Plant Physiology Preview. Published on June 14, 2016, … · The recombinant LcL/ODC preferentially...

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Running head: Lysine/ornithine Decarboxylase in Clubmosses 1

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All correspondence should be sent to: 3

Prof. Kazuki Saito and Assoc. Prof. Mami Yamazaki 4

Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 260-8675, Japan. 5

Tel: +81-43-226-2931; Fax: +81-43-226-2932 6

E-mail: [email protected], [email protected] 7

Requests for materials should be addressed to Mami Yamazaki (mamiy@ faculty.chiba-u.jp). 8

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Research area: Biochemistry and Metabolism 10

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Plant Physiology Preview. Published on June 14, 2016, as DOI:10.1104/pp.16.00639

Copyright 2016 by the American Society of Plant Biologists

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Molecular Evolution and Functional Characterization of a bifunctional Decarboxylase 25 Involved in Lycopodium Alkaloid Biosynthesis 26

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Somnuk Bunsupa, Kousuke Hanada, Akira Maruyama, Kaori Aoyagi, Kana Komatsu, Hideki Ueno, 28

Madoka Yamashita, Ryosuke Sasaki, Akira Oikawa, Kazuki Saito*, and Mami Yamazaki* 29

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Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan 31

(S.B., A.M., K.A., K.K., H.U., Mad.Y., K.S., M.Y.); Faculty of Pharmacy, Mahidol University, 32

Ratchathewi, Bangkok 10400, Thailand (S.B.); Kyushu Institute of Technology, Iizuka-shi, Fukuoka 33

820-8502, Japan (K.H.); RIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama 34

230-0045, Japan (R.S., A.O., K.S.); Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, 35

Japan (A.K.) 36

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Summary of significant findings 38

Production of plant lysine-derived alkaloids is due to convergent evolution of lysine 39

decarboxylase 40

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Footnotes 48

Author contributions K.S., M.Y., and S.B. designed the research; S.B., A.M., K.A., K.K., H.U., and 49

Mad. Y., cloned the constructs, performed recombinant protein purification and activity assays, 50

alkaloid metabolite profiles, gene expression, localization and analyzed the data; K.H. performed 51

evolutionary analyzes; R.S. and A.O. performed CE−MS analyzes; and S.B., K.H., and K.S. wrote 52

the paper. All authors discussed the results and commented on the manuscript. 53

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This study was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from The 55

Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and by Strategic Priority 56

Research Promotion Program, Chiba University. 57

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Corresponding author: Kazuki Saito ([email protected]), Mami Yamazaki 59

([email protected]) 60

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Abstract 72

Lycopodium alkaloids (LAs) are derived from Lys and are found mainly in Huperziaceae and 73

Lycopodiaceae. LAs are potentially useful against Alzheimer’s disease, schizophrenia, and 74

myasthenia gravis. Here, we cloned the bifunctional L/ODC (Lys/Orn decarboxylase), the first gene 75

involved in lycopodium alkaloid biosynthesis, from LA-producing plants, Lycopodium clavatum and 76

Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum 77

L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of 78

L-Lys over L-Orn by about five times. Transient expression of LcL/ODC fused with the N- or 79

C-terminal of green fluorescent protein, in onion epidermal cells and Nicotiana benthamiana leaves, 80

showed LcL/ODC localization in the cytosol. Transgenic Nicotiana tabacum hairy roots and 81

Arabidopsis thaliana plants expressing LcL/ODC enhanced the production of Lys-derived alkaloid, 82

anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In 83

addition, we present an example of convergent evolution of plant Lys decarboxylase that resulted in 84

the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). 85

This convergent evolution event probably occurred via the promiscuous functions of the ancestral 86

Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The 87

positive selection sites were detected by statistical analyses using phylogenetic trees and were 88

confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the 89

promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. 90

This study contributes to a better understanding of the LA biosynthesis and the molecular evolution 91

of plant Lys decarboxylase. 92



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Introduction 93

Since plants are sessile organisms, they produce a diverse range of defense chemicals, known as 94

specialized metabolites that contribute to the adaptation to their ecological niches (Pichersky and 95

Lewinsohn, 2011). Chemical compounds are important for plants, as they can serve as attractants for 96

insect pollinators or as defense against pathogens and herbivores (Pichersky and Gang, 2000). Many 97

plant species have been used in traditional medicines for the treatment of various human diseases 98

(Tang and Eisenbrand, 1992). Almost one-fourth of the modern medicines are derived from natural 99

sources (De Luca et al., 2012). Alkaloids are one of the most important specialized metabolites, and 100

are mostly derived from amino acids. Alkaloids display a vast variety of biological activities and 101

many of them are currently used for clinical purposes, examples include morphine as an analgesic, 102

artemisinin as antimalarial, and camptothecin as an antineoplastic (De Luca et al., 2012). 103

Lycopodium alkaloids (LAs) are Lys-derived alkaloids that have quinolizine or pyridine 104

and α-pyridine nuclei in their structures (Ma and Gang, 2004). LAs have been isolated primarily 105

from the genera Lycopodium and Huperzia, which are clubmosses (Ma and Gang, 2004). Whole 106

plants from the families Huperziaceae and Lycopodiaceae have been used in Chinese folk medicine 107

for the treatment of various symptoms (Ma et al., 2007). Huperzia serrata produces Huperzine A 108

(HupA), a promising candidate drug for the treatment of Alzheimer’s disease, owing to its function 109

as a potent acetylcholinesterase inhibitor (Wang et al., 2009; Qian and Ke, 2014). HupA and its 110

derivative ZT-1 have been evaluated in clinical trials for the treatment of Alzheimer’s disease (Ma et 111

al., 2007; Jia et al., 2013). 112

Owing to the difficulties in cultivation and in vitro propagation, the biosynthetic pathways 113

for LAs are not well documented and have been proposed based on tracer experiments using labelled 114

precursors and plants in their natural habitats (Ma and Gang, 2004 and references therein). LDC (Lys 115

decarboxylase) has been proposed as the entry-point enzyme in LA biosynthetic pathway, which 116



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catalyzes the decarboxylation of Lys to yield cadaverine (Fig. 1). Cadaverine is then catalyzed by 117

CuAO (copper amine oxidase) to produce 5-aminopentanal, which is spontaneously cyclized to the 118



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first intermediate for LA production, Δ1-piperideine (Ma and Gang, 2004). Based on analyses of the 119

expressed sequence tag data from LA-producing plants, several candidate genes for LA biosynthesis 120

have been proposed; however, no further investigation has been performed (Luo et al., 2010a; Luo et 121

al., 2010b). Recently, the CuAO gene from H. serrata was cloned and characterized, using 122

degenerate primers based on the conserved sequences of the known plant CuAO enzymes; however, 123

the cloned CuAO showed a broad substrate specificity (Sun et al., 2012). 124

Recently, we showed that bifunctional L/ODCs (Lys/Orn decarboxylases) in the 125

Lys-derived quinolizidine alkaloid (QA)-producing legumes were recruited by the ubiquitous 126

enzyme ODC (Orn decarboxylase) (Bunsupa et al., 2012a). ODC catalyzes the decarboxylation of 127

