33.9
5.78/33.6
13%
67
298c
At1g63000
Expressed protein
5.73/33.9
5.78/33.6
13%
72
Cell type differentiation (2)
22Ac
At2g19770
Profilin 4
5.02/14.6
4.84/14.5
50%
80
c
At4g29340
Profilin 3
5.02/14.6
4.84/14.4
57%
82
7.57/19.2
7.82/18.9
32%
100
5.54/20.1
5.65/19.9
40%
153
22B
Cell structure (12)
20
At1g48020
23c
At4g24640
87A
At3g12110
Invertase /pectin methylesterase
inhibitor family protein
Invertase /pectin methylesterase
inhibitor family protein
Actin 11 (ACT11)
5.23/41.9
5.05/41.6
31%
91
89c
At5g59370
Actin 4 (ACT4)
5.37/42.0
5.29/41.7
55%
213
94c
At3g02230
5.61/41.1
5.70/40.6
55%
224
96c
At5g15650
5.76/41.4
5.98/40.9
25%
111
126
At4g37990
6.79/39.4
7.24/38.9
16%
109
187c
At1g04820
Reversibly glycosylated polypeptide-1
(RGP1)
Reversibly glycosylated polypeptide-2
(RGP2)
Mannitol dehydrogenase, putative
(ELI3-2)
Tubulin alpha-2/alpha-4 chain (TUA4)
4.93/50.2
4.68/49.5
36%
128
196c
At4g25590
Actin-depolymerizing factor, putative
5.08/15.4
4.82/15.8
50%
70
198
c
At5g52360
Actin-depolymerizing factor, putative
5.57/15.4
5.31/15.9
55%
76
222Ac
At2g37620
Actin 1 (ACT1)
5.31/42.0
5.16/41.8
45%
190
c
At3g53750
Actin 3 (ACT3)
5.31/42.1
5.16/41.8
45%
190
Peroxiredoxin type 2, putative
5.33/17.6
5.23/17.4
65%
135
222B
Defense/stress responses (9)
3
At1g60740
3
At1g65970
Peroxiredoxin type 2, putative
5.33/17.6
5.23/17.4
65%
136
6
At2g30870
Glutathione S-transferase, putative
5.49/24.2
5.39/24.2
25%
68
7
At4g08390
L-ascorbate peroxidase, stromal (sAPX)
8.31/40.5
8.57/40.4
33%
133
12c
At4g11600
Glutathione peroxidase, putative
6.59/18.8
9.85/25.6
33%
85
29c
At4g11600
Glutathione peroxidase, putative
6.59/18.8
9.85/25.6
43%
82
c
At3g06050
Alkyl hydroperoxide reductase/thiol
specific antioxidant (AhpC/TSA)
8.99/21.3
9.39/21.4
52%
158
34
22
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
5.85/27.8
6.03/27.5
Sequence
coverage
Mascot
score
38%
103
5.85/27.8
6.03/27.5
60%
178
6.09/24.1
8.93/29.2
48%
92
6.09/24.1
8.93/29.2
75%
191
Monodehydroascorbate reductase,
putative
6.86/46.6
6.80/46.5
47%
205
Aldose 1-epimerase family protein
5.64/34.0
5.73/33.7
21%
77
Spot No.a
AGI name
44c
At1g07890
45c
At1g07890
48c
At2g47730
L-ascorbate peroxidase 1, cytosolic
(APX1)
L-ascorbate peroxidase 1, cytosolic
(APX1)
Glutathione S-transferase 6 (GST6)
49c
At2g47730
Glutathione S-transferase 6 (GST6)
122
At3g52880
Protein identity
Development (1)
216
At5g66530
Energy (59)
1c
5c
At3g52300
ATP synthase D chain-related
4.97/17.7
4.81/19.6
43%
87
At4g15530
5.25/93.9
6.06/104.2
36%
352
8c
At5g65690
5.98/69.3
6.37/72.9
14%
78
11c
At4g26970
6.71/108.9
7.15/108.5
21%
175
13c
At2g44350
Pyruvate phosphate dikinase family
protein
Phosphoenolpyruvate carboxykinase
[ATP], putative
Aconitate hydratase, cytoplasmic,
putative
