Molecular Genetics and Genomics

, Volume 275, Issue 1, pp 97–104 | Cite as

Identification of a new class of pistil-specific proteins of Petunia inflata that is structurally similar to, but functionally distinct from, the self-incompatibility factor HT

  • Hidenori Sassa
  • Hisashi Hirano
Original Paper


Pollen–pistil interactions are thought to involve a wide variety of intercellular recognition events controlled by diverse proteins and other molecules. One of the best characterized interactions is the S-RNase-based gametophytic self-incompatibility (GSI) system found in Solanaceae, Rosaceae and Scrophulariaceae. Although the S specificity of the pistil and the pollen in these families is determined by the S locus-encoded proteins S-RNase and SLF/SFB, respectively, these proteins alone are not sufficient for operation of the GSI reaction. Other factors are also required and are classified into three groups. To date, the only known factor is the pistil-expressed small asparagine-rich protein HT-B in three solanaceous genera Nicotiana, Lycopersicon and Solanum. HT-B is a Group 2 factor that is required for pollen rejection but do not affect S-RNase expression; factors in the other groups have not yet cloned. Here, we identified a new class of HT-like proteins in the style of Petunia inflata and named it HTL. Through alternative splicing, it was found that two isolated homologous HTL cDNAs, HTL-A and HTL-B, derived from a single gene. Like HT-B, HTL showed pistil-specific accumulation as well as significant sequence similarity to HT including conserved cystein residues at the C-terminal region and a signal peptide for extracellular localization. However, unlike HT-B, HTL lacked an asparagine-rich domain. Thus, it represents a new class of HT proteins. To determine whether HTL is involved in GSI function, RNA silencing constructs for HTL-A and HTL-B were introduced into self-incompatible P. inflata. Although several transgenic lines showed no detectable levels of both HTL-A and HTL-B transcripts, they retained normal GSI function and produced large fruits upon compatible pollination. This suggests that since silencing of the HTL gene alone is not sufficient to affect reproductive physiology, the gene is functionally distinct from the GSI factor HT-B.


Petunia Pollen–pistil interactions RNA silencing Self-incompatibility 



We thank Dr. J.B. Power for P. inflata seeds, Dr. P.M. Waterhouse for pHANNIBAL, Dr. A.P. Gleave for pART27 and Dr. W.J. Stiekema for pBINPLUS. Drs. Y. Ogawa, Y. Hoshino and H. Washida are acknowledged for their technical advice. We also thank Melody Kroll for proof reading this manuscript. This work was supported in part by the Grants-in-Aid for Scientific Research (C, 13660011) and the Grants-in-Aid for Young Scientists (A, 16688001) from the Ministry of Education, Science, Sports and Culture of Japan to H.S.