L-Orn to yield putrescine, which is the main precursor for the production of Orn-derived alkaloids. 128

In plant cells, putrescine and its derivative polyamines, spermidine and spermine, are essential for a 129

wide range of biological processes during the plant growth and development (Fuell et al., 2010). In 130

addition to its role in alkaloid biosynthesis, cadaverine has been implicated as a growth regulator and 131

stress-response compound in several plant species (Tomar et al., 2013). 132

In the present study, in order to elucidate the biosynthetic pathway of LAs and the 133

evolution of plant LDC, we cloned L/ODC from Lycopodium clavatum and H. serrata. We provide 134

results from both in vitro and in vivo experiments to confirm the functions of L/ODC in L. clavatum. 135

Using the tests for positive selection and assays of enzyme function, we then show the convergent 136

evolution of plant LDC in the Lys-derived alkaloid producing plants. Furthermore, we were able to 137

detect the substitution site that is under positive selection and is important for improving the LDC 138

function. 139



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Results 141

Cloning of Lys Decarboxylase from Lycopodium Alkaloids Producing Plants 142

To identify the LDC-encoding cDNAs in L. clavatum and H. serrata, we used degenerate primers 143

based on the sequence homology between the L/ODCs and other plant ODCs (Supplemental Fig. S1). 144

The full-length cDNA clones of L. clavatum and H. serrata L/ODCs (hereafter referred to as 145

LcL/ODC and HsL/ODC, respectively) were obtained using 5′- and 3′- RACEs. The LcL/ODC 146

contained a 1500-bp open reading frame (ORF), encoding 500 amino acids. Two homologues of 147

L/ODC from H. serrata, namely HsL/ODC1 and HsL/ODC2, were obtained. HsL/ODC1 and 148

HsL/ODC2 contained 1521 and 1527 bp ORFs, encoding 507 and 509 amino acids, respectively. The 149

deduced amino acid sequences of LcL/ODC, HsL/ODC1, and HsL/ODC2 were highly similar to one 150

another (82% identity between LcL/ODC and HsL/ODCs, and 97% identity between HsL/ODC1 151

and HsL/ODC2). Lower sequence identities of about 55% with other plant L/ODCs and ODCs were 152

observed. Sequence alignment of LcL/ODC with other eukaryotic ODCs and L/ODCs revealed that 153

all amino acid residues responsible for substrate binding were completely conserved (Supplemental 154

Fig. S1). The amino acid residue at position 344 of the narrow-leafed lupin LaL/ODC (Lupinus 155

angustifolius L/ODC) was Phe (F). This F344 residue is critical for enzymatic activities of both LDC 156

and ODC in LaL/ODC (Bunsupa et al., 2012a). Interestingly, this position in LcL/ODC (position 157

374), HsL/ODC1 (position 379), HsL/ODC2 (position 377) is Tyr (Y) (Supplemental Fig. S1). For 158

comparison, we also cloned the partial sequence of L/ODC from Thermopsis lupinoides (TlL/ODC), 159

which produces QAs. As expected, TlL/ODC had Phe at this position. 160

Phylogenetic analysis of the eukaryotic ODCs and LDCs provided good support for 161

monophyletic origin of the sequences belonging to their families (Fig. 2). LcL/ODC, HsL/ODC1, 162

and HsL/ODC2 formed a clade that was distant from the Leguminosae L/ODCs, indicating a 163

convergent evolution of the Lys-derived alkaloid production in distinct plant lineages. 164



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In Vitro Activity Assays of Recombinant LcL/ODC protein 165

To determine the biochemical function of the identified sequences, the ORFs of LcL/ODC and 166



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HsL/ODC1 were heterologously expressed in Escherichia coli, which were then affinity-purified and 167

assayed for LDC and ODC activities. However, we were unable to purify the recombinant 168

HsL/ODC1 because of its insoluble nature. A molecular mass of 54 kDa, in good agreement with the 169

predicted 54.21 kDa, was observed upon SDS-PAGE of the tag-purified/cleaved LcL/ODC protein 170

(Supplemental Fig. S2). This purified recombinant protein was used to test both LDC and ODC 171

activities, at optimal pH values of 8.0 and 7.0, respectively. LcL/ODC exhibited both LDC and ODC 172

activities to similar extents and at the same order of magnitude as the L/ODCs characterized 173

previously from QA-producing plants (Table I). The kcat values were calculated as 3.17 and 2.13 s−1 174

for L-Lys and L-Orn, respectively, while the Km values were 1.69 and 5.48 mM for L-Lys and L-Orn, 175

respectively. LcL/ODC preferentially catalyzed the decarboxylation of L-Lys over L-Orn by about 5 176

times the catalytic efficiency (kcat/Km). 177

A competition assay, performed by varying the concentration of L-Lys in the presence and 178

absence of L-Orn and vice versa, showed a competitive reaction pattern (Supplemental Fig. S3A and 179

S3B). The inhibitor assay, using α-difluoromethylornithine (α-DFMO), an ODC suicide inhibitor, 180

showed a dose-dependent inhibition of both LDC and ODC activities (Supplemental Fig. S3C and 181

S3D). These results suggest that the catalytic sites of LcL/ODC were identical in L-Orn and L-Lys, 182

and similar to that of previously studied L/ODCs (Bunsupa et al., 2012a). 183

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Overexpression of LcL/ODC in Tobacco Hairy Roots Significantly Increases Anabasine 185

Biosynthesis 186

To show that LcL/ODC functions as an LDC for alkaloid biosynthesis, LcL/ODC was expressed 187

under the control of the constitutive CaMV (Cauliflower mosaic virus) 35S promoter in tobacco 188

(Nicotiana tabacum) hairy roots, as well as a control GUS (β-glucuronidase). The expression of 189

LcL/ODC transcript was confirmed using quantitative PCR. The alkaloid levels in the transgenic 190



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tobacco lines were analyzed using HPLC-photodiode array detection and liquid 191

chromatography-mass spectrometry (LC−MS). The levels of anabasine, a Lys-derived alkaloid, in 192

the LcL/ODC transformed tobacco hairy roots significantly increased, showing an average 2.7-fold 193

increase (P < 0.05). In contrast, the levels of other tobacco alkaloids did not change significantly, 194



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compared with the control lines (P > 0.05; Fig. 3A). 195

