1 Long distance transport of thiamine and polyamines 2 ... · 91 derivatives (commonly referred to...

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Running head: Long distance transport of thiamine and polyamines 1

Address correspondence to: Teresa B. Fitzpatrick 2

Department of Botany and Plant Biology, 3

University of Geneva, 4

1211 Geneva, 5

Switzerland 6

Telephone: +41-22-3793016 7

Email: [email protected] 8

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Research area: Membranes, Transport and Biogenetics (Secondary area: Biochemistry and 10

Metabolism) 11

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Plant Physiology Preview. Published on March 22, 2016, as DOI:10.1104/pp.16.00009

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Long distance transport of thiamine (vitamin B1) is concomitant with that of 15

polyamines in Arabidopsis 16

17

Jacopo Martinis1†, Elisabet Gas-Pascual1†, Nicolas Szydlowski1†, Michèle Crèvecoeur1, Alexandra 18

Gisler1, Lukas Bürkle2 and Teresa B. Fitzpatrick1* 19 20

21 1Department of Botany and Plant Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 22

Geneva, Switzerland. 23

2The Institute of Agricultural Sciences, ETH Zurich, Universitätstrasse 2, 8092 Zurich, Switzerland. 24

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†These authors contributed equally. 27 28

One sentence summary: Long distance transport of thiamine is facilitated by PUT3 concomitant 29

with that of polyamines and is required for growth and development. 30

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1Financial support is gratefully acknowledged from the Swiss National Science Foundation (Grant 32

PPOOA_1191186 and 31003A-141117/1 to T.B.F.) as well as the University of Geneva. 33

34 2Present address: Department of Biochemistry and Molecular Biology, The University of Georgia, 35

Athens, GA 30602, U.S.A. (E.G.P.); Miniaturisation pour la Synthèse l'Analyse et la Protéomique, 36

Université Lille1 Sciences et Technologies, 59655 Villeneuve d'Ascq Cedex, France (N.S.); Stratec 37

Biomedical AG, 75217 Birkenfeld, Germany (L.B.). 38

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* Address correspondence to [email protected]



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ABSTRACT 41

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital 42

metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is 43

taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, 44

transport mechanisms are required to mediate the movement of this polar metabolite from source to 45

sink tissue to activate key enzymes in cellular energy generating pathways but are currently 46

unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for 47

diverse aspects of growth and development, the homeostasis of which necessitate long distance 48

transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify 49

Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, 50

physiological and genetic approaches permitted us to show that PUT3 mediates phloem transport of 51

both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution 52

of these metabolites is altered with growth and developmental consequences. The pivotal role of 53

PUT3 mediated thiamine and polyamine homeostasis in plants and its importance for plant fitness is 54

revealed through these findings. 55

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5

INTRODUCTION 57

Thiamine (vitamin B1) is essential for all organisms being well recognized in its diphosphorylated 58

form, thiamine diphosphate (TDP) (Fig. 1A), as a necessary cofactor for key metabolic enzymes 59

involved in glycolysis and the citric acid cycle (Fitzpatrick & Thore, 2014). In plants, it is also 60

necessary for the Calvin cycle, the biochemical route of carbon fixation (Khozaei et al., 2015). 61

Therefore, thiamine is vital for cell energy supply in all organisms. Interestingly, some additional 62

roles for thiamine have been proposed that include involvement in responses to DNA damage 63

(Machado et al., 1996), as well as the activation of plant defenses, resistance to pathogen attack and 64

attenuation of environmental stress responses (Ahn et al., 2005, Rapala-Kozik et al., 2012). Humans 65

cannot produce thiamine de novo with plants representing one of the most important sources of the 66

compound in the human diet. However, deficiency remains a critical global problem causing 67

detrimental neurological effects and cardiovascular problems particularly among populations that 68

rely on a single crop for sustenance, e.g. polished rice (Fitzpatrick et al., 2012). Thiamine is formed 69

by the condensation of the two separately biosynthesized heterocycle moieties 70

(hydroxyethylthiazole (HET) and hydroxymethylpyrimidine (HMP), respectively) (Supplemental 71

Fig. S1) and is later diphosphorylated to the cofactor form, TDP (reviewed in Fitzpatrick & Thore, 72

2014). Several previously elusive steps of thiamine biosynthesis de novo have been resolved 73

recently, one of which involves an unexpected suicide mechanism, where the catalytic protein 74

donates sulfur to the molecule in a single turnover reaction (Chatterjee et al., 2011) (Supplemental 75

Fig. S1). Remarkably, the majority of the enzymes involved in biosynthesis de novo are exclusively 76

found in the chloroplast, the exception being the kinase that diphosphorylates thiamine to TDP, 77

which is in the cytosol (Ajjawi et al., 2007a). Accordingly, the thiamine biosynthetic genes are 78

strongly expressed in green tissues but only at a very low level (if at all) in other non-79

photosynthetic tissues such as roots (Colinas & Fitzpatrick, 2015). However, thiamine is essential in 80

all actively dividing meristematic stem cells and organ initial cells (Wightman & Brown, 1953) and 81

thus in non-photosynthetic organs such as the roots. It is therefore relevant that a physiological 82

study in 1974 (Kruszewski & Jacobs, 1974) showed that thiamine moves through petiolar sections 83

of tomato with strong basipetal polarity and with kinetics similar to auxin and giberellin (at a 84

velocity of 3-5 mm per hour). Indeed, in the 1940s, thiamine was considered to fulfil the 85

requirements of a hormone in higher plants (Bonner, 1940, Bonner, 1942). This was due to the 86

observation that in order to grow aseptically, excised roots of many species require thiamine in 87

small amounts. Moreover, such observations were considered as confirmation of an hypothesis that 88

in the intact plant, thiamine is biosynthesized in the leaves and moves from there to the roots, which 89

cannot biosynthesize it sufficiently but need it for development. As thiamine and its phosphorylated 90

derivatives (commonly referred to as vitamin B1) are cationic molecules (i.e. charged) at 91



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physiological pH, the existence of transporters that can mediate their trafficking therefore become 92

apparent. Although the characterization of a homolog of the yeast mitochondrial transporter was 93

reported recently (Frelin et al., 2012), it remains uncharacterized in planta, while the proteins 94

participating in long distance transport of thiamine in plants have remained elusive. 95

Analogous to thiamine, polyamines are cationic molecules at physiological pH essential for the 96

growth and survival of all organisms. The diverse range of activities include regulation of cell 97

division, developmental processes such as those of root formation and flowering initiation, as well 98

as environmental stress responses (Alcázar et al., 2010, Kumar et al., 1997, Kusano et al., 2008). 99

Key representative molecules include putrescine (Put), spermidine (Spd) and spermine (Spm), all 100

three of which are derived from the amino acid arginine, via the intermediate agmatine (Agm), as 101

well as cadaverine (Cad) that is derived from lysine (Supplemental Fig. S1). Akin to other essential 102

multifunctional plant metabolites, polyamine contents must be carefully controlled to maintain 103

cellular homeostasis. This is achieved through biosynthesis, degradation, conjugation (to phenolic 104

acids, proteins, nucleic acids, membrane structures) and transport (Martin-Tanguy, 2001). Although 105

polyamine metabolism has been studied extensively, little is known of its transport in plants with 106

the exception of the recently identified PUT family (Fujita et al., 2012, Mulangi et al., 2012) and 107

the OCT1 transporter from Arabidopsis (Arabidopsis thaliana) that is implicated in Cad transport 108

(Strohm et al., 2015). The PUT family of proteins are localized at the cellular level to various 109

compartments including the plasma membrane, Golgi apparatus and endoplasmic reticulum (Fujita 110

et al., 2012, Li et al., 2013). However, polyamine homeostasis is also regulated by long distance 111

transport and indeed their presence in the vascular system of plants has been established in several 112

species (Friedman et al., 1986, Pommerrenig et al., 2011, Sood & Nagar, 2005, Yokota et al., 113

1994). To date nothing is known on the components of intercellular or long distance transport of 114

polyamines. 115

Here, a yeast-based screen allowed us to identify Arabidopsis PUT3 as a thiamine transporter. 116

Expression studies showed that PUT3 is localized to the phloem tissue in Arabidopsis and loss of 117

function studies showed that PUT3 is required for growth and development. A series of 118

micrografting and biochemical experiments demonstrated that PUT3 is important for long distance 119

transport of both thiamine and polyamines and thereby revealed its importance for homeostasis of 120

these essential molecules and maintenance of plant fitness. 121

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RESULTS 123

Identification of an Arabidopsis thiamine transporter 124

This study was initiated by performing a complementation screen employing a cDNA library from 125

