Dataset Information

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

ABSTRACT: Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

SUBMITTER: Reddy VA

PROVIDER: S-EPMC7826958 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Reddy Vaishnavi Amarr VA Li Chunhong C Nadimuthu Kumar K Tjhang Jessica Gambino JG Jang In-Cheol IC Rajani Sarojam S

International journal of molecular sciences 20210112 2

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in <i>E. coli</i> showed thei ...[more]

PMID: 33445552

Similar Datasets

Project description:BackgroundSesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.ResultsGlandular trichomes of sunflower (Helianthus annuus L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon delta-cadinene as one of the two main products with alpha-muuorlene, beta-caryophyllene, alpha-humulene and alpha-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.ConclusionThis study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.

Project description:Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets