Analysis of plant genes involved in aromatic volatile production
The cDNA CM-AATI from melon was expressed as a fusion protein in yeast, Saccharomyces cerevisiae. The protein exhibited alcohol acyl-transferase (AAT) activity, producing ester compounds from a wide range of alcohols and acyl-CoAs. A second cDNA clone, Le-AAT1 was identified by heterologous screening of a tomato fruit library with the melon CM-AAT1 probe. The amino acid sequence of the encoded protein showed some similarities to many proteins using acyl-CoAs as substrates, including CM-AAT1. The Le-AAT1 open reading frame (ORF) consists of 1329 nucleotides, encoding 442 amino acids, while the CM-AATI ORF is 1431 nucleotides in length with a deduced sequence of 476 amino acids. Although the Le-AAT1 showed 43% identity to the CM-AAT1 at the amino acid level, the yeast expressed protein demonstrated AAT activity. On the other hand, a second melon clone, CM-AAT2, encoding a 475 amino acid protein, which is 86% identical to the CM-AAT1 protein, did not show AAT activity. The CM-AAT1 fusion protein was active over pH 6.0 to pH 8.0 in vitro with activity being enhanced by Mg2+, whereas the Le-AAT1 protein performed efficiently at pH between 6.0 and 9.0 with Na+. The activity of the CM-AAT1 protein probably requires posttranslational modifications since the protein expressed in Escherichia coli was inactive. Northern analysis of RNA from a range of tissues including developing fruit showed that the melon CM-AAT1 and the tomato Le-AAT1 are fruit ripening specific genes. The endogenous CM-AAT1 mRNA was ethylene inducible and the expression dramatically reduced in transgenic low ethylene melon. The expression of Le-AAT1 mRNA was also enhanced by exogenous ethylene, but the levels were still high in low ethylene tomatoes. The ADH2 protein, which can transform aldehydes to alcohols, was found to be expressed in various organs of tomato and highly expressed at the late ripening stages. Exogenous ethylene could not induce high accumulation of the ADH2 mRNA. Tomato plants were transformed with gene constructs containing the CM-AATI sense and partial Le-AATI antisense cDNAs with either CaMV 35S promoter or tomato ACO I promoter. The production of all selected volatile compounds greatly increased during tomato fruit ripening. Although ester volatiles were rarely generated at the start of fruit development, they were produced in significant amounts as ripening proceeded, but were still low, compared to other volatiles. There was, however, no statistical difference of the ester concentrations in fruit between control and transgenic tomatoes.