Comparison of the LcL/ODC gene transcript levels and the tobacco alkaloid contents 196

revealed a significant positive correlation between the LcL/ODC transcript levels and anabasine 197

accumulation (Pearson’s correlation coefficient (r) = 0.858, P < 0.001; Fig. 3B). A significant 198

negative correlation between the LcL/ODC transcript levels and the levels of nicotine, an 199

Orn-derived alkaloid, was found (r = -0.636, P < 0.05; Fig. 3B). There was no significant correlation 200

between the LcL/ODC transcript levels and the levels of other alkaloids (Fig. 3B). 201

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Transgenic Arabidopsis Plants Expressing LcL/ODC Showed a Significant Increase in 203

Cadaverine Production 204

The levels of amines, including L-Lys, L-Orn, cadaverine, and putrescine, in the LcL/ODC- and 205

control (GUS)-transformed Arabidopsis plants were analyzed by capillary electrophoresis (CE)-MS. 206

The LcL/ODC-expressing Arabidopsis plants displayed significantly increased levels of cadaverine, 207

which were, on an average, 22-fold higher (P < 0.01), compared with the control plants. In contrast, 208

L-Lys, L-Orn, and putrescine levels did not change significantly (P > 0.05; Fig. 4A). Only the 209

cadaverine levels showed a significant positive correlation with the LcL/ODC transcript levels (r = 210

0.977, P < 0.001; Fig. 4B). 211

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Localization of LcL/ODC protein 213

The analysis of LcL/ODC nucleotide sequence indicated alternative translational initiation sites, 214

1AUG (LcL/ODC-Met1) and 47AUG (LcL/ODC-Met3) (http://www.cbs.dtu.dk/services/NetStart/). 215

The iPSORT program predicted that LcL/ODC-Met3 has a chloroplast transit peptide 216

(http://ipsort.hgc.jp/). 217

In order to determine the subcellular localization sites of the alternatively translated 218



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products of LcL/ODC, the full-length (LcL/ODC-Met1) and truncated (LcL/ODC-Met3) sequences 219

of LcL/ODC were fused to GFP (green fluorescent protein) at either the N- or the C- terminal, under 220



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the control of the 35S CaMV promoter. As a control, a vector for the expression of only GFP and 221

RFP (red fluorescent protein) from Discosoma sp. (DsRed) was used for cytosolic localization. Each 222



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resulting construct was simultaneously expressed with DsRed in onion epidermal cells and Nicotiana 223

benthamiana leaves using particle gun bombardment. The overlay of the green and red fluorescent 224



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images for all constructs localized the detected signal to the cytosol in both onion epidermal cells 225

and N. benthamiana leaves (Fig. 5A and 5B). These localization patterns were identical to the 226

cytosol localization references. 227

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L/ODCs and ODCs Transcript Levels and Metabolite Profiles of Alkaloid-producing and 229

Non-producing Plants 230

To determine the tissues where LcL/ODC is expressed, quantitative real-time PCR was performed 231

with the shoots and roots of L. clavatum, and the transcript levels in the roots were normalized to 232

that of the shoots. LcL/ODC expression levels were similar for both the tested organs (Fig. 5C). 233

In order to investigate the metabolite profiles and the gene expression patterns of plant 234

L/ODCs and ODCs, we determined the metabolite profiles of L. clavatum and H. serrata. In addition, 235

we assessed the transcript levels and metabolite profiles of alkaloid-free legumes: soybean (Glycine 236

max) and Lotus japonicus. In contrast with the transcript levels of LcL/ODC, which expressed 237

equally in the shoots and the roots, G. max ODC2 (GmODC2) and Lotus japonicus ODC (LjODC) 238

transcripts were expressed at higher levels in the roots (Fig. 5D and 5E). L-Lys and L-Orn were 239

mainly found in the shoots of the tested plants. On the other hand, cadaverine was detected only in G. 240

max, mainly in the roots (Supplemental Tables S1 and S2). LAs, such as lycodine and HupA, were 241

higher in the shoots than in the roots (Supplemental Table S1). 242

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Evolutionary Analyses of Plant ODCs and L/ODCs Detect Positive Selection Site at Amino 244

Acid 344 Position 245

There were three evolutionary events that led to the production of Lys-derived alkaloids in plants: 246

lycopodium alkaloids (branch A), nuphar alkaloids (branch B), and QAs (branch C) (Fig. 2). If these 247

events were advantageous, the branches representing them (branches A, B, and C) would likely be 248



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under positive selection (Fig. 2). To examine whether these branches were positively selected, we 249

first performed a codon site test. However, there were no positive selection sites found (Table II). 250

Since the positively selected site(s) might be found in only the three evolutionary events that led to 251

LAs in plants (Fig. 2), we simultaneously performed the branch-site test by selecting the branches A, 252

B, and C as the foreground, and the other branches as the background (Bielawski and Yang, 2005; 253

Zhang et al., 2005). The ratio of nonsynonymous (amino acid replacing) substitution rate (Ka) over 254

the synonymous (silent) substitution rate (Ks; ω = Ka/Ks) in the foreground (branches A, B, and C) 255

was 1.4 (Table II). We used the likelihood ratio test (LRT) to test the statistical significance of the 256

detection of positive selection (Zhang et al., 2005). The LRTs for positive selection in the selected 257

foreground branches yielded statistically significant results (P-value < 0.05, chi-square test, df = 1; 258

Table II). In the three branches, two amino acid residues, 112 and 344, were positively selected, as 259

shown using the Bayes Empirical Bayes method (posterior probability > 0.95; Table II; Bielawski 260

and Yang, 2005). 261

The homology modeling-based methods revealed that only the amino acid 344 was located 262

near the enzyme active site (Supplemental Fig. S4). Therefore, the amino acid substitutions at site 263

344 were predicted to enlarge the active site cavity of LDC in QA-producing plants to allow access 264

to L-Lys, which has one more carbon than L-Orn (Bunsupa et al., 2012a). 265

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Substitutions at Amino Acid 344 is Important for a Shift of ODC to LDC Activity 267

Since the amino acid at position 112 was not located near the active site and was not conserved 268

across LA-producing plants, we focused on the substitutions at amino acid 344 (Supplemental Table 269

S4). To investigate the catalytic importance of the substitutions at amino acid 344, an 270

LcL/ODC-Y344H mutant was constructed. In addition, N. tabacum ODC-3 (NtODC3) and its 271

mutants, NtODC3-H344F and NtODC3-H344Y, were cloned and prepared (Supplemental Fig. S2). 272



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The LcL/ODC-Y344H mutant exhibited a reduced catalytic efficiency (Kcat/Km) of LDC over ODC 273

activities, ranging from 4.84- to 0.08-fold, compared with the LcL/ODC-wild-type (Table I). The 274

NtODC3-H344F and NtODC3-H344Y mutants exhibited a reduced Kcat/Km towards ODC activity by 275

