Plant Molecular Biology

, Volume 57, Issue 4, pp 487–502 | Cite as

Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco

  • Roman A. Volkov
  • Irina I. Panchuk
  • Fritz SchöfflEmail author


In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, −3Cand -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.


Microspore embryogenesis Molecular chaperons Post-transcriptional regulation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

Supplementary material


  1. Albani, D., Robert, LS., Donaldson, PA., Altosaar, I., Arnison, P., Fabijanski, S. 1990Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore developmentPlant Mol Biol15605622CrossRefPubMedGoogle Scholar
  2. Albani, D., Sardana, R., Robert, LS., Altosaar, I., Arnison, P., Fabijanski, S. 1992A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plantsPlant J2331342PubMedGoogle Scholar
  3. Allen, R.L.., Lonsdale, D.M.. 1993Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen developmentPlant J3261271CrossRefPubMedGoogle Scholar
  4. Almoguera, C.., Jordano, J. 1992Developmental and environmental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAsPlant Mol Biol19781792CrossRefPubMedGoogle Scholar
  5. Almoguera, C., Prieto-Dapena, P., Jordano, J. 1998Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elementsPlant J13437446CrossRefPubMedGoogle Scholar
  6. Altschul, S.F., Madden, TL., Schäffer, AA., Zhang, J., Zhang, Z., Miller, W., Lipman, D. 1997Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Res2533893402CrossRefPubMedGoogle Scholar
  7. Apuya, N., Zimmerman, J. 1992Heat shock gene expression is controlled primarily at the translational level in carrot cells and somatic embryosPlant Cell4657665CrossRefPubMedGoogle Scholar
  8. Ariizumi, T., Amagai, M., Shibata, D., Hatakeyama, K., Watanabe, M., Toriyama, K. 2002Comparative study of promoter activity of three anther-specific genes encoding lipid transfer protein, xyloglucan endotransglucosylase/hydrolase and polygalacturonase in transgenic Arabidopsis thalianaPlant Cell Reports219096CrossRefGoogle Scholar
  9. Atanassov, I., Russinova, E., Antonov, L., Atanassov, A. 1998Expression of an anther-specific chalcone synthase-like gene is correlated with uninucleate microspore development in Nicotiana sylvestrisPlant Mol Biol3811691178CrossRefPubMedGoogle Scholar
  10. Atkinson, BG., Raizada, M., Bouchard, RA., Frappier, J., Walden, D. 1993The independent stage-specific expression of the 18-kDa heat shock protein genes during microsporogenesis in Zea maysL Dev Genet141526CrossRefGoogle Scholar
  11. Banzet, N., Richaud, C., Deveaux, Y., Kazmaier, M., Gagnon, J., Triantaphlides, C. 1998Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cellsPlant J13519527CrossRefPubMedGoogle Scholar
  12. Basha, E., Lee, GJ., Demeler, B., Vierling, E. 2004Chaperone activity of cytosolic small heat shock proteins from wheatEuropean J Biochem27114261436CrossRefGoogle Scholar
  13. Benito Moreno, RM., Macke, F., Hauser, M-T., Alwen, A., Heberle-Bors, E. 1988Sporophytes and male gametophytes from in vitrocultured, immature tobacco pollenCresti, M.Gori, P.Pacini, E. eds. Sexual Reproduction in Higher Plants.SpringerBerlin137142Google Scholar
  14. Berkelman, T., Stenstedt, T. 19982-D Electrophoresis using Immobilized pH Gradients: Principles and MethodsAmersham Pharmacia BiotechPiscatawayGoogle Scholar
  15. Bouchard, R. 1990Characterization of expressed meiotic prophase repeat transcript clones of Lilium: meiosis-specific expression, relatedness, and affinities to small heat-shock protein genesGenome336879PubMedGoogle Scholar
