Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Tobacco NUP1 transports both tobacco alkaloids and vitamin B6 Keita Kato a, Nobukazu Shitan b, Tsubasa Shoji a, Takashi Hashimoto a,⇑ a b
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0192, Japan Kobe Pharmaceutical University, Motoyama Kitamachi 4-19-1, Higashinada, Kobe, Hyogo 658-8558, Japan
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Nicotiana tabacum Solanaceae Tobacco Transport assays Transporter Nicotine Vitamin B6 PUP-family transporters
a b s t r a c t The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A Nicotiana tabacum (tobacco) PUP-family transporter, nicotine uptake permease 1 (NtNUP1), was previously shown to transport tobacco alkaloids and to affect both nicotine biosynthesis and root growth in tobacco plants. Since Arabidopsis PUP1, which belongs to the same subclade as NtNUP1, was recently reported to transport pyridoxine and its derivatives (vitamin B6), it was of interest to examine whether NtNUP1 could also transport these substrates. Direct uptake measurements in the yeast Saccharomyces cerevisiae demonstrated that NtNUP1 efficiently promoted the uptake of pyridoxamine, pyridoxine, anatabine, and nicotine. The naturally occurring (S)-isomer of nicotine was preferentially transported over the (R)-isomer. Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1, transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences. Therefore, the previously reported effects of NtNUP1 on tobacco physiology might involve bioactive metabolites other than tobacco alkaloids. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nicotine (1) (Fig. 1) and its related pyridine alkaloids occur mainly in the Nicotiana genus and contribute to the chemical defense against insects (Shoji and Hashimoto, 2013). In Nicotiana tabacum (tobacco), these alkaloids are exclusively synthesized in the root, translocated to the aerial parts via the xylem, and stored in the vacuoles of leaf cells. Since berberine bridge enzyme-like oxidoreductases, which catalyze the last or a late step of nicotine (1) biosynthesis, are localized in the vacuoles, nicotine (1) is thought to be formed in the vacuoles (Kajikawa et al., 2011). Nicotine (1) thus appears to travel from its site of synthesis in the root vacuole to its distant storage site in the leaf vacuole, via the root cytosol, root apoplast, xylem sap, leaf apoplast, and leaf cytosol. Besides this long-distance transport within the plant, tobacco alkaloids and their biosynthetic intermediates may be transported across endomembranes of cellular organelles or plasma membranes. Multidrug and toxic compound extrusion (MATE)-type transporters sequester nicotine (1) in the vacuoles of tobacco roots, thereby decreasing cytoplasmic its toxicity during active nicotine (1) synthesis in the root (Shoji et al. 2009). Another MATE-type ⇑ Corresponding author. Tel.: +81 743 72 5520; fax: +81 743 725529. E-mail address: [email protected] (T. Hashimoto).
transporter is proposed to sequester nicotine (1) into the vacuoles of the leaf (Morita et al. 2009). A plasma membrane-localized nicotine uptake permease (NtNUP1) was recently reported to import nicotine (1) from the apoplastic space, particularly in root tips (Hildreth et al. 2011). NtNUP1 belongs to a plant-specific class of purine permease-like transporters, which is classified into several subclades (Supplemental Fig. S1). RNAi-mediated suppression of NtNUP1 in tobacco hairy root cultures caused nicotine (1) levels in the culture medium to increase and those in the root cells to decrease, consistent with the nicotine (1) uptake activity of NtNUP1 (Hildreth et al. 2011). Surprisingly, the total nicotine (1) content in the leaves and roots of NtNUP1-suppressed tobacco plants was significantly reduced, and the roots of NtNUP1-suppressed seedlings grew better than those of the control (Hildreth et al. 2011). When tobacco plants with highly reduced nicotine (1) levels were generated by suppressing expression of its biosynthesis genes, growth promotion of these transgenic plants was not observed (e.g., Chintaparkorn and Hamill, 2003; Xie et al. 2004). The requirement of NtNUP1 for optimal nicotine (1) biosynthesis and the inhibitory effect of NtNUP1 on root growth (Hildreth et al. 2011) cannot readily be explained by its transport activity for nicotine (1). NtNUP1 belongs to the same subclade as Arabidopsis thaliana PUP1 in the PUP family of transporters (Supplemental Fig. S1).
http://dx.doi.org/10.1016/j.phytochem.2014.05.011 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
2
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
PUP1 was initially discovered as a plant transporter that complemented a yeast mutant deficient in adenine transport (Gillissen et al. 2000), and was shown to import adenine, cytosine, cytokinins, and related metabolites from the apoplast into the cytoplasm (Bürkle et al. 2003). More recently, PUP1 was shown to be a high affinity transporter of vitamin B6, which includes pyridoxine (6), pyridoxamine (5), and pyridoxal (7) (Szydlowski et al. 2013). The rather relaxed substrate specificity of PUP1 prompted us to investigate whether NtNUP1 transports vitamin B6 in addition to tobacco alkaloids. A better knowledge of NtNUP1 substrate preference is important for understanding the effects of NtNUP1 on plant metabolism and physiology.
2. Results 2.1. NtNUP1 transport assays in yeast Previously, Schizosaccharomyces pombe cells expressing NtNUP1 cDNA were used to measure the uptake of radioactive nicotine (1) into cells, and the inhibition of nicotine (1) uptake in the presence of excess amounts of non-radioactive competitors, such as other tobacco alkaloids and tropane alkaloids (Hildreth et al. 2011). Here, NtNUP1 cDNA was expressed in Saccharomyces cerevisiae and the uptake of various metabolites was assayed by directly measuring their cellular contents using high-performance liquid chromatography (HPLC) or gas chromatography (GC). As an initial experiment, a C-terminal green fluorescent protein (GFP) fusion of NtNUP1 (i.e., NtNUP1-GFP) was expressed in S. cerevisiae to analyze the subcellular localization of NtNUP1 (Fig. 1a). NtNUP1-GFP was localized mainly to the plasma membrane and to endomembranes, possibly the tonoplast. Substantial localization of NtNUP1-GFP to the plasma membrane suggests the feasibility of conducting uptake assays using transgenic yeast cells. Transient expression of NtNUP1-GFP in onion epidermal cells indicated that NtNUP1 was localized exclusively to the plasma membrane (Supplemental Fig. S2), in agreement with the results of a previous report (Hildreth et al. 2011). Next, S. cerevisiae cells expressing either an empty vector (control) or NtNUP1 cDNA was exposed to 250 lM of various plant metabolites (except for anatabine (2), which was administered at 50 lM), and the test metabolites was measured in yeast cells after a 4-h incubation period (Fig. 1b and c). (20 S)-Nicotine (1a) (the stereoisomer synthesized in tobacco plants) and anatabine (2) were taken up efficiently by NtNUP1expressing yeast cells, to reach a cellular concentration of ca. 0.16–0.18 lmol g1 fr. wt, whereas the contents of these alkaloids in the control cells were less than 0.02 lmol g1 fr. wt. Uptake of hyoscyamine (3) (atropine) and scopolamine (4) by the NtNUP1expressing cells was significantly enhanced (P < 0.05 and P < 0.01, respectively; Student’s t-test) compared to the control cells, but the NtNUP1-mediated accumulation of these tropane alkaloids was low (less than 0.03 lmol g1 fr. wt). The flavonoid rutin (10) was not taken up by either NtNUP1-expressing or control yeast cells. These uptake properties of NtNUP1 in S. cerevisiae are similar to those reported in S. pombe (Hildreth et al. 2011). Whether vitamin B6 could be transported by NtNUP1-expressing yeast cells, as reported for PUP1-expressing yeast cells (Szydlowski et al. 2013), was next investigated, Pyridoxine (6) and pyridoxamine (5) were both highly efficiently taken up by the NtNUP1-expressing cells, compared to control cells, reaching a concentration of 1.0 lmol g1 fr. wt during a 4-h incubation period (Fig. 1b and c). Uptake of pyridoxal (7), another form of vitamin B6, by NtNUP1 was significant (P < 0.01; Student’s t-test) but low (ca. 0.03 lmol g1 fr. wt). These results show that NtNUP1 mediates the efficient uptake of some vitamin B6 derivatives, in addition to tobacco alkaloids, when expressed in yeast cells.
To estimate initial uptake rates, tritium-labelled pyridoxine (6) and nicotine (1) at 50 lM were administered for 3 min. Yeast cells expressing either PUP1 or NtNUP1 were used to compare substrate preferences of these transporters. Since expression levels of functional PUP1 and NtNUP1 at the plasma membrane may be different, it is meaningful to compare uptake rates of two substrates in a particular yeast strain, but not to compare uptake rates of one substrate between two yeast strains. As shown in Table 1, both pyridoxine (6) and nicotine (1) were efficiently taken up by PUP1 and NtNUP1. PUP1’s ability to transport nicotine (1) has been inferred from an uptake competition study in yeasts (Gillissen et al. 2000). These results herein show that PUP1 prefers pyridoxine (6) over nicotine (1), whereas NtNUP1 transports nicotine (1) more efficiently than pyridoxine (6), under the short-term uptake conditions. Whether NtNUP1 displayed selectivity toward stereoisomers of nicotine (1) was also tested. (20 S)-Nicotine (1a) and (20 R)-nicotine (1b) (both at 125 lM) were mixed at a 1:1 ratio and supplied to the culture medium. After 4-h incubation, the nicotine (1) taken up by the yeast cells was analyzed using a chiral GC column, which separated the stereoisomers as distinct peaks (Fig. 2a). The amount of nicotine (1) taken up by the vector control cells was too low to be analyzed by this method. The nicotine (1) present in yeast cells expressing NtNUP1 or NtNUP1-GFP though was greatly enriched in the (S) isomer, (1a) representing ca. 81% of the total nicotine (1) present (Fig. 2b). Thus, NtNUP1 preferentially transports the naturally occurring isomer of (20 S)-nicotine (1a) over the unnatural (20 R) (1b) form.
