JXB Advance Access published online on October 5, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm199
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Proteomic analysis of tomato (Solanum lycopersicum, formerly hycopersicon esculentum) pollen
1Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada S7N 5E2
2National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9
* To whom correspondence should be addressed. E-mail: sawhney{at}admin.usask.ca
Received 5 July 2007; Revised 24 July 2007 Accepted 27 July 2007
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Abstract |
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In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca2+ signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.
Key words: Lycopersicon esculentum, MALDI-TOF MS, pollen, proteomics, Solanaceae, tomato, two-dimensional gel electrophoresis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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In angiosperms, pollen grains (male gametophytes) are the dispersal agents of sperm cells and are vital for successful sexual reproduction and subsequent seed and fruit production. After release from an anther, pollen grains are carried by insects, wind, or other agents to the stigma of a carpel, where they germinate and deliver sperm cells to the female gametophyte via the formation of pollen tubes. The development of pollen, microsporogenesis and microgametogenesis, involves the co-ordinated expression of several genes in different tissues of an anther (Koltunow et al., 1990; McCormick, 2004; Ma, 2005), and pollen grains at maturity contain a large number of transcripts with designated roles in cell wall metabolism, cytoskeleton formation, cell signalling, and vesicle transport (Becker et al., 2003; Honys and Twell, 2003, 2004; Pina et al., 2005).
Rapid advances in proteomic technologies, along with completion of the Arabidopsis and rice genome sequence projects and the availability of comprehensive public sequence databases, have provided tremendous impetus to plant proteomics research (Hirano et al., 2004; Rose et al., 2004; Rossignol et al., 2006). Proteomic analyses of various plant reproductive processes have been conducted, including the identification of sporophytic and gametophytic proteins in normal microspore development (Kerim et al., 2003; Miki-Hirosige et al., 2004), changes in anther proteins due to cold stress (Imin et al., 2006), proteins in relation to pollen germination and tube growth (Dai et al., 2006, 2007), and self-incompatibility (Kalinowski et al., 2002). The proteome analyses of mature Arabidopsis and rice pollen have also been conducted, and many proteins identified correspond to the known transcripts in pollen, in addition to several other proteins in each of these studies (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006).
The objective of this study was to analyse the proteome of tomato pollen. Tomato is an important crop grown around the world (Rick, 1980) and is also known to have significant effects on human health (Willcox et al., 2003; Omoni and Aluko, 2005). Tomato pollen grains are bi-cellular with a large vegetative cell and a small generative cell; the latter divides to form two sperm cells during pollen germination and tube growth, unlike the Arabidopsis and rice pollen which are tri-cellular. Pollen development in tomato has been studied extensively at both the light microscope and ultrastructural levels (Sawhney and Bhadula, 1988; Polowick and Sawhney, 1993a, b), and a number of male-sterile mutants are available in tomato which makes it an excellent model system for genetic and molecular investigations on pollen development, and in hybrid seed programmes (Sawhney, 1994; Gorman and McCormick, 1997).
By combining two-dimensional gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and by using the available databases for tomato and other species, as well as tomato expressed sequence tags (ESTs), a comprehensive analysis of the tomato pollen proteome has been performed. Many of the proteins identified in this study have designated roles in defence mechanisms, energy conversion, pollen germination, and pollen tube growth, and some possibly in sperm cell formation. To our knowledge, this is the first proteomic study on tomato pollen, and several of the proteins reported here have not been identified in the pollen of other species.
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Materials and methods |
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Plant growth
Seeds of tomato (cv. Rutgers) were germinated in 16 cm plastic pots containing Tera-lite Redi-earth mix. Young seedlings and plants were subsequently grown in a growth chamber at 26/23 °C (day/night) and under 16/8 h light/dark conditions. Illumination was provided by fluorescent tubes (F72T12/CW/VHO; Sylvania, USA) and incandescent bulbs at a photon flux density of 100–200 µmol m–2 s–1.