  16. Brander, K., Kuhlemeier, C. 1995A pollen-specific DEAD-box protein related to translation initiation factor eIF-4A from tobaccoPlant Mol Biol27637649CrossRefPubMedGoogle Scholar
  17. Carranco, R., Almoguera, C., Jordano, J. 1997A plant small heat shock protein gene expressed during zygotic embryogenesis but noninductible by heat stressJ Biol Chem2722747027475CrossRefPubMedGoogle Scholar
  18. Carranco, R., Almoguera, C., Jordano, J. 1999An imperfect heat shock element and different upstream sequences are required for the seed-specific expression of a small heat shock protein genePlant Physiol121723730CrossRefPubMedGoogle Scholar
  19. Carpenter, JL., Ploense, SE., Snustad, D., Silflow, C. 1992Preferential expression of an α-tubulin gene of Arabidopsis in pollenPlant Cell4557571CrossRefPubMedGoogle Scholar
  20. Christensen, HEM., Ramachandran, S., Tan, CT., Surana, U., Dong, C., Chua, N. 1996Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilinPlant J10269279CrossRefPubMedGoogle Scholar
  21. Coca, MA., Almoguera, C., Thomas, T., Jordano, J. 1996Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoterPlant Mol Biol31863876CrossRefPubMedGoogle Scholar
  22. Coleman, A., Goff, L. 1985Application of fluorochromes to pollen biology I Mithramycin and 4′ 6-diamidino-2-phenylindole (DAPI) as vital stains and for quantification of nuclear DNAStain Technol60145154PubMedGoogle Scholar
  23. Cordewener, JHG., Custers, JBM., Dons, HM., VanLookeren Campagne, M. 1996Molecular and biochemical events during the induction of microspore embryogenesisMohanjain, SM.Sopory, S.Veilleux, RE. eds. In vitro Haploid Production in Higher PlantsKluwer Academic PublishersDordrecht111124Google Scholar
  24. Cordewener, JHG., Hause, G., Gorgen, E., Busink, R., Hause, B., Dons, HJM., Van Lammeren, AAM., VanLookeren Campagne, M., Pechan, P. 1995Changes in synthesis and localization of members of the 70-kDa class of heat shock proteins accompany the induction of embryogenesis in Brassica napus L microsporesPlanta196747755CrossRefGoogle Scholar
  25. Custers, JBM., Oldenhof, MT., Schrauwen, JAM., Cordewener, JHG., Wullems, GJ., VanLookeren Campagne, M. 1997Analysis of microspore-specific promoters in transgenic tobaccoPlant Mol Biol35689699CrossRefPubMedGoogle Scholar
  26. Czarnecka, E., Gurley, WB., Nagao, RT., Mosquera, L., Key, J. 1985DNA sequence and transcript mapping of a soybean gene encoding a small heart shock proteinProc Natl Acad Sci USA8237263730Google Scholar
  27. Dzelzkàlns, VA., Thorsness, MK., Dwyer, KG., Baxter, JS., Balent, MA., Nasrallah, M., Nasrallah, J. 1993Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S locus glycoprotein gene promoterPlant Cell5855863CrossRefPubMedGoogle Scholar
  28. Engel, J. 1997Signifikante Schule der schlichten StatistikFilander VerlagFürthGoogle Scholar
  29. Gao, J., Kim, SR., Chung, YY., Lee, J., An, G. 1994Developmental and environmental regulation of two ribosomal protein genes in tobaccoPlant Mol Biol25761770CrossRefPubMedGoogle Scholar
  30. Gupta, R., Ting, JTL., Sokolov, LN., Johnson, S., Luan, S. 2002A tumor suppressor homolog, AtPTEN1, is essential for pollen development in ArabidopsisPlant Cell1424952507CrossRefPubMedGoogle Scholar
  31. Gustavsson, N., Kokke, BP., Harndahl, U., Silow, M., Bechtold, U., Poghosyan, Z., Murphy, D., Boelens, W., Sundby, C. 2002A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock proteinPlant J29545553CrossRefPubMedGoogle Scholar
