Skip to main content
SpringerLink
Log in

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

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
Fig. 7a–j
Fig. 8a–m

Similar content being viewed by others

Abbreviations

PCD :

Programmed cell death

PIN2 :

Serine proteinase inhibitor II

SaPIN2a :

Solanum americanum serine proteinase inhibitor IIa

SaPIN2b :

Solanum americanum serine proteinase inhibitor IIb

TdT :

Terminal deoxynucleotidyl transferase

TEM :

Transmission electron microscopy

TUNEL :

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling

References

  • 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 5:203–213

    Article  CAS  PubMed  Google Scholar 

  • Beers EP, Woffenden BJ, Zhao C (2000) Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol Biol 44:399–415

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brandstadter J, Roßbach C, Theres K (1996) Expression of genes for a defensin and a proteinase inhibitor in specific areas of the shoot apex and the developing flower in tomato. Mol Gen Genet 252:146–154

    Article  CAS  PubMed  Google Scholar 

  • Bryant J, Green TR, Gurusaddaiah T, Ryan CA (1976) Proteinase inhibitor II from potatoes: isolation and characterization of its promoter components. Biochemistry 15:3418–3424

    CAS  PubMed  Google Scholar 

  • Chen F, Foolad MR (1997) Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration. Plant Mol Biol 35:821–831

    CAS  PubMed  Google Scholar 

  • Chye ML, Huang BQ, Zee SY (1999) Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product. Plant J 18:205–214

    Article  CAS  PubMed  Google Scholar 

  • Cox KH, Goldberg RB (1988) Analysis of plant gene expression. In: Shaw CH (ed) Plant molecular biology: a practical approach. IRL Press, Oxford, pp 1–35

  • Drews GN, Okamuro JK (1996) In situ hybridization with nonradioactive probes. In: Cold Spring Harbor Arabidopsis molecular genetics manual. http://genome-www.stanford.edu/Arabidopsis/cshl-course

  • Domínguez F, Cejudo FJ (1998) Germination-related genes encoding proteolytic enzymes are expressed in the nucellus of developing wheat grains. Plant J 15:569–574

    Article  Google Scholar 

  • Domínguez F, González MC, Cejudo FJ (2002) A germination-related gene encoding a serine carboxypeptidase is expressed during the differentiation of the vascular tissue in wheat grains and seedlings. Planta 215:727–734

    Article  PubMed  Google Scholar 

  • Felton GW, Gatehouse JA (1996) Antinutritive plant defence mechanisms. In: Lehane MJ, Billingsley PF (eds) Biology of the insect midgut. Chapman and Hall, London, pp 373–416

  • Funk V, Kositsup B, Zhao C, Beers EP (2002) The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol 128:84–94

    CAS  PubMed  Google Scholar 

  • Gatehouse AMR (1999) Biotechnological applications of plant genes in the production of insect-resistant crops. In: Clement SL, Quisenberry SS (eds) Global plant genetic resources for insect-resistant crops. CRC Press, Boca Raton, pp 263–280

  • Golldack D, Vera P, Dietz KJ (2003) Expression of subtilisin-like serine proteases in Arabidopsis thaliana is cell-specific and responds to jasmonic acid and heavy metals with developmental differences. Physiol Plant 118:64–73

    CAS  PubMed  Google Scholar 

  • Groover A, Jones AM (1999) Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 119:375–384

    CAS  PubMed  Google Scholar 

  • Guerrero C, de la Calle M, Reid MS, Valpuesta V (1998) Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence. Plant Mol Biol 36:565–571

    CAS  PubMed  Google Scholar 

  • Gustafson G, Ryan CA (1976) Specificity of protein turnover in tomato leaves. J Biol Chem 251:7004–7010

    CAS  PubMed  Google Scholar 

  • Hendriks T, Vreugdenhil D, Stiekema WJ (1991) Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol Biol 17:385–394

    CAS  PubMed  Google Scholar 

  • Hilder VA, Gatehouse AMR, Boulter D (1993) Transgenic plants conferring insect tolerance: proteinase inhibitor approach. In: Kung SD, Wu R (eds) Transgenic plants, vol 1. Academic Press, New York, pp 317–338

  • Hollaender-Czytko H, Andersen JK, Ryan CA (1985) Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves. Plant Physiol 78:76–79

    Google Scholar 

  • Jones ML, Larsen PB, Woodson WR (1995) Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol Biol 28:505–512

    CAS  PubMed  Google Scholar 

  • Kush A, Goyvaerts E, Chye ML, Chua NH (1990) Laticifer-specific gene expression in Hevea brasiliensis. Proc Natl Acad Sci USA 87:1787–1790

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Lorberth R, Dammann C, Ebneth M, Amati S, Sanchez-Serrano JJ (1992) Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression. Plant J 2:477–486

    Article  CAS  PubMed  Google Scholar 

  • Nagy F, Kay SA, Chua NH (1988) Analysis of gene expression in transgenic plants. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp B4:1–29

