Skip to main content
SpringerLink
Log in

Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea

  • Original Article
  • Published:
Functional & Integrative Genomics Aims and scope Submit manuscript

Abstract

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al. 2014, Funct Integr Genomics 14:307–317).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Ahmed I, Yadav D, Shukla P, Vineeth TV, Sharma PC, Kirti PB (2017) Constitutive expression of Brassica juncea annexin, AnnBj2 confers salt tolerance and glucose and ABA insensitivity in mustard transgenic plants. Plant Sci 265:12–28

    Article  CAS  PubMed  Google Scholar 

  • Chichkova NV (2004) A plant caspase-like protease activated during the hypersensitive response. Plant Cell 16:157–171

  • Coupe SA, Sinclair BK, Watson LM, Heyes JA, Eason JR (2003) Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets. J Exp Bot 54:1045–1056

    Article  CAS  PubMed  Google Scholar 

  • Del Pozo O, Lam E (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Curr Biol 8

  • Denis M, Delourme R, Gourret JP, Mariani C, Renard M (1993) Expression of engineered nuclear male sterility in Brassica napus (genetics, morphology, cytology, and sensitivity to temperature). Plant Physiol 101:1295–1304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Drake R, John I, Farrell A, Cooper W, Schuch W, Grierson D (1996) Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant Mol Biol 30:755–767

    Article  CAS  PubMed  Google Scholar 

  • Goldberg RB, Beals TP, Sanders PM (1993) Anther development: basic principles and practical applications. Plant Cell 5:1217–1229

    CAS  PubMed  PubMed Central  Google Scholar 

  • Grudkowska M, Zagdanska B (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Pol 51:609–624

    CAS  PubMed  Google Scholar 

  • Jagannath A, Bandyopadhyay P, Arumugam N, Gupta V, Burma PK, Pental D (2001) The use of a spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breed 8:11–23

    Article  CAS  Google Scholar 

  • Kang L, Li P, Wang A, Ge X, Li Z (2017) A novel cytoplasmic male sterility in Brassica napus (inap CMS) with carpelloid stamens via protoplast fusion with Chinese Woad. Front Plant Sci 8

  • Kiba A, Tomiyama H, Takahashi H, Hamada H, Ohnishi K, Okuno T, Hikichi Y (2003) Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstoniasolanacearum. Plant Cell Physiol 44:287–295

    Article  CAS  PubMed  Google Scholar 

  • Kirti PB, Gaikwad K, Bhat SR, Dinesh Kumar V, Prakash S, Chopra VL (1998) Chloroplast substitution overcomes leaf chlorosis in Moricandia arvensis based cytoplasmic male sterility in Brassica juncea. Theor Appl Genet 97:1179–1182

    Article  Google Scholar 

  • Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

  • La Camera S, Balague C, Gobel C, Geoffroy P, Legrand M, Feussner I et al (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 22:469–481

    Article  CAS  PubMed  Google Scholar 

  • Lee S, Jung K-H, An G, Chung Y-Y (2004) Isolation and characterization of a rice cysteine Proteasegene, OsCP1, using the T-DNA gene-trap system. Plant Mol Biol 54:755–765

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Mariani C, Beuckeleer MD, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347:737–741

    Article  CAS  Google Scholar 

  • Mariani C, Gossele V, Beuckeleer MD, Block MD, Goldberg RB, Greef WD, Leemans J (1992) A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 737:741

    Google Scholar 

  • Martinez M, Cambra I, Carrillo L, Diaz-Mendoza M, Diaz I (2009) Characterization of the entire cystatin gene family in barley and their target Cathepsin L-like Cysteine-Proteases, partners in the hordein mobilization during seed germination. Plant Physiol 151:1531–1545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Martinez M, Cambra I, Gonzalez-Melendi P, Santamaria ME, Diaz I (2012) C1A cysteine-proteases and their inhibitors in plants. Physiol Plant 145:85–94

    Article  CAS  PubMed  Google Scholar 

  • Nandy S, Sinha R, Rajam MV (2013) Over-expression of arginine decarboxylase gene in tapetal tissue results in male sterility in tomato plants. Cell Dev Biol 2:117. https://doi.org/10.4172/2168-9296.1000117

    Article  CAS  Google Scholar 

  • Pontier D, Godiard L, Marco Y, Roby D (1994) hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions. Plant J 5:507–521

    Article  CAS  PubMed  Google Scholar 

  • Prakash S, Kirti PB, Gaikwad K, Bhat SR, Dinesh Kumar V, Chopra VL (1998) A Moricandia arvensis based CMS and fertility restoration in Brassica juncea through protoplast fusion. Theor Appl Genet 97:488–492

