Short Title: Pyrrolizidine alkaloid biosynthesis in ... · 2 Pyrrolizidine alkaloid biosynthesis in Comfrey 3 4 5 NOTE: 6 This paper is dedicated to Professor Thomas Hartmann on the

1

Short Title: 1

Pyrrolizidine alkaloid biosynthesis in Comfrey 2

3

4

NOTE: 5

This paper is dedicated to Professor Thomas Hartmann on the occasion of his 80th 6

birthday 7

(dedication is supported by the handling editor Jörg Bohlmann, see e-mail of 6th of 8

March 2017 to Ashton Wolf) 9

10

Corresponding Author: 11

Prof. Dr. Dietrich Ober 12

Botanical Institute and Botanical Gardens 13

Kiel University 14

Olshausenstr. 40 15

24098 Kiel 16

phone: +49 431 8804299 17

fax +49 431 8804500 18

e-mail [email protected] 19

20

Plant Physiology Preview. Published on March 8, 2017, as DOI:10.1104/pp.17.00265

Copyright 2017 by the American Society of Plant Biologists

www.plantphysiol.orgon April 22, 2019 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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2

Identification of a second site of pyrrolizidine alkaloid biosynthesis in Comfrey 21

to boost plant defense in floral stage 22

23

Lars H. Kruse, Thomas Stegemann, Christian Sievert, and Dietrich Ober 24

25

Botanisches Institut und Botanischer Garten, Universität Kiel, D–24098 Kiel, 26

Germany 27

28

One sentence summary 29

In Comfrey, Symphytum officinale, leaves subtending a young inflorescence have 30

been identified as an additional site of alkaloid biosynthesis to boost protection of 31

reproductive tissues. 32

33

AUTHOR CONTRIBUTIONS: L.H.K, and D.O. designed the study. L.H.K. conducted 34

the main part of the experiments. T.S. carried out HPLC and GC-MS analyses. C.S. 35

undertook the radioactive feeding of leaves and provided comments on the 36

manuscript. L.H.K. analyzed the data. L.H.K. and D.O. interpreted the results and 37

wrote the manuscript. 38

39

FUNDING INFORMATION: This work was supported by the Deutsche 40

Forschungsgemeinschaft (DFG) with a grant to DO. 41

42

CORRESPONDING AUTHOR: 43

Dietrich Ober, [email protected] 44



3

ABSTRACT 45

Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites that are found in 46

several, distantly related families of the angiosperms. The first specific step in PA 47

biosynthesis is catalyzed by homospermidine synthase (HSS), which has been 48

recruited several times independently by duplication of the gene encoding 49

deoxyhypusine synthase (DHS), an enzyme involved in the post-translational 50

activation of the eukaryotic initiation factor 5A. HSS shows highly diverse 51

spatiotemporal gene expression in various PA-producing species. In Symphytum 52

officinale (Boraginaceae), PAs are reported to be synthesized in the roots, with HSS 53

being localized in cells of the root endodermis. Here, we show that S. officinale plants 54

activate a second site of HSS expression when inflorescences start to develop. HSS 55

has been localized in the bundle sheath cells of specific leaves. Tracer feeding 56

experiments have confirmed that these young leaves not only express HSS, but the 57

whole PA biosynthetic route. This second site of PA biosynthesis results in drastically 58

increased PA levels within the inflorescences. The boost of PA biosynthesis is 59

proposed to guarantee optimal protection especially of the reproductive structures. 60

61



4

INTRODUCTION 62

Plant secondary metabolism is highly diverse on a chemical and regulatory level 63

(Grotewold, 2005; Pichersky et al., 2006; Weng et al., 2012; Cordell, 2013). It is often 64

characterized by a complex interplay of various cell types and enzymes (Facchini and 65

St-Pierre, 2005; Ziegler and Facchini, 2008). Well-characterized examples of the 66

involvement of various cell types in the biosynthesis of alkaloids can be found in the 67

benzylisoquinoline alkaloid biosynthesis of opium poppy (Bird et al., 2003), in the 68

tropane alkaloid biosynthesis of Hyoscyamus niger and Atropa belladonna (Nakajima 69

et al., 1999; Suzuki et al., 1999), and in the monoterpenoid indole alkaloid 70

biosynthesis of Catharanthus roseus (St-Pierre et al., 1999; Burlat et al., 2004). 71

Moreover, the transport from the site of synthesis to the site of accumulation requires 72

various cell types as described for nicotine in tobacco (Neumann, 1985; Morita et al., 73

2009) or pyrrolizidine alkaloids (PAs) in Senecio (Sander and Hartmann, 1989). 74

PAs are toxic compounds for the chemical defense of plants and show a scattered 75

occurrence in many distantly related angiosperms but have in common 76

homospermidine synthase (HSS), which is the first pathway-specific enzyme of PA 77

biosynthesis in all these lineages. HSS uses spermidine and putrescine as substrates 78

to catalyze the formation of homospermidine, an intermediate that has been shown to 79

be incorporated exclusively into the necine base moiety, the characteristic bicyclic 80

ring system of PAs (Böttcher et al., 1993; Böttcher et al., 1994). HSS has evolved by 81

gene duplication of deoxyhypusine synthase (DHS), an enzyme involved in the post-82

translational activation of the eukaryotic initiation factor 5A (Ober and Hartmann, 83

1999, 1999). Recent studies have shown that this duplication has occurred several 84

times independently during angiosperm evolution in lineages that are able to produce 85

PAs (Reimann et al., 2004; Anke et al., 2008; Kaltenegger et al., 2013; Irmer et al., 86

2015). This observation suggests that the whole pathway of PA biosynthesis also 87



5

evolved several times requiring repeated integration into the metabolism of the plant. 88

Expression analyses of HSS in PA-producing plants of various lineages show that 89

this integration resulted in different cellular organizations of PA biosynthesis in 90

different species. In the Asteraceae family, for example, in which independent 91

duplications have been described in two lineages, HSS expression has been 92

detected in Senecio (ragwort, tribe Senecioneae) only in cells of the endodermis and 93

adjacent root parenchyma, cells that lie opposite to the phloem, whereas, in 94

Eupatorium cannabinum (hemp-agrimony, tribe Eupatorieae), HSS expression has 95

been found in all cells of the root cortex, but not in the endodermis (Moll et al., 2002; 96

Anke et al., 2004). In orchids (Phalaenopsis species), HSS has been localized in 97

meristematic cells of the root apical meristem and in young flower buds (Anke et al., 98

2008). Within Boraginales, a single duplication event resulted in the evolution of 99

HSS. However, in this lineage, the expression pattern of HSS has been shown to be 100

multifaceted, suggesting independent scenarios of optimization and integration of the 101

gene duplicate into the metabolism of the plant. Within the Heliotropiaceae 102

(Heliotropium indicum, Indian heliotrope), HSS expression is found in the lower leaf 103

epidermis, whereas in Cynoglossum officinale (houndstongue, Boraginaceae), the 104

endodermis and pericycle of the root express HSS (Niemüller et al., 2012). In 105

Symphytum officinale (Comfrey, Boraginaceae), HSS has been localized exclusively 106

in cells of the endodermis (Niemüller et al., 2012) supporting previous studies by 107

Frölich et al. (2007) who have identified the roots of S. officinale as the site of PA 108

biosynthesis by tracer experiments with radiolabeled putrescine. Studies by 109

Niemüller et al. (2012) using protein blot analyses suggested that HSS expression 110

occurs during a restricted window of leaf development in S. officinale, an observation 111

that warrants a closer look at the PA biosynthetic capacity of leaves of this plant 112

species. 113



6

In this study, we show that S. officinale synthesizes PAs not only in the roots, but also 114

in young leaves subtending an inflorescence with unopened flower buds. We further 115

show that HSS is expressed in specific cells of the leaf during certain stages of 116

inflorescence development coinciding with PA production in the leaf. This second 117

auxiliary site of PA biosynthesis during flower development is discussed in 118

comparison with a similar mechanism previously observed in the orchid Phalaenopis 119

amabilis in which young flower buds produce PAs to boosts PA levels in the mature 120

flower in order to guarantee an efficient supply of deterrent PAs for the developing 121

reproductive structures (Anke et al., 2008). 122



7

RESULTS 123

Levels of hss Transcript and Protein Expression are Dependent on Leaf 124

Position in Relation to the Inflorescence 125

Analyzing three species of Boraginales, Niemüller et al. (2012) has shown that HSS 126

expression is species-specific. In S. officinale, in which the whole PA biosynthesis is 127

localized in the roots, their study has indicated that HSS is also expressed in leaves 128

of a specific developmental stage, viz., leaves that are directly beyond a terminal 129

inflorescence with closed flower buds. 130

In order to correlate leaf position with hss expression in S. officinale, we harvested 131

leaves from various positions in the shoot system of a plant just opening its first 132

flower buds (Fig. 1A). Analyses with RT-PCR and qRT-PCR showed that the highest 133

levels of hss transcript were detectable in young leaves directly next to 134

inflorescences with buds and flowers that had just opened (Fig. 1B and 135

Suppl. Fig. 2). The hss transcript level differed between the youngest and the oldest 136

leaf by almost a factor of 30 (Suppl. Fig. 2). To test whether hss transcript levels were 137

correlated with the expression of HSS protein, protein extracts of leaves from various 138

positions of the shoot system from another plant individual were analyzed by a 139

protein gel blot with an antibody specific for HSS of S. officinale (Niemüller et al., 140

2012). Like the hss transcript level, hss protein expression is strongest in young 141

leaves directly subtending an inflorescence (leaf 1 and 2) and fades away with 142

increasing distance of the leaves to the flower (Fig. 1C). Of note, in leaves that have 143

no axillary branch developing in their axils, HSS protein is not detectable (leaves 9, 144

and 10, blue in Fig. 1A), whereas leaves that possess axillary branches show distinct 145

HSS expression (leaves 3, 4, 6, 8, red in Fig. 1A). Congenital fusions of an axillary 146

branch with the main axis are common for S. officinalis (Kotelnikova et al., 2011) and 147



8

result in a lateral bud on the axis distant from its subtending leaf (e.g. leaves 5 and 7, 148

purple in Fig. 1A). 149

Immunolocalization of HSS in Leaves of S. officinale 150

Leaves that tested positive for HSS expression were used to identify, by 151

immunolocalization the cells expressing HSS. HSS expression was found to be 152

restricted to bundle sheath cells that formed a layer of compactly arranged 153

parenchyma around the vascular bundle (Fig. 2C and Fig. 2D). The labeled layer had 154

a thickness of one to two cells and was separated from the epidermis by at least a 155

single row of unlabeled mesophyll cells. Specificity of the label was tested by pre-156

incubation of the antibody with soluble, heterologously expressed HSS, DHS, or BSA 157

before labeling. Only pre-incubation with HSS resulted in a decrease of signal, 158

whereas pre-incubation with DHS or BSA had no effect (Suppl. Fig. 2). 159

The close vicinity of HSS expression and the vascular bundle is also observed in the 160

roots of S. officinale, in which HSS is only detectable in the endodermis that encloses 161

the root central cylinder with the vascular tissue (Niemüller et al., 2012). Both of 162

these tissues, namely the endodermis and the bundle sheath cells, play a key role in 163

controlling fluxes to and from the vascular bundle and are important for maintaining 164

the transport of nutrients and water in the plant (Steudle and Peterson, 1998; 165