2.2. NtNUP1 transport assays in tobacco BY-2 cells To validate the transport properties of NtNUP1, NtNUP1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in cultured tobacco BY-2 cells. Tobacco alkaloids (mainly anatabine (2)) are biosynthesized in BY-2 cell cultures only after jasmonate elicitation (Shoji and Hashimoto, 2008). In the present study, it was found that the expression level of NtNUP1 in BY-2 cells was low when the cells were cultured in the absence of jasmonates, but increased several fold after jasmonate treatment (Fig. 3a), and that alkaloid biosynthesis, as evidenced by robust induction of an alkaloid biosynthetic gene, PUTRESCINE N-METHYLTRANSFERASE (PMT), was induced by addition of methyl jasmonate (9) (MeJA; Fig. 3b). NtNUP1-mediated transport activities can thus be estimated by comparing the activities in NtNUP1-overexpressing cells with those in wild-type or vector-transformed cells, cultured in the absence of jasmonates. Quantitative RT-PCR showed that two independent BY-2 cell lines transformed with the CaMV 35S pro:: NtNUP1 vector had NtNUP1 transcript levels that were ca. 27-fold (OX10 line) or 64-fold (OX18 line) higher than those in the wild-type or vector control cell lines (Fig. 4a). Two tobacco BY-2 cell lines was also generated that overexpressed PUP1 (Fig. 4b), to compare the substrate specificity of NtNUP1 and PUP1. Tobacco cells were cultured in the presence of test compounds (50 lM), and the cellular concentrations of these compounds were measured after a 4-h period. The NtNUP1overexpressing tobacco cells took up (20 S)-nicotine (1a) and anatabine (2) more efficiently than did the wild-type and vector control cells, whereas the PUP1-overexpressing cells did not accumulate (20 S)-nicotine (1a) (Fig. 5a). In this uptake assay, NtNUP1 transported greater amounts of anatabine (2) (3–4 lmol g1 dry wt) than of nicotine (1) (0.5–0.6 lmol g1 dry wt). Tropane alkaloids were transported by NtNUP1 at low efficiency (ca. 0.2 lmol g1 dry wt for hyoscyamine (3), and less than 0.02 lmol g1 dry wt for scopolamine (4); data not shown). Rutin (10) was not transported by NtNUP1.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
3
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
GFP
(a)
Bright field
Merge
NtNUP1-GFP
(b)
pyridoxine (6)
pyridoxal (7)
**
hyoscyamine (3)
0.0
0.0
0.0
**
VC
0
NtNUP1
0
VC
1
*
0.1
**
0.0
2
* 1
0.2
pyridoxal (7)
pyridoxine (6) 2
NtNUP1
2
0.2
VC
0.1
NtNUP1
0.1
VC
0.1
NtNUP1
0.2
scopolamine (4)
NtNUP1
anatabine (2)
0.2
pyridoxamine (5) µmol g-1 fr. wt
rutin (10)
NtNUP1
(2’S)-nicotine (1a) **
VC
µmol g-1 fr. wt
(c)
adenine (8)
VC
methyl jasmonate (9)
scopolamine (4)
1
**
0
NtNUP1
pyridoxamine (5)
hyoscyamine (3)
anatabine (2)
VC
(2’S)-nicotine (1a)
Fig. 1. NtNUP1-expressing yeast cells import tobacco alkaloids and vitamin B6. (a) Subcellular localization of NtNUP1-GFP. GFP fluorescence (left) and bright-field (middle) images of NtNUP1-GFP-expressing yeast cells are merged in the right image. Scale bar: 100 lm. (b) Chemical structures of natural compounds evaluated in NtNUP1-mediated uptake assays. Adenine (8) was used in the experiments shown in Fig. 5a. (c) S. cereviseae cells transformed with either an empty vector or an NtNUP1-expression vector were incubated with a test substrate for 4 h, and the concentrations of the test compounds in the yeast cells were determined. (20 S)-Nicotine (1a), hyoscyamine (3), scopolamine (4), pyridoxamine (5), pyridoxine (6), and pyridoxal (7) were administered at 250 lM, whereas anatabine (2) was used at 50 lM. Error bars indicate s.d. for three biological replicates. Significant differences in NtNUP1-expressing cells compared to vector control (VC) cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄P < 0.05.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
4
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
Table 1 Uptake of radio-labelled substrates in short-term transport assays in yeasts. Substrate uptake (pmol mg fr wt-1 min-1)
Transporter expressed in yeasts
NtNUP1 PUP1
(20 S)-Nicotine (1a)
Pyridoxine (6)
Ratio of nicotine (1) to pyridoxine (6)
31.3 ± 3.9 5.2 ± 0.9
3.4 ± 0.6 12.3 ± 2.7
9.2:1 1:2.4
Tritium-labelled nicotine or tritium-labelled pyridoxine (6) (50 lM each) was incubated with yeast cells expressing either NtNUP1 or Arabidopsis PUP1 for 3 min, and the radio-labelled substrates taken up in the yeast cells were analyzed. The uptake values in the control yeast cells containing an empty vector were subtracted.
Next evaluated was whether pyridoxine (6) and structurally related compounds could serve as uptake substrates of NtNUP1. Pyridoxamine (5) was most efficiently transported by BY-2 cells expressing NtNUP1, followed by pyridoxine (6) (4–5 lmol g1 dry wt) and then pyridoxal (7) (ca. 1 lmol g1 dry wt) (Fig. 5a). BY-2 cells expressing PUP1 were also able to take up pyridoxamine (5) efficiently, in agreement with the findings of a previous yeastbased transport assay (Szydlowski et al. 2013). Furthermore, adenine (8) served as a transport substrate for PUP1, as previously reported (Gillissen et al. 2000; Bürkle et al. 2003; Szydlowski et al. 2013), but not for NtNUP1 (Fig. 5a). Whether nicotine (1) would compete with pyridoxamine (5) for uptake by NtNUP1 was next examined (Fig. 5b). Indeed, 50 lM or 500 lM nicotine (1) progressively inhibited uptake of 50 lM pyridoxamine (5) in NtNUP1-overexpressing tobacco cells, indicating that both nicotine (1) and pyridoxamine (5) share the same binding site in NtNUP1. Together, these results indicate that NtNUP1 efficiently transports both vitamin B6 and anatabine (2).
racemic nicotine NtNUP1 NtNUP1-GFP vector control
0
10
20
30
40
50
Elution time ( min ) Nicotine composition (%)
(b)
Membrane transporters often show relaxed substrate specificities, transporting multiple natural metabolites with related but distinct chemical structures. For example, Arabidopsis PUP1, a founding member of the plasma membrane-localized PUP family of transporters in plants, mediates the uptake of adenine (8) (Gillissen et al. 2000), trans-zeatin (Bürkle et al. 2003), and pyridoxine (6) (Szydlowski et al. 2013) when expressed in yeast. Although not directly measured, competition studies indicated that PUP1 could transport several purine derivatives, kinetin, and caffeine (Gillissen et al. 2000; Szydlowski et al. 2013). Nicotine (1) inhibited adenine (8) uptake by PUP1 in a yeast system to some extent (Gillissen et al. 2000). The finding that NtNUP1 belongs to the PUP1 subclade of the PUP superfamily (Suppl. Fig. 1) prompted us to examine whether NtNUP1 mediates the uptake of not only nicotine (1) but also of other nitrogen-containing metabolites. Competition studies in fis-
(2’S)-nicotine (1a) (2’R)-nicotine (1b) RT = 50.7 min RT = 51.4 min
Detector response (arbitrary unit)
(a)
3. Discussion
100 80
(2’R)-nicotine (1b)
60
(2’S)-nicotine (1a)
40 20 0
Racemic nicotine administered
NtNUP1
NtNUP1-GFP
Fig. 2. NtNUP1-expressing yeast cells import (20 S)-nicotine (1a) preferentially over (20 R)-nicotine (1b). (a) GC chromatograms of a 1:1 racemic mixture of (20 S)- and (20 R)nicotine (1a/b), and of extracts derived from yeast cells transformed with an empty vector, an NtNUP1 vector, or an NtNUP1-GFP vector. (20 S)-Nicotine (1a) and (20 R)-nicotine (1b) were eluted at a retention time of 50.7 min and 51.4 min, respectively. (b) Percentages of (20 S)- and (20 R)-nicotine (1a/b) in the racemic mixture added to the yeast cells (three independent measurements), and in the cell extracts derived from NtNUP1- or NtNUP1-GFP-expressing yeast cells (three biological replicates).
