Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit
Niels J. Nieuwenhuizen A , Lesley L. Beuning A , Paul W. Sutherland A , Neelam N. Sharma A , Janine M. Cooney B , Lara R. F. Bieleski A , Roswitha Schröder A , Elspeth A. MacRae A C and Ross G. Atkinson A DA The Horticulture and Food Research Institute of New Zealand, Mount Albert Research Centre, Private Bag 92 169, Auckland 1142, New Zealand.
B The Horticulture and Food Research Institute of New Zealand, Ruakura, Private Bag 3123, Hamilton 3240, New Zealand.
C Present address: Scion, Private Bag 3020, Rotorua, New Zealand.
D Corresponding author. Email: ratkinson@hortresearch.co.nz
Functional Plant Biology 34(10) 946-961 https://doi.org/10.1071/FP07121
Submitted: 16 May 2007 Accepted: 7 August 2007 Published: 13 September 2007
Abstract
Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa ‘Hayward’ (green-fleshed kiwifruit) and A. chinensis ‘Hort16A’ and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in ‘Hort16A’, the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.
Additional keywords: Actinidia, fruit.
Acknowledgements
We thank all members of the kiwifruit genomics programme team at HortResearch. Special thanks to Di Barraclough for advice on 2-D PAGE gels, Sean Bulley for preparing the protein samples used in Fig. 6 and William Laing and Richard Newcomb for critically reviewing the manuscript. This work was funded by the Foundation for Research, Science and Technology of New Zealand and the HortResearch Internal Investment Fund HII 06–04.
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