Leegood, 2008; Geldner, 2013). 166

Of note, nucleotide sequences encoding HSS amplified from the leaves proved to be 167

identical to those amplified from the roots, suggesting that a single hss gene is 168

responsible for the expression of HSS in the endodermis of the root and the bundle 169

sheath of specific leaves. 170

Capacity of S. officinale Leaves to Synthesize PAs 171



9

By applying 14C-labeled putrescine to young detached leaves expressing HSS, we 172

tested whether leaves expressed only HSS or the complete biosynthetic machinery to 173

produce PAs. After incubation for four days, the tracer was completely taken up by 174

the leaves, of which 16% of the applied radioactivity was detectable in the alkaloid-175

containing sulfuric acid crude extract. After reduction to convert all PA N-oxides to 176

their tertiary form, PAs were enriched by solid phase extraction. This alkaloid-177

enriched fraction containing 8% of the applied radioactivity was split into two fractions 178

that were treated differentially to confirm the identity of the labeled compounds as 179

PAs (Tab. 1). One fraction was analyzed directly by HPLC to detect radioactively 180

labeled PAs; several peaks of putative PAs were found (Fig. 3A). To establish that the 181

detected radioactive peaks were PAs that resulted from the incorporation of the tracer 182

putrescine, the second fraction was hydrolyzed to reduce all potential PAs to their 183

backbone, the necine base. S. officinale produces a PA bouquet of intermedine 184

derivatives including O9-monoesters, O7,O9-diesters, and 3'-acetyl derivatives such 185

as 7-acetylintermedine, 7-acetyllycopsamine, 7-senecioylintermedine (echiupinine), 186

or 7-tigloylintermedine (myoscorpine) (Hartmann and Witte, 1995; Frölich et al., 187

2007). The necine base of these PAs, all belonging to the so-called lycopsamine 188

type, is retronecine (Hartmann and Witte, 1995). If the radioactive peaks represent 189

PAs, hydrolyzation should result in a single peak that co-migrates with a retronecine 190

standard. In contrast to the several peaks detectable in the non-hydrolyzed extracts 191

(Fig. 3A), the hydrolyzed extract (Fig. 3B) showed only one prominent peak that had 192

the same retention time as retronecine (Fig. 3C). Spiking the hydrolyzed sample with 193

retronecine resulted only in an intensification of the signal indicating that the same 194

substance was being analyzed (Fig. 3D). As an additional proof of identity, the 195

feeding experiment was repeated as described, but with unlabeled putrescine. The 196

alkaloid-containing fraction was hydrolyzed and separated by thin layer 197



10

chromatography parallel to the hydrolyzed sample resulting from the tracer feeding 198

experiment. After the localization of radioactivity, the corresponding area in the lane 199

of the non-radioactive samples was scraped off the plate, and the compounds were 200

eluted and analyzed by GC-MS. In this spot, retronecine was detected as the major 201

compound, giving support to the interpretation that retronecine was the radioactive 202

compound detected after hydrolysis of the extract after tracer feeding (Suppl. Fig. 4). 203

Leaves Boost PA Levels in Inflorescences 204

To test the consequences of the capacity of leaves to produce PAs, we analyzed the 205

transcript and protein levels of HSS and the total PA content in leaves subtending an 206

inflorescence and in their associated inflorescences during development. Seven 207

different inflorescence developmental stages were defined with respect to size and 208

flowering state (Fig. 4A). Stage I was characterized by its small size (approx. 1 cm in 209

diameter), whereas stage II was much larger but had flower buds that were still 210

closed. Size increased until stage V. At stage IV, the flower bud tips became purple. 211

In stage V, the first buds started flowering, but most of the buds maintain closed, 212

whereas by stage VI, all the flowers had open, with those that had opened first were 213

beginning to darken. At stage VII, the flowers withered, and fruits began to develop. 214

We observed that HSS transcript and protein in leaves were detectable from stage I 215

onward, and that HSS expression levels reached their maximum at stage II and III, 216

after which they decreased to a low level and remained constant to the last stage 217

(Fig. 4B). PAs were not detectable in leaves of stage I. PA levels increased from 218

stage II to IV to a concentration of about 20 µg/g dry weight, and reached at stage VII 219

concentrations of about 180 µg/g dry weight (Fig. 4C), similar to that described for 220

leaves of S. officinale (Mattocks, 1980; Couet et al., 1996). As in the leaves, no PAs 221

were detectable in inflorescences at stage I. Inflorescences at stages II-V showed PA 222



11

concentrations of approx. 300-400 µg/g dry weight, increasing at stage VI to a 223

concentration of 3344 µg/g dry weight (Fig. 4C) with a total PA content of 700 µg per 224

inflorescence (Fig. 4D). These high concentrations correspond to PA levels described 225

for flowers of other PA-producing plant species (Muetterlein and Arnold, 1993; Couet 226

et al., 1996; Frölich et al., 2006; Frölich et al., 2007). In the last analyzed stage, 227

namely stage VII, the concentration dropped to approx. 500 µg/g dry weight. The 228

comparatively low PA concentrations in leaves compared with inflorescences suggest 229

that PAs are efficiently translocated from the leaves to the inflorescences. Thus, PA 230

biosynthesis in the young leaf subtending the inflorescence ensures a sufficient 231

supply of PAs (i) to keep alkaloid levels constant as long as the inflorescences 232

enlarge and (ii) to enable high PA levels to accumulate when the flowers open. 233

To test whether the inflorescence itself contributes to the high PA concentrations, we 234

analyzed HSS expression in inflorescences of various developmental stages (Suppl. 235

Fig. 7). Only very low levels of HSS protein were detectable at any stage of 236

inflorescence development. This observation is in accordance with Niemüller et al. 237

(2012) who have found low levels of HSS transcript but no protein in inflorescences. 238

In contrast to the inflorescences, roots showed a high HSS level of approx. 40-50 ng 239

per 20 µg of total protein, a value which is comparable with the highest HSS levels 240

detected in the leaf at stage II with a maximum value of 39 ng. The delay between 241

the highest levels of HSS (stage II) and the peak of PA accumulation (stage VI) 242

results most likely from two factors: (i) the need to transport PAs from their site of 243

synthesis in the young leaf into the inflorescence, and (ii) the finding that PA 244

biosynthesis in species of the Boraginales seems to be less channeled than in other 245

PA-producing species as shown by Frölich et al. (2007) using radioactive labeled 246

putrescine as a tracer. The maximal incorporation of tracer into PAs took more than 247

two weeks with a temporary accumulation of pathway intermediates such as 248



12

homospermidine or a polar uncharacterized compound. The drastic decrease of PA 249

levels in stage VII might have various causes. One explanation could be the 250

abscission of the petals, which was observed at this stage. PAs in the petals might be 251

a strategy to fend off nectar robbers such as short-tongued bumblebees of the genus 252

Bombus (Utelli and Roy, 2001; Irwin and Maloof, 2002). Nectar robbers bite a hole in 253

the petal tube to reach nectar without actually entering and pollinating the flower 254

(Castro et al., 2008). Another explanation for the drop of PA levels could be a 255

degradation of PAs in these tissues or the translocation of PAs from the 256

inflorescences to other tissues of the plant, as PA N-oxides are highly mobile and can 257

easily be translocated from one tissue to another (Hartmann, 1989 #36}. Further 258

studies should elucidate the dynamics of PA levels in these reproductive structures. 259

260

DISCUSSION 261

In this study, we have been able to show that S. officinale has two PA-biosynthetic 262

sites that differ with respect to their function, i.e., the roots as a well-characterized 263

organ of PA biosynthesis and young leaves that are in the direct vicinity of a 264

developing inflorescence. The roots in S. officinale are responsible for constitutive PA 265

production, most likely for the protection of the vegetative parts of the plant. 266

Confirmation that the whole PA pathway is expressed in the roots has been obtained 267

from root cultures that are able to produce PAs (Frölich et al., 2007). A link between 268

growth and PA biosynthesis should guarantee a constant ratio between PA and 269

biomass, a phenomenon that we have also observed for PA-producing species of 270

other lineages (Anke et al., 2004; Anke et al., 2008; Niemüller et al., 2012). In 271

contrast, the young leaves subtending an inflorescence with unopened flower buds 272

characterized in this study provide an additional PA supply for the reproductive 273



13

tissues within the flowers of S. officinale, a pattern previously observed for PA-274

producing Phalaenopsis (Orchidaceae). In Phalaenopsis, specific structures within 275

small flower buds, but not of open flowers, produce PAs during flower development, 276

boosting PA levels up to about 6 mg/g fresh weight (Anke et al., 2008). The 277

assumption that, in S. officinale PA biosynthesis in the young leaf is also of functional 278

importance is supported by the incorporation rate of labeled putrescine into PAs of 279

almost 10% (Tab. 1). This incorporation rate is remarkable bearing in mind that 280

putrescine is not only a precursor for PA biosynthesis, but also part of the highly 281

dynamic polyamine pool of primary metabolism, including essential functions such as 282

growth, development, or biotic and abiotic stress responses (Evans and Malmberg, 283

1989; Alcázar et al., 2010; Gill and Tuteja, 2010; Tiburcio et al., 2014). As HSS 284

evolved independently in Symphytum and Phalaenopsis (Reimann et al., 2004; 285

Nurhayati et al., 2009), the selection pressure that resulted from the need to protect 286

the reproductive structures against herbivores obviously resulted in a similar 287

regulation of PA biosynthesis in these two lineages. The protection of reproductive 288

structures is a phenomenon that is often observed in the plant kingdom and that is in 289

good accordance with the optimal defense theory (McKey, 1979). 290

The pattern of HSS expression in PA-producing angiosperms is highly variable. 291

Nonetheless, several PA-producing plants show similarities in HSS expression. The 292

vicinity of the vascular tissue to cells involved in PA biosynthesis is a motif not just 293

found in the roots and leaves of Symphytum, but also in other PA-producing plants 294

such as C. officinale (also Boraginaceae) expressing HSS in the endodermis and the 295

pericycle of the roots or in Senecio species (Asteraceae) in which HSS is expressed 296

in cells directly next to the phloem. In Senecio, the phloem has been shown to be the 297

tissue by which PAs are transported from the site of synthesis to the shoot in which 298



14

they are then efficiently allocated to the inflorescences, the major site of storage 299

(Hartmann et al., 1989; Moll et al., 2002). Moreover, a link between alkaloid 300

biosynthesis and the vascular tissue has been described for other plant systems, 301

e.g., for the early steps of monoterpene indole alkaloid biosynthesis (Burlat et al., 302

2004), for the biosynthesis of morphine in opium poppy (Bird et al., 2003; Weid et al., 303

2004), and for tropane alkaloid biosynthesis in solanaceous plants (Kanegae et al., 304

1994; Suzuki et al., 1999). 305

Despite the probability that HSS, and most likely, the complete pathway of PA 306

biosynthesis evolved several times independently in various lineages of the 307

angiosperms (Reimann et al., 2004; Anke et al., 2008; Kaltenegger et al., 2013), the 308

similarity between the PA structures produced is remarkable. Not only the bicyclic 309

ring system characteristic for all PAs, namely the necine bases, are identical with 310

respect to their structure and stereochemistry between unrelated plant lineages, but 311

also complete PA molecules (Hartmann and Witte, 1995). Similar selection pressures 312

probably forced the evolution of similar, almost identical traits (Pichersky et al., 2006). 313

In this study, we have been able to show that the strategy of using a second auxiliary 314

site for PA biosynthesis to protect floral structures also evolved independently in two 315

distantly related species, extending the aspect of convergent evolution from molecule 316

structures to the regulation of the whole pathway. Further work will be needed to 317

analyze the evolutionary mechanisms underlying the repeated recruitment, 318

optimization, and integration of PA biosynthesis into the metabolism of the plant. 319



