Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple

Abstract

Polyphenol oxidase (PPO), a copper-containing metalloprotein, catalyzes the oxidation of phenolics to quinones which make brown pigments in wounded tissues. Because the phenomena decrease fruit quality, PPO has been regarded to be a critical enzyme in food technology. In the course of expressed sequence tags (ESTs) analysis of the Fuji apple (Malus domesticus Borkh.), we identified two partial PPO cDNA clones; F114 corresponded to the previously isolated Granny Smith apple pAPO5 (Plant Mol. Biol. 27 (1995) 429), while F226 was a new clone. Using F226 as a probe, we isolated a full length PPO clone, pMD-PPO2, from a cDNA library prepared from young fruits of the Fuji apple. The deduced amino acid sequences of pMD-PPO2 and pAPO5 share 55% identity, and display a high degree of sequence identity (43–58%) with previously identified PPO from various species. RNA gel blot analysis using gene-specific probes showed that two apple PPO genes display unique patterns of expression in a tissue- and developmental-specific manner. In the process of flower development, the APO5 transcript was detectable only at the post-anthesis stage. In contrast, MD-PPO2 was expressed in all stages of flower development, with the abundance of mRNA being the highest at the pre-anthesis stage and then receding as the flower developed. Both genes are expressed in the early stages of fruit development. The expression was dramatically reduced as the fruit ripened. In leaf tissue, the APO5 gene was highly expressed in young and immature leaves, while MD-PPO2 was transcriptionally more active in both immature and mature leaves. Upon wounding, APO5 was significantly induced in leaves and fruits, whereas the level of MD-PPO2 mRNA was not affected by mechanical damage. Thus, it appears that two Fuji PPO genes are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple plants. The possible molecular mechanism of differential regulation of PPO gene expression in apple plant and its physiological significance are discussed.