Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit.
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.
New Zealand Institute for Plant and Food Research Limited, Mount Albert Research Centre, Auckland 1142, New Zealand.
Niels J
NJ
Nieuwenhuizen
Ratnasiri
R
Maddumage
Gianna K
GK
Tsang
Lena G
LG
Fraser
Janine M
JM
Cooney
H Nihal
HN
De Silva
Sol
S
Green
Kim A
KA
Richardson
Ross G
RG
Atkinson
D029042:Actinidia / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D002688:Chitinases / Q000378:metabolism; D002874:Chromosome Mapping; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary; D016368:Frameshift Mutation; D005780:Gelatin / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D040641:Quantitative Trait Loci; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D010940:Plant Proteins; D005780:Gelatin; D002688:Chitinases; D003546:Cysteine Endopeptidases; C018745:actinidain