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
Relative expression level
(a)
6
NtNUP1 + MeJA
4 2
control 0 0
20
(b) Relative expression level
40
Time after MeJA(9)treatment (h) 800
PMT
+ MeJA
600 400 200
control
0 0
20
40
Time after MeJA(9)treatment (h)
Fig. 3. Effects of exogenously added methyl jasmonate (9) on gene expression in tobacco cells. Expression levels of NtNUP1 (a) and PMT (b) in tobacco BY-2 cells cultured in the absence (triangles) or presence (squares) of 100 lM methyl jasmonate (9) were studied by quantitative RT-PCR. Error bars indicate s.d. for three biological replicates. Values are shown relative to the expression levels at the time of methyl jasmonate (9) (MeJA) treatment.
sion yeast indicated that anatabine (2), anabasine, kinetin, and atropine (a racemic mixture of hyoscyamine (3)) somewhat inhibited nicotine (1) uptake by NtNUP1, but that scopolamine (4) was a poor inhibitor of NtNUP1-mediated uptake (Hildreth et al., 2011). Our direct uptake assay in budding yeast confirmed that nicotine (1) and anatabine (2) were readily transported by NtNUP1, whereas NtNUP1 also facilitated the uptake of hyoscyamine (3) and, to a lesser extent, scopolamine (4). Tobacco BY-2 cells overexpressing NtNUP1 showed similar transport preferences toward
Relative NtNUP1 expression level
(a) 80 60 40 20 0
WT
VC4
VC9 VC VC
(b)
WT
OX10
OX18
NtNUP1ox PUP1ox
VC4 VC9 OX10 OX34
PUP1 EF1α Fig. 4. Expression levels of NtNUP1 and PUP1 in tobacco BY-2 cells. Tobacco cells were cultured in the absence of jasmonates. Two independent transgenic lines were used for each construct. (a) NtNUP1 transcript levels in wild-type tobacco cells and transgenic tobacco cells transformed with an empty vector (vector control; VC) or an NtNUP1-overexpressing vector (NtNUP1ox) were measured by quantitative RTPCR. Error bars indicate s.d. for three biological replicates. (b) PUP1 transcripts (after 28 cycles of amplification) and tobacco EF1a transcripts (after 23 cycles) were analyzed by RT-PCR in wild-type tobacco cells and transgenic tobacco cells transformed with an empty vector (VC) or a PUP1-overexpressing vector (PUP1ox). The bands were visualized by ethidium bromide staining. EF1a was used as a loading control.
5
these alkaloids, although nicotine (1) uptake by the tobacco cells was not as efficient as that by yeast cells, for unknown reasons. The import studies herein using yeast cells and tobacco cells demonstrated that NtNUP1 mediates the uptake of pyridoxamine (5) and pyridoxine (6) as efficiently as it mediates the uptake of tobacco alkaloids. Pyridoxamine (5) and pyridoxine (6) both contain a pyridine ring as a common structure. However, other structural features, including stereochemistry, must contribute to the substrate preference of NtNUP1, because (20 S)-nicotine (1a) was more efficiently transported than its stereoisomer, (20 R)-nicotine (1b). In this study, potential transport substrates were tested at the concentrations of either 50 lM or 250 lM. Because nicotine (1) concentrations in the apoplastic space of the tobacco root tissues and in the rhizosphere close to the tobacco roots are not known, and because the KM values of NtNUP1 for tobacco alkaloids are not available, it is difficult to assess physiological significance of NtNUP1’s nicotine (1) transport activity from these biochemical assays. On the other hand, PUP1 has a KM value of approximately 100 lM for pyridoxine (6), whereas its estimated concentration in guttation sap is 3 nM, a far lower concentration compared to the substrate affinity of PUP1 (Szydlowski et al. 2013). Nevertheless, knockout of PUP1 significantly increased concentrations of pyridoxine (6) and related compounds in the sap (Szydlowski et al. 2013), indicating physiological relevance of the uptake studies conducted under the high micromolar concentrations. Does the ability of NtNUP1 to transport vitamin B6 explain its knockdown phenotype, such as its decreased overall synthesis of tobacco alkaloids and increased root growth? Unlike auxotrophic animals, plants synthesize vitamin B6 de novo. Pyridoxal 50 -phosphate (PLP), a bioactive form of vitamin B6, is an essential cofactor for all transaminases and many decarboxylases and deaminases. A null Arabidopsis mutant of PDX2, a single gene encoding a subunit of an enzyme involved in PLP biosynthesis, is embryo lethal (Tambasco-Studart et al., 2007), reflecting the essential role of PLP in cellular metabolism. PUP1 is strongly expressed in the epithem of hydathodes in Arabidopsis leaves, where PUP1 is proposed to retrieve cytokinins and vitamin B6 from the guttation sap (Bürkle et al. 2003; Szydlowski et al. 2013). However, the biological significance of the PUP1-mediated retrieval of bioactive metabolites is not clear, since an Arabidopsis pup1 null mutant does not display any obvious growth or morphological defects (Szydlowski et al. 2013). In tobacco roots, NtNUP1 may import pyridoxine (6), a form of vitamin B6 included in various plant tissue culture media, or it may mediate the retrieval of vitamin B6 secreted into the rhizosphere. In this scenario, knockdown of NtNUP1 in tobacco roots might result in a shortage of PLP, leading to inefficient metabolic processes involving PLP-dependent enzymes. None of the enzymes known to be involved in the nicotine (1) biosynthetic pathway require PLP (Shoji and Hashimoto; 2013); however, the late pathway enzymes, which remain to be identified, may turn out to require PLP. Nonetheless, a cellular shortage of PLP would be detrimental to the metabolic activities and growth of plants. Thus, the uptake of vitamin B6 alone is not sufficient to explain the full spectrum of defects observed in NtNUP1-suppressed tobacco plants. Purine derivatives, including cytokinins, are also transported by PUP1 (Gillissen et al. 2000; Bürkle et al. 2003; Szydlowski et al. 2013). Since NtNUP1 does not show any uptake activity for adenine (2), the NtNUP1-supression phenotypes do not seem to be directly related to cellular abnormalities in cytokinin contents. Future studies should aim to decipher the molecular mechanisms underlying the NtNUP1-mediated control of cell metabolism and plant growth.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
pyridoxamine (5)
** **
10
2
0
0
pyridoxine (6)
10
** **
** **
0
10
WT
OX10
OX18
VC
**
NtNUP1ox
VC9
WT
adenine (8) **
pyridoxal (7)
20
0
20
** *
30
20 10
0 30
2
30
** **
20
4
VC4
30
4
OX18
0
hyoscyamine (3)
6
OX10
** **
**
**
NtNUP1ox
4 2
anatabine (2)
6
VC9
(2’S)-nicotine (1a)
VC
6
pyridoxamine (5) μmol g-1 dry wt
OX34
OX10
OX10
OX18
PUP1ox
(b)
NtNUP1ox
VC9
VC
VC4
0 WT
μmol g-1 dry wt
μmol g-1 dry wt
μmol g-1 dry wt
(a)
VC4
6
15 10
**
** 5
0 (2’S)-nicotine (µM)
**
** 0
0 50 500
0 50 500
WT
OX10
OX18
NtNUP1ox Fig. 5. Import assays in tobacco BY-2 cells expressing NtNUP1 or PUP1. (a) Cultured tobacco cells transformed with either an empty vector (EV), an NtNUP1-overexpressing construct (NtNUP1ox), or a PUP1-overexpressing construct (PUP1ox) were incubated with a test import substrate at 50 lM for 4 h, and the concentrations of the test compounds in the tobacco cells were determined. (20 S)-Nicotine (1a), pyridoxamine (5), and adenine (8) were tested in both NtNUP1ox cells and PUP1ox cells, whereas the import of anatabine (2), hyoscyamine (3), scopolamine (4), pyridoxine (5), and pyridoxal (6) was examined in NtNUP1ox cells. Error bars indicate s.d. for three biological replicates. Significant differences in NtNUP1- or PUP1-expressing cells compared to wild-type (non-transformed) cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄ P < 0.05. (b) Competition of pyridoxamine uptake by the simultaneous addition of various concentrations of (20 S)-nicotine (1a) in NtNUP1-overexpressing tobacco cells. Pyridoxamine (5) was added at 50 M, whereas nicotine (1) was supplied at 0, 50, or 500 lM. Error bars indicate s.d. for three biological replicates. Significant differences in nicotine (1)-supplied cells compared to non-supplied cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄P < 0.05.
4. Conclusions
5. Experimental
The uptake assays using budding yeast and cultured tobacco cells showed that NtNUP1 mediates the efficient uptake of not only tobacco alkaloids but also vitamin B6. Although its Arabidopsis homolog PUP1 transports vitamin B6, PUP1 additionally shows an uptake preference for purine derivatives, rather than nicotine (1). Thus, the PUP1/NtNUP1 subclade of the PUP family transporters appears to be characterized by the ability to transport vitamin B6, and members of this subclade additionally exhibit distinct substrate preferences for compounds with nitrogen-containing ring structures. Tobacco metabolites containing a pyridine ring may underlie the defects observed upon NtNUP1 suppression; however, further studies are required to pin-point the molecular mechanisms involved.
5.1. Transport assay in yeast cells An NtNUP1 cDNA fragment (accession number GU174267), encompassing the full-length coding region, its C-terminal fusion with GFP, and an Arabidopsis PUP1 cDNA (accession number At1g28230) were subcloned into pDONR/zeo (Invitrogen), and then transferred into pDR196GW using Gateway cloning technology (Invitrogen). These plasmids were introduced into the yeast (Saccharomyces cerevisiae) strain AD12345678 (MATa, PDR1-3, ura3, his1, Dyor1::hisG, Dsnq2::hisG, Dpdr5::hisG, Dpdr10::hisG, Dpdr11::hisG, Dycf1::hisG, Dpdr3::hisG, and Dpdr15::hisG) (Decottignies et al., 1998) by the lithium acetate method (Yazaki et al., 2002). Plasmid-harboring yeast cells were grown in synthetic
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
defined medium (minus uracil) at 30 °C. Yeast cells expressing NtNUP1 or NtNUP1-GFP grew slower than wild-type yeast cells. When the optical density of the culture at 600 nm reached 0.7, yeast was collected and resuspended in half-strength synthetic defined medium (minus uracil) supplemented with a test transport compound for 4 h. Nicotine (1), hyoscyamine (3), scopolamine (4), and rutin (10) were added at a final concentration of 250 lM, whereas anatabine (2) was used at 50 lM. Yeast cells were collected by centrifugation at 3500 rpm for 10 min, and washed twice with ice-cold deionized water to remove extracellular test compounds. For the uptake assay using radio-labelled substrates, yeasts were grown in the same medium until OD600 reached 1.3–1.8, the cells were collected and suspended to OD600 5 in 50 mM citrate phosphate buffer (pH4.0). 3H-labelled pyridoxine (6) (9.25 kBq) or 3 H-labelled nicotine (1) (9.25 kBq) in 5.25 ll was added to the yeast suspensions (520 ll) to a final concentration of 50 lM. The suspensions were incubated for 3 min at 30 °C. Aliquots (60 ll) were withdrawn from the suspensions, diluted with distilled H2O (5 ml), filtered through hydrophilic Glass Fibre filters (1.0 lm pore size, 25 mm diameter; Millipore), and washed with distilled H2O (5 ml), using a 1225 Sampling Manifold (Millipore). Filters were placed in vials containing LumaSafe Plus cocktail (3 ml) (PerkinElmer), and measured with a liquid scintillation counter (model LS6500, Beckman). To estimate uptake rates, values for yeasts expressing an empty vector were subtracted from those expressing PUP1 or NtNUP1. 5.2. Localization of NtNUP1-GFP in yeast cells Localization of NtNUP1-GFP in yeast cells was observed using a C2 Confocal Laser Microscope (Nikon). GFP and tagRFP were excited at 488 nm and 544 nm, respectively, and fluorescence was detected using a 514/30 nm band pass emission filter for GFP and a 585/65 nm band pass filter for tagRFP. All images were acquired from single optical sections. 5.3. Transport assay in tobacco cells The open reading frame (ORF) of NtNUP1 cDNA was flanked with BamHI and SacI sites by PCR, whereas the full-length ORF of PUP1 cDNA was flanked with HindIII and SacI sites. These clones were subcloned in pGEM-T (Promega), and then inserted into pBI121 (Clontech) to replace the GUS fragment. Tobacco BY-2 cells (N. tabacum cv. Bright Yellow-2) were subcultured in liquid MS medium supplemented with 20 mg l1 KH2PO4, 0.5 g l1 MES and 0.2 mg l1 2,4-D every week. Transgenic tobacco BY-2 cells were generated using the A. tumefaciens strain EHA105, harboring a binary vector as described (An, 1985). For jasmonate treatment, four-day-old BY-2 cells were first rinsed with and then transferred to fresh auxin-free MS medium containing 100 lM methyl jasmonate (MeJa 9). After NtNUP1- or PUP1-overexpressing BY-2 cells were cultured for 3 days, they were suspended in fresh BY-2 medium (30 ml) supplemented with a test transport substrate at a final concentration of 50 lM and incubated for 4 h. Tobacco cells were then harvested and washed with deionized H2O five times. 5.4. RT-PCR analysis Total RNA was isolated using an RNeasy Plant Mini Kit (Qiagen) from tobacco cells that had been ground in liq N2, and a portion of the total RNA (1 ng) was converted into cDNA using Super Script II Reverse Transcriptase (Invitrogen) and an oligo (dT) primer. cDNA templates were amplified using a LightCycler 480 (Roche) with SYBR Premix Ex Taq (Takara) for quantitative RT-PCR analysis
7
under the following conditions: 94 °C for 5 min, 55 cycles of 94 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s. To avoid saturated amplification, template amounts and cycle numbers were adjusted in individual experiments, and the specificity of the reactions was confirmed using the machine’s standard melt curve method. EF1a (accession number; AF120093) was used as a reference gene. The PCR primers used are listed in Supplementary Table S1. For semi-quantitative RT-PCR, cDNA templates were amplified using the GENE Amp@ PCR System 9700 (ABI) with ExTaq (Takara), under the following conditions: 94 °C for 5 min, 1 cycle of 94 °C for 10 s, 55 °C for 10 s, and 72 °C for 15 s. PUP1 cDNA was amplified for 28 cycles and EF1a cDNA for 23 cycles. Amplified products were separated on a 2% agarose gel and stained with ethidium bromide. 5.5. Quantification of test substrates For quantification of nicotine (1), anatabine (2), hyoscyamine (3), and scopolamine (4), lyophilized tobacco cells (50 mg dry wt) were homogenized in 0.1 N H2SO4 (4 ml), and the homogenate was sonicated for 60 min and then centrifuged at 880 g for 15 min. Fresh yeast cells were suspended in a 5-fold amount of 0.1 N H2SO4 (v/w), disrupted by vigorous vortexing with acidwashed glass beads, and centrifuged at 17,400 g for 10 min. The supernatant (either from tobacco samples or yeast samples) was neutralized by adding 25% NH4OH (0.4 ml). The mixture (1 ml) was loaded onto an Extrelut-1 column (Merck), and the alkaloids were eluted with CHCl3 (6 ml). The eluent was dried at 37 °C. The dry residues were dissolved in EtOH containing 0.1% dodecane as internal standard, and analyzed by gas chromatography (GC; GC-2010, Shimadzu) equipped with a Rtx-5 Amine capillary column (Restek, PA, US). The column temperature was held at 100 °C for 10 min, and then increased to 250 °C over a 35-min period. The injector and FID detector were held at 300 °C, and the flow rate of the carrier gas He was 0.99 ml min1, with a split ratio of 1:20. Chiral forms of nicotine (1a and 1b) were separated by GC with a capillary column (WCOT, 60 m 0.25 mm id. 0.25 lm df, fused silica, b-DEX™ 120, Supelco, PA, US). The injector and FID detector were held at 230 °C and 250 °C, respectively, whereas the column oven temperature was held at a constant temperature of 120 °C, and the carrier gas He was run at 0.95 ml min1, with a split ratio of 1:20. For vitamin B6 uptake analysis, yeast and tobacco cells were extracted in 25 mM phosphate buffer (pH. 4.0), homogenized, sonicated for 30 min, and incubated at 65 °C for 30 min. After centrifugation as described above, the supernatants were analyzed by high-performance liquid chromatography (HPLC), using the LC10 system (Shimadzu, Japan) equipped with a COSMOSIL Cholester column (4.6 mm id. 250 mm, Nacalai tesque, Japan). Pyridoxamine (5), pyridoxine (6), and pyridoxal (7) were eluted from the column with an isocratic mobile phase of 25 mM phosphate buffer (pH 4.0) at a flow rate of 1 ml min1 at 40 °C, and were detected with a fluorescence detector at an excitation of 328 nm and an emission of 393 nm. Adenine (8) was extracted from tobacco cells in 20 mM phosphate buffer (pH 7.0), homogenized, sonicated for 30 min, and incubated at 65 °C for 30 min. After centrifugation as described above, the supernatants were analyzed by HPLC as above. Adenine (8) was eluted from the column with an isocratic mobile phase of 20 mM phosphate buffer (pH. 7.0) at a flow rate of 1 ml min1 at 40 °C, and detected at UV 254 nm. Rutin (10) was extracted from yeast and tobacco cells with EtOH–H2O (1:1, v/v), and processed as described above. Supernatants from centrifugation were filtered using a COSMO Spin Filter H (Nacalai tesque, Japan) and analyzed by HPLC, using the MD-2018 system (Jasco, Japan), equipped with a COSMOSIL 5C18
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
8
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
MS-II column (5 lm, 4.6 mm 150 mm, Nacalai tesque, Japan). Rutin (10) was eluted from the column with a linear gradient program from 12% to 100% buffer (B), relative to buffer (A), in 15 min at a flow rate of 1 ml min1 at 40 °C, and detected at UV 358 nm. Buffer (A) consisted of H2O:HCOOH (9:1), whereas buffer (B) was composed of H2O:HCOOH:MeOH:CH3CN (40:10:22.5:22.5). 5.6. Chemicals (20 R)(+)-Nicotine (1b) (TRC, Canada), (20 S)()(1a)-nicotine (Wako Chemicals, Japan), anatabine (Wako Chemicals, Japan), hyoscyamine (3) (Sigma, Germany), scopolamine (4) (TOCRIS bioscience, UK), pyridoxine (6) (Nacalai tesque, Japan), pyridoxamine (5) (Sigma, Germany), pyridoxal (7) (Wako Chemicals, Japan), rutin (10) (Nacalai tesque, Japan), and adenine (8) (Sigma, Germany) were purchased commercially. To prepare a racemic mixture of nicotine (1a/b), equal molar amounts of (20 S)-nicotine (1a) and (20 R)-nicotine (1b) were mixed. [methylene-3H]-pyridoxine (6) (37 GBq ml1) and [N-methyl-3H]-(20 S)-nicotine (1a) (37 GBq ml1) were obtained from ARC Inc. (Saint Louis, MO).
Acknowledgments We thank W. Frommer (Carnegie Institution), K. Yazaki (Kyoto University), N. Inada (Nara institute of Science and Technology), and A. Goffeau (Université Catholique de Louvain) for providing us with pDR196, pDR196GW, pUGW2m-tagRFP, and the yeast strain AD12345678, respectively. This study was supported in part by a Grant from the Japan Society for the Promotion of Science to T.S. (Grant-in-Aid for Scientific Research (C), No. 23570035) and to N.S. (Grant-in-Aid for Young Scientists (A), No. 25712012).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 05.011.
