Abstract
Shikonin, a 1,4-naphthoquinone derivative produced by some limited Boraginaceae species, exhibits unique pharmacological properties and is also used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production, and the copper-requiring biosynthetic step is shikonin-specific, unlike the byproduct benzoquinones. Despite the long history of shikonin biosynthesis studies for over 40 years, the copper-involved reaction has been still unknown. Here, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Genes encoding polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted by the strong synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon (LePPOs), only LePPO1 showed a close correlation with shikonin production. Then, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines, while the content of deoxyshikonofuran, a hydroquinone-type shunt product that branches after 3''-hydroxygeranylhydroquinone in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings address the question why a copper ion is crucial for shikonin biosynthesis and suggest that LePPO1 participates in naphthalene ring formation. Interestingly, LePPO1 is localized in plastids, whereas shikonin is accumulated in the apoplast.
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Claims 7
Claim · model-written summary
PPO genes showed strong expression synchrony with shikonin production.
- Taxon
- Lithospermum erythrorhizon
Lithospermum erythrorhizont0 - Gene
- polyphenol oxidase (PPO)e0
- Outcome
- shikonin productione0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“Genes encoding polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted by the strong synchronous expression with shikonin production”
Claim · model-written summary
Among five expressed PPO genes, only LePPO1 closely correlated with shikonin production.
- Taxon
- L. erythrorhizonLithospermum erythrorhizone0
- Gene
- LePPO1e0
- Outcome
- shikonin productione0
- Value
- five PPO genese0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“of the five PPO genes expressed in L. erythrorhizon (LePPOs), only LePPO1 showed a close correlation with shikonin production”
Claim · model-written summary
LePPO1-edited lines had markedly reduced shikonin content.
- Taxon
- Lithospermum erythrorhizonLithospermum erythrorhizont0
- Treatment
- LePPO1-ge linese0
- Gene
- LePPO1e0
- Outcome
- shikonin contente0
- Direction
- reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“shikonin content was markedly reduced in all LePPO1-ge lines”
Claim · model-written summary
Deoxyshikonofuran content was unchanged in LePPO1-edited lines.
- Taxon
- Lithospermum erythrorhizonLithospermum erythrorhizont0
- Treatment
- LePPO1-ge linese0
- Gene
- LePPO1e0
- Outcome
- content of deoxyshikonofurane0
- Direction
- remained unaffectede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“content of deoxyshikonofuran, a hydroquinone-type shunt product that branches after 3''-hydroxygeranylhydroquinone in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines”
Claim · model-written summary
The authors suggest LePPO1 participates in naphthalene ring formation.
- Taxon
- Lithospermum erythrorhizonLithospermum erythrorhizont0
- Gene
- LePPO1e0
- Outcome
- naphthalene ring formatione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“suggest that LePPO1 participates in naphthalene ring formation”
Claim · model-written summary
LePPO1 was localized in plastids.
- Taxon
- Lithospermum erythrorhizonLithospermum erythrorhizont0
- Organ / stage
- plastidse0
- Gene
- LePPO1e0
- Outcome
- localized in plastidse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“LePPO1 is localized in plastids”
Claim · model-written summary
Shikonin accumulated in the apoplast.
- Taxon
- Lithospermum erythrorhizonLithospermum erythrorhizont0
- Organ / stage
- apoplaste0
- Outcome
- shikonin is accumulated in the apoplaste0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Lithospermum erythrorhizon”
e0“shikonin is accumulated in the apoplast”