A proteomic study of cysteine protease induced cell death in anthers of male sterile tobacco transgenic plants

  • Pawan ShuklaEmail author
  • Ranjana Gautam
  • Naveen Kumar Singh
  • Israr Ahmed
  • Pulugurtha Bharadwaja KirtiEmail author
Research Article


Manifestation of male sterility in plants is an important requirement for hybrid seed production. Tapetum cell layer of anther is a primary target for genetic manipulation for male sterility. In our previous report, the targeted expression of Arachis cysteine protease in tapetum led to premature degeneration of tapetal layer that resulted in complete male sterility in transgenic tobacco plants. To correlate cysteine protease mediated cell death of tapetum, transmission electron microscopy (TEM) and proteomic pattern of anthers of cysteine protease induced male sterile plant were compared with the untransformed control plant. TEM study revealed the abnormal growth of tapetal cells exhibiting excessive vacuolization that synchronized with irregular exine wall formation of the microspores. In anther proteome, a total 250 protein spots were detected that were reproducible and exhibited similar distribution pattern. Further, anther proteome of male sterile plant showed the significant upregulation (≥ 1.5) of 56 protein spots. Using Mass spectroscopy (MALDI TOF/TOF), we have identified 14 protein spots that were involved in several processes such as energy metabolism, protein synthesis, plastid protein, lipid metabolism, and cell wall assembly. Upregulation of patatin-like protein-2 homolog, carboxylesterase 17 and dicer like protein-4 in male sterile anthers that have been demonstrated to induce cell death, suggesting that cysteine protease mediated premature tapetal cell death might involve the lipid peroxidation pathway in coordination with gene silencing mechanism.


Cysteine protease Anther proteome Cell death Male sterility Patatin Dicer like protein 



