Molecular Evolution and Functional Characterization of a ......Lycopodium alkaloids (LAs) are Lys-derived alkaloids thathave quinolizine orpyridine and a-pyridinenucleiin their structures

Molecular Evolution and Functional Characterization of aBifunctional Decarboxylase Involved in LycopodiumAlkaloid Biosynthesis1[OPEN]

Somnuk Bunsupa, Kousuke Hanada, Akira Maruyama, Kaori Aoyagi, Kana Komatsu, Hideki Ueno,Madoka Yamashita, Ryosuke Sasaki, Akira Oikawa, Kazuki Saito*, and Mami Yamazaki*

Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan (S.B., A.M.,K.A., K.K., H.U., Mad.Y., K.S., Mam.Y.); Faculty of Pharmacy, Mahidol University, Ratchathewi, Bangkok10400, Thailand (S.B.); Kyushu Institute of Technology, Iizuka-shi, Fukuoka 820–8502, Japan (K.H.); RIKENCenter for Sustainable Resource Science, Tsurumi-ku, Yokohama 230-0045, Japan (R.S., A.O., K.S.); andFaculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan (A.O.)

ORCID ID: 0000-0001-6310-5342 (K.S.).

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs arepotentially useful against Alzheimer’s disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctionallysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodiumclavatum and Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC(LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of L-Lys over L-ornithine (L-Orn) by about5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Alliumcepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotianatabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derivedalkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present anexample of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae(legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of theancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites weredetected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance ofthose sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. Thisstudy contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

Since plants are sessile organisms, they produce a di-verse range of defense chemicals, known as specializedmetabolites, that contribute to the adaptation to their

ecological niches (Pichersky and Lewinsohn, 2011).Chemical compounds are important for plants, as theycan serve as attractants for insect pollinators or as de-fense against pathogens and herbivores (Pichersky andGang, 2000). Many plant species have been used in tra-ditional medicines for the treatment of various humandiseases (Tang andEisenbrand, 1992).Almost one-fourthof modern medicines are derived from natural sources(De Luca et al., 2012). Alkaloids are one of the most im-portant specialized metabolites and are mostly derivedfrom amino acids. Alkaloids display a vast variety of bio-logical activities, and many of them are currently used forclinical purposes; examples include morphine as an anal-gesic, artemisinin as an antimalarial, and camptothecin asan antineoplastic (De Luca et al., 2012).

Lycopodium alkaloids (LAs) are Lys-derived alkaloidsthat have quinolizine or pyridine and a-pyridine nuclei intheir structures (Ma and Gang, 2004). LAs have been iso-lated primarily from the genera Lycopodium andHuperzia,which are clubmosses (Ma andGang, 2004). Whole plantsfrom the families Huperziaceae and Lycopodiaceae havebeen used in Chinese folk medicine for the treatment ofvarious symptoms (Ma et al., 2007). Huperzia serrata pro-duces huperzine A (HupA), a promising candidate drug

1 This work was supported by Grants-in-Aid for Scientific Re-search from the Ministry of Education, Culture, Sports, Science,and Technology, JST, Strategic International Collaborative Re-search Program (SICORP), and by the Strategic Priority ResearchPromotion Program, Chiba University.

* Address correspondence to [email protected] or [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Mami Yamazaki ([email protected]).

K.S., Mam.Y., and S.B. designed the research; S.B., A.M., K.A.,K.K., H.U., and Mad.Y., cloned the constructs, performed recombi-nant protein purification and activity assays, alkaloid metabolite pro-files, gene expression, and localization, and analyzed the data; K.H.performed evolutionary analyses; R.S. and A.O. performed capillaryelectrophoresis-mass spectrometry analyses; S.B., K.H., and K.S.wrote the article; all authors discussed the results and commentedon the article.

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2432 Plant Physiology�, August 2016, Vol. 171, pp. 2432–2444, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved.

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for the treatment of Alzheimer’s disease, owing to itsfunction as a potent acetylcholinesterase inhibitor (Wanget al., 2009; Qian and Ke, 2014). HupA and its derivativeZT-1 have been evaluated in clinical trials for the treat-ment ofAlzheimer’s disease (Ma et al., 2007; Jia et al., 2013).Owing to the difficulties in cultivation and in vitro

propagation, the biosynthetic pathways for LAs are notwell documented and have been proposed based ontracer experiments using labeled precursors and plants intheir natural habitats (Ma and Gang, 2004, and refs.therein). Lysine decarboxylase (LDC) has been proposedas the entry-point enzyme in the LA biosynthetic path-way, which catalyzes the decarboxylation of Lys to yieldcadaverine (Fig. 1). Cadaverine is then catalyzed byCuAO to produce 5-aminopentanal, which is spontane-ously cyclized to thefirst intermediate for LAproduction,D1-piperideine (Ma and Gang, 2004). Based on analysesof the EST data from LA-producing plants, several can-didate genes for LA biosynthesis have been proposed;however, no further investigation has been performed(Luo et al., 2010a, 2010b). Recently, the CuAO gene fromH. serratawas cloned and characterized, using degenerateprimers based on the conserved sequences of the knownplant CuAO enzymes; however, the cloned CuAOshowed a broad substrate specificity (Sun et al., 2012).Recently,we showed that bifunctional lysine/ornithine

decarboxylases (L/ODCs) in the Lys-derived quinolizi-dine alkaloid (QA)-producing legumes were recruited bythe ubiquitous enzyme ornithine decarboxylase (ODC;Bunsupa et al., 2012a). ODC catalyzes the decarboxylationof L-Orn to yield putrescine, which is the main precur-sor for the production of Orn-derived alkaloids. In plantcells, putrescine and its derivative polyamines, spermi-dine and spermine, are essential for a wide range ofbiological processes during plant growth and devel-opment (Fuell et al., 2010). In addition to its role inalkaloid biosynthesis, cadaverine has been implicatedas a growth regulator and stress-response compoundin several plant species (Tomar et al., 2013).In this study, in order to elucidate the biosynthetic path-

way of LAs and the evolution of plant LDC, we clonedL/ODC from Lycopodium clavatum and H. serrata. We pro-vide results from both in vitro and in vivo experiments toconfirm the functions of L/ODC in L. clavatum. Using thetests for positive selection and assays of enzyme function,wethen show the convergent evolution of plant LDC in the Lys-derived alkaloid-producing plants. Furthermore, we wereable to detect the substitution site that is under positiveselection and is important for improving the LDC function.

