Opposite accumulation patterns of two glycoside hydrolase family 3 α-L-arabinofuranosidase proteins in avocado fruit during ripening

Abstract

Avocado fruit ripen with ethylene production after harvest and the flesh becomes soft and edible due to degradation of cell wall polysaccharides during ripening. α-L-Arabinofuranosidase is a hydrolytic enzyme known to digest arabinose-containing cell wall polysaccharides. It has been shown that its activity increased with fruit ripening. However, our previous study showed that an α-L-arabinofuranosidase gene (PaArf/Xyl3A) is expressed in the avocado fruit before ethylene production. In addition, the transcripts were detected in some organs in which the level of ethylene was extremely low. These results indicate that the gene expression is independent of ethylene. In the present study, we carried out immunoblot analyses of α-L-arabinofuranosidase at the protein level. Using a polyclonal antibody raised against Japanese pear α-L-arabinofuranosidase, two α-L-arabinofuranosidase proteins with molecular masses of 72 kDa and 68 kDa, presumably belonging to glycoside hydrolase family 3, were detected in ripening avocado fruit. The protein levels in ethylene or 1-methylcyclopropene (1-MCP)-treated fruits were examined and the results indicated that the two proteins responded to ethylene in opposite ways; the 68 kDa protein showed a temporary accumulation, whereas the 72 kDa protein exhibited dissipation possibly caused by a loss of stability. The total enzyme activity of α-L-arabinofuranosidase was elevated faster in the ethylene-treated fruit throughout ripening and was slower in the 1-MCP-treated fruit, suggesting the existence of another α-L-arabinofuranosidase, which did not cross-react with the antibody and was positively regulated by ethylene, in ripening avocado fruit.