15

EXPERIMENTAL 320

Plant Material 321

Symphytum officinale was grown in pots with a mixture of TKS2 (Floragard) and lava 322

granulate at a ratio of 3:1 in the Botanical Gardens Kiel from April to September. 323

RNA Isolation and Quantification of Transcripts 324

For transcript analyses of leaves in relation to their position in the shoot system, plant 325

samples were pulverized in liquid nitrogen with mortar and pestle before total RNA 326

was extracted with Trizol® (Invitrogen, Life Technologies) according to the 327

manufacturer’s protocol, but with two additional phase separation steps involving the 328

addition of 200 µl chloroform to the aqueous phase followed by phase separation via 329

centrifugation. Finally, RNA was washed twice with ice-cold ethanol (75%, v/v, in 330

water). A subsequent lithium chloride (2 M final concentration) precipitation (16 h, 331

4°C) and two additional washing steps with ice-cold ethanol were then performed to 332

remove possible inhibitors of the following reverse transcription reactions (RT). RNA 333

was dissolved in RNAse-free water, and RNA integrity and purity were tested by 334

agarose gel electrophoresis and by 260/280 nm and 260/230 nm ratio measurements 335

by using a NanoDrop 1000 UV/VIS spectrometer. One microgram of total RNA was 336

used as a template for reverse transcription with an Oligo(dT)17 primer (Suppl. 337

Tab. 1) and RevertAid® Premium Reverse Transcriptase (ThermoScientific). For the 338

transcript quantification of HSS in leaves subtending inflorescences at various 339

developmental stages, a slightly modified protocol for total RNA isolation was 340

employed. Instead of washes with ethanol, RNA was diluted 1:1 with 100 % ethanol 341

and loaded on spin columns from the Direct-zol™ RNA MiniPrep Kit (Zymo 342

Research). Samples were further processed as recommended by the manufacturer 343

including an optional on-column Dnase I digestion. For transcript quantification, 1.5 344



16

µg total RNA was used for reverse transcription as previously described. To test for 345

contaminating genomic DNA, control reactions were prepared without reverse 346

transcriptase and used as a template in control PCRs (Suppl. Fig. 4). 347

For semiquantitative PCR (sqPCR) GoTaq-DNA polymerase (Promega) with dNTPs 348

(0.2 mM each) and primers (0.2 µM each) was used, and aliquots were taken after 349

20, 25, 30, and 35 cycles to ensure sampling before PCR product formation reached 350

saturation (Suppl. Fig. 1). Products were analyzed on a 2% (w/v) agarose gel. 351

Quantitative real-time PCR was performed in a Rotor-Gene® Q System (Qiagen) 352

with GoTaq® qPCR Master Mix (Promega) following the manufacturer's protocol. 353

Melting curve analyses were undertaken to distinguish specific PCR products from 354

primer dimers or unspecific PCR products. The calculation of the relative transcript 355

levels of HSS was carried out by comparative CT methods (2-∆∆Ct for Fig. 1B, 2-∆Ct 356

for Fig. 4B) (Schmittgen and Livak, 2008). Actin served as the reference gene to 357

normalize expression levels. For both sqPCR and qRT-PCR, an annealing 358

temperature of 60°C was used. Primer pairs were: P1 and P2 (for HSS) and P3 and 359

P4 (for actin, Suppl. Tab. 1). The actin-specific primers resulted from an 360

actin-encoding sequence amplified with a pair of degenerate primers (P5/P6) 361

designed according to an alignment of the actin encoding sequences of Arabidopsis 362

thaliana as previously described (Sievert et al., 2015). For the cloning of PCR 363

products, the pGEM T-easy vector (Promega) was employed according to the 364

manufacturer’s protocol followed by transformation into chemically competent E.coli 365

TOP10 cells (Invitrogen) for vector propagation. Plasmids were sequenced at MWG 366

Eurofins to confirm the identity of the amplification products. Sequence data from this 367

article have been deposited in the EMBL/GenBank data libraries under the following 368

accession number: LT631489, Symphytum officinale partial mRNA for actin (act 369



17

gene). 370

Protein-Blot Analysis of S. officinale Leaves 371

Samples were pulverized in liquid nitrogen with pestle and mortar. Protein was 372

extracted with phosphate-buffered saline supplemented with 5% (m/v) 373

polyvinylpyrrolidone and 2.5% (w/v) sodium ascorbate to prevent protein precipitation 374

by polyphenols. 10 to 20 µg total protein per sample were mixed with SDS loading 375

buffer and separated by sodium dodecylsulfate polyacrylamide gel electrophoresis 376

(SDS-PAGE) followed by semidry blotting and immunodetection as described 377

previously (Anke et al., 2008). The polyclonal antibody was affinity-purified against 378

recombinant HSS of S. officinale (Niemüller et al., 2012). MultiMark Multi-Colored 379

Standard (NOVEX), PageRuler™ Plus Prestained Protein Ladder (Thermo 380

Scientific), and PageRuler™ Prestained Protein Ladder (Thermo Scientific) were 381

used as protein mass standards as indicated in the figure legends. After 382

immunodetection, PDVF membranes were stained with PageBlue™ Protein Staining 383

Solution (Thermo Scientific) to ensure that equal protein amounts were loaded on 384

each lane. For the relative quantification of protein levels by densitometry we used 385

the software ImageJ (version 1.48) with 25 ng of recombinant HSS protein as the 386

reference (Schneider et al., 2012). 387

Immunohistochemical Staining of HSS in Leaf Cross Sections 388

For the immunohistochemical localization of HSS in leaf cross-sections, young leaves 389

were harvested; and the tip and the base were was cut off to be used as “reference 390

tissue”, whereas the central part of the leaf was cut into pieces of approx. 1 cm side 391

length before being immersed in ice-cold fixation buffer according to Anke et al. 392

(2008). The “reference tissue” of each leaf was pulverized in liquid nitrogen, and total 393

RNA was extracted and transcribed to cDNA as described above. Each “reference 394



18

tissue” was tested by sqPCR for the presence of HSS transcripts. The fixated central 395

regions of positively tested leaves were dehydrated in an ethanol series and 396

embedded in Technovit 7100 (Heraeus-Kulzer). Sections of 3-4 µm thickness were 397

cut on a microtome (HM3555S, Microm) and mounted on adhesive microscope slides 398

(SuperFrost, Thermo Fisher Scientific). Immunodetection with HSS-specific 399

antibodies and specificity tests with recombinant HSS and DHS of S. officinale were 400

carried out as described previously (Niemüller et al., 2012). 401

Radioactive Tracer-Feeding Experiments and Product Analysis 402

To test the capacity of detached leaves of S. officinale to produce PAs, 403

[14C]putrescine (3.95 GBq/mmol, GE Healthcare) was used as tracer. Two young 404

leaves (approx. 3.5 cm in length) next to an inflorescence with developing flower 405

buds were cut from the plant and transferred into a 1.5 ml reaction tube containing 406

the tracer dissolved in 1 ml tap water (169 kBq). The leaves were incubated in a 407

light/dark regime of 12 h/12 h (light of approx. 1,000 lx) at room temperature. Once 408

the liquid was almost completely taken up, the same volume of tap water without 409

tracer was added. After 4 days, the leaves were frozen in liquid nitrogen and stored at 410

-50°C. Both leaves were pulverized in liquid nitrogen with mortar and pestle and each 411

extracted in 1.5 ml 0.05 M H2SO4 by being vortexed for 3 min at room temperature. 412

After centrifugation (10 min, 5000 x g) zinc dust was added in excess to the 413

supernatants, which were then stirred for 3 hours for the complete reduction of 414

alkaloid N-oxides. As SCX-SPE purification has been shown to be suitable for the 415

enrichment of PAs (Colegate et al., 2005), the samples were further centrifuged 416

(10 min, 5000 x g) and then applied to SCX-SPE cartridges (Phenomenex, 417

Aschaffenburg, Germany) equilibrated with 6 ml methanol and 6 ml of 0.05 M H2SO4. 418

After each column had been washed with 12 ml H2O and 12 ml methanol, the 419



19

alkaloids were eluted by applying 3 x 6 ml of methanol containing 5% (v/v) NH4OH. 420

All elution fractions were combined, the solvent vaporized, and the residue dissolved 421

in 2 ml methanol. The supernatant was transferred in equal parts into two vials, dried, 422

and stored at -20°C. For hydrolysis, one of the samples was incubated in 2 N sodium 423

hydroxide for 3 h at 60°C, and the solvent was vaporized. For HPLC analyses, the 424

hydrolyzed and the non-hydrolyzed samples were dissolved in 50 µl methanol. Each 425

step of the extraction process was monitored via scintillation counting (Tri-Carb LSC, 426

Perkin Elmer) to calculate the ratio of incorporated [14C]putrescine. 427

Radio-HPLC measurements were performed on a Merck-Hitachi L-6200 instrument 428

with solvent A (100 mM phosphate buffer pH 7.5) and solvent B (acetonitrile) at a 429

ratio of 85:15 with a flow rate of 1 ml per minute. Aliquots of 20 µl of sample were 430

injected, and fractions were collected every 30 s for comparison of the hydrolyzed 431

and the non-hydrolyzed extract and every 15 s for the comparison of the hydrolyzed 432

extract with retronecine and with hydrolyzed and non-hydrolyzed monocrotaline 433

standards (Roth, Karlsruhe, Germany). The radioactivity of the fractions was 434

quantified by scintillation counting. Radiolabeled retronecine was obtained by the 435

hydrolysis of [14C]senecionine (Lindigkeit et al., 1997). 436

For thin-layer chromatography, Silicagel G-25 TLC plates (Merck, Darmstadt) were 437

developed in a mobile phase containing ethyl acetate, isopropyl alcohol, and 438

ammonium hydroxide (25%, v/v) (45:35:20). Radioactivity was detected by using a 439

radioactivity thin-layer-chromatography detector (RITA, Raytest, Straubenhardt. 440

Germany). Compounds scraped from the TLC plate were eluted with the mobile 441

phase, dried, and dissolved in 50 µl methanol for GC-MS analysis. GC-MS data were 442

obtained with a Shimadzu GC-2010 chromatograph equipped with a 15 m Optima-1 443

MS capillary column coupled to a quadrupole mass spectrometer (Fisons MD800). 444



20

EI-mass spectra were recorded at 70 eV. GC conditions were: injector 250°C, 445

temperature program 60°C for 3 min, 60 to 300°C at 6°C/min, carrier gas helium 446

1 ml/min. GC-MS data for retronecine were: Ri 1478 (on Optima-1 MS), m/z 155 [M+]; 447

MS spectrum, m/z (rel. intensity): 80(100), 111(74), 155(45), 94(37), 68(34), 93(33), 448

82(31), 112(30), 67(29), 106(25). 449

450

Quantification of total PAs in inflorescences 451

Total PAs were extracted from lyophilized inflorescences, hydrolyzed, and reduced to 452

obtain the necine base retronecine as described for the tracer feeding experiment. 453

For quantification of the necine base, the sum parameter method involving silylation 454

of the necine base followed by GC-MS was applied according to Kempf et al. (2008). 455

