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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology 13, 513–520, May 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.chembiol.2006.03.005 Functional Genomic Analysis of Alkaloid Biosynthesis in Hyoscyamus niger Reveals a Cytochrome P450 Involved in Littorine Rearrangement Rong Li,1 Darwin W. Reed,1 Enwu Liu,1 Jacek Nowak,1 ing the types of enzymes involved in the conversion of Lawrence E. Pelcher,1 Jonathan E. Page,1 littorine to hyoscyamine. An enzyme with a mechanism 1, and Patrick S. Covello * and properties similar to vitamin B12-dependent isom- 1 Plant Biotechnology Institute erases has been suggested [1]. Retey and coworkers 110 Gymnasium Place [8–10] have provided evidence for such a littorine isom- Saskatoon, Saskatchewan S7N OW9 erase that requires S-adenosylmethionine (SAM) as a co- Canada factor. On the other hand, labeling and inhibitor studies and mechanistic considerations led Robins and co- workers to propose that a cytochrome P450 is involved, Summary and that hyoscyamine aldehyde is formed as an interme- diate [3, 11, 12]. Furthermore, Patterson and O’Hagan Tropane alkaloids are valuable pharmaceutical drugs presented results that were inconsistent with a vicinal derived from solanaceous plants such as Hyoscya- exchange mechanism expected for a SAM-dependent mus niger (black henbane). The biosynthesis of these reaction [13]. molecules, including the nature of the enigmatic rear- In order to settle the debate regarding the last steps rangement of (R)-littorine to (S)-hyoscyamine, is not in hyoscyamine biosynthesis, an investigation of the hy- completely understood. To test the hypothesis that a pothesis that a cytochrome P450 is involved was under- cytochrome P450 enzyme is involved in this rearrange- taken. Our functional genomics approach has included ment, we used virus-induced gene silencing to silence the generation of expressed sequence tags from an a cytochrome P450, CYP80F1, identified from H. niger H. niger L. cDNA library and the testing of candidate roots by EST sequencing. Silencing CYP80F1 resulted cytochrome P450 gene function by virus-induced gene in reduced hyoscyamine levels and the accumulation silencing (VIGS). of littorine. Hyoscyamine was observed in CYP80F1- VIGS exploits the RNA silencing pathway directed expressing tobacco hairy roots supplied with (R)-lit- against invading viruses to silence the expression of torine. Expression in yeast confirmed that CYP80F1 host genes [14] and has been used to silence genes catalyzes the oxidation of (R)-littorine with rearrange- involved in a variety of plant biosynthetic pathways ment to form hyoscyamine aldehyde, a putative pre- [15–19]. Tobacco rattle virus (TRV), a tobravirus that cursor to hyoscyamine, and without rearrangement silences genes in Nicotiana benthamiana [20, 21] and to form 30-hydroxylittorine. Our data strongly support other Solanaceae [22], has been reported to infect the involvement of CYP80F1 in the rearrangement of H. niger [23]. In related work, proof-of-concept experi- littorine to hyoscyamine. ments showed that TRV-mediated gene silencing was effective in H. muticus (Egyptian henbane) for phytoene Introduction desaturase and known alkaloid biosynthetic enzymes (J.E.P. et al., unpublished data). In this paper, we report Tropane alkaloids such as scopolamine and hyoscya- the use of VIGS to test the function of a candidate cyto- mine are found in various members of the Solanaceae chrome P450 gene from H. niger for its involvement in lit- family, including species of Hyoscyamus, Atropa, and torine rearrangement. This has lead to the successful Datura [1–4]. Such plants have a long history of use as identification of CYP80F1, a multifunctional cytochrome medicines and hallucinogens, and tropane alkaloids re- P450 capable of littorine rearrangement. main an important class of plant-derived anticholinergic drugs. Atropine (racemic hyoscyamine), for example, is Results used as a premedication for anesthesia to reduce bron- chial secretions and to block bradycardia associated Expressed Sequence Tags from Hyoscyamus niger with some anesthetic drugs. Given the relatively limited knowledge of the genes Despite considerable efforts over many years and involved in the biosynthesis, transport, and regulation good progress toward elucidating the biosynthetic of tropane alkaloids in the Solanaceae family, a collec- pathway leading to tropane alkaloids, knowledge of sev- tion of expressed sequence tags (ESTs) from H. niger eral key catalytic steps remains incomplete [3].An was assembled as an aid to addressing this issue. Since intriguing mystery is the nature of the carbon skeleton alkaloid biosynthesis in H. niger occurs in the roots [1], rearrangement that occurs during the conversion of a cDNA library was constructed so as to enhance the fre- (R)-littorine to (S)-hyoscyamine (see Figure 1). In this quency of cDNAs corresponding to genes expressed step, the 3-phenyllactate moiety of littorine is rear- root specifically. For this subtracted library, cDNA ranged to tropate. Isotope labeling studies have