Research articlePromoting scopolamine biosynthesis in transgenic Atropa belladonna plants with pmt and h6h overexpression under field conditions
Graphical abstract
T1 progeny of NtPMT-HnH6H overexpressing Atropa belladonna are shown enhanced scopolamine content.
Introduction
The tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are used medicinally as anticholinergic agents (Poupko et al., 2007, Yun et al., 1992), which are generally produced by the plant species of the Solanaceae family, including Atropa belladonna, Hyoscyamus niger, Datura species, and others (Yamada and Hashimoto, 1982). Among them, A. belladonna is one of the most important plant sources for TA production (Wang et al., 2011). Scopolamine is the most valuable TA because of more potent pharmaceutical activity and lesser side effects relative to those of hyoscyamine (Jaremicz et al., 2014, Wang et al., 2011). However, the scopolamine content in A. belladonna plants is much lower than that of hyoscyamine (Yun et al., 1992). Therefore, the development of scopolamine-rich A. belladonna is a common goal of the scopolamine industry. Over the past decades, traditional methods, including genetic breeding, polyploid breeding and radiation breeding, have failed to develop scopolamine-rich plants of A. belladonna (Wang et al., 2011). With the elucidation of several important TA biosynthetic steps at molecular and biochemical levels, the genes encoding some rate-limiting enzymes that can be used to genetically modify TA biosynthesis in planta have been functionally identified.
Putrescine N-methyltransferase (PMT) catalyzes the methylation of putrescine to form N-methylputrescine as the precursor for TA biosynthesis (Fig. 1). It is generally considered to be the first rate-limiting enzyme involved in TA biosynthetic pathway (Hibi et al., 1992, Zhang et al., 2007). Hyoscyamine 6β-hydroxylase (H6H) is the last rate-limiting enzyme involved in scopolamine biosynthesis, which catalyzes the 6β-hydroxylation of hyoscyamine to anisodamine, as well as the epoxidation of anisodamine to scopolamine (Fig. 1) (Kai et al., 2011, Matsuda et al., 1991, Zhang et al., 2004). Thus, PMT and H6H are often used to study TA biosynthesis using transgenic technology in hairy root cultures or plants. When a single PMT gene was overexpressed in transgenic hairy root cultures or plants of A. belladonna, the TA content was not altered (Rothe et al., 2003). However, overexpression of the same PMT gene in Datura metel resulted in TA contents that were significantly increased (Moyano et al., 2003). When H6H was overexpressed, biosynthesis of scopolamine was definitively enhanced because H6H efficiently converts hyoscyamine to scopolamine (Yun et al., 1992). Furthermore, simultaneous overexpression of both PMT and H6H coordinately promoted biosynthesis of scopolamine and made the scopolamine content very high in transgenic hairy root cultures of H. niger (Zhang et al., 2004). We established transgenic plants of A. belladonna in which both PMT and H6H were overexpressed for the first time and found that scopolamine biosynthesis was also strongly enhanced (Wang et al., 2011).
To study biosynthesis and translocation of TAs in the offspring of transgenic A. belladonna (T0 progeny), we generated T1 progeny of NtPMT-HnH6H-overexpressing plants of A. belladonna. In field conditions, we analyzed the tissue profiles of the two transgenes and the four endogenous TA biosynthetic genes in T1 progeny of transgenic A. belladonna. Further, we detected hyoscyamine, anisodamine and scopolamine in different organs of T1 progeny of transgenic A. belladonna. Moreover, we established the scopolamine-rich plants of transgenic A. belladonna that showed higher value for scopolamine industry.
Section snippets
Generation and plantation of T1 progeny of NtPMT-HnH6H overexpressing plants
The transgenic plants of A. belladonna were previously established in our laboratory in which both NtPMT and HnH6H were overexpressed (Wang et al., 2011). Self-pollination of A. belladonna (2n = 72) is available (Butcher, 1947) and the previous publication reported that true seeds were obtained (Yun et al., 1992). Three lines, including B1, D7 and D9 (T0 progeny), were used to generate T1 progeny by self-pollination. When the flower buds appeared, they were quarantined in parchment paper bags
Molecular detection of transgenic and wild-type plants of A. belladonna
In the transgenic plants of T1 progeny, the fragment of the marker gene NPTII was specifically amplified (Fig. 2), and the transgenes, including NtPMT with a fused 35S promoter fragment and HnH6H with a fused 35S promoter fragment, were simultaneously obtained (Fig. 2). The PCR results were consistent with that using pXI plasmid (the plant expression vector harboring both NtPMT and HnH6H) as the positive control. However, none of the three specific gene fragments were amplified from wild-type
Discussion
Biosynthesis of plant secondary metabolites is developmentally and environmentally regulated through spatial and temporal expression biosynthetic or regulatory genes. H6H was localized in root pericycle cells of A. belladonna (Suzuki et al., 1999). We discovered that AbTRI was almost specifically expressed in secondary roots of wild-type and transgenic plants and that AbPMT and AbCYP80F1 were almost exclusively expressed in roots with much higher level in secondary roots. The tissue profiles of
Conclusion
Overexpressing the rate-limiting enzymes has been successfully used for genetic improvement of metabolite biosynthesis in many cases. In this report, this strategy was also found to be effective. Metabolically engineering A. belladonna based on overexpressing NtPMT and HnH6H showed an impressive chemotype of rich scopolamine that greatly improved the commercial and pharmaceutical properties of A. belladonna.
Author contribution
Ke Xia, Xiaoqiang Liu and Qiaozhuo Zhang conducted the experiments, including plantation, managed and harvested the plant materials, and molecular analysis; Jianjun Guo and Xiaozhong Lan analyzed the data; Min Chen analyzed the alkaloids; Zhihua Liao conceived the experiments and wrote the paper. All authors reviewed the manuscript.
Acknowledgements
This research was financially supported by the NSFC Project (31370333), the National Hi-Tech Project (2011AA100605), the Program for New Century Excellent Talents in University (NCET-12-0930), and the Fundamental Research Funds for the Central Universities (XDJK2013A024). The authors declare no competing financial interests.
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These authors contributed equally to this work.