Pollen collection
Pollen grains were collected from freshly open flowers by shaking the anthers on a glass slide, checked under a dissecting microscope, and any debris removed with a needle. Pollen samples were pooled in an Eppendorf tube and the purity of pollen was again determined under a light microscope. Each pooled sample represented pollen from approximately 400 flowers. Pollen was either used immediately or stored at –80 °C until further use. The viability of each pollen sample was tested using an in vitro germination test (Shivanna and Sawhney, 1995) and pollen germination was in the range of 70–75%. Three separate batches of pooled pollen samples were used for protein extraction.
Protein extraction
The pollen samples (
50 mg each, collected from 150–200 flowers) were ground to a fine powder in a pestle and mortar in liquid nitrogen, and extracted with acetone containing 10% TCA and 1% DTT. The samples were kept at –20 °C for 2 h and centrifuged at 25 000 g for 20 min at 4 °C. The resulting pellet was washed by suspending in acetone containing 1% DTT, incubated at –20 °C for 2 h, and centrifuged; it was re-suspended in acetone, sonicated (3x15 s), and centrifuged at 25 000 g. The pellet was vacuum-dried and then dissolved in urea buffer comprising 8 M urea, 20 mM DTT, 4% CHAPS, and 2% ampholyte (pH 3–10). The solution was vortexed extensively for 1 h at room temperature, centrifuged at 20 °C for 20 min at 25 000 g, and the supernatant collected. The resulting pellets were re-extracted with urea buffer and the supernatant combined with that collected earlier. The resulting protein samples were centrifuged again for 20 min at 25 000 g, quantified (3–3.5 mg per 50 mg pollen) using the Bio-Rad DC protein Assay Kit (Bio-Rad, Hercules, CA, USA), and either used immediately or stored at –80 °C for later use.
Two-dimensional gel electrophoresis (2-DE)
2-DE was carried out as previously described (Sheoran et al., 2005, 2006). Isoelectric focusing (IEF) was performed using the Multiphor II horizontal electrophoresis system (Amersham Biosciences, Uppsala, Sweden) and 18 cm Immobiline Dry Strips of 4–7 or 3–10 linear pH gradients (Bio-Rad, Hercules, CA, USA). The strips were rehydrated overnight in a solution containing 8 M urea, 2% CHAPS, 20 mM DTT, 0.002% bromophenol blue, 2% IPG buffer (pH 3–10), and 600 µg of the protein sample. IEF was carried out by applying a voltage of 250 V for 1 h, increasing to 3500 V over 2 h, and holding at 3500 V until a total of 90 kVh was obtained.
Following IEF, the strips were equilibrated for 15 min in an equilibration buffer containing 0.05 M TRIS-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 20 mM DTT, followed by another 15 min equilibration in the same buffer containing 125 mM iodoacetamide without DTT. The equilibrated strips were applied to vertical SDS–polyacrylamide gels (12.5% resolving 5% stacking) and sealed with 0.5% agarose in SDS buffer containing bromophenol blue. Electrophoresis was performed for 30 min at 15 mA gel–1, and then at 20 mA gel–1 until the dye front reached the bottom of the gel, in an SDS electrophoresis buffer containing 25 mM TRIS base, 192 mM glycine, and 0.1% SDS, pH 8.3 in a PROTEAN II XL multi-cell (Bio-Rad, USA).
Gel staining and image analysis
Gels were fixed overnight in 50% (v/v) ethanol with 10% (v/v) orthophosphoric acid, washed with water (3x20 min), and stained with Colloidal Coomassie Blue G-250 (CCB) as described earlier (Sheoran et al., 2006). After washing with water, gels were scanned, annotated, and analysed for spot number using Phoretix 2D Image analysis software (UBI, Canada). Two replicate gels (for both pH 4–7 and 3–10) were run for each of three different pooled pollen samples collected from different batches of plants.
