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A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

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Abstract

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death.

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Acknowledgment

The authors thank JoAnn Trejo for advice and reagents. This work was supported by a grant from NSF, Developmental Mechanisms (IBN-9807801). Yair Heimer was supported by a UNC-Israel collaboration grant.

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Authors and Affiliations

  1. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 25799, USA

    Xiao-Hong Yu, Brian Jones, Alan M. Jones & Yair M. Heimer

  2. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 25799, USA

    Alan M. Jones

  3. Department of Dryland Biotechnologies, J. Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, 84990, Israel

    Yair M. Heimer

  4. Present address of X.-H. Yu: 111 Koshland Hall, Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, California 94720-3102, USA

    Xiao-Hong Yu

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  1. Xiao-Hong Yu
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  2. Brian Jones
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  3. Alan M. Jones
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  4. Yair M. Heimer
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Correspondence to Alan M. Jones or Yair M. Heimer.

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Yu, XH., Jones, B., Jones, A.M. et al. A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation. J Plant Growth Regul 23, 292–300 (2004). https://doi.org/10.1007/s00344-004-0409-4

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