RESULTS

Cloning of LDC from LA-Producing Plants

To identify the LDC-encoding cDNAs in L. clavatumand H. serrata, we used degenerate primers based onthe sequence homology between the L/ODCs and otherplant ODCs (Supplemental Fig. S1). The full-lengthcDNA clones of L. clavatum and H. serrata L/ODCs(hereafter referred to as LcL/ODC and HsL/ODC,

Figure 1. Putative biosynthetic pathway for LAs. Dotted arrows indicatemore than one catalytic conversion. CuAO, Copper amine oxidase.

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respectively) were obtained using 59- and 39-RACE. TheLcL/ODC contained a 1,500-bp open reading frame (ORF),encoding 500 amino acids. Two homologs of L/ODCfrom H. serrata, namely HsL/ODC1 and HsL/ODC2, wereobtained. HsL/ODC1 and HsL/ODC2 contained 1,521-and 1,527-bp ORFs, encoding 507 and 509 amino acids,respectively. The deduced amino acid sequences ofLcL/ODC, HsL/ODC1, and HsL/ODC2 were highlysimilar to one another (82% identity between LcL/ODCand HsL/ODCs, and 97% identity between HsL/ODC1and HsL/ODC2). Lower sequence identities of about55% with other plant L/ODCs and ODCs were observed.Sequence alignment of LcL/ODC with other eukaryoticODCs and L/ODCs revealed that all amino acid residuesresponsible for substrate binding were completelyconserved (Supplemental Fig. S1). The amino acid resi-due at position 344 of the narrow-leafed lupin (Lupinusangustifolius) L/ODC (LaL/ODC) was Phe. This Phe-344residue is critical for enzymatic activities of both LDC andODC in LaL/ODC (Bunsupa et al., 2012a). Interestingly,this position in LcL/ODC (position 374), HsL/ODC1(position 379), and HsL/ODC2 (position 377) is Tyr(Supplemental Fig. S1). For comparison,we also clonedthe partial sequence of L/ODC from Thermopsis lupinoides(TlL/ODC), which produces QAs. As expected, TlL/ODChad Phe at this position.

Phylogenetic analysis of the eukaryotic ODCs andLDCs provided good support for a monophyletic originof the sequences belonging to their families (Fig. 2).LcL/ODC, HsL/ODC1, and HsL/ODC2 formed a cladethat was distant from the Leguminosae L/ODCs, indi-cating a convergent evolution of the Lys-derived alkaloidproduction in distinct plant lineages.

In Vitro Activity Assays of Recombinant LcL/ODC Protein

To determine the biochemical functions of the identi-fied sequences, the ORFs of LcL/ODC and HsL/ODC1were heterologously expressed in Escherichia coli, whichwere then affinity purified and assayed for LDC andODC activities. However, we were unable to purify therecombinant HsL/ODC1 because of its insoluble nature.A molecular mass of 54 kD, in good agreement with thepredicted 54.21 kD,was observed upon SDS-PAGE of thetag-purified/cleaved LcL/ODC protein (SupplementalFig. S2). This purified recombinant protein was used totest both LDC and ODC activities, at optimal pH valuesof 8 and 7, respectively. LcL/ODC exhibited both LDCand ODC activities to similar extents and at the sameorder of magnitude as the L/ODCs characterized previ-ously fromQA-producing plants (Table I). The kcat valueswere calculated as 3.17 and 2.13 s21 for L-Lys and L-Orn,respectively, while the Km values were 1.69 and 5.48 mMfor L-Lys and L-Orn, respectively. LcL/ODC preferen-tially catalyzed the decarboxylation of L-Lys over L-Ornby about 5 times the catalytic efficiency (kcat/Km).

A competition assay, performed by varying the con-centration of L-Lys in the presence and absence of L-Ornand vice versa, showed a competitive reaction pattern

(Supplemental Fig. S3, A and B). The inhibitor assay,using a-difluoromethyl-Orn, an ODC suicide inhibitor,showed a dose-dependent inhibition of both LDC andODC activities (Supplemental Fig. S3, C and D). Theseresults suggest that the catalytic sites of LcL/ODC wereidentical in L-Orn and L-Lys and similar to that of pre-viously studied L/ODCs (Bunsupa et al., 2012a).

Overexpression of LcL/ODC in Tobacco Hairy RootsSignificantly Increases Anabasine Biosynthesis

To show that LcL/ODC functions as an LDC for al-kaloid biosynthesis, LcL/ODC was expressed under thecontrol of the constitutive cauliflower mosaic virus 35Spromoter in tobacco (Nicotiana tabacum) hairy roots aswell as a control GUS. The expression of LcL/ODCtranscript was confirmed using quantitative PCR.The alkaloid levels in the transgenic tobacco lineswere analyzed using HPLC-photodiode array detec-tion and liquid chromatography-mass spectrometry.The levels of anabasine, a Lys-derived alkaloid, in theLcL/ODC-transformed tobacco hairy roots increasedsignificantly, showing an average 2.7-fold increase(P , 0.05). In contrast, the levels of other tobacco al-kaloids did not change significantly compared withthe control lines (P . 0.05; Fig. 3A).

Comparison of the LcL/ODC gene transcript levels andthe tobacco alkaloid contents revealed a significant pos-itive correlation between the LcL/ODC transcript levelsand anabasine accumulation (Pearson’s correlation co-efficient [r] = 0.858, P , 0.001; Fig. 3B). A significantnegative correlation between the LcL/ODC transcriptlevels and the levels of nicotine, anOrn-derived alkaloid,was found (r = 20.636, P , 0.05; Fig. 3B). There was nosignificant correlation between the LcL/ODC transcriptlevels and the levels of other alkaloids (Fig. 3B).