Mass spectrometry and protein identification
Of the spots observed consistently on CCB-stained 2-DE gels, 190 were selected for mass spectrometric analysis from both pH 4–7 and 3–10 gels, as previously described (Sheoran et al., 2005, 2006). Excised protein spots were automatically de-stained, dehydrated, reduced with DTT, alkylated with iodoacetamide, and digested with trypsin using a MassPREP protein digest station (Micromass, Manchester, UK) according to the recommended procedure. The resulting tryptic digests were concentrated and desalted using C18 ZipTips (Millipore Corporation, Bedford, MA, USA) according to the manufacturer's instructions. Samples were then analysed by MALDI-TOF MS on a Voyager-DE STR instrument (Applied Biosystems, Framingham MA, USA) operating in the positive ion and reflectron modes as described earlier (Sheoran et al., 2005, 2006). Spectra were acquired in the 700–3000 m/z range, processed with Mascot Distiller 2.0.0 (www.matrixscience.com), and the resulting peak lists used to identify the corresponding proteins in NCBInr (non-redundant) and Swiss-Prot databases by peptide mass fingerprinting (PMF) using the Mascot (www.matrixscience.com) search engine. Searches were performed using the following parameters: trypsin as the proteolytic enzyme, allowing for one missed cleavage; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine as a variable modification. Proteins identified with a Mowse score greater than 66 (significant at 95% confidence interval) are reported. Because of the limited availability of tomato protein sequence information, database searches were also performed using the NCBI tomato EST database.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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The soluble proteins extracted from mature tomato pollen, separated by 2-DE on pH 4–7 and 3–10 IPG strips, and stained with CCB are shown in Fig. 1A and B, respectively. A total of 960 reproducible protein spots were detected using the pH 4–7 IPG strips (Fig. 1A) and 870 using the pH 3–10 strips (Fig. 1B), indicating better resolution on pH 4–7 gels, as previously observed for rice pollen (Dai et al., 2006). The number of protein spots observed in this study is comparable with that of our proteome analysis of Arabidopsis pollen (Sheoran et al., 2006) and is substantially higher than that reported in two other studies on Arabidopsis pollen (Holmes-Davis et al., 2005; Noir et al., 2005).
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One hundred and ninety protein spots were selected throughout the molecular mass and isoelectric point (pI) ranges of pH 4–7 and 3–10 gels and analysed by MALDI-TOF MS. Of these, 158 spots representing 133 distinct proteins were successfully analysed. Despite the limited availability of tomato protein sequence data, it was possible to identify 83% of the selected spots. This was achieved by concentrating and desalting the tryptic digests using C18 ZipTips, and by processing the MS data with Mascot Distiller 2.0.0. The identified proteins, along with the gene index (gi) number and Mowse score, are listed in Table 1.
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Some of the identified proteins were present as multiple spots on 2-DE gels. These may correspond to multiple isoforms, which could play an important role in pollen development and germination by diversifying the functions of proteins in the haploid genome. Multiple spots corresponding to the same protein have been reported in other proteomic studies (Kerim et al., 2003; Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006).
The predicted molecular masses and pIs for the majority of the identified proteins were generally consistent with the experimental data, as judged from the location of spots on 2-D gels; however, there were some exceptions. For example, spots 31, 47a, 65, 111, and 113 had an apparent molecular mass greater than the corresponding identified protein, whereas spots 5, 20, 24, 58, and 145 had a molecular mass lower than the predicted value. These deviations in molecular mass and pI, as well as multiple spots for the same protein, could be due to various factors, including post-translational modifications, protein degradation, and partial synthesis of proteins during pollen maturation, protein translation from alternatively spliced mRNAs, or protein homologues that may be unique to pollen (Sheoran et al., 2006).
