Proteomics 2012, 12, 1983–1998 1983 DOI 10.1002/pmic.201100407 RESEARCH ARTICLE Comparative proteomic analysis reveals similar and distinct features of proteins in dry and wet stigmas Ya Lin Sang, Meng Xu, Fang Fang Ma, Hao Chen, Xiao Hui Xu, Xin-Qi Gao and Xian Sheng Zhang State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong, China Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2DE and MS to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in “signal transduction” and “lipid metabolism,” relative expression levels of proteins containing signal peptides and proteins in “defense and stress response.” These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level. Keywords: 2DE / Nicotiana tabacum / Plant proteomics / Stigma / Zea Mays Received: August 3, 2011 Revised: February 25, 2012 Accepted: March 21, 2012 1 Introduction Stigma is essential for pollination process and functions as a “pollen sieve” [1]. Pollens landing on the stigma commu- nicate intensively with its surface cells to perform the first interaction between male and female reproductive plant tis- sues [2]. For compatible pollens, the stigma facilitates pollen germination and polarized tube growth through it and into the style [3, 4]. According to the presence or absence of sur- Correspondence: Professor Xian Sheng Zhang, State Key Labora- tory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, China E-mail: [email protected]Fax: +86-538-8226399 Abbreviations: ABA, abscisic acid; ANTH, AP180 N-terminal homology; APG, anther-specific proline-rich protein; BAR, Bin/amphiphysin/Rvs; GA, gibberellin; WDS, water deficit stress face exudate, stigmas are generally classified into two cate- gories, wet and dry [5]. Wet stigmas secrete the liquid exudate containing proteins, lipids, carbohydrates, and water to their surface, which has been shown to be necessary and sufficient for pollen–stigma interactions during tobacco pollination [6]. Wet stigmas also have smooth but discontinuous cuticles [7]. In contrast, dry stigmas do not deposit exudate on their sur- face. The epidermal cells of a dry stigma protrude as papillae and are covered by an intact cuticle and a proteinaceous pel- licle [8]. Also, pollens that land on wet stigmas, regardless of their origin, can be trapped by the exudate. The subsequent pollen hydration that occurs is largely passive and indiscrimi- nate, demonstrating that the recognition between pollen and wet stigma occurs after pollen hydration [9]. However, only the adhesion and hydration of compatible pollens are allowed in dry stigmas, indicating an initial pollen–stigma recogni- tion process that precedes adhesion [10, 11]. The different characteristics between the wet and the dry stigmas suggest a divergence of the pollen–stigma recognition mechanisms, C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Comparative proteomic analysis reveals similar and
distinct features of proteins in dry and wet stigmas
Ya Lin Sang, Meng Xu, Fang Fang Ma, Hao Chen, Xiao Hui Xu, Xin-Qi Gaoand Xian Sheng Zhang
State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Life Sciences,Shandong Agricultural University, Taian, Shandong, China
Angiosperm stigma supports compatible pollen germination and tube growth, resulting infertilization and seed production. Stigmas are mainly divided into two types, dry and wet,according to the absence or presence of exudates on their surfaces. Here, we used 2DE andMS to identify proteins specifically and preferentially expressed in the stigmas of maize (ZeaMays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed fromthe surface of the tobacco stigma. We found that the specifically and preferentially expressedproteins in maize and tobacco stigmas share similar distributions in functional categories.However, these proteins showed important difference between dry and wet stigmas in a fewaspects, such as protein homology in “signal transduction” and “lipid metabolism,” relativeexpression levels of proteins containing signal peptides and proteins in “defense and stressresponse.” These different features might be related to the specific structures and functionsof dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed.Our results provide important information on functions of proteins in dry and wet stigmas andreveal aspects of conservation and divergence between dry and wet stigmas at the proteomiclevel.
Received: August 3, 2011Revised: February 25, 2012
Accepted: March 21, 2012
1 Introduction
Stigma is essential for pollination process and functions asa “pollen sieve” [1]. Pollens landing on the stigma commu-nicate intensively with its surface cells to perform the firstinteraction between male and female reproductive plant tis-sues [2]. For compatible pollens, the stigma facilitates pollengermination and polarized tube growth through it and intothe style [3, 4]. According to the presence or absence of sur-
Correspondence: Professor Xian Sheng Zhang, State Key Labora-tory of Crop Biology, Shandong Key Laboratory of Crop Biology,College of Life Sciences, Shandong Agricultural University, Taian,Shandong 271018, ChinaE-mail: [email protected]: +86-538-8226399
face exudate, stigmas are generally classified into two cate-gories, wet and dry [5]. Wet stigmas secrete the liquid exudatecontaining proteins, lipids, carbohydrates, and water to theirsurface, which has been shown to be necessary and sufficientfor pollen–stigma interactions during tobacco pollination [6].Wet stigmas also have smooth but discontinuous cuticles [7].In contrast, dry stigmas do not deposit exudate on their sur-face. The epidermal cells of a dry stigma protrude as papillaeand are covered by an intact cuticle and a proteinaceous pel-licle [8]. Also, pollens that land on wet stigmas, regardless oftheir origin, can be trapped by the exudate. The subsequentpollen hydration that occurs is largely passive and indiscrimi-nate, demonstrating that the recognition between pollen andwet stigma occurs after pollen hydration [9]. However, onlythe adhesion and hydration of compatible pollens are allowedin dry stigmas, indicating an initial pollen–stigma recogni-tion process that precedes adhesion [10, 11]. The differentcharacteristics between the wet and the dry stigmas suggesta divergence of the pollen–stigma recognition mechanisms,
1984 Y. L. Sang et al. Proteomics 2012, 12, 1983–1998
which might be related to the proteins expressed within thestigmas [12].
Despite the significance of the stigma for plant reproduc-tion, the factors involved in its biological functions remainlargely elusive, with the exception of self-incompatibility [1,2].Few stigma mutants that block pollen–stigma signal commu-nication have been identified to date, which may be a resultof signaling pathway redundancy [8]. Because of this, the in-vestigation of stigma functions using genetic strategies hasbeen severely restricted. Thus, comparative genomic analysismight provide valuable information.