Retronecine from hydrolyzed monocrotaline served as the external standard. 456

457

List of Supplementary Material 458

Supplementary Figure 1. PCR cycle optimization. 459

Supplementary Figure 2. Relative HSS transcript levels with actin (ACT) as reference 460

gene. 461

Supplementary Figure 3. Specificity tests of antibody labeling by pre - incubation of 462

the HSS-specific antibody with soluble protein. 463

Supplementary Figure 4. MS-identification of the product accumulating in the leaf 464

after feeding with putrescine. 465

Supplementary Figure 5. Tests for contaminating gDNA in cDNA samples. 466

Supplementary Figure 6. Protein blot analyses of leaves subtending inflorescences of 467

various developmental stages. 468

Supplementary Figure 7. Protein blot analyses of various developmental stages of 469



21

inflorescences. 470

Supplementary Table 1. Primer sequences. 471

472

473

Acknowledgements: We thank Dr. Dorothee Langel for help in designing the 474

radioactive tracer feeding experiments and Jan Baur, Margret Doose, Brigitte 475

Schemmerling, and Karina Thöle for support in the laboratory. We are grateful 476

Dr. Elisabeth Kaltenegger, Dr. Jessica Garzke, and Annika Jonathas for helpful 477

comments on the manuscript. 478



22

Table 1. Tracer-feeding experiment. Recovery of applied radioactivity. 479

radioactivity [kBq] % of recovered

radioactivity

Total radioactivity applied 169 100

Crude extract (sulfuric acid) 25 16

Crude extract after reduction 27 16

Extract purified via SCX-SPE (PA enrichment) 15 8

480

481

482

Supplementary Table 1. Primer sequences 483

primer sequence

Oligo(dT) 17 5’-GTCGACTCGAGAATTCTTTTTTTTTTTTTTTTT-3’

P1 5’-AGTGCTATGGACAATGAATCAGTGA-3’

P2 5’-AGCAAAATCAGCGCCTCCA-3’

P3 5’-CAAGGCTAACAGGGAGAAAATGAC-3’

P4 5’-ATCACCAGAATCCAGCACAATACC-3’

P5 5’-WSNAAYTGGGAYGAYATGGA-3’

P6 5’-TCRBHYTTNGTDATCCACA-3’

For degenerate primers P5 and P6, the following code is used: B = C + G + T, D = A 484 + G + T, H = A + T + C, N = A + T + C + G, R = A + G, S = C + G, W = A + T, and Y = 485 T + C. 486



23

Figure 1. Transcript and protein blot analyses of leaves at various 487

developmental stages. A, Habitus of a S. officinale shoot. Numbers indicate leaves 488

that were analyzed by protein blot analysis. The color code indicates the level of HSS 489

expression according to the protein blot analysis given in C (red, high expression; 490

purple, medium expression; blue, no expression). B, Semi quantitative RT-PCR 491

shows the highest expression of HSS transcripts in leaf type I. Leaf types I to IV 492

comply with leaves 1, 4, 8, and 10 in Fig. 1A, respectively, but were harvested from 493

another plant individual as independent proof. RNA integrity was confirmed by 494

detection of two distinct bands of 28 S and 18 S rRNA. Actin served as the reference 495

gene. Closed arrow heads show marker bands at 150 bp, open arrows indicate 496

marker bands at 100 bp. C, Protein blot analysis of protein extracts from leaves 497

numbered according to A. Crude protein extracts (10 µg protein per lane) were 498

separated via SDS-PAGE and blotted onto a PDVF membrane. Affinity-purified HSS-499

specific antibody was used for detection in combination with a secondary goat-anti-500

rabbit antibody conjugated with horseradish peroxidase. A protein extract of a root of 501

S. officinale served as a positive control (sample 11). 502

503

Figure 2. Immunolabeling of HSS in cross-sections of a young leaf subtending 504

an inflorescence with unopened flower buds of S. officinale. A, Plant habitus. B, 505

Brightfield microscopy of a leaf cross-section. C, Detail of B. D, Leaf cross-section 506

with a vascular bundle cut longitudinally. In C and D, HSS was immunolabeled by 507

incubation with an affinity-purified HSS-specific antibody and visualized with a 508

secondary antibody conjugated with AlexaFluor488 under UV-light. Size bars refer to 509

10 µm. ep, epidermis; bs, bundle sheath cells; cu, cuticle; pp, palisade parenchyma; 510

sm, spongy mesophyll; ph, phloem; t, trichome; ve, vessel element; xy, xylem; gc, 511



24

guard cell. 512

513

Figure 3. HPLC elution profiles of alkaloid extracts of leaves after tracer 514

feeding. Young leaves subtending an inflorescence with unopened flower buds were 515

fed with [14C]putrescine as tracer. Extracts were enriched for PAs by SPE purification 516

before separation. A, Non-hydrolyzed leaf extract. B, Hydrolyzed leaf extract. C, 517

Radiolabeled retronecine standard. D, Mixture of hydrolyzed extract and retronecine 518

standard. 519

520

Figure 4. PA content and HSS expression in leaves and inflorescences of 521

various developmental stages. A, Inflorescences at various developmental stages. 522

B, Protein and transcript levels of HSS in leaves subtending inflorescences. C, PA 523

concentrations in the inflorescences and in the subtending leaves at the respective 524

developmental stages. D, Total PA content and dry weight of the inflorescence. 525

Inflorescences and corresponding subtending leaves were harvested, and 526

inflorescences at the developmental stages I-VII were weighed. PAs from 527

inflorescences and leaves were extracted and analyzed according to the sum 528

parameter method (C and D). For relative transcript quantification, qRT-PCR was 529

used with actin (ACT) as the reference gene (B). For relative protein quantification, 530

total protein was extracted and separated via SDS-PAGE before being transferred to 531

a PDVF membrane. Affinity-purified HSS-specific antibody was used for detection in 532

combination with a secondary goat-anti-rabbit antibody conjugated with horseradish 533

peroxidase. Protein level was calculated in relation to 25 ng recombinant HSS that 534

was used as a standard (B). Error bars indicate the standard error of the mean (SEM) 535

of three independent biological replicates for each developmental stage. Error bars of 536



25

PA concentration in leaves of stage II and III are missing in C because of insufficient 537

sample material. 538



26

Supplementary Figure S1. PCR cycle optimization. Levels of HSS- and actin 539

(ACT)-encoding transcripts in the various leaf types were estimated by 540

semi-quantitative RT-PCR. To ensure quantification during the exponential phase, 541

amplification aliquots were analyzed after 20, 25, 30, and 35 cycles by gel 542

electrophoresis. Arrowheads indicate PCR products with the expected size between 543

100 and 150 bp. Roman numerals refer to leaf types given in Fig. 1C. M, DNA ladder; 544

C, no template control. 545

546

Supplementary Figure 2. Relative HSS transcript levels with actin (ACT) as 547

reference gene. HSS transcript quantification in leaves subtending inflorescences of 548

various developmental stages by using the same cDNA as for qRT-PCR given in 549

Fig. 1B. The HSS transcript level of leaf type IV was set to one. Error bars indicate 550

the standard deviation of three technical replicates (n=1). 551

552

Supplementary Figure 3. Specificity tests of antibody labeling by 553

pre-incubation of the HSS-specific antibody with soluble protein. Leaf cross-554

sections were labeled with HSS-specific antibody after pre-incubation with HSS (A, 555

B), DHS (D, E), and bovine serum albumin (BSA) (G, H) at a molar ratio of antibody 556

to added protein of 10:1 (B, E H) and 1:3 (A, D, G). A section labeled with HSS-557

specific antibody without pre-incubation (C) and a section labeled without primary 558

antibody incubation to show unspecific binding of secondary antibody (F) served as 559

controls. Only pre-incubation with soluble HSS resulted in a significant reduction of 560

signal. 561

562



27

Supplementary Figure 4. MS-identification of the product accumulating in the 563

leaf after feeding with putrescine. A, Total ion chromatogram of the spot co-564

migrating on TLC with the radioactive signal resulting from the [14C]putrescine 565

feeding experiment. The largest peak shows a retention index (Kovats) at 1478. The 566

two minor peaks with similar retention times show a typical fragmentation pattern of 567

softeners (plasticizers). B, Extracted ion chromatogram of the same sample as in A 568

for the [M+] of retronecine and its stereoisomer heliotridine with an m/z of 155. The 569

peak is identical to the peak in A. C, Mass spectrum of the largest peak in A with the 570

retention index of 1478. The peak was identified via the NIST database as being 571

retronecine. 572

573

Supplementary Figure 5. Tests for contaminating gDNA in cDNA samples. 574

Agarose gel electrophoresis of total RNA and PCR products resulting from 575

amplification of RNA used for reverse transcription (no RT-control). Contamination of 576

RNA with gDNA should result in fragments as shown in the positive control amplified 577

with cDNA as the template in P, as the amplified sequence does not contain introns. 578

Arrowheads indicate PCR products with the expected size between 100 and 150 bp. 579

Roman numerals refer to the leaf stages from Fig. 4. The three biological replicates 580

are indicated with a, b, and c. M, DNA ladder; NTC, no template control. 581

582

Supplementary Figure 6. Protein blot analyses of leaves subtending 583

inflorescences of various developmental stages. Leaves were sampled in 584

triplicate and protein extracts were blotted on separate membranes. A, C, and E, 585

PDVF membranes stained with colloidal Coomassie G-250 dye to test for comparably 586

loaded lanes. B, D, and F, X-ray film of the membranes corresponding to those given 587



28

in A, C, and E, respectively, after ECL detection of horseradish-peroxidase generated 588

bio-luminescence for quantification of HSS as illustrated in Fig. 4B. Crude protein 589

extracts of leaves and the root (20 µg protein per lane) were separated via SDS-590

PAGE and blotted onto a PDVF membrane. Affinity-purified HSS-specific antibody 591

was used for detection in combination with a secondary goat-anti-rabbit antibody 592

conjugated to horseradish peroxidase. Recombinant HSS protein (25 ng) served as 593

positive a control. The observed size difference between heterologous protein and 594

the native protein is caused by His-tag fusion. As the antibody was generated against 595

the protein heterologously expressed in E. coli, unspecific binding to contaminating 596

E. coli proteins in lane “control” is expected. The apparent additional signal in root 597

extracts was described earlier (Niemueller et al., 2012) and is caused by unspecific 598

protein cleavage by proteases of soil bacteria. M, protein mass standard (A-D: 599

PageRuler™ Plus Prestained Protein Ladder, E-F: PageRuler™ Prestained Protein 600

Ladder); I-VII represent the various developmental stages defined in Fig. 4A; 601

numbers indicate the mass of the corresponding bands of a protein standard in kDa; 602

white arrow head, indicates size of native HSS protein; black arrow head, size of 603

recombinant HSS (with His-tag). 604

605

Supplementary Figure 7. Protein blot analyses of various developmental stages 606

of inflorescences. A, PDVF membrane stained with colloidal Coomassie G-250 dye. 607

B, ECL detection of horseradish-peroxidase generated bio-luminescence on an X-ray 608

film. Protein extracts of inflorescences and roots collected from three independent 609

plant individuals were pooled and separated via SDS-PAGE (20 µg protein per lane) 610

and blotted onto a PDVF membrane. Affinity-purified HSS-specific antibody was used 611

for detection in combination with a secondary goat-anti-rabbit antibody conjugated to 612



29

horseradish peroxidase. Recombinant HSS protein (25 ng), which was 613

heterologously expressed in E. coli, served as positive a control. The additional 614

signal in root extract is attributable to unspecific protein cleavage (Niemüller et al., 615

2012). All analyzed developmental stages of inflorescences show only low protein 616

levels of HSS. M, protein mass standard (PageRuler™ Prestained Protein Ladder); I-617

VII represents the various developmental stages defined in Fig. 4A; numbers indicate 618

the mass of the corresponding bands of protein standard in kDa; white arrow heads 619

indicate the size of native HSS and black arrow heads that of recombinant HSS (with 620