Citrate synthase, mitochondrial, putative
6.41/53.0
6.88/52.6
14%
79
24c
At5g47030
ATP synthase delta chain, mitochondrial
6.20/21.5
6.68/21.5
39%
70
33
At1g08480
Expressed protein/unknown protein
6.28/15.8
6.80/15.8
19%
61
39c
At3g55440
5.24/27.4
5.16/27.1
44%
137
40c
At3g55440
5.24/27.4
5.16/27.1
82%
234
47
At5g54500
Triosephosphate isomerase, cytosolic,
putative
Triosephosphate isomerase, cytosolic,
putative
Quinone reductase, putative
5.96/21.8
6.35/21.8
50%
105
57
At4g02580
7.55/27.6
8.03/28.4
30%
115
60c
At1g53240
8.54/36.0
8.58/35.8
15%
72
69c
At3g59480
5.12/35.2
4.99/35.0
65%
291
70
At5g50850
5.67/39.4
5.55/39.2
38%
133
71c
At2g31390
5.31/35.4
5.13/35.3
32%
99
72c
At2g31390
5.31/35.4
5.13/35.3
53%
230
76c
At3g15020
8.3/36.0
8.43/35.8
26%
87
80c
At3g47520
8.66/42.6
8.81/42.4
23%
107
82
At2g01140
8.19/42.5
8.27/42.3
17%
64
91
At1g43670
NADH-ubiquinone oxidoreductase 24
kDa subunit, putative
Malate dehydrogenase [NAD],
mitochondrial
Pfkb-type carbohydrate kinase family
protein
Pyruvate dehydrogenase E1 component
beta subunit, mitochondrial / PDHE1-B
(PDH2)
Pfkb-type carbohydrate kinase family
protein
Pfkb-type carbohydrate kinase family
protein
Malate dehydrogenase [NAD],
mitochondrial, putative
Malate dehydrogenase [NAD],
chloroplast (MDH)
Fructose-bisphosphate aldolase,
putative
Fructose-1,6-bisphosphatase, putative
5.28/37.7
5.12/37.3
26%
94
23
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
5.49/42.2
6.30/45.6
5.33/42.1
6.68/45.3
28%
48%
106
188
6.11/35.9
6.51/35.6
46%
171
6.05/38.9
6.40/38.5
52%
203
At5g43330
Phosphoglycerate kinase, putative
Succinyl-coa ligase [GDP-forming]
beta-chain, mitochondrial, putative
Malate dehydrogenase, cytosolic,
putative
Fructose-bisphosphate aldolase,
putative
Cytosolic malate dehydrogenase
6.33/36.0
6.77/35.6
40%
84
107
At5g43330
Cytosolic malate dehydrogenase
6.33/36.0
6.77/35.6
31%
78
108c
At5g43330
Cytosolic malate dehydrogenase
6.33/36.0
6.77/35.6
31%
74
c
109
At3g04120
6.62/37.0
7.14/36.9
33%
120
110c
At1g13440
6.67/37.0
7.21/36.9
49%
161
111
At1g79530
8.75/45.0
8.97/44.8
18%
81
113
At5g08300
8.55/36.6
8.39/36.1
23%
74
115
At1g01090
Glyceraldehyde-3-phosphate
dehydrogenase,cytosolic(GAPC)
Glyceraldehyde 3-phosphate
dehydrogenase, cytosolic, putative
Glyceraldehyde 3-phosphate
dehydrogenase, cytosolic, putative
Succinyl-coa ligase [GDP-forming]
alpha-chain, mitochondrial, putative
Pyruvate dehydrogenase E1 component
alpha subunit, chloroplast
Isocitrate dehydrogenase, putative
7.16/47.6
7.54/47.2
17%
74
6.13/46.1
6.52/45.7
26%
100
Spot No.a
AGI name
92c
93Ac
At1g79550
At2g20420
101c
At1g04410
102c
At3g52930
106c
c
120c
c
At1g65930
Protein identity
121
At2g35840
Sucrose-phosphatase 1 (SPP1)
6.24/47.8
6.57/47.8
33%
170
123