Arabidopsis (Minet et al., 1992) with the yeast mutant CVY4, deficient in both the biosynthesis and 126

the transport of thiamine (Vogl et al., 2008). This strain grows either in the presence of strong 127

thiamine supplementation (120 µM) or can be complemented with the yeast thiamine biosynthesis 128

gene THI4 (Vogl et al., 2008). In line with this, several clones harboring the ortholog of the latter 129

from Arabidopsis (THI1) were found to complement the growth phenotype even in the absence of 130

thiamine supplementation in our screen. On the other hand, a single Arabidopsis locus (At5g05630) 131

encoding a protein belonging to the amino acid permease family was found to restore growth in the 132

presence of low levels of thiamine (1.2 µM), thus complementing the impairment in thiamine 133

transport in this yeast strain (Fig. 1A). This protein has been previously reported to facilitate the 134

uptake of polyamines in a yeast based study and was accordingly named POLYAMINE UPTAKE 135

TRANSPORTER 3 (PUT3) (Mulangi et al., 2012) and mediated paraquat uptake in Arabidopsis for 136

which it was given the name RESISTANT TO METHYL VIOLOGEN 1 (RMV1)(Fujita et al., 137

2012). The PUT3/RMV1 locus, which we will refer to as PUT3 from here on, encodes a protein of 138

490 amino acid residues and is predicted to have 12 transmembrane domains with the N- and C-139

terminal hydrophilic domains residing on the inside of the membrane (Fig. 1B). It has 4 paralogs in 140

Arabidopsis, At1g31820 (68% identity), At1g31830 (63% identity), At3g19553 (52% identity) and 141

At3g13620 (43% identity), respectively. None of the latter paralogs were found in our screen and 142

presumably cannot transport thiamine (at least in yeast) under the conditions used. Additional close 143

homologs of PUT3 were identified in several plants including Vitis vinifera (grapevine), Populus 144

trichocarpa (poplar), Oryza sativa (rice), Zea mays (corn), Sorghum bicolor (Sorghum), as well as 145

Physcomitrella patens (moss), while weaker homologs could be identified in Chlorella variabilis 146

(green algae). Notably, an extensive recent phylogenetic study hypothesized that this family of 147

transporters was acquired by horizontal transfer from green algae (Mulangi et al., 2012). 148

149

Kinetics of PUT3 facilitated thiamine transport 150

The kinetics of thiamine transport by PUT3 in yeast was characterized using the tritiated vitamers, 151

[3H]-thiamine and [3H]-TDP. Note, vitamers are related chemical substances that fulfil the same 152

specific vitamin function. Uptake occurred at an optimal pH of 5.5 (Supplemental Fig. S2A). Both 153

vitamers could be transported at similar rates (Fig. 1C). In addition, the vitamers displayed similar 154

Michaelis-Menten parameters (Vmax:169.3 ± 12.3 pmol min-1.OD cells-1, KM:43.3 ± 7.7 µM for 155

thiamine and Vmax:146.5 ± 13.7 pmol min-1.OD cells-1, KM of 34.2 ± 8.6 µM for TDP, respectively 156



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(Fig. 1D and 1E)). Competition experiments showed that B1 vitamers, TDP, thiamine 157

monophosphate (TMP) and thiamine were able to compete for TDP transport, whereas HET or 158

HMP (the thiamine biosynthesis precursors) were not (Fig. 1F). Interestingly, the thiamine 159

antagonist, pyrithiamine, also competed with TDP uptake (Fig. 1F). As anticipated, the polyamine 160

Spd, shown previously to be transported by PUT3 in a yeast based assay (Mulangi et al., 2012), 161

competed strongly with TDP transport (Fig. 1G), indicating that thiamine uses the same transport 162

pathway as polyamines. The withdrawal of glucose or addition of the protonophore, 163

carbonylcyanide-3-chlorophenylhydrazone, abolishes the transport activity, suggesting that 164

transport occurs via H+ symport (Fig. 1H). Since PUT3 was originally annotated as a putative 165

amino acid transporter (Jack et al., 2000), we also tested for competition of TDP uptake in the 166

presence of unlabeled cyclic amino-acids, but none showed significant modulation of transport 167

(Supplemental Fig. S2B). In the same context and as the human thiamine transporter belongs to the 168

folate transporter family (Zhao & Goldman, 2013), we also tested folate, but it did not compete for 169

TDP transport (Supplemental Fig. S2B). Similar results were obtained when the competition studies 170

were performed with thiamine instead of TDP. It can therefore be concluded that PUT3 is a 171

polyspecific transporter than mediates thiamine as well as polyamine transport, at least in yeast. 172

173

PUT3 is localized to the phloem and mediates transport of B1 vitamers 174

Given the demonstrated ability of PUT3 to import thiamine/TDP in yeast, we wanted to establish its 175

localization in planta, to complement the previous cellular localization studies of this protein (Fujita 176

et al., 2012). To this end, we generated plants expressing the β-GLUCURONIDASE (GUS) 177

reporter gene under control of the 850 base-pair region immediately upstream of the transcriptional 178

start site of PUT3 (pPUT3-GUS). Representative pictures of 20 independent transgenic lines 179

analyzed are shown in Fig. 2A. In all lines examined, PUT3 promoter driven GUS activity was 180

predominant in the vascular tissue of leaves and the central cylinder of roots. In particular, two 181

strands of GUS activity could be observed in the central stele of the differentiation region of the 182

root. Moreover, transverse sections of the root tissue established localization to the phloem tissue 183

(Fig. 2B). Weak GUS activity was also observed in the anthers of flowers, as well as the base and 184

tip of mature siliques (Supplemental Fig. S3). At the subcellular level, fusion of the full-length 185

PUT3 protein sequence to YFP and examination of the corresponding fluorescence upon transient 186

expression in Arabidopsis mesophyll protoplasts confirmed the exclusive location of this protein to 187

the plasma membrane (Supplemental Fig. S4) in agreement with a previous report (Fujita et al., 188

2012). 189

Since B1 vitamers need to be translocated from sites of biosynthesis (source) to sites where they 190

cannot be biosynthesized but are required (sink), we were prompted to investigate if they are found 191



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in the phloem sap. Indeed, the three B1 vitamers could be detected in the phloem sap of Arabidopsis 192

(Fig. 3A) and thereby implies that a transport system is in place for translocation into this tissue. It 193

is worth noting that the ratio between TDP and either TMP or thiamine in the phloem sap is 194

drastically reduced when compared with extracts from whole rosette leaves (Supplemental Fig. S5). 195

In order to test the contribution of PUT3 to the vitamin B1 transport process, the B1 vitamer profiles 196

of phloem exudates from wild-type plants were compared with that of the previously characterized 197

T-DNA insertion mutant line rmv1 (GT_3_3436) (Fujita et al., 2012). Downregulation of PUT3 198

expression was confirmed by real-time quantitative PCR (qPCR) (Supplemental Fig. S6). Indeed, 199

the total vitamin B1 content was decreased in the rmv1 mutant phloem exudates compared to that of 200

the wild-type (Fig. 3B). Importantly, B1 vitamer contents were restored to wild-type levels in 201

phloem exudates of rmv1 carrying the PUT3 transgene under control of its own promoter (Fig. 3B, 202

rmv1/pPUT3:PUT3). These observations suggest that PUT3 is involved in transport of vitamin B1 203

in the phloem. 204

205

PUT3 also mediates phloem transport of polyamines 206

The localization of PUT3 to the phloem tissue and its previous reported ability to facilitate 207

polyamine transport, at least in yeast (Mulangi et al., 2012), prompted us to next investigate the 208

polyamine profiles of phloem exudates from Arabidopsis. In particular, we adapted a protocol 209

(Fontaniella et al., 2001) to profile the diamines Cad and Put, the triamine Spd, the tetramine Spm, 210

as well as the Put/Spd/Spm precursor Agm. All of these compounds could be detected in phloem 211

exudates from Arabidopsis (Fig. 4A). Put was the most abundant polyamine detected followed by 212

Spd and Cad, while Spm accumulated at much lower levels (Fig. 4B). Significantly, there was a 213

considerable decrease in the levels of Put, in particular, in phloem exudates of rmv1 (Fig. 4B). 214

Importantly, the polyamine profile was restored to that of wild-type in phloem exudates of rmv1 215

carrying the PUT3 transgene under control of its own promoter (Fig. 4B, rmv1/pPUT3:PUT3), 216

validating the role of PUT3 in this molecular phenotype. Taken together, this implies that PUT3 is 217

required for transport of both polyamines and vitamin B1 in phloem tissue. 218

219

Alterations in growth and developmental processes are associated with PUT3 220

As B1 vitamers and polyamines are essential for development in plants, we next examined the 221

phenotypic consequences of loss of function of PUT3 on plant growth. Notably, previous studies on 222

PUT3 in Arabidopsis were restricted to quantifying its association with paraquat resistance (Fujita 223

et al., 2012). Interestingly, here we observed that the rmv1 mutant showed stunted growth under 224

standard growth conditions that was not reported on in previous studies (Fig. 5A), and was 225



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corroborated by a slower rate of root growth compared to wild-type (Fig. 5B). Furthermore, rmv1 226

entered the reproductive phase later than wild-type (Fig. 5C), bolting at day 67 instead of day 62 227

(Fig. 5D), concomitant with an increase in the number of rosette leaves at this developmental stage 228