    Article  CAS  Google Scholar 

  • Premi OP, Kandpal BK, Rathore SS, Shekhawat K, Chauhan JS (2013) Green manuring, mustard residue recycling and fertilizer application affects productivity and sustainability of Indian mustard (Brassica juncea L.) in Indian semi-arid tropics. Ind Crop Prod 41:423–429

    Article  Google Scholar 

  • 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 96:35–51

    Article  CAS  PubMed  Google Scholar 

  • Reape TJ, Molony EM, Mc Cabe PF (2008) Programmed cell death in plants: distinguishing between different modes. J Exp Bot 59:435–444

    Article  CAS  PubMed  Google Scholar 

  • Schrauwen JAM, Mettenmeyer T, Croes AF, Wullems GJ (1996) Tapetum-specific genes: what role do they play in male gametophyte development? Acta Bot Neerlandica 45:1–15

    Article  CAS  Google Scholar 

  • Shindo T, Misas-Villamil JC, Hörger AC, Song J, van der Hoorn RAL (2012) A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14. PLoS One 7:e29317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 Genomics 14:307–317

    Article  CAS  PubMed  Google Scholar 

  • 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 246:52–61

    Article  CAS  PubMed  Google Scholar 

  • 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 59:445–457

    Article  CAS  PubMed  Google Scholar 

  • Shukla P, Gautam R, Ahmed I, Kirti PB (2019) A Proteomic study of cysteine protease induced cell death in anthers of male sterile tobacco transgenic plants. Physiol Mol Biol Plants 1–10. https://doi.org/10.1007/s12298-019-00642-y

  • Song L, Zhou Z, Tang S, Zhang Z, Xia S, Qin M, Li B, Wen J, Yi B, Shen J, Ma C, Fu T, Tu J (2016) Ectopic expression of BnaC.CP20.1 results in premature tapetal programmed cell death in Arabidopsis. Plant Cell Physiol 57:1972–1984

  • Takahashi Y, Uehara Y, Berberich T, Ito A, Saitoh H, Miyazaki A, Terauchi R, Kusano T (2004) A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco. Plant J 40:586–595

  • Trobacher CP, Senatore A, Greenwood JS (2006) Masterminds or minions? Cysteine proteinases in plant programmed cell death. Can J Bot 84:651–667

    Article  CAS  Google Scholar 

  • Wan L, Xia Q, Qiu X, Selvaraj G (2002) Early stages of seed development in Brassica napus: a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. Plant J 30:1–10

    Article  CAS  PubMed  Google Scholar 

  • Wang XQ, Shen X, He YM, Ren TN, Wu WT, Xi T (2011) An optimized freeze-thaw method for transformation of Agrobacterium tumefaciens EHA105 and LBA4404. Pharm Biotechnol 18:382–386

    CAS  Google Scholar 

  • Xu FX, Chye ML (2002) Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J 17:321–327

    Article  Google Scholar 

  • Zhang X-M, Wang Y, Lv X-M, Li H, Sun P, Lu H, Li FL (2009) NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development. J Exp Bot 60:1569–1577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Ranjana Gautam especially thanks UGC for providing fellowship to carry out this research work. The authors also thank to the Head, Department of Plant Sciences, University of Hyderabad, Hyderabad, for facilities available under DST-FIST and UGC-SAP-DRS while performing the experiments.

Funding

This work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, under a research project no- 38(1393)/14/EMR-II) to one of the authors (PBK).

Author information

Author notes

  1. Pawan Shukla

    Present address: Central Sericultural Research and Training Institute, Central Silk Board, NH-1A, Gallandar, Pampore, Jammu and Kashmir, 192121, India

  2. P. B. Kirti

    Present address: Rajendra Prasad Central Agricultural University, Pusa-Samsthipur, Bihar, India

Authors and Affiliations

  1. Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, 500046, India

    Ranjana Gautam, Pawan Shukla & P. B. Kirti

  2. Agri Biotech Foundation, Rajendranagar, Hyderabad, Telangana, India

    P. B. Kirti

Authors
  1. Ranjana Gautam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pawan Shukla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. B. Kirti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. B. Kirti.

Ethics declarations

The authors have no conflicts of interest to declare.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 65 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gautam, R., Shukla, P. & Kirti, P.B. Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea. Funct Integr Genomics 19, 703–714 (2019). https://doi.org/10.1007/s10142-019-00674-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-019-00674-3

Keywords

Search

Navigation