  32. Harada, H., Kyo, M., Imamura, J. 1988The induction of embryogenesis in Nicotiana immature pollen cultureBock, G.Marsh, J. eds. Application of Plant Cell and Tissue Culture.WileyChichester5974Google Scholar
  33. Harlow, E., Lane, D. 1988Antibodies: A Laboratory ManualCold Spring Harbor LaboratoryNew YorkGoogle Scholar
  34. Hartwell, J., Jenkins, GI., Wilkins, M., Nimmo, H. 1999The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translationPlant Cell Environ22883889CrossRefGoogle Scholar
  35. Hoch B, Lutsch G, Schlegel WP, Stahl J, Wallukat G, Bartel S, Krause EG, Benndorf R, Karczewski P (1996) HSP25 in isolated perfused rat hearts: localization and response to hyperthermia. Mol Cell Biochem 160–161:231–239Google Scholar
  36. Honys D, Twell D (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5:R851–13Google Scholar
  37. Huang, S., An, YQ., McDowell, JM., McKinney, E., Meagher, R. 1996The Arabidopsis thalianaACT4/ACT12 actin gene subclass is strongly expressed throughout pollen developmentPlant J10189202CrossRefPubMedGoogle Scholar
  38. Jia, Y., Ransom, RF., Shibanuma, M., Liu, C., Welsh, M., Smoyer, W. 2001Identification and characterization of hic-5/ARA55 as an hsp27 binding proteinJ Biol Chem2763991139918CrossRefPubMedGoogle Scholar
  39. Kandasamy, MK., McKinney, EC., Meagher, R. 1999The late pollen-specific actins in angiospermsPlant J18681691CrossRefPubMedGoogle Scholar
  40. Key, JL., Lin, C., Chen, Y. 1981Heat shock proteins of higher plantsProc Natl Acad Sci USA7835263530Google Scholar
  41. Kim, SY., Hong, C., Lee, I. 2001Heat shock stress causes stage-specific male sterility in Arabidopsis thalianaJ Plant Res114301307Google Scholar
  42. Kobayashi, T., Kobayashi, E., Sato, S., Hotta, Y., Miyajima, N., Tanaka, A., Tabata, S. 1994Characterization of cDNAs induced in meiotic prophase in Lily microsporocytesDNA Res11526PubMedGoogle Scholar
  43. Kyo, M., Harada, H. 1986Control of the developmental pathway of tobacco pollen in vitroPlanta168427432CrossRefGoogle Scholar
  44. Lee, JH., Hübel, A., Schöffl, F. 1995Depression of the activity of genetically enginered heat shock factor causes constitutive synthesis of heat shock proteins and increased thermal tolerance in transgenic ArabidopsisPlant J8603612CrossRefPubMedGoogle Scholar
  45. Lee, GJ., Roseman, AM., Saibil, H., Vierling, E. 1997A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent stateEMBO J16659671PubMedGoogle Scholar
  46. Lee, G., Vierling, E. 2000A small heat shock protein cooperates with heat shock protein 70 system to reactivate a heat-denatured proteinPlant Physiol122189197CrossRefPubMedGoogle Scholar
  47. Löw, D., Brandle, K., Nover, L., Forreiter, C. 2000Cytosolic heat-stress proteins Hsp177 class I and Hsp173 class II of tomato act as molecular chaperones in vivoPlanta211575582CrossRefPubMedGoogle Scholar
  48. Marchetti, S., Zaina, G., Chiabà, C., Pappalardo, C., Pitotti, A. 2001Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L) uninucleate microsporesPlanta213199206CrossRefPubMedGoogle Scholar
  49. Mascarenhas, J. 1990Gene activity during pollen developmentAnn Rev Plant Physiol Plant Mol Biol41317338CrossRefGoogle Scholar
  50. Mascarenhas, J., Crone, D. 1996Pollen and heat-shock responseSex Plant Reprod9370374CrossRefGoogle Scholar
  51. Mittermann, I., Swoboda, I., Pierson, E., Eller, N., Kraft, D., Valenta, R., Heberle-Bors, E. 1995Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturationPlant Mol Biol27137146CrossRefPubMedGoogle Scholar
  52. Mu, JH., Lee, H., Kao, T. 1994Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinasePlant Cell6709721CrossRefPubMedGoogle Scholar