At4g35650
Isocitrate dehydrogenase, putative
7.08/40.3
7.49/39.9
23%
72
124
At1g24180
7.62/43.7
8.01/43.3
25%
128
125
At4g35260
Pyruvate dehydrogenase Ela-like
subunit IAR4
Isocitrate dehydrogenase subunit 1
8.12/40.0
8.27/39.6
21%
104
128c
At2g47510
Fumarate hydratase, putative
7.98/53.4
7.98/53.0
16%
72
131c
At1g48030
6.96/54.0
7.45/54.0
23%
105
132c
At5g25880
Dihydrolipoamide dehydrogenase 1,
mitochondrial
Malate oxidoreductase, putative
6.55/65.0
6.98/64.6
27%
177
133
At5g63680
Pyruvate kinase, putative
6.24/55.6
6.62/55.0
18%
79
134d
At2g07698
6.23/55.3
5.23/85.9
37%
229
141c
At4g35830
ATP synthase alpha chain,
mitochondrial, putative
Aconitate hydratase, cytoplasmic
5.98/98.8
6.35/98.1
20%
141
c
142
At2g05710
6.72/108.8
7.12/108.2
16%
103
143c
At5g37510
6.24/82.2
6.59/81.2
31%
220
144c
At1g23190
5.82/63.2
6.21/63.2
20%
156
145c
At5g65690
5.98/69.3
6.37/72.9
14%
109
146c
At2g45290
Aconitate hydratase, cytoplasmic,
putative
NADH-ubiquinone dehydrogenase,
mitochondrial, putative
Phosphoglucomutase, cytoplasmic,
putative
Phosphoenolpyruvate carboxykinase
[ATP], putative
Transketolase, putative
5.63/69.3
6.55/79.9
12%
70
c
152
At3g03250
5.80/51.9
5.98/51.7
30%
111
155c
At5g17310
5.73/52.1
5.79/51.9
31%
100
156c
At3g03250
UTP-glucose-1-phosphate
uridylyltransferase, putative
UTP-glucose-1-phosphate
uridylyltransferase, putative
UTP-glucose-1-phosphate
uridylyltransferase, putative
5.80/51.9
5.98/51.7
37%
228
24
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
6.32/63.6
6.53/63.3
20%
93
5.53/60.9
5.62/60.7
51%
261
5.53/60.9
5.62/60.7
32%
148
5.54/48.0
5.51/47.7
44%
189
6.52/63.6
6.52/59.6
62%
377
6.18/59.8
6.52/59.6
48%
219
7.55/58.9
7.75/58.4
36%
158
5.36/63.0
5.20/60.5
27%
119
5.41/48.3
6.52/59.6
21%
84
5.41/48.3
6.52/59.6
37%
187
At1g12240
D-3-phosphoglycerate dehydrogenase,
putative
2,3-biphosphoglycerate-independent
phosphoglycerate mutase, putative
2,3-biphosphoglycerate-independent
phosphoglycerate mutase, putative
Enolase (2-phospho-D-glycerate
hydroylase)
ATP synthase beta chain 1,
mitochondrial
ATP synthase beta chain 1,
mitochondrial
Dihydrolipoamide S-acetyltransferase,
putative
2,3-bisphosphoglycerate-independent
phosphoglycerate mutase, putative
ATP synthase beta chain 1,
mitochondrial
ATP synthase beta chain 1,
mitochondrial
Beta-fructosidase (BFRUCT4)
5.39/73.7
5.30/73.8
18%
149
At2g21870
Expressed protein
9.0/25.1
6.59/27.6
21%
84
228
At1g30120
5.92/44.7
6.31/44.2
12%
68
241Ac
At5g08670
6.18/59.8
6.52/59.6
26%
98
241Bc
At5g08680
6.06/60.0
6.45/59.8
26%
98
241Cc
At5g08690
6.18/59.8
6.59/59.7
26%
98
243
At2g27860
Pyruvate dehydrogenase E1 component
beta subunit, chloroplast
ATP synthase beta chain 1,
mitochondrial
ATP synthase beta chain, mitochondrial,
putative
