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

Abstract 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.