(Fig. 5E and 5F). The observed phenotypes were most pronounced under an 8-hour photoperiod and 229

were significantly attenuated under longer photoperiods (data not shown). All of the growth and 230

developmental phenotypes were abrogated in rmv1 upon introduction of the PUT3 transgene (Fig. 231

5A-5F, rmv1/pPUT3:PUT3). It must therefore be concluded that PUT3 is required for proper 232

growth and development in Arabidopsis. 233

234

Long distance transport of a shoot-derived metabolite by PUT3 is responsible for the root 235

development phenotype in rmv1 236

To further probe the growth and development phenotype of rmv1 and the possible physiological 237

relevance of PUT3 in long distance transport, we performed a series of grafting experiments. 238

Notably, grafted plants with the same genetic combinations exhibited the same phenotypes as non-239

grafted plants, i.e. grafting of an rmv1 scion onto its corresponding rootstock (rmv1/rmv1) led to 240

retention of the stunted growth phenotype compared to grafting of the wild-type scion onto its 241

corresponding rootstock (wild-type/wild-type) (Fig. 6). Remarkably, grafting of the wild-type scion 242

onto the rmv1 rootstock (wild-type/rmv1) led to recovery of the stunted root growth phenotype to 243

that of the corresponding grafted wild-type plants (wild-type/wild-type) (Fig. 6). On the other hand, 244

the reciprocal graft of the rmv1 scion onto the wild-type rootstock (rmv1/wild-type) did not lead to 245

recovery of root growth impairment (Fig. 6). Indeed, root growth was even further impaired than 246

rmv1/rmv1. This data supports the notion that PUT3 is required for long distance transport of a 247

shoot-derived metabolite that is essential for proper root growth. 248

249

Shoot to root partitioning of vitamin B1 as a function of PUT3 250

Given the implication of PUT3 in long distance transport, we next sought to obtain a molecular 251

explanation for the growth impairment phenotypes observed. In the first instance, we therefore 252

examined shoot and root partitioning of B1 vitamers in seedlings. Intriguingly, B1 vitamer levels in 253

shoots and roots (albeit to a much lesser extent) of rmv1 were lower than those of wild-type (Fig. 254

7A). While the slight depletion in root tissue (sink tissue) is consistent with the role of PUT3 in 255

long distance transport of B1 vitamers from source to sink, we expected a corresponding 256

accumulation of B1 vitamers in shoots (source tissue) rather than a depletion. Interestingly, rmv1 257

does not show the well-characterized pale shoot phenotype associated with a deficiency in B1 258

vitamers (Ajjawi et al., 2007a, Ajjawi et al., 2007b, Raschke et al., 2007). Nonetheless, levels of B1 259



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vitamers in shoots and roots of rmv1 recovered upon reintroduction of the PUT3 transgene (Fig. 260

7A). To provide evidence that shoot-derived vitamin B1 is important for root growth in particular, 261

another series of grafting experiments was performed with the Arabidopsis mutant line th1-201 262

(Ajjawi et al., 2007b). In this line, the THIAMIN SYNTHASE enzyme TH1 (encoded at locus 263

At1g22940), which condenses the thiazole and pyrimidine heterocycles to form TMP, is impaired, 264

preventing thiamine biosynthesis de novo and resulting in overall stunted shoot and root growth and 265

pale leaves. Remarkably, grafting of the wild-type scion onto the th1-201 rootstock (wild-type/th1-266

201) led to recovery of the stunted root growth phenotype that is observed with th1-201 (Fig. 7B). 267

However, grafting of the rmv1 scion onto the th1-201 rootstock (rmv1/th1-201) did not lead to 268

recovery of the short-root phenotype in th1-201. Indeed, root growth was even more impaired than 269

for the th1-201/th1-201 genetic combination, corroborating the notion that loss of function of PUT3 270

in the shoot, in particular, has a negative impact on root growth (Fig. 7B). Taken together, the data 271

demonstrate that long distance transport of B1 vitamers from the shoot is necessary for root growth 272

and that impairment of PUT3 in the shoot has a negative impact on root growth. 273

274

Shoot to root partitioning of polyamines as a function of PUT3 275

Given that PUT3 is also implicated in polyamine transport, we next looked at shoot to root 276

partitioning of polyamine contents. In the first instance, we noted that the total polyamine content is 277

very similar between shoots and roots in wild-type plants (Fig. 8A). A significant reduction of total 278

polyamine levels was observed in the roots of rmv1, whereas shoot content remained similar to that 279

of wild-type (Fig. 8A). Notably, levels of root polyamines recovered upon reintroduction of the 280

PUT3 transgene in rmv1 (Fig. 8A). Intriguingly, a closer look at the polyamines measured indicates 281

that the predominant polyamine in shoots and roots is Spd and that it is mainly this polyamine that 282

is depleted in roots of rmv1 (Fig. 8B-8E). This is in contrast to the polyamine profile of phloem 283

exudates, where Put was the predominant polyamine detected and the one most depleted in rmv1 284

(Fig. 4B). 285

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DISCUSSION 287

A fundamental understanding of phloem transport with its important role in plant development, 288

reproduction, signaling and growth is central to plant biology. This is further emphasized by the 289

core set of compounds, such as biological cofactors and growth regulators, required in all living 290

cells to drive key metabolic processes, the biosynthesis of which may be restricted to certain organs 291

and therefore must be transported to the site of requirement. As central components of metabolic 292

function, the supply of many of these molecules must be strictly regulated, such that provision is 293

coordinated with metabolic needs. However, despite their critical roles, surprisingly little is known 294

on the intercellular transport of such molecules in plants. In this study, we demonstrated the 295

importance of long distance transport of vitamin B1 and polyamines as mediated by the phloem 296

localized transporter PUT3. Disruption of PUT3 led to a deficit of B1 vitamers and polyamines in 297

phloem exudates that perturbed the homeostasis of these compounds in the organs where they are 298

required resulting in a negative impact on growth and development in Arabidopsis. 299

Long distance transporters of the vital cofactor vitamin B1 have been supposed for almost a 300

century (Bonner, 1940, Bonner, 1942), yet the proteins carrying out these processes and evidence 301

for the presence of this family of compounds in the vascular system have remained elusive. Only a 302

single transporter has been reported for vitamin B1 in plants and operates at the subcellular level in 303

the mitochondrial membrane (Frelin et al., 2012). Polyamines, on the other hand, were detected in 304

the phloem sap several decades ago (Friedman et al., 1986). While they can be biosynthesized to a 305

certain extent in the phloem (Pommerrenig et al., 2011), the factors facilitating transport of these 306

important cationic molecules in the vascular system to maintain homeostasis were unknown. Here 307

we have demonstrated the involvement of PUT3 in these transport processes in plants. Although 308

PUT3 was previously implicated in subcellular polyamine transport based on a yeast 309

complementation assay (Mulangi et al., 2012), as well as paraquat transport in Arabidopsis (Fujita 310

et al., 2012), it went unnoted that it is localized to the phloem and mediates long distance transport. 311

Here we have shown that the affinity of PUT3 for B1 vitamers is similar to those reported for 312

polyamines and paraquat, i.e. low μM range (Fujita et al., 2012), classifying it as a polyspecific 313

high affinity cationic transporter. Indeed, exogenous thiamine reportedly enhances tolerance to 314

paraquat (Tunc-Ozdemir et al., 2009), supporting the notion that all of these compounds use the 315

same trafficking pathway. The functional significance of PUT3 for phloem transport of B1 vitamers 316

and polyamines became evident from the deficit of these compounds in the phloem exudates of the 317

PUT3 mutant line rmv1, previously characterized because of its resistance to paraquat (Fujita et al., 318

2012). The implication of PUT3 in these transport processes is validated upon restoration of the B1 319

vitamer and polyamine contents in phloem exudates of the transgenic rmv1 line expressing PUT3 320

under the control of its own promoter sequence (rmv1/pPUT3:PUT3). We acknowledge that the 321



13

EDTA-based method employed here for the collection of phloem sap has well-documented pitfalls 322

(Turgeon and Wolf, 2009), including that the procedure requires the cutting of a surface and 323

extended exposure of cells to EDTA causes damage, leakage from which may contaminate the sap. 324

Therefore, we chose the EDTA-facilitated phloem exudation method described by Tetyuk et al. that 325

has been optimized for Arabidopsis (Tetyuk et al., 2013), in which the samples are exposed to 326

EDTA for 1 hour, washed with water, followed by collection of the phloem exudate into water. 327

This strives to minimize cell damage and downstream interference from EDTA, while removing 328

compounds that have accumulated from damaged or cut cells. Indeed, assuming no metabolite 329

degradation occurs during the procedure, the divergent B1 vitamer and polyamine profiles of 330

phloem exudates versus tissue extracts could serve to support the predominant recovery of 331

metabolites being transported in the phloem with this method rather than that from damaged cells. 332

Moreover, the collection of material directly into water without prior EDTA-treatment was free of 333