ATP synthase beta chain 2,
mitochondrial
Expressed protein
5.49/44.1
5.47/43.6
14%
63
247c
At4g10260
6.90/35.0
7.36/34.6
21%
74
267c
At5g65690
5.98/69.3
6.37/72.9
13%
100
268c
At5g13450
Pfkb-type carbohydrate kinase family
protein
Phosphoenolpyruvate carboxykinase
[ATP], putative
ATP synthase delta chain,
mitochondrial, putative
9.12/26.3
9.86/26.3
28%
102
Inorganic pyrophosphatase, putative
[soluble]
Delta-1-pyrroline-5-carboxylate
dehydrogenase (P5CDH)
Carbonic anhydrase, putative
5.58/26.7
5.96/24.5
32%
75
6.26/62.1
6.71/61.7
17%
141
6.54/29.2
6.99/28.8
38%
112
Spot No.a
AGI name
161c
At4g34200
162c
At3g08590
163c
At3g08590
164c
At2g36530
165c
At5g08670
169Ac
At5g08670
171c
At3g13930
176c
At1g09780
181c
At5g08670
182c
At5g08670
186
220c
Protein identity
Metabolism (47)
9
At1g01050
14
At5g62530
17c
21c
At1g23730
At1g23730
Carbonic anhydrase, putative
6.54/29.2
6.99/28.8
31%
97
30
At4g09320
6.84/15.8
8.47/18.8
33%
70
38c
At5g26667
5.79/22.6
5.83/22.5
50%
181
41c
At1g75270
Nucleoside diphosphate kinase 1
(NDK1)
Uridylate kinase, uridine
monophosphate kinase (PYR6)
Dehydroascorbate reductase, putative
6.0/23.5
6.03/23.4
24%
73
25
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
5.55/25.1
5.63/24.9
30%
65
5.55/25.1
5.63/24.9
43%
105
5.92/31.4
8.75/36.7
23%
80
6.71/30.2
7.27/30.0
38%
119
5.06/39.0
4.82/38.6
27%
116
At3g22850
Inorganic pyrophosphatase, putative
[soluble]
Inorganic pyrophosphatase, putative
[soluble]
Short-chain dehydrogenase / reductase
(SDR) family protein
Bacterial transferase hexapeptide
repeat-containing protein
Alpha-1,4-glucan-protein synthase
(UDP-forming)
Expressed protein
5.84/27.5
6.12/27.1
27%
83
At5g01410
Stress-responsive protein, putative
5.79/33.4
5.98/33.2
25%
122
5.79/33.4
5.98/33.2
24%
92
9.19/41.5
9.36/41.2
40%
170
Spot No.a
AGI name
54c
At2g46860
55c
At2g46860
58
At1g54870
62c
At1g47260
66c
At5g16510
73
74c
78c
At5g01410
Stress-responsive protein, putative
79c
At2g05990
Enoyl-[acyl-carrier protein] reductase
[NADH], chloroplast, putative
Expressed protein
6.15/36.8
6.41/36.4
38%
144
Bacterial transferase hexapeptide
repeat-containing protein
Adenosine kinase 2 (ADK2)
6.75/28.0
7.30/27.8
33%
107
5.14/38.2
4.90/37.8
31%
75
Monodehydroascorbate reductase,
putative
Monodehydroascorbate reductase,
putative
Adenosine kinase 1 (ADK1)
5.18/47.4
4.97/47.4
22%
84
5.18/47.4
4.97/47.4
27%
87
5.29/38.3
5.11/37.8
72%
271
Semialdehyde dehydrogenase family
protein
Glutamine synthetase (GS1)
5.39/36.9
7.00/40.7
26%
96
5.72/38.8
5.93/38.6
46%
125
81c
At2g45600
83
At5g66510
84
At5g03300
c
86
At5g03630
87Bc
At5g03630
88c
At3g09820
90
At1g14810
95
At3g17820
c
Protein identity
At3g17940
Aldose 1-epimerase family protein
5.88/37.3
6.28/37.2
62%
240
100
At1g48470