Recently, specifically and preferentially expressed genes inthe dry stigmas of Arabidopsis (Arabidopsis thaliana) and rice(Oryza sativa) were identified using genome-wide Affymetrixarrays or cDNA subtraction strategies [13–15]. Moreover, arecent genome-scale cDNA library of the tobacco (Nicotianatabacum) stigma/style has provided an abundant database ofgenes expressed in the wet stigma [12]. These studies haveprovided a general overview of gene expression in both wetand dry stigmas and also revealed differential patterns of geneexpression between the two kinds of stigmas. However, thebiological characterization of both dry and wet stigmas at aproteomic level remains poor [1]. Because proteins are themain building blocks and executants of biological functionsin living organisms [16, 17], proteomic analysis is critical forfull elucidation of the biological functions of both dry and wetstigmas.
Maize is a dry stigma species that has been used as a modelfor the investigation of pollen tube growth and guidance be-cause of its morphological and genomic advantages [18]. To-bacco is a wet stigma plant species. Many pioneering works inexploring the mechanisms of wet-stigma function have beenperformed in this species [1,6,12,19,20]. In our current study,we have analyzed the proteins expressed in the dry stigmas(silk) of maize (Zea Mays) and the wet stigmas of tobaccoby 2DE and MALDI-TOF MS. These two groups of proteinsshowed similar and distinct features between dry and wetstigmas. Our results suggest that the molecular mechanismsunderlying the biological functions of dry and wet stigmasdiffer considerably.
2 Materials and methods
2.1 Plant materials and sampling
Maize (Z. mays L.) plants were grown under natural condi-tions during the maize growing season (April to August) inTaian (36�11′39′′N, 117�6′43′′E). Maize silks (stigmas) at 3days after silking with a length of approximately 5 cm outof the husk were harvested as mature stigmas, and ovarieswere harvested at the same stage. Tobacco (Nicotiana tabacumNC89) plants were grown in a greenhouse at 28�C/25�C. Stig-mas and ovaries at stage 12 were harvested [21]. All sampleswere immediately frozen in liquid nitrogen and stored at−70�C for protein extraction.
2.2 Preparation of proteins
Plant samples were fine powdered in liquid nitrogen andthen suspended in ice-cold 10% (w/v) trichloroacetic acid inacetone containing 0.07% (v/v) �-mercaptoethanol. After in-cubation at −20�C for 2 h, the pellet was collected by centrifu-gation at 18 000 × g for 20 min at 4�C, and then washed twicewith ice-cold 80% (v/v) acetone containing 0.07% (v/v) �-mercaptoethanol. After collection by centrifugation at 15 000× g for 20 min at 4�C, the pellet was vacuum dried. Subse-quently, the pelleted proteins were dissolved in lysis buffer(7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 1% [v/v] TritonX-100, 25 mM dithiothreitol, and 2% [v/v] pharmalyte 4–7) atroom temperature. After the debris was removed by centrifu-gation at 40 000 × g for 20 min, the proteins were cleanedup using a commercial clean-up kit (GE Healthcare, Uppsala,Sweden) following the manufacturer’s protocol and then dis-solved in lysis buffer described before. Finally, proteins werequantified by the Bradford method [22] using a GeneQuantpro UV/Vis spectrophotometer (Biochrom Ltd., Cambridge,UK). BSA was used as the standard. For protein preparationsfrom each sample, three biological replicates were generated.For one biological replicate, maize or tobacco stigmas wereharvested from a group of plants and combined. Stigma sam-ples used in three biological replicates were obtained fromthree different plant groups, respectively.
2.3 Rinsing of proteins from the surface of the
tobacco stigma
Tobacco pistils at stage 12 were excised from plants, and theirstigmas were submerged in the lysis buffer described abovefor 2 min, with gentle shaking. Approximately 1 mL of lysisbuffer was used to rinse 1000 stigmas, after which the eluatewas cleaned up and quantified, as described above. Tripli-cate biological preparations were made from each sample.Biological replicates were defined as described above.
2.4 2DE and imaging analysis
For each sample, 1.2 mg of protein was diluted in a rehy-dration buffer (8 M urea, 4% [w/v] CHAPS, 18 mM DTT,0.5% immobilized pH gradient [IPG] buffer 4–7, and 0.002%[w/v] bromophenol blue) to a final volume of 450 mL, andloaded onto a 24-cm pH 4–7 linear gradient IPG strip (GEHealthcare). Isoelectric focusing (IEF) was then performedusing an Ettan IPGphor system according to the manufac-turer’s instructions. Following IEF, the IPG strips were equi-librated in Eq buffer (50 mM Tris, pH 8.8, 6 M urea, 30%[v/v] glycerol, 2% [w/v] SDS, and 0.002% [w/v] bromophenolblue) containing 1% (w/v) DTT for 15 min and then equili-brated in Eq buffer containing 2.5% (w/v) iodoacetamide for15 min. For resolution in the second dimension, the equi-librated IPG strips were then transferred onto 12.5% SDS-PAGE gels using an Ettan DALT Six Electrophoresis Unit
(GE Healthcare). Low-molecular mass protein markers (GEHealthcare) were coelectrophoresed as standards (Mr). Gelswere stained with CBB G-250. All experiments were per-formed in triplicate using biological replicate separate sam-ples.
After visualization, images were obtained using an Im-ageScanner (GE Healthcare) at 300 dpi and 16-bit grayscalepixel depth, and then analyzed with ImageMaster 2D version5.0 software (GE Healthcare). The experimental Mr of eachprotein spot was determined by comparison with the coelec-trophoresed standard markers. The experimental isoelectricpoint (pI) of each protein spot was calculated using its mi-gration on the IPG strip. Spot detection, quantification, andbackground subtractions were preformed, and the relativevolume (RV) of every spot was obtained by the ratio of thevolume of each spot to that of all spots in one gel. After thegels were matched, spot groups that existed in at least twogels were selected for further analysis. Spots showing at leasta twofold increase in their RV in the stigma gels and with aStudent’s t-test p-value of <0.05 were considered to be pref-erentially expressed stigma proteins. Spots expressed in thestigma gels but not in the ovary gels were considered to bestigma-specific proteins.
2.5 Protein identification with MALDI-TOF/TOF MS
All selected protein spots were excised manually from the2DE gels, destained in 25 mM ammonium bicarbonate and50% (v/v) acetonitrile, and then dehydrated using acetoni-trile and spun dry. The dried gel spots were rehydrated in 25mM ammonium bicarbonate containing 10 ng/�L sequenc-ing grade modified trypsin (Roche, Mannheim, Germany)for enzyme digestions. After incubation for 16 h at 37�C, pep-tides were extracted by sonication. For MALDI-TOF MS, 1 �Lof the peptide extracts was spotted onto a sample plate withmatrix solution (8 mg/mL �-cyano-4-hydroxcinnamic acid in70% ACN, 0.1% TFA). MALDI-TOF MS were carried outon Ultraflex II MALDI TOF/TOF MS spectrometer (BrukerDaltonics, Bremen, Germany) using Flexanalysis 2.4 soft-ware (Bruker Daltonics). To ensure the accuracy of MALDI-TOF/TOF MS, standard peptide mixture (Bruker Daltonics)was used for external calibration, the deviations of externalcalibration peaks were no more than 10 ppm. Trypsin au-tolysis peptides (Roche, m/z observed: 805.4163, 1020.5030,1111.5605, 1433.7206, 2163.0564, 2273.1595) and keratins(m/z observed: 1179.6010, 1300.5302, 1716.8517, 1993.9767,1165.5853, 2825.4056) were used for internal calibration, andthe maximum deviation was limited in 10 ppm.
After the removal of peaks of trypsin autolysis peptidesand keratins as well as peaks with signal/noise (S/N) < 6,peptide mass finger printing results were transferred to theBioTools 3.0 software and searched against the National Cen-ter for Biotechnology Information (NCBInr) nonredundantprotein databases (http://www.ncbi.nlm.nih.gov/; NCBInr20111203; 16 392 747 sequences; 5 641 810 382 residues) with
the Mascot software 2.2.03 (http://www.matrixscience.com).Green plant (1 015 289 sequences in December 2011) was se-lected as the taxonomic category. All peptide masses were as-sumed to be monoisotopic and [M + H]+ (protonated molecu-lar ions). Searches involved a mass tolerance of 100 ppm, withone missing cleavage site was allowed. Carbamidomethyl (C)was set as a fixed modification whereas oxidation (M) andpyro-glu (N-termQ) were set as variable modifications. Toimprove the confidence of the identification, identified pro-teins must meet the following criteria: (i) probability-basedMOWSE p < 0.05, (ii) if peptides matched multiple homol-ogous proteins, the one with the highest score was selectedas the identified protein, (iii) the identified protein has tomatch more than 5 peptides that covered more than 10% ofthe protein sequences.
3 Results and discussion
3.1 Characteristics of proteins specifically or
preferentially expressed in the stigma versus the
ovary in maize
In the pistil, the stigma rather than the ovary functions pri-marily to receive and then discriminate pollens. Hence, pro-teins specifically or preferentially expressed in the stigmaversus the ovary are likely to confer specialized functionswith respect to interaction between pollen and stigma [15]. Toidentify these proteins in the stigma of maize, we comparedthe protein expression profiles between the stigma and ovaryof maize plants at anthesis using 2DE. Based on the data ob-tained from three biological replicates, we found that 42 spotsspecifically expressed and 60 spots preferentially expressed inthe stigma (Fig. 1A and Supporting Information Fig. S1A).Thus, we identified a total of 102 stigma-specific/preferentialspots in comparison with the ovary. Ninety-four of theseprotein spots could be successfully detected using MALDI-TOF MS. After excluding four spots containing two proteinseach (Supporting Information Table S7), we identified 90stigma-specific/preferential proteins representing 67 uniqueproteins (Supporting Information Fig. S2 and Table S1).
The 90 identified stigma-specific/preferential proteinswere classified into 14 groups according to their anno-tated functions (Fig. 1B). Of them, 71 proteins (78.02%) be-longed to the six most abundant categories including defenseand stress response, carbohydrate and energy metabolism,protein metabolism and folding, cell wall remodeling andmetabolism, signal transduction, and photosynthesis (Fig.1B). We also analyzed the relative abundance of each categoryamong our sample cohort by summing the relative volumevalues (RV values) of each spot. Proteins within the six mostabundant categories represented a large proportion of eitherthe spot number or relative abundance (Fig. 1B and C). Inaddition, the 20 most abundant unique proteins were listedaccording to their RV volume (Supporting Information Fig.S3A).
1986 Y. L. Sang et al. Proteomics 2012, 12, 1983–1998
Figure 1. Proteomic analysis of maizestigma-specific/preferential proteins at an-thesis. (A) Representative 2DE image ofmature maize stigma at anthesis. Arrowsindicate spots specifically or preferentiallyexpressed in the maize stigma versusovary. Ms, stigma specifically expressedspots; Mp, stigma preferentially expressedspots. Proteins were separated by 2DE-PAGE and stained with Coomassie Bril-liant Blue. The relative molecular mass(Mr) in kilodaltons and isoelectric point (pI)of the proteins are shown on the left andtop of each image, respectively. (B) Func-tional classification of 94 identified maizestigma-specific/preferentially proteins. (C)Relative abundance of each functional cat-egory shown in (B) (ratio of the relativeabundance of each category to the totalabundance of all identified proteins). Theabundance of each category was calcu-lated as the summation of the relative ex-pression level of all identified factors.
Several recent proteomic studies showed that some plantproteins had isoforms, which may result from distinct en-coding genes, alternative splicing of transcripts, or PTM[23–27]. In our results, 29 of the identified spots represented10 stigma-specific/preferential proteins with isoforms (Sup-porting Information Fig. S4A). Five proteins with differentisoforms belong to 20 most abundant unique ones (Support-ing Information Figs. S3A and S4A). Two members of thisgroup were assigned as an anther-specific proline-rich pro-tein (APG) and secretory peroxidase, respectively. Both APGand secretory peroxidase were predicted to contain signalpeptides by SignalP program analysis (SignalP 3.0 server,http://www.cbs.dtu.dk/services/SignalP/; Table 1) [28,29]. Itwas thought that APG protein was a secretory esterase/lipase.Esterase activity was observed to be localized at the regionsof contact between pollen and stigma during pollination inBrassica, implying these enzymes were involved in pollen–stigma interactions [30]. The activity of surface peroxidasesand esterases were used to determine the stigma receptivityfor pollens because these proteins showed the highest activitywhen the stigma is ready for pollination [31]. The cuticle of adry stigma is covered with a thin pellicle containing proteinsessential for compatible pollen–stigma interactions [9]. Con-sidering their high expression levels and the containing of
signal peptides, we hypothesize that APG and secretory per-oxidase may be important components of the maize stigmapellicle.
Other six proteins were also predicted to contain sig-nal peptides and may be the components of the pellicle.Five of which were found to be cell wall-related (Table 1).Among them, beta-expansins have been reported to func-tion in cell wall loosening and play roles in pollen tubepenetration into the stigma/style [32]. Alpha-galactosidase,beta-galactosidase, alpha-L-fucosidase have also been shownto function in cell wall loosening and expansion [33–37].These proteins may either facilitate the penetration of thepollen tube into the stigma, or direct its polar elongationalong the extracellular matrix within the stigma/style tis-sue since reorganization of the cell wall is required for bothprocesses. Additionally, a polygalacturonase inhibiting pro-tein was predicted to be a secreted protein (Table 1). Re-cent studies showed that plant polygalacturonase-inhibitingproteins can interact with polygalacturonase in both plantsand fungi in vivo and inhibit their activity [38, 39]. Becausepolygalacturonase is an abundantly expressed excreted en-zyme in maize pollen [40], it is possible that pollen poly-galacturonase interacts with the polygalacturonase inhibitorsecreted by the stigma at the cell surface, and that this
interaction may be a recognition signal between the pollenand the stigma.
The involvement of phytohormones in the pollination pro-cess has been widely studied [41–43]. For instance, gibberellin
(GA) is required for pollen tube growth both in vivo and invitro [44]. Recent study showed the sharp increase in the GAlevels in pollen tubes soon after germination is closely relatedto the pollen–pistil interactions in Pyrus pyrifolia [45]. In ourcurrent analyses, a GA receptor (Mp41, Supporting Informa-tion Table S1) was identified to be preferentially expressedin the maize stigma. We speculate therefore that this recep-tor may receive GA signals from elongating pollen tubes andthus may play a role in the penetration of these tubes intothe pistil tissues. Additionally, we identified a stigma prefer-entially expressed ethylene receptor (Mp54, Supporting In-formation Table S1). Because ethylene promotes early pollentube growth in the petunia (Petunia inflata) pistil, and initi-ates perianth senescence, pigmentation changes, and ovuledifferentiation in the orchid [46,47], we propose that the ethy-lene signal transduction mediated by this receptor participatein either the pollen tube growth or floral organ development.
3.2 Characteristics of specifically/preferentially
expressed proteins in the tobacco stigma
To characterize proteins specifically or preferentially ex-pressed in the wet stigma of the tobacco, we compared theprotein expression profile of the stigma with that of theovary at stage 12 (Fig. 2A and Supporting Information
Figure 2. Proteomic analysis of tobaccostigma-specific/preferential proteins at an-thesis. (A) Representative 2DE image oftobacco stigma at stage 12. Arrows indi-cate spots that are specifically or prefer-entially expressed in the tobacco stigmaversus the ovary. Ts, stigma specificallyexpressed spots; Tp, stigma preferentiallyexpressed spots. Proteins were separatedby 2DE PAGE and stained with CoomassieBrilliant Blue. Molecular mass (Mr in kilo-daltons) and the isoelectric point (pI) ofthe proteins are shown on the left andtop of each image, respectively. (B) Func-tional categorization of 71 tobacco stigma-specific/preferential proteins. (C) Relativeabundance of each functional categoryshown in (B).
1988 Y. L. Sang et al. Proteomics 2012, 12, 1983–1998
Figure 3. Proteomic identification of pro-teins rinsed from the tobacco stigma.(A) Representative 2DE image of pro-teins rinsed from the tobacco stigma.Arrows indicate putative secreted orplasma membrane-localized proteins. Pro-teins were separated by 2DE PAGE andstained with Coomassie Brilliant Blue. Themolecular mass (Mr in kilodaltons) and iso-electric point (pI) of the proteins are shownon the left and top of each image, respec-tively. (B) Functional categorization of theproteins rinsed from the tobacco stigma.(C) Relative abundance of each functionalcategory shown in (B).
Fig. S1B). Seventy-one proteins were identified from69 stigma-specific/preferential spots. Two spots contain-ing two proteins each were excluded from stigma-specific/preferential proteins (Supporting Information Ta-ble S7). Thus, we identified 67 stigma-specific/preferentialproteins representing 47 unique proteins (Supporting In-formation Fig. S2 and Table S2). These 67 identities werethen classified into 12 functional categories (Fig. 2B). Sim-ilar to the situation in the maize stigma, the spot numberand expression levels of proteins in six of these categoriesaccounted for 83.58% and 88.02% of the total, respectively.These functional groups included photosynthesis, defenseand stress response, carbohydrate and energy metabolism,protein metabolism and folding, lipid metabolism, and signaltransduction, suggesting the importance of these processesin the tobacco stigma (Fig. 2B and C).