B1 vitamers and polyamines and used as a negative control. Nonetheless, the use of another method 334

designed to exclusively measure mobile elements in the phloem sap could serve to validate the 335

findings. 336

As vitamin B1 biosynthesis de novo is predominantly limited to photosynthesizing tissue 337

(reviewed in Colinas & Fitzpatrick, 2015), it can be suggested that PUT3 activity in its capacity as 338

long distance B1 vitamer transporter is mainly restricted to shoot tissue. On the one hand, the 339

requirement for shoot to root long distance transport of vitamin B1 is supported by the recovery of 340

root growth in the biosynthesis mutant th1-201 upon grafting of a wild-type scion. On the other 341

hand, the failure to restore root growth upon grafting a PUT3 mutant scion (rmv1) onto th1-201 342

rootstock demonstrates the important role of PUT3 in this process. In line with this, we predict that 343

PUT3 is involved in phloem loading of B1 vitamers and is supported firstly by the decrease in B1 344

vitamer contents in phloem exudates and secondly by the decrease (albeit slight) in the root tissue 345

of rmv1. We noted above that the ratio between TDP and either TMP or thiamine in the phloem sap 346

is drastically reduced when compared with extracts from whole rosette leaves. Therefore, either a 347

selective transport system takes place in the leaves for phloem loading, and/or vitamers can be 348

interconverted within the phloem tissues by phosphatases that may be non-specific. The deficit in 349

shoot tissue B1 vitamer contents of rmv1 cannot be explained at present and requires further 350

investigation. Nonetheless, recovery towards wild-type shoot to root partitioning of B1 vitamers is 351

observed in the transgenic rmv1/pPUT3:PUT3 line implicating PUT3 in the process. The retention 352

of considerable levels of vitamin B1 in the roots even in the absence of PUT3 suggests that more 353

components of long distance transport or perhaps salvaging of the vitamin in the root remain to be 354

discovered in future studies. As noted previously, PUT3 has four additional homologs in 355

Arabidopsis, which although not found in our yeast screen may add a level of redundancy to this 356



14

transporter family and should be assessed for their contribution to the transport of vitamin B1 in 357

planta. Interestingly, the Arabidopsis PUT family appears to have a divergent pattern of tissue 358

expression as assessed by RT-PCR (Mulangi et al., 2012), as well as divergent intracellular 359

localization patterns (Li et al., 2013), which may suggest specialization in a spatial manner. 360

Notwithstanding, the small deficit of B1 vitamers observed in rmv1 may contribute to the stunted 361

root phenotype but is likely to have a stronger contribution from the disruption in polyamine 362

homeostasis (see below). 363

In contrast to vitamin B1, polyamines can be biosynthesized and interconverted ubiquitously in 364

tissues (Urano et al., 2003) including that of the phloem (Pommerrenig et al., 2011). Nonetheless, 365

their involvement in diverse cellular activities warrants that their content is carefully regulated to 366

maintain cellular homeostasis. Intercellular transport is an important part of this process and is 367

validated here through the characterization of the role of PUT3 in phloem transport of polyamines. 368

As for the B1 vitamers, PUT3 can be implicated in phloem loading of polyamines based on the 369

observation that the total polyamine content decreased in phloem exudates of the PUT3 mutant 370

rmv1 and were restored to wild-type levels in the transgenic line rmv1/pPUT3:PUT3. Notably, a 371

closer look at the individual polyamines revealed that Put is the most predominant polyamine in 372

phloem exudates and was considerably decreased along with its polyamine derivatives, Spd and 373

Spm in the rmv1 mutant line. Therefore, PUT3 may either load all three polyamines into the phloem 374

or it may have a preference for Put, which can be metabolized to Spd and Spm by the 375

corresponding synthases in the phloem. On the other hand, there is no change in the levels of Cad in 376

phloem exudates of rmv1 compared to wild-type, implying that PUT3 does not transport this 377

polyamine. This would be consistent with the recent implication of OCT1 in the transport of Cad 378

(Strohm et al., 2015) but remains to be verified in planta. 379

The striking reduction in total polyamine content in the root tissue of rmv1 can be used to 380

provide a molecular explanation for the stunted root growth observed. It has long been 381

demonstrated that polyamines play a major role in cell proliferation and cell division (Fariduddin et 382

al., 2013, Galston & Kaur-Sawhney, 1987, Jang et al., 2002, Kaur-Sawhney et al., 2003). Moreover, 383

alterations in polyamine levels can be correlated with modifications in root development. In 384

particular, increased and decreased levels of the polyamine Spd translate to enhanced and stunted 385

root growth, respectively (Hummel et al., 2002, Jang et al., 2002, Watson et al., 1998). Spd was by 386

far the most abundant polyamine found in roots of Arabidopsis in this study, having been found at 387

approximately 20-fold higher levels than Put, Spm or Cad. Notably, this is in contrast to phloem 388

exudates where Put was the most prevalent polyamine and is significantly reduced in rmv1 but 389

levels are restored to that of wild-type in rmv1/pPUT3:PUT3. Spd levels were strongly reduced in 390

roots of rmv1, thereby contributing the most to the overall deficit in polyamines seen in the roots of 391



15

this mutant. Spm levels are also reduced in root tissue of rmv1, which may be due either to reduced 392

transport into the root or a consequence of the deficit in Spd, as it is the precursor to Spm. Both Spd 393

and Spm levels are restored to wild-type levels in the transgenic line rmv1/pPUT3:PUT3, as is the 394

stunted growth phenotype. Taken together, we hypothesize from this study that Put is the 395

predominant polyamine carried in the phloem by PUT3 and as the direct precursor to Spd and Spm 396

may contribute to the pool of the latter polyamines in the root. Notably, there is no significant 397

change in the levels of Put or Cad in root tissue of rmv1 compared to the wild-type. 398

It is striking on the other hand that Put levels decreased in shoots of rmv1 and may account for 399

the delayed bolting phenotype, as elevated Put levels serve as a marker for the transition to 400

flowering (Applewhite et al., 2000, Havelange et al., 1996). Put levels recovered towards that of 401

wild-type in the transgenic line rmv1/pPUT3:PUT3 concomitant with the restoration of the bolting 402

time. Cad levels were also increased in shoots of rmv1 but appeared to be independent of PUT3, as 403

homeostasis was not restored in rmv1/pPUT3:PUT3. It is unlikely that the reduced B1 vitamer 404

levels in the shoots of rmv1 contribute to the delayed bolting time, as foliar application of thiamine 405

had no effect on bolting time (data not shown). Taken together the changes in polyamine 406

homeostasis upon disruption of PUT3 may account for the growth and developmental phenotypes 407

observed in rmv1. 408

In summary, this study identifies a long sought component of vitamin B1 shoot to root 409

translocation and highlights the necessity of maintaining the balance of two sets of vital 410

metabolites, vitamin B1 and polyamines as a function of PUT3, and the importance of their 411

appropriate tissue distribution for proper growth and development in plants. 412

413



16

MATERIALS AND METHODS 414

Yeast studies 415

The complementation screen was performed employing the Saccharomyces cerevisiae knock out 416

mutant CVY4 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 thi7Δ::kanMX4 thi71Δ::LEU2 thi72Δ::LYS2 417

thi4Δ::his5+) deficient in the biosynthesis and transport of vitamin B1 (Vogl et al., 2008) and which 418

only grows in the presence of high concentrations of thiamine (≥ 120 µM). The strain was 419

transformed with an Arabidopsis cDNA expression library (Minet et al., 1992) and transformants 420

were selected on media containing 1.2 µM thiamine. S. cerevisiae BY4742 (MATα his3Δ1 leu2Δ0 421

lys2Δ0 ura3Δ0) was used as the wild-type strain. Synthetic dextrose medium lacking vitamin B1 422

and uracil (Formedium, U.K.) and containing 2% glucose was used as the basic growth medium and 423

supplemented with the indicated concentrations of thiamine. The cDNA corresponding to 424

Arabidopsis PUT3 was amplified using the primer pairs listed in Supplemental Table S1 and after 425

cloning by homologous recombination into pGREG506-TEF (Jansen et al., 2005) to generate 426

pGREG506-TEF-PUT3 was used for the analysis shown in Fig. 1. Yeast uptake assays were carried 427

out with strain CVY3 (thi10-1Δ::KanMX thi71Δ::LEU2 thi72Δ::LYS2) (Vogl et al., 2008) 428

transformed with pGREG506-TEF-PUT3 or pGREG506-TEF as control, essentially as described by 429

(Stolz & Vielreicher, 2003). Notably, in contrast to strain CVY4, the THI4 gene is not disrupted in 430

strain CVY3, which can therefore grow in the absence of thiamine supplementation making it 431

suitable for uptake studies. Briefly, cells were grown to mid-logarithmic phase in SD-URA medium 432

(Formedium, U.K.) containing 2% glucose, 0.67% yeast nitrogen base and amino acids, washed 433

twice in water and resuspended in citrate/phosphate buffer at pH 5.5. Five hundred µl of cells (at 434