  53. Murashige, T., Skoog, F. 1962A revised medium for rapid growth and bioassays with tobacco tissue culturesPhysiol Plant15437497Google Scholar
  54. Nagao, RT., Czarnecka, E., Gurley, WB., Schoeffl, F., Key, JL. 1985Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene familyMol Cell Biol534173428PubMedGoogle Scholar
  55. Newman, TC., Ohme-Takagi, M., Taylor, C., Green, P. 1993DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobaccoPlant Cell5701714CrossRefPubMedGoogle Scholar
  56. Oldenhof, MT., Groot, PFM., Visser, JH., Schrauwen, JM., Wullems, G. 1996Isolation and characterization of a microspore-specific gene from tobaccoPlant Mol Biol31213225CrossRefPubMedGoogle Scholar
  57. Park, S., Hong, C. 1998Comparison of the structure and expression pattern for a low molecular weight heat-shock protein cDNA clone from Nicotiana tabacumMol Cells8594599PubMedGoogle Scholar
  58. Prändl, R., Kloske, E., Schöffl, F. 1995Developmental regulation and tissue specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plantsPlant Mol Biol287382CrossRefPubMedGoogle Scholar
  59. Raghavan, V. 1986Emryogenesis in Angiosperms: A Developmental and Experimental StudyCambridge University PressNew YorkGoogle Scholar
  60. Ramakrishna, W., Deng, ZP., Ding, CK., Handa, A., Ozminkowski, R. 2003A novel small heat shock protein gene, vis1, contributes to pectin depolymerization and juice viscosity in tomato fruitPlant Physiol131725735CrossRefPubMedGoogle Scholar
  61. Raschke, E., Baumann, G., Schöffl, F. 1988Nucleotide sequence analysis of soybean small heat shock protein genes belonging to two different multigene familiesJ Mol Biol199549557CrossRefPubMedGoogle Scholar
  62. Reynolds, T. 1997Pollen embryogenesisPlant Mol Biol33110CrossRefPubMedGoogle Scholar
  63. Roberts, MR., Foster, GD., Blundell, RP., Robinson, SW., Kumar, A., Draper, J., Scott, R. 1993Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana genePlant J3111120CrossRefPubMedGoogle Scholar
  64. Sabehat, A., Lurie, S., Weiss, D. 1998Expression of small heat-shock proteins at low temperaturesPlant Physiol117651658CrossRefPubMedGoogle Scholar
  65. Sambrook, J., Fritsch, E., Maniatis, T. 1989Molecular cloningCold Spring Harbor Laboratory PressNew YorkGoogle Scholar
  66. Scharf, KD., Siddique, M., Vierling, E. 2001The expending family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing α-crystallin domainsCell Stress Chaperones6225238PubMedGoogle Scholar
  67. Schöffl, F., Key, J. 1982An analysis of mRNAs for a group of heat shock proteins of soybean using cloned cDNAsJ Mol Appl Genet1301314PubMedGoogle Scholar
  68. Schöffl, F., Prändl, R., Reindl, A. 1998Regulation of the heat shock responsePlant Physiol11711351141CrossRefPubMedGoogle Scholar
  69. Schöffl, F., Prändl, R., Reindl, A. 1998Molecular responses to heat stressShinozaki, K.Yamaguchi-Shinozaki, K. eds. Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants.RG Landes CompanyAustin TX8198Google Scholar
  70. Segui-Simarro, JM., Testillano, P., Risueno, MC. 2003Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassicanapus LJ Struct Biol142379391CrossRefPubMedGoogle Scholar
  71. Singh, M., Blialla, PL., Xu, H., Singh, M. 2003Isolation and characterization of a flowering plant male gametic cell-specific promoterFEBS Lett5424752CrossRefPubMedGoogle Scholar
  72. Smykal, P., Masin, J., Hrdy, I., Konopasek, I., Zarsky, V. 2000Chaperone activity of tobacco HSP18, a small heat-shock protein, is inhibited by ATPPlant J23703713CrossRefPubMedGoogle Scholar