The 20 most abundant proteins in our sample cohort, andproteins with isoforms, were listed in Supporting Informa-tion Figs. S3B and S4B. Glucan endo-1,3-beta-glucosidase andtriosephosphate isomerase have the most isoforms (Support-ing Information Fig. S4B). Glucan endo-1,3-beta-glucosidaseand thaumatin-like protein SE39b were previously reported tobe present in the stigma exudate [48]. Glucan endo-1,3-beta-
glucosidase constitutes a major component of proteins inthe tobacco transmitting tract and belongs to a pathogenesis-related protein superfamily. The maximal accumulation ofthis protein during anthesis implies its function in the re-productive process [49]. In contrast, the biological function ofthaumatin-like protein SE39b has not been reported to dateas we know. In addition, a lipid transfer protein (LTP) wasidentified (Supporting Information Table S2). Recent studyshowed an LTP in the tobacco stigma to facilitate cell wallextension, suggesting its roles in facilitating either the pollentube growth or the wall-loosening of the cells in secretoryzone [20].
3.3 Identification of proteins rinsed from the
tobacco stigma
The absence or presence of a secreted exudate is the majordifference between dry and wet stigmas [5]. Moreover, theimportance of the stigma exudate for pollen–stigma interac-tions in wet stigmas has been well demonstrated [6]. Here,we analyzed the proteins that could be rinsed from the to-bacco stigma surface with lysis buffer using 2DE (Fig. 3A).
Three independent biological replicates were performed andspots that appeared on at least two gels were further used forMALDI-TOF MS analysis. A total of 177 proteins were identi-fied from 172 spots, 167 spots contained a single protein each,and 5 spots contained two proteins each (Supporting Infor-mation Fig. S2 and Table S3). These 177 proteins represented132 unique proteins and fell into 14 functional categories. Theproteins involved in “defense and stress response” and “cellwall remodeling and metabolism” accounted for 40.62% and26.26% of the total cohort, respectively (Fig. 3B and C). Thesignificant abundance of these proteins suggested that theirfunctions are positively involved in the biological processesthat take place on the tobacco stigma. However, althoughlipids have been shown to be important components of thestigma exudate and are critical for the pollen–stigma interac-tions [19], no proteins participating in lipid metabolism wereidentified in the eluate in this analysis (Supporting Informa-tion Table S3).
As illustrated in Figs. 3A and 2A, the 2DE map of the pro-teins eluted from the stigma surface differed greatly fromthat of the tobacco stigma proteome. Furthermore, longitu-dinal sections showed that only the outermost cells of thetobacco stigma were damaged by the rinse with lysis buffer(Supporting Information Fig. S5). We concluded thereforethat proteins in the eluate originated from the exudate and
the outermost layers of the stigma cells. The three mostabundant proteins (glucan endo-1,3-beta-glucosidase, beta-expansin-like protein, thaumatin-like protein SE39b) repre-senting 46.44% of the relative expression levels have beenfound in the tobacco stigma exudate, highlighting the factthat the exudate proteins were enriched (Supporting Infor-mation Fig. S3C).
Through a combination of SignalP program analysis withpublished results, we identified 30 proteins containing sig-nal peptides and/or locating at the plasma membrane amongthe eluted identities (Table 2) [50–60]. These proteins wererepresented by 63 spots and accounted for 35.6% of the to-tal rinsed protein content. Among the proteins in this list,glucan endo-1,3-beta-glucosidase, beta-expansin-like protein,thaumatin-like protein SE39b, and acidic chitinase PR-P havebeen shown previously to be present in the tobacco stigmaexudate [48]. Besides them, we identified some proteins in-cluding squalene monooxygenase, several cell wall-relatedproteins, proteases, and unknown function proteins (Table2). Squalene monooxygenase catalyzes the rate-limiting stepduring the biosynthesis of sterol, an important componentof lipid rafts [61]. Many studies have indicated that lipid raftsfunction on a variety of cellular processes, such as signaltransduction, stress responses, and membrane trafficking[62]. Thus, it is likely that this enzyme participates in thesynthesis of plasma membrane lipid rafts of the outermostcells and is important for signal transduction as well as de-fense activity at the surface of the tobacco stigma. Previousstudies showed that alpha-galactosidase, alpha-mannosidase,cell wall invertase INV5, and cellulase function on cell wallloosening and expansion [33, 58, 63, 64]. These four proteinswere detectable in the eluate of the tobacco stigma, sug-gesting that they may facilitate pollen tube growth throughtheir cell wall loosening activity (Table 2). 1,3-beta-glucan syn-thase (Table 2) is responsible for the biosynthesis of callosethat accumulates in the plant cell in response to incompati-ble pollination and stress stimuli such as a pathogen attack[65–67].
Some emerging evidences have revealed the importanceof vesicular transport during pollen–pistil interactions [68].A group of 18 vesicle trafficking-related proteins was identi-fied from the stigma surface eluate (Table 3). Among them,Rab2, SEC8, and Exo70 have been shown to be involved inpollen tube growth or pollen–stigma interactions. Mutationof Rab2 in tobacco causes a block in the delivery of cellmembrane and in the secretion of proteins, and manifestinhibited pollen tube growth [69]. SEC8 facilitates pollen ger-mination and competitive pollen tube growth in Arabidop-sis [51]. Exo70A1 is a subunit of the exocyst complex Exo70and is important for targeted secretion at the plasma mem-brane. Loss of Exo70A1 results in the rejection of compatiblepollen in both Arabidopsis and Brassica stigmas, whereasthe increased expression of Exo70A1 partially overcomesthe self-incompatibility in Brassica, indicating the direct in-volvement of vesicle transport in pollen–stigma interactions[56, 70, 71].
