* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Long distance transport of thiamine (vitamin B1
Survey
Document related concepts
Cultivated plant taxonomy wikipedia , lookup
History of botany wikipedia , lookup
Venus flytrap wikipedia , lookup
Plant stress measurement wikipedia , lookup
Plant defense against herbivory wikipedia , lookup
Plant use of endophytic fungi in defense wikipedia , lookup
Plant secondary metabolism wikipedia , lookup
Arabidopsis thaliana wikipedia , lookup
Plant physiology wikipedia , lookup
Plant morphology wikipedia , lookup
Plant evolutionary developmental biology wikipedia , lookup
Transcript
Plant Physiology Preview. Published on March 22, 2016, as DOI:10.1104/pp.16.00009 1 Running head: Long distance transport of thiamine and polyamines 2 Address correspondence to: Teresa B. Fitzpatrick 3 Department of Botany and Plant Biology, 4 University of Geneva, 5 1211 Geneva, 6 Switzerland 7 Telephone: +41-22-3793016 8 Email: [email protected] 9 10 Research area: Membranes, Transport and Biogenetics (Secondary area: Biochemistry and 11 Metabolism) 12 13 14 Downloaded from on August 3, 20171- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Copyright 2016 by the American Society of Plant Biologists 15 Long distance transport of thiamine (vitamin B1) is concomitant with that of 16 polyamines in Arabidopsis 17 18 Jacopo Martinis1†, Elisabet Gas-Pascual1†, Nicolas Szydlowski1†, Michèle Crèvecoeur1, Alexandra 19 Gisler1, Lukas Bürkle2 and Teresa B. Fitzpatrick1* 20 21 1 22 Department of Botany and Plant Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 23 24 Geneva, Switzerland. 2 The Institute of Agricultural Sciences, ETH Zurich, Universitätstrasse 2, 8092 Zurich, Switzerland. 25 26 27 † These authors contributed equally. 28 29 One sentence summary: Long distance transport of thiamine is facilitated by PUT3 concomitant 30 with that of polyamines and is required for growth and development. 31 Downloaded from on August 3, 20172- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 32 1 33 PPOOA_1191186 and 31003A-141117/1 to T.B.F.) as well as the University of Geneva. 34 35 2 36 Athens, GA 30602, U.S.A. (E.G.P.); Miniaturisation pour la Synthèse l'Analyse et la Protéomique, 37 Université Lille1 Sciences et Technologies, 59655 Villeneuve d'Ascq Cedex, France (N.S.); Stratec 38 Biomedical AG, 75217 Birkenfeld, Germany (L.B.). Financial support is gratefully acknowledged from the Swiss National Science Foundation (Grant Present address: Department of Biochemistry and Molecular Biology, The University of Georgia, 39 40 * Address correspondence to [email protected] Downloaded from on August 3, 20173- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 41 ABSTRACT 42 Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital 43 metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is 44 taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, 45 transport mechanisms are required to mediate the movement of this polar metabolite from source to 46 sink tissue to activate key enzymes in cellular energy generating pathways but are currently 47 unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for 48 diverse aspects of growth and development, the homeostasis of which necessitate long distance 49 transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify 50 Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, 51 physiological and genetic approaches permitted us to show that PUT3 mediates phloem transport of 52 both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution 53 of these metabolites is altered with growth and developmental consequences. The pivotal role of 54 PUT3 mediated thiamine and polyamine homeostasis in plants and its importance for plant fitness is 55 revealed through these findings. 56 Downloaded from on August 3, 20174- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 57 INTRODUCTION 58 Thiamine (vitamin B1) is essential for all organisms being well recognized in its diphosphorylated 59 form, thiamine diphosphate (TDP) (Fig. 1A), as a necessary cofactor for key metabolic enzymes 60 involved in glycolysis and the citric acid cycle (Fitzpatrick & Thore, 2014). In plants, it is also 61 necessary for the Calvin cycle, the biochemical route of carbon fixation (Khozaei et al., 2015). 62 Therefore, thiamine is vital for cell energy supply in all organisms. Interestingly, some additional 63 roles for thiamine have been proposed that include involvement in responses to DNA damage 64 (Machado et al., 1996), as well as the activation of plant defenses, resistance to pathogen attack and 65 attenuation of environmental stress responses (Ahn et al., 2005, Rapala-Kozik et al., 2012). Humans 66 cannot produce thiamine de novo with plants representing one of the most important sources of the 67 compound in the human diet. However, deficiency remains a critical global problem causing 68 detrimental neurological effects and cardiovascular problems particularly among populations that 69 rely on a single crop for sustenance, e.g. polished rice (Fitzpatrick et al., 2012). Thiamine is formed 70 by 71 (hydroxyethylthiazole (HET) and hydroxymethylpyrimidine (HMP), respectively) (Supplemental 72 Fig. S1) and is later diphosphorylated to the cofactor form, TDP (reviewed in Fitzpatrick & Thore, 73 2014). Several previously elusive steps of thiamine biosynthesis de novo have been resolved 74 recently, one of which involves an unexpected suicide mechanism, where the catalytic protein 75 donates sulfur to the molecule in a single turnover reaction (Chatterjee et al., 2011) (Supplemental 76 Fig. S1). Remarkably, the majority of the enzymes involved in biosynthesis de novo are exclusively 77 found in the chloroplast, the exception being the kinase that diphosphorylates thiamine to TDP, 78 which is in the cytosol (Ajjawi et al., 2007a). Accordingly, the thiamine biosynthetic genes are 79 strongly expressed in green tissues but only at a very low level (if at all) in other non- 80 photosynthetic tissues such as roots (Colinas & Fitzpatrick, 2015). However, thiamine is essential in 81 all actively dividing meristematic stem cells and organ initial cells (Wightman & Brown, 1953) and 82 thus in non-photosynthetic organs such as the roots. It is therefore relevant that a physiological 83 study in 1974 (Kruszewski & Jacobs, 1974) showed that thiamine moves through petiolar sections 84 of tomato with strong basipetal polarity and with kinetics similar to auxin and giberellin (at a 85 velocity of 3-5 mm per hour). Indeed, in the 1940s, thiamine was considered to fulfil the 86 requirements of a hormone in higher plants (Bonner, 1940, Bonner, 1942). This was due to the 87 observation that in order to grow aseptically, excised roots of many species require thiamine in 88 small amounts. Moreover, such observations were considered as confirmation of an hypothesis that 89 in the intact plant, thiamine is biosynthesized in the leaves and moves from there to the roots, which 90 cannot biosynthesize it sufficiently but need it for development. As thiamine and its phosphorylated 91 derivatives (commonly referred to as vitamin B1) are cationic molecules (i.e. charged) at the condensation of the two separately biosynthesized heterocycle Downloaded from on August 3, 20175- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. moieties 92 physiological pH, the existence of transporters that can mediate their trafficking therefore become 93 apparent. Although the characterization of a homolog of the yeast mitochondrial transporter was 94 reported recently (Frelin et al., 2012), it remains uncharacterized in planta, while the proteins 95 participating in long distance transport of thiamine in plants have remained elusive. 96 Analogous to thiamine, polyamines are cationic molecules at physiological pH essential for the 97 growth and survival of all organisms. The diverse range of activities include regulation of cell 98 division, developmental processes such as those of root formation and flowering initiation, as well 99 as environmental stress responses (Alcázar et al., 2010, Kumar et al., 1997, Kusano et al., 2008). 100 Key representative molecules include putrescine (Put), spermidine (Spd) and spermine (Spm), all 101 three of which are derived from the amino acid arginine, via the intermediate agmatine (Agm), as 102 well as cadaverine (Cad) that is derived from lysine (Supplemental Fig. S1). Akin to other essential 103 multifunctional plant metabolites, polyamine contents must be carefully controlled to maintain 104 cellular homeostasis. This is achieved through biosynthesis, degradation, conjugation (to phenolic 105 acids, proteins, nucleic acids, membrane structures) and transport (Martin-Tanguy, 2001). Although 106 polyamine metabolism has been studied extensively, little is known of its transport in plants with 107 the exception of the recently identified PUT family (Fujita et al., 2012, Mulangi et al., 2012) and 108 the OCT1 transporter from Arabidopsis (Arabidopsis thaliana) that is implicated in Cad transport 109 (Strohm et al., 2015). The PUT family of proteins are localized at the cellular level to various 110 compartments including the plasma membrane, Golgi apparatus and endoplasmic reticulum (Fujita 111 et al., 2012, Li et al., 2013). However, polyamine homeostasis is also regulated by long distance 112 transport and indeed their presence in the vascular system of plants has been established in several 113 species (Friedman et al., 1986, Pommerrenig et al., 2011, Sood & Nagar, 2005, Yokota et al., 114 1994). To date nothing is known on the components of intercellular or long distance transport of 115 polyamines. 116 Here, a yeast-based screen allowed us to identify Arabidopsis PUT3 as a thiamine transporter. 117 Expression studies showed that PUT3 is localized to the phloem tissue in Arabidopsis and loss of 118 function studies showed that PUT3 is required for growth and development. A series of 119 micrografting and biochemical experiments demonstrated that PUT3 is important for long distance 120 transport of both thiamine and polyamines and thereby revealed its importance for homeostasis of 121 these essential molecules and maintenance of plant fitness. 