  1. Ai Y, Singh A, Coleman CE, Ioerger TR, Kheyr-Pour A, Kao T-H (1990) Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins. Sex Plant Reprod 3:130–138CrossRefGoogle Scholar
  2. Ai Y, Kron E, Kao T-H (1991) S-alleles are retained and expressed in a self-compatible cultivar of Petunia hybrida. Mol Gen Genet 230:353–358CrossRefPubMedGoogle Scholar
  3. Anderson MA, Cornish EC, Mau S-L et al (1986) Cloning of cDNA for a style glycoprotein associated with expression of self-incompatibility in Nicotiana alata. Nature 321:38–44CrossRefGoogle Scholar
  4. Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA (1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5(2):203–213CrossRefPubMedGoogle Scholar
  5. Bernatzky R, Glaven RH, Rivers BA (1995) S-related protein can be recombined with self-compatibility in interspecific derivatives of Lycopersicon (tomato). Biochem Genet 33:215–225PubMedCrossRefGoogle Scholar
  6. Bradford M (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  7. Broothaerts W, Janssens GA, Proost P, Broekaert WF (1995) cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Mol Biol 27:499–511CrossRefPubMedGoogle Scholar
  8. Cruz-Garcia F, Hancock N, Kim D, McClure B (2005) Stylar glycoproteins bind to S-RNase in vitro. Plant J 42:295–304CrossRefPubMedGoogle Scholar
  9. de Nettancourt D (2001) Incompatibility and incongruity in wild and cultivated plants. Springer, Berlin Heidelberg New YorkGoogle Scholar
  10. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  11. Entani T, Iwano M, Shiba H, Che F-S, Isogai A, Takayama S (2003) Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: identification of a pollen expressed F-box gene with allelic diversity. Genes Cells 8:203–213CrossRefPubMedGoogle Scholar
  12. Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002PubMedCrossRefGoogle Scholar
  13. Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conductive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207CrossRefPubMedGoogle Scholar
  14. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  15. Hosaka K, Hannemen RE (1998a) Genetics of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 1. Detection of an S locus inhibitor (Sli) gene. Euphytica 99:191–197CrossRefGoogle Scholar
  16. Hosaka K, Hannemen RE (1998b) Genetics of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 2. Localization of an S locus inhibitor (Sli) gene on the potato genome using DNA markers. Euphytica 103:265–271CrossRefGoogle Scholar
  17. Hugot K, Ponchet M, Marais A, Ricci P, Galiana E (2002) A tobacco S-like RNase inhibits hyphal elongation of plant pathogens. Mol Plant Microbe Interact 15:243–250PubMedCrossRefGoogle Scholar
  18. Kondo K, Yamamoto M, Itahashi R, Sato T, Egashira H, Hattori T, Kowyama Y (2002a) Insights into the evolution of self-incompatibility in Lycopersicon from a study of stylar factors. Plant J 30:143–154CrossRefGoogle Scholar
  19. Kondo K, Yamamoto M, Matton DP, Sato T, Hirai M, Norioka S, Hattori T, Kowyama Y (2002b) Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility. Plant J 29:627–636CrossRefGoogle Scholar
  20. Lai Z, Ma W, Han B, Liang L, Zhang Y, Hong G, Xue Y (2002) An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum. Plant Mol Biol 50:29–42CrossRefPubMedGoogle Scholar
  21. Lee H-S, Singh A, Kao T-H (1992) RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequence similarity with solanaceous S proteins. Plant Mol Biol 20:1131–1141CrossRefPubMedGoogle Scholar
  22. Lee H-S, Huang S, Kao T-H (1994) S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367:560–563CrossRefPubMedGoogle Scholar
  23. McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE (1989) Style self-incompatibility products of Nicotiana alata are ribonucleases. Nature 342:955–957CrossRefPubMedGoogle Scholar
  24. McClure BA, Gray JE, Anderson MA, Clarke AE (1990) Self-incompatibility in Nicotiana alata involves degradation of pollen rRNA. Nature 347:757–760CrossRefGoogle Scholar
  25. McClure B, Mou B, Canevascini S, Bernatzky R (1999) A small asparagine -rich protein required for S-allele-specific pollen rejection in Nicotiana. Proc Natl Acad Sci USA 96:13548–13553CrossRefPubMedGoogle Scholar
  26. McClure BA, Cruz-Garcia F, Beecher BS, Sulaman W (2000) Factors affecting inter- and intra-specific pollen rejection in Nicotiana. Ann Bot 85:113–123CrossRefGoogle Scholar