0.5 OD600 units/ml) were incubated at 30°C and energized with 1% (w/v) glucose one minute prior 435

to starting the experiment. Then, 5 µl of a mixture of tritiated thiamine or TDP (both at 20 Ci mmol-436 1; American Radiolabeled Chemicals, USA) and unlabeled thiamine or TDP at 500 µM were added 437

to give a final concentration of 5 µM (specific activity 0.1 nCi µl-1). At given times, 90 µl aliquots 438

were withdrawn, diluted with 5 ml of water and filtered through GN-6 metricel membrane filters 439

(0.45 µm pore size, Pall Corporation, USA). After washing with excess water, the radioactivity in 440

the filters was quantified in 4 ml Emulsifier Safe scintillation cocktail (Perkin Elmer, USA) in a 441

Beckman LS6500 liquid scintillation counter for 5 minutes. Uptake velocities were determined 442

from the slope of the linear fit of time points covering a period of 10 min after substrate addition. 443

The optimum pH of PUT3 was determined with citrate:phosphate buffers adjusted to the indicated 444

values. Competitors or inhibitors were all used at 20-fold excess (100 µM final concentration), 445

tested at pH 5.5 and added 30 s before starting the experiment. The KM value was determined with 446

substrate solutions containing a constant amount of labeled thiamine or TDP and various 447



17

concentrations of unlabeled thiamine or TDP. The KM value was calculated from a Michaelis-448

Menten plot. The energy requirement of thiamine uptake was determined in the absence or presence 449

of 1% (w/v) glucose. The protonophore carbonyl cyanide m-chlorophenylhydrazone was used at a 450

final concentration of 100 µM. It was added 2 min before the labeled substrate and tested in assays 451

that also contained 1% (w/v) D-glucose. 452

453

Generation of plant material and growth conditions 454

The mutant line used was GT_3.3436 recently annotated as rmv1 (Fujita et al., 2012), obtained 455

from the European Arabidopsis Stock Centre and isolated to homozygosity (primer pairs used are 456

given in Supplemental Table S1), as well as its corresponding wild-type (Ler-1). The cloning of 457

PUT3 for plant transformation was carried out by amplification of the full-length sequence from 458

complementary DNA using the primer pairs PUT3.attB1.F/PUT3.attB2.R (Supplemental Table S1). 459

The resulting PCR product were used to generate pENTR221-PUT3 Gateway®-compatible vectors 460

(Life Technologies, Europe). PUT3 was recombined into the pUBC-YFP-DEST vector (Grefen et 461

al., 2010) to generate UBQ:PUT3-YFP. The UBQ10 promoter was replaced by an EcoRI/NcoI 462

restriction/ligation with the 850 bp intergenic region upstream of the PUT3 start codon, amplified 463

from genomic DNA using the primer pairs promPUT3:PUT3.EcoR1.F and promPUT3:PUT3.R, 464

indicated in Supplemental Table S1, to generate promPUT3:PUT3-YFP for complementation of 465

rmv1. For GUS expression, the 850 bp region upstream of the PUT3 start codon was amplified by 466

PCR from genomic DNA using primer pairs promPUT3.SalI.F and promPUT3.NcoI.R, inserted 467

into the pENTR/D-TOPO vector (Life Technologies, Europe) followed by recombination upstream 468

of the GUS coding sequence of pMDC162 (Curtis & Grossniklaus, 2003) to generate pPUT3:GUS. 469

Agrobacterium-mediated transformation of Arabidopsis by the floral dip method was used (Clough 470

& Bent, 1998). For PUT3 subcellular localization, UBQ:PUT3-YFP was used. Transient expression 471

in Arabidopsis mesophyll protoplasts was performed essentially as described in (Szydlowski et al., 472

2013) using 3- to 4-week-old plants maintained on half-strength Murashige and Skoog (Murashige 473

& Skoog, 1962) medium (Duchefa, Holland) containing 1% sucrose grown under 100 µmol 474

photons.m-2 s-1 for 16 hours at 22°C and 8 hours dark at 18°C with some modifications: incubation 475

in the enzyme solution was performed overnight, in the dark, and without agitation. Cells were 476

allowed to recover 24-48 hours before analysis by confocal laser scanning microscopy (Leica 477

Microsystems, Germany) using an ArKr laser at 488 nm. YFP fluorescence was recorded between 478

503 and 550 nm. In all cases, transformants were selected on the appropriate antibiotic, either 479

kanamycin or hygromycin (50 mg l-1), followed by segregation analysis to isolate lines with a single 480

insertion and were confirmed by PCR analysis and sequencing (Microsynth, Switzerland). Unless 481

stated otherwise, plants were grown either on soil or in sterile culture plates (after surface 482



18

sterilization) on half-strength Murashige and Skoog medium (Murashige & Skoog, 1962) under 483

standard growth conditions (150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark at 484

18°C). 485

486

Gene expression analyses 487

Histochemical GUS expression analyses was carried out essentially as described by (Boycheva 488

et al., 2015). Images were captured with a Leica MZ16 stereomicroscope equipped with an Infinity 489

2 digital camera (Leica Microsystems, Germany). To visualize cross-sections of GUS stained 490

seedlings, they were fixed for 5 hours at room temperature in 100 mM phosphate buffer (pH 7.2) 491

supplemented with 4% formaldehyde and 1% glutaraldehyde, followed by extensive washing in 492

phosphate buffer at 4°C, embedded in 1.5% agarose and dehydrated in a graded ethanol series 493

(50%, 70%, 90% and 3 times in 100% ethanol) in steps of 20 min each. Samples were then 494

progressively infiltrated with Technovit 7100 resin (Heraeus, Germany) supplemented with 1% 495

(w/v) hardener I (benzoyl peroxide) in three separate steps using different mixtures with ethanol at 496

50%, 33% and 0% respectively), then embedded in moulds filled with embedding medium (1 part 497

of hardener II mixed with 11 parts of infiltration medium), left for 30 min at room temperature and 498

then at 60°C for an additional 30 min until complete polymerization. Serial cross sections of 4 µm 499

thickness were prepared using a Leica Ultracut UCT microtome (Leica Microsystems, Switzerland) 500

equipped with glass knives. The sections were collected on superfrost glass slides and 501

counterstained for cell walls with 0.05% ruthenium red. Roots were observed using a Nikon eclipse 502

80i microscope (Nikon, Japan) and pictures were taken with a Nikon Digital Camera Sight DS-Fi-1 503

(Nikon, Japan). 504

For quantification of RNA, total RNA was extracted from plant samples using the NucleoSpin® 505

RNA Plant Kit (Macherey-Nagel, Germany) following the manufacturer’s protocols and treated 506

with RNase-free DNase (Promega, Switzerland) to remove traces of DNA. RNA (2 μg) was 507

reverse-transcribed into complementary DNA using the Superscript II Reverse Transcriptase and 508

oligo(dT)20 primers (Life Technologies, Europe) according to the manufacturer’s recommendations 509

and stored at -20°C. Quantification analyses were performed with a 7900 HT Fast Real-Time PCR 510

system using the Power SYBR® Green PCR Master Mix (Applied Biosystems, USA). Relative 511

mRNA abundance was calculated using the comparative ΔCt method and normalized against the 512

housekeeping gene UBC21 (At5g25760). The list of primer pairs used is given in Supplemental 513

Table S1. 514

515

Phloem exudate collection 516



19

Six-week old Arabidopsis plants were used to collect phloem exudates essentially as described in 517

(Tetyuk et al., 2013) with minor modifications. Rosette leaf numbers 8-15 were used and exudates 518

collected in doubly distilled water spiked with internal standards pyrithiamine or 519

hexamethylenediamine for B1 vitamer or polyamine determination, respectively, after EDTA 520

treatment. Exudates were collected for 5 hours and then lyophilized. Samples were resuspended in 521

doubly distilled water for metabolite determination and normalized to the protein content (Bradford, 522

1976). 523

524

Determination of vitamin B1 and polyamine content 525

B1 vitamer content was carried out as described in (Moulin et al., 2013) with the exception that 526

Arabidopsis tissues (50-100 mg) were used as indicated, extracted in 1% trichloroacetic acid (v/v) 527

and pyrithiamine was used as an internal standard. Soluble polyamines were extracted and their 528

conjugates hydrolyzed as described in (Fontaniella et al., 2001) with minor modifications. Briefly, 529

free polyamines were isolated from plant extracts or phloem exudates by mixing with an equal 530

volume of doubly distilled water and directly derivatized. Conjugated polyamines were first 531

hydrolysed to free polyamines by treatment with hydrochloric acid for 18 hours at room 532

temperature and the pH was raised to ≅12. Derivatization was performed by mixing samples with 533

two volumes of a saturated sodium carbonate solution, followed by four volumes of 7.5 mg ml-1 534

dansyl chloride in 98% (v/v) acetone and incubation in darkness for 1 hour at 60°C; supplemented 535

with one volume of 100 mg ml-1 L-proline and incubated for a further 30 min at room temperature. 536