References An, G., 1985. High efficiency transformation of cultured tobacco cells. Plant Physiol. 79, 568–570. Bürkle, L., Cedzich, A., Döpke, C., Stransky, H., Okumoto, S., Gillissen, B., Kühn, C., Frommer, W.B., 2003. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J. 34, 13–26. Chintaparkorn, Y., Hamill, J.D., 2003. Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine. Plant Mol. Biol. 53, 87–105. Decottignies, A., Grant, A.M., Nichols, J.W., de Wet, H., McIntosh, D.B., Goffeau, A., 1998. ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J. Biol. Chem. 273, 12612–12622. Gillissen, B., Bürkle, L., André, B., Kühn, C., Rentsch, D., Brandl, B., Frommer, W.B., 2000. A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis. Plant Cell 12, 291–300. Hildreth, S.B., Gehman, E.A., Yang, H., Lu, R.-H., Rithish, K.C., Harich, K.C., Yu, S., Lin, J., Sandoe, J.L., Okumoto, S., Murphy, A.S., Jelesko, J.G., 2011. Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism. Proc. Natl. Acad. Sci. U.S.A. 108, 18179–18184. Kajikawa, M., Shoji, T., Kato, A., Hashimoto, T., 2011. Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco. Plant Physiol. 155, 2010–2022. Morita, M., Shitan, N., Sawada, K., Montagu, M.C.E.V., Inzé, D., Rischer, H., Goossens, A., Oksman-Caldentey, K.-M., Moriyama, Y., Yazaki, K., 2009. Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc. Natl. Acad. Sci. U.S.A. 106, 2447–2452. Shoji, T., Hashimoto, T., 2008. Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells? Plant Cell Physiol. 49, 1209– 1216. Shoji, T., Hashimoto, T., 2013. Smoking out the masters: transcriptional regulators for nicotine biosynthesis in tobacco. Plant Biotechnol. 30, 217–224. Shoji, T., Inai, K., Yazaki, Y., Sato, Y., Takase, H., Shitan, N., Yazaki, K., Goto, Y., Toyooka, K., Matsuoka, K., Hashimoto, T., 2009. Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots. Plant Physiol. 149, 708–718. Szydlowski, N., Bürkle, L., Pourcel, L., Moulin, M., Stolz, J., Fitzpatrick, T.B., 2013. Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis. Plant J. 75, 40–52. Tambasco-Studart, M., Tews, I., Amrhein, N., Fitzpatrick, T.B., 2007. Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiol. 144, 915–925. Xie, J., Song, W., Maksymowicz, W., Jin, W., Cheah, K., Chen, W., Carnes, C., Ke, J., Conkling, M.A., 2004. Biotechnology: A tool for reduced risk tobacco products. Recent Adv. Tobacco Sci. 30, 17–37. Yazaki, K., Kunihisa, M., Fujisaki, T., Sato, F., 2002. Geranyl diphosphate:4hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon: Cloning and characterization of a key enzyme in shikonin biosynthesis. J. Biol. Chem. 277, 6240–6246.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Tobacco NUP1 transports both tobacco alkaloids and vitamin B6 Keita Kato a, Nobukazu Shitan b, Tsubasa Shoji a, Takashi Hashimoto a,⇑ a b
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0192, Japan Kobe Pharmaceutical University, Motoyama Kitamachi 4-19-1, Higashinada, Kobe, Hyogo 658-8558, Japan
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Nicotiana tabacum Solanaceae Tobacco Transport assays Transporter Nicotine Vitamin B6 PUP-family transporters
a b s t r a c t The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A Nicotiana tabacum (tobacco) PUP-family transporter, nicotine uptake permease 1 (NtNUP1), was previously shown to transport tobacco alkaloids and to affect both nicotine biosynthesis and root growth in tobacco plants. Since Arabidopsis PUP1, which belongs to the same subclade as NtNUP1, was recently reported to transport pyridoxine and its derivatives (vitamin B6), it was of interest to examine whether NtNUP1 could also transport these substrates. Direct uptake measurements in the yeast Saccharomyces cerevisiae demonstrated that NtNUP1 efficiently promoted the uptake of pyridoxamine, pyridoxine, anatabine, and nicotine. The naturally occurring (S)-isomer of nicotine was preferentially transported over the (R)-isomer. Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1, transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences. Therefore, the previously reported effects of NtNUP1 on tobacco physiology might involve bioactive metabolites other than tobacco alkaloids. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nicotine (1) (Fig. 1) and its related pyridine alkaloids occur mainly in the Nicotiana genus and contribute to the chemical defense against insects (Shoji and Hashimoto, 2013). In Nicotiana tabacum (tobacco), these alkaloids are exclusively synthesized in the root, translocated to the aerial parts via the xylem, and stored in the vacuoles of leaf cells. Since berberine bridge enzyme-like oxidoreductases, which catalyze the last or a late step of nicotine (1) biosynthesis, are localized in the vacuoles, nicotine (1) is thought to be formed in the vacuoles (Kajikawa et al., 2011). Nicotine (1) thus appears to travel from its site of synthesis in the root vacuole to its distant storage site in the leaf vacuole, via the root cytosol, root apoplast, xylem sap, leaf apoplast, and leaf cytosol. Besides this long-distance transport within the plant, tobacco alkaloids and their biosynthetic intermediates may be transported across endomembranes of cellular organelles or plasma membranes. Multidrug and toxic compound extrusion (MATE)-type transporters sequester nicotine (1) in the vacuoles of tobacco roots, thereby decreasing cytoplasmic its toxicity during active nicotine (1) synthesis in the root (Shoji et al. 2009). Another MATE-type ⇑ Corresponding author. Tel.: +81 743 72 5520; fax: +81 743 725529. E-mail address: [email protected] (T. Hashimoto).
transporter is proposed to sequester nicotine (1) into the vacuoles of the leaf (Morita et al. 2009). A plasma membrane-localized nicotine uptake permease (NtNUP1) was recently reported to import nicotine (1) from the apoplastic space, particularly in root tips (Hildreth et al. 2011). NtNUP1 belongs to a plant-specific class of purine permease-like transporters, which is classified into several subclades (Supplemental Fig. S1). RNAi-mediated suppression of NtNUP1 in tobacco hairy root cultures caused nicotine (1) levels in the culture medium to increase and those in the root cells to decrease, consistent with the nicotine (1) uptake activity of NtNUP1 (Hildreth et al. 2011). Surprisingly, the total nicotine (1) content in the leaves and roots of NtNUP1-suppressed tobacco plants was significantly reduced, and the roots of NtNUP1-suppressed seedlings grew better than those of the control (Hildreth et al. 2011). When tobacco plants with highly reduced nicotine (1) levels were generated by suppressing expression of its biosynthesis genes, growth promotion of these transgenic plants was not observed (e.g., Chintaparkorn and Hamill, 2003; Xie et al. 2004). The requirement of NtNUP1 for optimal nicotine (1) biosynthesis and the inhibitory effect of NtNUP1 on root growth (Hildreth et al. 2011) cannot readily be explained by its transport activity for nicotine (1). NtNUP1 belongs to the same subclade as Arabidopsis thaliana PUP1 in the PUP family of transporters (Supplemental Fig. S1).
http://dx.doi.org/10.1016/j.phytochem.2014.05.011 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
2
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
PUP1 was initially discovered as a plant transporter that complemented a yeast mutant deficient in adenine transport (Gillissen et al. 2000), and was shown to import adenine, cytosine, cytokinins, and related metabolites from the apoplast into the cytoplasm (Bürkle et al. 2003). More recently, PUP1 was shown to be a high affinity transporter of vitamin B6, which includes pyridoxine (6), pyridoxamine (5), and pyridoxal (7) (Szydlowski et al. 2013). The rather relaxed substrate specificity of PUP1 prompted us to investigate whether NtNUP1 transports vitamin B6 in addition to tobacco alkaloids. A better knowledge of NtNUP1 substrate preference is important for understanding the effects of NtNUP1 on plant metabolism and physiology.