Functional grouping of identified proteins
The identified proteins were categorized into 12 functional groups (Fig. 2) based on predicted protein function and defined criteria (Berardini et al., 2004; Sheoran et al., 2006). More than half of the identified proteins were in three major groups, i.e. energy (19%), defence-related (18%), and protein synthesis and processing (18%). The other groups included proteins involved in cytoskeleton (cell biogenesis and organization), membrane transport, hormone metabolism and signalling, Ca2+ binding and signalling, pollen allergens, other metabolism, and those of unknown function (Fig. 2). Proteins associated with hormone metabolism and signalling, Ca2+ binding and signalling, pollen allergens, and glycine-rich proteins (GRPs) were grouped separately in Fig. 2 because of their established roles in pollen function. In Arabidopsis and rice pollen also, proteins belonging to most, but not all, of these groups were identified and the majority of them were associated with energy, defence, and protein synthesis and processing (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). For comparison purposes, proteins in tomato pollen which are common with Arabidopsis and/or rice pollen are indicated in the last column in Table 1.
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Each group of proteins in tomato pollen and their potential roles in pollen function are discussed separately below.
Defence-related proteins
Proteins included in this group are associated with both biotic and abiotic stresses. The biotic stress-related proteins included the Pto-disease resistance protein, Pto-like serine/threonine kinase, I2 (disease resistance protein), tomato mosaic viral (ToMV) coat protein, and a hypothetical protein PGEC 13.19. Plants expressing the Pto gene encoding a serine/threonine kinase protein were shown to be resistant to both bacterial and fungal pathogens (Tang et al., 1999; Pedley and Martin, 2003), and the Pto-mediated resistance has been used to control the bacterial speck disease in different tomato cultivars (Martin, et al., 2003). The presence of ToMV coat protein in tomato pollen could provide resistance to viruses, since the constitutive expression of viral coat protein genes is known to be effective in producing virus-resistant plants (Koo et al., 2004). The Pto-like serine/threonine kinase and ToMV coat protein were not reported in rice and Arabidopsis pollen (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006) although they were present in the embryo and endosperm of tomato seed (Sheoran et al., 2005).
Pollen grains are free-floating structures and are subject to various abiotic stresses, including drought and extreme temperatures. Plants produce reactive oxygen species (ROS) in responses to abiotic and biotic stresses. Although ROS are known to serve as second messengers in many developmental processes (Foyer and Noctor, 2005), the excessive production of ROS causes oxidative damage to cellular components (Apel and Hirt, 2004; Gechev et al., 2006). Plants have evolved a strategy to combat the ROS by inducing various protective enzymes. Many enzymes known to play a role in the detoxification of ROS were identified in tomato pollen, including superoxide dismutase, thioredoxin peroxidase, ascorbate peroxidase, mono-dehydroascorbate reductase, glutathione transferase, glutaredoxin, and catalase (Table 1). These proteins were also reported in rice and Arabidopsis pollen (Table 1; Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006).
Heat shock proteins (HSPs) and luminal-binding proteins play key roles in defence mechanisms, in addition to their roles as molecular chaperones in protein processing. In tomato pollen, HSP 60, HSP 70, HSP 70-cognate, chaperonin 60, and luminal-binding proteins were identified. HSPs are known to act as protectants of protein function (Vierling, 1991; Wang et al., 2004) and their accumulation in response to heat stress has been reported in developing pollen (Mascarenhas and Crone, 1996). Other proteins present in tomato pollen, such as temperature stress-induced lipocalin, ripening-regulated protein DDTFR10, and the heat stress DnaK homologue, might also have a role in combating abiotic stresses. One spot representing LEA proteins, which are known to have a role in desiccation tolerance (Park et al., 2005), was identified in tomato pollen, whereas 4–7 LEA protein spots were observed in Arabidopsis pollen (Noir et al., 2005; Sheoran et al., 2006).
The defence-related proteins identified in tomato pollen could be part of the survival strategy of these small two-celled structures, which are independent of the parental tissues and, therefore, particularly susceptible to biotic and abiotic stresses.