  27. McCubbin AG, Kao T-H (2000) Molecular recognition and response in pollen and pistil interactions. Annu Rev Cell Dev Biol 16:333–364CrossRefPubMedGoogle Scholar
  28. Murfett J, Atherton TL, Mou B, Gasser CS, McClure BA (1994) S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection. Nature 367:563–566CrossRefPubMedGoogle Scholar
  29. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6CrossRefPubMedGoogle Scholar
  30. O’Brien M, Kapfer C, Major G, Laurin M, Bertrand C, Kondo K, Kowyama Y, Matton DP (2002) Molecular analysis of the stylar-expressed Solanum chacoense small asparagine-rich protein family related to the HT modifier of gametophytic self-incompatibility in Nicotiana. Plant J 32:985–996CrossRefPubMedGoogle Scholar
  31. Olmstead RG, Sweere JA, Spangler RE, Bohs L, Palmer JD (1999) Phylogenetic and provisional classification of the Solanaceae based on chloroplast DNA. In: Nee M, Symon DE, Lester PN, Jessop JP (eds) Solanaceae IV. Royal Botanic Gardens, Kew, pp 111–137Google Scholar
  32. Qiao H, Wang H, Zhao L, Zhou J, Huang J, Zhang Y, Xue Y (2004) The F-box protein AhSLF-S2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum. Plant Cell 16:571–581CrossRefPubMedGoogle Scholar
  33. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  34. Sassa H, Hirano H (1998) Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR 5-like protein in Japanese pear (Pyrus serotina Rehd.). Planta 205:514 521CrossRefPubMedGoogle Scholar
  35. Sassa H, Hirano H, Ikehashi H (1992) Self-incompatibility-related RNases in styles of Japanese pear (Pyrus serotina Rehd.). Plant Cell Physiol 33:811–814Google Scholar
  36. Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H (1996) Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S-ribonuclease superfamily. Mol Gen Genet 250:547–557PubMedGoogle Scholar
  37. Sassa H, Hirano H, Nishio T, Koba T (1997) Style-specific self-compatible mutation caused by deletion of the S-RNase gene in Japanese pear (Pyrus serotina). Plant J 12:223–227CrossRefGoogle Scholar
  38. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  39. Sessa G, Fluhr R (1995) The expression of an abundunt transmitting tract-specific endoglucanase (Sp41) is promoter-dependent and not essential for the reproductive physiology of tobacco. Plant Mol Biol 29:969–982CrossRefPubMedGoogle Scholar
  40. Sijacic P, Wang X, Skirpan AL, Wang Y, Dowd PE, McCubbin AG, Huang S, Kao T-H (2004) Identification of the pollen determinant of S-RNase-mediated self-incompatibility. Nature 429:302–305CrossRefPubMedGoogle Scholar
  41. Singh A, Ai Y, Kao T-H (1991) Characterization of ribonuclease activity of three S-allele-associated proteins of Petunia inflata. Plant Physiol 96:61–68PubMedCrossRefGoogle Scholar
  42. Sonneveld T, Tobutt KR, Vaughan SP, Robbins T (2005) Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene. Plant Cell 17:37–51CrossRefPubMedGoogle Scholar
  43. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedGoogle Scholar
  44. Ushijima K, Sassa H, Tamura M, Kusaba M, Tao R, Gradziel TM, Dandekar AM, Hirano H (2001) Characterization of the S-locus region of almond (Prunus dulcis): analysis of a somaclonal mutant and a cosmid contig for an S haplotype. Genetics 158:379–386PubMedGoogle Scholar
  45. Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H (2003) Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism. Plant Cell 15:771–781CrossRefPubMedGoogle Scholar
  46. Ushijima K, Yamane H, Watari A, Kakehi E, Ikeda K, Hauck NR, Iezzoni AF, Tao R (2004) The S haplotype-specific F-box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume. Plant J 39:573–586CrossRefPubMedGoogle Scholar
  47. van Engelen FA, Molthoff JW, Conner AJ, Nap J-P, Pereira A, Stiekema WJ (1995) pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res 4:288–290CrossRefPubMedGoogle Scholar
  48. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590CrossRefPubMedGoogle Scholar
  49. Xue Y, Carpenter R, Dickinson H, Coen ES (1996) Origin of allelic diversity in Antirrhinum S locus RNases. Plant Cell 8:805–814CrossRefPubMedGoogle Scholar
  50. Yamane H, Ikeda K, Ushijima K, Sassa H, Tao R (2003) A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry, Prunus cerasus and P. avium. Plant Cell Physiol 44:764–769PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Faculty of HorticultureChiba UniversityMatsudoJapan
  2. 2.Kihara Institute for Biological ResearchYokohama City UniversityYokohamaJapan

Personalised recommendations