Endocytosis15 Putative sorting nexin 145 ANTH domain family protein124 Putative clathrin-adaptor medium
chain apm 4177 Bin/Amphiphysin/Rvs (BAR) domain
Vesicle trafficking18.2 KEULE Sec1 family192 Vesicle tethering family protein
Motor protein49 Dynein-1-beta heavy chain63 Kinesin-II motor protein, flagellar
associated131 Chromosome segregation protein,
kinesin motor domain147 Kinesin motor domain172 Kinesin motor domain
We identified four proteins including sorting nexin1, AP180 N-terminal homology (ANTH) domain familyprotein, putative clathrin-adaptor medium chain 4, andBin/amphiphysin/Rvs (BAR) domain proteins that are in-volved in endocytosis (Table 3). Sorting nexin 1 regulates theendosomal sorting of internalized receptors in mammals [72].In Arabidopsis, this protein participates in the auxin signal-ing pathway through the endocytic sorting of PIN-FORMED2 [73]. The function of sorting nexin 1 in organizing internal-ized receptors indicates its potential involvement in signaltransduction regulation at the plasma membrane of the out-ermost cells in the tobacco stigma, which may be relatedto the interaction of the stigma with pollen. ANTH domainfamily protein and putative clathrin-adaptor medium chain 4were previously annotated to be involved in clathrin-mediatedendocytosis [53]. Protein containing BAR domain was foundto sense the curvature of the membrane during endocytosis[74].
We also identified seven proteins involved in exocyto-sis, such as SEC14, EMP24/GP25L domain-containing pro-tein, the small G protein SAR1, etc. (Table 3). SEC14 inArabidopsis and yeast stimulates phosphatidylinositol 4,5-bisphosphate synthesis and modulates its homoeostasis at theplasma membrane, suggesting that this protein may functionin signal transduction pathways at the cell surface [54,75,76].Indeed, mutations in AtSFH1, that is, the SEC14 PITP ho-molog in Arabidopsis, disturbs vesicle trafficking, Ca2+ sig-
naling, and cytoskeleton functions, resulting in a restrainedpolarized root hair expansion [54]. Since root hair growthand pollen tube elongation share similar mechanism, it islikely that SEC14 also functions in the pollen tube growth instigma tissues [77]. The EMP24/GP25L domain-containingprotein and the small G protein SAR1 have been shown to benecessary components of coated vesicles and are responsiblefor the sorting of secretory proteins into membrane vesicles[78, 79]. Expression of ER43, a homolog of SAR1 in tomato,was found to be down-regulated after the application of ethy-lene [55, 80], suggesting the vesicular trafficking in tobaccostigma is mediated by ethylene.
The presence of vesicle transport-related proteins and mo-tor proteins indicates an active and highly regulated vesiculartrafficking system within the outermost cells of the tobaccostigma (Table 3). This further suggests that the fine modu-lation of vesicle transport plays important roles in pollen–stigma interactions. After landing on the stigma surface,recognition molecules released from pollen grains may beinternalized into stigma cells through endocytosis at the out-ermost cells. Alternatively, the resources necessary for pollengermination and tube growth, such as proteins, nutrients,and other factors, have been shown to be transported to thecontract points of compatible pollen at the stigma cell surface[68].
Tobacco is a typical self-compatible species, and it wastherefore unexpected to identify locus S-RNase S15 that is re-lated to self-incompatiblility (Supporting Information TableS3) [81, 82]. Sorting nexin 1 has been shown to interact withS-locus receptor kinase, which acts as the female element ofthe self-incompatibility response, in Brassica oleracea (Sup-porting Information Table S3) [81]. We thus speculate that,in a similar manner to Arabidopsis, the tobacco plant hasevolved from a self-incompatible ancestor and has retainedseveral proteins involved in self-incompatible signal trans-duction [83]. However, whether these proteins possess newfunctions is largely unknown.
3.4 The stigma-specific/preferential proteins in
maize and tobacco share similar distributions in
functional categories
Maize and tobacco plants used in this study were grown un-der their normal growth conditions, with efficient fertilizationand seed production. Stigma samples from both maize and to-bacco were harvested at the stage with the highest receptivityfor pollen grains [84–86]. Thus, we suggest that a comparisonbetween the stigma-specific/preferential proteins from maizeand tobacco under these circumstances could reveal commonor different features between the biological characteristics ofthe two kinds of stigmas. For this purpose, we comparedthe functional categories of the stigma-specific/preferentialproteins between maize and tobacco. The spot numberprofiles for the functional categories of these two sets ofproteins were compared using a histogram (Fig. 4A). The
Figure 4. Comparison of the functionaldistribution of maize and tobacco stigma-specifically/preferentially expressed pro-teins. (A) Comparison of the proportionof each functional category to the totalcohort between maize and tobacco stigma-specifically/preferentially expressed pro-teins. (B) Comparison of the relative abun-dance of different functional categories ofproteins between maize and tobacco.
relative expression levels of the proteins in each of these func-tional categories were also compared in the same way (Fig.4B). As shown in Fig. 4, the proportions and abundance ofstigma proteins in each functional category were comparablebetween dry and wet stigmas, indicating that the processesactivated by proteins specifically or preferentially expressedin maize and tobacco stigmas are common at a broad level.The high similarity in terms of the functional protein pro-file is consistent with the fact that these organs have verysimilar roles, such as discrimination of pollen grains, guid-ance of pollen tube growth, and defense against abiotic/bioticdamage.
The same functional categories of stigma-specific/preferential proteins in maize and tobacco were also foundto contain the highest numbers of protein spots. Notably,the categories include “defense and stress response,” “carbo-hydrate and energy metabolism,” and “protein metabolismand folding,” highlighting the importance of these processesin both dry and wet stigmas (Fig. 4A and B). Recent tran-scriptomic research in rice and tobacco suggested that thestress/defense and pollination response pathways shared ex-tensive gene sets [12, 87]. Consistent with this, a number ofdefense-related proteins identified from either maize or to-bacco in our current analysis may respond to pollination and
play key roles in the interactions between pollen grains andthe stigma.
