Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal
Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copiou...
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| Format: | Article |
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Frontiers Media S.A.
2019-07-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2019.00842/full |
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| author | Arti Sharma Gulzar A. Rather Prashant Misra Manoj K. Dhar Surrinder K. Lattoo |
| author_facet | Arti Sharma Gulzar A. Rather Prashant Misra Manoj K. Dhar Surrinder K. Lattoo |
| author_sort | Arti Sharma |
| collection | DOAJ |
| description | Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of WsCYP85A69 in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of WsCYP85A69 resulted in the identification of several potential cis-regulatory elements. Elicitations, given on the basis of identified cis-regulatory elements, demonstrated methyl jasmonate as an effective inducer of WsCYP85A69. Overall, these empirical findings suggest that functional characterization of WsCYP85A69 may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering. |
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| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-13T08:16:07Z |
| publishDate | 2019-07-01 |
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| spelling | doaj.art-3df1b3f0e6dc45358339608e10bdf5bb2022-12-21T23:54:07ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2019-07-011010.3389/fpls.2019.00842447055Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) DunalArti Sharma0Gulzar A. Rather1Prashant Misra2Manoj K. Dhar3Surrinder K. Lattoo4Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, IndiaPlant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, IndiaPlant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, IndiaSchool of Biotechnology, Faculty of Life Sciences, University of Jammu, Jammu, IndiaPlant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, IndiaWithania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of WsCYP85A69 in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of WsCYP85A69 resulted in the identification of several potential cis-regulatory elements. Elicitations, given on the basis of identified cis-regulatory elements, demonstrated methyl jasmonate as an effective inducer of WsCYP85A69. Overall, these empirical findings suggest that functional characterization of WsCYP85A69 may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering.https://www.frontiersin.org/article/10.3389/fpls.2019.00842/fullWithania somniferabrassinosteroidsstigmasterolwithanolidescytochrome P450 monooxygenase |
| spellingShingle | Arti Sharma Gulzar A. Rather Prashant Misra Manoj K. Dhar Surrinder K. Lattoo Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal Frontiers in Plant Science Withania somnifera brassinosteroids stigmasterol withanolides cytochrome P450 monooxygenase |
| title | Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal |
| title_full | Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal |
| title_fullStr | Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal |
| title_full_unstemmed | Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal |
| title_short | Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal |
| title_sort | gene silencing and over expression studies in concurrence with promoter specific elicitations reveal the central role of wscyp85a69 in biosynthesis of triterpenoids in withania somnifera l dunal |
| topic | Withania somnifera brassinosteroids stigmasterol withanolides cytochrome P450 monooxygenase |
| url | https://www.frontiersin.org/article/10.3389/fpls.2019.00842/full |
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