Glutamine synthetase, putative
6.20/38.4
6.63/38.9
32%
148
114
At5g54160
Quercetin 3-O-methyltransferase 1
5.73/40.1
5.81/39.6
22%
72
116c
At1g02500
5.50/43.6
5.60/43.1
39%
124
117
At3g17390
5.51/43.2
5.60/42.8
30%
103
118c
At4g01850
5.67/43.6
5.94/43.2
25%
78
119c
At2g36880
5.76/42.9
6.09/42.5
46%
179
127c
At2g30970
S-adenosylmethionine synthetase 1
(SAM1)
S-adenosylmethionine synthetase,
putative
S-adenosylmethionine synthetase 2
(SAM2)
S-adenosylmethionine synthetase,
putative
Aspartate aminotransferase (ASP1)
8.36/48.1
8.34/47.7
40%
143
135
At1g23800
6.21/56.8
7.35/58.1
29%
185
138c
At1g53500
6.04/75.7
6.40/75.2
32%
257
140c
At5g17920
6.09/84.6
6.47/84.3
33%
224
147c
At5g17920
6.09/84.6
6.47/84.3
16%
88
150c
At5g15490
Aldehyde dehydrogenase, mitochondrial
(ALDH3)
NAD-dependent epimerase/dehydratase
family protein
5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase
5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase
UDP-glucose-6-dehydrogenase,
putative
5.76/53.7
5.82/53.1
31%
185
98
26
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
5.69/53.7
5.77/53.1
35%
139
At1g19920
UDP-glucose-6-dehydrogenase,
putative
Sulfate adenylyltransferase 2
6.15/53.7
6.58/53.6
13%
74
At3g06580
Galactokinase (GAL1)
5.67/55.1
5.81/54.3
23%
123
At3g29360
UDP-glucose 6-dehydrogenase, putative
5.69/53.7
5.77/53.2
13%
64
204c
At5g40370
Glutaredoxin, putative
6.71/11.9
7.34/117.5
62%
82
214
At3g22850
Expressed protein
5.84/27.5
6.12/27.1
22%
78
Spot No.a
AGI name
153
At3g29360
157
159
160
c
Protein identity
224
At5g19550
Aspartate aminotransferase 2 (ASP2)
6.80/44.5
7.32/44.3
28%
81
225
At4g31990
Aspartate aminotransferase, chloroplast
8.18/50.0
8.38/49.8
19%
87
226
c
At5g07440
Glutamate dehydrogenase 2 (GDH2)
6.07/45.0
6.51/44.7
24%
79
227c
At4g13930
6.80/52.1
7.25/51.7
44%
226
232
At4g23100
Glycine hydroxymethyltransferase,
putative
Glutamate-cysteine ligase
6.16/58.9
6.52/58.5
14%
100
c
234
At5g57655
Xylose isomerase family protein
5.74/53.5
7.88/32.4
21%
69
242
At5g39320
UDP-glucose 6-dehydrogenase, putative
5.60/53.5
5.56/53.1
19%
86
250c
At5g15490
UDP-glucose 6-dehydrogenase, putative
5.76/53.7
5.82/53.1
13%
61
253c
At5g15490
UDP-glucose 6-dehydrogenase, putative
5.76/53.7
5.82/53.1
13%
81
254
At1g79440
6.10/55.6
6.90/56.5
39%
175
255
At5g35360
Succinate-semialdehyde dehydrogenase
(SSADH1)
Acetyl-coa carboxylase, biotin
carboxylase subunit (CAC2)
Expressed protein
8.26/54.6
7.28/58.4
24%
143
5.84/27.5
6.12/27.1
27%
76
6.09/84.6
6.47/84.3
16%
125
269
At3g22850
c
272
At5g17920
273c
At2g17420
5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase
Thioredoxin reductase 2
6.26/40.2
5.81/54.3
22%
77
276
At3g22200
4-aminobutyrate aminotransferase
6.33/51.3
8.07/55.2
17%
85