Energy-related proteins
The presence of a high percentage (19%) of proteins related to energy metabolism correlates well with the large number of mitochondria observed in mature tomato pollen (Polowick and Sawhney, 1993b). These include proteins associated with glycolysis, for example phosphoglucomutase, glyceraldehyde 3-P dehydrogenase, triose phosphate isomerase, enolase, and fructokinase; with the TCA cycle, for example MDH, succinyl CoA ligase, dihydrolipoamide dehydrogenase; and with the electron transport chain, for example cytochrome c oxidase and reductase, and various subunits of ATP synthase (Table 1). Cytochrome c oxidase and reductase were identified only in tomato pollen, and a relatively high number of spots was observed for fructokinase and enolase in tomato compared with Arabidopsis and rice pollen. However, most of the energy-related proteins in tomato were also reported in rice and Arabidopsis pollen (Table 1; Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). Pollen germination and tube growth are high-energy-requiring processes and it seems that most of the proteins required for these events are in place in mature tomato pollen. Although the transcriptome of tomato pollen is not yet available, in Arabidopsis pollen, the transcripts of energy-related proteins are under-represented (Becker et al., 2003; Honys and Twell, 2003, 2004; Pina et al., 2005). Indeed, Holmes-Davis et al. (2005) showed an inverse relationship of high abundance energy-related proteins between and the corresponding mRNA in Arabidopsis pollen.
Protein synthesis and processing
Three spots corresponding to a putative elongation factor and elongation factor-1ß involved in protein synthesis were identified in tomato pollen. Proteins involved in proper protein folding, assembly, and localization, including chaperonin 60, cyclophilin, HSPs, luminal-binding proteins, 78 kDa glucose-regulated protein homologue 1, and HSP68 heat stress DnaK homologue, were also identified (Table 1). In addition, a large number of proteins involved in protein degradation such as cystatin, transitional endoplasmic ATPase, mitochondrial processing peptidase-like, proteasome subunits, ubiquitin-conjugating enzyme, cell division cycle protein 48-related, and translationally controlled tumour protein-like were identified (Table 1). There were relatively few proteins involved in protein synthesis compared with those in protein processing and degradation in tomato pollen, as in pollen of other species (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). This is consistent with the relatively small number of polysomes observed in mature tomato pollen (Polowick and Sawhney, 1993b). However, the mature pollen has abundant stored mRNA (Schrauwen et al., 1990; Honys et al., 2000) and translational apparatus (Mascarenhas, 1989), indicating rapid protein synthesis at the onset of pollen germination and tube growth.
Cell biogenesis and organization
Actin cytoskeleton is an essential component of pollen tube growth as it transports new cell wall materials to the growing tip region (Drobak et al., 2004). Actin was identified in tomato pollen (Table 1), as in the pollen of other species (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). In addition, profilin, a major actin-binding protein involved in pollen tube growth (Taylor and Hepler, 1997), was also identified in tomato pollen (Table 1).
Cell wall loosening and synthesis are essential for pollen germination and rapid pollen tube growth, and various proteins associated with these processes were identified in tomato pollen, including pectin methylesterase (PME), pectin methylesterase inhibitor (PMEI), UTP-glucose-1-phosphate uridylyltransferase, UDP-glucose pyrophosphorylase, UDP-glucose:protein transglucosylase-like, microtubule-associated dTDP-glucose 4–6-dehydratase, and reversibly glycosylated protein. The PME enzyme catalyses the demethylesterification of homogalacturonans and plays an important role in pollen tube growth (Bosch et al., 2005; Chen and Ye, 2007). The post-translational modulation of PME activity is regulated by the enzyme PMEI, and both these enzymes were also reported in rice and Arabidopsis pollen (Table 1). Three GRPs were identified in tomato pollen, and the cell wall GRPs are suggested to have a structural function, probably acting as a scaffold or agglutinating agent for the deposition of cell wall constituents (Mousavi and Hotta, 2005). The presence of GRPs in tomato pollen could reflect their requirement during germination and tube growth.