  • Pearce G, Ryan CA, Liljegren D (1988) Proteinase inhibitors I and II in fruit of wild tomato species: transient components of a mechanism for defense and seed dispersal. Planta 175:527–531

    CAS  Google Scholar 

  • Pearce G, Johnson S, Ryan CA (1993) Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family. Plant Physiol 102:639–644

    Article  CAS  PubMed  Google Scholar 

  • Pena-Cortes H, Sanchez-Serrano J, Rocha-Sosa M, Willmitzer L (1988) Systemic induction of proteinase-inhibitor II gene expression in potato plants by wounding. Planta 174:84–89

    CAS  Google Scholar 

  • Pena-Cortes H, Willmitzer L, Sanchez-Serrano JJ (1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell 3:963–972

    Google Scholar 

  • Reeck GR, Kramer KJ, Baker JE, Kanost MR, Fabrick JA, Behnke CA (1997) Proteinase inhibitors and resistance of transgenic plants to insects. In: Carozzi N, Koziel M (eds) Advances in insect control: the role of transgenic plants. Taylor and Francis, London, pp 157–183

  • Roberts IN, Murray PF, Caputo CP, Passeron S, Barneix AJ (2003) Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leaves. Physiol Plant 118:483–490

    Article  CAS  Google Scholar 

  • Rosahl S, Schell EJ, Willmitzer L (1986) Organ-specific gene expression in potato: isolation and characterization of tuber-specific cDNA sequences. Mol Gen Genet 202:368–373

    CAS  Google Scholar 

  • Runeberg-Roos P, Saarma M (1998) Phytepsin, a barley vacuolar aspartic proteinases, is highly expressed during autolysis of developing tracheary elements and sieve cells. Plant J 15:139–145

    CAS  PubMed  Google Scholar 

  • Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. Bioessays 10:20–24

    CAS  PubMed  Google Scholar 

  • Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28:425–449

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sanchez-Serrano J, Schmidt R, Schell J, Willmitzer L (1986) Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol Gen Genet 203:15–20

    CAS  Google Scholar 

  • Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443

    CAS  PubMed  Google Scholar 

  • Varagona MJ, Raikhel NV (1994) Immunocytochemistry for light and electron microscopy. In: Freeling M, Walbot V (eds) The maize handbook. Springer, New York Berlin Heidelberg, pp 149–157

  • Walker-Simmons M, Ryan CA (1977) Immunological identification of proteinase inhibitors I and II in isolated tomato leaf vacuoles. Plant Physiol 60:61–63

    CAS  Google Scholar 

  • Wingate VPM, Franceschi VR, Ryan CA (1991) Tissue and cellular localization of proteinase inhibitors I and II in the fruit of the wild tomato, Lycopersicon peruvianum (L.) Mill. Plant Physiol 97:490–495

    CAS  Google Scholar 

  • Wu HM, Cheung AY (2000) Programmed cell death in plant reproduction. Plant Mol Biol 44:267–281

    PubMed  Google Scholar 

  • Xu FX, Chye ML (1999) Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J 17:321–327

    Article  CAS  PubMed  Google Scholar 

  • Xu H, Kermode AR (2003) Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds. Plant Mol Biol 52:729–744

    Article  PubMed  Google Scholar 

  • Xu ZF, Qi WQ, Ouyang XZ, Yeung E, Chye ML (2001) A proteinase inhibitor II of Solanum americanum is expressed in phloem. Plant Mol Biol 47:727–738

    Article  CAS  PubMed  Google Scholar 

  • Xu ZF, Teng WL, Chye ML (2004) Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing hetergenous proteinase inhibitor SaPIN2a. Planta 218:623–629

    Article  CAS  PubMed  Google Scholar 

  • Yano A, Suzuki K, Shinshi H (1999) A signaling pathway, independent of the oxidative burst, that leads to hypersensitive cell death in cultured tobacco cells includes a serine protease. Plant J 18:105–109

    Article  Google Scholar 

Download references

Acknowledgements

We thank W.T. Tam for excellent assistance in ultra-thin sectioning and photography, X.Z. Ouyang for technical assistance in immunolocalization, Prof. E. Yeung for helpful discussions and Prof. S.Y. Zee for provision of a microtome. This project was funded by the Department of Botany, University of Hong Kong. S.-F. S. is supported by a postgraduate studentship from the University of Hong Kong and the Sir Edward Youde Memorial Foundation.

Author information

Authors and Affiliations

  1. Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China

    Suk-Fong Sin & Mee-Len Chye

Authors
  1. Suk-Fong Sin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mee-Len Chye
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mee-Len Chye.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sin, SF., Chye, ML. Expression of proteinase inhibitor II proteins during floral development in Solanum americanum . Planta 219, 1010–1022 (2004). https://doi.org/10.1007/s00425-004-1306-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-004-1306-6

Keywords

Search

Navigation