Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit1[C][W][OA]

• Published in: Plant physiology (Bethesda) Vol. 158; no. 1; pp. 376 - 388
• Main Authors: Nieuwenhuizen, Niels J., Maddumage, Ratnasiri, Tsang, Gianna K., Fraser, Lena G., Cooney, Janine M., De Silva, H. Nihal, Green, Sol, Richardson, Kim A., Atkinson, Ross G.
• Format: Journal Article
• Language: English
• Published: American Society of Plant Biologists 28-10-2011
• Subjects: Genetics, Genomics, and Molecular Evolution
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.111.187989

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of
Actinidia
species (kiwifruit) exhibit a range of CP activities (e.g. the
Actinidia
chinensis
variety YellowA shows less than 2% of the activity of
Actinidia
deliciosa
variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of
A. chinensis
. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the
ACT1A-1
allele. Sequence analysis indicated that the
ACT1A
locus in the segregating
A. chinensis
population contained one functional allele (
A-2
) and three nonfunctional alleles (
a-3
,
a-4
, and
a-5
) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles:
a-6
, which was nonfunctional because of a large insertion, and
a-7
, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional
ACT1A-1
cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.