122 Downloaded from on August 3, 20176- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 123 RESULTS 124 Identification of an Arabidopsis thiamine transporter 125 This study was initiated by performing a complementation screen employing a cDNA library from 126 Arabidopsis (Minet et al., 1992) with the yeast mutant CVY4, deficient in both the biosynthesis and 127 the transport of thiamine (Vogl et al., 2008). This strain grows either in the presence of strong 128 thiamine supplementation (120 µM) or can be complemented with the yeast thiamine biosynthesis 129 gene THI4 (Vogl et al., 2008). In line with this, several clones harboring the ortholog of the latter 130 from Arabidopsis (THI1) were found to complement the growth phenotype even in the absence of 131 thiamine supplementation in our screen. On the other hand, a single Arabidopsis locus (At5g05630) 132 encoding a protein belonging to the amino acid permease family was found to restore growth in the 133 presence of low levels of thiamine (1.2 µM), thus complementing the impairment in thiamine 134 transport in this yeast strain (Fig. 1A). This protein has been previously reported to facilitate the 135 uptake of polyamines in a yeast based study and was accordingly named POLYAMINE UPTAKE 136 TRANSPORTER 3 (PUT3) (Mulangi et al., 2012) and mediated paraquat uptake in Arabidopsis for 137 which it was given the name RESISTANT TO METHYL VIOLOGEN 1 (RMV1)(Fujita et al., 138 2012). The PUT3/RMV1 locus, which we will refer to as PUT3 from here on, encodes a protein of 139 490 amino acid residues and is predicted to have 12 transmembrane domains with the N- and C- 140 terminal hydrophilic domains residing on the inside of the membrane (Fig. 1B). It has 4 paralogs in 141 Arabidopsis, At1g31820 (68% identity), At1g31830 (63% identity), At3g19553 (52% identity) and 142 At3g13620 (43% identity), respectively. None of the latter paralogs were found in our screen and 143 presumably cannot transport thiamine (at least in yeast) under the conditions used. Additional close 144 homologs of PUT3 were identified in several plants including Vitis vinifera (grapevine), Populus 145 trichocarpa (poplar), Oryza sativa (rice), Zea mays (corn), Sorghum bicolor (Sorghum), as well as 146 Physcomitrella patens (moss), while weaker homologs could be identified in Chlorella variabilis 147 (green algae). Notably, an extensive recent phylogenetic study hypothesized that this family of 148 transporters was acquired by horizontal transfer from green algae (Mulangi et al., 2012). 149 150 Kinetics of PUT3 facilitated thiamine transport 151 The kinetics of thiamine transport by PUT3 in yeast was characterized using the tritiated vitamers, 152 [3H]-thiamine and [3H]-TDP. Note, vitamers are related chemical substances that fulfil the same 153 specific vitamin function. Uptake occurred at an optimal pH of 5.5 (Supplemental Fig. S2A). Both 154 vitamers could be transported at similar rates (Fig. 1C). In addition, the vitamers displayed similar 155 Michaelis-Menten parameters (Vmax:169.3 ± 12.3 pmol min-1.OD cells-1, KM:43.3 ± 7.7 µM for 156 thiamine and Vmax:146.5 ± 13.7 pmol min-1.OD cells-1, KM of 34.2 ± 8.6 µM for TDP, respectively Downloaded from on August 3, 20177- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 157 (Fig. 1D and 1E)). Competition experiments showed that B1 vitamers, TDP, thiamine 158 monophosphate (TMP) and thiamine were able to compete for TDP transport, whereas HET or 159 HMP (the thiamine biosynthesis precursors) were not (Fig. 1F). Interestingly, the thiamine 160 antagonist, pyrithiamine, also competed with TDP uptake (Fig. 1F). As anticipated, the polyamine 161 Spd, shown previously to be transported by PUT3 in a yeast based assay (Mulangi et al., 2012), 162 competed strongly with TDP transport (Fig. 1G), indicating that thiamine uses the same transport 163 pathway as polyamines. The withdrawal of glucose or addition of the protonophore, 164 carbonylcyanide-3-chlorophenylhydrazone, abolishes the transport activity, suggesting that 165 transport occurs via H+ symport (Fig. 1H). Since PUT3 was originally annotated as a putative 166 amino acid transporter (Jack et al., 2000), we also tested for competition of TDP uptake in the 167 presence of unlabeled cyclic amino-acids, but none showed significant modulation of transport 168 (Supplemental Fig. S2B). In the same context and as the human thiamine transporter belongs to the 169 folate transporter family (Zhao & Goldman, 2013), we also tested folate, but it did not compete for 170 TDP transport (Supplemental Fig. S2B). Similar results were obtained when the competition studies 171 were performed with thiamine instead of TDP. It can therefore be concluded that PUT3 is a 172 polyspecific transporter than mediates thiamine as well as polyamine transport, at least in yeast. 173 174 PUT3 is localized to the phloem and mediates transport of B1 vitamers 175 Given the demonstrated ability of PUT3 to import thiamine/TDP in yeast, we wanted to establish its 176 localization in planta, to complement the previous cellular localization studies of this protein (Fujita 177 et al., 2012). To this end, we generated plants expressing the β-GLUCURONIDASE (GUS) 178 reporter gene under control of the 850 base-pair region immediately upstream of the transcriptional 179 start site of PUT3 (pPUT3-GUS). Representative pictures of 20 independent transgenic lines 180 analyzed are shown in Fig. 2A. In all lines examined, PUT3 promoter driven GUS activity was 181 predominant in the vascular tissue of leaves and the central cylinder of roots. In particular, two 182 strands of GUS activity could be observed in the central stele of the differentiation region of the 183 root. Moreover, transverse sections of the root tissue established localization to the phloem tissue 184 (Fig. 2B). Weak GUS activity was also observed in the anthers of flowers, as well as the base and 185 tip of mature siliques (Supplemental Fig. S3). At the subcellular level, fusion of the full-length 186 PUT3 protein sequence to YFP and examination of the corresponding fluorescence upon transient 187 expression in Arabidopsis mesophyll protoplasts confirmed the exclusive location of this protein to 188 the plasma membrane (Supplemental Fig. S4) in agreement with a previous report (Fujita et al., 189 2012). 190 Since B1 vitamers need to be translocated from sites of biosynthesis (source) to sites where they 191 cannot be biosynthesized but are required (sink), we were prompted to investigate if they are found Downloaded from on August 3, 20178- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 192 in the phloem sap. Indeed, the three B1 vitamers could be detected in the phloem sap of Arabidopsis 193 (Fig. 3A) and thereby implies that a transport system is in place for translocation into this tissue. It 194 is worth noting that the ratio between TDP and either TMP or thiamine in the phloem sap is 195 drastically reduced when compared with extracts from whole rosette leaves (Supplemental Fig. S5). 196 In order to test the contribution of PUT3 to the vitamin B1 transport process, the B1 vitamer profiles 197 of phloem exudates from wild-type plants were compared with that of the previously characterized 198 T-DNA insertion mutant line rmv1 (GT_3_3436) (Fujita et al., 2012). Downregulation of PUT3 199 expression was confirmed by real-time quantitative PCR (qPCR) (Supplemental Fig. S6). Indeed, 200 the total vitamin B1 content was decreased in the rmv1 mutant phloem exudates compared to that of 201 the wild-type (Fig. 3B). Importantly, B1 vitamer contents were restored to wild-type levels in 202 phloem exudates of rmv1 carrying the PUT3 transgene under control of its own promoter (Fig. 3B, 203 rmv1/pPUT3:PUT3). These observations suggest that PUT3 is involved in transport of vitamin B1 204 in the phloem. 205 206 PUT3 also mediates phloem transport of polyamines 207 The localization of PUT3 to the phloem tissue and its previous reported ability to facilitate 208 polyamine transport, at least in yeast (Mulangi et al., 2012), prompted us to next investigate the 209 polyamine profiles of phloem exudates from Arabidopsis. In particular, we adapted a protocol 210 (Fontaniella et al., 2001) to profile the diamines Cad and Put, the triamine Spd, the tetramine Spm, 211 as well as the Put/Spd/Spm precursor Agm. All of these compounds could be detected in phloem 212 exudates from Arabidopsis (Fig. 4A). Put was the most abundant polyamine detected followed by 213 Spd and Cad, while Spm accumulated at much lower levels (Fig. 4B). Significantly, there was a 214 considerable decrease in the levels of Put, in particular, in phloem exudates of rmv1 (Fig. 4B). 215 Importantly, the polyamine profile was restored to that of wild-type in phloem exudates of rmv1 216 carrying the PUT3 transgene under control of its own promoter (Fig. 4B, rmv1/pPUT3:PUT3), 217 validating the role of PUT3 in this molecular phenotype. Taken together, this implies that PUT3 is 218 required for transport of both polyamines and vitamin B1 in phloem tissue. 219 220 Alterations in growth and developmental processes are associated with PUT3 221 As B1 vitamers and polyamines are essential for development in plants, we next examined the 222 phenotypic consequences of loss of function of PUT3 on plant growth. Notably, previous studies on 223 PUT3 in Arabidopsis were restricted to quantifying its association with paraquat resistance (Fujita 224 et al., 2012). Interestingly, here we observed that the rmv1 mutant showed stunted growth under 225 standard growth conditions that was not reported on in previous studies (Fig. 5A), and was Downloaded from on August 3, 20179- Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 226 corroborated by a slower rate of root growth compared to wild-type (Fig. 5B). Furthermore, rmv1 227 entered the reproductive phase later than wild-type (Fig. 5C), bolting at day 67 instead of day 62 228 (Fig. 5D), concomitant with an increase in the number of rosette leaves at this developmental stage 229 (Fig. 5E and 5F). The observed phenotypes were most pronounced under an 8-hour photoperiod and 230 were significantly attenuated under longer photoperiods (data not shown). All of the growth and 231 developmental phenotypes were abrogated in rmv1 upon introduction of the PUT3 transgene (Fig. 232 5A-5F, rmv1/pPUT3:PUT3). It must therefore be concluded that PUT3 is required for proper 233 growth and development in Arabidopsis. 234 235 Long distance transport of a shoot-derived metabolite by PUT3 is responsible for the root 236 development phenotype in rmv1 237 To further probe the growth and development phenotype of rmv1 and the possible physiological 238 relevance of PUT3 in long distance transport, we performed a series of grafting experiments. 