Transgenic Arabidopsis Plants Expressing LcL/ODCShowed a Significant Increase in Cadaverine Production

The levels of amines, including L-Lys, L-Orn, cadav-erine, and putrescine, in the LcL/ODC- and control(GUS)-transformed Arabidopsis (Arabidopsis thaliana)plants were analyzed by capillary electrophoresis-massspectrometry. The LcL/ODC-expressing Arabidopsisplants displayed significantly increased levels of ca-daverine, which were, on average, 22-fold higher (P ,0.01) compared with the control plants. In contrast,L-Lys, L-Orn, and putrescine levels did not change signif-icantly (P . 0.05; Fig. 4A). Only the cadaverine levelsshowed a significant positive correlation with theLcL/ODC transcript levels (r = 0.977, P, 0.001; Fig. 4B).

Localization of LcL/ODC Protein

The analysis of LcL/ODC nucleotide sequence in-dicated alternative translational initiation sites,1AUG (LcL/ODC-Met-1) and 47AUG (LcL/ODC-Met-3;

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http://www.cbs.dtu.dk/services/NetStart/). The iPSORTprogram predicted that LcL/ODC-Met-3 has a chloroplasttransit peptide (http://ipsort.hgc.jp/).In order to determine the subcellular localization sites

of the alternatively translated products of LcL/ODC, thefull-length (LcL/ODC-Met-1) and truncated (LcL/ODC-

Met-3) sequences of LcL/ODC were fused to GFP ateither the N or the C terminus under the control of the35S cauliflower mosaic virus promoter. As a control, avector for the expression of only GFP and red fluorescentprotein (RFP) from Discosoma sp. (DsRed) was used forcytosolic localization. Each resulting construct was

Figure 2. Rooted phylogenetic tree of ODC and LDC amino acid sequences from eukaryotes. From an alignment of highlyconserved amino acids without gaps built using MEGA version 6 (Supplemental Data S1), the phylogenetic tree was constructedby PhyML3.0 using the best-fit mode. The divergence node derived from nonplant eukaryote genes is defined to be the root of thephylogenetic tree. Asterisks represent enzymes whose biochemical properties have been investigated. The blue branch linesindicate the Lys-derived alkaloid-producing plants. Uppercase letters next to the taxa represent the amino acid at position344 (LaL/ODC numbering). The bootstrap values (1,000 replicates) are shown. Letters A, B, and C indicate the branches that arelikely to be under positive selection for the production of Lys-derived alkaloids in plants: LAs (branch A), nuphar alkaloids (branchB), and QAs (branch C). Bootstrap values greater than 50% are shown. The accession numbers of the enzymes are listed inSupplemental Table S3.



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expressed simultaneously with DsRed in onion (Alliumcepa) epidermal cells and Nicotiana benthamiana leavesusing particle gun bombardment. The overlay of thegreen and red fluorescent images for all constructs lo-calized the detected signal to the cytosol in both onionepidermal cells and N. benthamiana leaves (Fig. 5, A andB). These localization patterns were identical to the cy-tosol localization references.

L/ODC and ODC Transcript Levels and Metabolite Profilesof Alkaloid-Producing and Nonproducing Plants

To determine the tissues where LcL/ODC is ex-pressed, quantitative real-time PCR was performedwith the shoots and roots of L. clavatum, and the tran-script levels in the roots were normalized to that of theshoots. LcL/ODC expression levels were similar for boththe tested organs (Fig. 5C).

In order to investigate the metabolite profiles and thegene expression patterns of plant L/ODCs and ODCs,we determined the metabolite profiles of L. clavatumand H. serrata. In addition, we assessed the transcriptlevels and metabolite profiles of two alkaloid-free le-gumes: soybean (Glycine max) and Lotus japonicus. Incontrast with the transcript levels of LcL/ODC, whichwere expressed equally in the shoots and the roots,soybean ODC2 (GmODC2) and L. japonicus ODC(LjODC) transcripts were expressed at higher levels inthe roots (Fig. 5, D and E). L-Lys and L-Orn were foundmainly in the shoots of the tested plants. On the otherhand, cadaverine was detected only in soybean,mainly in the roots (Supplemental Tables S1 and S2).LAs, such as lycodine and HupA, were higher in theshoots than in the roots (Supplemental Table S1).

Evolutionary Analyses of Plant ODCs and L/ODCs Detecta Positive Selection Site at Amino Acid Position 344

There were three evolutionary events that led to theproduction of Lys-derived alkaloids in plants: LAs(branch A), nuphar alkaloids (branch B), and QAs(branch C; Fig. 2). If these events were advantageous,the branches representing them (branches A, B, and C)would likely be under positive selection (Fig. 2).To examine whether these branches were positively

selected, we first performed a codon site test. However,there were no positive selection sites found (Table II).Since the positively selected site(s) might be found inonly the three evolutionary events that led to LAs inplants (Fig. 2), we simultaneously performed thebranch-site test by selecting the branches A, B, and C asthe foreground and the other branches as the back-ground (Bielawski and Yang, 2005; Zhang et al., 2005).The ratio of nonsynonymous (amino acid replacing)substitution rate (Ka) over the synonymous (silent)substitution rate (Ks; v = Ka/Ks) in the foreground(branches A, B, and C) was 1.4 (Table II). We used thelikelihood ratio test (LRT) to test the statistical signifi-cance of the detection of positive selection (Zhang et al.,2005). The LRTs for positive selection in the selectedforeground branches yielded statistically significantresults (P , 0.05, x2 test, degrees of freedom = 1; TableII). In the three branches, two amino acid residues,112 and 344, were positively selected, as shown usingthe Bayes empirical Bayes method (posterior probabil-ity . 0.95; Table II; Bielawski and Yang, 2005).

The homology modeling-based methods revealedthat only the amino acid 344 was located near the en-zyme active site (Supplemental Fig. S4). Therefore, theamino acid substitutions at site 344 were predicted toenlarge the active site cavity of LDC in QA-producingplants to allow access to L-Lys, which has one morecarbon than L-Orn (Bunsupa et al., 2012a).

Substitutions at Amino Acid 344 Are Important for a Shiftof ODC to LDC Activity

Since the amino acid at position 112 was not locatednear the active site and was not conserved across LA-producing plants, we focused on the substitutions atamino acid 344 (Supplemental Table S4). To investigatethe catalytic importance of the substitutions at aminoacid 344, an LcL/ODC-Y344H mutant was constructed.In addition, tobacco ODC-3 (NtODC3) and its mutants,NtODC3-H344F and NtODC3-H344Y, were clonedand prepared (Supplemental Fig. S2). The LcL/ODC-Y344H mutant exhibited a reduced catalytic efficiency(Kcat/Km) of LDC over ODC activities, ranging from4.84- to 0.08-fold, compared with the LcL/ODC-wildtype (Table I). The NtODC3-H344F and NtODC3-H344Y mutants exhibited a reduced Kcat/Km toward

Table I. Kinetic parameters of L/ODC and its mutant proteins

All experiments were performed in 50 mM potassium phosphate buffer (at optimal pH for each enzyme). Kinetic parameters were calculated frommean values (n = 3–4). ND, Not detected.

ProteinKm Vmax kcat kcat/Km

LDC/ODC Ratio of kcat/KmLDC ODC LDC ODC LDC ODC LDC ODC

mM nmol min21mg21 s21 m21 s21

LcL/ODC-wild type 1.69 5.48 3.65 2.46 3.17 2.13 1,878 388 4.84LcL/ODC-Y344H 22.39 8.21 0.51 2.47 0.44 2.14 20 261 0.08NtODC3-wild type ND 1.44 ND 30.30 ND 23.54 ND 16,351 NDNtODC3-H344Y ND 0.75 ND 14.33 ND 11.33 ND 14,794 NDNtODC3-H344F ND 0.61 ND 3.42 ND 2.66 ND 4,368 ND


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ODC activity by 1.1- and 3.7-fold, respectively, com-pared with the NtODC3-wild type (Table I). However,LDC activity was not detected in either the wild type orthe NtODC3 mutants.These results strongly suggest that the amino acid

substitution of His to Tyr in clubmosses, or from His toPhe/Tyr in legumes, is an important event that allowsLDC activity, although further substitutions are re-quired to optimize the LDC activity. In addition,

putative ODCs from Nuphar avena and Nelumbo nucifera,which produce LAs, have Tyr at position 344 (Forrestand Ray, 1971). In chickpea (Cicer arietinum), putrescineand cadaverine are accumulated and degraded in asimilar manner during seed germination and seedlingdevelopment, and the presence of LDC in this plant wasconfirmed by feeding experiments using labeled [14C]Lys (Torrigiani and Scoccianti, 1995). The chickpeaL/ODC has Phe at position 344. Taken together, these

Figure 3. Major alkaloid levels and correlations between the relativeabundance of LcL/ODC transcript and the alkaloid levels in tobaccohairy roots overexpressing LcL/ODC. A, Abundance of tobacco alka-loids in six and five independent hairy roots for LcL/ODC- and GUS-overexpressing lines, respectively (biological replicates, n = 4 for eachline). Values are means6 SE. Lowercase a indicates that the mean valueis statistically different from the corresponding GUS control, based on aone-tailed Student’s t test: P, 0.05. B, Correlations between each of thetobacco alkaloids and the relative LcL/ODC transcripts in tobacco hairyroots overexpressing LcL/ODC (orange circles) andGUS (blue triangles)lines. Pearson correlation coefficients (r) with the number of testedsamples in parentheses and the corresponding P values are shown. FW,Fresh weight.

Figure 4. Amine levels and correlations between the relative abun-dance of LcL/ODC transcript and the amine levels in Arabidopsis plantsexpressing LcL/ODC. A, Abundance of amines in pooled samples (six toeight plants) of Arabidopsis plants expressing LcL/ODC or GUS, witheight independent lines for each. Values are means 6 SE. Lowercase aindicates that the mean value is statistically different from the corre-sponding GUS control, based on a one-tailed Student’s t test: P, 0.01.B, Correlations between each of the amines and the relative LcL/ODCtranscripts in tobacco hairy roots overexpressing LcL/ODC (gray circles)and GUS (magenta triangles) lines. Pearson correlation coefficients (r)with the number of tested samples in parentheses and P values areshown. FW, Fresh weight.



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results support the importance of amino acid substitu-tions from His to Tyr or Phe at position 344.

DISCUSSION

Lys-derived alkaloids are widely distributed through-out the plant kingdom, from clubmosses to floweringplants (Bunsupa et al., 2012b). Based on the skeleton

structure, the Lys-derived alkaloids can be subdividedinto four main groups: quinolizidine, lycopodium, pi-peridine, and indolizidine alkaloids. With the exceptionof indolizidine alkaloids, LDC is the enzyme involvedin the first step of Lys-derived alkaloid biosynthesis(Bunsupa et al., 2012b). In previous studies, we reportedthe cloning and characterization of LDC, which is re-sponsible for the production of QAs (Bunsupa et al.,

Figure 5. Subcellular localization of LcL/ODC fused with GFP in onion epidermal cells and N. benthamiana leaves, and therelative abundance of LcL/ODC, GmODC, and LjODC transcript levels. A and B, LcL/ODC from the first (Met-1) and the third(Met-3) start codons were fused with GFP at the N-terminal (GFP-Met-1 and GFP-Met-3) or C-terminal (Met-1-GFP and Met-3-GFP) end. The resultant constructs were simultaneously and transiently expressed withDiscosoma sp. (DsRed). RFP from DsRedand GFP were used as references for cytosolic localization. GFP (green; top row), RFP (red; middle row), and merged (green andred; bottom row) fluorescence observed in onion epidermal cells (A) and N. benthamiana leaves (B) expressing the indicatedtarget constructs are shown. Bars = 50 mm and 20 mm for the onion epidermal cells (A) and the N. benthamiana leaves (B),respectively. C to E, Quantitative real-time PCR analysis of ODC or L/ODC transcript levels in the shoots and roots of L. clavatum(C), soybean (D), and L. japonicus (E). Values shown are means 6 SD of analytical replicates; n = 3 to 4.


