Kiran, N. R. | Search bioRxiv

bioRxiv2024

Phylogenetically distant enzymes localized in cytosol and plastids drive citral biosynthesis in lemongrass

Except for the genetic basis of citral-forming alcohol dehydrogenases (ADHs) in Litsea cubeba tree, and biochemical studies on citral-forming enzymes from select plants, knowledge regarding in-planta biosynthesis of citral and its metabolic origin remains limited. Here, we have elucidated the functions of an ADH (CfADH1) and an aldoketo-reductase (CfAKR2b) in citral biosynthesis in lemongrass (Cymbopogon flexuosus), one of the most cultivated aromatic crops for its citral-rich essential oil. Expression of both CfADH1 and CfAKR2b showed correlation with citral accumulation in different developmental stages. Recombinant CfADH1 and CfAKR2b, despite their sequence unrelatedness, exhibited similar kinetic properties and formed citral from geraniol with NADP cofactor. Virus-induced gene silencing in lemongrass, and transient expression in lemon balm (Melissa officinalis), demonstrated the in-planta involvement of CfADH1 and CfAKR2b in citral biosynthesis. While CfADH1 exhibited a dual cytosolic/plastidial localization, CfAKR2b was localized to cytosol. Moreover, feeding lemongrass seedlings with mevalonate- and methylerythritol-phosphate-pathway specific inhibitors combined with volatile profiling supported the role of both pathways in citral formation. Our results demonstrate phylogenetically distant enzymes localized in cytosol and plastids drive citral biosynthesis in lemongrass, indicating an evolutionary scenario aimed at maximizing the utilization of precursor pools from both cytosolic and plastidial pathways for high citral production.

plant biology↗

bioRxiv2023

Analysis of root volatiles and functional characterization of a root-specific germacrene A synthase in Artemisia pallens

Davana (Artemisia pallens) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatiles and their specific composition, and genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields [~]0.05% of EO, which is primarily composed of sesquiterpenes {beta}-elemene, neryl isovalerate, {beta}-selinene, and -selinene, and trace amounts of monoterpenes {beta}-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of {beta}-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Further, detailed analysis of assay products revealed that {beta}-elemene was the thermal rearrangement product of germacrene A. Furthermore, the functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo {beta}-elemene/germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in root, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of {beta}-elemene. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile. Main conclusionThe study demonstrated that Artemisia pallens roots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene-A contributing to major root volatiles.

plant biology↗