239 Notably, grafted plants with the same genetic combinations exhibited the same phenotypes as non- 240 grafted plants, i.e. grafting of an rmv1 scion onto its corresponding rootstock (rmv1/rmv1) led to 241 retention of the stunted growth phenotype compared to grafting of the wild-type scion onto its 242 corresponding rootstock (wild-type/wild-type) (Fig. 6). Remarkably, grafting of the wild-type scion 243 onto the rmv1 rootstock (wild-type/rmv1) led to recovery of the stunted root growth phenotype to 244 that of the corresponding grafted wild-type plants (wild-type/wild-type) (Fig. 6). On the other hand, 245 the reciprocal graft of the rmv1 scion onto the wild-type rootstock (rmv1/wild-type) did not lead to 246 recovery of root growth impairment (Fig. 6). Indeed, root growth was even further impaired than 247 rmv1/rmv1. This data supports the notion that PUT3 is required for long distance transport of a 248 shoot-derived metabolite that is essential for proper root growth. 249 250 Shoot to root partitioning of vitamin B1 as a function of PUT3 251 Given the implication of PUT3 in long distance transport, we next sought to obtain a molecular 252 explanation for the growth impairment phenotypes observed. In the first instance, we therefore 253 examined shoot and root partitioning of B1 vitamers in seedlings. Intriguingly, B1 vitamer levels in 254 shoots and roots (albeit to a much lesser extent) of rmv1 were lower than those of wild-type (Fig. 255 7A). While the slight depletion in root tissue (sink tissue) is consistent with the role of PUT3 in 256 long distance transport of B1 vitamers from source to sink, we expected a corresponding 257 accumulation of B1 vitamers in shoots (source tissue) rather than a depletion. Interestingly, rmv1 258 does not show the well-characterized pale shoot phenotype associated with a deficiency in B1 259 vitamers (Ajjawi et al., 2007a, Ajjawi et al., 2007b, Raschke et al., 2007). Nonetheless, levels of B1 10- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 260 vitamers in shoots and roots of rmv1 recovered upon reintroduction of the PUT3 transgene (Fig. 261 7A). To provide evidence that shoot-derived vitamin B1 is important for root growth in particular, 262 another series of grafting experiments was performed with the Arabidopsis mutant line th1-201 263 (Ajjawi et al., 2007b). In this line, the THIAMIN SYNTHASE enzyme TH1 (encoded at locus 264 At1g22940), which condenses the thiazole and pyrimidine heterocycles to form TMP, is impaired, 265 preventing thiamine biosynthesis de novo and resulting in overall stunted shoot and root growth and 266 pale leaves. Remarkably, grafting of the wild-type scion onto the th1-201 rootstock (wild-type/th1- 267 201) led to recovery of the stunted root growth phenotype that is observed with th1-201 (Fig. 7B). 268 However, grafting of the rmv1 scion onto the th1-201 rootstock (rmv1/th1-201) did not lead to 269 recovery of the short-root phenotype in th1-201. Indeed, root growth was even more impaired than 270 for the th1-201/th1-201 genetic combination, corroborating the notion that loss of function of PUT3 271 in the shoot, in particular, has a negative impact on root growth (Fig. 7B). Taken together, the data 272 demonstrate that long distance transport of B1 vitamers from the shoot is necessary for root growth 273 and that impairment of PUT3 in the shoot has a negative impact on root growth. 274 275 Shoot to root partitioning of polyamines as a function of PUT3 276 Given that PUT3 is also implicated in polyamine transport, we next looked at shoot to root 277 partitioning of polyamine contents. In the first instance, we noted that the total polyamine content is 278 very similar between shoots and roots in wild-type plants (Fig. 8A). A significant reduction of total 279 polyamine levels was observed in the roots of rmv1, whereas shoot content remained similar to that 280 of wild-type (Fig. 8A). Notably, levels of root polyamines recovered upon reintroduction of the 281 PUT3 transgene in rmv1 (Fig. 8A). Intriguingly, a closer look at the polyamines measured indicates 282 that the predominant polyamine in shoots and roots is Spd and that it is mainly this polyamine that 283 is depleted in roots of rmv1 (Fig. 8B-8E). This is in contrast to the polyamine profile of phloem 284 exudates, where Put was the predominant polyamine detected and the one most depleted in rmv1 285 (Fig. 4B). 286 11- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 287 DISCUSSION 288 A fundamental understanding of phloem transport with its important role in plant development, 289 reproduction, signaling and growth is central to plant biology. This is further emphasized by the 290 core set of compounds, such as biological cofactors and growth regulators, required in all living 291 cells to drive key metabolic processes, the biosynthesis of which may be restricted to certain organs 292 and therefore must be transported to the site of requirement. As central components of metabolic 293 function, the supply of many of these molecules must be strictly regulated, such that provision is 294 coordinated with metabolic needs. However, despite their critical roles, surprisingly little is known 295 on the intercellular transport of such molecules in plants. In this study, we demonstrated the 296 importance of long distance transport of vitamin B1 and polyamines as mediated by the phloem 297 localized transporter PUT3. Disruption of PUT3 led to a deficit of B1 vitamers and polyamines in 298 phloem exudates that perturbed the homeostasis of these compounds in the organs where they are 299 required resulting in a negative impact on growth and development in Arabidopsis. 300 Long distance transporters of the vital cofactor vitamin B1 have been supposed for almost a 301 century (Bonner, 1940, Bonner, 1942), yet the proteins carrying out these processes and evidence 302 for the presence of this family of compounds in the vascular system have remained elusive. Only a 303 single transporter has been reported for vitamin B1 in plants and operates at the subcellular level in 304 the mitochondrial membrane (Frelin et al., 2012). Polyamines, on the other hand, were detected in 305 the phloem sap several decades ago (Friedman et al., 1986). While they can be biosynthesized to a 306 certain extent in the phloem (Pommerrenig et al., 2011), the factors facilitating transport of these 307 important cationic molecules in the vascular system to maintain homeostasis were unknown. Here 308 we have demonstrated the involvement of PUT3 in these transport processes in plants. Although 309 PUT3 was previously implicated in subcellular polyamine transport based on a yeast 310 complementation assay (Mulangi et al., 2012), as well as paraquat transport in Arabidopsis (Fujita 311 et al., 2012), it went unnoted that it is localized to the phloem and mediates long distance transport. 312 Here we have shown that the affinity of PUT3 for B1 vitamers is similar to those reported for 313 polyamines and paraquat, i.e. low μM range (Fujita et al., 2012), classifying it as a polyspecific 314 high affinity cationic transporter. Indeed, exogenous thiamine reportedly enhances tolerance to 315 paraquat (Tunc-Ozdemir et al., 2009), supporting the notion that all of these compounds use the 316 same trafficking pathway. The functional significance of PUT3 for phloem transport of B1 vitamers 317 and polyamines became evident from the deficit of these compounds in the phloem exudates of the 318 PUT3 mutant line rmv1, previously characterized because of its resistance to paraquat (Fujita et al., 319 2012). The implication of PUT3 in these transport processes is validated upon restoration of the B1 320 vitamer and polyamine contents in phloem exudates of the transgenic rmv1 line expressing PUT3 321 under the control of its own promoter sequence (rmv1/pPUT3:PUT3). We acknowledge that the 12- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 322 EDTA-based method employed here for the collection of phloem sap has well-documented pitfalls 323 (Turgeon and Wolf, 2009), including that the procedure requires the cutting of a surface and 324 extended exposure of cells to EDTA causes damage, leakage from which may contaminate the sap. 325 Therefore, we chose the EDTA-facilitated phloem exudation method described by Tetyuk et al. that 326 has been optimized for Arabidopsis (Tetyuk et al., 2013), in which the samples are exposed to 327 EDTA for 1 hour, washed with water, followed by collection of the phloem exudate into water. 328 This strives to minimize cell damage and downstream interference from EDTA, while removing 329 compounds that have accumulated from damaged or cut cells. Indeed, assuming no metabolite 330 degradation occurs during the procedure, the divergent B1 vitamer and polyamine profiles of 331 phloem exudates versus tissue extracts could serve to support the predominant recovery of 332 metabolites being transported in the phloem with this method rather than that from damaged cells. 333 Moreover, the collection of material directly into water without prior EDTA-treatment was free of 334 B1 vitamers and polyamines and used as a negative control. Nonetheless, the use of another method 335 designed to exclusively measure mobile elements in the phloem sap could serve to validate the 336 findings. 