Characterization and functional analysis of eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L.

Abstract

Eugenol-O-methyltransferase (EOMT) catalyzes the conversion of eugenol to methyleugenol in one of the final steps of phenylpropanoid pathway. There are no comprehensive reports on comparative EOMT gene expression and developmental stage specific accumulation of phenylpropenes in Ocimum tenuiflorum. Seven chemotypes, rich in eugenol and methyleugenol, were selected by assessment of volatile metabolites through multivariate data analysis. Isoeugenol accumulated in higher levels during juvenile stage (36.86 ng g(-1)), but reduced sharply during preflowering (8.04 ng g(-1)), flowering (2.29 ng g(-1)) and postflowering stages (0.17 ng g(-1)), whereas methyleugenol content gradually increased from juvenile (12.25 ng g(-1)) up to preflowering (16.35 ng g(-1)) and then decreased at flowering (7.13 ng g(-1)) and post flowering (5.95 ng g(-1)) from fresh tissue. Extreme variations of free intracellular and alkali hydrolysable cell wall released phenylpropanoid compounds were observed at different developmental stages. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90 bp intron. The translated proteins had eight catalytic domains, the major two being dimerisation superfamily and methyltransferase_2 superfamily. A validated 3D structure of EOMT protein was also determined. The chemotype Ot7 had a reduced reading frame that lacked both dimerisation domains and one of the two protein-kinase-phosphorylation sites; this was also reflected in reduced accumulation of methyleugenol compared to other chemotypes. EOMT transcripts showed enhanced expression in juvenile stage that increased further during preflowering but decreased at flowering and further at postflowering. The expression patterns may possibly be compared and correlated to the amounts of eugenol/isoeugenol and methyleugenol in different developmental stages of all chemotypes.

Composition variation of the essential oil from Ocimum basilicum L. cv. Genovese Gigante in response to Glomus intraradices and mild water stress at different stages of growth

“…The linalool compound in the samples of the control and the treatments was the main compound observed, varying between 46 and 55%, which is in accordance with the relative percentage of linalool obtained in samples from this cultivar during the flowering stage (Marotti et al, 1996). Regarding eugenol, Renu et al (2014) reported that there is a higher level of eugenol-o-methyltransferase (EOMT) transcripts during the juvenile and pre-flowering stage and that, conversely, a remarkable decrease of EOMT transcripts occurs during the post-flowering stages of several chemotypes, which indicates an extensive transcriptional reprogramming associated with decreased accumulation of the metabolites. Similarly, during the flowering stage, when the 2nd harvest was carried out, the essential oil samples presented relatively low amounts of methyleugenol and eugenol, which decreased from the 1st to the 2nd harvest, independent of the treatment applied.…”

Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq.

“…Furthermore, eugenol synthase genes (EGS) have also been reported in some species such as Rose, Ocimum, and Gymnadenia [28,29,30]. The genes coding O -methyltransferases (EOMT) involved in methyleugenol have been isolated and functionally characterized in loquat ( Eriobotrya japonica ), Ocimum tenuiflorum , Clarkia breweri , sweet basil ( Ocimum basilicum ), and ripe apple fruit [31,32,33,34,35]. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195), a member of alcohol dehydrogenase, functions in converting hydroxycinnamyl aldehydes to corresponding alcohols, the various direct precursors of lignin [36], among which the coniferyl alcohol also acts as the precursor in methyleugenol boosynthesis.…”

Development of a rapid and high frequency Agrobacterium rhizogenes mediated transformation protocol for Ocimum tenuiflorum

“…Green forma of O. tenuiflorum showed high transformation frequency and leaves were the best among the explants in terms of both transformation efficiency and frequency. Though it is hard to explain as to why hairy roots developed more in green forma leaves, in an earlier study (Renu et al 2014) it was observed that green plants accumulated more signaling phenolics like phenylpropenes whereas red plants accumulated more anthocyanins. These phenylpropenes, like acetosyringone might influence more hairy root formation in the green forma.…”

“…The red forma accumulates the pigment anthocyanin that is very reactive towards most reactive oxygen species (ROS) because of their electron deficiency (Galvano et al 2004) and thus contributes towards health promoting properties as potent antioxidants. Both red and green forma plants harbor a wide diversity of volatile essential oils rich in phenylpropanoids and terpenoids (Jirovetz et al 2003;Renu et al 2014). These metabolites individually or in combination impart aroma, fragrance, UV protection and have important roles in plant defence or serve as signal molecules between plants and microbes (Viña & Murillo 2003).…”