Figures (6)  Tables (2)

    • Figure 1. 

      Collection of pollen mother cells using laser-capture microdissection from cross-sections of frozen flower buds of tomato 'Maxifort'. (a) Transference of sectioned tissues onto a pre-coated adhesive slide; (b) flower bud cross-sections visualized under Zeiss PALM system; (c) images of DAPI stained PMCs showing the blue florescent nuclei.

    • Figure 2. 

      Correlation between flower size and male gametophyte development stages in tomato 'Maxifort'. Frozen cross section of tomato 'Maxifort' flower buds were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were taken under an upright Zeiss imager M2 with EC PlnN 40X objective and a BP335-383 excitation DAPI filter. The inflorescence of 'Maxifort' flower were labeled 1−11. Nine distinct developmental stages were identified including: 1) sporogenous cells (SC); 2) pollen mother cells (PMCs); 3) meiosis I to dyads; 4) meiosis II to tetrads; 5) tetrads to early microspore; 6) microspore (MSP); 7) polarized MSPs; 8) mitosis I; 9) mitosis II. Zoom-in image of PMCs, meiosis I, meiosis II and tetrads with merged bright field and DAPI channel are displayed to show pollen cell structure.

    • Figure 3. 

      Protein distribution in the quantified proteomes from pollen mother cells of tomato 'Maxifort'. (a) Protein distribution based on the number of unique peptides assigned to each protein; (b) protein distribution based on the fold change from HS to N-HS conditions. HS-regulated proteins: the number and fold distribution of proteins showing significant differences in abundance level from HS-treated to non-HS-treated conditions; Pollen mother cell proteome: the quantified proteome with two or more peptides assigned to the identified protein from the HS-treated and non-HS-treated groups.

    • Figure 4. 

      Enrichment gene ontology (GO) analysis of heat stress regulated DEPs (HS-DEPs) in pollen mother cells (PMCs), which include 158 DEPs-down-regulated protein and 96 DEPs-up-regulated proteins. (a) GO terms of molecular functions; (b) GO terms of biological processes. Positive value indicates HS-up-regulated DEPs and negative value indicates HS-down-regulated DEPs. The classification analysis was conducted using multivariant test at FDR adjusted P ≤ 0.05 in Plant MetGenMap (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi).

    • Figure 5. 

      Pie chart of functional groups enriched with DEPs using Plant MetGenMap. Some proteins were placed in more than one functional group. GO terms are: Nuclease activity (GO:0004518); Protein binding (GO:0005515); Translation factor activity (GO:0008135); Enzyme regulator activity (GO:0030234); Transcription regulator activity (GO:0030528); Hydrolase activity (GO:0016787); Receptor binding (GO:0005102); Transferase activity (GO:0016740); Transcription factor activity (GO:0003700); Signal transducer activity (GO:0004871); Kinase activity (GO:0016301).

    • Figure 6. 

      Protein interaction networks of proteins showing significant change from heat stress (HS) to non-heat stress (N-HS) conditions in pollen mother cells from tomato 'Maxifort'. The network was constructed based on functional and physical interactions at medium confidence (0.400) level in STRING (https://string-db.org). String clusters, protein accession, and fold change are listed in Table 2. The image was generated using Cystoscope associated with the STRING database. The red color nodes are HS-up-regulated proteins, and the blue color nodes are HS-down-regulated proteins. The color depth corresponds to the protein Fold (HS/N-HS) values.

    • Flower size (stamen length mm)Pollen developmental stageNote
      Flower size 1−6 (3−6 mm)Sporogenous cells (SCs), Pollen mother cells (PMCs), Meiosis I to dyads, Meiosis II to tetrads, Tetrads to early microsporesMicrosporogenesis: PMCs undergo meiosis I and II to produce haploid tetrad.
      Flower size 7−8 (7−9 mm)Microspores (MSPs), Polarized MSPsUnicellular microspores release, and polarization.
      Flower size 9−11 (9−12 mm)Mitosis I, Mitosis IIMicrogametogenesis: Microspores undergo mitosis I and II to produce pollen grain.

      Table 1. 

