Dataset Information

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.

SUBMITTER: Dhar N

PROVIDER: S-EPMC7307857 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Dhar Niha N Sarangapani Sreelatha S Reddy Vaishnavi Amarr VA Kumar Nadimuthu N Panicker Deepa D Jin Jingjing J Chua Nam-Hai NH Sarojam Rajani R

Journal of experimental botany 20200601 12

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 ...[more]

PMID: 32198522

Similar Datasets

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.

Project description:Prunus mume, a traditional Chinese flower, is the only species of Prunus known to produce a strong floral fragrance, of which eugenol is one of the principal components. To explore the molecular mechanism of eugenol biosynthesis in P. mume, patterns of dynamic, spatial and temporal variation in eugenol were analysed using GC-MS. Coniferyl alcohol acetyltransferase (CFAT), a member of the BAHD acyltransferase family, catalyses the substrate of coniferyl alcohol to coniferyl acetate, which is an important substrate for synthesizing eugenol. In a genome-wide analysis, we found 90 PmBAHD genes that were phylogenetically clustered into five major groups with motif compositions relatively conserved in each cluster. The phylogenetic tree showed that the PmBAHD67-70 proteins were close to the functional CFATs identified in other species, indicating that these four proteins might function as CFATs. In this work, 2 PmCFAT genes, named PmCFAT1 and PmCFAT2, were cloned from P. mume 'Sanlunyudie', which has a strong fragrance. Multiple sequences indicated that PmCFAT1 contained two conserved domains, HxxxD and DFGWG, whereas DFGWG in PmCFAT2 was changed to DFGFG. The expression levels of PmCFAT1 and PmCFAT2 were examined in different flower organs and during the flowering stages of P. mume 'Sanlunyudie'. The results showed that PmCFAT1 was highly expressed in petals and stamens, and this expression increased from the budding stage to the full bloom stage and decreased in the withering stage, consistent with the patterns of eugenol synthesis and emission. However, the peak of gene expression appeared earlier than those of eugenol synthesis and emission. In addition, the expression level of PmCFAT2 was higher in pistils and sepals than in other organs and decreased from the budding stage to the blooming stage and then increased in the withering stage, which was not consistent with eugenol synthesis. Subcellular localization analysis indicated that PmCFAT1 and PmCFAT2 were located in the cytoplasm and nucleus, while enzyme activity assays showed that PmCFAT1 is involved in eugenol biosynthesis in vitro. Overall, the results suggested that PmCFAT1, but not PmCFAT2, contributed to eugenol synthesis in P. mume.

Project description:Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium-mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1-bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9-mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene-free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.

Dataset Information

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Publications

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets