Download Long distance transport of thiamine (vitamin B1

Plant Physiology Preview. Published on March 22, 2016, as DOI:10.1104/pp.16.00009
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Running head: Long distance transport of thiamine and polyamines
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Address correspondence to: Teresa B. Fitzpatrick
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Department of Botany and Plant Biology,
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University of Geneva,
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1211 Geneva,
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Switzerland
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Telephone: +41-22-3793016
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Email: [email protected]
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Research area: Membranes, Transport and Biogenetics (Secondary area: Biochemistry and
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Metabolism)
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Copyright 2016 by the American Society of Plant Biologists
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Long distance transport of thiamine (vitamin B1) is concomitant with that of
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polyamines in Arabidopsis
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Jacopo Martinis1†, Elisabet Gas-Pascual1†, Nicolas Szydlowski1†, Michèle Crèvecoeur1, Alexandra
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Gisler1, Lukas Bürkle2 and Teresa B. Fitzpatrick1*
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Department of Botany and Plant Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211
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Geneva, Switzerland.
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The Institute of Agricultural Sciences, ETH Zurich, Universitätstrasse 2, 8092 Zurich, Switzerland.
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†
These authors contributed equally.
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One sentence summary: Long distance transport of thiamine is facilitated by PUT3 concomitant
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with that of polyamines and is required for growth and development.
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1
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PPOOA_1191186 and 31003A-141117/1 to T.B.F.) as well as the University of Geneva.
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2
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Athens, GA 30602, U.S.A. (E.G.P.); Miniaturisation pour la Synthèse l'Analyse et la Protéomique,
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Université Lille1 Sciences et Technologies, 59655 Villeneuve d'Ascq Cedex, France (N.S.); Stratec
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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,
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*
Address correspondence to [email protected]
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ABSTRACT
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Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital
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metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is
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taking place predominantly in green tissues and is furthermore limited to plastids. Therefore,
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transport mechanisms are required to mediate the movement of this polar metabolite from source to
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sink tissue to activate key enzymes in cellular energy generating pathways but are currently
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unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for
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diverse aspects of growth and development, the homeostasis of which necessitate long distance
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transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify
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Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical,
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physiological and genetic approaches permitted us to show that PUT3 mediates phloem transport of
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both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution
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of these metabolites is altered with growth and developmental consequences. The pivotal role of
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PUT3 mediated thiamine and polyamine homeostasis in plants and its importance for plant fitness is
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revealed through these findings.
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INTRODUCTION
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Thiamine (vitamin B1) is essential for all organisms being well recognized in its diphosphorylated
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form, thiamine diphosphate (TDP) (Fig. 1A), as a necessary cofactor for key metabolic enzymes
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involved in glycolysis and the citric acid cycle (Fitzpatrick & Thore, 2014). In plants, it is also
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necessary for the Calvin cycle, the biochemical route of carbon fixation (Khozaei et al., 2015).
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Therefore, thiamine is vital for cell energy supply in all organisms. Interestingly, some additional
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roles for thiamine have been proposed that include involvement in responses to DNA damage
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(Machado et al., 1996), as well as the activation of plant defenses, resistance to pathogen attack and
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attenuation of environmental stress responses (Ahn et al., 2005, Rapala-Kozik et al., 2012). Humans
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cannot produce thiamine de novo with plants representing one of the most important sources of the
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compound in the human diet. However, deficiency remains a critical global problem causing
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detrimental neurological effects and cardiovascular problems particularly among populations that
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rely on a single crop for sustenance, e.g. polished rice (Fitzpatrick et al., 2012). Thiamine is formed
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by
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(hydroxyethylthiazole (HET) and hydroxymethylpyrimidine (HMP), respectively) (Supplemental
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Fig. S1) and is later diphosphorylated to the cofactor form, TDP (reviewed in Fitzpatrick & Thore,
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2014). Several previously elusive steps of thiamine biosynthesis de novo have been resolved
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recently, one of which involves an unexpected suicide mechanism, where the catalytic protein
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donates sulfur to the molecule in a single turnover reaction (Chatterjee et al., 2011) (Supplemental
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Fig. S1). Remarkably, the majority of the enzymes involved in biosynthesis de novo are exclusively
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found in the chloroplast, the exception being the kinase that diphosphorylates thiamine to TDP,
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which is in the cytosol (Ajjawi et al., 2007a). Accordingly, the thiamine biosynthetic genes are
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strongly expressed in green tissues but only at a very low level (if at all) in other non-
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photosynthetic tissues such as roots (Colinas & Fitzpatrick, 2015). However, thiamine is essential in
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all actively dividing meristematic stem cells and organ initial cells (Wightman & Brown, 1953) and
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thus in non-photosynthetic organs such as the roots. It is therefore relevant that a physiological
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study in 1974 (Kruszewski & Jacobs, 1974) showed that thiamine moves through petiolar sections
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of tomato with strong basipetal polarity and with kinetics similar to auxin and giberellin (at a
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velocity of 3-5 mm per hour). Indeed, in the 1940s, thiamine was considered to fulfil the
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requirements of a hormone in higher plants (Bonner, 1940, Bonner, 1942). This was due to the
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observation that in order to grow aseptically, excised roots of many species require thiamine in
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small amounts. Moreover, such observations were considered as confirmation of an hypothesis that
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in the intact plant, thiamine is biosynthesized in the leaves and moves from there to the roots, which
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cannot biosynthesize it sufficiently but need it for development. As thiamine and its phosphorylated
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derivatives (commonly referred to as vitamin B1) are cationic molecules (i.e. charged) at
the
condensation
of
the
two
separately
biosynthesized
heterocycle
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moieties
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physiological pH, the existence of transporters that can mediate their trafficking therefore become
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apparent. Although the characterization of a homolog of the yeast mitochondrial transporter was
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reported recently (Frelin et al., 2012), it remains uncharacterized in planta, while the proteins
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participating in long distance transport of thiamine in plants have remained elusive.
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Analogous to thiamine, polyamines are cationic molecules at physiological pH essential for the
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growth and survival of all organisms. The diverse range of activities include regulation of cell
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division, developmental processes such as those of root formation and flowering initiation, as well
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as environmental stress responses (Alcázar et al., 2010, Kumar et al., 1997, Kusano et al., 2008).
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Key representative molecules include putrescine (Put), spermidine (Spd) and spermine (Spm), all
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three of which are derived from the amino acid arginine, via the intermediate agmatine (Agm), as
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well as cadaverine (Cad) that is derived from lysine (Supplemental Fig. S1). Akin to other essential
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multifunctional plant metabolites, polyamine contents must be carefully controlled to maintain
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cellular homeostasis. This is achieved through biosynthesis, degradation, conjugation (to phenolic
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acids, proteins, nucleic acids, membrane structures) and transport (Martin-Tanguy, 2001). Although
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polyamine metabolism has been studied extensively, little is known of its transport in plants with
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the exception of the recently identified PUT family (Fujita et al., 2012, Mulangi et al., 2012) and
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the OCT1 transporter from Arabidopsis (Arabidopsis thaliana) that is implicated in Cad transport
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(Strohm et al., 2015). The PUT family of proteins are localized at the cellular level to various
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compartments including the plasma membrane, Golgi apparatus and endoplasmic reticulum (Fujita
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et al., 2012, Li et al., 2013). However, polyamine homeostasis is also regulated by long distance
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transport and indeed their presence in the vascular system of plants has been established in several
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species (Friedman et al., 1986, Pommerrenig et al., 2011, Sood & Nagar, 2005, Yokota et al.,
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1994). To date nothing is known on the components of intercellular or long distance transport of
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polyamines.
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Here, a yeast-based screen allowed us to identify Arabidopsis PUT3 as a thiamine transporter.
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Expression studies showed that PUT3 is localized to the phloem tissue in Arabidopsis and loss of
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function studies showed that PUT3 is required for growth and development. A series of
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micrografting and biochemical experiments demonstrated that PUT3 is important for long distance
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transport of both thiamine and polyamines and thereby revealed its importance for homeostasis of
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these essential molecules and maintenance of plant fitness.
