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Plants have evolved a plethora of chemical solutions to help them cope with dynamic biotic and abiotic environments, and much of this diversity was used throughout human history for medicinal and hedonistic purposes (seasoning, preservatives, etc.). Despite the enormous progress made in the last decade in elucidating the genes and enzymes involved in these metabolic pathways using classical biochemical approaches and biochemical genomic methods, very few studies applied targeted and non-targeted mutagenesis for isolating novel genes to study their hitherto unknown function. This minireview will discuss recent examples of forward and reverse genetics approaches taken in some medicinal plants and herbs, and will present data that we obtained from an ethylmethane sulfate mutagenesis project in Ocimum basilicum (sweet basil), aiming to identify new genes that are involved in the biosynthesis of aroma compounds. [ABSTRACT FROM AUTHOR]

Surface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (US) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frame shift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes. [ABSTRACT FROM AUTHOR]

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn[sup 2+] as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. K[sub m] values of 21 and 51 µm were obtained for geranyl diphosphate and Mn[sup 2+], respectively. In the presence of [sup 18]O-labeled water, GES catalyzed the formation of [sup 18]O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. AGES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral. [ABSTRACT FROM AUTHOR]

Abstract: Basil glands of the Sweet Dani cultivar contain high levels of citral, a mixture of geranial and its cis-isomer neral, as well as low levels of geraniol and nerol. We have previously reported the identification of a cDNA from Sweet Dani that encodes an enzyme responsible for the formation of geraniol from geranyl diphosphate in the glands, and that these glands cannot synthesize nerol directly from geranyl diphosphate. Here, we report the identification of two basil cDNAs encoding NADP+-dependent dehydrogenases that can use geraniol as the substrate. One cDNA, designated CAD1, represents a gene whose expression is highly specific to gland cells of all three basil cultivars examined, regardless of their citral content, and encodes an enzyme with high sequence similarity to known cinnamyl alcohol dehydrogenases (CADs). The enzyme encoded by CAD1 reversibly oxidizes geraniol to produce geranial (which reversibly isomerizes to neral via keto–enol tautomerization) at half the efficiency compared with its activity with cinnamyl alcohol. CAD1 does not use nerol and neral as substrates. A second cDNA, designated GEDH1, encodes an enzyme with sequence similarity to CAD1 that is capable of reversibly oxidizing geraniol and nerol in equal efficiency, and prolonged incubation of geraniol with GEDH1 in vitro produces not only geranial and neral, but also nerol. GEDH1 is also active, although at a lower efficiency, with cinnamyl alcohol. However, GEDH1 is expressed at low levels in glands of all cultivars compared with its expression in leaves. These and additional data presented indicate that basil glands may contain additional dehydrogenases capable of oxidizing geraniol. [Copyright &y& Elsevier]