The review, led by Eliakira Kisetu Nassary of Sokoine University of Agriculture in Tanzania together with colleagues, followed the PRISMA-ScR framework, the recognised reporting standard for scoping reviews. The team searched Scopus, Web of Science, PubMed, ScienceDirect and Google Scholar for publications from January 2010 through February 2026, screening 286 records and ultimately including 95 peer-reviewed studies in the evidence synthesis. Rather than simply summarising the literature, the authors assigned each study to a specific segment of the citral biosynthetic pathway and scored the depth of experimental validation using a purpose-built tool they call the Evidence Maturity Index, or EMI. The index ranks evidence hierarchically: transcriptomic prediction scores lowest at 1.0, integrated transcriptome-metabolome studies score 1.5, recombinant enzyme characterisation scores 2.0, and in planta functional validation, the strongest form of proof, scores 2.5.

The biosynthetic story begins in the chloroplast. The plastidial methylerythritol phosphate, or MEP, pathway supplies the isoprenoid building blocks that are assembled into geranyl diphosphate, the immediate precursor of all monoterpenes. Transcriptome assemblies and comparative expression analyses have repeatedly flagged genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS), its reductoisomerase partner DXR, and geranyl diphosphate synthase (GPPS) as central players in precursor production. In the review’s evidence map, the MEP pathway was the single most heavily studied segment, accounting for 24 of the 95 included studies, with GPPS-related work adding another 16. But here lies the review’s central warning: sheer volume of publications did not translate into mechanistic certainty, because most of these investigations relied on gene expression patterns rather than direct measurements of enzyme activity or metabolic flux.

The best-resolved step in the entire pathway turns out to be the conversion of geranyl diphosphate into geraniol. Despite being represented by only 13 studies, geraniol synthase achieved one of the highest validation scores in the EMI analysis. Recent work combining recombinant enzyme assays, subcellular localisation and functional analyses revealed something unexpected: a dual-localised geraniol synthase operating in both plastids and cytosol, working alongside a previously uncharacterised cytosolic geranyl pyrophosphatase. This cooperation between cellular compartments refines the older picture of monoterpenes being built exclusively inside chloroplasts and demonstrates that citral formation depends on metabolic exchange across cellular boundaries. Comparable compartmental coordination has since been documented in camphor trees, suggesting the pattern may be widespread among aromatic plants.

The final oxidation step, in which geraniol is converted into the aldehydes geranial and neral, has historically been the murkiest part of the pathway. For years, alcohol dehydrogenases were implicated purely on the basis of transcript abundance and correlations with citral content. That changed when recent functional studies in lemongrass deployed recombinant enzyme assays, virus-induced gene silencing and transient overexpression to demonstrate a direct enzymatic contribution to citral accumulation. The review notes that these findings require an update to previous classifications that treated ADH-mediated oxidation as merely predictive. Even so, comparative enzyme kinetics among ADH isoforms, their substrate preferences and their relative contributions to geranial versus neral formation remain unexplored, leaving the terminal chemistry only partially resolved.

The weakest link, according to the EMI, is transcriptional regulation. Only nine studies examined regulatory factors, and nearly all relied on co-expression networks and candidate gene prediction. Transcriptomic analyses have associated terpene accumulation with transcription factors of the AP2/ERF and MYB families, but direct evidence of promoter binding or transcriptional activation in Cymbopogon is essentially absent. A compelling comparator exists: in the laurel family, a MYB44 transcription factor was experimentally shown to regulate alcohol dehydrogenase expression, providing a mechanistic framework that lemongrass researchers could replicate using chromatin immunoprecipitation, yeast one-hybrid assays and promoter-reporter systems. Until such experiments are performed in Cymbopogon itself, the regulatory hierarchy controlling citral production remains an inference rather than a demonstrated fact.

Beyond the pathway itself, the review highlights how environmental conditions, agronomic practices and post-harvest handling shape citral accumulation in ways that are still poorly connected to molecular mechanisms. Drought studies have revealed significant genotype-by-environment interactions for essential oil yield, and transcriptomic evidence suggests water stress alters expression of upstream isoprenoid genes, including GPPS-related components. Harvest interval, cutting frequency and developmental stage all modify oil yield and composition, likely through changes in glandular structure, precursor availability and enzymatic activity. Crucially, the authors urge a clear separation between biosynthetic regulation and post-harvest chemistry: drying conditions, storage duration and distillation procedures can shift the geranial-to-neral ratio through volatilisation, oxidation or chemical conversion, meaning that two samples of the same cultivar may differ chemically without any difference in genetic capacity.

The practical implications reach into breeding and biotechnology. Current cultivar improvement relies heavily on phenotypic evaluation, oil yield and chemical profiling, with molecular markers directly tied to citral biosynthesis still scarce. The review argues that genome-wide association studies and quantitative trait locus mapping across diverse Cymbopogon germplasm, combined with multi-environment phenotyping, could identify genomic regions controlling citral concentration, geranial-neral ratios and environmental stability. Emerging technologies could accelerate this transition: long-read sequencing promises better genome annotation and resolution of terpene gene families, single-cell transcriptomics could reveal which specialised cells actually manufacture citral, spatial metabolomics can map where intermediates accumulate within tissues, and artificial intelligence-assisted multi-omics integration may predict pathway behaviour under contrasting production environments.

The overarching lesson of the evidence map is that research volume and mechanistic resolution are not the same thing. Pathway segments drowning in transcriptomic datasets scored lower on the Evidence Maturity Index than segments supported by fewer but functionally decisive experiments, a pattern the authors say should redirect future effort toward enzyme kinetics, metabolic flux analysis, targeted gene perturbation using RNA interference or CRISPR/Cas editing, and integrated studies that simultaneously measure gene expression, protein activity, metabolite flux and final oil composition. As global demand for naturally derived bioactive compounds pushes lemongrass cultivation into new regions and production systems, the gap between knowing which genes are present and knowing which genes control the chemistry becomes the decisive bottleneck. Closing it, the review concludes, will determine whether citral-rich lemongrass cultivars of the future are selected by trial and error or engineered by design.

Subject of Research: Molecular pathways and validation status of citral biosynthesis in Cymbopogon species

Article Title: Evidence mapping of molecular pathways regulating citral biosynthesis and validation status in Cymbopogon spp.

Article References: Nassary, E. K., Magubika, A. J., Mhagama, E. A., Salimu, A. I., Kilasi, N. L., Tryphone, G. M., & Shitindi, M. J. (2026). Evidence mapping of molecular pathways regulating citral biosynthesis and validation status in Cymbopogon spp.. Discover Plants, 3(1), Article 378. https://doi.org/10.1007/s44372-026-00852-1

Image Credits: AI Generated

DOI: 10.1007/s44372-026-00852-1

Keywords: citral, Cymbopogon, lemongrass, monoterpene biosynthesis, MEP pathway, geraniol synthase, alcohol dehydrogenase, evidence mapping, transcriptomics, metabolomics, essential oil, plant breeding