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RESULTS
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Identification of an Arabidopsis thiamine transporter
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This study was initiated by performing a complementation screen employing a cDNA library from
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Arabidopsis (Minet et al., 1992) with the yeast mutant CVY4, deficient in both the biosynthesis and
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the transport of thiamine (Vogl et al., 2008). This strain grows either in the presence of strong
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thiamine supplementation (120 µM) or can be complemented with the yeast thiamine biosynthesis
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gene THI4 (Vogl et al., 2008). In line with this, several clones harboring the ortholog of the latter
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from Arabidopsis (THI1) were found to complement the growth phenotype even in the absence of
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thiamine supplementation in our screen. On the other hand, a single Arabidopsis locus (At5g05630)
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encoding a protein belonging to the amino acid permease family was found to restore growth in the
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presence of low levels of thiamine (1.2 µM), thus complementing the impairment in thiamine
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transport in this yeast strain (Fig. 1A). This protein has been previously reported to facilitate the
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uptake of polyamines in a yeast based study and was accordingly named POLYAMINE UPTAKE
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TRANSPORTER 3 (PUT3) (Mulangi et al., 2012) and mediated paraquat uptake in Arabidopsis for
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which it was given the name RESISTANT TO METHYL VIOLOGEN 1 (RMV1)(Fujita et al.,
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2012). The PUT3/RMV1 locus, which we will refer to as PUT3 from here on, encodes a protein of
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490 amino acid residues and is predicted to have 12 transmembrane domains with the N- and C-
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terminal hydrophilic domains residing on the inside of the membrane (Fig. 1B). It has 4 paralogs in
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Arabidopsis, At1g31820 (68% identity), At1g31830 (63% identity), At3g19553 (52% identity) and
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At3g13620 (43% identity), respectively. None of the latter paralogs were found in our screen and
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presumably cannot transport thiamine (at least in yeast) under the conditions used. Additional close
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homologs of PUT3 were identified in several plants including Vitis vinifera (grapevine), Populus
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trichocarpa (poplar), Oryza sativa (rice), Zea mays (corn), Sorghum bicolor (Sorghum), as well as
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Physcomitrella patens (moss), while weaker homologs could be identified in Chlorella variabilis
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(green algae). Notably, an extensive recent phylogenetic study hypothesized that this family of
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transporters was acquired by horizontal transfer from green algae (Mulangi et al., 2012).
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Kinetics of PUT3 facilitated thiamine transport
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The kinetics of thiamine transport by PUT3 in yeast was characterized using the tritiated vitamers,
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[3H]-thiamine and [3H]-TDP. Note, vitamers are related chemical substances that fulfil the same
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specific vitamin function. Uptake occurred at an optimal pH of 5.5 (Supplemental Fig. S2A). Both
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vitamers could be transported at similar rates (Fig. 1C). In addition, the vitamers displayed similar
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Michaelis-Menten parameters (Vmax:169.3 ± 12.3 pmol min-1.OD cells-1, KM:43.3 ± 7.7 µM for
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thiamine and Vmax:146.5 ± 13.7 pmol min-1.OD cells-1, KM of 34.2 ± 8.6 µM for TDP, respectively
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(Fig. 1D and 1E)). Competition experiments showed that B1 vitamers, TDP, thiamine
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monophosphate (TMP) and thiamine were able to compete for TDP transport, whereas HET or
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HMP (the thiamine biosynthesis precursors) were not (Fig. 1F). Interestingly, the thiamine
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antagonist, pyrithiamine, also competed with TDP uptake (Fig. 1F). As anticipated, the polyamine
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Spd, shown previously to be transported by PUT3 in a yeast based assay (Mulangi et al., 2012),
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competed strongly with TDP transport (Fig. 1G), indicating that thiamine uses the same transport
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pathway as polyamines. The withdrawal of glucose or addition of the protonophore,
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carbonylcyanide-3-chlorophenylhydrazone, abolishes the transport activity, suggesting that
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transport occurs via H+ symport (Fig. 1H). Since PUT3 was originally annotated as a putative
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amino acid transporter (Jack et al., 2000), we also tested for competition of TDP uptake in the
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presence of unlabeled cyclic amino-acids, but none showed significant modulation of transport
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(Supplemental Fig. S2B). In the same context and as the human thiamine transporter belongs to the
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folate transporter family (Zhao & Goldman, 2013), we also tested folate, but it did not compete for
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TDP transport (Supplemental Fig. S2B). Similar results were obtained when the competition studies
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were performed with thiamine instead of TDP. It can therefore be concluded that PUT3 is a
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polyspecific transporter than mediates thiamine as well as polyamine transport, at least in yeast.