2. Results 2.1. NtNUP1 transport assays in yeast Previously, Schizosaccharomyces pombe cells expressing NtNUP1 cDNA were used to measure the uptake of radioactive nicotine (1) into cells, and the inhibition of nicotine (1) uptake in the presence of excess amounts of non-radioactive competitors, such as other tobacco alkaloids and tropane alkaloids (Hildreth et al. 2011). Here, NtNUP1 cDNA was expressed in Saccharomyces cerevisiae and the uptake of various metabolites was assayed by directly measuring their cellular contents using high-performance liquid chromatography (HPLC) or gas chromatography (GC). As an initial experiment, a C-terminal green fluorescent protein (GFP) fusion of NtNUP1 (i.e., NtNUP1-GFP) was expressed in S. cerevisiae to analyze the subcellular localization of NtNUP1 (Fig. 1a). NtNUP1-GFP was localized mainly to the plasma membrane and to endomembranes, possibly the tonoplast. Substantial localization of NtNUP1-GFP to the plasma membrane suggests the feasibility of conducting uptake assays using transgenic yeast cells. Transient expression of NtNUP1-GFP in onion epidermal cells indicated that NtNUP1 was localized exclusively to the plasma membrane (Supplemental Fig. S2), in agreement with the results of a previous report (Hildreth et al. 2011). Next, S. cerevisiae cells expressing either an empty vector (control) or NtNUP1 cDNA was exposed to 250 lM of various plant metabolites (except for anatabine (2), which was administered at 50 lM), and the test metabolites was measured in yeast cells after a 4-h incubation period (Fig. 1b and c). (20 S)-Nicotine (1a) (the stereoisomer synthesized in tobacco plants) and anatabine (2) were taken up efficiently by NtNUP1expressing yeast cells, to reach a cellular concentration of ca. 0.16–0.18 lmol g1 fr. wt, whereas the contents of these alkaloids in the control cells were less than 0.02 lmol g1 fr. wt. Uptake of hyoscyamine (3) (atropine) and scopolamine (4) by the NtNUP1expressing cells was significantly enhanced (P < 0.05 and P < 0.01, respectively; Student’s t-test) compared to the control cells, but the NtNUP1-mediated accumulation of these tropane alkaloids was low (less than 0.03 lmol g1 fr. wt). The flavonoid rutin (10) was not taken up by either NtNUP1-expressing or control yeast cells. These uptake properties of NtNUP1 in S. cerevisiae are similar to those reported in S. pombe (Hildreth et al. 2011). Whether vitamin B6 could be transported by NtNUP1-expressing yeast cells, as reported for PUP1-expressing yeast cells (Szydlowski et al. 2013), was next investigated, Pyridoxine (6) and pyridoxamine (5) were both highly efficiently taken up by the NtNUP1-expressing cells, compared to control cells, reaching a concentration of 1.0 lmol g1 fr. wt during a 4-h incubation period (Fig. 1b and c). Uptake of pyridoxal (7), another form of vitamin B6, by NtNUP1 was significant (P < 0.01; Student’s t-test) but low (ca. 0.03 lmol g1 fr. wt). These results show that NtNUP1 mediates the efficient uptake of some vitamin B6 derivatives, in addition to tobacco alkaloids, when expressed in yeast cells.
To estimate initial uptake rates, tritium-labelled pyridoxine (6) and nicotine (1) at 50 lM were administered for 3 min. Yeast cells expressing either PUP1 or NtNUP1 were used to compare substrate preferences of these transporters. Since expression levels of functional PUP1 and NtNUP1 at the plasma membrane may be different, it is meaningful to compare uptake rates of two substrates in a particular yeast strain, but not to compare uptake rates of one substrate between two yeast strains. As shown in Table 1, both pyridoxine (6) and nicotine (1) were efficiently taken up by PUP1 and NtNUP1. PUP1’s ability to transport nicotine (1) has been inferred from an uptake competition study in yeasts (Gillissen et al. 2000). These results herein show that PUP1 prefers pyridoxine (6) over nicotine (1), whereas NtNUP1 transports nicotine (1) more efficiently than pyridoxine (6), under the short-term uptake conditions. Whether NtNUP1 displayed selectivity toward stereoisomers of nicotine (1) was also tested. (20 S)-Nicotine (1a) and (20 R)-nicotine (1b) (both at 125 lM) were mixed at a 1:1 ratio and supplied to the culture medium. After 4-h incubation, the nicotine (1) taken up by the yeast cells was analyzed using a chiral GC column, which separated the stereoisomers as distinct peaks (Fig. 2a). The amount of nicotine (1) taken up by the vector control cells was too low to be analyzed by this method. The nicotine (1) present in yeast cells expressing NtNUP1 or NtNUP1-GFP though was greatly enriched in the (S) isomer, (1a) representing ca. 81% of the total nicotine (1) present (Fig. 2b). Thus, NtNUP1 preferentially transports the naturally occurring isomer of (20 S)-nicotine (1a) over the unnatural (20 R) (1b) form.
2.2. NtNUP1 transport assays in tobacco BY-2 cells To validate the transport properties of NtNUP1, NtNUP1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in cultured tobacco BY-2 cells. Tobacco alkaloids (mainly anatabine (2)) are biosynthesized in BY-2 cell cultures only after jasmonate elicitation (Shoji and Hashimoto, 2008). In the present study, it was found that the expression level of NtNUP1 in BY-2 cells was low when the cells were cultured in the absence of jasmonates, but increased several fold after jasmonate treatment (Fig. 3a), and that alkaloid biosynthesis, as evidenced by robust induction of an alkaloid biosynthetic gene, PUTRESCINE N-METHYLTRANSFERASE (PMT), was induced by addition of methyl jasmonate (9) (MeJA; Fig. 3b). NtNUP1-mediated transport activities can thus be estimated by comparing the activities in NtNUP1-overexpressing cells with those in wild-type or vector-transformed cells, cultured in the absence of jasmonates. Quantitative RT-PCR showed that two independent BY-2 cell lines transformed with the CaMV 35S pro:: NtNUP1 vector had NtNUP1 transcript levels that were ca. 27-fold (OX10 line) or 64-fold (OX18 line) higher than those in the wild-type or vector control cell lines (Fig. 4a). Two tobacco BY-2 cell lines was also generated that overexpressed PUP1 (Fig. 4b), to compare the substrate specificity of NtNUP1 and PUP1. Tobacco cells were cultured in the presence of test compounds (50 lM), and the cellular concentrations of these compounds were measured after a 4-h period. The NtNUP1overexpressing tobacco cells took up (20 S)-nicotine (1a) and anatabine (2) more efficiently than did the wild-type and vector control cells, whereas the PUP1-overexpressing cells did not accumulate (20 S)-nicotine (1a) (Fig. 5a). In this uptake assay, NtNUP1 transported greater amounts of anatabine (2) (3–4 lmol g1 dry wt) than of nicotine (1) (0.5–0.6 lmol g1 dry wt). Tropane alkaloids were transported by NtNUP1 at low efficiency (ca. 0.2 lmol g1 dry wt for hyoscyamine (3), and less than 0.02 lmol g1 dry wt for scopolamine (4); data not shown). Rutin (10) was not transported by NtNUP1.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
3
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
GFP
(a)
Bright field
Merge
NtNUP1-GFP
(b)
pyridoxine (6)
pyridoxal (7)
**
hyoscyamine (3)
0.0
0.0
0.0
**
VC
0
NtNUP1
0
VC
1
*
0.1
**
0.0
2
* 1
0.2
pyridoxal (7)
pyridoxine (6) 2
NtNUP1
2
0.2
VC
0.1
NtNUP1
0.1
VC
0.1
NtNUP1
0.2
scopolamine (4)
NtNUP1
anatabine (2)
0.2
pyridoxamine (5) µmol g-1 fr. wt
rutin (10)
NtNUP1
(2’S)-nicotine (1a) **
VC
µmol g-1 fr. wt
(c)
adenine (8)
VC
methyl jasmonate (9)
scopolamine (4)
1
**
0
NtNUP1
pyridoxamine (5)
hyoscyamine (3)
anatabine (2)
VC
(2’S)-nicotine (1a)
Fig. 1. NtNUP1-expressing yeast cells import tobacco alkaloids and vitamin B6. (a) Subcellular localization of NtNUP1-GFP. GFP fluorescence (left) and bright-field (middle) images of NtNUP1-GFP-expressing yeast cells are merged in the right image. Scale bar: 100 lm. (b) Chemical structures of natural compounds evaluated in NtNUP1-mediated uptake assays. Adenine (8) was used in the experiments shown in Fig. 5a. (c) S. cereviseae cells transformed with either an empty vector or an NtNUP1-expression vector were incubated with a test substrate for 4 h, and the concentrations of the test compounds in the yeast cells were determined. (20 S)-Nicotine (1a), hyoscyamine (3), scopolamine (4), pyridoxamine (5), pyridoxine (6), and pyridoxal (7) were administered at 250 lM, whereas anatabine (2) was used at 50 lM. Error bars indicate s.d. for three biological replicates. Significant differences in NtNUP1-expressing cells compared to vector control (VC) cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄P < 0.05.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
4
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
Table 1 Uptake of radio-labelled substrates in short-term transport assays in yeasts. Substrate uptake (pmol mg fr wt-1 min-1)
Transporter expressed in yeasts
NtNUP1 PUP1
(20 S)-Nicotine (1a)
Pyridoxine (6)
Ratio of nicotine (1) to pyridoxine (6)
31.3 ± 3.9 5.2 ± 0.9
3.4 ± 0.6 12.3 ± 2.7
9.2:1 1:2.4
Tritium-labelled nicotine or tritium-labelled pyridoxine (6) (50 lM each) was incubated with yeast cells expressing either NtNUP1 or Arabidopsis PUP1 for 3 min, and the radio-labelled substrates taken up in the yeast cells were analyzed. The uptake values in the control yeast cells containing an empty vector were subtracted.
