Detection of a plant enzyme exhibiting chlorogenate-dependant caffeoyltransferase activity in methanolic extracts of arbuscular mycorrhizal tomato roots

Abstract

When Glomus intraradices-colonised tomato roots were extracted in methanol at 6 °C, chlorogenic acid (5-caffeoylquinic acid), naturally present in the extract, was slowly converted by transesterification into methyl caffeate. The progress of the reaction could be monitored by HPLC. The reaction only occurred when the ground roots were left in contact with the hydro-alcoholic extract and required the presence of 15-35% water in the mixture. When the roots were extracted in ethanol, chlorogenic acid was transformed to ethyl caffeate in the same conditions. The reaction was also detected in Glomus mosseae-colonised tomato root extracts. It was also detectable in non-mycorrhizal root extracts but was 10-25 times slower. By contrast it was undetectable in extracts of the aerial parts of tomato plants, which also contain high amounts of chlorogenic acid, whether or not these plants were inoculated by the arbuscular mycorrhizal fungus. We found that this transesterification reaction is catalysed by a tomato enzyme, which remains active in hydro-alcoholic mixtures and exhibits chlorogenate-dependant caffeoyltransferase activity in the presence of methanol or ethanol. This transferase activity is inhibited by phenylmethanesulfonyl fluoride. The 4- and 3-caffeoylquinic acid isomers were also used as substrates but were less active than chlorogenic acid. Highest activity was detected in mycorrhizal roots of nutrient-deprived tomato plants. Surprisingly this caffeoyltransferase activity could also be detected in hydro-alcoholic extracts of G. intraradices-colonised roots of leek, sorghum or barrel medic.