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PUT3 is localized to the phloem and mediates transport of B1 vitamers
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Given the demonstrated ability of PUT3 to import thiamine/TDP in yeast, we wanted to establish its
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localization in planta, to complement the previous cellular localization studies of this protein (Fujita
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et al., 2012). To this end, we generated plants expressing the β-GLUCURONIDASE (GUS)
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reporter gene under control of the 850 base-pair region immediately upstream of the transcriptional
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start site of PUT3 (pPUT3-GUS). Representative pictures of 20 independent transgenic lines
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analyzed are shown in Fig. 2A. In all lines examined, PUT3 promoter driven GUS activity was
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predominant in the vascular tissue of leaves and the central cylinder of roots. In particular, two
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strands of GUS activity could be observed in the central stele of the differentiation region of the
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root. Moreover, transverse sections of the root tissue established localization to the phloem tissue
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(Fig. 2B). Weak GUS activity was also observed in the anthers of flowers, as well as the base and
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tip of mature siliques (Supplemental Fig. S3). At the subcellular level, fusion of the full-length
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PUT3 protein sequence to YFP and examination of the corresponding fluorescence upon transient
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expression in Arabidopsis mesophyll protoplasts confirmed the exclusive location of this protein to
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the plasma membrane (Supplemental Fig. S4) in agreement with a previous report (Fujita et al.,
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2012).
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Since B1 vitamers need to be translocated from sites of biosynthesis (source) to sites where they
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cannot be biosynthesized but are required (sink), we were prompted to investigate if they are found
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in the phloem sap. Indeed, the three B1 vitamers could be detected in the phloem sap of Arabidopsis
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(Fig. 3A) and thereby implies that a transport system is in place for translocation into this tissue. It
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is worth noting that the ratio between TDP and either TMP or thiamine in the phloem sap is
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drastically reduced when compared with extracts from whole rosette leaves (Supplemental Fig. S5).
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In order to test the contribution of PUT3 to the vitamin B1 transport process, the B1 vitamer profiles
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of phloem exudates from wild-type plants were compared with that of the previously characterized
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T-DNA insertion mutant line rmv1 (GT_3_3436) (Fujita et al., 2012). Downregulation of PUT3
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expression was confirmed by real-time quantitative PCR (qPCR) (Supplemental Fig. S6). Indeed,
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the total vitamin B1 content was decreased in the rmv1 mutant phloem exudates compared to that of
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the wild-type (Fig. 3B). Importantly, B1 vitamer contents were restored to wild-type levels in
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phloem exudates of rmv1 carrying the PUT3 transgene under control of its own promoter (Fig. 3B,
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rmv1/pPUT3:PUT3). These observations suggest that PUT3 is involved in transport of vitamin B1
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in the phloem.
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PUT3 also mediates phloem transport of polyamines
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The localization of PUT3 to the phloem tissue and its previous reported ability to facilitate
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polyamine transport, at least in yeast (Mulangi et al., 2012), prompted us to next investigate the
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polyamine profiles of phloem exudates from Arabidopsis. In particular, we adapted a protocol
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(Fontaniella et al., 2001) to profile the diamines Cad and Put, the triamine Spd, the tetramine Spm,
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as well as the Put/Spd/Spm precursor Agm. All of these compounds could be detected in phloem
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exudates from Arabidopsis (Fig. 4A). Put was the most abundant polyamine detected followed by
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Spd and Cad, while Spm accumulated at much lower levels (Fig. 4B). Significantly, there was a
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considerable decrease in the levels of Put, in particular, in phloem exudates of rmv1 (Fig. 4B).
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Importantly, the polyamine profile was restored to that of wild-type in phloem exudates of rmv1
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carrying the PUT3 transgene under control of its own promoter (Fig. 4B, rmv1/pPUT3:PUT3),
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validating the role of PUT3 in this molecular phenotype. Taken together, this implies that PUT3 is
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required for transport of both polyamines and vitamin B1 in phloem tissue.