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2012a). In this study, we isolated the LcL/ODC genefrom LA-producing plants, thus supporting the im-portant role of LDC in the production of alkaloids. Ourresults also provide a better understanding of the evo-lution of plant LDC.

Physiological Importance of LDC for Alkaloid Production

The recombinant LcL/ODC preferentially catalyzedthe decarboxylation of L-Lys over L-Orn, with a 5-foldincrease in efficiency in vitro, unlike LaL/ODC, whichcatalyzes both substrates nearly equally (Bunsupa et al.,2012a). The cellular abundance of Lys is expected toplay an important role in the production Lys-derivedalkaloids. The L-Lys level was about 15 times higherthan that of L-Orn in L. clavatum and 45 times higher inthe narrow-leafed lupin (Supplemental Table S1;Bunsupa et al., 2012a). LaL/ODC is localized in thechloroplast, where the last step of Lys biosynthesis isthought to take place, whereas LcL/ODC is localizedin the cytosol (Mazelis et al., 1976; Bunsupa et al.,2012a). These results suggest that the subcellulartrafficking of Lys to the cytosol may play a role in theefficient production of LAs. However, it is difficultto differentiate between a cytosolic localizationand localization in the plasma membrane or endo-plasmic reticulum. Further experiments, such as thoseemploying fluorescence recovery after photobleachingand colocalization studies with membrane markers,are needed to provide additional evidence of cytosoliclocalization, to further address this issue of compart-mentation of biosynthesis. The similar transcript levels ofL/ODC observed in the shoots and roots of L. clavatumwere inconsistent with the fact that major accumulationof the LAs happens in the stems and leaves. Thus, thedownstream enzymes in LA biosynthesis might belocalized in the shoots, or the transportation of thealkaloids produced might play a role in the differen-tial accumulation of LAs.The functions of LcL/ODC in vivo were character-

ized using a stable transformation in tobacco hairyroots and Arabidopsis plants, because of the difficultyin the transformation of L. clavatum. Analysis ofthe transgenic Arabidopsis plants and tobacco hairyroots expressing LcL/ODC showed a significant in-crease in cadaverine and the Lys-derived alkaloid,anabasine, respectively (Figs. 3 and 4). Furthermore,

the correlation analysis showed a significant correla-tion between the expression of LcL/ODC and thecadaverine levels in transgenic Arabidopsis as well asbetween LcL/ODC and anabasine in the transgenictobacco hairy roots. Anabasine is composed of tworings, a piperidine ring derived from Lys and a pyr-idine ring derived from nicotinic acid. The two ringsin nicotine are a pyrrolidine ring derived from Ornand a pyridine ring (Bunsupa et al., 2014). A negativecorrelation between LcL/ODC transcript and nicotinewasfound, but the nicotine levels did not decrease signifi-cantly. This result suggests a tight regulation of nicotinebiosynthesis in tobacco. Taken together, these resultsclearly support the function of LcL/ODC in plants.

Evolution of Plant LDC for the Production of Alkaloids

ODC, L/ODC, arginine decarboxylase (ADC),and diaminopimelate decarboxylase are pyridoxal-59-phosphate (PLP)-dependent enzymes that belong tothe Ala racemase family (Christen and Mehta, 2001).The functional specialization of most PLP-dependentenzymes occurred more than 1,500 million years ago,before the divergence of eukaryotes, archaebacteria,and eubacteria; their substrate specificities were alteredby the substitution of specific amino acids in the en-zyme active site (Christen and Mehta, 2001). Plants arethe only eukaryotes that possess the Arg pathway thatare not dependent on ODC (Fig. 6; Fuell et al., 2010).Interestingly, the protozoa Trypanosoma cruzi lacksODC activity and cannot grow in a medium withoutputrescine (Algranati, 2010).

In this study, we addressed the evolution of pro-miscuous functions, focusing on the activities of twoenzymes: ODC and LDC. Our data suggest that pro-miscuous activities existed in an ancestral gene, becausemost of the functionally characterized ODC genesexhibited both ODC and LDC activities, although amajority of them had a minor (promiscuous) LDCactivity and a major ODC activity. In the two distantlineages, legumes and clubmosses, the LDC activitywas reinforced independently via at least one eventof positive selection at amino acid position 344. Theindependent occurrence of the same event is likelyto be a consequence of natural selection rather thangenetic drift.

Table II. Molecular evolutionary analysis of eukaryotic ODCs and LDCs

Model ts/tvaNo. of

Genes

No. of

Codon Sites

vb in the Background

Branches

vb in the Foreground

BranchesLog Likelihood P

Positively Selected

Sitesc

Branch-site model 1.764 156 675 0.094 1 248,716.1203 Not applicable1.763 156 675 0.094 1.43 248,714.1402 0.028 112 (0.980)

344 (0.951)d

aTransversion:transition ratio. bv value is the ratio of nonsynonymous (amino acid replacing) substitution rate (Ka) over the synonymous (silent)substitution rate (Ks), Ka/Ks. cThe amino acid position is based on LaL/ODC amino acid numbering. The sites that have posterior proba-bilities . 0.9, by Bayes empirical Bayes analysis, are shown with the posterior probabilities in parentheses. dBoldface numbers indicatepositively selected sites (posterior probabilities . 0.95).



