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First published online September 30, 2005; 10.1105/tpc.105.034678

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The Plant Cell 17:3176-3189 (2005)
© 2005 American Society of Plant Biologists

A Continent of Plant Defense Peptide Diversity: Cyclotides in Australian Hybanthus (Violaceae){boxw}

Shane M. Simonsen, Lillian Sando, David C. Ireland, Michelle L. Colgrave, Rekha Bharathi, Ulf Göransson1 and David J. Craik2

Institute for Molecular Bioscience, University of Queensland, Australian Research Council Special Research Centre for Functional and Applied Genomics, Brisbane 4072, Australia

2 To whom correspondence should be addressed. E-mail d.craik{at}imb.uq.edu.au; fax 61-7-3346 2029.

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.




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