Abstract:The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interfere… Show more
“…This indicated that G10H and 12OPR unigenes should be up-expressed in all RR lines from above three Lithospermeae plants, and probably related to shikonin biosynthesis in this present study. Moreover, the functional validation of the G10H and 12OPR unigenes is now underway, according to the similar strategy as reported in our previous papers 14, 31, 32 .Figure 6The qRT-PCR verification of the G10H and 12OPR unigenes’ expression profiles between GL and RR lines in Lithospermum erythrorhizon ( Le ), Arnebia euchroma ( Ae ) and Echium plantagineum ( Ep ). ( a ) G10H - LeCL2572 .…”
Section: Resultsmentioning
confidence: 83%
“…euchroma and E . plantagineum were germinated according to the methods, as previously reported 14, 26 . The germinated seeds were then transferred into square plastic pots (50 * 30 * 30 cm) with peat growing medium (Pindstrup, Denmark) and were cultured in a greenhouse at 23 ± 1 °C under a 16 h/day photoperiod for approximately 60 to 90 days.…”
Section: Methodsmentioning
confidence: 99%
“…Contig2 (Supplementary Table S5), were selected for representing 12OPR unigenes in this study. The glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ) was used as internal reference as previously reported 14, 32 . All primers were listed in Supplementary Table S8.…”
Section: Methodsmentioning
confidence: 99%
“…erythrorhizon dark-inducible gene 1 , 2 , 3 , 4 , 5a/b/c ( LeDI - 1 , 2 , 3 , 4 , 5a/b/c ) and L . erythrorhizon 1-aminocyclopropane-1-carboxylate synthase gene ( LeACS ) 12–14 . Moreover, several factors, such as light, methyl jasmonate, nitric oxide, ethylene and fungal elicitor are crucial regulators of shikonin biosynthesis 12, 15–19 .…”
Shikonin and its derivatives extracted from Lithospermeae plants’ red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in “shikonin downstream biosynthesis” and “methyl jasmonate biosynthesis” were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.
“…This indicated that G10H and 12OPR unigenes should be up-expressed in all RR lines from above three Lithospermeae plants, and probably related to shikonin biosynthesis in this present study. Moreover, the functional validation of the G10H and 12OPR unigenes is now underway, according to the similar strategy as reported in our previous papers 14, 31, 32 .Figure 6The qRT-PCR verification of the G10H and 12OPR unigenes’ expression profiles between GL and RR lines in Lithospermum erythrorhizon ( Le ), Arnebia euchroma ( Ae ) and Echium plantagineum ( Ep ). ( a ) G10H - LeCL2572 .…”
Section: Resultsmentioning
confidence: 83%
“…euchroma and E . plantagineum were germinated according to the methods, as previously reported 14, 26 . The germinated seeds were then transferred into square plastic pots (50 * 30 * 30 cm) with peat growing medium (Pindstrup, Denmark) and were cultured in a greenhouse at 23 ± 1 °C under a 16 h/day photoperiod for approximately 60 to 90 days.…”
Section: Methodsmentioning
confidence: 99%
“…Contig2 (Supplementary Table S5), were selected for representing 12OPR unigenes in this study. The glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ) was used as internal reference as previously reported 14, 32 . All primers were listed in Supplementary Table S8.…”
Section: Methodsmentioning
confidence: 99%
“…erythrorhizon dark-inducible gene 1 , 2 , 3 , 4 , 5a/b/c ( LeDI - 1 , 2 , 3 , 4 , 5a/b/c ) and L . erythrorhizon 1-aminocyclopropane-1-carboxylate synthase gene ( LeACS ) 12–14 . Moreover, several factors, such as light, methyl jasmonate, nitric oxide, ethylene and fungal elicitor are crucial regulators of shikonin biosynthesis 12, 15–19 .…”
Shikonin and its derivatives extracted from Lithospermeae plants’ red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in “shikonin downstream biosynthesis” and “methyl jasmonate biosynthesis” were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.
“…The shikonin and its derivative content reached the highest level on day 6 (Fang et al . ). The expression level of LeMRP in hairy roots was determined through real‐time PCR to confirm the transgenic effects.…”
Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.
Production of secondary metabolites from in vitro cell and hairy root cultures (CHRC) of medicinal and aromatic plants (MAP) is considered a promising alternative to gathering plant material from MAP natural populations, often a reason for their overexploitation and even extinction. However, most of the valuable secondary metabolites extracted from different MAP species are present in very low amounts in the respective CHRC. Plant metabolic engineering offers an attractive opportunity to increase the content of target secondary metabolites in engineered transgenic CHRC for production at feasible levels. Moreover, applying metabolic engineering makes it possible to redirect target metabolic pathway K. Rusanov (*) • I.
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