1.1- and 3.7-fold, respectively, compared with the NtODC3-wild-type (Table I). However, LDC 276

activity was not detected in either the wild-type or the NtODC3 mutants. 277

These results strongly suggest that the amino acid substitution of H to Y in clubmosses, or 278

from H to F/Y in legumes, is an important event that allows LDC activity, although further 279

substitutions are required to optimize the LDC activity. In addition, putative ODCs from Nuphar 280

avena and Nelumbo nucifera, which produce LA-alkaloids, have Y at position 344 (Forrest and Ray, 281

1971). In chickpeas (Cicer arietinum), putrescine and cadaverine are accumulated and degraded in a 282

similar manner during seed germination and seedling development, and the presence of LDC in this 283

plant was confirmed by feeding experiments using labeled 14C-Lys (Torrigiani and Scoccianti, 1995). 284

The C. arietinum L/ODC has Phe at position 344. Taken together, these results support the 285

importance of amino acid substitutions from H to Y or F at the 344th position. 286

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Discussion 288

Lys-derived alkaloids are widely distributed throughout the plant kingdom, from clubmosses to 289

flowering plants (Bunsupa et al., 2012b). Based on the skeleton structure, the Lys-derived alkaloids 290

can be subdivided into four main groups: quinolizidine, lycopodium, piperidine, and indolizidine 291

alkaloids. With the exception of indolizidine alkaloids, LDC is the enzyme involved in the first step 292

of Lys-derived alkaloid biosynthesis (Bunsupa et al., 2012b). In the previous studies, we have 293

reported the cloning and characterization of LDC, which is responsible for the production of QAs 294

(Bunsupa et al., 2012a). In this study, we isolated the LcL/ODC gene from lycopodium alkaloid 295

producing plants, thus, supporting the important role of LDC in the production of alkaloids. Our 296

results also provide a better understanding of the evolution of plant LDC. 297

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Physiological importance of LDC for alkaloids production 299

The recombinant LcL/ODC preferentially catalyzed the decarboxylation of L-Lys over L-Orn, with a 300

5-fold increase in efficiency in vitro, unlike LaL/ODC, which catalyzes both the substrates nearly 301

equally (Bunsupa et al., 2012a). The cellular abundance of Lys is expected to play an important role 302

in the production Lys-derived alkaloids. The L-Lys level was about 15 times higher than that of 303

L-Orn in L. clavatum and 45 times higher in the narrow-leafed lupin (Supplemental Table S1; 304

Bunsupa et al., 2012a). LaL/ODC is localized in the chloroplast, where the last step of Lys 305

biosynthesis is thought to take place, whereas LcL/ODC is localized in the cytosol (Mazelis et al., 306

1976; Bunsupa et al., 2012a). These results suggest that the subcellular trafficking of Lys to the 307

cytosol may play a role in the efficient production of LAs. However, it is difficult to differentiate 308

between a cytosolic localization and localization in the plasma membrane or endoplasmic reticulum. 309

Further experiments, such as the ones employing fluorescence recovery after photobleaching 310

(FRAP) and co-localization studies with membrane markers, are needed to provide additional 311



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evidence of cytosolic localization, to further address this issue of compartmentation of biosynthesis. 312

The similar transcript levels of L/ODC observed in the shoots and roots of L. clavatum were 313

inconsistent with the fact that major accumulation of the LAs happens in the stems and leaves. Thus, 314

the downstream enzymes in LA biosynthesis might be localized in the shoots, or the transportation of 315

the produced alkaloids might play a role in the differential accumulation of LAs. 316

The functions of LcL/ODC in vivo were characterized using a stable transformation in 317

tobacco hairy roots and Arabidopsis plants, because of the difficulty in transformation of L. clavatum. 318

Analysis of the transgenic Arabidopsis plants and tobacco hairy roots expressing LcL/ODC showed a 319

significant increase in cadaverine and Lys-derived alkaloid, anabasine, respectively (Fig. 3 and 4). 320

Furthermore, the correlation analysis showed a significant correlation between the expression of 321

LcL/ODC and the cadaverine levels in transgenic Arabidopsis, as well as between LcL/ODC and 322

anabasine in the transgenic tobacco hairy roots. Anabasine is composed of two rings, a piperidine 323

ring derived from Lys and a pyridine ring derived from nicotinic acid. The two rings in nicotine are: 324

a pyrrolidine ring derived from Orn, and a pyridine ring (Bunsupa et al., 2014). The negative 325

correlation between LcL/ODC transcript and nicotine was found, but the nicotine levels did not 326

decrease significantly. This result suggests a tight regulation of nicotine biosynthesis in tobacco. 327

Taken together, these results clearly support the function of LcL/ODC in plants. 328

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Evolution of Plant LDC for the production of Alkaloids 330

ODC, L/ODC, ADC (arginine decarboxylase), and DAPC (diaminopimelate decarboxylase) are 331

pyridoxal-5′-phosphate (PLP)-dependent enzymes that belong to the alanine racemase family 332

(Christen and Mehta, 2001). The functional specialization of most PLP-dependent enzymes occurred 333

more than 1500 million years ago, before the divergence of eukaryotes, archaebacteria, and 334

eubacteria; their substrate specificities were altered by the substitution of specific amino acids in the 335



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enzyme active site (Christen and Mehta, 2001). Plants are the only eukaryotes that possess the 336

arginine pathway that is not dependent on ODC (Fig. 6; Fuell et al., 2010). Interestingly, the 337

protozoa Trypanosoma cruzi lacks ODC activity and cannot grow in a medium without putrescine 338

(Algranati, 2010). 339

In the present study, we addressed the evolution of promiscuous functions, focusing on the 340

activities of two enzymes: ODC and LDC. Our data suggest that promiscuous activities existed in an 341



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ancestral gene, because most of the functionally characterized ODC genes exhibited both ODC and 342

LDC activities, although a majority of them had a minor (promiscuous) LDC activity and a major 343

ODC activity. In the two distant lineages, legumes and clubmosses, the LDC activity was reinforced 344

independently via at least one event of positive selection at the amino acid position 344. The 345

independent occurrence of the same event is likely to be a consequence of natural selection rather 346

than genetic drift. 347

Bifunctional L/ODC could be advantageous for both the lineages because both primary 348

(putrescine for polyamine production) and specialized (cadaverine for alkaloid production) 349

metabolisms are important for cell growth and differentiation, and for protection against pathogens 350

and herbivores (Pichersky and Gang, 2000), respectively. In contrast, the orthologous ODC gene 351

disappeared in other plant lineages, such as Arabidopsis thaliana and the moss, Physcomitrella 352

patens (Fuell et al., 2010). Plants possess an arginine pathway consisting of enzymes derived from a 353

cyanobacterial ancestor (Illingworth et al., 2003), for the complementation of putrescine production 354