His-tag). 621



30

Literature Cited 622

Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, 623

Tiburcio AF (2010) Polyamines: molecules with regulatory functions in plant 624

abiotic stress tolerance. Planta 231: 1237-1249 625

Anke S, Gonde D, Kaltenegger E, Hansch R, Theuring C, Ober D (2008) 626

Pyrrolizidine alkaloid biosynthesis in Phalaenopsis orchids: developmental 627

expression of alkaloid-specific homospermidine synthase in root tips and 628

young flower buds. Plant Physiology 148: 751-760 629

Anke S, Niemüller D, Moll S, Hänsch R, Ober D (2004) Polyphyletic origin of 630

pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue 631

expression of homospermidine synthase. Plant Physiology 136: 4037-4047 632

Bird DA, Franceschi VR, Facchini PJ (2003) A tale of three cell types: alkaloid 633

biosynthesis is localized to sieve elements in opium poppy. Plant Cell 15: 634

2626-2635 635

Böttcher F, Adolph RD, Hartmann T (1993) Homospermidine synthase, the first 636

pathway-specific enzyme in pyrrolizidine alkaloid biosynthesis. Phytochemistry 637

32: 679-689 638

Böttcher F, Ober D, Hartmann T (1994) Biosynthesis of pyrrolizidine alkaloids: 639

putrescine and spermidine are essential substrates of enzymatic 640

homospermidine formation. Canadian Journal of Chemistry 72: 80-85 641

Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of 642

three MEP pathway genes and geraniol 10-hydroxylase in internal phloem 643

parenchyma of Catharanthus roseus implicates multicellular translocation of 644

intermediates during the biosynthesis of monoterpene indole alkaloids and 645

isoprenoid-derived primary metabolites. Plant Journal 38: 131-141 646

Castro S, Silveira P, Navarro L (2008) Consequences of nectar robbing for the 647

fitness of a threatened plant species. Plant Ecology 199: 201-208 648

Colegate SM, Edgar JA, Knill AM, Lee ST (2005) Solid-phase extraction and 649

HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study 650

of Echium plantagineum. Phytochemical Analysis 16: 108-119 651

Cordell GA (2013) Fifty years of alkaloid biosynthesis in Phytochemistry. 652

Phytochemistry 91: 29-51 653



31

Couet CE, Crews C, Hanley AB (1996) Analysis, separation, and bioassay of 654

pyrrolizidine alkaloids from comfrey (Symphytum officinale). Natural toxins 4: 655

163-167 656

Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? 657

Annual Review of Plant Physiology and Plant Molecular Biology 40: 235-269 658

Facchini PJ, St-Pierre B (2005) Synthesis and trafficking of alkaloid biosynthetic 659

enzymes. Current Opinion in Plant Biology 8: 657-666 660

Frölich C, Hartmann T, Ober D (2006) Tissue distribution and biosynthesis of 1,2-661

saturated pyrrolizidine alkaloids in Phalaenopsis hybrides (Orchidaceae). 662

Phytochemistry 67: 1493-1502 663

Frölich C, Ober D, Hartmann T (2007) Tissue distribution, core biosynthesis and 664

diversification of pyrrolizidine alkaloids of the lycopsamine type in three 665

Boraginaceae species. Phytochemistry 68: 1026-1037 666

Geldner N (2013) The endodermis. Annual Review of Plant Biology 64: 531-558 667

Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant 668

Signaling & Behavior 5: 26-33 669

Grotewold E (2005) Plant metabolic diversity: a regulatory perspective. Trends in 670

Plant Science 10: 57-62 671

Hartmann T, Ehmke A, Eilert U, von Borstel K, Theuring C (1989) Sites of 672

synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in 673

Senecio vulgaris L. Planta 177: 98-107 674

Hartmann T, Witte L (1995) Chemistry, biology and chemoecology of the 675

pyrrolizidine alkaloids. In SW Pelletier, ed, Alkaloids: Chemical and Biological 676

Perspectives, Vol 9. Pergamon Press, Oxford, pp 155-233 677

Irmer S, Podzun N, Langel D, Heidemann F, Kaltenegger E, Schemmerling B, 678

Geilfus C-M, Zorb C, Ober D (2015) New aspect of plant-rhizobia interaction: 679

Alkaloid biosynthesis in Crotalaria depends on nodulation. Proceedings of the 680

National Academy of Sciences of the United States of America 112: 4164-681

4169 682

Irwin RE, Maloof JE (2002) Variation in nectar robbing over time, space, and 683

species. Oecologia 133: 525-533 684

Kaltenegger E, Eich E, Ober D (2013) Evolution of homospermidine synthase in the 685

Convolvulaceae: a story of gene duplication, gene loss, and periods of various 686

selection pressures. Plant Cell 25: 1213-1227 687



32

Kanegae T, Kajiya H, Amano Y, Hashimoto T, Yamada Y (1994) Species-688

dependent expression of the hyoscyamine 6-b-hydroxylase gene in the 689

pericycle. Plant Physiology 105: 483-490 690

Kempf M, Beuerle T, Buhringer M, Denner M, Trost D, von der Ohe K, Bhavanam 691

VB, Schreier P (2008) Pyrrolizidine alkaloids in honey: Risk analysis by gas 692

chromatography-mass spectrometry. Molecular Nutrition and Food Research 693

52: 1193-1200 694

Kotelnikova KV, Tretjakova DJ, Nuraliev MS (2011) Shoot structure of Symphytum 695

officinale L. (Boraginaceae) in relation to the nature of its axially shiftes lateral 696

branches. Wulfenia - Mitteilungen des Kärntner Botanikzentrums Klagenfurt 697

18: 63-79 698

Leegood RC (2008) Roles of the bundle sheath cells in leaves of C3 plants. Journal 699

of Experimental Botany 59: 1663-1673 700

Lindigkeit R, Biller A, Buch M, Schiebel HM, Boppré M, Hartmann T (1997) The 701

two faces of pyrrolizidine alkaloids: The role of the tertiary amine and its N-702

oxide in chemical defense of insects with acquired plant alkaloids. European 703

Journal of Biochemistry 245: 626-636 704

Mattocks AR (1980) Toxic pyrrolizidine alkaloids in comfrey. Lancet (London, 705

England) 2: 1136-1137 706

McKey D (1979) The distribution of secondary compounds within plants. In GA 707

Rosenthal, DH Janzen, eds, Herbivores: Their Interaction with Secondary 708

Plant Metabolites. Academic Press, New York, pp 55-133 709

Moll S, Anke S, Kahmann U, Hänsch R, Hartmann T, Ober D (2002) Cell-specific 710

expression of homospermidine synthase, the entry enzyme of the pyrrolizidine 711

alkaloid pathway in Senecio vernalis, in comparison with its ancestor, 712

deoxyhypusine synthase. Plant Physiology 130: 47-57 713

Morita M, Shitan N, Sawada K, Van Montagu MCE, Inze D, Rischer H, Goossens 714

A, Oksman-Caldentey K-M, Moriyama Y, Yazaki K (2009) Vacuolar transport 715

of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) 716

transporter in Nicotiana tabacum. Proceedings of the National Academy of 717

Sciences of the United States of America 106: 2447-2452 718

Muetterlein R, Arnold C-G (1993) Investigations concerning the content and the 719

pattern of pyrrolizidine alkaloids in Symphytum officinale L. (Comfrey). 720

Pharmazeutische Zeitung 138: 119-125 721



33

Nakajima K, Oshita Y, Kaya M, Yamada Y, Hashimoto T (1999) Structures and 722

expression patterns of two tropinone reductase genes from Hyoscyamus 723

niger. Bioscience, Biotechnology and Biochemistry 63: 1756-1764 724

Neumann D (1985) Storage of alkaloids. In K Mothes, HR Schütte, M Luckner, eds, 725

Biochemistry of Alkaloids. VCH, Weilheim, pp 49-55 726

Niemüller D, Reimann A, Ober D (2012) Distinct cell-specific expression of 727

homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in 728

three species of the Boraginales. Plant Physiology 159: 920-929 729

Nurhayati N, Gondé D, Ober D (2009) Evolution of pyrrolizidine alkaloids in 730

Phalaenopsis orchids and other monocotyledons: Identification of 731

deoxyhypusine synthase, homospermidine synthase and related 732

pseudogenes. Phytochemistry 70: 508-516 733

Ober D, Hartmann T (1999) Deoxyhypusine synthase from tobacco. cDNA isolation, 734

characterization, and bacterial expression of an enzyme with extended 735

substrate specificity. Journal of Biological Chemistry 274: 32040-32047 736

Ober D, Hartmann T (1999) Homospermidine synthase, the first pathway-specific 737

enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine 738

synthase. Proceedings of the National Academy of Sciences of the United 739

States of America 96: 14777-14782 740

Pichersky E, Noel JP, Dudareva N (2006) Biosynthesis of plant volatiles: nature's 741

diversity and ingenuity. Science (New York, N.Y.) 311: 808-811 742

Reimann A, Nurhayati N, Backenköhler A, Ober D (2004) Repeated evolution of 743

the pyrrolizidine alkaloid-mediated defense system in separate angiosperm 744

lineages. Plant Cell 16: 2772-2784 745

Sander H, Hartmann T (1989) Site of synthesis, metabolism and translocation of 746

senecionine N-oxide in cultured roots of Senecio erucifolius. Plant Cell, Tissue 747

and Organ Culture 18: 19-32 748

Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative 749

C(T) method. Nature Protocols 3: 1101-1108 750

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of 751

image analysis. Nature methods 9: 671-675 752

Sievert C, Beuerle T, Hollmann J, Ober D (2015) Single cell subtractive 753

transcriptomics for identification of cell-specifically expressed candidate genes 754

of pyrrolizidine alkaloid biosynthesis. Phytochemistry 117: 17-24 755



34

St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of 756

Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation 757

of a pathway intermediate. Plant Cell 11: 887-900 758

Steudle E, Peterson CA (1998) How does water get through roots? Journal of 759

Experimental Botany 49: 775-788 760

Suzuki KI, Yamada Y, Hashimoto T (1999) Expression of Atropa belladonna 761

putrescine N-methyltransferase gene in root pericycle. Plant and Cell 762

Physiology 40: 289-297 763

Suzuki KI, Yun DJ, Chen XY, Yamada Y, Hashimoto T (1999) An Atropa belladonna 764

hyoscyamine 6b-hydroxylase gene is differentially expressed in the root 765

pericycle and anthers. Plant Molecular Biology 40: 141-152 766

Tiburcio AF, Altabella T, Bitrian M, Alcazar R (2014) The roles of polyamines 767

during the lifespan of plants: from development to stress. Planta 240: 1-18 768

Utelli AB, Roy BA (2001) Causes and consequences of floral damage in Aconitum 769

lycoctonum at high and low elevations in Switzerland. Oecologia 127: 266-273 770

Weid M, Ziegler J, Kutchan TM (2004) The roles of latex and the vascular bundle in 771

morphine biosynthesis in the opium poppy, Papaver somniferum. Proceedings 772

of the National Academy of Sciences of the United States of America 101: 773

13957-13962 774

Weng J-K, Philippe RN, Noel JP (2012) The rise of chemodiversity in plants. 775

Science 336: 1667-1670 776

Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. 777

Annual Review of Plant Biology 59: 735-769 778

779





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Parsed CitationsAlcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010) Polyamines: molecules withregulatory functions in plant abiotic stress tolerance. Planta 231: 1237-1249

