Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit

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.

Differential Constitution in Promoter Region Leads to a Phenotype with a Lower Allergic Actinidin Level in Yellow-fleshed Kiwifruit (<i>Actinidia chinensis</i>)

“…On the other hand, actinidin was almost absent from two A. chinensis cultivars, 'Kohi' and 'Hort16A', and A. eriantha. Although the lower actinidin level in 'Hort16A' has already been reported (Maddumage et al, 2013;Nieuwenhuizen et al, 2007), 'Kohi' was newly found to contain only a little actinidin in this study. In the CBB-stained gel, a protein with slightly lower molecular mass than actinidin was detected in 'Koshin', 'Kohi', and 'Hort16A' (Fig.…”

“…S1), this cultivar could be a valuable source for breeding. To address the molecular basis for the lower actinidin level in 'Kohi', we first analyzed the gene expression of the actinidin-coding genes, Act1a and Act2a, because they have been shown to be detected in the ripe fruits of A. deliciosa 'Hayward' and A. chinensis 'Hort16A' (Nieuwenhuizen et al, 2007). Genomic PCR first confirmed the presence of Act1a and Act2a in 'Kohi' as well as in 'Hayward' and 'Hort16A' (Fig.…”

“…For instance, A. deliciosa cultivars in general contain actinidin as a major protein, but many of the A. eriantha and A. rufa genotypes do not accumulate a lot of the protein (Maddumage et al, 2013;Nishiyama, 2007). A great diversity in one species has been found in yellowfleshed A. chinensis (Maddumage et al, 2013;Nieuwenhuizen et al, 2007;Zhang et al, 2017 (Nieuwenhuizen et al, 2007). A recent genetic study using a segregating population of A. chinensis determined a region containing Act1a, an actinidin-coding gene, as a major quantitative trait locus for Cys protease activity (Nieuwenhuizen et al, 2012).…”

“…A. chinensis 'Hort16A', commercially known as "Zespri Gold", has become globally popular because of its yellow-fleshed sweet and tropical flavored fruit. Another big advantage of this cultivar is its low actinidin level (Nieuwenhuizen et al, 2007). Given that global production of kiwifruit has increased over the last few decades (Ward and Courtney, 2013), introduction of low actinidin cultivars will help to grow the market share of kiwifruit.…”

“…Because of its significant content, the protein has been extensively studied from biochemical and application aspects (Boland, 2013;Nishiyama, 2007). Actinidin possesses cysteine (Cys) protease activity and belongs to a papain-like subfamily that includes papain from papaya (Carica papaya), bromelain from pineapple (Ananas (Nieuwenhuizen et al, 2007), suggesting its unique role in kiwifruit. While actinidin is not thought to be an essential protein for fruit development because no phenotypic defect has been found in actinidin-deficient varieties (Boland, 2013), some studies suggested its potential roles in pathogen defense through processing of a class IV chitinase (Nieuwenhuizen et al, 2012) and insect resistance (Malone et al, 2005).…”