(Fig. 6). Therefore, it is likely that ODC is not truly required in plants. 355

The eukaryotic ODC forms a homodimer, the subunits of which interact in a head-to-tail 356

manner, producing two active sites at their interphase (Lee et al., 2007). The fact that only ODC or 357

LDC is found in plants could be explained by dominant-negative mutations, which lead to mutant 358

enzymes that disrupt the original activity (Veitia, 2007). Thus, the spatial expression of duplicated 359

copies, an ancestral and novel/improve LDC functions of ODC, might release these two copies from 360

molecular constraints, which was reported during the evolution of homospermidine synthase for the 361

production of pyrrolizidine alkaloids (Kaltenegger et al., 2013). The proteins encoded by ODC and 362

LDC might form heterodimers that are less efficient, or even inactive. Therefore, either the native or 363

the evolved enzymes could become fixed in the population, via natural selection. 364

We used the amino acid sequences of LaL/ODC and LcL/ODC as the query sequences to 365



24

perform BLAST searches against the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Phytozome 366

(http://www.phytozome.net/), and OneKP (Johnson et al., 2012; Matasci et al., 2014; Wickett et al., 367

2014; Xie et al., 2014; https://www.bioinfodata.org/Blast4OneKP/) databases. We identified 368

two copies of ODC from the foxtail millet (S. italica) on the same scaffold with a distance of ~24 kb 369

(hereafter referred to as SiODC1 and SiODC2). SiODC1 and SiODC2 have H and Y at position 344, 370

respectively. Although SiODC1 had no intron in its genomic sequence, SiODC2 contained one intron 371

and lacked 64 nucleotides in the coding region, which resulted in the loss of 22 amino acids 372

(Supplemental Fig. S5). This was probably accomplished via a pseudoexonization mechanism, 373

during which an exon sequence becomes intronic (Xu et al., 2012; Supplemental Fig. S1 and S5). 374

These specific amino acids are very important for the ODC and LDC enzymes to bind to their 375

cofactor, PLP (Lee et al., 2007). Therefore, it is likely that SiODC2 is a pseudogene. However, 376

functional analysis of SiODC1 and SiODC2 is needed to support this hypothesis. 377

In addition, recent draft genome sequence studies on the narrow-leafed lupin revealed the 378

presence of a single LDC gene (Conant and Wolfe, 2008; Yang et al., 2013). As active copies of both 379

ODC and LDC were not found in the same plant, divergence in the regulatory regions due to 380

changes in the expression patterns of the LDC and ODC copies to reduce the dominant-negative 381

effect might not have occurred during the plant LDC evolution. Therefore, either ODC or LDC were 382

selected during the evolution and were maintained in the population. 383

The results presented here indicate that an adaptive change from ODC to LDC occurred in 384

the plants that produce Lys-derived alkaloids and cadaverine, via their promiscuous functions. The 385

LDC activity could be gained independently within the Leguminosae and clubmoss lineages. There 386

are several models that could explain the possible routes by which the plant ODC diverged to obtain 387

an LDC function. First, the promiscuous LDC activity from the ancestral ODC, which was mainly 388

involved in the primary metabolism, could have evolved gradually via several mutations and 389



25

selections because of its physiological advantage for the production of Lys-derived alkaloids. This 390

would have increased the LDC function without drastically altering the original ODC function (i.e., 391

a bifunctional enzyme). Additionally, the alternative metabolic ADC pathway could also produce 392

putrescine; thus, the ancestral ODC was likely not constrained to maintain its original function. 393

Therefore, the process of LDC evolution could have started prior to the gene duplication of ODC. 394

This kind of evolutionary process has been termed as a weak negative trade-off, where the 395

divergence of a novel enzyme function occurs via a generalist intermediate (Khersonsky and Tawfik, 396

2010). Second, when environmental changes made the promiscuous LDC function beneficial for 397

plants, gene duplication would have been advantageous to increase the dose of the ancestral ODC 398

gene, thus, resulting in increased protein levels. This process would have allowed a wider variety of 399

function-altering mutations to accumulate, including potentially beneficial mutations that increase 400

the LDC function, and get fixed in the population. In contrast, the less functional copies and those 401

containing deleterious mutations, including the parental gene, could have been lost. This 402

evolutionary process has been proposed as the Innovation, Amplification, and Divergence (IAD) 403

model (Bergthorsson et al., 2007). This model is supported by the identification of ODC-like and 404

LDC-like sequences from the Lys-derived alkaloid-free S. italica; however, the LDC-like sequences 405

show signatures of pseudogenization. In both the models, subsequent gene duplication could have 406

helped resolve the adaptive conflict between the ODC and LDC activities by allowing the 407

optimization of each activity in two separate copies. However, our data suggest that the divergence 408

path toward a newly specialized LDC enzyme has not been completed; therefore, the present-day 409

enzymes exhibit only ODC or L/ODC (bi-functional) activity. 410

411

Conclusions 412

Overall, our results describe a clear case of the evolutionary innovation that uses promiscuous 413



26

activities as the starting point for the divergence of novel enzymes. In addition, the occurrence of an 414

alternative metabolic pathway might increase the evolutionary adaptability of the related enzymes. 415

These findings contribute to a better understanding of how the enzymes in the primary metabolism, 416

which are under a strong purifying selection, could evolve to have a novel function for the 417

specialized metabolism. The molecular cloning and characterization of LcL/ODC shed the light on 418

LA biosynthesis and can serve as a basis for further biotechnological production of LAs for human 419

benefit. 420

421

422

423

424

425

426

427

428

429

430

431

432

433



27

Materials and Methods 434

Plant materials 435

G. max (B01151) and Lotus japonicus (Gifu) seeds were obtained from the National BioResource 436

Project (Miyazaki, Japan) and Dr. Hiroshi Sudo (Hoshi University, Japan), respectively. T. lupinoides 437

(synonym Thermopsis fabacea), a QA-producing plant, was obtained from the Medicinal Plant 438

Gardens of the Graduate School of Pharmaceutical Sciences at Chiba University, Japan. L. clavatum 439

and H. serrata were purchased from plant markets in Japan. N. tabacum cv Petit Havana line SR1 440

was obtained from Ghent University, Belgium. 441

442

Metabolite profiling 443

G. max and Lotus japonicus were cultured on Murashige and Skoog medium (Murashige and Skoog, 444

1962) containing 1% (w/v) Suc with 0.8% agar in a growth chamber at 25°C under 16 h/8 h light 445

(~3000 lux)/dark cycles for 30 days before metabolite analysis. L. clavatum and H. serrata were 446

maintained in a growth chamber at the same condition as G. max and Lotus japonicus. Alkaloids, 447

amines, and amino acids were extracted from the different organs of L. clavatum, H. serrata, G. max, 448

and Lotus japonicus and analyzed using CE−MS as described previously (Oikawa et al., 2011). The 449

