Shikonin and its derivatives are produced mainly in the roots of Boraginaceae plants and have attracted interest for their reported antimicrobial, anti-inflammatory, antiviral, wound-healing and pigment properties. Yet the pathway behind their formation is complex, involving upstream metabolic routes, downstream enzymatic steps and transcriptional regulation. Research has also been slowed by the lack of high-quality reference genomes for Arnebia species and by long-running taxonomic debate over A. tschimganica. Based on these challenges, a deeper investigation of the genome, metabolome and regulatory network behind shikonin biosynthesis is needed.
The study was led by researchers from the State Key Laboratory of Pharmaceutical Biotechnology, Institute for Plant Molecular Biology, School of Life Sciences, Nanjing University, with collaborators from Nanjing Forestry University and other institutions. It was published (DOI: 10.1093/hr/uhag077) on 3 March 2026 in Horticulture Research . The team assembled a chromosome-level genome, clarified taxonomic relationships, and mapped the molecular basis of shikonin biosynthesis regulation.
Using high-fidelity (HiFi) sequencing and high-throughput chromosome conformation capture (Hi-C), the team assembled a 611.54-megabase genome anchored to 14 chromosomes and predicted 35,059 protein-coding genes. Comparative genomic analysis suggested that A. tschimganica is more closely aligned with Lithospermum than with Arnebia, supporting a revised taxonomic view. The researchers also found evidence of a shared recent whole-genome duplication (WGD) event that helped expand shikonin-related gene families. Metabolomic profiling identified 1,156 metabolites in A. tschimganica roots and 1,137 in L. erythrorhizon roots, but shikonin derivatives were far less abundant in A. tschimganica. Transcriptome analysis linked this low accumulation to weak expression of key pathway genes, including GHQ3H1, DSH1 and SAT1. In dark-cultured callus cells, these genes were reactivated, and shikonin production increased. Functional assays further showed that the AtsDSH1 gene encodes an enzyme catalyzing the conversion of deoxyshikonin to shikonin, while two ethylene-responsive factor (ERF) transcription factors can activate DSH1 promoters.
The authors said the work turns a long-standing plant classification puzzle into a practical molecular roadmap. By comparing a low-shikonin species with a high-producing close relative, they said, the study reveals not only where the pathway sits in the genome, but also why it may be quiet in one plant and active in another. They said the discovery of AtsDSH1 and conserved ERF regulators provides clear entry points for testing how shikonin production can be strengthened without relying only on wild plant resources.
The findings provide genomic resources for conserving A. tschimganica, a vulnerable medicinal species, while also offering candidate genes and regulators for future metabolic engineering. In the longer term, the work could support more sustainable production of shikonin and related natural pigments for pharmaceutical, cosmetic and dyeing applications. Because the study links taxonomy, evolution and biosynthesis in one framework, it also gives researchers a model for exploring other medicinal plants whose valuable compounds remain poorly understood. Further validation will be needed before these molecular targets can be translated into scalable production systems, but the pathway is now much clearer.
###
References
DOI
10.1093/hr/uhag077
Original Source URL
https://doi.org/10.1093/hr/uhag077
Funding information
This research was supported by the National Natural Science Foundation of China (U1903201, 31970321, 31771413, 32400305), the Natural Science Foundation of Jiangsu Bureau of Science and Technology (BK20191254), the Open Project Program from the MOE Key Laboratory of Molecular Epigenetics of China, and the Program for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (IRT_14R27).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.