Plant Molecular Biology

, Volume 82, Issue 1–2, pp 169–180 | Cite as

RNAi silencing of three homologues of S-adenosylmethionine decarboxylase gene in tapetal tissue of tomato results in male sterility

  • Ranjita Sinha
  • Manchikatla Venkat Rajam


Polyamines play very important role in various cellular metabolic functions, including floral induction, floral differentiation and fertility regulation. In the present study, S-adenosylmethionine decarboxylase (SAMDC), a key gene involved in polyamine biosynthesis, has been targeted in tapetal tissue of tomato using RNAi to examine its effect on tapetum development and pollen viability. The target SAMDC gene fragments of three homologues were cloned in a hairpin RNA construct under the control of tapetal-specific A9 promoter, which was used to generate several RNAi tomato plants. These RNAi lines expressed the intended small interfering RNAs in the anther and showed the aborted and sterile pollen exhibiting shrunken and distorted morphology. These RNAi tomato plants having sterile pollen, failed to set fruits but female fertility of the plants remained unaffected as cross pollination resulted in fruit setting. Expression profiling of SAMDC genes showed considerable decrease in transcripts of SAMDC1 (5–8 fold) and SAMDC2 and SAMDC3 (2–3 fold) in the anthers of RNAi plants. The other polyamine biosynthesis genes, ADC and SPDSYN exhibited ~1.5 fold decrease in their transcript levels. Presence of siRNA molecules specific to SAMDC homologues in anther and tapetal-specific activity of A9 promoter as shown with GUS reporter system of RNAi plants suggested down-regulation of the target genes in tapetum by RNAi. These observations indicate the importance of SAMDC, in turn polyamines in pollen development, and thus tapetum-specific down-regulation of SAMDC genes using RNAi can be used for developing male sterile plants.


Solanumlycopersicum Male sterility Polyamines S-Adenosylmethionine decarboxylase RNAi Pollen development 



This work was carried out by a grant from Department of Biotechnology (DBT), New Delhi. Ranjita Sinha is grateful to DBT for senior research fellowship. We also thank University Grant Commission for special assistance program and Department of Science and Technology for FIST program. We thank Ms. Aarti Gupta for providing RNAi-ACS lines, which were used as unrelated control for the present study.

Conflict of interest

The authors declare that they have no conflict of interests.

Supplementary material

11103_2013_51_MOESM1_ESM.jpg (132 kb)
Supplementary material 1 (JPG 132 kb)
11103_2013_51_MOESM2_ESM.jpg (49 kb)
Supplementary material 2 (JPG 50 kb)
11103_2013_51_MOESM3_ESM.jpg (37 kb)
Supplementary material 3 (JPG 38 kb)
11103_2013_51_MOESM4_ESM.jpg (34 kb)
Supplementary material 4 (JPG 34 kb)
11103_2013_51_MOESM5_ESM.jpg (44 kb)
Supplementary material 5 (JPG 44 kb)
11103_2013_51_MOESM6_ESM.jpg (194 kb)
Supplementary material 6 (JPG 194 kb)
11103_2013_51_MOESM7_ESM.jpg (98 kb)
Supplementary material 7 (JPG 99 kb)
11103_2013_51_MOESM8_ESM.jpg (86 kb)
Supplementary material 8 (JPG 86 kb)
11103_2013_51_MOESM9_ESM.doc (42 kb)
Supplementary material 9 (DOC 42 kb)