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Anke S, Gonde D, Kaltenegger E, Hansch R, Theuring C, Ober D (2008) Pyrrolizidine alkaloid biosynthesis in Phalaenopsisorchids: developmental expression of alkaloid-specific homospermidine synthase in root tips and young flower buds. PlantPhysiology 148: 751-760


Anke S, Niemüller D, Moll S, Hänsch R, Ober D (2004) Polyphyletic origin of pyrrolizidine alkaloids within the Asteraceae. Evidencefrom differential tissue expression of homospermidine synthase. Plant Physiology 136: 4037-4047


Bird DA, Franceschi VR, Facchini PJ (2003) A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opiumpoppy. Plant Cell 15: 2626-2635


Böttcher F, Adolph RD, Hartmann T (1993) Homospermidine synthase, the first pathway-specific enzyme in pyrrolizidine alkaloidbiosynthesis. Phytochemistry 32: 679-689


Böttcher F, Ober D, Hartmann T (1994) Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substratesof enzymatic homospermidine formation. Canadian Journal of Chemistry 72: 80-85


Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates duringthe biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant Journal 38: 131-141


Castro S, Silveira P, Navarro L (2008) Consequences of nectar robbing for the fitness of a threatened plant species. Plant Ecology199: 201-208


Colegate SM, Edgar JA, Knill AM, Lee ST (2005) Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and theirN-oxides: a case study of Echium plantagineum. Phytochemical Analysis 16: 108-119


Cordell GA (2013) Fifty years of alkaloid biosynthesis in Phytochemistry. Phytochemistry 91: 29-51Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Couet CE, Crews C, Hanley AB (1996) Analysis, separation, and bioassay of pyrrolizidine alkaloids from comfrey (Symphytumofficinale). Natural toxins 4: 163-167


Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? Annual Review of Plant Physiology and PlantMolecular Biology 40: 235-269


Facchini PJ, St-Pierre B (2005) Synthesis and trafficking of alkaloid biosynthetic enzymes. Current Opinion in Plant Biology 8: 657-666

Pubmed: Author and TitleCrossRef: Author and Title


http://www.ncbi.nlm.nih.gov/pubmed?term=Alc�zar R%2C Altabella T%2C Marco F%2C Bortolotti C%2C Reymond M%2C Koncz C%2C Carrasco P%2C Tiburcio AF %282010%29 Polyamines%3A molecules with regulatory functions in plant abiotic stress tolerance%2E Planta 231%3A 1237%2D1249&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Alc�zar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010) Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231: 1237-1249

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http://search.crossref.org/?page=1&rows=2&q=Anke S, Gonde D, Kaltenegger E, Hansch R, Theuring C, Ober D (2008) Pyrrolizidine alkaloid biosynthesis in Phalaenopsis orchids: developmental expression of alkaloid-specific homospermidine synthase in root tips and young flower buds. Plant Physiology 148: 751-760

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

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http://search.crossref.org/?page=1&rows=2&q=Anke S, Niem�ller D, Moll S, H�nsch R, Ober D (2004) Polyphyletic origin of pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expression of homospermidine synthase. Plant Physiology 136: 4037-4047

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

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http://search.crossref.org/?page=1&rows=2&q=Bird DA, Franceschi VR, Facchini PJ (2003) A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell 15: 2626-2635

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

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http://search.crossref.org/?page=1&rows=2&q=B�ttcher F, Adolph RD, Hartmann T (1993) Homospermidine synthase, the first pathway-specific enzyme in pyrrolizidine alkaloid biosynthesis. Phytochemistry 32: 679-689

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http://search.crossref.org/?page=1&rows=2&q=B�ttcher F, Ober D, Hartmann T (1994) Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substrates of enzymatic homospermidine formation. Canadian Journal of Chemistry 72: 80-85

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

http://scholar.google.com/scholar?as_q=Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substrates of enzymatic homospermidine formation&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

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http://search.crossref.org/?page=1&rows=2&q=Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant Journal 38: 131-141

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

http://scholar.google.com/scholar?as_q=Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites&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

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http://search.crossref.org/?page=1&rows=2&q=Castro S, Silveira P, Navarro L (2008) Consequences of nectar robbing for the fitness of a threatened plant species. Plant Ecology 199: 201-208

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

http://scholar.google.com/scholar?as_q=Consequences of nectar robbing for the fitness of a threatened plant species&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

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http://www.ncbi.nlm.nih.gov/pubmed?term=Colegate SM%2C Edgar JA%2C Knill AM%2C Lee ST %282005%29 Solid%2Dphase extraction and HPLC%2DMS profiling of pyrrolizidine alkaloids and their N%2Doxides%3A a case study of Echium plantagineum%2E Phytochemical Analysis 16%3A 108%2D119&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Colegate SM, Edgar JA, Knill AM, Lee ST (2005) Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum. Phytochemical Analysis 16: 108-119

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

http://scholar.google.com/scholar?as_q=Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum&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=Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Colegate&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cordell GA %282013%29 Fifty years of alkaloid biosynthesis in Phytochemistry%2E Phytochemistry 91%3A 29%2D51&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cordell GA (2013) Fifty years of alkaloid biosynthesis in Phytochemistry. Phytochemistry 91: 29-51

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

http://scholar.google.com/scholar?as_q=Fifty years of alkaloid biosynthesis in Phytochemistry&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=Fifty years of alkaloid biosynthesis in Phytochemistry&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cordell&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Couet CE%2C Crews C%2C Hanley AB %281996%29 Analysis%2C separation%2C and bioassay of pyrrolizidine alkaloids from comfrey %28Symphytum officinale%29%2E Natural toxins 4%3A 163%2D167&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Couet CE, Crews C, Hanley AB (1996) Analysis, separation, and bioassay of pyrrolizidine alkaloids from comfrey (Symphytum officinale). Natural toxins 4: 163-167

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

http://scholar.google.com/scholar?as_q=Analysis, separation, and bioassay of pyrrolizidine alkaloids from comfrey (Symphytum officinale)&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=Analysis, separation, and bioassay of pyrrolizidine alkaloids from comfrey (Symphytum officinale)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Couet&as_ylo=1996&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Evans PT%2C Malmberg RL %281989%29 Do polyamines have roles in plant development%3F Annual Review of Plant Physiology and Plant Molecular Biology 40%3A 235%2D269&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? Annual Review of Plant Physiology and Plant Molecular Biology 40: 235-269

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

http://scholar.google.com/scholar?as_q=Do polyamines have roles in plant development&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=Do polyamines have roles in plant development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Evans&as_ylo=1989&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Facchini PJ%2C St%2DPierre B %282005%29 Synthesis and trafficking of alkaloid biosynthetic enzymes%2E Current Opinion in Plant Biology 8%3A 657%2D666&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Facchini PJ, St-Pierre B (2005) Synthesis and trafficking of alkaloid biosynthetic enzymes. Current Opinion in Plant Biology 8: 657-666


Google Scholar: Author Only Title Only Author and Title

Frölich C, Hartmann T, Ober D (2006) Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsishybrides (Orchidaceae). Phytochemistry 67: 1493-1502


Frölich C, Ober D, Hartmann T (2007) Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of thelycopsamine type in three Boraginaceae species. Phytochemistry 68: 1026-1037


Geldner N (2013) The endodermis. Annual Review of Plant Biology 64: 531-558Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signaling & Behavior 5: 26-33Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Grotewold E (2005) Plant metabolic diversity: a regulatory perspective. Trends in Plant Science 10: 57-62Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hartmann T, Ehmke A, Eilert U, von Borstel K, Theuring C (1989) Sites of synthesis, translocation and accumulation of pyrrolizidinealkaloid N-oxides in Senecio vulgaris L. Planta 177: 98-107


Hartmann T, Witte L (1995) Chemistry, biology and chemoecology of the pyrrolizidine alkaloids. In SW Pelletier, ed, Alkaloids:Chemical and Biological Perspectives, Vol 9. Pergamon Press, Oxford, pp 155-233


Irmer S, Podzun N, Langel D, Heidemann F, Kaltenegger E, Schemmerling B, Geilfus C-M, Zorb C, Ober D (2015) New aspect ofplant-rhizobia interaction: Alkaloid biosynthesis in Crotalaria depends on nodulation. Proceedings of the National Academy ofSciences of the United States of America 112: 4164-4169


Irwin RE, Maloof JE (2002) Variation in nectar robbing over time, space, and species. Oecologia 133: 525-533Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kaltenegger E, Eich E, Ober D (2013) Evolution of homospermidine synthase in the Convolvulaceae: a story of gene duplication,gene loss, and periods of various selection pressures. Plant Cell 25: 1213-1227


Kanegae T, Kajiya H, Amano Y, Hashimoto T, Yamada Y (1994) Species-dependent expression of the hyoscyamine 6-b-hydroxylasegene in the pericycle. Plant Physiology 105: 483-490


Kempf M, Beuerle T, Buhringer M, Denner M, Trost D, von der Ohe K, Bhavanam VB, Schreier P (2008) Pyrrolizidine alkaloids inhoney: Risk analysis by gas chromatography-mass spectrometry. Molecular Nutrition and Food Research 52: 1193-1200


Kotelnikova KV, Tretjakova DJ, Nuraliev MS (2011) Shoot structure of Symphytum officinale L. (Boraginaceae) in relation to thenature of its axially shiftes lateral branches. Wulfenia - Mitteilungen des Kärntner Botanikzentrums Klagenfurt 18: 63-79


Leegood RC (2008) Roles of the bundle sheath cells in leaves of C3 plants. Journal of Experimental Botany 59: 1663-1673Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title www.plantphysiol.orgon April 22, 2019 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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

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

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http://search.crossref.org/?page=1&rows=2&q=Fr�lich C, Ober D, Hartmann T (2007) Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species. Phytochemistry 68: 1026-1037

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

http://scholar.google.com/scholar?as_q=Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species&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

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http://www.ncbi.nlm.nih.gov/pubmed?term=Geldner N %282013%29 The endodermis%2E Annual Review of Plant Biology 64%3A 531%2D558&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Geldner N (2013) The endodermis. Annual Review of Plant Biology 64: 531-558

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

http://scholar.google.com/scholar?as_q=The endodermis&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=The endodermis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Geldner&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Gill SS%2C Tuteja N %282010%29 Polyamines and abiotic stress tolerance in plants%2E Plant Signaling %26 Behavior 5%3A 26%2D33&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signaling & Behavior 5: 26-33

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

http://scholar.google.com/scholar?as_q=Polyamines and abiotic stress tolerance in plants&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=Polyamines and abiotic stress tolerance in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gill&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Grotewold E %282005%29 Plant metabolic diversity%3A a regulatory perspective%2E Trends in Plant Science 10%3A 57%2D62&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Grotewold E (2005) Plant metabolic diversity: a regulatory perspective. Trends in Plant Science 10: 57-62

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

http://scholar.google.com/scholar?as_q=Plant metabolic diversity: a regulatory perspective&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=Plant metabolic diversity: a regulatory perspective&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Grotewold&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hartmann T%2C Ehmke A%2C Eilert U%2C von Borstel K%2C Theuring C %281989%29 Sites of synthesis%2C translocation and accumulation of pyrrolizidine alkaloid N%2Doxides in Senecio vulgaris L%2E Planta 177%3A 98%2D107&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hartmann T, Ehmke A, Eilert U, von Borstel K, Theuring C (1989) Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L. Planta 177: 98-107