      The correlation between flower size and pollen developmental stages

    • Cluster numberyProtein #xProtein accessionvProtein descriptionuFold changew
      Cluster 1: Protein folding/ unfolding/ targeting1Solyc06g051650.2.1Peptidyl-prolyl cis-trans isomerase–0.60
      3Solyc05g056230.2.1Calreticulin0.8
      8Solyc01g104740.2.1Ethylene-responsive transcriptional coactivator0.7
      14Solyc06g076520.1.1HSP201.11
      15Solyc03g082420.2.1Small heat shock protein1.05
      16Solyc11g020040.1.1HSP 700.96
      18Solyc02g088610.2.1ClpA/Clp HSP0.78
      29Solyc01g086740.2.1Chaperone DnaJ0.6
      36Solyc01g099660.2.1HSP 700.67
      54Solyc02g077670.2.1Chaperone protein dnaJ 21.41
      96Solyc11g006170.1.1DnaJ0.62
      100Solyc11g071830.1.1Chaperone protein dnaj1.36
      109Solyc07g053620.2.1Chaperone protein dnaJ0.63
      115Solyc03g005700.1.1Mitochondrial import receptor subunit TOM220.67
      116Solyc10g083970.1.1S-adenosylmethionine synthase–0.60
      Cluster 2: Protein translation9Solyc06g048410.2.1Superoxide dismutase0.64
      31Solyc01g094560.2.1Eukaryotic ribosomal protein eL36 family0.79
      35Solyc01g097760.2.1Ribosomal protein L7a0.63
      39Solyc01g103510.2.1Ribosomal protein L3 family0.91
      45Solyc02g038650.1.1Tetratricopeptide repeat protein 5–1.15
      46Solyc02g038660.1.1Tetratricopeptide-like helical–0.64
      47Solyc02g038670.1.1Tetratricopeptide-like helical–0.83
      48Solyc02g038680.1.1Tetratricopeptide-like helical–1.04
      49Solyc02g038700.1.1Tetratricopeptide repeat protein 5–1.00
      61Solyc03g112360.1.1Ribosomal protein0.61
      82Solyc06g064460.2.1Ribosomal protein L7a0.64
      94Solyc10g061970.1.160S ribosomal protein L37a0.63
      126Solyc10g085480.1.160S ribosomal protein L240.66
      132Solyc05g008420.2.1Tetratricopeptide repeat protein 5–0.62
      Cluster 3: Defense against cell wall stress factors5Solyc04g072160.2.1Prostaglandin E synthase 30.78
      33Solyc01g095320.2.1BCL-2-associated athanogene 60.91
      50Solyc02g065170.2.1Laccase-22–0.61
      66Solyc03g117630.1.1HSP701.02
      67Solyc03g118430.2.1Peptidase family M16–0.74
      74Solyc04g014480.2.1HSP20-like chaperones1.29
      79Solyc05g056560.2.1C11orf73 homolog0.95
      80Solyc06g053380.2.1Chitinase–1.11
      87Solyc07g005820.2.1Heat shock protein0.89
      91Solyc09g075950.1.1Heat shock protein 70 family.1.35
      103Solyc09g092690.2.1Peptidyl-prolyl cis-trans isomerase1.27
      107Solyc10g007220.1.1Hsp40, DnaJ–1.05
      Cluster 4: Cytosolic heat stress response/ programmed cell death13Solyc08g062340.2.1Cytosolic class II small heat shock protein HCT21.56
      21Solyc08g062450.1.1Class II small heat shock protein Le-HSP17.61.09
      22Solyc08g078700.2.1HSP201.38
      37Solyc01g102960.2.1HSP201.3
      62Solyc03g113930.1.1HSP201.29
      90Solyc09g015000.2.1HSP201.86
      64Solyc03g115230.2.1ClpA/ClpB HSP0.61
      120Solyc10g084170.1.1Bcl-2-associated athanogene1.04
      133Solyc09g015020.1.1Small heat shock protein (HSP20)1.91
      Cluster 5: Rerouting the carbohydrate flux6Solyc04g082200.2.1Bcl-2-associated athanogene1.37
      30Solyc01g091200.2.1NAD dependent epimerase/ dehydratase–0.79
      44Solyc01g112210.2.1Hexosyltransferase–0.61
      68Solyc03g120310.2.1Glycosyl transferase–0.99
      77Solyc05g054060.2.1UTP-glucose 1 phosphate uridylyltransferase–0.65
