Skip to main content
SpringerLink
Log in

Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

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

Abstract

In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

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. 7

Similar content being viewed by others

Abbreviations

p-CMPS:

p-Chloromercuriphenylsulfonic acid

CBZ:

N-Carbobenzyloxy

DAI:

Days after imbibition

E-64:

l-Trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane

EDTA:

Ethylenediaminetetra-acetic acid

leu:

Leupeptin

β-ME:

β-Mercaptoethanol

o-phen:

o-Phenantroline

pep-A:

Pepstatin A

PMSF:

Phenylmethylsulfonyl fluoride

BPB:

Bromophenol blue

References

  • Allona I, Collada C, Casado R, Aragoncillo C (1994) Electrophoretic analysis of seed storage proteins from gymnosperms. Electrophoresis 15:1062–1067

    Article  PubMed  CAS  Google Scholar 

  • Antão CM, Malcata FX (2005) Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol Biochem 43:637–650

    Article  PubMed  Google Scholar 

  • Azeez A, Sane AP, Bhatnagar D, Nath P (2007) Enhanced expression of serine proteases during floral senescence in Gladiolus. Phytochemistry 68:1352–1357

    Article  PubMed  CAS  Google Scholar 

  • Belozersky MA, Sarbakanova ST (1989) Aspartic proteinase from wheat seeds—isolation, properties and action on gliadin. Planta 177:321–326

    Article  Google Scholar 

  • Belozersky MA, Dunaevsky YE, Voskoboynikova NE (1990) Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds. Biochem J 272:677–682

    PubMed  CAS  Google Scholar 

  • Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250

    Article  PubMed  CAS  Google Scholar 

  • Bond HM, Bowles DJ (1983) Characterization of soybean endopeptidase activity using exogenous and endogenous substrates. Plant Physiol 72:345–350

    Article  PubMed  CAS  Google Scholar 

  • Burrows GE, Boag TS, Stockey RA (1992) A morphological investigation of the unusual cryptogeal germination strategy of bunya pine (Araucaria bidwillii)-an Australian rain forest conifer. Int J Plant Sci 153:503–512

    Article  Google Scholar 

  • Capocchi A, Cinollo M, Galleschi L, Saviozzi F, Calucci L, Pinzino C, Zandomeneghi M (2000) Degradation of gluten by protease from dry and germinating wheat (Triticum durum) seeds: an in vitro approach to storage protein mobilization. J Agric Food Chem 48:6271–6279

    Article  PubMed  CAS  Google Scholar 

  • Casani S, Fontanini D, Capocchi A, Lombardi L, Galleschi L (2009) Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook post-germinated seeds. Plant Physiol Biochem 47:599–607

    Article  PubMed  CAS  Google Scholar 

  • Chichkova NV, Shaw J, Galiullina RA, Drury GE, Tuzhikov AI, Kim SH, Kalkum M, Hong TB, Gorshkova EN, Torrance L, Vartapetian AB, Talianski M (2010) Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. EMBO J 29:1149–1161

    Article  PubMed  CAS  Google Scholar 

  • Coffeen WC, Wolpert TJ (2004) Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa. Plant Cell 16:857–873

    Article  PubMed  CAS  Google Scholar 

  • Del Zoppo M, Galleschi L, Saviozzi F (1998) Long-term storage of Araucaria bidwillii Hook seeds. Seed Sci Technol 26:267–270

    Google Scholar 

  • Del Zoppo M, Galleschi L, Onnis A, Pardossi A, Saviozzi F (1999) Effect of salinity on water relations, sodium accumulation, chlorophyll content and proteolytic enzymes in a wild wheat. Biol Plant 42:97–104

    Article  Google Scholar 

  • Doi E, Shibata D, Matoba T, Yonezawa D (1980) Characterization of pepstatin-sensitive acid protease in resting rice seeds. Agric Biol Chem 44:741–747

