Proteomic and biochemical evidence links the callose synthase in Nicotiana alata pollen tubes to the product of the NaGSL1 gene

Authors

  • Lynette Brownfield,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia
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    • Present address: Department of Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK.

  • Kris Ford,

    1. Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, Victoria 3010, Australia
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  • Monika Susanne Doblin,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia
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  • Ed Newbigin,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia
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  • Steve Read,

    1. School of Forest and Ecosystem Science, University of Melbourne, Creswick, Victoria 3363, Australia
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    • Present address: Division of Forest Research & Development, Forestry Tasmania, Hobart, Tamania 7001, Australia.

  • Antony Bacic

    Corresponding author
    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia
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(fax +61 3 9347 1071; e-mail abacic@unimelb.edu.au).

Summary

The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-β-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa β-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.

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