(±)-HupA standard was purchased from Sigma-Aldrich, St-Louis, MO. 450

451

Measurement of RNA levels 452

The total RNA was extracted (RNeasy kit; Qiagen, Hilgen, Germany) and reverse-transcribed into 453

cDNA as described elsewhere (Bunsupa et al., 2012a). Real-time PCR was performed using the 454

SYBR Green master mix (Applied Biosystems, Carlsbad, CA) at a final volume of 25 μL, including 455

the appropriate primer pairs for each target (Supplemental Table S4). Assays were run in 456

quadruplicate in a StepOnePlus Real-Time PCR system (Applied Biosciences). The amplification 457



28

program consisted of 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min. Relative 458

quantification of the gene expression was performed using the comparative Ct (threshold cycle) 459

method. β-tubulin was used as an endogenous reference (Supplemental Table S4). 460

461

Cloning ODC and L/ODCs from plants 462

The cDNAs encoding LcL/ODC, HsL/ODC1, HsL/ODC2, and T. lupinoides ODC (TlL/ODC) were 463

isolated using the degenerate primers as described elsewhere (Bunsupa et al., 2012a). The full-length 464

cDNAs were obtained using the 5′- and 3′-RACEs (TaKaRa Bio, Shiga, Japan). However, only a 465

partial sequence for TlL/ODC was obtained. The full-length sequence for NtODC3 was isolated from 466

N. tabacum by using the specific primers designed from NtODC (GenBank_AB031066). 467

468

Heterologous expression of recombinant proteins 469

The LcL/ODC and NtODC3 ORFs were amplified using gene-specific primers with overhangs 470

containing restriction sites (Supplemental Table S4). The mutants were then prepared by PCR-based 471

mutagenesis (Higuchi et al., 1988) using the primers listed in Supplemental Table S4. The amplified 472

fragments were inserted in-frame into the same restriction sites within the pGEX-6P-2 expression 473

vector (GE-healthcare, Pittsburgh, PA), which yielded recombinant gene products with N-terminal 474

GST protein tags. The complete constructs were sequenced to confirm the correct orientation, 475

expressed in E. coli, and purified as described elsewhere (Bunsupa et al., 2012). We also cloned and 476

expressed HseL/ODC1 in E. coli; however, we were unable to purify the recombinant HseL/ODC1 477

protein because of its insoluble nature. The ratio of the targeted recombinant protein to other 478

co-eluted proteins, quantified by densitometry using Image J software (http://imagej.nih.gov/ij/), was 479

used for calculating the protein concentration. 480

481



29

LDC and ODC activity assays 482

LDC and ODC enzyme activities were determined by measuring the CO2 released from 14C-L-Lys 483

and 14C-L-Orn, respectively (Gaines et al., 1988). The decarboxylase activities were assayed in 50 484

mM potassium phosphate, 5 mM EDTA, 4 mM DTT, 0.3 mM PLP, 0.5 to 3.0 mM L-[1-14C] Lys (40 485

μCi) or L-[1-14C] Orn (40 μCi), and 0.5-1.0 μg purified enzyme, at pH 7.5 (except the LDC activity 486

assay for LcL/ODC, which was performed at pH 7.0), in a final volume of 500 μL. The released 487

labeled CO2 is trapped on Whatman 3MM filter paper soaked in Soluene® 350 (PerkinElmer) which 488

put to the top of a glass tube and closed with rubber cap. Each reaction was performed at 37 °C for 489

30 min. The ODC and LDC activities were then determined by measuring 14CO2 released from 490

L-[1-14C] Orn and L-[1-14C] Lys, respectively, by a liquid scintillation counting. The kinetics of 491

decarboxylation of both L-Lys and L-Orn were analyzed by measuring the initial velocities over a 492

range of substrate concentrations (0.5 to 2.0 mM). The competition assays were performed using 2 493

and 4 mM L-Orn or L-Lys, while 10 and 20 μM α-DFMO were used for inhibitor assays. 494

495

Molecular modeling 496

The three-dimensional model structures of LcL/ODC were predicted using SWISS-MODEL (Arnold 497

et al., 2006) and the published human-ODC-putrescine complex (Protein Data Bank entry 2000) as 498

the template (Dufe et al., 2007). The modeled protein was visualized using PyMOL 499

(www.pymol.org). 500

501

Phylogenetic analysis 502

LaL/ODC and LcL/ODC were used as queries and blasted with “tblastn” against the NCBI 503

(http://blast.ncbi.nlm.nih.gov/Blast.cgi), Phytozome (http://www.phytozome.net/), and OneKP 504

(Johnson et al., 2012; Matasci et al., 2014; Wickett et al., 2014; Xie et al., 2014; 505



30

https://www.bioinfodata.org/Blast4OneKP/) databases. The ODCs and L/ODCs from plants that had 506

E values < 10 e-06 and had important catalytic residues which are important for both ODC and LDC 507

activities (Supplemental Fig. S1), especially aspartic acid (D) at position 343 (LaL/ODC numbering), 508

were selected (Grishin et al., 1999; Kern et al., 1999). Other eukaryotic ODCs, such as the ones from 509

yeast and human, were also included in the phylogenetic tree. The accession numbers of each ODC 510

and L/ODC are shown in Supplemental Table S3. Amino acid alignments were performed using 511

MEGA version 6 and manually adjusted to improve the reliability of the alignment (Supplemental 512

Data S2; Tamura et al., 2013). If a codon site has at least a gap in the generated alignment, the codon 513

site with a gap was not used for generating phylogenetic tree. Only highly conserved amino acids 514

without gaps were used for further analysis (Supplemental Data S1). To generate a phylogenetic tree, 515

the best-fit model for the amino acid replacements was searched using ProtTest (Abascal et al., 516

2005), Chosen the best-fit model is LG (An Improved General Amino Acid Replacement Matrix) + I 517

(Invariable sites) + G (Gamma shape). The gamma shape is 1.116 in 4 rate categories. The 518

proportion of invariable sites was 0.08. Using the best-fit mode, we generated the phylogenetic tree 519

in PhyML3.0 (Guindon et al., 2010). 520

521

Test for positive selection 522

The ORFs corresponding to all available ODC and L/ODC amino acid sequences, as described 523

above, were aligned. The resulting alignments were used for further analyses. We performed two 524

analyses, the codon site test and the branch site test, in ‘codeml’ of PAML (v.4) package (Yang, 525

2007). In the codon site test, we performed two analyses, using models M7 (model = 0 and NSsites = 526

7) and M8 (model = 0 and NSsites = 8). The likelihood ratio test (LRT) was used to compare the two 527

models, assuming that twice the log-likelihood difference between the two models (2∆L) follows a 528