337 As vitamin B1 biosynthesis de novo is predominantly limited to photosynthesizing tissue 338 (reviewed in Colinas & Fitzpatrick, 2015), it can be suggested that PUT3 activity in its capacity as 339 long distance B1 vitamer transporter is mainly restricted to shoot tissue. On the one hand, the 340 requirement for shoot to root long distance transport of vitamin B1 is supported by the recovery of 341 root growth in the biosynthesis mutant th1-201 upon grafting of a wild-type scion. On the other 342 hand, the failure to restore root growth upon grafting a PUT3 mutant scion (rmv1) onto th1-201 343 rootstock demonstrates the important role of PUT3 in this process. In line with this, we predict that 344 PUT3 is involved in phloem loading of B1 vitamers and is supported firstly by the decrease in B1 345 vitamer contents in phloem exudates and secondly by the decrease (albeit slight) in the root tissue 346 of rmv1. We noted above that the ratio between TDP and either TMP or thiamine in the phloem sap 347 is drastically reduced when compared with extracts from whole rosette leaves. Therefore, either a 348 selective transport system takes place in the leaves for phloem loading, and/or vitamers can be 349 interconverted within the phloem tissues by phosphatases that may be non-specific. The deficit in 350 shoot tissue B1 vitamer contents of rmv1 cannot be explained at present and requires further 351 investigation. Nonetheless, recovery towards wild-type shoot to root partitioning of B1 vitamers is 352 observed in the transgenic rmv1/pPUT3:PUT3 line implicating PUT3 in the process. The retention 353 of considerable levels of vitamin B1 in the roots even in the absence of PUT3 suggests that more 354 components of long distance transport or perhaps salvaging of the vitamin in the root remain to be 355 discovered in future studies. As noted previously, PUT3 has four additional homologs in 356 Arabidopsis, which although not found in our yeast screen may add a level of redundancy to this 13- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 357 transporter family and should be assessed for their contribution to the transport of vitamin B1 in 358 planta. Interestingly, the Arabidopsis PUT family appears to have a divergent pattern of tissue 359 expression as assessed by RT-PCR (Mulangi et al., 2012), as well as divergent intracellular 360 localization patterns (Li et al., 2013), which may suggest specialization in a spatial manner. 361 Notwithstanding, the small deficit of B1 vitamers observed in rmv1 may contribute to the stunted 362 root phenotype but is likely to have a stronger contribution from the disruption in polyamine 363 homeostasis (see below). 364 In contrast to vitamin B1, polyamines can be biosynthesized and interconverted ubiquitously in 365 tissues (Urano et al., 2003) including that of the phloem (Pommerrenig et al., 2011). Nonetheless, 366 their involvement in diverse cellular activities warrants that their content is carefully regulated to 367 maintain cellular homeostasis. Intercellular transport is an important part of this process and is 368 validated here through the characterization of the role of PUT3 in phloem transport of polyamines. 369 As for the B1 vitamers, PUT3 can be implicated in phloem loading of polyamines based on the 370 observation that the total polyamine content decreased in phloem exudates of the PUT3 mutant 371 rmv1 and were restored to wild-type levels in the transgenic line rmv1/pPUT3:PUT3. Notably, a 372 closer look at the individual polyamines revealed that Put is the most predominant polyamine in 373 phloem exudates and was considerably decreased along with its polyamine derivatives, Spd and 374 Spm in the rmv1 mutant line. Therefore, PUT3 may either load all three polyamines into the phloem 375 or it may have a preference for Put, which can be metabolized to Spd and Spm by the 376 corresponding synthases in the phloem. On the other hand, there is no change in the levels of Cad in 377 phloem exudates of rmv1 compared to wild-type, implying that PUT3 does not transport this 378 polyamine. This would be consistent with the recent implication of OCT1 in the transport of Cad 379 (Strohm et al., 2015) but remains to be verified in planta. 380 The striking reduction in total polyamine content in the root tissue of rmv1 can be used to 381 provide a molecular explanation for the stunted root growth observed. It has long been 382 demonstrated that polyamines play a major role in cell proliferation and cell division (Fariduddin et 383 al., 2013, Galston & Kaur-Sawhney, 1987, Jang et al., 2002, Kaur-Sawhney et al., 2003). Moreover, 384 alterations in polyamine levels can be correlated with modifications in root development. In 385 particular, increased and decreased levels of the polyamine Spd translate to enhanced and stunted 386 root growth, respectively (Hummel et al., 2002, Jang et al., 2002, Watson et al., 1998). Spd was by 387 far the most abundant polyamine found in roots of Arabidopsis in this study, having been found at 388 approximately 20-fold higher levels than Put, Spm or Cad. Notably, this is in contrast to phloem 389 exudates where Put was the most prevalent polyamine and is significantly reduced in rmv1 but 390 levels are restored to that of wild-type in rmv1/pPUT3:PUT3. Spd levels were strongly reduced in 391 roots of rmv1, thereby contributing the most to the overall deficit in polyamines seen in the roots of 14- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 392 this mutant. Spm levels are also reduced in root tissue of rmv1, which may be due either to reduced 393 transport into the root or a consequence of the deficit in Spd, as it is the precursor to Spm. Both Spd 394 and Spm levels are restored to wild-type levels in the transgenic line rmv1/pPUT3:PUT3, as is the 395 stunted growth phenotype. Taken together, we hypothesize from this study that Put is the 396 predominant polyamine carried in the phloem by PUT3 and as the direct precursor to Spd and Spm 397 may contribute to the pool of the latter polyamines in the root. Notably, there is no significant 398 change in the levels of Put or Cad in root tissue of rmv1 compared to the wild-type. 399 It is striking on the other hand that Put levels decreased in shoots of rmv1 and may account for 400 the delayed bolting phenotype, as elevated Put levels serve as a marker for the transition to 401 flowering (Applewhite et al., 2000, Havelange et al., 1996). Put levels recovered towards that of 402 wild-type in the transgenic line rmv1/pPUT3:PUT3 concomitant with the restoration of the bolting 403 time. Cad levels were also increased in shoots of rmv1 but appeared to be independent of PUT3, as 404 homeostasis was not restored in rmv1/pPUT3:PUT3. It is unlikely that the reduced B1 vitamer 405 levels in the shoots of rmv1 contribute to the delayed bolting time, as foliar application of thiamine 406 had no effect on bolting time (data not shown). Taken together the changes in polyamine 407 homeostasis upon disruption of PUT3 may account for the growth and developmental phenotypes 408 observed in rmv1. 409 In summary, this study identifies a long sought component of vitamin B1 shoot to root 410 translocation and highlights the necessity of maintaining the balance of two sets of vital 411 metabolites, vitamin B1 and polyamines as a function of PUT3, and the importance of their 412 appropriate tissue distribution for proper growth and development in plants. 413 15- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 414 MATERIALS AND METHODS 415 Yeast studies 416 The complementation screen was performed employing the Saccharomyces cerevisiae knock out 417 mutant CVY4 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 thi7Δ::kanMX4 thi71Δ::LEU2 thi72Δ::LYS2 418 thi4Δ::his5+) deficient in the biosynthesis and transport of vitamin B1 (Vogl et al., 2008) and which 419 only grows in the presence of high concentrations of thiamine (≥ 120 µM). The strain was 420 transformed with an Arabidopsis cDNA expression library (Minet et al., 1992) and transformants 421 were selected on media containing 1.2 µM thiamine. S. cerevisiae BY4742 (MATα his3Δ1 leu2Δ0 422 lys2Δ0 ura3Δ0) was used as the wild-type strain. Synthetic dextrose medium lacking vitamin B1 423 and uracil (Formedium, U.K.) and containing 2% glucose was used as the basic growth medium and 424 supplemented with the indicated concentrations of thiamine. The cDNA corresponding to 425 Arabidopsis PUT3 was amplified using the primer pairs listed in Supplemental Table S1 and after 426 cloning by homologous recombination into pGREG506-TEF (Jansen et al., 2005) to generate 427 pGREG506-TEF-PUT3 was used for the analysis shown in Fig. 1. Yeast uptake assays were carried 428 out with strain CVY3 (thi10-1Δ::KanMX thi71Δ::LEU2 thi72Δ::LYS2) (Vogl et al., 2008) 429 transformed with pGREG506-TEF-PUT3 or pGREG506-TEF as control, essentially as described by 430 (Stolz & Vielreicher, 2003). Notably, in contrast to strain CVY4, the THI4 gene is not disrupted in 431 strain CVY3, which can therefore grow in the absence of thiamine supplementation making it 432 suitable for uptake studies. Briefly, cells were grown to mid-logarithmic phase in SD-URA medium 433 (Formedium, U.K.) containing 2% glucose, 0.67% yeast nitrogen base and amino acids, washed 434 twice in water and resuspended in citrate/phosphate buffer at pH 5.5. Five hundred µl of cells (at 435 0.5 OD600 units/ml) were incubated at 30°C and energized with 1% (w/v) glucose one minute prior 436 to starting the experiment. Then, 5 µl of a mixture of tritiated thiamine or TDP (both at 20 Ci mmol- 437 1 438 to give a final concentration of 5 µM (specific activity 0.1 nCi µl-1). At given times, 90 µl aliquots 439 were withdrawn, diluted with 5 ml of water and filtered through GN-6 metricel membrane filters 440 (0.45 µm pore size, Pall Corporation, USA). After washing with excess water, the radioactivity in 441 the filters was quantified in 4 ml Emulsifier Safe scintillation cocktail (Perkin Elmer, USA) in a 442 Beckman LS6500 liquid scintillation counter for 5 minutes. Uptake velocities were determined 443 from the slope of the linear fit of time points covering a period of 10 min after substrate addition. 444 The optimum pH of PUT3 was determined with citrate:phosphate buffers adjusted to the indicated 445 values. Competitors or inhibitors were all used at 20-fold excess (100 µM final concentration), 446 tested at pH 5.5 and added 30 s before starting the experiment. The KM value was determined with 447 substrate solutions containing a constant amount of labeled thiamine or TDP and various ; American Radiolabeled Chemicals, USA) and unlabeled thiamine or TDP at 500 µM were added 16- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 448 concentrations of unlabeled thiamine or TDP. The KM value was calculated from a Michaelis- 449 Menten plot. The energy requirement of thiamine uptake was determined in the absence or presence 450 of 1% (w/v) glucose. The protonophore carbonyl cyanide m-chlorophenylhydrazone was used at a 451 final concentration of 100 µM. It was added 2 min before the labeled substrate and tested in assays 452 that also contained 1% (w/v) D-glucose. 453 454 Generation of plant material and growth conditions 455 The mutant line used was GT_3.3436 recently annotated as rmv1 (Fujita et al., 2012), obtained 456 from the European Arabidopsis Stock Centre and isolated to homozygosity (primer pairs used are 457 given in Supplemental Table S1), as well as its corresponding wild-type (Ler-1). The cloning of 458 PUT3 for plant transformation was carried out by amplification of the full-length sequence from 459 complementary DNA using the primer pairs PUT3.attB1.F/PUT3.attB2.R (Supplemental Table S1). 