Five volumes of ethyl acetate were then added, centrifuged for 5 min and the supernatant was 537

lyophilized. Samples were resuspended in methanol before analysis. Separation was performed on a 538

C18 Ultrasphere column (250 mm x 4.6 mm, 2.5 μm, Hichrom, UK) under the following conditions: 539

Solvent A = doubly distilled water; Solvent B = 100% methanol; 60-95% B in 23 min, 90-100% B 540

in 2 min, 100% B for 5 min, returned to 60% B in 1 min and re-equilibrated for an additional 9 min 541

at a flow rate of 1.0 ml min-1 and 27°C. Dansylated polyamines were identified by fluorescence 542

(λex=365 nm; λem=515 nm) and quantified by comparison with commercial standards. 543

544

Arabidopsis micrografting 545

Grafting of young Arabidopsis seedlings was carried out according to the methods described in 546

(Bainbridge et al., 2006) and (Marsch-Martinez et al., 2013) with minor modifications. Seeds were 547

surface-sterilized, plated on half-strength Murashige and Skoog medium (Murashige & Skoog, 548

1962) (Duchefa, Holland) containing 0.8% (w/v) agar and stratified for 4 days in the dark at 4°C, 549

then plates were incubated vertically in the dark at 28°C for an additional 2 days to promote 550

hypocotyl etiolation before being moved to standard growth conditions (150 µmol photons.m-2 s-1 551



20

for 8 hours at 22°C and 16 hours dark at 18°C). Grafting was performed under sterile conditions 4-5 552

days after germination using a binocular stereoscope. Hypocotyls were cut using a sharp razor blade 553

and freshly cut scions and rootstocks were moved to a new plate, aligned and joined. Sterile silicon 554

tubing with an internal diameter of 0.3 mm (HelixMark, Germany) was cut in half longitudinally 555

and transversely into segments of about 2 mm, laid on top of the union site of the grafted seedling 556

and gently pushed so that it would adhere tightly to both the reconstituted hypocotyl and the gel 557

surface. Plates were then placed vertically and plants were allowed to heal and grow under standard 558

conditions for an additional 5-7 days before grafting efficiency was evaluated. The use of C-shaped 559

sheaths represents an intermediate solution between collar and collar-free grafting procedures in 560

which scions and rootstocks are less damaged during manipulation and more precisely aligned 561

compared to when they are forced into a solid collar, while maintaining the presence of a solid cast 562

in addition to the gel surface, thus limiting movement of the two extremities during the healing 563

process. 564

565

Sequence data from this article are as follows: At5g05630 (PUT3/RMV1), At5g25760 (UBC21), 566

At1g13320 (GAPDH), At1g13320 (PP2AA3), At5g54770 (THI1), At1g22940 (TH1). 567

568

Supplemental Data 569

The following supplemental materials are available: 570

Supplemental Figure S1. Simplified schemes of thiamine and polyamine biosynthesis in plants. 571

Supplemental Figure S2. Optimum pH and TDP competition assays in yeast. 572

Supplemental Figure S3. Histochemical localization of pPUT3-GUS expression activity. 573

Supplemental Figure S4. Subcellular localization of PUT3-YFP to the plasma membrane. 574

Supplemental Figure S5. Proportion of B1 vitamers in phloem exudates compared to leaves. 575

Supplemental Figure S6. qPCR of PUT3 expression in rmv1 versus wild-type. 576

Supplemental Table S1. List of oligonucleotide primers used in this study. 577

578

ACKNOWLEDGMENTS 579

We thank the European Arabidopsis Stock Centre (NASC) for seeds of line GT_3_3436. We are 580

also indebted to Mireille De Meyer-Fague and Christian Megies for technical support. 581

582

AUTHOR CONTRIBUTIONS 583

T.B.F. conceived the study. All authors designed and performed various aspects of the research as 584



21

well as analyzing and interpreting the data. T.B.F. wrote the paper with assistance from J.M., E.G. 585

and N.S. 586

587



22

FIGURE LEGENDS 588

Figure 1. Identification of an Arabidopsis locus that mediates transport of thiamine in yeast. A, 589

Complementation of S. cerevisiae CVY4 that is unable to biosynthesize or transport thiamine with 590

the Arabidopsis locus At5g05630 (PUT3). Pictures were captured after 3 days of growth on 591

synthetic dextrose medium supplemented with the indicated thiamine concentrations. The 592

individual rows of yeast growth represent 10-fold serial dilutions of wild-type (WT, BY4742), 593

CVY4, or CVY4 transformed with PUT3. B, Cartoon of the predicted topology of PUT3 as 594

determined by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). C, Time-course 595

of thiamine (■) or thiamine diphosphate (□) transport (5 μM each) by S. cerevisiae CVY3 harboring 596

PUT3 in citrate-phosphate buffer pH 5.5. S. cerevisiae CVY3 transformed with the empty vector 597

serves as control (). D, Representative Michaelis-Menten plot (f(x)=Vmax*X/(KM+X)) of the rates 598

of thiamine (Thi) uptake as a function of the indicated concentrations of substrate. E, as in (D) but 599

rates are as a function of the indicated concentrations of thiamine diphosphate (TDP). F, 600

Competition studies with the thiamine precursors hydroxyethylthiazole (HET), 601

hydroxymethylpyrimidine (HMP) or the anti-vitamin pyrithiamine (Pyr), or B1 vitamers thiamine 602

(Thi), thiamine monophosphate (TMP) or TDP. TDP uptake activity was determined at a 603

concentration of 5 μM in the presence of a 20-fold excess of competitor. Activities are expressed as 604

a percentage (% activity) compared with the activity measured with TDP alone (control). EV refers 605

to the empty vector. G, as in (F) but measured with spermidine as the competitor. H, Energy 606

requirement of PUT3 uptake activity in yeast. The assays were performed as described in (C) in the 607

absence or presence of 1% (w/v) D-glucose (control). The protonophore carbonyl cyanide 3-608

chlorophenylhydrazone (CCCP) was added to a final concentration of 100 μM 2 min before the 609

labeled substrate. The data suggest that PUT3 is a proton symporter. For (C)-(H) values shown are 610

the mean ± S.D from ≥ 3 independent experiments. 611

612

Figure 2. PUT3 is localized to the phloem. A, Histochemical localization of GUS activity in 613

Arabidopsis. Representative images of Arabidopsis expressing GUS under the control of the 614

promoter of PUT3: 7-day old whole seedling (left); enlarged image of a mature rosette leaf margin 615

(top center), differentiation zone of a mature root (top right) and 7-day old seedling root tip (bottom 616

right). B, Transverse section of the root of a GUS stained transgenic seedling (left). Arrows point to 617

the position of the phloem, whereas arrowheads indicate the position of the xylem axis. The scale 618

bar represents 50 μm. A scheme of the cross-sectional layers observed is illustrated on the right. 619

620

Figure 3. Transport of B1 vitamers in the phloem requires PUT3. A, Representative HPLC profile 621



23

of B1 vitamers in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The 622

commercially available B1 standards (Stds) are thiamine diphosphate (TDP), thiamine 623

monophosphate (TMP) and thiamine (Thi). The chemical derivative pyrithiamine (Pyr) was used as 624

an internal standard. Two pmol of each compound was injected. B, Quantification of the B1 vitamer 625

content of phloem exudates of rmv1 and the complementation line rmv1/pPUT3:PUT3 versus wild-626

type (WT). Values are the average of four biological replicates with error bars representing SD. 627

Statistically significant changes for total B1 vitamers compared to wild-type were calculated by a 628

two tailed Student’s t-test for P < 0.05 and are indicated by an asterisk. 629

630

Figure 4. Transport of polyamines in the phloem requires PUT3. A, Representative HPLC profile 631

of polyamines in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The 632

commercially available standards (Stds) used are putrescine (Put, 8 pmol), cadaverine (Cad, 2 633

pmol), spermidine (Spd, 40 pmol), spermine (Spm, 4 pmol), the polyamine precursor agmatine 634

(Agm, 20 pmol) and the degradation product 1,3-diaminopropane (Dap, 8 pmol). 635

Hexamethylenediamine (Hdm, 2 pmol) was used as an internal standard. B, Quantification of the 636

polyamine contents of phloem exudates from rmv1 and the complementation line 637

rmv1/pPUT3:PUT3 versus wild-type (WT). C, Quantification of the precursor Agm, the 638

degradation product Dap could not be detected. The values for (B) and (C) are the average of four 639

biological replicates with errors bars representing SD. Statistically significant changes for total 640

polyamines compared to wild-type were calculated by a two tailed Student’s t-test for P < 0.05 and 641

are indicated by an asterisk. 642

643

Figure 5. PUT3 is required for proper root and timely reproductive development in Arabidopsis. A, 644