  1. Applewhite PB, Kaur-Sawhney R, Galston AW (2000) A role for spermidine in the bolting and flowering of Arabidopsis. Physiol Plant 108:314–320CrossRefGoogle Scholar
  2. Aribaud M, Martin-Tanguy J (1994) Polyamine metabolism in normal and sterile Chrysanthemum morifolium. Phytochemistry 37:927–932CrossRefGoogle Scholar
  3. Bajaj S, Rajam MV (1996) Polyamine accumulation and near-loss of morphogenesis in long-term callus cultures of rice: restoration of plant regeneration by manipulation of cellular polyamine levels. Plant Physiol 112:1343–1348PubMedGoogle Scholar
  4. Bitrian M, Zarza X, Altabella T, Tiburcio AF, Alcazar R (2012) Polyamines under abiotic stress: metabolic crossroads and hormonal crosstalks in plants. Metabolites 2:516–528CrossRefGoogle Scholar
  5. Bokern M, Witte L, Wray V, Nimtz M, Meurer-Grimes B (1995) Trisubstituted hydroxycinnamic acid spermidines from Quercus dentata pollen. Phytochemistry 39:1371–1375CrossRefGoogle Scholar
  6. Brukhin V, Hernould M, Gonzalez N, Mouras CCA (2003) Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry. Sex Plant Reprod 15:311–320Google Scholar
  7. Cetin E, Yildirim C, Palavan-Unsal N, Unal M (2000) Effect of spermine and cyclohexylamine on in vitro pollen germination and tube growth in Helianthus annuus. Can J Plant Sci 80:241–245CrossRefGoogle Scholar
  8. Chibi F, Matilla AJ, Angosto T, Garrido D (1994) Changes in polyamine synthesis during anther development and pollen germination in tobacco (Nicotiana tabacum). Physiol Plant 92:61–68CrossRefGoogle Scholar
  9. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  10. Duvick DN (1999) Heterosis, feeding people and protecting natural resources. Crop Science Society of America/Soil Science Society of America, MadisonGoogle Scholar
  11. Duvick DN (2005) The contribution of breeding to yield advances in maize (Zea mays L.). Elsevier Academic Press, San DiegoGoogle Scholar
  12. Edlund AF, Swanson R, Preuss D (2004) Pollen and stigma structure and function, the role of diversity in pollination. Plant Cell 16:S84–S97PubMedCrossRefGoogle Scholar
  13. Falasca G, Franceschetti M, Bagni N, Altamura MM, Biasi R (2010) Polyamine biosynthesis and control of the development of functional pollen in kiwifruit. Plant Physiol Biochem 48:565–573PubMedCrossRefGoogle Scholar
  14. Fellenberg C, Milkowski C, Hause B, Lange PR, Böttcher C, Vogt T (2008) Tapetum specific location of a cation-dependent O-methyltransferase in A. thaliana. Plant J 56:132–145PubMedCrossRefGoogle Scholar
  15. Fellenberg C, Ziegler J, Handrick V, Vogt T (2012) Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamen. Front Plant Sci 180:1–11Google Scholar
  16. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedCrossRefGoogle Scholar
  17. Grienenberger E, Besseau S et al (2009) A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines. Plant J 58:246–259PubMedCrossRefGoogle Scholar
  18. Guo DP, Sun YZ, Chen ZJ (2003) Involvement of polyamines in cytoplasmic male sterility of stem mustard (Brassica juncea var. tsatsai). Plant Growth Regul 41:33–40CrossRefGoogle Scholar
  19. Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence, intracellular hydrolysis of fluorescein diacetate. Stain Technol 145:115–120Google Scholar
  20. Huang CK, Chang BS et al (2004) Changes in polyamine pattern are involved in floral initiation and development in Polianthes tuberosa. J Plant Physiol 161:709–713PubMedCrossRefGoogle Scholar
  21. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol 42:39–51PubMedCrossRefGoogle Scholar
  22. Jackson D (1991) In situ hybridization in plants. In: Bowles DJ, Gurr SJ, McPherson M (eds) Molecular plant pathology, a practical approach. Oxford University Press, OxfordGoogle Scholar
  23. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  24. Kakkar RK, Sawhney VK (2002) Polyamine research in plants—a changing perspective. Physiol Plant 116:281–288CrossRefGoogle Scholar
  25. Kawanabe T, Ariizumi T, Kawai-Yamada M, Uchimiya H, Toriyama K (2006) Abolition of the tapetum suicide program ruins microsporogenesis. Plant Cell Physiol 47:784–787PubMedCrossRefGoogle Scholar
  26. Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258PubMedCrossRefGoogle Scholar
  27. Kumar A, Taylor MA, Madrif SA, Davies HV (1996) Potato plants expressing antisense and sense S-adenosylmethionine decarboxylase (SAMDC) transgenes showed altered levels of polyamines ethylene: antisense plants display abnormal phenotypes. Plant J 9:147–158CrossRefGoogle Scholar
  28. Kumar SV, Sharma ML, Rajam MV (2006) Polyamine biosynthetic pathway as a novel target for potential applications in plant biotechnology. Physiol Mol Biol Plants 12:13–28Google Scholar
  29. Kusano T, Yamaguchi K, Berberich T, Takahashi Y (2007) Advances in polyamine research in 2007. J Plant Res 120:345–350PubMedCrossRefGoogle Scholar
  30. Li SF, Iacuone S, Parish RW (2007) Suppression and restoration of male fertility using a transcription factor. Plant Biotechnol J 5:297–312PubMedCrossRefGoogle Scholar
  31. Lu C, Blake CM, Pamela JG (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117PubMedCrossRefGoogle Scholar
