, Volume 250, Issue 2, pp 623–629 | Cite as

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements

  • Maria Mulisch
  • Torben AspEmail author
  • Karin Krupinska
  • Julien Hollmann
  • Preben Bach Holm
Short Communication


Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.


Cysteine protease Endoplasmic reticulum Immunolocalization Tracheary element differentiation Trifolium repens 



Sanne Seval and Ole Braad Hansen, Aarhus University, are acknowledged for technical and horticultural assistance, respectively. Anke Schäfer, University of Kiel, is acknowledged for purification of the antibody. Marita Beese is acknowledged for assistance in the electron microscopical preparations. The project was supported by the Danish Food Industry Agency through the programme “Biotechnology in Food Research”.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Asp T, Bowra S, Borg S, Holm P (2004a) Cloning and characterisation of three groups of cysteine protease genes expressed in the senescing zone of white clover (Trifolium repens) nodules. Plant Sci 167:825–837CrossRefGoogle Scholar
  2. Asp T, Bowra S, Borg S, Holm P (2004b) Molecular cloning, functional expression in Escherichia coli and enzymatic characterisation of a cysteine protease from white clover (Trifolium repens). Biochim Biophys Acta 1699:111–122PubMedGoogle Scholar
  3. Avci U, Petzold HE, Ismail IO, Beers EP, Haigler CH (2008) Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots. Plant J 56:303–315PubMedCrossRefGoogle Scholar
  4. Beers EP, Jones AM, Dickerman AW (2004) The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis. Phytochemistry 65:43–58PubMedCrossRefGoogle Scholar
  5. Cacas J-L (2010) Devil inside: does plant programmed cell death involve the endomembrane system? Plant Cell Environ 33:1453–1473PubMedGoogle Scholar
  6. Chiba A, Ishida H, Nishizawa NK, Makino A, Mae T (2003) Exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol 44:914–921PubMedCrossRefGoogle Scholar
  7. Esteban-Garcia B, Antonio Garrido-Cardenas J, Lopez Alonso D, Garcia-Maroto F (2010) A distinct subfamily of papain-like cystein proteinases regulated by senescence and stresses in Glycine max. J Plant Physiol 167:1101–1108PubMedCrossRefGoogle Scholar
  8. Fukuda H (1996) Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol 47:299–325CrossRefGoogle Scholar
  9. Fukuda H (2000) Programmed cell death of tracheary elements as a paradigm in plants. Plant Mol Biol 44:245–253PubMedCrossRefGoogle Scholar
  10. Fukuda H (2004) Signals that control plant vascular cell differentiation. Nat Rev Mol Cell Biol 5:379–391PubMedCrossRefGoogle Scholar
  11. Fukuda H, Komamine A (1980a) Direct evidence for cyto-differentiation to tracheary elements without intervening mitosis in a culture of single cells isolated from the mesophyll of Zinnia elegans. Plant Physiol 65:61–64PubMedCrossRefGoogle Scholar
  12. Fukuda H, Komamine A (1980b) Establishment of an experimental system for the tracheary element differentiation from single cells isolated from mesophyll of Zinnia elegans. Plant Physiol 65:57–60PubMedCrossRefGoogle Scholar
  13. Funk V, Kositsup B, Zhao C, Beers E (2002) The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol 128:84–94PubMedCrossRefGoogle Scholar
  14. Gan S, Amasino R (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986–1988PubMedCrossRefGoogle Scholar
  15. Gietl C, Schmid M (2001) Ricinosomes: an organelle for develpmentally regulated programmed cell death in senescing plant tissues. Naturwissenschaften 88:49–58PubMedCrossRefGoogle Scholar
  16. Groover A, Jones AM (1999) Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 119:375.384PubMedCrossRefGoogle Scholar
  17. Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouzé P, Brunak S (1996) Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res 24:3439–3452PubMedCrossRefGoogle Scholar
  18. Horak M, Wenthold RJ (2009) Different roles of C-terminal cassettes in the trafficking of full-length NR1 subunits to the cell surface. J Biol Chem 284:9683–9691PubMedCrossRefGoogle Scholar
  19. Ito J, Fukuda H (2002) ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements. Plant Cell 14:3201–3211PubMedCrossRefGoogle Scholar