Luminal binding protein 2 (BiP-2)
(BP2)
FK506-binding protein 2-2 (FKBP15-2)
20S proteasome beta subunit A (PBA1)
(PRCD)
20S proteasome beta subunit F1 (PBF1)
Lactoylglutathione lyase, putative
5.11/73.8
4.84/73.5
31%
273
4.90/17.8
5.31/25.2
5.05/17.6
5.24/25.1
22%
33%
64
89
6.95/24.9
5.11/32.0
7.49/24.6
9.75/62.7
53%
25%
154
79
4.99/30.7
4.73/30.5
45%
94
Protein fate (25)
2c
At5g42020
25
42
At5g48580
At4g31300
50
52
At3g60820
At1g18840
65
At5g42790
97-2c
At2g47470
20S proteasome alpha subunit F1
(PAF1)
Thioredoxin family protein
5.52/29.5
5.86/39.5
35%
84
136c
At3g11830
Chaperonin, putative
6.03/60.2
6.29/59.8
15%
73
149c
At3g02090
6.29/59.2
6.76/59.1
37%
190
154c
At5g20890
Mitochondrial processing peptidase beta
subunit, putative
Chaperonin, putative T-complex protein
1, beta subunit
5.59/57.8
5.66/57.3
29%
130
27
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
Cytosol aminopeptidase
Luminal binding protein 1 (bip-1) (BP1)
5.66/54.8
5.08/73.9
5.72/54.5
4.81/73.6
25%
11%
157
62
At5g28540
Luminal binding protein 1 (bip-1) (BP1)
5.08/73.9
4.81/73.6
17%
93
168c
At5g02500
5.03/71.7
4.75/71.3
20%
128
169Bc
At5g28540
Heat shock cognate 70 kDa protein 1
(HSC70-1) (HSP70-1)
Luminal binding protein 1 (bip-1) (BP1)
5.08/73.9
4.81/73.6
20%
97
173c
At3g23990
Chaperonin (CPN60) (HSP60)
5.66/61.6
5.41/61.3
28%
125
174
c
At5g09590
5.63/73.2
5.44/73.0
11%
74
178c
At1g51980
5.94/54.5
6.26/54.4
35%
215
180
At3g13860
Heat shock protein 70/HSP70
(HSP70-5)
Mitochondrial processing peptidase
alpha subunit, putative
Chaperonin, putative
7.57/46.0
5.89/60.4
11%
68
184
At5g58290
5.42/45.9
5.25/45.7
21%
95
191
At3g05530
4.91/47.7
4.65/47.5
37%
132
212
At2g27020
5.77/27.5
6.25/27.3
29%
92
218
At1g11910
26S proteasome AAA-ATPase subunit
(RPT3)
26S proteasome AAA-ATPase subunit
(RPT5a)
20S proteasome alpha subunit G
(PAG1) (PRC8)
Aspartyl protease family protein
5.29/53.0
5.20/54.6
17%
101
219
At5g01660
Ferritin 1 (FER1)
5.73/28.2
4.79/68.5
13%
74
Spot No.a
AGI name
Protein identity
158
166c
At2g24200
At5g28540
167c
235c
At1g21750
Protein disulfide isomerase, putative
4.81/55.9
4.54/55.6
54%
301
239A
At2g05850
5.46/55.2
5.55/54.7
17%
65
288
At2g05840
Serine carboxypeptidase S10 family
protein
20S proteasome alpha subunit A2
(PAA2)
5.75/27.4
5.91/27.3
30%
108
60S acidic ribosomal protein P0
(RPP0B)
60S acidic ribosomal protein P0
(RPP0B)
Elongation factor Tu, putative/EF-Tu,
putative
5.0/34.2
4.69/34.1
27%
63
5.0/34.2
4.69/34.1
30%
87
6.25/49.6
6.68/49.4
39%
195
Protein synthesis (2)
67c
At3g09200
68c
At3g09200
99c
At4g02930
Signal transduction (11)
4c
At1g35720
Annexin 1 (ANN1)
5.21/36.1
5.01/36.2
41%
124
10A
At1g15470
5.72/36.7
6.07/36.3
42%
107
10B
At5g65020
Transducin family protein / WD-40
repeat family protein