Representative photographs of 12-day old seedlings illustrating root growth impairment of rmv1 645

compared to the respective wild-type (WT) growing vertically in culture. Complementation of the 646

phenotype is observed in transgenic line rmv1/pPUT3:PUT3. B, Root growth rate of the same lines 647

shown in (A). C, Photographs of the plant lines as in (A) grown on soil 65-days after germination, 648

illustrating the late bolting phenotype of rmv1 compared to WT but which recovers in 649

rmv1/pPUT3:PUT3. D, Bolting time of soil grown lines as indicated. E, Representative 650

photographs of the lines as indicated during the late vegetative stage. Pictures were captured 55-651

days after germination. F, Number of rosette leaves at the time of bolting illustrating the increased 652

number of leaves in rmv1 but which returns to that of WT in rmv1/pPUT3:PUT3. In the case of (B), 653

(D) and (F), values shown are the mean ± SD from ≥ three independent experiments. Statistically 654

significant changes compared to wild-type were calculated by a two tailed Student’s t-test for P < 655

0.05 and are indicated by an asterisk. In all cases, plants were grown under an 8-hour photoperiod 656



24

(150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C. 657

658

Figure 6. Micrografting illustrates the importance of long distance transport mediated by PUT3. 659

Representative pictures of micrografts of scion and rootstock of the wild-type (WT) and rmv1 660

genetic combinations as indicated. Pictures were captured 7 days after the procedure from 661

micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark 662

at 18°C. The scale bar represents 1 cm. 663

664

Figure 7. PUT3 is required for long distance transport of vitamin B1. A, B1 vitamer levels in shoots 665

(S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the transgenic 666

line rmv1/pPUT3:PUT3. The vitamers measured are thiamine diphosphate (TDP), thiamine 667

monophosphate (TMP) and thiamine (Thi). Values shown are the mean ± SD from ≥ four 668

independent experiments. Significant differences of total B1 vitamer contents were determined by 669

one-way ANOVA (P < 0.05). B, Micrografting of wild-type (WT), rmv1 and the thiamine 670

biosynthesis mutant th1-201 as indicated. Pictures were captured 7 days after the procedure from 671

micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark 672

at 18°C. The scale bar represents 1 cm. 673

674

Figure 8. PUT3 is required for long distance transport of polyamines. A, Total polyamine levels in 675

shoots (S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the 676

transgenic line rmv1/pPUT3:PUT3. B-E, Levels of the individual polyamines cadaverine (Cad), 677

putrescine (Put), spermidine (Spd) and spermine (Spm) of the same samples shown in (A). The 678

values shown are the mean ± SD from four biological replicates. Significant differences for each 679

polyamine were determined by a one-way ANOVA (P < 0.05). In all cases, plants were grown 680

under an 8-hour photoperiod (150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C. 681

682

683



25

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812



B

N-term (1) C-term (490)

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inside

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CVY4

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Figure 1. Identification of an Arabidopsis locus that mediates transport of thiamine in yeast. A, Complementation of S. cerevisiae CVY4 that is unable to biosynthesize or transport thiamine with the Arabidopsis locus At5g05630 (PUT3). Pictures were captured after 3 days of growth on synthetic dextrose medium supplemented with the indicated thiamine concentrations. The individual rows of yeast growth represent 10-fold serial dilutions of wild-type (WT, BY4742), CVY4, or CVY4 transformed with PUT3. B, Cartoon of the predicted topology of PUT3 as determined by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). C, Time-course of thiamine ( ) or thiamine diphosphate ( ) transport (5 μM each) by S. cerevisiae CVY3 harboring PUT3 in citrate-phosphate buffer pH 5.5. S. cerevisiae CVY3 transformed with the empty vector serves as control ( ). D, Representative Michaelis-Menten plot (f(x)=Vmax*X/(KM+X)) of the rates of thiamine (Thi) uptake as a function of the indicated concentrations of substrate. E, as in (D) but rates are as a function of the indicated concentrations of thiamine diphosphate (TDP). F, Competition studies with the thiamine precursors hydroxyethylthiazole (HET), hydroxymethylpyrimidine (HMP) or the anti-vitamin pyrithiamine (Pyr), or B1 vitamers thiamine (Thi), thiamine monophosphate (TMP) or TDP. TDP uptake activity was determined at a concentration of 5 μM in the presence of a 20-fold excess of competitor. Activities are expressed as a percentage (% activity) compared with the activity measured with TDP alone (control). EV refers to the empty vector. G, as in (F) but measured with spermidine as the competitor. H, Energy requirement of PUT3 uptake activity in yeast. The assays were performed as described in (C) in the absence or presence of 1% (w/v) D-glucose (control). The protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to a final concentration of 100 µM 2 min before the labeled substrate. The data suggest that PUT3 is a proton symporter. For (C)-(H) values shown are the mean ± S.D from 3 independent experiments.



A

pPU

T3:G

US Epidermis

Cortex

Endodermis

Phloem Xylem

B

Figure 2. PUT3 is localized to the phloem. A, Histochemical localization of GUS activity in Arabidopsis. Representative images of Arabidopsis expressing GUS under the control of the promoter of PUT3: 7-day old whole seedling (left); enlarged image of a mature rosette leaf margin (top center), differentiation zone of a mature root (top right) and 7-day old seedling root tip (bottom right). B, Transverse section of the root of a GUS stained transgenic seedling (left). Arrows point to the position of the phloem, whereas arrowheads indicate the position of the xylem axis. The scale bar represents 50 mm. A scheme of the cross-sectional layers observed is illustrated on the right.



A

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rmv1

Figure 3. Transport of B1 vitamers in the phloem requires PUT3. A, Representative HPLC profile of B1 vitamers in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The commercially available B1 standards (Stds) are thiamine diphosphate (TDP), thiamine monophosphate (TMP) and thiamine (Thi). The chemical derivative pyrithiamine (Pyr) was used as an internal standard. Two pmol of each compound was injected. B, Quantification of the B1 vitamer content of phloem exudates of rmv1 and the complementation line rmv1/pPUT3:PUT3 versus wild-type (WT). Values are the average of four biological replicates with error bars representing SD. Statistically significant changes for total B1 vitamers compared to wild-type were calculated by a two tailed Student’s t-test for P < 0.05 and are indicated by an asterisk.

*

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rmv1 0

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WT rmv1/pPUT3:PUT3

rmv1

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pmol

µg

prot

ein-

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Figure 4. Transport of polyamines in the phloem requires PUT3. A, Representative HPLC profile of polyamines in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The commercially available standards (Stds) used are putrescine (Put, 8 pmol), cadaverine (Cad, 2 pmol), spermidine (Spd, 40 pmol), spermine (Spm, 4 pmol), the polyamine precursor agmatine (Agm, 20 pmol) and the degradation product 1,3-diaminopropane (Dap, 8 pmol). Hexamethylenediamine (Hdm, 2 pmol) was used as an internal standard. B, Quantification of the polyamine contents of phloem exudates from rmv1 and the complementation line rmv1/pPUT3:PUT3 versus wild-type (WT). C, Quantification of the precursor Agm, the degradation product Dap could not be detected. The values for (B) and (C) are the average of 4 biological replicates with errors bars representing SD. Statistically significant changes for total polyamines compared to wild-type were calculated by a two tailed Student’s t-test for P < 0.05 and are indicated by an asterisk.

*



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s)

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ette

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es a

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*

*

*

WT

rmv1

rm

v1/

pPU

T3 :P

UT3

A

C

E

WT rmv1 rmv1/ pPUT3: PUT3

Figure 5. PUT3 is required for proper root and timely reproductive development in Arabidopsis. A, Representative photographs of 12-day old seedlings illustrating root growth impairment of rmv1 compared to the respective wild-type (WT) growing vertically in culture. Complementation of the phenotype is observed in transgenic line rmv1/pPUT3:PUT3. B, Root growth rate of the same lines shown in (A). C, Photographs of the plant lines as in (A) grown on soil 65-days after germination, illustrating the late bolting phenotype of rmv1 compared to WT but which recovers in rmv1/pPUT3:PUT3. D, Bolting time of soil grown lines as indicated. E, Representative photographs of the lines as indicated during the late vegetative stage. Pictures were captured 55-days after germination. F, Number of rosette leaves at the time of bolting illustrating the increased number of leaves in rmv1 but which returns to that of WT in rmv1/pPUT3:PUT3. In the case of (B), (D) and (F), values shown are the mean ± SD from ≥ 3 independent experiments. Statistically significant changes compared to wild-type were calculated by a two tailed Student’s t-test for P < 0.05 and are indicated by an asterisk. In all cases, plants were grown under an 8-hour photoperiod (150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C.



rmv1

WT rmv1

Scion

Rootstock

WT WT

rmv1

rmv1

WT

Figure 6. Micrografting illustrates the importance of long distance transport mediated by PUT3. Representative pictures of micrografts of scion and rootstock of the wild-type (WT) and rmv1 genetic combinations as indicated. Pictures were captured 7 days after the procedure from micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark at 18°C. The scale bar represents 1 cm.



rmv1

WT rmv1

Scion

Rootstock

WT th1-201

th1-201 th1-201 th1-201

rmv1 WT

B A

WT rmv1

Figure 7. PUT3 is required for long distance transport of vitamin B1. A, B1 vitamer levels in shoots (S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the transgenic line rmv1/pPUT3:PUT3. The vitamers measured are thiamine diphosphate (TDP), thiamine monophosphate (TMP) and thiamine (Thi). Values shown are the mean ± SD from ≥ 4 independent experiments. Significant differences of total B1 vitamer contents were determined by one-way ANOVA (p < 0.05). B, Micrografting of wild-type (WT), rmv1 and the thiamine biosynthesis mutant th1-201 as indicated. Pictures were captured 7 days after the procedure from micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark at 18°C. The scale bar represents 1 cm.

