RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

Ritesh Kumar,Ajit Kumar Shasany,Prashant Kumar Rout,Md Qussen Akhtar,Divya Vashisth,Madan Mohan Gupta,Amita Misra,Syed Uzma Jalil,Karuna Shanker,Anil Kumar Gupta

doi:10.1038/srep26458

Ritesh Kumar, Ajit Kumar Shasany + Show 8 more

Open Access

https://doi.org/10.1038/srep26458

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Abstract

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.

Highlights

The outer layer of glandular trichome (GT), while the phenolic substances were detected in the neck and gland cells
GT density is highest at the maximum size of leaves, after which density decreases rapidly, suggesting the rupture over time in the older leaves
Similar decrease in the GT number in Mentha arvensis from upper expanding young, to the lower level leaves proceeding for senescence is reported[21]

Summary

Introduction

The outer layer of GTs, while the phenolic substances were detected in the neck and gland cells. Though a comparison cannot be made between Arabidopsis and higher plants glandular trichomes, the role of lignin cannot be ruled out in the architecture of the glandular peltate trichomes This specialized tissue follows a specific pattern of arrangement of cells providing unique architecture to prevent the premature loss of metabolites. Earlier during the characterization of Cytochrome P450 monooxygenases (CYPs), we observed that the AaCYP71AV1 transcript of artemisinin biosynthesis pathway overexpressed significantly with trans-cinnamic acid treatment[13]. Considering all these aspects, the functional significance of the C4H was investigated in detail and compared in normal as well as RNAi plants. This report demonstrates experimentally, the link between lignin/phenylpropanoid and artemisinin biosynthesis pathway at the level of trans-cinnamic acid for the first time, indicating the diversion of carbon flux of one pathway for the biosynthesis of signal molecule inducing the biosynthesis of commercially and medicinally important molecule of a different pathway

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