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Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata

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Abstract

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.

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Abbreviations

PI:

Proteinase inhibitor

TGN:

trans-Golgi network

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Acknowledgments

We thank Greg Neumann (La Trobe University) for performing the mass spectrometry and Ingrid Bönig and Dr. Simon Crawford from the University of Melbourne for their expert assistance with electron microscopy. The anti-BiP antibodies were the kind gift of Maarten Chrispeels (University of California). We thank Marcus Lee and Fung Lay from La Trobe University for critically reading the manuscript and for assistance with preparation of the figures. This research was supported by a grant from the Australian Research Council (grant no. 80896) to M.A.A. and E.D.J. and an Australian Postgraduate Award to E.A.M.

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Authors and Affiliations

  1. Department of Biochemistry, La Trobe University, 3086, Melbourne, VIC, Australia

    Elizabeth D. Johnson & Marilyn A. Anderson

  2. Department of Biological Sciences, Columbia University, New York, NY, USA

    Elizabeth A. Miller

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  1. Elizabeth D. Johnson
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  2. Elizabeth A. Miller
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Correspondence to Elizabeth D. Johnson.

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Johnson, E.D., Miller, E.A. & Anderson, M.A. Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata . Planta 225, 1265–1276 (2007). https://doi.org/10.1007/s00425-006-0418-6

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