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Abstract Detail


Suttiyut, Thiti [1], Auber, Robert [2], Ghaste, Manoj [3], Wisecaver, Jennifer [2], Widhalm, Joshua [4].

Investigating the biochemical evolution of the shikonin pathway in red gromwell (Lithospermum erythrorhizon).

Shikonin, its enantiomer alkannin, and dozens of structurally diverse shikonin/alkannin derivatives are produced exclusively in Boraginaceae species. These 1,4-naphthoquinones are predominantly made in roots where they function in mediating plant-plant (allelopathic) and plant-microbe interactions. Moreover, excretion of shikonins into the rhizosphere has been attributed to the invasion success of some species. Our recent assembly and analysis of the red gromwell (Lithospermum erythrorhizon) genome uncovered metabolic innovation events that contributed to evolution of the shikonin/alkannin pathway. However, many open questions remain about the evolution of the shikonin/alkannin pathway, including the metabolic origin of its 4-hydroxybenzoic acid (4-HBA) and geranyl diphosphate (GPP) precursors. To gain further insight, we used a transcriptome-based network analysis approach which uncovered a shikonin gene network model that predicts strong associations between primary metabolic pathway genes and known shikonin biosynthesis genes, as well as links with uncharacterized enzyme-coding genes. Among those found in the shikonin metabolic network model was L. erythrorhizon GPP synthase (LeGPPS), which encodes a cytoplasmic enzyme shown in vitro to produce GPP. To test if LeGPPS is involved in supplying GPP for shikonin biosynthesis, we used RNAi-downregulation to knockdown expression of LeGPPS in L. erythrorhizon hairy roots. Downregulation of LeGPPS by over 95% reduced shikonin content more than 90% without affecting expression of the canonical plastid-localized GPPS. These data provide functional evidence demonstrating that cytoplasmic LeGPPS supplies GPP precursor to the shikonin pathway. Downregulation of LeGPPS also increased ubiquinone content, further supporting our hypothesis on the metabolic and evolutionary connection between the shikonin/alkannin and ubiquinone pathways. RNA-seq analysis of empty-vector control and LeGPPS-RNAi lines revealed that downregulating LeGPPS reduces expression of upstream genes in the mevalonic acid (MVA) pathway. The MVA pathway, and the parallel methylerythritol phosphate (MEP) pathway in plastids, supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) building blocks for producing terpenoids. Our transcriptional data, combined with MVA- and MEP-pathway inhibitor experiments performed in this study, corroborate previous labeling studies and demonstrate that the MVA pathway supplies IPP/DMAPP to LeGPPS. RNA-seq analysis also showed that downregulating LeGPPS significantly reduces expression of benzenoid/phenylpropanoid genes, indicating the presence of factors that coordinately regulate the pathways providing the 4-HBA and GPP precursors to the shikonin/alkannin pathway. In addition to answering questions specific to the biochemical evolution of the shikonin/alkannin pathway, our study demonstrates that global coexpression analysis with limited transcriptomic data generated from well-designed experiments is effective for uncovering gene associations within a defined metabolic network.

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1 - Purdue University, Department Of Horticulture And Landscape Architecture, 625 Agriculture Mall Dr, West Lafayette, IN, 47907, United States
2 - Purdue University, Biochemistry, 175 South University St., West Lafayette, IN, 47907, United States
3 - Purdue University, Horticulture and Landscape Architecture , 625 Agriculture mall dr, West Lafayette, IN, 47907, United States
4 - Purdue University, Horticulture And Landscape Architecture, 625 Agricultural Mall Drive, West Lafayette, IN, 47907, United States

Shikonin Biosynthesis
Plant Specialized Metabolism
Transcriptome-based Network Analysis.

Presentation Type: Oral Paper
Session: PHYT2, Phyochemistry II
Location: /
Date: Tuesday, July 20th, 2021
Time: 1:15 PM(EDT)
Number: PHYT2004
Abstract ID:491
Candidate for Awards:Phytochemical Best Oral Presentation Award

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