3.5 The stigma-specific/preferential proteins exhibit
different features between maize and tobacco
Although with similar functional categories, the spe-cific/preferential proteins of dry and wet stigmas showedpoor conservation and distinct features. Sequence compar-ison analysis revealed that there are only 11 homologous pro-teins (18.03%), represented by 16 spots in maize and 19 spotsin tobacco (p-value < 2e-56) (Table 4). Except a few categoriesinvolved in basic metabolisms, the proteins in other cate-gories were found to be highly diverse. For example, in thecategories of “signal transduction” and “lipid metabolism,”no proteins with significant homology were found betweenthese two groups. This is consistent with the idea that thegenes involved in regulating sexual reproduction are fasterevolving, and that genes maintaining species boundaries arespecies-specific, and therefore highly divergent [10]. Thesedifferences may originate from the distinct nature of wet anddry stigmas, and the evolutionary divergence between Poaceaeand Solanaceae. However, the poor conservation of enriched
1992 Y. L. Sang et al. Proteomics 2012, 12, 1983–1998
Table 4. Homologues between maize and tobacco stigma-specific/preferential proteins
Spots number Spots number Protein name Categoryin maize in tobacco
Mp2/Mp23/Ms32/Ms58 Tp8 Oxygen-evolving enhancer protein 1 PhotosynthesisMp14 Ts6/Ts23 Phosphoribulokinase PhotosynthesisMs47 Tp44/Ts7/Ts11 23kDa polypeptide of photosystem II PhotosynthesisMs38 Tp2/Tp19/Tp23/Tp34 Triosephosphate isomerase Carbohydrate and energy metabolismMp55 Ts27 Putative ATP synthase subunit beta Carbohydrate and energy metabolismMs56 Ts2 Fructose-1,6-bisphosphate Carbohydrate and energy metabolismMp57/Mp58 Ts17 Malate dehydrogenase Carbohydrate and energy metabolismMp33 Tp33/Ts19/Ts20 Beta-expansin Cell wall remodeling and metabolismMp48 Ts4 Cinnamyl alcohol dehydrogenase Cell wall remodeling and metabolismMp36 Ts12 Harpin binding protein 1 Defense and stress responseMp65/Mp66 Ts29 Molecular chaperone Protein metabolism and folding
proteins from dry and wet stigmas does indicate a divergenceof functional mechanisms between the two types of stigmas.
The specifically or preferentially expressed proteins inmaize and tobacco stigmas exhibit different features thatmight be related to the specific structures and functions ofdry and wet stigmas, respectively. For instance, the relativeexpression level of proteins containing signal peptides tooka much larger proportion in tobacco (37.38%) than that inmaize (12.53%). This might partially result from the pres-ence of the exudate on tobacco stigma, because some pu-tative secretory proteins are important components of thestigma exudate. In addition, five proteins involved in cell wallremodeling and metabolism were identified out of the 8 pu-tative secretory proteins in maize, while only two were foundin that of tobacco (Table 1 and Supporting Information TableS4). The well represented cell wall-related proteins in maizecorresponded to the finding that pollen tubes grow throughthe papillar cell wall in dry stigmas. But in wet stigma specieswith solid style, such as tobacco, germinated pollen tubeselongate in the intercellular spaces of the stigma secretoryzone [30]. Thus, the putative secretory proteins involved incell wall remodeling and metabolism seem to be importantfor the pollen tube growth through the papillar cell wall indry stigmas [66].
Furthermore, in the category “defense and stress re-sponse,” four proteins involved in the response to droughtstress represented by five 2DE spots were identified in maize(Supporting Information Table S1). Of them, two proteinsbelonging to the ABA/WDS family can be induced by waterdeficit stress (WDS), abscisic acid (ABA), or ripening [88]. Theplastid–lipid-associated proteins in this group were found tobe accumulated in response to water deficits in various plantspecies [89]. In Arabidopsis, the genes encoding arginine de-carboxylase are induced under drought stress. Furthermore,over-expressed arginine decarboxylase 2 gene enhances thedrought tolerance of transgenic Arabidopsis through the pro-motion of putrescine production [90]. However, no proteinswith similar functions were found in the tobacco stigma inour analysis. The pollen grain is a significantly dehydratedmicrospore [91], and the rehydration process during polli-
nation may induce a WDS response in the dry stigma. Incontrast, this would not occur in the wet stigma because it iscovered by an aqueous exudate. Consequently, some droughtresponse proteins were found to be specifically or preferen-tially expressed in the dry rather than the wet stigma, as areaction to the osmotic adjustment that occurs during pollenhydration and the germination of pollen grains [87].
The presence of a nutritious exudate makes the wet stigmaprone to pathogen attack. As a result, to ensure success-ful pollination, certain proteins may be expressed to protectthe wet stigmas from pathogens. This hypothesis was sup-ported by our data showing that the proportion of disease-resistance proteins was much higher in tobacco than inmaize (Supporting Information Tables S1 and S2). Elevenidentified factors were found to be disease resistance-relatedproteins in tobacco, which represented 85% of the “de-fense and stress response” category (Supporting InformationTable S2).
Anthocyanin biosynthesis-related proteins accounting fora large proportion of the defense- and stress response-relatedproteins in the maize stigmas were not found in tobacco(Supporting Information Tables S1 and S2). Different fromthat of tobacco, maize stigmas bear bright colors, whichmight be a result of anthocyanin accumulation. Whetherthe differential expression of these anthocyanin biosynthesis-related proteins between maize and tobacco stigmas leads todifferences in pollination mechanisms remains unknown.Chalcone-flavanone isomerase and chalcone synthase are in-volved in the biosynthesis of flavonol, and it was found thatmaize mutants with a disrupted flavonol biosynthesis mani-fest a failure of pollen tube development, resulting in sterility[92]. However, the application of flavonol during pollinationin these mutants is sufficient to restore fertility, indicatingthat the flavonols control maize reproduction by regulatingpollen tube navigation [92–94]. Nevertheless, flavonol agly-cones (a functional component of flavonols) are undetectablein petunia stigma unless pollination or wounding occurs[94]. Thus, we proposed that flavonols may function at dif-ferent stages between dry and wet stigmas during pollinationprocess.
Possibly because tobacco has a secretory stigma, all of theidentified factors related to lipid metabolism among our panelof tobacco stigma-specific/preferential proteins were found tobe involved in fatty acid biosynthesis, which is quite differentfrom the situation in maize (Supporting Information TablesS1 and S2). It is thus possible that proteins with other func-tions in lipid metabolism are also specifically or preferentiallyexpressed in the tobacco stigma. However, we failed to detectsuch factors due to a sensitivity limitation of 2DE. Neverthe-less, the presence of these fatty acid biosynthesis-related pro-teins in our panel at high abundance may reflect the synthesisof specific lipids that comprise the stigma surface exudate.