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Alterations in growth and developmental processes are associated with PUT3
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As B1 vitamers and polyamines are essential for development in plants, we next examined the
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phenotypic consequences of loss of function of PUT3 on plant growth. Notably, previous studies on
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PUT3 in Arabidopsis were restricted to quantifying its association with paraquat resistance (Fujita
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et al., 2012). Interestingly, here we observed that the rmv1 mutant showed stunted growth under
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standard growth conditions that was not reported on in previous studies (Fig. 5A), and was
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corroborated by a slower rate of root growth compared to wild-type (Fig. 5B). Furthermore, rmv1
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entered the reproductive phase later than wild-type (Fig. 5C), bolting at day 67 instead of day 62
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(Fig. 5D), concomitant with an increase in the number of rosette leaves at this developmental stage
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(Fig. 5E and 5F). The observed phenotypes were most pronounced under an 8-hour photoperiod and
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were significantly attenuated under longer photoperiods (data not shown). All of the growth and
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developmental phenotypes were abrogated in rmv1 upon introduction of the PUT3 transgene (Fig.
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5A-5F, rmv1/pPUT3:PUT3). It must therefore be concluded that PUT3 is required for proper
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growth and development in Arabidopsis.
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Long distance transport of a shoot-derived metabolite by PUT3 is responsible for the root
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development phenotype in rmv1
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To further probe the growth and development phenotype of rmv1 and the possible physiological
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relevance of PUT3 in long distance transport, we performed a series of grafting experiments.
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Notably, grafted plants with the same genetic combinations exhibited the same phenotypes as non-
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grafted plants, i.e. grafting of an rmv1 scion onto its corresponding rootstock (rmv1/rmv1) led to
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retention of the stunted growth phenotype compared to grafting of the wild-type scion onto its
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corresponding rootstock (wild-type/wild-type) (Fig. 6). Remarkably, grafting of the wild-type scion
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onto the rmv1 rootstock (wild-type/rmv1) led to recovery of the stunted root growth phenotype to
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that of the corresponding grafted wild-type plants (wild-type/wild-type) (Fig. 6). On the other hand,
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the reciprocal graft of the rmv1 scion onto the wild-type rootstock (rmv1/wild-type) did not lead to
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recovery of root growth impairment (Fig. 6). Indeed, root growth was even further impaired than
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rmv1/rmv1. This data supports the notion that PUT3 is required for long distance transport of a
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shoot-derived metabolite that is essential for proper root growth.
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Shoot to root partitioning of vitamin B1 as a function of PUT3
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Given the implication of PUT3 in long distance transport, we next sought to obtain a molecular
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explanation for the growth impairment phenotypes observed. In the first instance, we therefore
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examined shoot and root partitioning of B1 vitamers in seedlings. Intriguingly, B1 vitamer levels in
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shoots and roots (albeit to a much lesser extent) of rmv1 were lower than those of wild-type (Fig.
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7A). While the slight depletion in root tissue (sink tissue) is consistent with the role of PUT3 in
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long distance transport of B1 vitamers from source to sink, we expected a corresponding
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accumulation of B1 vitamers in shoots (source tissue) rather than a depletion. Interestingly, rmv1
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does not show the well-characterized pale shoot phenotype associated with a deficiency in B1
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vitamers (Ajjawi et al., 2007a, Ajjawi et al., 2007b, Raschke et al., 2007). Nonetheless, levels of B1
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vitamers in shoots and roots of rmv1 recovered upon reintroduction of the PUT3 transgene (Fig.
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7A). To provide evidence that shoot-derived vitamin B1 is important for root growth in particular,
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another series of grafting experiments was performed with the Arabidopsis mutant line th1-201
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(Ajjawi et al., 2007b). In this line, the THIAMIN SYNTHASE enzyme TH1 (encoded at locus
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At1g22940), which condenses the thiazole and pyrimidine heterocycles to form TMP, is impaired,
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preventing thiamine biosynthesis de novo and resulting in overall stunted shoot and root growth and
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pale leaves. Remarkably, grafting of the wild-type scion onto the th1-201 rootstock (wild-type/th1-
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201) led to recovery of the stunted root growth phenotype that is observed with th1-201 (Fig. 7B).
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However, grafting of the rmv1 scion onto the th1-201 rootstock (rmv1/th1-201) did not lead to
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recovery of the short-root phenotype in th1-201. Indeed, root growth was even more impaired than
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for the th1-201/th1-201 genetic combination, corroborating the notion that loss of function of PUT3
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in the shoot, in particular, has a negative impact on root growth (Fig. 7B). Taken together, the data
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demonstrate that long distance transport of B1 vitamers from the shoot is necessary for root growth
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and that impairment of PUT3 in the shoot has a negative impact on root growth.