Next evaluated was whether pyridoxine (6) and structurally related compounds could serve as uptake substrates of NtNUP1. Pyridoxamine (5) was most efficiently transported by BY-2 cells expressing NtNUP1, followed by pyridoxine (6) (4–5 lmol g1 dry wt) and then pyridoxal (7) (ca. 1 lmol g1 dry wt) (Fig. 5a). BY-2 cells expressing PUP1 were also able to take up pyridoxamine (5) efficiently, in agreement with the findings of a previous yeastbased transport assay (Szydlowski et al. 2013). Furthermore, adenine (8) served as a transport substrate for PUP1, as previously reported (Gillissen et al. 2000; Bürkle et al. 2003; Szydlowski et al. 2013), but not for NtNUP1 (Fig. 5a). Whether nicotine (1) would compete with pyridoxamine (5) for uptake by NtNUP1 was next examined (Fig. 5b). Indeed, 50 lM or 500 lM nicotine (1) progressively inhibited uptake of 50 lM pyridoxamine (5) in NtNUP1-overexpressing tobacco cells, indicating that both nicotine (1) and pyridoxamine (5) share the same binding site in NtNUP1. Together, these results indicate that NtNUP1 efficiently transports both vitamin B6 and anatabine (2).
racemic nicotine NtNUP1 NtNUP1-GFP vector control
0
10
20
30
40
50
Elution time ( min ) Nicotine composition (%)
(b)
Membrane transporters often show relaxed substrate specificities, transporting multiple natural metabolites with related but distinct chemical structures. For example, Arabidopsis PUP1, a founding member of the plasma membrane-localized PUP family of transporters in plants, mediates the uptake of adenine (8) (Gillissen et al. 2000), trans-zeatin (Bürkle et al. 2003), and pyridoxine (6) (Szydlowski et al. 2013) when expressed in yeast. Although not directly measured, competition studies indicated that PUP1 could transport several purine derivatives, kinetin, and caffeine (Gillissen et al. 2000; Szydlowski et al. 2013). Nicotine (1) inhibited adenine (8) uptake by PUP1 in a yeast system to some extent (Gillissen et al. 2000). The finding that NtNUP1 belongs to the PUP1 subclade of the PUP superfamily (Suppl. Fig. 1) prompted us to examine whether NtNUP1 mediates the uptake of not only nicotine (1) but also of other nitrogen-containing metabolites. Competition studies in fis-
(2’S)-nicotine (1a) (2’R)-nicotine (1b) RT = 50.7 min RT = 51.4 min
Detector response (arbitrary unit)
(a)
3. Discussion
100 80
(2’R)-nicotine (1b)
60
(2’S)-nicotine (1a)
40 20 0
Racemic nicotine administered
NtNUP1
NtNUP1-GFP
Fig. 2. NtNUP1-expressing yeast cells import (20 S)-nicotine (1a) preferentially over (20 R)-nicotine (1b). (a) GC chromatograms of a 1:1 racemic mixture of (20 S)- and (20 R)nicotine (1a/b), and of extracts derived from yeast cells transformed with an empty vector, an NtNUP1 vector, or an NtNUP1-GFP vector. (20 S)-Nicotine (1a) and (20 R)-nicotine (1b) were eluted at a retention time of 50.7 min and 51.4 min, respectively. (b) Percentages of (20 S)- and (20 R)-nicotine (1a/b) in the racemic mixture added to the yeast cells (three independent measurements), and in the cell extracts derived from NtNUP1- or NtNUP1-GFP-expressing yeast cells (three biological replicates).
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
Relative expression level
(a)
6
NtNUP1 + MeJA
4 2
control 0 0
20
(b) Relative expression level
40
Time after MeJA(9)treatment (h) 800
PMT
+ MeJA
600 400 200
control
0 0
20
40
Time after MeJA(9)treatment (h)
Fig. 3. Effects of exogenously added methyl jasmonate (9) on gene expression in tobacco cells. Expression levels of NtNUP1 (a) and PMT (b) in tobacco BY-2 cells cultured in the absence (triangles) or presence (squares) of 100 lM methyl jasmonate (9) were studied by quantitative RT-PCR. Error bars indicate s.d. for three biological replicates. Values are shown relative to the expression levels at the time of methyl jasmonate (9) (MeJA) treatment.
sion yeast indicated that anatabine (2), anabasine, kinetin, and atropine (a racemic mixture of hyoscyamine (3)) somewhat inhibited nicotine (1) uptake by NtNUP1, but that scopolamine (4) was a poor inhibitor of NtNUP1-mediated uptake (Hildreth et al., 2011). Our direct uptake assay in budding yeast confirmed that nicotine (1) and anatabine (2) were readily transported by NtNUP1, whereas NtNUP1 also facilitated the uptake of hyoscyamine (3) and, to a lesser extent, scopolamine (4). Tobacco BY-2 cells overexpressing NtNUP1 showed similar transport preferences toward
Relative NtNUP1 expression level
(a) 80 60 40 20 0
WT
VC4
VC9 VC VC
(b)
WT
OX10
OX18
NtNUP1ox PUP1ox
VC4 VC9 OX10 OX34
PUP1 EF1α Fig. 4. Expression levels of NtNUP1 and PUP1 in tobacco BY-2 cells. Tobacco cells were cultured in the absence of jasmonates. Two independent transgenic lines were used for each construct. (a) NtNUP1 transcript levels in wild-type tobacco cells and transgenic tobacco cells transformed with an empty vector (vector control; VC) or an NtNUP1-overexpressing vector (NtNUP1ox) were measured by quantitative RTPCR. Error bars indicate s.d. for three biological replicates. (b) PUP1 transcripts (after 28 cycles of amplification) and tobacco EF1a transcripts (after 23 cycles) were analyzed by RT-PCR in wild-type tobacco cells and transgenic tobacco cells transformed with an empty vector (VC) or a PUP1-overexpressing vector (PUP1ox). The bands were visualized by ethidium bromide staining. EF1a was used as a loading control.
5
these alkaloids, although nicotine (1) uptake by the tobacco cells was not as efficient as that by yeast cells, for unknown reasons. The import studies herein using yeast cells and tobacco cells demonstrated that NtNUP1 mediates the uptake of pyridoxamine (5) and pyridoxine (6) as efficiently as it mediates the uptake of tobacco alkaloids. Pyridoxamine (5) and pyridoxine (6) both contain a pyridine ring as a common structure. However, other structural features, including stereochemistry, must contribute to the substrate preference of NtNUP1, because (20 S)-nicotine (1a) was more efficiently transported than its stereoisomer, (20 R)-nicotine (1b). In this study, potential transport substrates were tested at the concentrations of either 50 lM or 250 lM. Because nicotine (1) concentrations in the apoplastic space of the tobacco root tissues and in the rhizosphere close to the tobacco roots are not known, and because the KM values of NtNUP1 for tobacco alkaloids are not available, it is difficult to assess physiological significance of NtNUP1’s nicotine (1) transport activity from these biochemical assays. On the other hand, PUP1 has a KM value of approximately 100 lM for pyridoxine (6), whereas its estimated concentration in guttation sap is 3 nM, a far lower concentration compared to the substrate affinity of PUP1 (Szydlowski et al. 2013). Nevertheless, knockout of PUP1 significantly increased concentrations of pyridoxine (6) and related compounds in the sap (Szydlowski et al. 2013), indicating physiological relevance of the uptake studies conducted under the high micromolar concentrations. Does the ability of NtNUP1 to transport vitamin B6 explain its knockdown phenotype, such as its decreased overall synthesis of tobacco alkaloids and increased root growth? Unlike auxotrophic animals, plants synthesize vitamin B6 de novo. Pyridoxal 50 -phosphate (PLP), a bioactive form of vitamin B6, is an essential cofactor for all transaminases and many decarboxylases and deaminases. A null Arabidopsis mutant of PDX2, a single gene encoding a subunit of an enzyme involved in PLP biosynthesis, is embryo lethal (Tambasco-Studart et al., 2007), reflecting the essential role of PLP in cellular metabolism. PUP1 is strongly expressed in the epithem of hydathodes in Arabidopsis leaves, where PUP1 is proposed to retrieve cytokinins and vitamin B6 from the guttation sap (Bürkle et al. 2003; Szydlowski et al. 2013). However, the biological significance of the PUP1-mediated retrieval of bioactive metabolites is not clear, since an Arabidopsis pup1 null mutant does not display any obvious growth or morphological defects (Szydlowski et al. 2013). In tobacco roots, NtNUP1 may import pyridoxine (6), a form of vitamin B6 included in various plant tissue culture media, or it may mediate the retrieval of vitamin B6 secreted into the rhizosphere. In this scenario, knockdown of NtNUP1 in tobacco roots might result in a shortage of PLP, leading to inefficient metabolic processes involving PLP-dependent enzymes. None of the enzymes known to be involved in the nicotine (1) biosynthetic pathway require PLP (Shoji and Hashimoto; 2013); however, the late pathway enzymes, which remain to be identified, may turn out to require PLP. Nonetheless, a cellular shortage of PLP would be detrimental to the metabolic activities and growth of plants. Thus, the uptake of vitamin B6 alone is not sufficient to explain the full spectrum of defects observed in NtNUP1-suppressed tobacco plants. Purine derivatives, including cytokinins, are also transported by PUP1 (Gillissen et al. 2000; Bürkle et al. 2003; Szydlowski et al. 2013). Since NtNUP1 does not show any uptake activity for adenine (2), the NtNUP1-supression phenotypes do not seem to be directly related to cellular abnormalities in cytokinin contents. Future studies should aim to decipher the molecular mechanisms underlying the NtNUP1-mediated control of cell metabolism and plant growth.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
pyridoxamine (5)
** **
10
2
0
0
pyridoxine (6)
10
** **
** **
0
10
WT
OX10
OX18
VC
**
NtNUP1ox
VC9
WT
adenine (8) **
pyridoxal (7)
20
0
20
** *
30
20 10
0 30
2
30
** **
20
4
VC4
30
4
OX18
0
hyoscyamine (3)
6
OX10
** **
**
**
NtNUP1ox
4 2
anatabine (2)
6
VC9
(2’S)-nicotine (1a)
VC
6
pyridoxamine (5) μmol g-1 dry wt
OX34
OX10
OX10
OX18
PUP1ox
(b)
NtNUP1ox
VC9
VC
VC4
0 WT
μmol g-1 dry wt
μmol g-1 dry wt
μmol g-1 dry wt
(a)
VC4
6
15 10
**
** 5
0 (2’S)-nicotine (µM)
**
** 0
0 50 500
0 50 500
WT
OX10
OX18
NtNUP1ox Fig. 5. Import assays in tobacco BY-2 cells expressing NtNUP1 or PUP1. (a) Cultured tobacco cells transformed with either an empty vector (EV), an NtNUP1-overexpressing construct (NtNUP1ox), or a PUP1-overexpressing construct (PUP1ox) were incubated with a test import substrate at 50 lM for 4 h, and the concentrations of the test compounds in the tobacco cells were determined. (20 S)-Nicotine (1a), pyridoxamine (5), and adenine (8) were tested in both NtNUP1ox cells and PUP1ox cells, whereas the import of anatabine (2), hyoscyamine (3), scopolamine (4), pyridoxine (5), and pyridoxal (6) was examined in NtNUP1ox cells. Error bars indicate s.d. for three biological replicates. Significant differences in NtNUP1- or PUP1-expressing cells compared to wild-type (non-transformed) cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄ P < 0.05. (b) Competition of pyridoxamine uptake by the simultaneous addition of various concentrations of (20 S)-nicotine (1a) in NtNUP1-overexpressing tobacco cells. Pyridoxamine (5) was added at 50 M, whereas nicotine (1) was supplied at 0, 50, or 500 lM. Error bars indicate s.d. for three biological replicates. Significant differences in nicotine (1)-supplied cells compared to non-supplied cells were determined by Student’s t-test. ⁄⁄P < 0.01, ⁄P < 0.05.