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Bifunctional L/ODC could be advantageous for bothlineages, because both primary (putrescine for poly-amine production) and specialized (cadaverine for al-kaloid production) metabolisms are important for cellgrowth and differentiation and for protection againstpathogens and herbivores (Pichersky and Gang, 2000),respectively. In contrast, the orthologous ODC genedisappeared in other plant lineages, such as Arabi-dopsis and the moss Physcomitrella patens (Fuell et al.,2010). Plants possess an Arg pathway consisting ofenzymes derived from a cyanobacterial ancestor(Illingworth et al., 2003) for the complementation ofputrescine production (Fig. 6). Therefore, it is likelythat ODC is not truly required in plants.

The eukaryotic ODC forms a homodimer, the sub-units of which interact in a head-to-tail manner, pro-ducing two active sites at their interphase (Lee et al.,2007). The fact that only ODC or LDC is found in plantscould be explained by dominant-negative mutations,which lead to mutant enzymes that disrupt the originalactivity (Veitia, 2007). Thus, the spatial expressionof duplicated copies, ancestral and novel/improvedLDC functions of ODC, might release these two copiesfrommolecular constraints, which was reported duringthe evolution of homospermidine synthase for the

production of pyrrolizidine alkaloids (Kalteneggeret al., 2013). The proteins encoded by ODC and LDCmight form heterodimers that are less efficient or eveninactive. Therefore, either the native or the evolvedenzymes could become fixed in the population vianatural selection.

We used the amino acid sequences of LaL/ODC andLcL/ODC as the query sequences to perform BLASTsearches against the National Center for Biotech-nology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Phytozome (http://www.phytozome.net/), andOneKP (Johnson et al., 2012; Matasci et al., 2014; Wickettet al., 2014; Xie et al., 2014; https://www.bioinfodata.org/Blast4OneKP/) databases. We identified two copies ofODC from foxtail millet (Setaria italica) on the samescaffold with a distance of approximately 24 kb (here-after referred to as SiODC1 and SiODC2). SiODC1 andSiODC2 have His and Tyr at position 344, respectively.Although SiODC1 had no intron in its genomic se-quence, SiODC2 contained one intron and lacked64 nucleotides in the coding region, which resulted inthe loss of 22 amino acids (Supplemental Fig. S5). Thiswas probably accomplished via a pseudoexonizationmechanism, during which an exon sequence becomesintronic (Xu et al., 2012; Supplemental Figs. S1 and S5).

Figure 6. Biosynthetic pathways for Lys- and Orn-derived alkaloids in plants.


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These specific amino acids are very important for theODC and LDC enzymes to bind to their cofactor, PLP(Lee et al., 2007). Therefore, it is likely that SiODC2 is apseudogene. However, functional analysis of SiODC1and SiODC2 is needed to support this hypothesis.In addition, recent draft genome sequence studies on

narrow-leafed lupin revealed the presence of a singleLDC gene (Conant and Wolfe, 2008; Yang et al., 2013).As active copies of both ODC and LDC were not foundin the same plant, divergence in the regulatory regionsdue to changes in the expression patterns of the LDCand ODC copies to reduce the dominant-negative effectmight not have occurred during plant LDC evolution.Therefore, either ODC or LDC was selected duringevolution and were maintained in the population.The results presented here indicate that an adaptive

change from ODC to LDC occurred in plants that pro-duce Lys-derived alkaloids and cadaverine, via theirpromiscuous functions. The LDC activity could havebeen gained independently within the Leguminosaeand clubmoss lineages. There are several models thatcould explain the possible routes by which the plantODC diverged to obtain an LDC function. First, thepromiscuous LDC activity from the ancestral ODC,which was involvedmainly in the primarymetabolism,could have evolved gradually via several mutationsand selections because of its physiological advantagefor the production of Lys-derived alkaloids. This wouldhave increased the LDC function without drasticallyaltering the original ODC function (i.e. a bifunctionalenzyme). Additionally, the alternative metabolic ADCpathway also could produce putrescine; thus, the an-cestral ODC was likely not constrained to maintain itsoriginal function. Therefore, the process of LDC evo-lution could have started prior to the gene duplicationof ODC. This kind of evolutionary process has beentermed a weak negative tradeoff, where the divergenceof a novel enzyme function occurs via a generalist in-termediate (Khersonsky and Tawfik, 2010). Second,when environmental changes made the promiscuousLDC function beneficial for plants, gene duplicationwould have been advantageous to increase the dose ofthe ancestral ODC gene, thus resulting in increasedprotein levels. This process would have allowed awider variety of function-altering mutations to accu-mulate, including potentially beneficial mutationsthat increase the LDC function, and get fixed in thepopulation. In contrast, the less functional copies andthose containing deleterious mutations, including theparental gene, could have been lost. This evolutionaryprocess has been proposed as the innovation, amplifi-cation, and divergence model (Bergthorsson et al., 2007).This model is supported by the identification of ODC-like and LDC-like sequences from the Lys-derivedalkaloid-free foxtail millet; however, the LDC-like se-quences show signatures of pseudogenization. In bothmodels, subsequent gene duplication could havehelped resolve the adaptive conflict between theODC and LDC activities by allowing the optimizationof each activity in two separate copies. However, our

data suggest that the divergence path toward a newlyspecialized LDC enzyme has not been completed;therefore, present-day enzymes exhibit only ODC orL/ODC (bifunctional) activity.

CONCLUSION

Overall, our results describe a clear case of theevolutionary innovation that uses promiscuous activ-ities as the starting point for the divergence of novelenzymes. In addition, the occurrence of an alternativemetabolic pathway might increase the evolutionaryadaptability of the related enzymes. These findingscontribute to a better understanding of how the en-zymes in the primary metabolism, which are under astrong purifying selection, could evolve to have anovel function for the specialized metabolism. Themolecular cloning and characterization of LcL/ODCshed light on LA biosynthesis and can serve as a basisfor further biotechnological production of LAs forhuman benefit.