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

http://scholar.google.com/scholar?as_q=Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L&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=Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hartmann&as_ylo=1989&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hartmann T%2C Witte L %281995%29 Chemistry%2C biology and chemoecology of the pyrrolizidine alkaloids%2E In SW Pelletier%2C ed%2C Alkaloids%3A Chemical and Biological Perspectives%2C Vol 9%2E Pergamon Press%2C Oxford%2C pp 155%2D233&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hartmann T, Witte L (1995) Chemistry, biology and chemoecology of the pyrrolizidine alkaloids. In SW Pelletier, ed, Alkaloids: Chemical and Biological Perspectives, Vol 9. Pergamon Press, Oxford, pp 155-233

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

http://scholar.google.com/scholar?as_q=&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=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hartmann&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Irmer S%2C Podzun N%2C Langel D%2C Heidemann F%2C Kaltenegger E%2C Schemmerling B%2C Geilfus C%2DM%2C Zorb C%2C Ober D %282015%29 New aspect of plant%2Drhizobia interaction%3A Alkaloid biosynthesis in Crotalaria depends on nodulation%2E Proceedings of the National Academy of Sciences of the United States of America 112%3A 4164%2D4169&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Irmer S, Podzun N, Langel D, Heidemann F, Kaltenegger E, Schemmerling B, Geilfus C-M, Zorb C, Ober D (2015) New aspect of plant-rhizobia interaction: Alkaloid biosynthesis in Crotalaria depends on nodulation. Proceedings of the National Academy of Sciences of the United States of America 112: 4164-4169

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

http://scholar.google.com/scholar?as_q=New aspect of plant-rhizobia interaction: Alkaloid biosynthesis in Crotalaria depends on nodulation&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=New aspect of plant-rhizobia interaction: Alkaloid biosynthesis in Crotalaria depends on nodulation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Irmer&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Irwin RE%2C Maloof JE %282002%29 Variation in nectar robbing over time%2C space%2C and species%2E Oecologia 133%3A 525%2D533&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Irwin RE, Maloof JE (2002) Variation in nectar robbing over time, space, and species. Oecologia 133: 525-533

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

http://scholar.google.com/scholar?as_q=Variation in nectar robbing over time, space, and species&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=Variation in nectar robbing over time, space, and species&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Irwin&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kaltenegger E%2C Eich E%2C Ober D %282013%29 Evolution of homospermidine synthase in the Convolvulaceae%3A a story of gene duplication%2C gene loss%2C and periods of various selection pressures%2E Plant Cell 25%3A 1213%2D1227&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kaltenegger E, Eich E, Ober D (2013) Evolution of homospermidine synthase in the Convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures. Plant Cell 25: 1213-1227

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

http://scholar.google.com/scholar?as_q=Evolution of homospermidine synthase in the Convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures&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=Evolution of homospermidine synthase in the Convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kaltenegger&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kanegae T%2C Kajiya H%2C Amano Y%2C Hashimoto T%2C Yamada Y %281994%29 Species%2Ddependent expression of the hyoscyamine 6%2Db%2Dhydroxylase gene in the pericycle%2E Plant Physiology 105%3A 483%2D490&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kanegae T, Kajiya H, Amano Y, Hashimoto T, Yamada Y (1994) Species-dependent expression of the hyoscyamine 6-b-hydroxylase gene in the pericycle. Plant Physiology 105: 483-490

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

http://scholar.google.com/scholar?as_q=Species-dependent expression of the hyoscyamine 6-b-hydroxylase gene in the pericycle&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=Species-dependent expression of the hyoscyamine 6-b-hydroxylase gene in the pericycle&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kanegae&as_ylo=1994&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kempf M%2C Beuerle T%2C Buhringer M%2C Denner M%2C Trost D%2C von der Ohe K%2C Bhavanam VB%2C Schreier P %282008%29 Pyrrolizidine alkaloids in honey%3A Risk analysis by gas chromatography%2Dmass spectrometry%2E Molecular Nutrition and Food Research 52%3A 1193%2D1200&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kempf M, Beuerle T, Buhringer M, Denner M, Trost D, von der Ohe K, Bhavanam VB, Schreier P (2008) Pyrrolizidine alkaloids in honey: Risk analysis by gas chromatography-mass spectrometry. Molecular Nutrition and Food Research 52: 1193-1200

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

http://scholar.google.com/scholar?as_q=Pyrrolizidine alkaloids in honey: Risk analysis by gas chromatography-mass spectrometry&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=Pyrrolizidine alkaloids in honey: Risk analysis by gas chromatography-mass spectrometry&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kempf&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kotelnikova KV%2C Tretjakova DJ%2C Nuraliev MS %282011%29 Shoot structure of Symphytum officinale L%2E %28Boraginaceae%29 in relation to the nature of its axially shiftes lateral branches%2E Wulfenia %2D Mitteilungen des K�rntner Botanikzentrums Klagenfurt 18%3A 63%2D79&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kotelnikova KV, Tretjakova DJ, Nuraliev MS (2011) Shoot structure of Symphytum officinale L. (Boraginaceae) in relation to the nature of its axially shiftes lateral branches. Wulfenia - Mitteilungen des K�rntner Botanikzentrums Klagenfurt 18: 63-79

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

http://scholar.google.com/scholar?as_q=Shoot structure of Symphytum officinale L&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=Shoot structure of Symphytum officinale L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kotelnikova&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Leegood RC %282008%29 Roles of the bundle sheath cells in leaves of C3 plants%2E Journal of Experimental Botany 59%3A 1663%2D1673&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Leegood RC (2008) Roles of the bundle sheath cells in leaves of C3 plants. Journal of Experimental Botany 59: 1663-1673

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

http://scholar.google.com/scholar?as_q=Roles of the bundle sheath cells in leaves of C3 plants&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=Roles of the bundle sheath cells in leaves of C3 plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Leegood&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1


Lindigkeit R, Biller A, Buch M, Schiebel HM, Boppré M, Hartmann T (1997) The two faces of pyrrolizidine alkaloids: The role of thetertiary amine and its N-oxide in chemical defense of insects with acquired plant alkaloids. European Journal of Biochemistry 245:626-636


Mattocks AR (1980) Toxic pyrrolizidine alkaloids in comfrey. Lancet (London, England) 2: 1136-1137Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

McKey D (1979) The distribution of secondary compounds within plants. In GA Rosenthal, DH Janzen, eds, Herbivores: TheirInteraction with Secondary Plant Metabolites. Academic Press, New York, pp 55-133


Moll S, Anke S, Kahmann U, Hänsch R, Hartmann T, Ober D (2002) Cell-specific expression of homospermidine synthase, the entryenzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. PlantPhysiology 130: 47-57


Morita M, Shitan N, Sawada K, Van Montagu MCE, Inze D, Rischer H, Goossens A, Oksman-Caldentey K-M, Moriyama Y, Yazaki K(2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotianatabacum. Proceedings of the National Academy of Sciences of the United States of America 106: 2447-2452


Muetterlein R, Arnold C-G (1993) Investigations concerning the content and the pattern of pyrrolizidine alkaloids in Symphytumofficinale L. (Comfrey). Pharmazeutische Zeitung 138: 119-125


Nakajima K, Oshita Y, Kaya M, Yamada Y, Hashimoto T (1999) Structures and expression patterns of two tropinone reductasegenes from Hyoscyamus niger. Bioscience, Biotechnology and Biochemistry 63: 1756-1764


Neumann D (1985) Storage of alkaloids. In K Mothes, HR Schütte, M Luckner, eds, Biochemistry of Alkaloids. VCH, Weilheim, pp 49-55


Niemüller D, Reimann A, Ober D (2012) Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidinealkaloid biosynthesis in three species of the Boraginales. Plant Physiology 159: 920-929


Nurhayati N, Gondé D, Ober D (2009) Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons:Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes. Phytochemistry 70: 508-516


Ober D, Hartmann T (1999) Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of anenzyme with extended substrate specificity. Journal of Biological Chemistry 274: 32040-32047


Ober D, Hartmann T (1999) Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis,evolved from deoxyhypusine synthase. Proceedings of the National Academy of Sciences of the United States of America 96:14777-14782


Pichersky E, Noel JP, Dudareva N (2006) Biosynthesis of plant volatiles: nature's diversity and ingenuity. Science (New York, N.Y.)311: 808-811

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http://www.ncbi.nlm.nih.gov/pubmed?term=Lindigkeit R%2C Biller A%2C Buch M%2C Schiebel HM%2C Boppr� M%2C Hartmann T %281997%29 The two faces of pyrrolizidine alkaloids%3A The role of the tertiary amine and its N%2Doxide in chemical defense of insects with acquired plant alkaloids%2E European Journal of Biochemistry 245%3A 626%2D636&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lindigkeit R, Biller A, Buch M, Schiebel HM, Boppr� M, Hartmann T (1997) The two faces of pyrrolizidine alkaloids: The role of the tertiary amine and its N-oxide in chemical defense of insects with acquired plant alkaloids. European Journal of Biochemistry 245: 626-636

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http://www.ncbi.nlm.nih.gov/pubmed?term=Mattocks AR %281980%29 Toxic pyrrolizidine alkaloids in comfrey%2E Lancet %28London%2C England%29 2%3A 1136%2D1137&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Mattocks AR (1980) Toxic pyrrolizidine alkaloids in comfrey. Lancet (London, England) 2: 1136-1137

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

http://scholar.google.com/scholar?as_q=Toxic pyrrolizidine alkaloids in comfrey&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

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http://www.ncbi.nlm.nih.gov/pubmed?term=McKey D %281979%29 The distribution of secondary compounds within plants%2E In GA Rosenthal%2C DH Janzen%2C eds%2C Herbivores%3A Their Interaction with Secondary Plant Metabolites%2E Academic Press%2C New York%2C pp 55%2D133&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=McKey D (1979) The distribution of secondary compounds within plants. In GA Rosenthal, DH Janzen, eds, Herbivores: Their Interaction with Secondary Plant Metabolites. Academic Press, New York, pp 55-133

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http://www.ncbi.nlm.nih.gov/pubmed?term=Moll S%2C Anke S%2C Kahmann U%2C H�nsch R%2C Hartmann T%2C Ober D %282002%29 Cell%2Dspecific expression of homospermidine synthase%2C the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis%2C in comparison with its ancestor%2C deoxyhypusine synthase%2E Plant Physiology 130%3A 47%2D57&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Moll S, Anke S, Kahmann U, H�nsch R, Hartmann T, Ober D (2002) Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. Plant Physiology 130: 47-57

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

http://scholar.google.com/scholar?as_q=Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase&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=Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Moll&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Morita M%2C Shitan N%2C Sawada K%2C Van Montagu MCE%2C Inze D%2C Rischer H%2C Goossens A%2C Oksman%2DCaldentey K%2DM%2C Moriyama Y%2C Yazaki K %282009%29 Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion %28MATE%29 transporter in Nicotiana tabacum%2E Proceedings of the National Academy of Sciences of the United States of America 106%3A 2447%2D2452&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Morita M, Shitan N, Sawada K, Van Montagu MCE, Inze D, Rischer H, Goossens A, Oksman-Caldentey K-M, Moriyama Y, Yazaki K (2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proceedings of the National Academy of Sciences of the United States of America 106: 2447-2452

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

http://scholar.google.com/scholar?as_q=Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum&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=Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Morita&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Muetterlein R, Arnold C-G (1993) Investigations concerning the content and the pattern of pyrrolizidine alkaloids in Symphytum officinale L. (Comfrey). Pharmazeutische Zeitung 138: 119-125