0

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1400

1600 TDP TMP Thi

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pPUT3 :PUT3

a

a c

b b d

pmol

g F

W-1



A B C

Figure 8. PUT3 is required for long distance transport of polyamines. A, Total polyamine levels in shoots (S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the transgenic line rmv1/pPUT3:PUT3. B-E, Levels of the individual polyamines cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm) of the same samples shown in (A). The values shown are the mean ± SD from four biological replicates. Significant differences for each polyamine were determined by a one-way ANOVA (P < 0.05). In all cases, plants were grown under an 8-hour photoperiod (150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C.

0.0

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 138%5BVolume%5D AND 1505%5BPagination%5D AND Ahn IP%2C Kim S%2C Lee YH%5BAuthor%5D&dopt=abstract

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

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http://search.crossref.org/?page=1&rows=2&q=Ajjawi I, Rodriguez Milla MA, Cushman J, Shintani DK (2007a) Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis. Plant Mol Biol 65: 151-162

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

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http://search.crossref.org/?page=1&rows=2&q=Ajjawi I, Tsegaye Y, Shintani D (2007b) Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1. Arch Biochem Biophys 459: 107-114

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Planta%5BTitle%5D%29 AND 231%5BVolume%5D AND 1237%5BPagination%5D AND Alc�zar R%2C Altabella T%2C Marco F%2C et al%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 54%5BVolume%5D AND 310%5BPagination%5D AND Kruszewski SP%2C Jacobs WP%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kruszewski SP, Jacobs WP (1974) Polarity of thiamin movement through tomato petioles. Plant Physiol 54: 310-311

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

http://scholar.google.com/scholar?as_q=Polarity of thiamin movement through tomato petioles&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=%28Trends Plant Sci%5BTitle%5D%29 AND 2%5BVolume%5D AND 124%5BPagination%5D AND Kumar A%2C Taylor M%2C Triburcio AF%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Physiol Plant%5BTitle%5D%29 AND 15%5BVolume%5D AND 473%5BPagination%5D AND Murashige T%2C Skoog F%5BAuthor%5D&dopt=abstract

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Sood S, Nagar PK (2005) Xylem and phloem derived polyamines during flowering in two diverse rose species. J Plant GrowthRegul 24: 36-40


Stolz J, Vielreicher M (2003) Tpn1p, the plasma membrane vitamin B-6 transporter of Saccharomyces cerevisiae. J Biol Chem 278:18990-18996


Strohm AK, Vaughn LM, Masson PH (2015) Natural variation in the expression of ORGANIC CATION TRANSPORTER 1 affects rootlength responses to cadaverine in Arabidopsis. J Exp Bot 66: 853-862


Szydlowski N, Bürkle L, Pourcel L, Moulin M, Stolz J, Fitzpatrick TB (2013) Recycling of pyridoxine (vitamin B6) by PUP1 inArabidopsis. Plant J. 75: 40-52


Tetyuk O, Benning UF, Hoffmann-Benning S (2013) Collection and analysis of Arabidopsis phloem exudates using the EDTA-facilitated method. J Visual Exp 80.


Tunc-Ozdemir M, Miller G, Song LH, et al (2009) Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis. PlantPhysiol 151: 421-432


Turgeon R, Wolf, S (2009) Phloem transport:Cellular pathways and molecular trafficking. Annu Rev Plant Biol 60: 207-221Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Urano K, Yoshiba Y, Nanjo T, et al (2003) Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abioticstress responses and developmental stages. Plant Cell Environ 26: 1917-1926


Vogl C, Klein CM, Batke AF, Schweingruber ME, Stolz J (2008) Characterization of Thi9, a novel thiamine (vitamin B-1) transporterfrom Schizosaccharomyces pombe. J Biol Chem 283: 7379-7389


Watson MB, Emory KK, Piatak RM, Malmberg RL (1998) Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsisthaliana exhibit altered root growth. Plant J 13: 231-239


Wightman F, Brown R (1953) The effects of thiamin and nicotinic acid on meristematic activity in pea roots. J Exp Bot 4: 184-196Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title


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http://scholar.google.com/scholar?as_q=Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Raschke&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Plant Growth Regul%5BTitle%5D%29 AND 24%5BVolume%5D AND 36%5BPagination%5D AND Sood S%2C Nagar PK%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sood S, Nagar PK (2005) Xylem and phloem derived polyamines during flowering in two diverse rose species. J Plant Growth Regul 24: 36-40

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

http://scholar.google.com/scholar?as_q=Xylem and phloem derived polyamines during flowering in two diverse rose 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=Xylem and phloem derived polyamines during flowering in two diverse rose species&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sood&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Biol Chem%5BTitle%5D%29 AND 278%5BVolume%5D AND 18990%5BPagination%5D AND Stolz J%2C Vielreicher M%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Stolz J, Vielreicher M (2003) Tpn1p, the plasma membrane vitamin B-6 transporter of Saccharomyces cerevisiae. J Biol Chem 278: 18990-18996

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

http://scholar.google.com/scholar?as_q=Tpn1p, the plasma membrane vitamin B-6 transporter of Saccharomyces cerevisiae&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=Tpn1p, the plasma membrane vitamin B-6 transporter of Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Stolz&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Exp Bot%5BTitle%5D%29 AND 66%5BVolume%5D AND 853%5BPagination%5D AND Strohm AK%2C Vaughn LM%2C Masson PH%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Strohm AK, Vaughn LM, Masson PH (2015) Natural variation in the expression of ORGANIC CATION TRANSPORTER 1 affects root length responses to cadaverine in Arabidopsis. J Exp Bot 66: 853-862

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

http://scholar.google.com/scholar?as_q=Natural variation in the expression of ORGANIC CATION TRANSPORTER 1 affects root length responses to cadaverine in Arabidopsis&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://scholar.google.com/scholar?as_q=Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Szydlowski&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

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

http://scholar.google.com/scholar?as_q=Phloem transport:Cellular pathways and molecular 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

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http://scholar.google.com/scholar?as_q=Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages&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=Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Urano&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth&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=Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Watson&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Exp Bot%5BTitle%5D%29 AND 4%5BVolume%5D AND 184%5BPagination%5D AND Wightman F%2C Brown R%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=The effects of thiamin and nicotinic acid on meristematic activity in pea 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

http://scholar.google.com/scholar?as_q=The effects of thiamin and nicotinic acid on meristematic activity in pea roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wightman&as_ylo=1953&as_allsubj=all&hl=en&c2coff=1


Yokota T, Nakayama M, Harasawa I, Sato M, Katsuhara M, Kawabe S (1994) Polyamines, indole-3-acetic-acid and abscisic-acid inrice phloem sap. Plant Growth Regul 15: 125-128


Zhao R, Goldman ID (2013) Folate and thiamine transporters mediated by facilitative carriers (SLC19A1-3 and SLC46A1) and folatereceptors. Mol Aspects Med 34: 373-385



http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Growth Regul%5BTitle%5D%29 AND 15%5BVolume%5D AND 125%5BPagination%5D AND Yokota T%2C Nakayama M%2C Harasawa I%2C Sato M%2C Katsuhara M%2C Kawabe S%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Yokota T, Nakayama M, Harasawa I, Sato M, Katsuhara M, Kawabe S (1994) Polyamines, indole-3-acetic-acid and abscisic-acid in rice phloem sap. Plant Growth Regul 15: 125-128

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http://scholar.google.com/scholar?as_q=Polyamines, indole-3-acetic-acid and abscisic-acid in rice phloem sap&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, indole-3-acetic-acid and abscisic-acid in rice phloem sap&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yokota&as_ylo=1994&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Aspects Med%5BTitle%5D%29 AND 34%5BVolume%5D AND 373%5BPagination%5D AND Zhao R%2C Goldman ID%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhao R, Goldman ID (2013) Folate and thiamine transporters mediated by facilitative carriers (SLC19A1-3 and SLC46A1) and folate receptors. Mol Aspects Med 34: 373-385

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

http://scholar.google.com/scholar?as_q=Folate and thiamine transporters mediated by facilitative carriers (SLC19A1-3 and SLC46A1) and folate receptors&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=Folate and thiamine transporters mediated by facilitative carriers (SLC19A1-3 and SLC46A1) and folate receptors&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1


1 Long distance transport of thiamine and polyamines 2 ... · 91 derivatives (commonly referred to...

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