Another important difference revealed by our data was thatmany proteins related to vesicle trafficking were identifiedfrom the eluate of the tobacco stigma (Supporting Informa-tion Tables S1, S2, and S3). Our results indicate that vesicletrafficking is an active process in the tobacco stigma, consis-tent with the features of a secretory stigma [12].
3.6 Comparisons between transcriptomic and
proteomic data reveal that posttranscriptional
and posttranslational regulation occur in both
maize and tobacco stigmas
To investigate the relationship between stigma-enriched pro-teins and their corresponding coding genes, the stigma-specific/preferential proteins we identified in maize andtobacco were compared with previously reported transcrip-tomic data [12–14]. The stigma proteins in our panel werecompared with maize stigma-enriched ESTs obtained byan Affymetrix maize genome array, which were submit-ted to ArrayExpress (http://www.ebi.ac.uk/arrayexpress; ac-cession number E-MEXP-3292). We blasted the sequencesof these maize stigma-enriched proteins against a maizecoding sequence (CDS) database from phytozome v 6.0(http://www.phytozome.net/maize.php). The best three CDShits for each stigma-enriched protein were further selectedusing E-values < 1e-35 (Supporting Information Table S5).These sequences were then blasted against stigma-enrichedESTs (alignment score > 90%, sequence length > 100 bp). Foreach unique protein, only the best hit was selected. Finally,31 proteins (46.27%) represented by 45 spots showed cor-responding sequences in the CDS database. These proteinsmainly fell into the functional categories “defense and stressresponse,” “cell wall remodeling and metabolism,” “signaltransduction,” and basic metabolism proteins (Table 5). Theproteins involved in anthocyanin biosynthesis were well rep-resented (Table 5). The correlation between the mRNA andprotein levels of these 31 factors indicated a steady expres-sion in the mature stigma at both the transcriptional andtranslational level, implying a continuous supply of theseproducts in the maize stigma. For instance, the presence ofthe peroxidase and APG proteins described above may reflectthe receptive state of the stigma to receive pollen grains. TheDREPP plasma membrane polypeptide and Jacalin-like lectin
domain containing protein were previously hypothesized tobe involved in inter- or intracellular signal transduction in thestigma [95, 96].
The low correlation (46.27%) between specifically or pref-erentially expressed proteins and enriched ESTs in maizestigma pointed to an extensive activity of posttranscriptionaland/or posttranslational regulatory mechanisms. It has beenfound in this regard that the relationship between the mRNAlevel and protein abundance is determined by the synthe-sis and degradation of both mRNA and protein. Posttran-scriptional and posttranslational regulation during these pro-cesses thus plays important roles in modulating gene expres-sion [97]. We speculated that some mRNAs in the stigmawere transcribed but not translated until the stimulation ofexogenous signals, which may be caused by pollen grainslanding on the stigma surface, or by environmental stressessuch as pathogen attack. Alternatively, other mRNAs maybe degraded through the posttranscriptional regulation sys-tem. Moreover, PTM of certain proteins may lead to theirdegradation depending on the demands of particular stigmafunctions, leaving a relatively higher level of mRNA. As aresult, the abundance of a protein was not always tightlylinked to its corresponding mRNA level. Thus, the pro-teomic data we obtained for exclusively expressed stigma pro-teins showed discrepancies with respect to the transcriptome.Moreover, correlation variations were observed between thedifferent functional categories. For instance, proteins in the“defense and stress response,” “lipid metabolism,” and “sig-nal transduction” categories correlated only moderately tothe transcriptomic data (64.3%, 66.7%, and 50.0%, respec-tively; Table 5, Supporting Information Table S1). In con-trast, the factors were assigned to the “cell wall remodel-ing and metabolism” and “protein folding and metabolism”category showed a poor correlation between their transcrip-tional and translational levels (37.5% and 22.2%; Table 5,Supporting Information Table S1) suggesting that the expres-sion of distinct functional categories of genes is differentiallyregulated
In the case of tobacco stigma-enriched proteins, we com-pared our results with previously reported transcriptomic datafor the tobacco stigma/style [12]. Our analysis showed that thegenes encoding stigma-enriched proteins were absent fromthe earlier transcriptomic data. This may be a consequenceof small data sets in both cases. However, even the com-parison of tobacco stigma enriched proteins identified herewith stigma-specific/preferential genes isolated previously inArabidopsis revealed some commonality (Supporting Infor-mation Table S6). Thus, it is possible that posttranscriptionaland posttranslational regulation is more active in wet stigmasthan in dry ones.
4 Concluding remarks
We reported here the identification of 67 and 47 proteinsspecifically or preferentially expressed in maize and tobacco
Mp53 gi|45567358 GRMZM2G110504_T01 99 498 Hypothetical protein Function unknown
stigmas versus their ovaries, respectively. The distinct fea-tures of these proteins corresponded well with the differingstructure of the dry or wet stigma. Furthermore, the analysisof proteins rinsed from the exudate of the tobacco stigmarevealed proteins correlated with the typical characteristics ofsecretory stigmas, especially those involved in vesicle trans-port. These findings suggested the both common and distinctmolecular mechanisms underlie the biological processes thatfunction in dry and wet stigmas, and provided a better un-derstanding of the biological mechanisms of two kinds ofstigmas. Moreover, some proteins identified in our analysisare candidates for pollination-related factors in either the dryor wet stigma. These results gave us a substantial clue to in-vestigate the pollen–stigma interaction on the female side.Genetic and biochemical analysis for revealing roles of thesecandidate proteins in the pollination process will be our fu-ture focus.
We thank Dr. Sharman D. O’Neill for her critical reading forthe manuscript (Section of Plant Biology, University of California,Davis, CA 95616, USA). This study is funded by the MajorResearch Plan from the Ministry of Science and Technology ofChina (No. 2007CB947600).
The authors have declared no conflict of interest.
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