Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes

Abstract

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.

Figure 1

Functional genomics approach to evaluate ESTs from a peppermint oil gland secretory cell cDNA library. (A) Flow diagram of program. (B) Light micrograph of oil gland secretory cell clusters isolated from peppermint leaves (Bar = 200 μm).

Figure 2

Functional classification of ESTs from the peppermint oil gland secretory cell cDNA library. Secondary metabolism accounts for about 35% of total metabolism.

Figure 3

(A) Outline of monoterpene biosynthesis in peppermint glandular trichomes. The enzymes indicated are: (−)-limonene synthase (1), (−)-limonene-3-hydroxylase (2), (−)-isopiperitenone reductase (3), (+)-pulegone reductase (4), (+)-menthofuran synthase (5), (−)-menthol acetyltransferase (6), and 1,8-cineole synthase (7). (B) Structures of selected sesquiterpenes of peppermint oil.