      125Solyc06g074670.2.1NAD-dependent epimerase/ dehydratase–0.76
      88Solyc07g055300.2.1Alpha alpha-trehalose-phosphate synthase–0.62
      119Solyc07g007790.2.1Sucrose phosphate synthase0.6
      Cluster 6: Transcription/ translation11Solyc10g051390.1.1RNA-binding glycine-rich protein-1b–0.99
      59Solyc03g059010.2.1Glucose-repressible alcohol dehydrogenase transcriptional effector CCR4–0.83
      75Solyc04g074750.2.1RNA-binding (RRM/RBD/RNP motifs)–0.60
      78Solyc05g054240.2.1Protein arginine N-methyltransferase–0.64
      81Solyc06g062800.2.1DEAD box helicase family0.63
      84Solyc06g071560.2.1Phosphatase 2A regulatory subunit B–1.45
      89Solyc09g008620.1.1Polyadenylate-binding protein–1.14
      Cluster 7: Cytosolic protein degradation and secretion28Solyc01g079450.2.1Prolyl oligopeptidase family protein–0.68
      40Solyc01g105990.2.1Os01g0107900 protein–0.64
      53Solyc02g069160.2.1Synaptobrevin family1.28
      83Solyc06g071430.2.1T17H3.1 protein–0.74
      111Solyc11g066830.1.1Splicing factor U2af subunit0.66
      Cluster 8: Chromatin status/ gene transcriptional activity12Solyc06g074790.1.1Histone H2B.10.71
      17Solyc01g099410.2.1Histone H2A.10.61
      112Solyc03g116770.2.1DNA-binding protein–1.44
      113Solyc06g062690.2.1Nucleosome assembly protein– 0.69
      114Solyc11g066160.1.1Histone H40.65
      Cluster 9: Ubiquitin-mediated degradation of intracellular proteins4Solyc09g074680.2.1Cullin family–0.63
      43Solyc01g111680.2.1Ubiquitin-conjugating enzyme–0.61
      104Solyc05g052960.2.1BTB/POZ domain containing protein–0.72
      105Solyc06g073820.1.1Ubiquitin carboxyl-terminal hydrolase–1.10
      106Solyc11g072070.1.1BTB/POZ domain containing protein–0.63
      Cluster 10: Synthesis of secondary metabolites26Solyc01g010900.2.1Cytochrome P450–0.78
      123Solyc03g053130.2.1Calcium-dependent phosphotriesterase–0.96
      122Solyc07g065770.2.1ABC transporter-like–0.85
      124Solyc11g071800.1.1Strictosidine synthase–0.68
      Cluster 11: Stress-response20Solyc09g065180.2.1mRNA binding protein–0.80
      25Solyc06g076570.1.1HSP202.4
      63Solyc03g114930.2.1PsbP-like protein 1–0.67
      118Solyc08g080190.2.1Glucose-methanol-choline (GMC) oxidoreductase–0.99
      Cluster 12: Protecting cells against oxidative damage7Solyc03g111720.2.1Peptide methionine sulfoxide reductase–1.02
      72Solyc04g010010.1.1Thioredoxin-like protein 1–1.05
      73Solyc04g010030.1.1Thioredoxin-like protein 1–1.32
      93Solyc10g007230.1.1Tetratricopeptide repeat protein 2-like–1.64
      Cluster 13: Programmed cell death/ protein targeting38Solyc01g103080.2.1RNA helicase, ATP-dependent1.15
      58Solyc03g025740.2.1VASCULAR-RELATED NAC-DOMAIN 60.64
      98Solyc11g011790.1.1Importin beta-3–1.09
      99Solyc11g011800.1.1Importin beta–0.90
      Cluster 14: Lipidome10Solyc08g076470.2.1Glycerol-3-phosphate acyltransferase–0.76
      32Solyc01g094700.2.1Glycerol-3-phosphate acyltransferase–0.81
      42Solyc01g109580.2.1Adenylyl cyclase-associated protein–0.74
      110Solyc07g005580.2.1Phospholipid/glycerol acyltransferase–0.75
      Cluster 15: TFs and signaling pathways27Solyc01g057270.2.1Calmodulin-binding transcription factor SR4–0.95
      86Solyc06g083150.2.1U-box domain-containing protein–0.82
      137Solyc07g055130.2.1Kinase family protein–0.75