    CAS  Google Scholar 

  • Dunaevsky YE, Sarbakanova SH, Belozersky MA (1989) Wheat seed carboxypeptidase and joint action on gliadin of proteases from dry and germinating seeds. J Exp Bot 40:1323–1329

    Article  Google Scholar 

  • Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278

    Article  PubMed  CAS  Google Scholar 

  • Fontanini D, Capocchi A, Saviozzi F, Galleschi L (2007) Simplified electrophoretic assay for human salivary α-amylase inhibitor detection in cereal seed flours. J Agric Food Chem 55:4334–4339

    Article  PubMed  CAS  Google Scholar 

  • Galleschi L (1998) Proteolysis in germinating cereal grains. Recent Res Dev Phytochem 2:95–106

    CAS  Google Scholar 

  • Galleschi L, Pellegrini L (1989) Purification and some properties of an aminopeptidase from endosperms of xHaynaldoticum sardoum Meletti et Onnis. New Phytol 113:301–306

    Article  CAS  Google Scholar 

  • Galleschi L, Capocchi A, Giannoni P, Floris C (1989) Proteinase activities in quiescent and germinating seeds of xHaynaldoticum sardoum. Physiol Plant 75:1–6

    Article  CAS  Google Scholar 

  • Galleschi L, Capocchi A, Ghiringhelli S, Saviozzi F (2002) Some properties of proteolytic enzymes and storage proteins in recalcitrant and orthodox seeds of Araucaria. Biol Plant 45:539–544

    Article  CAS  Google Scholar 

  • Gifford DJ (1988) An electrophoretic analysis of the seed proteins from Pinus monticola and eight other species of pine. Can J Bot 66:1808–1812

    CAS  Google Scholar 

  • Gifford DJ, Tolley C (1989) The seed proteins of white spruce and their mobilization following germination. Physiol Plant 77:254–261

    Article  CAS  Google Scholar 

  • Gifford DJ, Greenwood JS, Bewley DJ (1982) Deposition of matrix and crystalloid storage proteins during protein body development in the endosperm of Ricinus communis L cv. Hale seeds. Plant Physiol 69:1471–1478

    Article  PubMed  CAS  Google Scholar 

  • Gifford DJ, Wenzel KA, Lammer DL (1989) Lodgepole pine seed germination I. Changes in peptidase activity in the megagametophyte and embryonic axis. Can J Bot 67:2539–2543

    Article  CAS  Google Scholar 

  • Groome MC, Axler SR, Gifford DJ (1991) Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic pattern following imbibition. Physiol Plant 83:99–106

    Article  CAS  Google Scholar 

  • Hara-Nishimura I, Hatsugai N, Nakaune S, Kuroyanagi M, Nishimura M (2005) Vacuolar processing enzyme: an executor of plant cell death. Curr Opin Plant Biol 8:1–5

    Article  Google Scholar 

  • He X, 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  CAS  Google Scholar 

  • Jensen U, Lixue C (1991) Abies seed protein profile divergent from other Pinaceae. Taxon 40:435–440

    Article  Google Scholar 

  • King JE, Gifford DJ (1997) Amino acid utilization in seeds of loblolly pine during germination and early seedling growth I. Arginine and arginase activity. Plant Physiol 113:1125–1135

    PubMed  CAS  Google Scholar 

  • Kumar S (2007) Caspase function in programmed cell death. Cell Death Differ 14:32–43

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Lammer DL, Gifford DJ (1989) Lodgepole pine seed germination II. The seed proteins and their mobilization in the megagametophyte and embryonic axis. Can J Bot 67:2544–2551

    Article  CAS  Google Scholar 

  • Mikkonen A (1992) Purification and characterization of leucine aminopeptidase from kidney bean cotyledons. Physiol Plant 84:393–398

    Article  CAS  Google Scholar 

  • Mikola L (1983) Germinating barley grains contain five acid carboxypeptidases with complementary substrate specificities. Biochim Biophys Acta 747:241–252