χ2 distribution with a number of degrees of freedom. In the branch-site model, we selected two 529



31

branches that led to Lys-derived alkaloids as foreground branches and searched for the positively 530

selected sites (model = 2, NSsites = 2 and fix_omega = 0 [Ka/Ks=free]). For the null hypothesis, we 531

used the branch site model with following parameters: model = 2, NSsites = 2, and fix_omega = 1 532

[Ka/Ks=1]. The likelihood ratio test (LRT) was used to compare the two models, assuming that 533

twice the log-likelihood difference between the two models (2∆L) follows a χ2 distribution with a 534

number of degrees of freedom. 535

536

Protein localization analysis 537

The chimeric gene constructs of 35Spro:LcL/ODC-Met1 and -Met3 fused with GFP at N- or C- 538

terminal were created using the primers presented in Supplemental Table 4, and subsequently, cloned 539

into the pTH2 vector (Chiu et al., 1996). An empty vector fused with RFP from Discosoma sp. 540

(DsRed), 35Spro:DsRed, was used as a reference for the localization to cytosol (Kitajima et al., 541

2009). The resulting plasmids were expressed transiently in the onion epidermal cells and N. 542

benthamiana leaves (8-week-old plants), using a Helios gene gun (Bio-Rad, Hercules, CA) as 543

described elsewhere (Bunsupa et al., 2012a). The GFP and RFP signals were observed using a 544

confocal laser-scanning microscope, LSM700 (Zeiss, Oberkochen, Germany). For GFP, we used an 545

argon laser with excitation at 488 nm with FSet38 wf filter. Argon laser with excitation at 555 nm 546

with Fset43 wf filter was used for RFP. All images were acquired from single optical sections and 547

were merged using the ZEN 2012 lite imaging software (Zeiss, Oberkochen, Germany). 548

549

Plasmid construction and plant transformation 550

To construct pGWB2-LcL/ODC (35Spro:LcL/ODC), the full-length sequence for LcL/ODC was 551

cloned into the binary vector pGWB2 (Nakagawa et al., 2007; see Supplemental Table S4 for the 552

primer sequences) via Gateway technology (Invitrogen, CA, USA). The transgenic tobacco (N. 553



32

tabacum cv Petit Havana line SR1) hairy roots and A. thaliana were generated as described 554

elsewhere (Bunsupa et al., 2012a). Tobacco alkaloids and amines were measured as described 555

elsewhere (Bunsupa et al., 2012a). 556

557

Statistical analysis 558

Student’s one-tailed t test was used to identify statistically significant differences in the metabolite 559

levels of transgenic Arabidopsis plants and tobacco hairy roots. Pearson correlation analysis was 560

performed to calculate the correlation between the metabolite levels and the expression levels of 561

LcL/ODC in transgenic Arabidopsis plants and tobacco hairy roots. For all statistical tests, 562

significance was determined at P < 0.05. 563

564

Accession numbers The new DNA sequences reported here are deposited in the DNA Data Bank of 565

Japan (DDBJ) under accession numbers AB915695 (LcL/ODC), AB915696 (HsL/ODC1), 566

AB915697 (HsL/ODC2), AB915698 (TlL/ODC), and LC030209 (NtODC3). 567

568

569

570

571

572

573

574

575

576

577



33

578



34

Table I. Kinetic parameters of L/ODCs and its mutant proteins 579

580

Protein

Km

(mM)

Vmax

nmol min-1μg-1

kcat

(s-1)

kcat/Km

(M-1 s-1)

LDC/ODC

ratio of

kcat/Km LDC ODC LDC ODC LDC ODC LDC ODC

LcL/ODC_wild-type

LcL/ODC-Y344H

NtODC3_wild-type

NtODC3-H344Y

NtODC3-H344F

1.69

22.39

ND

ND

ND

5.48

8.21

1.44

0.75

0.61

3.65

0.51

ND

ND

ND

2.46

2.47

30.30

14.33

3.42

3.17

0.44

ND

ND

ND

2.13

2.14

23.54

11.33

2.66

1878

20

ND

ND

ND

388

261

16351

14794

4368

4.84

0.08

ND

ND

ND

All experiments were performed in 50 mM potassium phosphate buffer (at optimal pH of each 581

enzyme). Kinetic parameters were calculated from mean values (n = 3 to 4); ND, not detected. 582

583

584

585

586

587

588

589

590

591

592

593



35

Table II. Molecular evolutionary analysis of eukaryotic ODCs and LDCs 594

Model ts/tva Number of genes

Number of codon

sites

ωb in the background branches

ωb in the foreground branches

Log likelihood P-value Positively

selected sitesc

Branch-site model

1.764

156 675 0.094 1 -48716.1203 0.028

not applicable

1.763 156 675 0.094 1.43

-48714.1402 112 (0.980) 344 (0.951)d

595 a, Transversion/Transition ratio 596 b, ω value is the ratio of nonsynonymous (amino acid replacing) substitution rate (Ka) over the 597 synonymous (silent) substitution rate (Ks) (Ka/Ks) 598 c, The amino acid position is based on La-L/ODC amino acid numbering. The sites that have the 599 posterior probabilities > 0.90, by Bayes Empirical Bayes analysis, are shown with the posterior 600 probabilities in the parentheses. 601 d, Bold numbers indicate positively selected sites (posterior probabilities > 0.95) 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635



36

Supplemental Materials 636

The following supplemental materials are available. 637

Supplemental Figure S1. Alignment of selected eukaryotics ODCs and L/ODCs amino acid 638

sequences. 639

Supplemental Figure S2. SDS-PAGE of the recombinant LcL/ODC, NtODC3, and their mutant 640

proteins purified from E. coli. 641

Supplemental Figure S3. Competition and inhibition studies of LcL/ODC. 642

Supplemental Figure S4. Overview of predicted protein structure of LcL/ODC complex homology 643

model with the Schiff base intermediate of putrescine (PUT) and pyridoxol-5′-phosphate (PLP) at 644

the active site. 645

Supplemental Figure S5. Alignment of genomic sequences of putative ODCs from S. italica. 646

Supplemental Table S1. Levels of amines and lycopodium alkaloids in each organ of L. clavatum 647

and H. serrata. 648

Supplemental Table S2. Levels of amines in each organ of G. max and Lotus japonicus. 649

Supplemental Table S3. Accession numbers of sequences used for phylogenetic analysis. 650

Supplemental Table S4. List of primers used in this study 651

Supplemental Data S1. Highly conserved amino acid alignment without gaps for the construction 652

of phylogenetic tree in Figure 2 653

Supplemental Data S2. Original amino acid alignment by using ClustalW in MEGA6 program 654