Arachis cysteine protease


Transmission electron microscopy




Isoelectric focusing


Isoelectric point


Programmed cell death



The authors are grateful to Council of Scientific and Industrial Research, Government of India for a Research Grant [38 (1393/EMR-II)] to one of the authors (PBK), DST-FIST, UGC-SAP, Government of India, for the facilities provided to the Department of Plant Sciences, University of Hyderabad.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ahmadovich Bozorov T, Prakash Pandey S, Dinh ST, Kim SG, Heinrich M, Gase K, Baldwin IT (2012) DICER-like proteins and their role in plant-herbivore interactions in nicotiana attenuata. J Integr Plant Biol 54:189–206CrossRefGoogle Scholar
  2. Anderson NL, Anderson NG (1998) Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 19:1853–1861CrossRefGoogle Scholar
  3. Bézier A, Lambert B, Baillieul F (2002) Cloning of a grapevine Botrytis-responsive gene that has homology to the tobacco hypersensitivity-related hsr203J. J Exp Bot 53:2279–2280CrossRefGoogle Scholar
  4. Bolwell GP, Wojtaszek P (1997) Mechanisms for the generation of reactive oxygen species in plant defence: a broad perspective. Physiol Mol Plant Pathol 51:347–366CrossRefGoogle Scholar
  5. Camera SL, Balagué C, Göbel C, Geoffroy P, Legrand M, Feussner I, Roby D, Heitz T (2009) The Arabidopsis patatin-like protein 2 (PLP2) plays an essential role in cell death execution and differentially affects biosynthesis of oxylipins and resistance to pathogens. Mol Plant Microbe Interact 22:469–481CrossRefGoogle Scholar
  6. Dhondt S, Geoffroy P, Stelmach BA, Legrand M, Heitz T (2000) Soluble phospholipase A2 activity is induced before oxylipin accumulation in tobacco mosaic virus-infected tobacco leaves and is contributed by patatin-like enzymes. Plant J 23:431–440CrossRefGoogle Scholar
  7. Drews GN, Beals TP, Bui AQ, Goldberg RB (1992) Regional and cell-specific gene expression patterns during petal development. Plant Cell 4:1383–1404CrossRefGoogle Scholar
  8. Dunoyer P, Himber C, Voinnet O (2005) DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat Genet 37:1356CrossRefGoogle Scholar
  9. Konagaya K-I, Ando S, Kamachi S, Tsuda M, Tabei Y (2008) Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa. Plant Cell Rep 27:1741–1754CrossRefGoogle Scholar
  10. Kumar KR, Kirti PB (2011) Differential gene expression in Arachis diogoi upon interaction with peanut late leaf spot pathogen, Phaeoisariopsis personata and characterization of a pathogen induced cyclophilin. Plant Mol Biol 75:497–513CrossRefGoogle Scholar
  11. Kumar D, Kirti PB (2015) Transcriptomic and proteomic analyses of resistant host responses in Arachis diogoi challenged with late leaf spot pathogen, Phaeoisariopsis personata. PLoS ONE 10:e0117559CrossRefGoogle Scholar
  12. La Camera S, Geoffroy P, Samaha H, Ndiaye A, Rahim G, Legrand M, Heitz T (2005) A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis. Plant J 44:810–825CrossRefGoogle Scholar
  13. Lee S, Jung K-H, An G, Chung Y-Y (2004) Isolation and characterization of a rice cysteine protease gene, OsCP1, using T-DNA gene-trap system. Plant Mol Biol 54:755–765CrossRefGoogle Scholar
  14. Liu F, Bakht S, Dean C (2012) Cotranscriptional role for Arabidopsis DICER-LIKE 4 in transcription termination. Science 335:1621–1623CrossRefGoogle Scholar
  15. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  16. Marco YJ, Ragueh F, Godiard L, Froissard D (1990) Transcriptional activation of 2 classes of genes during the hypersensitive reaction of tobacco leaves infiltrated with an incompatible isolate of the phytopathogenic bacterium Pseudomonas solanacearum. Plant Mol Biol 15:145–154CrossRefGoogle Scholar
  17. 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
  18. Marshall SD, Putterill JJ, Plummer KM, Newcomb RD (2003) The carboxylesterase gene family from Arabidopsis thaliana. J Mol Evol 57:487–500CrossRefGoogle Scholar
  19. May C, Preisig-Müller R, Höhne M, Gnau P, Kindl H (1998) A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies1Enzymes: lipoxygenase (EC; phospholipase A2 (EC Biochim Biophys Acta (BBA) Lipids Lipid Metab 1393:267–276CrossRefGoogle Scholar
  20. Melan MA, Dong X, Endara ME, Davis KR, Ausubel FM, Peterman TK (1993) An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant Physiol 101:441–450CrossRefGoogle Scholar
  21. Nizampatnam NR, Doodhi H, Kalinati NY, Mulpuri S, Viswanathaswamy DK (2009) Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants. Planta 229:987–1001CrossRefGoogle Scholar
  22. Pontier D, Tronchet M, Rogowsky P, Lam E, Roby D (1998) Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death. Mol Plant Microbe Interact 11:544–554CrossRefGoogle Scholar
  23. Rancé I, Fournier J, Esquerré-Tugayé M-T (1998) The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences. Proc Natl Acad Sci 95:6554–6559CrossRefGoogle Scholar
  24. Rao GS, Tyagi AK, Rao KV (2017) Development of an inducible male-sterility system in rice through pollen-specific expression of l-ornithinase (argE) gene of E. coli. Plant Sci 256:139–147CrossRefGoogle Scholar
  25. Rao GS, Deveshwar P, Sharma M, Kapoor S, Rao KV (2018) Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties. Plant Mol Biol 96:35–51CrossRefGoogle Scholar
  26. Saravanan RS, Rose JK (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4:2522–2532CrossRefGoogle Scholar
  27. Scherer GF, Ryu SB, Wang X, Matos AR, Heitz T (2010) Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants. Trends Plant Sci 15:693–700CrossRefGoogle Scholar
  28. Sengupta D, Kannan M, Reddy AR (2011) A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in Vigna radiata (L.) Wilczek. Planta 233:1111–1127CrossRefGoogle Scholar
  29. Seo H-H, Park AR, Lee H-H, Park S, Han Y-J, Hoang QT, Choi GJ, Kim J-C, Kim YS, Kim J-I (2018) A fungus-inducible pepper carboxylesterase exhibits antifungal activity by decomposing the outer layer of fungal cell walls. Mol Plant Microbe Interact 31:505–515CrossRefGoogle Scholar
  30. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858CrossRefGoogle Scholar
  31. Shukla P, Singh NK, Kumar D, Vijayan S, Ahmed I, Kirti PB (2014) Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco. Funct Integr Genom 14:307–317CrossRefGoogle Scholar
  32. Shukla P, Subhashini M, Singh NK, Ahmed I, Trishla S, Kirti PB (2016) Targeted expression of cystatin restores fertility in cysteine protease induced male sterile tobacco plants. Plant Sci 246:52–61CrossRefGoogle Scholar
  33. Shukla P, Singh NK, Gautam R, Ahmed I, Yadav D, Sharma A, Kirti PB (2017) Molecular approaches for manipulating male sterility and strategies for fertility restoration in plants. Mol Biotechnol 59:445–457CrossRefGoogle Scholar
  34. 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 8:1005–1022CrossRefGoogle Scholar
  35. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43CrossRefGoogle Scholar
  36. Sueldo D, Ahmed A, Misas-Villamil J, Colby T, Tameling W, Joosten MH, Hoorn RA (2014) Dynamic hydrolase activities precede hypersensitive tissue collapse in tomato seedlings. New Phytol 203:913–925CrossRefGoogle Scholar
  37. Vancanneyt G, Sonnewald U, Hofgen R, Willmitzer L (1989) Expression of a patatin-like protein in the anthers of potato and sweet pepper flowers. Plant Cell 1:533–540CrossRefGoogle Scholar
  38. Walden AR, Walter C, Gardner RC (1999) Genes expressed in Pinus radiata male cones include homologs to anther-specific and pathogenesis response genes. Plant Physiol 121:1103–1116CrossRefGoogle Scholar
  39. Zhang X-M, Wang Y, Lv X-M, Li H, Sun P, Lu H, Li F-L (2009) NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development. J Exp Bot 60:1569–1577CrossRefGoogle Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  1. 1.Department of Plant Sciences, School of Life SciencesUniversity of HyderabadHyderabadIndia
  2. 2.Central Sericultural Research and Training Institute (CSR&TI)PamporeIndia
  3. 3.Agricultural Research Organization-The Volcani CenterRishon LeZionIsrael
  4. 4.Rajendra Prasad Central Agricultural University, PusaSamastipurIndia
  5. 5.Agri Biotech FoundationHyderabadIndia

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