MATERIALS AND METHODS

Plant Materials

Soybean (Glycine max; B01151) and Lotus japonicus (Gifu) seeds wereobtained from the National BioResource Project and Dr. Hiroshi Sudo (HoshiUniversity), respectively. Thermopsis lupinoides (syn. Thermopsis fabacea), aQA-producing plant, was obtained from the Medicinal Plant Gardens of theGraduate School of Pharmaceutical Sciences at Chiba University. Lycopodiumclavatum and Huperzia serrata were purchased from plant markets in Japan.Tobacco (Nicotiana tabacum ‘Petit Havana’ line SR1) was obtained from GhentUniversity.

Metabolite Profiling

Soybean and L. japonicus were cultured on Murashige and Skoog medium(Murashige and Skoog, 1962) containing 1% (w/v) Suc with 0.8% agar in agrowth chamber at 25°C under 16-h/8-h light (approximately 3,000 lx)/darkcycles for 30 d before metabolite analysis. L. clavatum and H. serrata weremaintained in a growth chamber in the same conditions as soybean andL. japonicus. Alkaloids, amines, and amino acids were extracted from thedifferent organs of L. clavatum, H. serrata, soybean, and L. japonicus and ana-lyzed using capillary electrophoresis-mass spectrometry as described previ-ously (Oikawa et al., 2011). The (6)-HupA standard was purchased fromSigma-Aldrich.

Measurement of RNA Levels

The total RNA was extracted (RNeasy kit; Qiagen) and reverse transcribedinto cDNA as described elsewhere (Bunsupa et al., 2012a). Real-time PCR wasperformed using the SYBR Green master mix (Applied Biosystems) at a finalvolume of 25 mL, including the appropriate primer pair for each target(Supplemental Table S4). Assays were run in quadruplicate in a StepOnePlusreal-time PCR system (Applied Biosciences). The amplification program con-sisted of 40 cycles of 95°C for 15 s followed by 60°C for 1 min. The relativequantification of gene expression was performed using the comparativethreshold cycle method. b-TUBULIN was used as an endogenous reference(Supplemental Table S4).

Cloning ODC and L/ODCs from Plants

The cDNAs encoding LcL/ODC,HsL/ODC1,HsL/ODC2, and TlL/ODCwereisolated using degenerate primers as described elsewhere (Bunsupa et al.,



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2012a). The full-length cDNAs were obtained using 59- and 39-RACE (TaKaRaBio). However, only a partial sequence for TlL/ODC was obtained. The full-length sequence for NtODC3 was isolated from tobacco using specific primersdesigned from NtODC (GenBank accession no. AB031066).

Heterologous Expression of Recombinant Proteins

The LcL/ODC and NtODC3 ORFs were amplified using gene-specificprimers with overhangs containing restriction sites (Supplemental Table S4).The mutants were then prepared by PCR-based mutagenesis (Higuchi et al.,1988) using the primers listed in Supplemental Table S4. The amplified frag-ments were inserted in frame into the same restriction sites within the pGEX-6P-2expression vector (GE Healthcare), which yielded recombinant gene productswithN-terminal glutathione S-transferase protein tags. The complete constructswere sequenced to confirm the correct orientation, expressed in Escherichia coli,and purified as described elsewhere (Bunsupa et al., 2012a). We also clonedand expressed HseL/ODC1 in E. coli; however, we were unable to purify therecombinant HseL/ODC1 protein because of its insoluble nature. The ratio ofthe targeted recombinant protein to other coeluted proteins, quantified bydensitometry using ImageJ software (http://imagej.nih.gov/ij/), was used tocalculate the protein concentration.

LDC and ODC Activity Assays

LDC and ODC enzyme activities were determined by measuring the CO2released from [14C]L-Lys and [14C]L-Orn, respectively (Gaines et al., 1988). Thedecarboxylase activities were assayed in 50 mM potassium phosphate, 5 mMEDTA, 4 mM dithiothreitol, 0.3 mM PLP, 0.5 to 3 mM L-[1-14C]Lys (40 mCi) orL-[1-14C]Orn (40 mCi), and 0.5 to 1 mg of purified enzyme, at pH 7.5 (except forthe LDC activity assay for LcL/ODC, which was performed at pH 7), in a finalvolume of 500 mL. The released labeled CO2 was trapped on Whatman 3MMfilter paper soaked in Soluene 350 (Perkin-Elmer), which was put to the top of aglass tube and closed with a rubber cap. Each reaction was performed at 37°Cfor 30 min. The ODC and LDC activities were then determined by measuring14CO2 released from L-[1-

14C]Orn and L-[1-14C]Lys, respectively, by liquid scintil-lation counting. The kinetics of decarboxylation of both L-Lys and L-Orn were an-alyzed by measuring the initial velocities over a range of substrate concentrations(0.5–2 mM). The competition assays were performed using 2 and 4 mM L-Orn orL-Lys, while 10 and 20 mM a-difluoromethyl-Orn were used for inhibitor assays.

Molecular Modeling

The three-dimensional model structures of LcL/ODC were predicted usingSWISS-MODEL (Arnold et al., 2006) and the published human-ODC-putrescinecomplex (Protein Data Bank entry 2000) as the template (Dufe et al., 2007). Themodeled protein was visualized using PyMOL (www.pymol.org).

Phylogenetic Analysis

LaL/ODC and LcL/ODC were used as queries and blasted with TBLASTNagainst the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Phytozome (http://www.phytozome.net/), andOneKP (Johnson et al., 2012; Matasci et al., 2014; Wickett et al., 2014; Xie et al.,2014; https://www.bioinfodata.org/Blast4OneKP/) databases. The ODCs andL/ODCs from plants that had E values , 10 e206 and had important catalyticresidues that are important for both ODC and LDC activities (SupplementalFig. S1), especially Asp at position 343 (LaL/ODC numbering), were selected(Grishin et al., 1999; Kern et al., 1999). Other eukaryotic ODCs, such as thosefrom yeast and human, also were included in the phylogenetic tree. The ac-cession numbers of each ODC and L/ODC are shown in Supplemental TableS3. Amino acid alignments were performed using MEGA version 6 and man-ually adjusted to improve the reliability of the alignment (Supplemental DataS2; Tamura et al., 2013). If a codon site has at least a gap in the generated align-ment, the codon site with a gapwas not used to generate the phylogenetic tree.Only highly conserved amino acids without gaps were used for further analysis(Supplemental Data S1). To generate a phylogenetic tree, the best-fit model forthe amino acid replacements was searched using ProtTest (Abascal et al., 2005);chosen for the best-fit model is LG (an improved general amino acid replace-ment matrix) + I (invariable sites) + G (g shape). The g shape is 1.116 in four ratecategories. The proportion of invariable sites was 0.08. Using the best-fit mode,we generated the phylogenetic tree in PhyML3.0 (Guindon et al., 2010).