  32. Ma H (2005) Molecular genetic analysis of microsporogenesis and microgametogenesis in flowering plants. Annu Rev Plant Biol 56:393–434PubMedCrossRefGoogle Scholar
  33. Ma W, Wang J, Hu J, Guan Y, Li Y, Zheng Y (2012) Relation between changes in polyamine, protective enzyme activity and pollen vigor of tobacco in different flowering stages. Afr J Agric Res 7:5491–5497Google Scholar
  34. Madhulatha P, Pandey R, Hazarika P, Rajam MV (2007) High transformation frequency in Agrobacterium mediated genetic transformation of tomato by using polyamines and maltose in shoot regeneration medium. Physiol Mol Biol Plants 13:191–198Google Scholar
  35. Malmberg RL (1980) Biochemical, cellular, and developmental characterization of a temperature sensitive mutant of Nicotiana tabacum and its second site revertant. Cell 22:603–609PubMedCrossRefGoogle Scholar
  36. Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737–741CrossRefGoogle Scholar
  37. Martin-Tanguy J, Perdrizet E, Prevost J, Martin C (1982) The distribution of hydroxycinnamic acid amides in fertile and cytoplasmic male sterile lines of maize. Phytochemistry 21:1939–1945CrossRefGoogle Scholar
  38. Moon HS, Li Y, Stewart CN Jr (2010) Keeping the genie in the bottle, a transgene biocontainment by excision in pollen. Trends Biotechnol 28:3–8PubMedCrossRefGoogle Scholar
  39. Moritoh S, Miki D, Akiyama M, Kawahara M, Izawa T, Maki H, Shimamoto K (2005) RNAi-mediated silencing of OsGEN-L (OsGEN-like), a new member of the RAD2/XPG nuclease family, causes male sterility by defect of microspore development in rice plant. Cell Physiol 46:699–715CrossRefGoogle Scholar
  40. Parish RW, Li SF (2010) Death of a tapetum, a programme of developmental altruism. Plant Sci 178:73–89CrossRefGoogle Scholar
  41. Prabhavathi V, Rajam MV (2007) Mannitol accumulating transgenic eggplants exhibit enhanced resistance to fungal wilts. Plant Sci 173:50–54CrossRefGoogle Scholar
  42. Rajam MV (1989) Restriction of pollen germination and tube growth in lily pollen by inhibitors of polyamine metabolism. Plant Sci 59:53–56CrossRefGoogle Scholar
  43. Rastogi R, Sawhney VK (1990) Polyamines and flower development in the male sterile stainless-2 mutant of tomato (Lycopersicon esculentum Mill.). I. Level of polyamines and their biosynthesis in normal and mutant flowers. Plant Physiol 93:439–445PubMedCrossRefGoogle Scholar
  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New YorkGoogle Scholar
  45. Shaya F, Gaiduk S, Keren I, Shevtsov S, Zemah H, Belausov E, Evenor D, Reuveni M, Ostersetzer-Biran O (2012) Expression of mitochondrial gene fragments within the tapetum induce male sterility by limiting the biogenesis of the respiratory machinery in transgenic tobacco. J Integr Plant Biol 54:115–130PubMedCrossRefGoogle Scholar
  46. Singh SP, Pandey T, Srivastava R, Verma PC, Singh PK, Tuli R, Sawant SV (2010) BECLIN1 from Arabidopsis thaliana under the generic control of regulated expression systems, a strategy for developing male sterile plants. Plant Biotechnol J 8:1005–1022PubMedCrossRefGoogle Scholar
  47. Song J, Nada K, Tachibana S (2001) The early increase of S-adenosylmethionine decarboxylase activity is essential for the normal germination and tube growth in tomato (Lycopersicon esculentum Mill.) pollen. Plant Sci 161:507–515CrossRefGoogle Scholar
  48. Tarenghi E, Martin-Tanguy J (1995) Polyamines, floral induction and floral development of strawberry (Fragaria ananassa Dutch.). Plant Growth Regul 17:157–165CrossRefGoogle Scholar
  49. Tehseen M, Imran M, Hussain M, Irum S, Ali S, Mansoor S, Zafar Y (2010) Development of male sterility by silencing Bcp1 gene of Arabidopsis through RNA interference. Afr J Biotechnol 9:2736–2741Google Scholar
  50. Tsuchiya T, Toriyama K, Yoshikawa M, Ejiri S, Hinata K (1995) Tapetum-specific expression of the gene for an endo-β-1, 3-glucanase causes male sterility in transgenic tobacco. Plant Cell Physiol 36:487–494PubMedGoogle Scholar
  51. Van der Meer IM, Stam ME, van Tunen AJ, Mol JNM, Stuitje AR (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4:253–262PubMedGoogle Scholar
  52. Wang Y, Xue Y, Li J (2005) Towards molecular breeding and improvement of rice in China. Trends Plant Sci 10:610–614PubMedCrossRefGoogle Scholar
  53. Wang X, Singer SD, Liu Z (2012) Silencing of meiosis-critical genes for engineering male sterility in plants. Plant Cell Rep 31:747–756PubMedCrossRefGoogle Scholar
  54. Wolukau JN, Zhang SL, Xu GH, Chen D (2004) The effect of temperature, polyamines and polyamine synthesis inhibitor on in vitro pollen germination and pollen tube growth of Prunus mume. Sci Hortic 99:289–299CrossRefGoogle Scholar
  55. Wu J, Shang Z, Wu J, Jiang X, Moschou PN, Sun W, Roubelakis-Angelakis KA, Zhang S (2010) Spermidine oxidase-derived H2O2 regulates pollen plasma membrane hyperpolarization-activated Ca2+-permeable channels and pollen tube growth. Plant J 63:1042–1053PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Plant Polyamine, Transgenic and RNAi Laboratory, Department of GeneticsUniversity of DelhiNew DelhiIndia

Personalised recommendations