  20. Jung J-H, Kim S-G, Seo PJ, Park C-M (2008) Molecular mechanisms underlying vascular development. Adv Bot Res 48:1–68CrossRefGoogle Scholar
  21. Kim J, Johannes L, Goud B, Antony C, Lingwood C, Daneman R, Grinstein S (1998) Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release. Proc Natl Acad Sci USA 95:2997–3002PubMedCrossRefGoogle Scholar
  22. Kuriyama H, Fukuda H (2001) Regulation of tracheary element differentiation. J Plant Growth Regul 20:35–51CrossRefGoogle Scholar
  23. Lehmann K, Hause B, Altmann D, Kock M (2001) Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence. Plant Physiol 127:436–449PubMedCrossRefGoogle Scholar
  24. Minami A, Fukuda H (1995) Transient and specific expression of a cysteine endopeptidase associated with autolysis during differentiation of Zinnia mesophyll cells into tracheary elements. Plant Cell Physiol 36:1599–1606PubMedGoogle Scholar
  25. Obara K, Kuriyama H, Fukuda H (2001) Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia. Plant Physiol 125:615–626PubMedCrossRefGoogle Scholar
  26. Okamoto T, Minamikawa T (1998) A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: characterization, regulation of gene expression, and posttranslational processing. J Plant Physiol 152:675–682CrossRefGoogle Scholar
  27. Okamoto T, Toyooka K, Minamikawa T (2001) Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole. J Biol Chem 276:742–751PubMedCrossRefGoogle Scholar
  28. Pennell R, Lamb C (1997) Programmed cell death in plants. Plant Cell 9:1157–1168PubMedCrossRefGoogle Scholar
  29. Pyo H, Demura T, Fukuda H (2004) Spatial and temporal tracing of vessel differentiation in young Arabidopsis seedlings by the expression of an immature tracheary element-specific promoter. Plant Cell Physiol 45:1529–1536PubMedCrossRefGoogle Scholar
  30. Reynolds R (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–212PubMedCrossRefGoogle Scholar
  31. Schmid M, Simpson D, Gietl C (1999) Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes. Proc Natl Acad Sci USA 96:14159–14164PubMedCrossRefGoogle Scholar
  32. Taylor MA, Baker KC, Briggs GS, Connerton IF, Cummings NJ, Pratt KA, Revell DF, Freedman RB, Goodenough PW (1995) Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes. Protein Eng 8:59–62PubMedCrossRefGoogle Scholar
  33. Toyooka K, Okamoto T, Minamikawa T (2000) Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmic reticulum-derived vesicle is involved in protein mobilization in germinating seeds. J Cell Biol 148:453–464PubMedCrossRefGoogle Scholar
  34. Tsuru-Furuno A, Okamoto T, Minamikawa T (2001) Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings. Plant Cell Physiol 42:1062–1070PubMedCrossRefGoogle Scholar
  35. Turner S, Gallois P, Brown D (2007) Tracheary element differentiation. Annu Rev Plant Biol 58:407–433PubMedCrossRefGoogle Scholar
  36. van der Hoorn RAL (2008) Plant proteases: from phenotypes to molecular mechanisms. Annu Rev Plant Biol 59:191–223PubMedCrossRefGoogle Scholar
  37. Watson M (1958) Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4(4):475–478PubMedCrossRefGoogle Scholar
  38. Woffenden B, Freeman T, Beers E (1998) Proteasome inhibitors prevent tracheary element differentiation in Zinnia mesophyll cell cultures. Plant Physiol 118:419–429PubMedCrossRefGoogle Scholar
  39. Yamamoto R, Demura T, Fukuda H (1997) Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol 38:980–983PubMedCrossRefGoogle Scholar
  40. Ye Z, Varner J (1996) Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol 30:1233–1246PubMedCrossRefGoogle Scholar
  41. Zhao C, Johnson B, Kositsup B, Beers E (2000) Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases. Plant Physiol 123:1185–1196PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Maria Mulisch
    • 2
  • Torben Asp
    • 1
    Email author
  • Karin Krupinska
    • 2
  • Julien Hollmann
    • 2
  • Preben Bach Holm
    • 1
  1. 1.Faculty of Science and Technology, Department of Molecular Biology and Genetics, Research Centre FlakkebjergAarhus UniversitySlagelseDenmark
  2. 2.Institute of Botany and Central MicroscopyChristian-Albrechts-University KielKielGermany

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