Test for Positive Selection

The ORFs corresponding to all available ODC and L/ODC amino acid se-quences, as described above, were aligned. The resulting alignments were usedfor further analyses. We performed two analyses, the codon-site test and thebranch-site test, in codeml of the PAML (version 4) package (Yang, 2007). In thecodon site test, we performed two analyses, using models M7 (model = 0 andNSsites = 7) and M8 (model = 0 and NSsites = 8). The LRT was used to comparethe twomodels, assuming that twice the log-likelihood difference between thetwo models (2ΔL) follows a x2 distribution with a number of degrees offreedom. In the branch-site model, we selected two branches that led to Lys-derived alkaloids as foreground branches and searched for the positivelyselected sites (model = 2, NSsites = 2, and fix_omega = 0 [Ka/Ks = free]). Forthe null hypothesis, we used the branch-site model with following parame-ters: model = 2, NSsites = 2, and fix_omega = 1 [Ka/Ks = 1]. The LRT was usedto compare the two models, assuming that twice the log-likelihood differencebetween the two models (2ΔL) follows a x2 distribution with a number ofdegrees of freedom.

Protein Localization Analysis

The chimeric gene constructs of 35Spro:LcL/ODC-Met-1 and 35Spro:LcL/ODC-Met-3 fused with GFP at the N or C terminus were created using theprimers presented in Supplemental Table S4 and subsequently cloned into thepTH2 vector (Chiu et al., 1996). An empty vector fused with RFP from Discosomasp. (DsRed), 35Spro:DsRed, was used as a reference for the localization to cytosol(Kitajima et al., 2009). The resulting plasmids were expressed transiently inonion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves (8-week-oldplants) using a Helios gene gun (Bio-Rad) as described elsewhere (Bunsupaet al., 2012a). The GFP and RFP signals were observed using a confocal laser-scanning microscope (LSM700; Zeiss). For GFP, we used an argon laser with ex-citation at 488 nm with FSet38 wf filter. An argon laser with excitation at 555 nmwith FSet43 wf filter was used for RFP. All images were acquired from single op-tical sections and were merged using the ZEN 2012 lite imaging software (Zeiss).

Plasmid Construction and Plant Transformation

To construct pGWB2-LcL/ODC (35Spro:LcL/ODC ), the full-length sequencefor LcL/ODCwas cloned into the binary vector pGWB2 (Nakagawa et al., 2007;for primer sequences, see Supplemental Table S4) via Gateway technology(Invitrogen). Transgenic tobacco (‘Petit Havana’ line SR1) hairy roots andArabidopsis (Arabidopsis thaliana) were generated as described elsewhere(Bunsupa et al., 2012a). Tobacco alkaloids and amines were measured asdescribed elsewhere (Bunsupa et al., 2012a).

Statistical Analysis

Student’s one-tailed t test was used to identify statistically significant dif-ferences in the metabolite levels of transgenic Arabidopsis plants and tobaccohairy roots. Pearson correlation analysis was performed to calculate the cor-relation between the metabolite levels and the expression levels of LcL/ODC intransgenic Arabidopsis plants and tobacco hairy roots. For all statistical tests,significance was determined at P , 0.05.

Accession Numbers

The new DNA sequences reported here are deposited in the DNA DataBank of Japan under accession numbers AB915695 (LcL/ODC), AB915696(HsL/ODC1), AB915697 (HsL/ODC2), AB915698 (TlL/ODC ), and LC030209(NtODC3).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Alignment of selected eukaryotic ODC andL/ODC amino acid sequences.

Supplemental Figure S2. SDS-PAGE of the recombinant LcL/ODC,NtODC3, and their mutant proteins purified from E. coli.

Supplemental Figure S3. Competition and inhibition studies of LcL/ODC.


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Supplemental Figure S4. Overview of the predicted protein structure ofthe LcL/ODC complex homology model with the Schiff base interme-diate of putrescine and PLP at the active site.

Supplemental Figure S5. Alignment of genomic sequences of putativeODCs from foxtail millet.

Supplemental Table S1. Levels of amines and LAs in each organ ofL. clavatum and H. serrata.

Supplemental Table S2. Levels of amines in each organ of soybean andL. japonicus.

Supplemental Table S3. Accession numbers of the sequences used forphylogenetic analysis.

Supplemental Table S4. List of primers used in this study.

Supplemental Data S1. Highly conserved amino acid alignment withoutgaps for the construction of the phylogenetic tree in Figure 2.

Supplemental Data S2. Original amino acid alignment using ClustalW inthe MEGA6 program.

ACKNOWLEDGMENTS

We thank Dr. Ryo Nakabayashi (RIKEN Center for Sustainable ResourceScience) for preliminary analyses of LAs by liquid chromatography-mass spec-trometry; Satoko Sugawara (RIKEN Center for Sustainable Resource Science)for excellent technical support in the preparation of transgenic Arabidopsisplants; Tsuyoshi Nakagawa (Shimane University) for providing the destinationvector pGWB2; Toshiaki Mitsui (Niigata University) for providing pWx-TP-DsRed vector; and all 1000 Plants Project contributors for gene sequencing data.

Received April 25, 2016; accepted June 9, 2016; published June 14, 2016.

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Molecular Evolution and Functional Characterization of a ......Lycopodium alkaloids (LAs) are Lys-derived alkaloids thathave quinolizine orpyridine and a-pyridinenucleiin their structures