indi- from H. niger root cultures was used as the tester, and cated that C10 and C30 form the phenylacetate moiety leaf cDNA was used as the driver in a suppression of hyoscyamine, and that C20 forms the hydroxylmethyl PCR procedure. To generate ESTs from the library, bac- group in a stereochemically controlled reaction [5–7].In terial colonies representing cDNA clones were picked recent years, there has been some controversy regard- randomly, and their plasmid inserts were sequenced. After sequence analysis, the 2286 EST sequences repre- senting 214 clusters and 1299 singletons were sub- *Correspondence: [email protected] jected to similarity searches with BLAST. Chemistry & Biology 514 Figure 1. The Proposed Tropane Alkaloid Biosynthetic Pathway in Members of the Solanaceae Two different routes for the conversion of littorine to hyoscyamine are shown. Route a involves the SAM-dependent conversion proposed by Retey et al. [8], while route b, which is supported by the results of this paper, is a two-step process catalyzed by a cytochrome P450 and an alcohol dehydrogenase. The 20 carbon atom of littorine is labeled with a black dot to indicate its new position as the 30 carbon atom in hyoscyamine after rearrangement. As a starting point to investigate the possibility that not shown). Significantly, silencing of the gene corre- a cytochrome P450 was involved in the conversion of lit- sponding to pRL011 by using the VIGS construct torine to hyoscyamine, the H. niger ESTs showing simi- pRL033 had a profound and reproducible effect on alka- larity to cytochrome P450s were examined. A total of 25 loid quality and quantity, as shown in Figure 2A. In this ESTs (4 clusters and 11 singletons) showed similarity to case, the total tropane alkaloid content, and, notably, cytochrome P450 sequences. By virtue of the involve- that of hyoscyamine, was decreased, yet littorine levels ment of its homologs in plant secondary metabolism, increased in the same plants. Since littorine is thought to a clone (pRL011) representing the CYP80B subfamily be a precursor of hyoscyamine, this indicates a role for was one of the candidates chosen for functional analysis the cytochrome P450 gene corresponding to pRL011 with VIGS. in the conversion of littorine to hyoscyamine. Suppression of a Hyoscyamus Cytochrome P450 Cloning and Sequence Analysis of Full-Length by Virus-Induced Gene Silencing H. niger CYP80F1 cDNA A TRV-based VIGS system was found to be an effective The cDNA clone pRL011 is one of five ESTs that were tool for transient loss-of-function experiments in Hyos- found to be similar to the plant CYP80B subfamily mem- cyamus. Proof-of-concept experiments showed that bers. Based on sequence data in comparison with a full- TRV-mediated silencing of phytoene desaturase was length cDNA (see below), the five clones appeared to more effective in H. muticus (Egyptian henbane) than represent RsaI restriction fragments of cDNAs of the in H. niger, and, consequently, the former species was same gene. used as the VIGS host plant. The two Hyoscyamus spe- A full-length clone, pRL037, corresponding to the par- cies have similar leaf alkaloid levels and profiles (data tial ORF of pRL011 was obtained by 50 and 30 RACE and not shown). The silencing of several known genes in tro- PCR amplification. The cDNA insert of 1745 nt has an pane alkaloid biosynthesis in H. muticus, as evidenced ORF of 510 amino acids, predicting a polypeptide of by reduced alkaloid levels in infected plants (unpub- molecular mass 58.6 kDa. The predicted amino acid lished data), demonstrated that recombinant TRV could sequence shares 41%, 39%, and 38% sequence identity successfully silence the tropane alkaloid pathway in with the Coptis japonica, Papaver somniferum, and roots, the site of alkaloid biosynthesis in this species [1]. Eschscholzia californica (S)-N-methylcoclaurine-30- For functional identification, the DNA insert of pRL011 hydroxylases, respectively [24, 25]. It is notable that was subcloned into the VIGS binary vector pTRV2 [21]. the N-methylcoclaurine-30-hydroxylases of the CYP80B The resulting plasmid (pRL033) was used to transform subfamily hydroxylate an aromatic position of an Agrobacterium tumefaciens, and gene silencing was N-methylated intermediate in the benzylisoquinoline effected by infiltration of the transformants into the alkaloid pathway. Sequence alignment of the H. niger leaves of H. muticus plants (see Experimental Proce- enzyme with the C. japonica N-methylcoclaurine-30- dures for details). hydroxylase, CYP80B2, shows that the sequences Negative control experiments showed no change in share conserved eukaryotic cytochrome P450 regions: alkaloid profiles in plants subjected to VIGS (see Fig- a helix K region, an aromatic region, and a heme binding ure 2A). Vector control plants appeared healthy, and region at the C-terminal end (see Figure S3 in the Sup- plants undergoing silencing of the phytoene desaturase plemental Data available with this article online). Based gene showed the expected bleached phenotype (data on sequence comparisons, the H.

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