655

Acknowledgements 656

We thank Dr. Ryo Nakabayashi (RIKEN Center for Sustainable Resource Science (CSRS), Japan) 657

for preliminary analyzes of LAs by LC−MS; Satoko Sugawara (RIKEN CSRS, Japan) for her 658

excellent technical support for preparation of transgenic Arabidopsis plants; Tsuyoshi Nakagawa 659



37

(Shimane University, Japan) for providing the destination vector pGWB2; Toshiaki Mitsui (Niigata 660

University, Japan) for providing pWx-TP-DsRed vector; and all The 1000 Plants Project (oneKP) 661

contributors for gene sequencing data. 662

663

Figure legends 664

Figure 1. Putative biosynthetic pathway for lycopodium alkaloids. Dotted arrows indicate more than 665

one catalytic conversion. Abbreviations: LDC, Lys decarboxylase; CuAO, copper amine oxidase. 666

667

Figure 2. Rooted phylogenetic tree of ODC and LDC amino acid sequences from eukaryotes. From 668

an alignment of highly conserved amino acid without gaps built using MEGA version 6 669

(Supplemental Data S1), the phylogenetic tree was constructed by PhyML3.0 using the best-fit mode. 670

The divergence node derived from non-plant eukaryote genes is defined to be the root of 671

phylogenetic tree. The asterisks represent the enzymes whose biochemical properties have been 672

investigated. The blue branch lines indicate the Lys-derived alkaloid producing plants. The capital 673

letters next to the taxa represent the amino acid at position 344 (LaL/ODC numbering). The 674

bootstrap values (1000 replicates) are shown. Letters A, B, and C indicate the branches which are 675

likely to be under positive selection for the production of Lys-derived alkaloids in plants: 676

lycopodium alkaloids (branch A), nuphar alkaloids (branch B), and QAs (branch C). The bootstrap 677

values more than 50% are shown. The accession numbers of the enzymes are listed in Supplemental 678

Table S3. 679

680

Figure 3. Major alkaloid levels and correlations between the relative abundance of LcL/ODC 681

transcript and the alkaloid levels in tobacco hairy roots overexpressing LcL/ODC. A, Abundance of 682

tobacco alkaloids in six and five independent hairy roots for LcL/ODC- and GUS-overexpressing 683



38

lines, respectively (biological replicates, n = 4 for each line). Values are means ± SEM. Letters above 684

the bars indicate that the mean values are statistically different from the corresponding GUS controls, 685

based on Student’s one-tailed t test: aP < 0.05. B, Correlations between each of the tobacco alkaloids 686

and the relative LcL/ODC transcripts in tobacco hairy roots overexpressing LcL/ODC (filled orange 687

circles) and GUS (blue triangles) lines. Pearson correlation coefficients (r) with the number of tested 688

samples in the bracket and the corresponding P values are shown. 689

690

Figure 4. Amine levels and correlations between the relative abundance of LcL/ODC transcript and 691

the amine levels in Arabidopsis plants expressing LcL/ODC. A, Abundance of amines in pooled 692

samples (6 to 8 plants) of Arabidopsis plants expressing LcL/ODC or GUS, with eight independent 693

lines for each. Values are means ± SEM. Letters above the bars indicate that the mean values are 694

statistically different from the corresponding GUS control, based on Student’s t test (one-tailed): aP 695

< 0.01. B, Correlations between each of the amines and the relative LcL/ODC transcripts in tobacco 696

hairy roots overexpressing LcL/ODC (filled gray circles) and GUS (magenta triangles) lines. Pearson 697

correlation coefficients (r) with the number of tested samples in the bracket and P values are shown. 698

699

Figure 5. Subcellular localization of L. clavatum L/ODC (LcL/ODC) fused with GFP in onion 700

epidermal cells and N. benthamiana leaves, and the relative abundance of LcL/ODC, GmODC, and 701

LjODC transcript levels. LcL/ODC from the first (Met1) and the third (Met3) start codons were 702

fused with the GFP at N- (GFP-Met1 and GFP-Met3) or C-terminal (Met1-GFP and Met3-GFP). The 703

resultant constructs were simultaneously and transiently expressed with Discosoma sp. (DsRed). Red 704

fluorescent protein (RFP) from DsRed and the GFP were used as a reference for cytosolic 705

localization. GFP (green, top row), RFP (red, middle row), and merged (green and red, bottom row) 706

fluorescence observed in the onion epidermal cells (A) and the N. benthamiana leaves (B) 707



39

expressing the indicated target constructs are shown. Scale bars represent 50 μm and 20 μm for the 708

onion epidermal cells (A) and the N. benthamiana leaves (B), respectively. Quantitative RT-PCR 709

analysis of ODC or L/ODC transcript levels in the shoots and roots of L. clavatum (C), G. max (D), 710

and Lotus japonicus (E). Bars represent mean ± SD of analytical replicates, n = 3 to 4. 711

712

Figure 6. Biosynthetic pathways for Lys- and Orn-derived alkaloids in plants. ODC, Orn 713

decarboxylase; LDC, Lys decarboxylase; ADC, arginine decarboxylase. 714

715

716

717

718

719

720

721

722

723

724

725

726

727

728



40

729



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Tang W, Eisenbrand G (1992) Chinese Drugs of Plant Origin: Chemistry, Pharmacology, and Use in Traditional and ModernMedicine, Ed 1. Springer-Verlag, Berlin.


Tomar PC, Lakra N, Mishra SN (2013) Cadaverine: a lysine catabolite involved in plant growth and development. Plant SignalBehav 8: 10. PMCID: PMC4091120


Torrigiani P, Scoccianti V (1995) Regulation of cadaverine and putrescine levels in different organs of chick-pea seed andseedlings during germination. Physiol Plant 93: 512-518


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Wang B, Wang H, Wei Z, Song Y, Zhang L, Chen H (2009) Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A inthe treatment of Alzheimer's disease: an updated meta-analysis. J Neural Transm 116: 457-465


Wickett NJ, Mirarab S, Nguyen N, Warnow T, Carpenter E, Matasci N, Ayyampalayam S, Barker MS, Burleigh JG, Gitzendanner MA,Ruhfel BR (2014) Phylotranscriptomic analysis of the origin and early diversification of land plants. Proc Natl Acad Sci USA 111:E4859(4868


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Xu G, Guo C, Shan H, Kong H (2012) Divergence of duplicate genes in exon-intron structure. Proc Natl Acad Sci USA 109:1187(1192


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Yang Z (2007) PAML 4: Phylogenetic analysis by Maximum Likelihood. Mol Biol Evol 24: 1586-1591Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang J, Nielsen R, Yang Z (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at themolecular level. Mol Biol Evol 22: 2472-2479



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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Torrigiani&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Torrigiani&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Exploring the molecular etiology of dominant-negative mutations&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Exploring the molecular etiology of dominant-negative mutations&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Veitia&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A in the treatment of Alzheimer's disease: an updated meta-analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1


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