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

http://scholar.google.com/scholar?as_q=Investigations concerning the content and the pattern of pyrrolizidine alkaloids in Symphytum officinale L&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=Investigations concerning the content and the pattern of pyrrolizidine alkaloids in Symphytum officinale L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Muetterlein&as_ylo=1993&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Nakajima K%2C Oshita Y%2C Kaya M%2C Yamada Y%2C Hashimoto T %281999%29 Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger%2E Bioscience%2C Biotechnology and Biochemistry 63%3A 1756%2D1764&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Nakajima K, Oshita Y, Kaya M, Yamada Y, Hashimoto T (1999) Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger. Bioscience, Biotechnology and Biochemistry 63: 1756-1764

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

http://scholar.google.com/scholar?as_q=Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger&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=Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakajima&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Neumann D %281985%29 Storage of alkaloids%2E In K Mothes%2C HR Sch�tte%2C M Luckner%2C eds%2C Biochemistry of Alkaloids%2E VCH%2C Weilheim%2C pp 49%2D55&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Neumann D (1985) Storage of alkaloids. In K Mothes, HR Sch�tte, M Luckner, eds, Biochemistry of Alkaloids. VCH, Weilheim, pp 49-55

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

http://scholar.google.com/scholar?as_q=Storage of alkaloids.&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=Storage of alkaloids.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Neumann&as_ylo=1985&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Niem�ller D%2C Reimann A%2C Ober D %282012%29 Distinct cell%2Dspecific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the Boraginales%2E Plant Physiology 159%3A 920%2D929&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Niem�ller D, Reimann A, Ober D (2012) Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the Boraginales. Plant Physiology 159: 920-929

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

http://scholar.google.com/scholar?as_q=Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the Boraginales&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=Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the Boraginales&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Niem�ller&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Nurhayati N%2C Gond� D%2C Ober D %282009%29 Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons%3A Identification of deoxyhypusine synthase%2C homospermidine synthase and related pseudogenes%2E Phytochemistry 70%3A 508%2D516&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Nurhayati N, Gond� D, Ober D (2009) Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes. Phytochemistry 70: 508-516

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

http://scholar.google.com/scholar?as_q=Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes&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=Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nurhayati&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ober D%2C Hartmann T %281999%29 Deoxyhypusine synthase from tobacco%2E cDNA isolation%2C characterization%2C and bacterial expression of an enzyme with extended substrate specificity%2E Journal of Biological Chemistry 274%3A 32040%2D32047&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ober D, Hartmann T (1999) Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of an enzyme with extended substrate specificity. Journal of Biological Chemistry 274: 32040-32047

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

http://scholar.google.com/scholar?as_q=Deoxyhypusine synthase from tobacco&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=Deoxyhypusine synthase from tobacco&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ober&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ober D%2C Hartmann T %281999%29 Homospermidine synthase%2C the first pathway%2Dspecific enzyme of pyrrolizidine alkaloid biosynthesis%2C evolved from deoxyhypusine synthase%2E Proceedings of the National Academy of Sciences of the United States of America 96%3A 14777%2D14782&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ober D, Hartmann T (1999) Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proceedings of the National Academy of Sciences of the United States of America 96: 14777-14782

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

http://scholar.google.com/scholar?as_q=Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase&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=Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ober&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Pichersky E%2C Noel JP%2C Dudareva N %282006%29 Biosynthesis of plant volatiles%3A nature%27s diversity and ingenuity%2E Science %28New York%2C N%2EY%2E%29 311%3A 808%2D811&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pichersky E, Noel JP, Dudareva N (2006) Biosynthesis of plant volatiles: nature's diversity and ingenuity. Science (New York, N.Y.) 311: 808-811

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

http://scholar.google.com/scholar?as_q=Biosynthesis of plant volatiles: nature's diversity and ingenuity&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=Biosynthesis of plant volatiles: nature's diversity and ingenuity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pichersky&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1


Reimann A, Nurhayati N, Backenköhler A, Ober D (2004) Repeated evolution of the pyrrolizidine alkaloid-mediated defense systemin separate angiosperm lineages. Plant Cell 16: 2772-2784


Sander H, Hartmann T (1989) Site of synthesis, metabolism and translocation of senecionine N-oxide in cultured roots of Senecioerucifolius. Plant Cell, Tissue and Organ Culture 18: 19-32


Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3: 1101-1108Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature methods 9: 671-675Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sievert C, Beuerle T, Hollmann J, Ober D (2015) Single cell subtractive transcriptomics for identification of cell-specificallyexpressed candidate genes of pyrrolizidine alkaloid biosynthesis. Phytochemistry 117: 17-24


St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesispredicts intercellular translocation of a pathway intermediate. Plant Cell 11: 887-900


Steudle E, Peterson CA (1998) How does water get through roots? Journal of Experimental Botany 49: 775-788Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Suzuki KI, Yamada Y, Hashimoto T (1999) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle.Plant and Cell Physiology 40: 289-297


Suzuki KI, Yun DJ, Chen XY, Yamada Y, Hashimoto T (1999) An Atropa belladonna hyoscyamine 6b-hydroxylase gene isdifferentially expressed in the root pericycle and anthers. Plant Molecular Biology 40: 141-152


Tiburcio AF, Altabella T, Bitrian M, Alcazar R (2014) The roles of polyamines during the lifespan of plants: from development tostress. Planta 240: 1-18


Utelli AB, Roy BA (2001) Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations inSwitzerland. Oecologia 127: 266-273


Weid M, Ziegler J, Kutchan TM (2004) The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy,Papaver somniferum. Proceedings of the National Academy of Sciences of the United States of America 101: 13957-13962


Weng J-K, Philippe RN, Noel JP (2012) The rise of chemodiversity in plants. Science 336: 1667-1670Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. Annual Review of Plant Biology 59: 735-769Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title


http://www.ncbi.nlm.nih.gov/pubmed?term=Reimann A%2C Nurhayati N%2C Backenk�hler A%2C Ober D %282004%29 Repeated evolution of the pyrrolizidine alkaloid%2Dmediated defense system in separate angiosperm lineages%2E Plant Cell 16%3A 2772%2D2784&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Reimann A, Nurhayati N, Backenk�hler A, Ober D (2004) Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16: 2772-2784

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

http://scholar.google.com/scholar?as_q=Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages&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=Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Reimann&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sander H%2C Hartmann T %281989%29 Site of synthesis%2C metabolism and translocation of senecionine N%2Doxide in cultured roots of Senecio erucifolius%2E Plant Cell%2C Tissue and Organ Culture 18%3A 19%2D32&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sander H, Hartmann T (1989) Site of synthesis, metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius. Plant Cell, Tissue and Organ Culture 18: 19-32

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

http://scholar.google.com/scholar?as_q=Site of synthesis, metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius&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=Site of synthesis, metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sander&as_ylo=1989&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schmittgen TD%2C Livak KJ %282008%29 Analyzing real%2Dtime PCR data by the comparative C%28T%29 method%2E Nature Protocols 3%3A 1101%2D1108&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3: 1101-1108

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

http://scholar.google.com/scholar?as_q=Analyzing real-time PCR data by the comparative C(T) method&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=Analyzing real-time PCR data by the comparative C(T) method&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schmittgen&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schneider CA%2C Rasband WS%2C Eliceiri KW %282012%29 NIH Image to ImageJ%3A 25 years of image analysis%2E Nature methods 9%3A 671%2D675&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature methods 9: 671-675

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

http://scholar.google.com/scholar?as_q=NIH Image to ImageJ: 25 years of image analysis&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=NIH Image to ImageJ: 25 years of image analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schneider&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sievert C%2C Beuerle T%2C Hollmann J%2C Ober D %282015%29 Single cell subtractive transcriptomics for identification of cell%2Dspecifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis%2E Phytochemistry 117%3A 17%2D24&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sievert C, Beuerle T, Hollmann J, Ober D (2015) Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis. Phytochemistry 117: 17-24

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

http://scholar.google.com/scholar?as_q=Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis&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=Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sievert&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=St%2DPierre B%2C Vazquez%2DFlota FA%2C De Luca V %281999%29 Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate%2E Plant Cell 11%3A 887%2D900&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11: 887-900

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

http://scholar.google.com/scholar?as_q=Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate&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=Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=St-Pierre&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Steudle E%2C Peterson CA %281998%29 How does water get through roots%3F Journal of Experimental Botany 49%3A 775%2D788&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Steudle E, Peterson CA (1998) How does water get through roots? Journal of Experimental Botany 49: 775-788

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

http://scholar.google.com/scholar?as_q=How does water get through roots&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

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http://www.ncbi.nlm.nih.gov/pubmed?term=Suzuki KI%2C Yamada Y%2C Hashimoto T %281999%29 Expression of Atropa belladonna putrescine N%2Dmethyltransferase gene in root pericycle%2E Plant and Cell Physiology 40%3A 289%2D297&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Suzuki KI, Yamada Y, Hashimoto T (1999) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant and Cell Physiology 40: 289-297

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

http://scholar.google.com/scholar?as_q=Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle&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=Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Suzuki&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Suzuki KI%2C Yun DJ%2C Chen XY%2C Yamada Y%2C Hashimoto T %281999%29 An Atropa belladonna hyoscyamine 6b%2Dhydroxylase gene is differentially expressed in the root pericycle and anthers%2E Plant Molecular Biology 40%3A 141%2D152&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Suzuki KI, Yun DJ, Chen XY, Yamada Y, Hashimoto T (1999) An Atropa belladonna hyoscyamine 6b-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plant Molecular Biology 40: 141-152

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

http://scholar.google.com/scholar?as_q=An Atropa belladonna hyoscyamine 6b-hydroxylase gene is differentially expressed in the root pericycle and anthers&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=An Atropa belladonna hyoscyamine 6b-hydroxylase gene is differentially expressed in the root pericycle and anthers&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Suzuki&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Tiburcio AF%2C Altabella T%2C Bitrian M%2C Alcazar R %282014%29 The roles of polyamines during the lifespan of plants%3A from development to stress%2E Planta 240%3A 1%2D18&dopt=abstract

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

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http://scholar.google.com/scholar?as_q=The roles of polyamines during the lifespan of plants: from development to stress&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tiburcio&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Utelli AB%2C Roy BA %282001%29 Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations in Switzerland%2E Oecologia 127%3A 266%2D273&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Utelli AB, Roy BA (2001) Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations in Switzerland. Oecologia 127: 266-273

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

http://scholar.google.com/scholar?as_q=Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations in Switzerland&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=Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations in Switzerland&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Utelli&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Weid M%2C Ziegler J%2C Kutchan TM %282004%29 The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy%2C Papaver somniferum%2E Proceedings of the National Academy of Sciences of the United States of America 101%3A 13957%2D13962&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Weid M, Ziegler J, Kutchan TM (2004) The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum. Proceedings of the National Academy of Sciences of the United States of America 101: 13957-13962

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

http://scholar.google.com/scholar?as_q=The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum&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=The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Weid&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Weng J%2DK%2C Philippe RN%2C Noel JP %282012%29 The rise of chemodiversity in plants%2E Science 336%3A 1667%2D1670&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Weng J-K, Philippe RN, Noel JP (2012) The rise of chemodiversity in plants. Science 336: 1667-1670

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

http://scholar.google.com/scholar?as_q=The rise of chemodiversity in plants&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=The rise of chemodiversity in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Weng&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ziegler J%2C Facchini PJ %282008%29 Alkaloid biosynthesis%3A metabolism and trafficking%2E Annual Review of Plant Biology 59%3A 735%2D769&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. Annual Review of Plant Biology 59: 735-769

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

http://scholar.google.com/scholar?as_q=Alkaloid biosynthesis: metabolism and trafficking&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=Alkaloid biosynthesis: metabolism and trafficking&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ziegler&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1


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