  73. Sun, W., Bernard, C., Vande Cotte, B., Van Montagu, M., Verbruggen, N. 2001At-HSP176A, encoding a small heat-shock protein in Arabidopsis, can enhance osmotolerance upon overexpressionPlant J27407415CrossRefPubMedGoogle Scholar
  74. Sunderland, N., Wicks, F. 1973Embryoid formation in pollen grains of Nicotiana tabacumJ Exp Bot22213226Google Scholar
  75. Tanaka, I. 1997Differentiation of generative and vegetative cells in angiosperm pollenSex Plant Reprod1017CrossRefGoogle Scholar
  76. Tebbutt, SJ., Rogers, H., Lonsdale, D. 1994Characterization of a tobacco gene encoding a pollen-specific polygalacturonasePlant Mol Biol25283297CrossRefPubMedGoogle Scholar
  77. Thangavelu, M., Belostotsky, D., Bevan, MW., Flavell, RB., Rogers, H., Lonsdale, D. 1993Partial characterization of the Nicotiana tabacum actin gene family: evidence for pollen-specific expression of one of the gene family membersMol Gen Genet240290295PubMedGoogle Scholar
  78. Touraev, A., Heberle-Bors, E. 1999Microspore embryogenesis and In vitro pollen maturation in tobaccoMethods Mol Biol111281292PubMedGoogle Scholar
  79. Volkov, RA., Panchuk, I., Schöffl, F. 2003Heat-stress dependent and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCRJ Exp Bot5423432349CrossRefPubMedGoogle Scholar
  80. Waters, E. 1995The molecular evolution of the small heat-shock proteins in plantsGenetics141785795PubMedGoogle Scholar
  81. Waters, E., Vierling, E. 1999The diversification of plant cytosolic small heart shock proteins preceded the divergence of mossesMol Biol Evol16127139PubMedGoogle Scholar
  82. Wehmeyer, N., Hernandez, LD., Finkelstein, R., Vierling, E. 1996Synthesis of small heat-shock proteins is part of the developmental program of late seed maturationPlant Physiol112747757CrossRefPubMedGoogle Scholar
  83. Wehmeyer, N., Vierling, E. 2000The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerancePlant Physiol12210991108CrossRefPubMedGoogle Scholar
  84. Xu, H., Swoboda, I., Bhalla, PL., Sijbers, AM., Zhao, C., Ong, EK., Hoeijmakers, J., Singh, M. 1998Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanismsPlant J13823829CrossRefPubMedGoogle Scholar
  85. Xu, HL., Swoboda, I., Bhalla, P., Singh, M. 1999Male gametic cell-specific expression of H2A and H3 histone genesPlant Mol Biol39607614CrossRefPubMedGoogle Scholar
  86. Xu, HL., Swoboda, I., Bhalla, P., Singh, M. 1999Male gametic cell-specific gene expression in flowering plantsProc Natl Acad Sci USA9625542558CrossRefPubMedGoogle Scholar
  87. Ylstra, B., McCormick, S. 1999Analysis of mRNA stabilities during pollen development and in BY2 cellsPlant J20101108CrossRefPubMedGoogle Scholar
  88. Young, L., Wilen, R., Bonham-Smith, P. 2004High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed productionJ Exp Bot55485495CrossRefPubMedGoogle Scholar
  89. Zarsky, V., Garrido, D., Eller, N., Tupy, J., Vicente, O., Schöffl, F., Heberle-Bors, E. 1995The expression of a small heat shock gene is activated during induction of tobacco pollen embryogenesis by starvationPlant Cell Environ18139147Google Scholar
  90. Zhao, JP., Newcomb, W., Simmonds, D. 2003Heat-shock proteins 70 kDa and 19 kDa are not requiredfor induction of embryogenesis of Brassica napus L cv topas microsporesPlant Cell Physiol4414171421CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Roman A. Volkov
    • 1
  • Irina I. Panchuk
    • 1
  • Fritz Schöffl
    • 1
    Email author
  1. 1.Zentrum für Molekularbiologie der Pflanzen–Allgemeine GenetikUniversität TübingenTübingenGermany

Personalised recommendations