460 The resulting PCR product were used to generate pENTR221-PUT3 Gateway®-compatible vectors 461 (Life Technologies, Europe). PUT3 was recombined into the pUBC-YFP-DEST vector (Grefen et 462 al., 2010) to generate UBQ:PUT3-YFP. The UBQ10 promoter was replaced by an EcoRI/NcoI 463 restriction/ligation with the 850 bp intergenic region upstream of the PUT3 start codon, amplified 464 from genomic DNA using the primer pairs promPUT3:PUT3.EcoR1.F and promPUT3:PUT3.R, 465 indicated in Supplemental Table S1, to generate promPUT3:PUT3-YFP for complementation of 466 rmv1. For GUS expression, the 850 bp region upstream of the PUT3 start codon was amplified by 467 PCR from genomic DNA using primer pairs promPUT3.SalI.F and promPUT3.NcoI.R, inserted 468 into the pENTR/D-TOPO vector (Life Technologies, Europe) followed by recombination upstream 469 of the GUS coding sequence of pMDC162 (Curtis & Grossniklaus, 2003) to generate pPUT3:GUS. 470 Agrobacterium-mediated transformation of Arabidopsis by the floral dip method was used (Clough 471 & Bent, 1998). For PUT3 subcellular localization, UBQ:PUT3-YFP was used. Transient expression 472 in Arabidopsis mesophyll protoplasts was performed essentially as described in (Szydlowski et al., 473 2013) using 3- to 4-week-old plants maintained on half-strength Murashige and Skoog (Murashige 474 & Skoog, 1962) medium (Duchefa, Holland) containing 1% sucrose grown under 100 µmol 475 photons.m-2 s-1 for 16 hours at 22°C and 8 hours dark at 18°C with some modifications: incubation 476 in the enzyme solution was performed overnight, in the dark, and without agitation. Cells were 477 allowed to recover 24-48 hours before analysis by confocal laser scanning microscopy (Leica 478 Microsystems, Germany) using an ArKr laser at 488 nm. YFP fluorescence was recorded between 479 503 and 550 nm. In all cases, transformants were selected on the appropriate antibiotic, either 480 kanamycin or hygromycin (50 mg l-1), followed by segregation analysis to isolate lines with a single 481 insertion and were confirmed by PCR analysis and sequencing (Microsynth, Switzerland). Unless 482 stated otherwise, plants were grown either on soil or in sterile culture plates (after surface 17- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 483 sterilization) on half-strength Murashige and Skoog medium (Murashige & Skoog, 1962) under 484 standard growth conditions (150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark at 485 18°C). 486 487 Gene expression analyses 488 Histochemical GUS expression analyses was carried out essentially as described by (Boycheva 489 et al., 2015). Images were captured with a Leica MZ16 stereomicroscope equipped with an Infinity 490 2 digital camera (Leica Microsystems, Germany). To visualize cross-sections of GUS stained 491 seedlings, they were fixed for 5 hours at room temperature in 100 mM phosphate buffer (pH 7.2) 492 supplemented with 4% formaldehyde and 1% glutaraldehyde, followed by extensive washing in 493 phosphate buffer at 4°C, embedded in 1.5% agarose and dehydrated in a graded ethanol series 494 (50%, 70%, 90% and 3 times in 100% ethanol) in steps of 20 min each. Samples were then 495 progressively infiltrated with Technovit 7100 resin (Heraeus, Germany) supplemented with 1% 496 (w/v) hardener I (benzoyl peroxide) in three separate steps using different mixtures with ethanol at 497 50%, 33% and 0% respectively), then embedded in moulds filled with embedding medium (1 part 498 of hardener II mixed with 11 parts of infiltration medium), left for 30 min at room temperature and 499 then at 60°C for an additional 30 min until complete polymerization. Serial cross sections of 4 µm 500 thickness were prepared using a Leica Ultracut UCT microtome (Leica Microsystems, Switzerland) 501 equipped with glass knives. The sections were collected on superfrost glass slides and 502 counterstained for cell walls with 0.05% ruthenium red. Roots were observed using a Nikon eclipse 503 80i microscope (Nikon, Japan) and pictures were taken with a Nikon Digital Camera Sight DS-Fi-1 504 (Nikon, Japan). 505 For quantification of RNA, total RNA was extracted from plant samples using the NucleoSpin® 506 RNA Plant Kit (Macherey-Nagel, Germany) following the manufacturer’s protocols and treated 507 with RNase-free DNase (Promega, Switzerland) to remove traces of DNA. RNA (2 μg) was 508 reverse-transcribed into complementary DNA using the Superscript II Reverse Transcriptase and 509 oligo(dT)20 primers (Life Technologies, Europe) according to the manufacturer’s recommendations 510 and stored at -20°C. Quantification analyses were performed with a 7900 HT Fast Real-Time PCR 511 system using the Power SYBR® Green PCR Master Mix (Applied Biosystems, USA). Relative 512 mRNA abundance was calculated using the comparative ΔCt method and normalized against the 513 housekeeping gene UBC21 (At5g25760). The list of primer pairs used is given in Supplemental 514 Table S1. 515 516 Phloem exudate collection 18- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 517 Six-week old Arabidopsis plants were used to collect phloem exudates essentially as described in 518 (Tetyuk et al., 2013) with minor modifications. Rosette leaf numbers 8-15 were used and exudates 519 collected 520 hexamethylenediamine for B1 vitamer or polyamine determination, respectively, after EDTA 521 treatment. Exudates were collected for 5 hours and then lyophilized. Samples were resuspended in 522 doubly distilled water for metabolite determination and normalized to the protein content (Bradford, 523 1976). in doubly distilled water spiked with internal standards pyrithiamine or 524 525 Determination of vitamin B1 and polyamine content 526 B1 vitamer content was carried out as described in (Moulin et al., 2013) with the exception that 527 Arabidopsis tissues (50-100 mg) were used as indicated, extracted in 1% trichloroacetic acid (v/v) 528 and pyrithiamine was used as an internal standard. Soluble polyamines were extracted and their 529 conjugates hydrolyzed as described in (Fontaniella et al., 2001) with minor modifications. Briefly, 530 free polyamines were isolated from plant extracts or phloem exudates by mixing with an equal 531 volume of doubly distilled water and directly derivatized. Conjugated polyamines were first 532 hydrolysed to free polyamines by treatment with hydrochloric acid for 18 hours at room 533 temperature and the pH was raised to ≅12. Derivatization was performed by mixing samples with 534 two volumes of a saturated sodium carbonate solution, followed by four volumes of 7.5 mg ml-1 535 dansyl chloride in 98% (v/v) acetone and incubation in darkness for 1 hour at 60°C; supplemented 536 with one volume of 100 mg ml-1 L-proline and incubated for a further 30 min at room temperature. 537 Five volumes of ethyl acetate were then added, centrifuged for 5 min and the supernatant was 538 lyophilized. Samples were resuspended in methanol before analysis. Separation was performed on a 539 C18 Ultrasphere column (250 mm x 4.6 mm, 2.5 μm, Hichrom, UK) under the following conditions: 540 Solvent A = doubly distilled water; Solvent B = 100% methanol; 60-95% B in 23 min, 90-100% B 541 in 2 min, 100% B for 5 min, returned to 60% B in 1 min and re-equilibrated for an additional 9 min 542 at a flow rate of 1.0 ml min-1 and 27°C. Dansylated polyamines were identified by fluorescence 543 (λex=365 nm; λem=515 nm) and quantified by comparison with commercial standards. 544 545 Arabidopsis micrografting 546 Grafting of young Arabidopsis seedlings was carried out according to the methods described in 547 (Bainbridge et al., 2006) and (Marsch-Martinez et al., 2013) with minor modifications. Seeds were 548 surface-sterilized, plated on half-strength Murashige and Skoog medium (Murashige & Skoog, 549 1962) (Duchefa, Holland) containing 0.8% (w/v) agar and stratified for 4 days in the dark at 4°C, 550 then plates were incubated vertically in the dark at 28°C for an additional 2 days to promote 551 hypocotyl etiolation before being moved to standard growth conditions (150 µmol photons.m-2 s-1 19- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 552 for 8 hours at 22°C and 16 hours dark at 18°C). Grafting was performed under sterile conditions 4-5 553 days after germination using a binocular stereoscope. Hypocotyls were cut using a sharp razor blade 554 and freshly cut scions and rootstocks were moved to a new plate, aligned and joined. Sterile silicon 555 tubing with an internal diameter of 0.3 mm (HelixMark, Germany) was cut in half longitudinally 556 and transversely into segments of about 2 mm, laid on top of the union site of the grafted seedling 557 and gently pushed so that it would adhere tightly to both the reconstituted hypocotyl and the gel 558 surface. Plates were then placed vertically and plants were allowed to heal and grow under standard 559 conditions for an additional 5-7 days before grafting efficiency was evaluated. The use of C-shaped 560 sheaths represents an intermediate solution between collar and collar-free grafting procedures in 561 which scions and rootstocks are less damaged during manipulation and more precisely aligned 562 compared to when they are forced into a solid collar, while maintaining the presence of a solid cast 563 in addition to the gel surface, thus limiting movement of the two extremities during the healing 564 process. 565 566 Sequence data from this article are as follows: At5g05630 (PUT3/RMV1), At5g25760 (UBC21), 567 At1g13320 (GAPDH), At1g13320 (PP2AA3), At5g54770 (THI1), At1g22940 (TH1). 568 569 Supplemental Data 570 The following supplemental materials are available: 571 Supplemental Figure S1. Simplified schemes of thiamine and polyamine biosynthesis in plants. 572 Supplemental Figure S2. Optimum pH and TDP competition assays in yeast. 573 Supplemental Figure S3. Histochemical localization of pPUT3-GUS expression activity. 574 Supplemental Figure S4. Subcellular localization of PUT3-YFP to the plasma membrane. 575 Supplemental Figure S5. Proportion of B1 vitamers in phloem exudates compared to leaves. 576 Supplemental Figure S6. qPCR of PUT3 expression in rmv1 versus wild-type. 577 Supplemental Table S1. List of oligonucleotide primers used in this study. 578 579 ACKNOWLEDGMENTS 580 We thank the European Arabidopsis Stock Centre (NASC) for seeds of line GT_3_3436. We are 581 also indebted to Mireille De Meyer-Fague and Christian Megies for technical support. 