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Shoot to root partitioning of polyamines as a function of PUT3
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Given that PUT3 is also implicated in polyamine transport, we next looked at shoot to root
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partitioning of polyamine contents. In the first instance, we noted that the total polyamine content is
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very similar between shoots and roots in wild-type plants (Fig. 8A). A significant reduction of total
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polyamine levels was observed in the roots of rmv1, whereas shoot content remained similar to that
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of wild-type (Fig. 8A). Notably, levels of root polyamines recovered upon reintroduction of the
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PUT3 transgene in rmv1 (Fig. 8A). Intriguingly, a closer look at the polyamines measured indicates
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that the predominant polyamine in shoots and roots is Spd and that it is mainly this polyamine that
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is depleted in roots of rmv1 (Fig. 8B-8E). This is in contrast to the polyamine profile of phloem
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exudates, where Put was the predominant polyamine detected and the one most depleted in rmv1
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(Fig. 4B).
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DISCUSSION
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A fundamental understanding of phloem transport with its important role in plant development,
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reproduction, signaling and growth is central to plant biology. This is further emphasized by the
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core set of compounds, such as biological cofactors and growth regulators, required in all living
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cells to drive key metabolic processes, the biosynthesis of which may be restricted to certain organs
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and therefore must be transported to the site of requirement. As central components of metabolic
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function, the supply of many of these molecules must be strictly regulated, such that provision is
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coordinated with metabolic needs. However, despite their critical roles, surprisingly little is known
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on the intercellular transport of such molecules in plants. In this study, we demonstrated the
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importance of long distance transport of vitamin B1 and polyamines as mediated by the phloem
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localized transporter PUT3. Disruption of PUT3 led to a deficit of B1 vitamers and polyamines in
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phloem exudates that perturbed the homeostasis of these compounds in the organs where they are
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required resulting in a negative impact on growth and development in Arabidopsis.
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Long distance transporters of the vital cofactor vitamin B1 have been supposed for almost a
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century (Bonner, 1940, Bonner, 1942), yet the proteins carrying out these processes and evidence
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for the presence of this family of compounds in the vascular system have remained elusive. Only a
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single transporter has been reported for vitamin B1 in plants and operates at the subcellular level in
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the mitochondrial membrane (Frelin et al., 2012). Polyamines, on the other hand, were detected in
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the phloem sap several decades ago (Friedman et al., 1986). While they can be biosynthesized to a
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certain extent in the phloem (Pommerrenig et al., 2011), the factors facilitating transport of these
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important cationic molecules in the vascular system to maintain homeostasis were unknown. Here
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we have demonstrated the involvement of PUT3 in these transport processes in plants. Although
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PUT3 was previously implicated in subcellular polyamine transport based on a yeast
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complementation assay (Mulangi et al., 2012), as well as paraquat transport in Arabidopsis (Fujita
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et al., 2012), it went unnoted that it is localized to the phloem and mediates long distance transport.
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Here we have shown that the affinity of PUT3 for B1 vitamers is similar to those reported for
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polyamines and paraquat, i.e. low μM range (Fujita et al., 2012), classifying it as a polyspecific
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high affinity cationic transporter. Indeed, exogenous thiamine reportedly enhances tolerance to
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paraquat (Tunc-Ozdemir et al., 2009), supporting the notion that all of these compounds use the
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same trafficking pathway. The functional significance of PUT3 for phloem transport of B1 vitamers
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and polyamines became evident from the deficit of these compounds in the phloem exudates of the
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PUT3 mutant line rmv1, previously characterized because of its resistance to paraquat (Fujita et al.,
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2012). The implication of PUT3 in these transport processes is validated upon restoration of the B1
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vitamer and polyamine contents in phloem exudates of the transgenic rmv1 line expressing PUT3
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under the control of its own promoter sequence (rmv1/pPUT3:PUT3). We acknowledge that the
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
684
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Ahn IP, Kim S, Lee YH (2005) Vitamin B-1 functions as an activator of plant disease resistance.
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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
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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.
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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.
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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.
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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
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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.
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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.
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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.
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