Phragmoplastin, which is known to function in cell division and cell plate formation (Hong et al., 2003), was also identified in tomato pollen. The presence of phragmoplastin in mature tomato pollen could be related to its role in generative cell division, i.e. sperm cell formation, during pollen germination. Phragmoplastin was not reported in tri-cellular Arabidopsis and rice pollen.
Ca2+ binding and signalling
Ca2+ binding and signalling proteins such as annexin, calreticulin, and calmodulin were identified in tomato pollen, as in rice and Arabidopsis pollen (Table 1; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). Calcium and Ca2+-binding proteins play important roles in pollen germination and tube growth (Taylor and Hepler, 1997; Golovkin and Reddy, 2003; Rato et al., 2004), and the presence of such proteins in mature pollen is indicative of the ready availability of Ca2+ required for these processes.
Hormone metabolism and signalling
The proteins aminocyclopropane-1-carboxylate (ACC) oxidase, IAA5 transcription factor, auxin-responsive calmodulin binding, and 
-G protein (involved in hormone metabolism and signalling) were identified in tomato pollen (Table 1). ACC oxidase is one of the key enzymes in ethylene biosynthesis, and IAA5 transcription factor and auxin-responsive calmodulin-binding proteins play a significant role in auxin signalling. Both ethylene and auxin are known to regulate cell elongation, and these proteins could be required for pollen tube growth. Ethylene and auxin, or their precursors, have been reported in mature pollen (Singh and Sawhney, 1992; Holden et al., 2003). G proteins, which are heterotrimeric with , ß, and 
subunits, have a role in several plant developmental processes (Perfus-Barbeoch et al., 2005; Pandey et al., 2006) including modulation of cell division (Ullah et al., 2001; Chen et al., 2006). The presence of GPA1 in tomato pollen could be another factor involved in generative cell division. G proteins were not identified in Arabidopsis and rice pollen.
Pollen allergens
Pollen grains have been studied for allergen proteins in a number of species because of their allergenic activity toward humans (Mohapatra et al., 2005; Radauer and Breiteneder, 2006). A number of proteins in tomato pollen, such as profilins, luminal-binding proteins, and some of the Ca2+-binding proteins discussed earlier, are also known to act as allergens. Allergenic proteins were also reported in rice and Arabidopsis pollen (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006).
Other proteins
A number of proteins associated with nucleic acid, amino acid, lipid, and various other metabolic processes were identified. For example, six spots representing porins, which are localized in the outer mitochondrial membrane in various organisms and play a crucial role in the transport of metabolites between mitochondria and cytoplasm (Benz, 1994), were identified (Table 1). Seven spots were identified as hypothetical/unknown proteins with no well-defined function, as in other pollen proteomic studies (Holmes-Davis et al., 2005; Noir et al., 2005; Dai et al., 2006; Sheoran et al., 2006). An anther-specific protein, LAT52, with an established role in pollen development (Muschietti et al., 1994; McCormick, 2004), was also identified in tomato pollen.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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This study has shown that tomato pollen contain various proteins with designated roles in defence mechanisms, energy metabolism, protein synthesis and processing, cytoskeleton formation, Ca2+ binding and signalling, and hormone signalling. The presence of these proteins in mature pollen is reflective both of the survival strategies of this small, two-celled independent structure and of the requirement for, and participation in, subsequent pollen germination and tube growth. Although no proteins specific for sperm cell formation were identified in mature tomato pollen, phragmoplastin and the
-G-protein may have a role in this process. Several new proteins not reported in the pollen of other species were identified in tomato pollen, including Pto-like serine/threonine kinase, coat protein (ToMV), HSP68 heat stress DnaK homologue, proteasome subunit-4, cystatin, IAA5 transcription factor, phragmoplastin, and -G protein. Hence, this study (along with others) represents a significant contribution towards the construction of a comprehensive pollen proteome database encompassing many different species, which could serve as a valuable resource for researchers in plant biology in general, and in sexual plant reproduction in particular.
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Acknowledgements |
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This research was supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada to VKS, and by funding for mass spectrometry and proteomics equipment from the National Research Council of Canada.
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