    Article  CAS  Google Scholar 

  • Muccilli V, Licciardello C, Fontanini D, Russo MP, Cunsolo V, Saletti R, Reforgiato Recupero G, Foti S (2009) Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time. J Proteomics 73:134–152

    Article  PubMed  CAS  Google Scholar 

  • Müntz K (1996) Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. J Exp Bot 47:605–622

    Article  Google Scholar 

  • Pak C, van Doorn WG (2005) Delay of Iris flower senescence by protease inhibitors. New Phytol 165:473–480

    Article  PubMed  CAS  Google Scholar 

  • Piszczek E, Gutman W (2007) Caspase-like proteases and their role in programmed cell death in plants. Acta Physiol Plant 29:391–398

    Article  CAS  Google Scholar 

  • Qi X, Wilson KA, Tan-Wilson A (1992) Characterization of the major proteinase involved in the soybean β-conglicinin storage protein mobilization. Plant Physiol 99:725–733

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Rotari VI, He R, Gallois P (2005) Death by proteases in plants: whodunit. Physiol Plant 123:376–385

    Article  CAS  Google Scholar 

  • Ryan CA, Walker-Simmons M (1981) Plant proteinases. In: Marcus A (ed) The biochemistry of plants. Proteins and nucleic acids. Academic Press, New York, pp 321–349

    Google Scholar 

  • Salmia MA, Mikola JJ (1976a) Localization and activity of a carboxypeptidase in germinating seeds of Scots pine, Pinus sylvestris. Physiol Plant 36:388–392

    Article  CAS  Google Scholar 

  • Salmia MA, Mikola JJ (1976b) Localization and activity of naphtylamidases in germinating seeds of scots pine, Pinus sylvestris. Physiol Plant 38:73–77

    Article  CAS  Google Scholar 

  • Salmia MA, Nyman SA, Mikola JJ (1978) Characterization of the proteinases present in germinating seeds of Scots pine, Pinus sylvestris. Physiol Plant 42:252–256

    Article  CAS  Google Scholar 

  • Sarath G, De La Motte RS, Wagner FW (1989) Protease assay methods. In: Beynon RJ, Bond JS (eds) Proteolytic enzymes: a practical approach. IRL Press, Oxford, pp 25–55

    Google Scholar 

  • Schlereth A, Standhardt D, Mock HP, Müntz K (2001) Stored cysteine proteinases start globulin breakdown in protein bodies of embryonic axes and cotyledons of germinating vetch (Vicia sativa L.) seeds. Planta 212:718–727

    Article  PubMed  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  PubMed  CAS  Google Scholar 

  • Shutov AD, Vaintraub IA (1987) Degradation of storage proteins in germinating seeds. Phytochemistry 26:1557–1566

    Article  CAS  Google Scholar 

  • Tishinov K, Stambolieva N, Petrova S, Galunsky B, Nedkov P (2009) Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase. Acta Physiol Plant 31:199–205

    Article  CAS  Google Scholar 

  • Wrobel R, Jones BL (1992) Appearance of endoproteolytic enzymes during the germination of barley. Plant Physiol 100:1508–1516

    Article  PubMed  CAS  Google Scholar 

  • Zhang N, Jones B (1999) Polymorphism of aspartic proteinases in resting and germinating barley seeds. Cereal Chem 76:134–138

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The Authors thank Mr. Franco Saviozzi for his precious technical assistance in the preparation of the present work.

Author information

Authors and Affiliations

  1. Department of Biology, University of Pisa, Via L. Ghini 5, 56126, Pisa, Italy

    Antonella Capocchi, Simone Casani, Luciano Galleschi & Debora Fontanini

  2. Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy

    Vera Muccilli & Salvatore Foti

Authors
  1. Antonella Capocchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vera Muccilli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simone Casani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Salvatore Foti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luciano Galleschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Debora Fontanini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Antonella Capocchi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Capocchi, A., Muccilli, V., Casani, S. et al. Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds. Planta 233, 817–830 (2011). https://doi.org/10.1007/s00425-010-1342-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1342-3

Keywords

Search

Navigation