Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis

Abstract

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf–PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil.

The genome sequence of tetraploid sweet basil, Ocimum basilicum L., provides tools for advanced genome editing and molecular breeding

“…We used BLASTN to assign the known genes to their genomic location and to their Gene ID. The coding genes of the four enzymes, p -coumaroyl shikimate 3′-hydroxylase (CS3′H), 50 eugenol synthase (EGS), 51 coniferyl alcohol acetyltransferase (CAAT) 52 and eugenol O -methyl-transferase (EOMT), 53 were characterized for eugenol/methyl eugenol biosynthesis.…”

“…Values are means of three biological repeats ± SE (leaves and flowers), or of one biological repeat (stem and roots). ObCS3 ′ H1 / 2 are the genes characterized by Gang et al 50 ; ObEGS1 is the gene characterized by Koeduka et al 51 ; and ObCAAT1 is the gene sequenced and silenced by Dhar et al 52 …”

Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

“…Apart from ObCAAT2 , RNA-seq data of root revealed several other BAHD family AAT transcripts like ObCAAT2 . BAHD enzymes are known to display substrate versatility by accepting other alcohol substrates and thereby functioning in multiple pathways [ 21 , 49 ]. In aseptically grown roots expression of ObCAAT2 is observed.…”

“…The first functionally characterized CAAT was PhCFAT from petunia [ 20 ]. Recently, two CAATs, ObCAAT1 and ObCAAT2 (PhCFAT homologue) were identified and characterized from sweet basil involved in eugenol synthesis [ 21 ]. Apart from ObEGS1, EGSs have been characterized from few other plants also such as Petunia (PhEGS1) [ 19 ], Gymnadenia odoratissima (GoEGS1 and GoEGS2) [ 22 ], Clarkia breweri (CbEGS1 and CbEGS2) [ 23 ], rose (RcEGS1) [ 24 ], strawberry (FaEGS1a and FaEGS1b) [ 25 ], and carrot (DcE(I)GS1) [ 26 ].…”

“…To gain a comprehensive knowledge about eugenol production in sweet basil and eugenol synthases involved in its synthesis in PGTs and roots, a functional genomics approach was pursued. In our lab we have previously performed RNA-Seq of four tissues of sweet basil [leaf (L), leaf stripped of PGTs (L-T), roots (R) and PGTs (T)] [ 21 ]. The RNA seq data showed the absence of ObEGS1 expression in roots.…”