Annexin 2 (ANN2)
5.76/36.4
5.96/36.3
50%
154
18
At5g63400
Adenylate kinase
6.91/27.1
7.45/26.9
41%
119
104c
At5g16760
Inositol 1,3,4-Trisphosphate 5/6 kinase
5.89/36.4
6.43/36.2
36%
119
c
c
105
At5g16760
Inositol 1,3,4-Trisphosphate 5/6 kinase
5.89/36.4
6.43/36.2
39%
135
112
At2g46280
6.50/36.7
6.99/36.4
38%
198
139
At1g50480
Eukaryotic translation initiation factor 3
subunit 2
10-formyltetrahydrofolate synthetase
(THFS)
6.26/68.3
6.69/67.8
42%
302
28
Table 1. continued
pI/MW (kDa)d
Experimental Theoretical
Sequence
coverage
Mascot
score
Rab GDP dissociation inhibitor, putative
5.11/50.0
4.91/49.5
34%
121
Guanine nucleotide-binding family
protein
Expression protein,putative GTPbinding protein
Cyclase-associated protein (cap1)
7.07/36.3
7.12/35.8
34%
131
6.35/44.7
6.79/44.5
50%
215
6.23/51.5
6.62/50.9
14%
66
At1g07750
Cupin family protein
5.83/38.5
6.06/38.3
20%
62
At2g28680
Cupin family protein
6.25/38.7
6.68/38.4
31%
88
Dehydratase family
5.85/65.6
6.15/64.9
23%
123
Pentatricopeptide (PPR) repeatcontaining protein
Glycine cleavage T family protein /
aminomethyl transferase family protein
9.25/56.0
9.66/55.6
17%
72
6.30/43.6
6.69/43.5
30%
75
6.97/18.7
7.89/18.6
64%
146
8.09/16.9
7.89/18.6
25%
62
5.42/32.8
5.53/32.6
30%
79
6.36/54.3
6.78/53.8
18%
64
Spot No.a
AGI name
Protein identity
172c
At5g09550
246
At1g48630
270c
At1g30580
283
At4g34490
Storage protein (2)
97-1c
103
c
Subcellular localization (1)
239B
At3g23940
Transcription (2)
32
At2g01860
93Bc
At4g12130
Transport (7)
36c
At4g16160
37c
At4g16160
75
At2g30050
129c
At3g24170
Mitochondrial import inner membrane
translocase subunit Tim17/Tim22/Tim23
family protein
Mitochondrial import inner membrane
translocase subunit Tim17/Tim22/Tim23
family protein
Transducin family protei/WD-40 repeat
family protein
Glutathione reductase, putative
c
130
At3g24170
Glutathione reductase, putative
6.36/54.3
6.78/53.8
36%
175
151c
At1g80460
Glycerol kinase, putative
6.11/53.0
6.20/56.4
23%
69
c
170
At1g78900
5.11/69.1
4.86/68.8
19%
117
188c
At4g38510
5.03/54.4
4.77/54.3
35%
146
189c
At1g20260
Vacuolar ATP synthase catalytic subunit
A
Vacuolar ATP synthase subunit B,
putative
Vacuolar ATP synthase subunit B,
putative
5.11/54.4
4.55/36.3
53%
235
Hypothetical protein
7.74/15.0
8.18/14.8
17%
68
Unclassified protein (4)
31
At2g10360
43
At2g31985
Expressed protein
5.79/26.9
6.03/26.7
33%
72
183
At1g72880
5.29/38.3
5.13/40.6
21%
75
207
At1g10590
Acid phosphatase survival protein surE,
putative
DNA-binding protein-related
6.60/15.5
7.36/15.4
32%
63
a
Total protein spots identified from mature pollen an pollen tubes gels. The annotation of spot number with ‘A’, ‘B’
or ‘C’ indicates that two or three different proteins were identified from one protein spot.