582 583 AUTHOR CONTRIBUTIONS 584 T.B.F. conceived the study. All authors designed and performed various aspects of the research as 20- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 585 well as analyzing and interpreting the data. T.B.F. wrote the paper with assistance from J.M., E.G. 586 and N.S. 587 21- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 588 FIGURE LEGENDS 589 Figure 1. Identification of an Arabidopsis locus that mediates transport of thiamine in yeast. A, 590 Complementation of S. cerevisiae CVY4 that is unable to biosynthesize or transport thiamine with 591 the Arabidopsis locus At5g05630 (PUT3). Pictures were captured after 3 days of growth on 592 synthetic dextrose medium supplemented with the indicated thiamine concentrations. The 593 individual rows of yeast growth represent 10-fold serial dilutions of wild-type (WT, BY4742), 594 CVY4, or CVY4 transformed with PUT3. B, Cartoon of the predicted topology of PUT3 as 595 determined by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). C, Time-course 596 of thiamine (■) or thiamine diphosphate (□) transport (5 μM each) by S. cerevisiae CVY3 harboring 597 PUT3 in citrate-phosphate buffer pH 5.5. S. cerevisiae CVY3 transformed with the empty vector 598 serves as control (). D, Representative Michaelis-Menten plot (f(x)=Vmax*X/(KM+X)) of the rates 599 of thiamine (Thi) uptake as a function of the indicated concentrations of substrate. E, as in (D) but 600 rates are as a function of the indicated concentrations of thiamine diphosphate (TDP). F, 601 Competition 602 hydroxymethylpyrimidine (HMP) or the anti-vitamin pyrithiamine (Pyr), or B1 vitamers thiamine 603 (Thi), thiamine monophosphate (TMP) or TDP. TDP uptake activity was determined at a 604 concentration of 5 μM in the presence of a 20-fold excess of competitor. Activities are expressed as 605 a percentage (% activity) compared with the activity measured with TDP alone (control). EV refers 606 to the empty vector. G, as in (F) but measured with spermidine as the competitor. H, Energy 607 requirement of PUT3 uptake activity in yeast. The assays were performed as described in (C) in the 608 absence or presence of 1% (w/v) D-glucose (control). The protonophore carbonyl cyanide 3- 609 chlorophenylhydrazone (CCCP) was added to a final concentration of 100 μM 2 min before the 610 labeled substrate. The data suggest that PUT3 is a proton symporter. For (C)-(H) values shown are 611 the mean ± S.D from ≥ 3 independent experiments. studies with the thiamine precursors hydroxyethylthiazole (HET), 612 613 Figure 2. PUT3 is localized to the phloem. A, Histochemical localization of GUS activity in 614 Arabidopsis. Representative images of Arabidopsis expressing GUS under the control of the 615 promoter of PUT3: 7-day old whole seedling (left); enlarged image of a mature rosette leaf margin 616 (top center), differentiation zone of a mature root (top right) and 7-day old seedling root tip (bottom 617 right). B, Transverse section of the root of a GUS stained transgenic seedling (left). Arrows point to 618 the position of the phloem, whereas arrowheads indicate the position of the xylem axis. The scale 619 bar represents 50 μm. A scheme of the cross-sectional layers observed is illustrated on the right. 620 621 Figure 3. Transport of B1 vitamers in the phloem requires PUT3. A, Representative HPLC profile 22- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 622 of B1 vitamers in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The 623 commercially available B1 standards (Stds) are thiamine diphosphate (TDP), thiamine 624 monophosphate (TMP) and thiamine (Thi). The chemical derivative pyrithiamine (Pyr) was used as 625 an internal standard. Two pmol of each compound was injected. B, Quantification of the B1 vitamer 626 content of phloem exudates of rmv1 and the complementation line rmv1/pPUT3:PUT3 versus wild- 627 type (WT). Values are the average of four biological replicates with error bars representing SD. 628 Statistically significant changes for total B1 vitamers compared to wild-type were calculated by a 629 two tailed Student’s t-test for P < 0.05 and are indicated by an asterisk. 630 631 Figure 4. Transport of polyamines in the phloem requires PUT3. A, Representative HPLC profile 632 of polyamines in phloem exudates (PEs) collected from Arabidopsis leaf petioles. The 633 commercially available standards (Stds) used are putrescine (Put, 8 pmol), cadaverine (Cad, 2 634 pmol), spermidine (Spd, 40 pmol), spermine (Spm, 4 pmol), the polyamine precursor agmatine 635 (Agm, 636 Hexamethylenediamine (Hdm, 2 pmol) was used as an internal standard. B, Quantification of the 637 polyamine 638 rmv1/pPUT3:PUT3 versus wild-type (WT). C, Quantification of the precursor Agm, the 639 degradation product Dap could not be detected. The values for (B) and (C) are the average of four 640 biological replicates with errors bars representing SD. Statistically significant changes for total 641 polyamines compared to wild-type were calculated by a two tailed Student’s t-test for P < 0.05 and 642 are indicated by an asterisk. 20 pmol) and contents of the degradation phloem exudates product 1,3-diaminopropane from rmv1 and the (Dap, 8 pmol). complementation line 643 644 Figure 5. PUT3 is required for proper root and timely reproductive development in Arabidopsis. A, 645 Representative photographs of 12-day old seedlings illustrating root growth impairment of rmv1 646 compared to the respective wild-type (WT) growing vertically in culture. Complementation of the 647 phenotype is observed in transgenic line rmv1/pPUT3:PUT3. B, Root growth rate of the same lines 648 shown in (A). C, Photographs of the plant lines as in (A) grown on soil 65-days after germination, 649 illustrating the late bolting phenotype of rmv1 compared to WT but which recovers in 650 rmv1/pPUT3:PUT3. D, Bolting time of soil grown lines as indicated. E, Representative 651 photographs of the lines as indicated during the late vegetative stage. Pictures were captured 55- 652 days after germination. F, Number of rosette leaves at the time of bolting illustrating the increased 653 number of leaves in rmv1 but which returns to that of WT in rmv1/pPUT3:PUT3. In the case of (B), 654 (D) and (F), values shown are the mean ± SD from ≥ three independent experiments. Statistically 655 significant changes compared to wild-type were calculated by a two tailed Student’s t-test for P < 656 0.05 and are indicated by an asterisk. In all cases, plants were grown under an 8-hour photoperiod 23- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 657 (150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C. 658 659 Figure 6. Micrografting illustrates the importance of long distance transport mediated by PUT3. 660 Representative pictures of micrografts of scion and rootstock of the wild-type (WT) and rmv1 661 genetic combinations as indicated. Pictures were captured 7 days after the procedure from 662 micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark 663 at 18°C. The scale bar represents 1 cm. 664 665 Figure 7. PUT3 is required for long distance transport of vitamin B1. A, B1 vitamer levels in shoots 666 (S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the transgenic 667 line rmv1/pPUT3:PUT3. The vitamers measured are thiamine diphosphate (TDP), thiamine 668 monophosphate (TMP) and thiamine (Thi). Values shown are the mean ± SD from ≥ four 669 independent experiments. Significant differences of total B1 vitamer contents were determined by 670 one-way ANOVA (P < 0.05). B, Micrografting of wild-type (WT), rmv1 and the thiamine 671 biosynthesis mutant th1-201 as indicated. Pictures were captured 7 days after the procedure from 672 micrografts grown vertically under 150 µmol photons.m-2 s-1 for 8 hours at 22°C and 16 hours dark 673 at 18°C. The scale bar represents 1 cm. 674 675 Figure 8. PUT3 is required for long distance transport of polyamines. A, Total polyamine levels in 676 shoots (S) and roots (R) of 14-day old seedlings of rmv1 compared to wild-type (WT) and the 677 transgenic line rmv1/pPUT3:PUT3. B-E, Levels of the individual polyamines cadaverine (Cad), 678 putrescine (Put), spermidine (Spd) and spermine (Spm) of the same samples shown in (A). The 679 values shown are the mean ± SD from four biological replicates. Significant differences for each 680 polyamine were determined by a one-way ANOVA (P < 0.05). In all cases, plants were grown 681 under an 8-hour photoperiod (150 µmol photons.m-2 s-1 at 22°C) and 16 hours of darkness at 18°C. 682 683 24- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 684 LITERATURE CITED 685 Ahn IP, Kim S, Lee YH (2005) Vitamin B-1 functions as an activator of plant disease resistance. 686 Plant Physiol 138: 1505-1515 687 Ajjawi I, Rodriguez Milla MA, Cushman J, Shintani DK (2007a) Thiamin pyrophosphokinase is 688 required for thiamin cofactor activation in Arabidopsis. Plant Mol Biol 65: 151-162 689 Ajjawi I, Tsegaye Y, Shintani D (2007b) Determination of the genetic, molecular, and 690 biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1. Arch Biochem Biophys 459: 691 107-114 692 Alcázar R, Altabella T, Marco F, et al (2010) Polyamines, molecules with regulatory functions in 693 planta biotic stress tolerance. Planta 231: 1237-1249 694 Applewhite PB, Kaur-Sawhney R, Galston AW (2000) A role for spermidine in the bolting and 695 flowering of Arabidopsis. Physiol Plant 108: 314-320 696 Bainbridge K, Bennett T, Turnbull C, Leyser O (2006) Grafting. In: Salinas J, Sanchez-Serrano 697 JJ, eds. Arabidopsis Protocols. Humana Press, 39-44 (Methods in Molecular Biology; vol. 323) 698 Bonner J (1940) On the growth factor requirements of isolated roots. Amer J Bot 27: 692-701 699 Bonner J (1942) Transport of thiamine in the tomato plant. Amer J Bot 29: 136-142 700 Boycheva S, Dominguez A, Rolcik J, Boller T, Fitzpatrick TB (2015) Consequences of a deficit 701 in pyridoxal 5'-phosphate (vitamin B6) biosynthesis de novo for hormone homeostasis and root 702 development in Arabidopsis. Plant Physiol 167: 102-117 703 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of 704 protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 705 Chatterjee A, Abeydeera ND, Bale S, et al (2011) Saccharomyces cerevisiae THI4p is a suicide 706 thiamine thiazole synthase. Nature 478: 542-546 707 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated 708 transformation of Arabidopsis thaliana. Plant J 16: 735-743 