Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

Hongzhen Wang,Junli Han,Anneli Lundgren,Peter E. Brodelius,Selvaraju Kanagarajan,Christian Schönbach

doi:10.1371/journal.pone.0080643

Abstract

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

Highlights

Secondary metabolites released from aerial organs into air and roots into soil play important roles in plant responsiveness to various external signals, such as direct and indirect defenses, plant reproduction and plant-plant interactions
B-caryophyllene synthase (CPS) and (3R)-linalool synthase (LIS) were found to be involved in responsiveness to wounding and fungal elicitors in Artemisia annua [1,2]. (E)-b-CPS is suggested to be involved in indirect defenses of maize and rice, even though it is not expressed in all varieties of maize [7,8]. (E)-bFarnesene synthase (FS), which can be induced by herbivore in some maize genotypes, was cloned and characterized from maize [9]
The four cloned sesquiterpene synthase promoters show different expression patterns as reported by the histochemical GUS staining. It seems that CPS and epi-cedrol synthase (ECS) do not influence the biosynthesis of artemisinin to any great extent due to their low expression in glandular trichomes (Figures 4 and 5)

Summary

Introduction

Secondary metabolites released from aerial organs into air and roots into soil play important roles in plant responsiveness to various external signals, such as direct and indirect defenses, plant reproduction and plant-plant interactions. Many of these secondary products are terpenoids. (E)-bFarnesene synthase (FS), which can be induced by herbivore in some maize genotypes, was cloned and characterized from maize [9] This enzyme has been cloned from a number of plants, such as A. annua [10], Norway spruce [11], peppermint [12], and Citrus junos [13]

Methods

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Conclusion