29
b
Functional category analysis was conducted using the website tools provided by MIPS, BBC, PIR, and Plant
Energy Biology.
c
Proteins reported by other pollen proteomic researches (Holmes-Davis et al. 2005; Noir et al. 2005; Sheoran et al.
2006).
d
The theoretical pI and MW of each protein was from the data of TAIR8 (http://www.arabidopsis.org/index.jsp).
30
Table 2. Proteins differentially expressed between pollen grains and pollen tubes of Arabidopsis.
Spot No.
AGI name
Fold change (n=3)a
in proteome
Pollen Specificityb
Reported in other
proteome studies
Subcellular Localizationc
Up-regulated protein
3
At1g60740
Appeared
No
No
Unknown
3
At1g65970
Appeared
No
No
Cytosol
267
At5g65690
Appeared
No data
Yes
Unknown
33
At1g08480
↑2.0 ± 0.3
No
No
Mitochondrion
36
At4g16160
↑2.0 ± 0.2
No
Yes
Plastid outer membrane
37
At4g16160
↑3.7 ± 0.4
No
Yes
Plastid outer membrane
269
At3g22850
↑3.0 ± 0.3
No
No
Unknown
Down-regulated protein
18
At5g63400
Disappeared
No
Yes
Mitochondrion
20
At1g48020
Disappeared
No data
No
Endomembrane system
214
At3g22850
Disappeared
No
No
Unknown
1
At3g52300
↓6.4 ± 0.3
No
Yes
Mitochondrion, cytoplasm
4
At1g35720
↓2.6 ± 0.4
No
Yes
Cytosol, membrane
6
At2g30870
↓2.3 ± 0.1
No
No
Cytoplasm
7
At4g08390
↓2.6 ± 0.3
No
No
Mitochondrion
9
At1g01050
↓3.1 ± 0.6
No
No
Cytoplasm, nucleus
17
At1g23730
↓2.2 ± 0.3
No
Yes
Cytoplasm
23
At4g24640
↓2.8 ± 0.5
Yes
Yes
Endomembrane system
28
At1g26630
↓3.5 ± 1.2
No
Yes
Unknown
31
At2g10360
↓3.0 ± 0.1
No data
No
Endomembrane system
32
At2g01860
↓5.1 ± 0.4
No
No
Chloroplast
38
At5g26667
↓2.4 ± 0.2
No
Yes
Unknown
41
At1g75270
↓2.0 ± 0.2
No
No
Unknown
73
At3g22850
↓2.2 ± 0.2
No
No
Unknown
270
At1g30580
↓4.0 ± 1.2
No
Yes
Unknown
a
The fold change values were determined by comparing the values between pollen tube and pollen.
b
Pollen specificity determined according to Genevestigator (https://www.genevestigator.ethz.ch/).
c
Subcellular localization determined by using the TAIR database (http://www.arabidopsis.org).
31
Figure 1
32
Figure 2
33
Figure 3
34
Figure 4
35
Figure 5
36
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