709 Colinas M, Fitzpatrick TB (2015) Natures balancing act: examining biosynthesis de novo, 710 recycling and processing damaged vitamin B metabolites. Curr Opin Plant Biol 25: 98-106 711 Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional 712 analysis of genes in planta. Plant Physiol 133: 462-469 713 Fariduddin Q, Varshney P, Yusuf M, Ahmad A (2013) Polyamines: potent modulators of plant 714 responses to stress. J Plant Interact 8: 1-16 715 Fitzpatrick TB, Basset GJ, Borel P, et al (2012) Vitamin deficiencies in humans: can plant 716 science help? Plant Cell 24: 395-414 717 Fitzpatrick TB, Thore S (2014) Complex behavior: from cannibalism to suicide in the vitamin B1 718 biosynthesis world. Curr Opin Struct Biol 29: 34-43 25- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 719 Fontaniella B, Mateos JL, Vicente C, Legaz ME (2001) Improvement of the analysis of 720 dansylated derivatives of polyamines and their conjugates by high-performance liquid 721 chromatography. J Chromatography 919: 283-288 722 Frelin O, Agrimi G, Laera VL, et al (2012) Identification of mitochondrial thiamin diphosphate 723 carriers from Arabidopsis and maize. Funct Integr Genomics 12: 317-326 724 Friedman R, Levin N, Altman A (1986) Presence and identification of polyamines in xylem and 725 phloem exudates of plants. Plant Physiol 82: 1154-1157 726 Fujita M, Fujita Y, Luchi S, et al (2012) Natural variation in a polyamine transporter determines 727 paraquat tolerance in Arabidopsis. Proc Natl Acad Sci U S A 109: 6343-6347 728 Galston A, Kaur-Sawhney R (1987) Polyamines as Endogenous Growth Regulators. In: Davies, 729 P. eds Plant hormones and their role in plant growth and development, 280-295 Netherlands: 730 Springer 731 Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A ubiquitin-10 732 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native 733 protein distribution in transient and stable expression studies. Plant J 64: 355-365 734 Havelange A, Lejeune P, Bernier G, Kaursawhney R, Galston AW (1996) Putrescine export 735 from leaves in relation to floral transition in Sinapis alba. Physiol Plant 96: 59-65 736 Hummel I, Couee I, El Amrani A, Martin-Tanguy J, Hennion F (2002) Involvement of 737 polyamines in root development at low temperature in the subantarctic cruciferous species Pringlea 738 antiscorbutica. J Exp Bot 53: 1463-1473 739 Jack DL, Paulsen IT, Saier MH (2000) The amino acid/polyamine/organocation (APC) 740 superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 741 146: 1797-1814 742 Jang SJ, Choi YJ, Park KY (2002) Effects of polyamines on shoot and root development in 743 Arabidopsis seedlings and carnation cultures. J Plant Biol 45: 230-236 744 Jansen G, Wu CL, Schade B, Thomas DY, Whiteway M (2005) Drag and drop cloning in yeast. 745 Gene 344: 43-51 746 Kaur-Sawhney R, Tiburcio AF, Altabella T, Galston AW (2003) Polyamines in plants: an 747 overview. J Cell Mol Biol 2: 1-12 748 Khozaei M, Fisk S, Lawson T, et al (2015) Overexpression of plastid transketolase in tobacco 749 results in a thiamine auxotrophic phenotype. Plant Cell 27: 432-447 750 Kruszewski SP, Jacobs WP (1974) Polarity of thiamin movement through tomato petioles. Plant 751 Physiol 54: 310-311 752 Kumar A, Taylor M, Triburcio AF (1997) Recent advances in polyamine research. Trends Plant 753 Sci 2: 124-130 26- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 754 Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines: essential factors for growth 755 and survival. Planta 228: 367-381 756 Li J, Mu J, Bai J, et al (2013) Paraquat resistant 1, a Golgi-localized putative transporter protein, 757 is involved in intracellular transport of paraquat. Plant Physiol 162: 470-483 758 Machado CR, De Oliveira RL, Boiteux S, Praekelt UM, Meacock PA, Menck CF (1996) Thi1, 759 a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair. 760 Plant Mol Biol 31: 585-593 761 Marsch-Martinez N, Franken J, Gonzalez-Aguilera K, De Folter S, Angenent G, Alvarez- 762 Buylla E (2013) An efficient flat-surface collar-free grafting method for Arabidopsis thaliana 763 seedlings. Plant Methods 9: 14 764 Martin-Tanguy J (2001) Metabolism and function of polyamines in plants: recent development 765 (new approaches). Plant Growth Regul 34: 135-148 766 Minet M, Dufour ME, Lacroute F (1992) Complementation of Saccharomyces cerevisiae 767 auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J 2: 417-422 768 Moulin M, Nguyen GT, Scaife MA, Smith AG, Fitzpatrick TB (2013) Analysis of 769 Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity. Proc Natl Acad Sci USA 770 110: 14622-14627 771 Mulangi V, Chibucos MC, Phuntumart V, Morris PF (2012) Kinetic and phylogenetic analysis 772 of plant polyamine uptake transporters. Planta 236: 1261-1273 773 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco 774 tissue cultures. Physiol Plant 15: 473-497 775 Pommerrenig B, Feussner K, Zierer W, et al (2011) Phloem-specific expression of yang cycle 776 genes and identification of novel yang cycle enzymes in Plantago and Arabidopsis. Plant Cell 23: 777 1904-1919 778 Rapala-Kozik M, Wolak N, Kujda M, Banas AK (2012) The upregulation of thiamine (vitamin 779 B-1) biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is 780 mediated by abscisic acid at the early stages of this stress response. BMC Plant Biol 12. 781 Raschke M, Burkle L, Muller N, et al (2007) Vitamin B1 biosynthesis in plants requires the 782 essential iron sulfur cluster protein, THIC. Proc Natl Acad Sci USA 104: 19637-19642 783 Sood S, Nagar PK (2005) Xylem and phloem derived polyamines during flowering in two diverse 784 rose species. J Plant Growth Regul 24: 36-40 785 Stolz J, Vielreicher M (2003) Tpn1p, the plasma membrane vitamin B-6 transporter of 786 Saccharomyces cerevisiae. J Biol Chem 278: 18990-18996 27- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. 787 Strohm AK, Vaughn LM, Masson PH (2015) Natural variation in the expression of ORGANIC 788 CATION TRANSPORTER 1 affects root length responses to cadaverine in Arabidopsis. J Exp Bot 789 66: 853-862 790 Szydlowski N, Bürkle L, Pourcel L, Moulin M, Stolz J, Fitzpatrick TB (2013) Recycling of 791 pyridoxine (vitamin B6) by PUP1 in Arabidopsis. Plant J. 75: 40-52 792 Tetyuk O, Benning UF, Hoffmann-Benning S (2013) Collection and analysis of Arabidopsis 793 phloem exudates using the EDTA-facilitated method. J Visual Exp 80. 794 Tunc-Ozdemir M, Miller G, Song LH, et al (2009) Thiamin confers enhanced tolerance to 795 oxidative stress in Arabidopsis. Plant Physiol 151: 421-432 796 Turgeon R, Wolf, S (2009) Phloem transport:Cellular pathways and molecular trafficking. Annu 797 Rev Plant Biol 60: 207-221 798 Urano K, Yoshiba Y, Nanjo T, et al (2003) Characterization of Arabidopsis genes involved in 799 biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant Cell Environ 800 26: 1917-1926 801 Vogl C, Klein CM, Batke AF, Schweingruber ME, Stolz J (2008) Characterization of Thi9, a 802 novel thiamine (vitamin B-1) transporter from Schizosaccharomyces pombe. J Biol Chem 283: 803 7379-7389 804 Watson MB, Emory KK, Piatak RM, Malmberg RL (1998) Arginine decarboxylase (polyamine 805 synthesis) mutants of Arabidopsis thaliana exhibit altered root growth. Plant J 13: 231-239 806 Wightman F, Brown R (1953) The effects of thiamin and nicotinic acid on meristematic activity in 807 pea roots. J Exp Bot 4: 184-196 808 Yokota T, Nakayama M, Harasawa I, Sato M, Katsuhara M, Kawabe S (1994) Polyamines, 809 indole-3-acetic-acid and abscisic-acid in rice phloem sap. Plant Growth Regul 15: 125-128 810 Zhao R, Goldman ID (2013) Folate and thiamine transporters mediated by facilitative carriers 811 (SLC19A1-3 and SLC46A1) and folate receptors. Mol Aspects Med 34: 373-385 812 28- Published by www.plantphysiol.org Downloaded from on August 3, 2017 Copyright © 2016 American Society of Plant Biologists. All rights reserved. A F Thiamine 1.2 µM 12 µM 120 µM 0 µM + HET WT + HMP + Pyr CVY4 + TDP + TMP CVY4 + PUT3 + Thi Control outside B EV 0 20 40 60 80 PUT3 Spermidine Control N-term (1) EV C-term (490) D pmol.min-1.OD cells-1 200 160 120 80 40 0 5 10 Time (min) E 150 pmol.min-1.OD cells-1 C pmol vitamer.OD cells-1 120 G inside 0 100 % Activity 100 50 0 0 50 Thi (µM) 100 0 20 40 60 80 100 120 140 % Activity 150 H 100 - Glucose 50 Control + CCCP 0 0 50 100 TDP (µM) 0 20 40 60 80 100 120 % Activity 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) absence or presence of 1% (w/v) D-glucose (control). The protonophore carbonyl cyanide 3Downloaded frominonthe August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights chlorophenylhydrazone (CCCP) was added to a final concentrationreserved. 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. B Epidermis pPUT3:GUS A Cortex Endodermis Phloem Xylem 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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. B 500 TDP 400 TMP Pyr 300 200 PEs Stds 100 0 0 5 10 15 Time (min) 2.0 Thi 20 25 pmol µg protein-1 Fluorescence units A TDP TMP Thi 1.6 * 1.2 0.8 0.4 0.0 WT rmv1 rmv1/ pPUT3: PUT3 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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. B C 25 Dap Agm 500 Spm Put 35 Cad Hdm Spd 250 PEs Stds 0 0 5 10 15 20 Time (min) 25 30 Cad Put Spd 30 25 * 20 Agm Spm pmol µg protein-1 750 pmol µg protein-1 Fluorescence units A 15 10 20 15 10 5 5 0 0 WT rmv1 rmv1/ pPUT3: PUT3 WT rmv1 rmv1/ pPUT3: PUT3 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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 0.5 B Root growth rate (cm day-1) A 0.4 0.3 * 0.2 0.1 D C Bolting time (days) 0.0 100 80 * 60 40 20 rmv1 rmv1/ pPUT3: PUT3 * rmv1 rmv1/ pPUT3 :PUT3 WT 80 75 70 65 60 55 50 45 40 WT F E Rosette leaves at bolting 0 Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 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. Scion WT rmv1 WT rmv1 Rootstock 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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 1600 A pmol g FW-1 1400 TDP TMP Thi B a a 1200 c 1000 Scion WT rmv1 th1-201 WT Rootstock WT rmv1 th1-201 th1-201 rmv1 th1-201 800 600 b d S R WT S R rmv1 b 400 200 0 S R rmv1 pPUT3 :PUT3 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 wildtype (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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. A Total polyamines 500.0 a nmol g FW-1 14.0 a a a b 300.0 C Cad 16.0 a 400.0 B 10.0 10.0 b b 6.0 4.0 100.0 b a SR WT SR SR rmv1 rmv1 pPUT3 :PUT3 0.0 S R S R WT rmv1 350.0 c 6.0 b b 4.0 2.0 SR rmv1 pPUT3 :PUT3 a 0.0 S R S R S R WT rmv1 rmv1 pPUT3 :PUT3 a a 12.0 a a a 10.0 250.0 8.0 200.0 6.0 b 4.0 100.0 2.0 50.0 0.0 a a b 150.0 a 14.0 a 300.0 b Spm 16.0 400.0 d 8.0 b 2.0 0.0 14.0 450.0 12.0 8.0 200.0 500.0 a E Spd 16.0 12.0 b D Put SR WT S R S R 0.0 S R S R S R rmv1 rmv1 WT rmv1 rmv1 pPUT3 pPUT3 :PUT3 :PUT3 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. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Parsed Citations Ahn IP, Kim S, Lee YH (2005) Vitamin B-1 functions as an activator of plant disease resistance. Plant Physiol 138: 1505-1515 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title 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 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title 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 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Alcázar R, Altabella T, Marco F, et al (2010) Polyamines, molecules with regulatory functions in planta biotic stress tolerance. Planta 231: 1237-1249 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Applewhite PB, Kaur-Sawhney R, Galston AW (2000) A role for spermidine in the bolting and flowering of Arabidopsis. Physiol Plant 108: 314-320 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bainbridge K, Bennett T, Turnbull C, Leyser O (2006) Grafting. In: Salinas J, Sanchez-Serrano JJ, eds. Arabidopsis Protocols. Humana Press, 39-44 (Methods in Molecular Biology; vol. 323) Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bonner J (1940) On the growth factor requirements of isolated roots. Amer J Bot 27: 692-701 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bonner J (1942) Transport of thiamine in the tomato plant. Amer J Bot 29: 136-142 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Boycheva S, Dominguez A, Rolcik J, Boller T, Fitzpatrick TB (2015) Consequences of a deficit in pyridoxal 5'-phosphate (vitamin B6) biosynthesis de novo for hormone homeostasis and root development in Arabidopsis. Plant Physiol 167: 102-117 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Chatterjee A, Abeydeera ND, Bale S, et al (2011) Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase. Nature 478: 542-546 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Colinas M, Fitzpatrick TB (2015) Natures balancing act: examining biosynthesis de novo, recycling and processing damaged vitamin B metabolites. Curr Opin Plant Biol 25: 98-106 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133: 462-469 Pubmed: Author and Title Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fariduddin Q, Varshney P, Yusuf M, Ahmad A (2013) Polyamines: potent modulators of plant responses to stress. J Plant Interact 8: 1-16 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fitzpatrick TB, Basset GJ, Borel P, et al (2012) Vitamin deficiencies in humans: can plant science help? Plant Cell 24: 395-414 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fitzpatrick TB, Thore S (2014) Complex behavior: from cannibalism to suicide in the vitamin B1 biosynthesis world. Curr Opin Struct Biol 29: 34-43 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fontaniella B, Mateos JL, Vicente C, Legaz ME (2001) Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography. J Chromatography 919: 283-288 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Frelin O, Agrimi G, Laera VL, et al (2012) Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize. Funct Integr Genomics 12: 317-326 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Friedman R, Levin N, Altman A (1986) Presence and identification of polyamines in xylem and phloem exudates of plants. Plant Physiol 82: 1154-1157 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fujita M, Fujita Y, Luchi S, et al (2012) Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis. Proc Natl Acad Sci U S A 109: 6343-6347 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Galston A, Kaur-Sawhney R (1987) Polyamines as Endogenous Growth Regulators. In: Davies, P. eds Plant hormones and their role in plant growth and development, 280-295 Netherlands: Springer Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J 64: 355-365 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Havelange A, Lejeune P, Bernier G, Kaursawhney R, Galston AW (1996) Putrescine export from leaves in relation to floral transition in Sinapis alba. Physiol Plant 96: 59-65 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Hummel I, Couee I, El Amrani A, Martin-Tanguy J, Hennion F (2002) Involvement of polyamines in root development at low temperature in the subantarctic cruciferous species Pringlea antiscorbutica. J Exp Bot 53: 1463-1473 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Jack DL, Paulsen IT, Saier MH (2000) The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146: 1797-1814 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Jang SJ, Choi YJ, Park KY (2002) Effects of polyamines on shoot and root development in Arabidopsis seedlings and carnation cultures. J Plant Biol 45: 230-236 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Jansen G, Wu CL, Schade B, Thomas DY, Whiteway M (2005) Drag and drop cloning in yeast. Gene 344: 43-51 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Kaur-Sawhney R, Tiburcio AF, Altabella T, Galston AW (2003) Polyamines in plants: an overview. J Cell Mol Biol 2: 1-12 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Khozaei M, Fisk S, Lawson T, et al (2015) Overexpression of plastid transketolase in tobacco results in a thiamine auxotrophic phenotype. Plant Cell 27: 432-447 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Kruszewski SP, Jacobs WP (1974) Polarity of thiamin movement through tomato petioles. Plant Physiol 54: 310-311 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Kumar A, Taylor M, Triburcio AF (1997) Recent advances in polyamine research. Trends Plant Sci 2: 124-130 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines: essential factors for growth and survival. Planta 228: 367-381 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Li J, Mu J, Bai J, et al (2013) Paraquat resistant 1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat. Plant Physiol 162: 470-483 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Machado CR, De Oliveira RL, Boiteux S, Praekelt UM, Meacock PA, Menck CF (1996) Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair. Plant Mol Biol 31: 585-593 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Marsch-Martinez N, Franken J, Gonzalez-Aguilera K, De Folter S, Angenent G, Alvarez-Buylla E (2013) An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings. Plant Methods 9: 14 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Martin-Tanguy J (2001) Metabolism and function of polyamines in plants: recent development (new approaches). Plant Growth Regul 34: 135-148 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Minet M, Dufour ME, Lacroute F (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J 2: 417-422 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Moulin M, Nguyen GT, Scaife MA, Smith AG, Fitzpatrick TB (2013) Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity. Proc Natl Acad Sci USA 110: 14622-14627 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Mulangi V, Chibucos MC, Phuntumart V, Morris PF (2012) Kinetic and phylogenetic analysis of plant polyamine uptake transporters. Planta 236: 1261-1273 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473497 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Pommerrenig B, Feussner K, Zierer W, et al (2011) Phloem-specific expression of yang cycle genes and identification of novel yang cycle enzymes in Plantago andDownloaded Arabidopsis. Plant Cell 23: 1904-1919 from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Rapala-Kozik M, Wolak N, Kujda M, Banas AK (2012) The upregulation of thiamine (vitamin B-1) biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response. BMC Plant Biol 12. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Raschke M, Burkle L, Muller N, et al (2007) Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC. Proc Natl Acad Sci USA 104: 19637-19642 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Sood S, Nagar PK (2005) Xylem and phloem derived polyamines during flowering in two diverse rose species. J Plant Growth Regul 24: 36-40 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Stolz J, Vielreicher M (2003) Tpn1p, the plasma membrane vitamin B-6 transporter of Saccharomyces cerevisiae. J Biol Chem 278: 18990-18996 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title 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 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Szydlowski N, Bürkle L, Pourcel L, Moulin M, Stolz J, Fitzpatrick TB (2013) Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis. Plant J. 75: 40-52 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Tetyuk O, Benning UF, Hoffmann-Benning S (2013) Collection and analysis of Arabidopsis phloem exudates using the EDTAfacilitated method. J Visual Exp 80. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Tunc-Ozdemir M, Miller G, Song LH, et al (2009) Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis. Plant Physiol 151: 421-432 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Turgeon R, Wolf, S (2009) Phloem transport:Cellular pathways and molecular trafficking. Annu Rev Plant Biol 60: 207-221 Pubmed: Author and Title CrossRef: Author and Title Google 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 abiotic stress responses and developmental stages. Plant Cell Environ 26: 1917-1926 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Vogl C, Klein CM, Batke AF, Schweingruber ME, Stolz J (2008) Characterization of Thi9, a novel thiamine (vitamin B-1) transporter from Schizosaccharomyces pombe. J Biol Chem 283: 7379-7389 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Watson MB, Emory KK, Piatak RM, Malmberg RL (1998) Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth. Plant J 13: 231-239 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Wightman F, Brown R (1953) The effects of thiamin and nicotinic acid on meristematic activity in pea roots. J Exp Bot 4: 184-196 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 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 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title 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 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.