      Cluster 16: Cell Wall degradation/ PMC separation34Solyc01g097000.2.1Glycosyl hydrolase family protein–0.77
      85Solyc06g073750.2.1Beta-D-glucosidase–0.80
      127Solyc11g071640.1.1Beta-D-glucosidase–0.65
      Cluster 17: Elasticity of cell surface structures70Solyc03g123540.2.1HSP20-like chaperones1.78
      101Solyc09g092160.2.1Beta-galactosidase–0.76
      108Solyc05g014280.2.1Small heat shock protein1.59
      Cluster 18: Attenuating the apoptotic effects and mitochondrial damages102Solyc11g020330.1.1Small heat shock protein2.18
      41Solyc01g106820.2.1Peptidase M50 family1.1
      92Solyc09g091470.2.13-ketoacyl CoA thiolase 20.68
      Cluster 19: Meiotic process57Solyc03g007800.2.1Topoisomerase 1-associated factor 1/Timeless family protein0.7
      71Solyc04g008600.2.1Chromosome segregation in meiosis protein 30.7
      136Solyc11g020780.1.1DCK/dGK-like deoxyribonucleoside kinase–0.60
      Cluster 20: Cell surface activities on premeiotic PMCs affecting fate of the cells51Solyc02g068400.2.1Pectin lyase-like superfamily protein–1.31
      128Solyc08g077860.2.1Meiotic serine proteinase–1.14
      Cluster 21: N/A23Solyc07g054210.2.1Protochlorophyllide reductase like protein–0.92
      121Solyc10g008740.2.1Mg-protoporphyrin IX chelatase-like–0.62
      Cluster 22: Folding and targeting of nascent proteins52Solyc02g069120.2.1Zinc-finger protein ZPR10.61
      56Solyc02g090480.2.1Peptidyl-prolyl cis-trans isomerase D–0.61
      Cluster 23: Pre-meiotic chromosome69Solyc03g120900.1.1Belongs to the WD repeat SEC13 family–0.64
      135Solyc10g007680.2.1Regulator of chromosome condensation–0.64
      Cluster 24: Maintenance of acquired thermos-tolerance2Solyc08g077980.2.1Bax inhibitor; Belongs to the BI1 family0.64
      24Solyc02g079930.2.1Phosphosulfolactate synthase-related protein0.66
      Cluster 25: Cell wall95Solyc11g005490.1.1Fasciclin-like arabinogalactan family protein–1.12
      138Solyc12g013900.1.1CT0990.61
      z Proteins clustered on the STRING networks in Fig. 6. These proteins were identified as differentially expressed proteins (DEPs) in the heat stress (HS)-treated pollen mother cells which were significantly up-regulated or down-regulated compared to to the non-HS-treated control condition. The relative difference in abundance of each protein, measured by the intensity of its constituent peptides, was compared between the two treatment conditions. The protein has passed the t test with false discovery rate (FDR) corrections (P ≤ 0.05) and with a fold change greater than 0.60-fold (±). Statistical analyses were performed using SAS (Version 9.3; SAS Institute, Cary, NC)
      y Cluster number in Fig. 6, which were constructed in STRING protein−protein interaction networks using Markov cluster (MCL) algorithm
      x Protein # in Fig. 6
      w Protein accession number in the ITAG Protein database (version 2.40; Sol Genomics Network, Boyce Thompson Institute, Ithaca, NY).
      v Protein description in the ITAG Protein database (version 2.40; Sol Genomics Network, Boyce Thompson Institute, Ithaca, NY)
      u The fold (HS/N-HS) for each protein is the log2 ratio of the protein abundance level between heat stress (HS) and non-HS-treated (N-HS) conditions

      Table 2. 

      Proteins clustered on STRING association networksz