4. Conclusions
5. Experimental
The uptake assays using budding yeast and cultured tobacco cells showed that NtNUP1 mediates the efficient uptake of not only tobacco alkaloids but also vitamin B6. Although its Arabidopsis homolog PUP1 transports vitamin B6, PUP1 additionally shows an uptake preference for purine derivatives, rather than nicotine (1). Thus, the PUP1/NtNUP1 subclade of the PUP family transporters appears to be characterized by the ability to transport vitamin B6, and members of this subclade additionally exhibit distinct substrate preferences for compounds with nitrogen-containing ring structures. Tobacco metabolites containing a pyridine ring may underlie the defects observed upon NtNUP1 suppression; however, further studies are required to pin-point the molecular mechanisms involved.
5.1. Transport assay in yeast cells An NtNUP1 cDNA fragment (accession number GU174267), encompassing the full-length coding region, its C-terminal fusion with GFP, and an Arabidopsis PUP1 cDNA (accession number At1g28230) were subcloned into pDONR/zeo (Invitrogen), and then transferred into pDR196GW using Gateway cloning technology (Invitrogen). These plasmids were introduced into the yeast (Saccharomyces cerevisiae) strain AD12345678 (MATa, PDR1-3, ura3, his1, Dyor1::hisG, Dsnq2::hisG, Dpdr5::hisG, Dpdr10::hisG, Dpdr11::hisG, Dycf1::hisG, Dpdr3::hisG, and Dpdr15::hisG) (Decottignies et al., 1998) by the lithium acetate method (Yazaki et al., 2002). Plasmid-harboring yeast cells were grown in synthetic
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
defined medium (minus uracil) at 30 °C. Yeast cells expressing NtNUP1 or NtNUP1-GFP grew slower than wild-type yeast cells. When the optical density of the culture at 600 nm reached 0.7, yeast was collected and resuspended in half-strength synthetic defined medium (minus uracil) supplemented with a test transport compound for 4 h. Nicotine (1), hyoscyamine (3), scopolamine (4), and rutin (10) were added at a final concentration of 250 lM, whereas anatabine (2) was used at 50 lM. Yeast cells were collected by centrifugation at 3500 rpm for 10 min, and washed twice with ice-cold deionized water to remove extracellular test compounds. For the uptake assay using radio-labelled substrates, yeasts were grown in the same medium until OD600 reached 1.3–1.8, the cells were collected and suspended to OD600 5 in 50 mM citrate phosphate buffer (pH4.0). 3H-labelled pyridoxine (6) (9.25 kBq) or 3 H-labelled nicotine (1) (9.25 kBq) in 5.25 ll was added to the yeast suspensions (520 ll) to a final concentration of 50 lM. The suspensions were incubated for 3 min at 30 °C. Aliquots (60 ll) were withdrawn from the suspensions, diluted with distilled H2O (5 ml), filtered through hydrophilic Glass Fibre filters (1.0 lm pore size, 25 mm diameter; Millipore), and washed with distilled H2O (5 ml), using a 1225 Sampling Manifold (Millipore). Filters were placed in vials containing LumaSafe Plus cocktail (3 ml) (PerkinElmer), and measured with a liquid scintillation counter (model LS6500, Beckman). To estimate uptake rates, values for yeasts expressing an empty vector were subtracted from those expressing PUP1 or NtNUP1. 5.2. Localization of NtNUP1-GFP in yeast cells Localization of NtNUP1-GFP in yeast cells was observed using a C2 Confocal Laser Microscope (Nikon). GFP and tagRFP were excited at 488 nm and 544 nm, respectively, and fluorescence was detected using a 514/30 nm band pass emission filter for GFP and a 585/65 nm band pass filter for tagRFP. All images were acquired from single optical sections. 5.3. Transport assay in tobacco cells The open reading frame (ORF) of NtNUP1 cDNA was flanked with BamHI and SacI sites by PCR, whereas the full-length ORF of PUP1 cDNA was flanked with HindIII and SacI sites. These clones were subcloned in pGEM-T (Promega), and then inserted into pBI121 (Clontech) to replace the GUS fragment. Tobacco BY-2 cells (N. tabacum cv. Bright Yellow-2) were subcultured in liquid MS medium supplemented with 20 mg l1 KH2PO4, 0.5 g l1 MES and 0.2 mg l1 2,4-D every week. Transgenic tobacco BY-2 cells were generated using the A. tumefaciens strain EHA105, harboring a binary vector as described (An, 1985). For jasmonate treatment, four-day-old BY-2 cells were first rinsed with and then transferred to fresh auxin-free MS medium containing 100 lM methyl jasmonate (MeJa 9). After NtNUP1- or PUP1-overexpressing BY-2 cells were cultured for 3 days, they were suspended in fresh BY-2 medium (30 ml) supplemented with a test transport substrate at a final concentration of 50 lM and incubated for 4 h. Tobacco cells were then harvested and washed with deionized H2O five times. 5.4. RT-PCR analysis Total RNA was isolated using an RNeasy Plant Mini Kit (Qiagen) from tobacco cells that had been ground in liq N2, and a portion of the total RNA (1 ng) was converted into cDNA using Super Script II Reverse Transcriptase (Invitrogen) and an oligo (dT) primer. cDNA templates were amplified using a LightCycler 480 (Roche) with SYBR Premix Ex Taq (Takara) for quantitative RT-PCR analysis
7
under the following conditions: 94 °C for 5 min, 55 cycles of 94 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s. To avoid saturated amplification, template amounts and cycle numbers were adjusted in individual experiments, and the specificity of the reactions was confirmed using the machine’s standard melt curve method. EF1a (accession number; AF120093) was used as a reference gene. The PCR primers used are listed in Supplementary Table S1. For semi-quantitative RT-PCR, cDNA templates were amplified using the GENE Amp@ PCR System 9700 (ABI) with ExTaq (Takara), under the following conditions: 94 °C for 5 min, 1 cycle of 94 °C for 10 s, 55 °C for 10 s, and 72 °C for 15 s. PUP1 cDNA was amplified for 28 cycles and EF1a cDNA for 23 cycles. Amplified products were separated on a 2% agarose gel and stained with ethidium bromide. 5.5. Quantification of test substrates For quantification of nicotine (1), anatabine (2), hyoscyamine (3), and scopolamine (4), lyophilized tobacco cells (50 mg dry wt) were homogenized in 0.1 N H2SO4 (4 ml), and the homogenate was sonicated for 60 min and then centrifuged at 880 g for 15 min. Fresh yeast cells were suspended in a 5-fold amount of 0.1 N H2SO4 (v/w), disrupted by vigorous vortexing with acidwashed glass beads, and centrifuged at 17,400 g for 10 min. The supernatant (either from tobacco samples or yeast samples) was neutralized by adding 25% NH4OH (0.4 ml). The mixture (1 ml) was loaded onto an Extrelut-1 column (Merck), and the alkaloids were eluted with CHCl3 (6 ml). The eluent was dried at 37 °C. The dry residues were dissolved in EtOH containing 0.1% dodecane as internal standard, and analyzed by gas chromatography (GC; GC-2010, Shimadzu) equipped with a Rtx-5 Amine capillary column (Restek, PA, US). The column temperature was held at 100 °C for 10 min, and then increased to 250 °C over a 35-min period. The injector and FID detector were held at 300 °C, and the flow rate of the carrier gas He was 0.99 ml min1, with a split ratio of 1:20. Chiral forms of nicotine (1a and 1b) were separated by GC with a capillary column (WCOT, 60 m 0.25 mm id. 0.25 lm df, fused silica, b-DEX™ 120, Supelco, PA, US). The injector and FID detector were held at 230 °C and 250 °C, respectively, whereas the column oven temperature was held at a constant temperature of 120 °C, and the carrier gas He was run at 0.95 ml min1, with a split ratio of 1:20. For vitamin B6 uptake analysis, yeast and tobacco cells were extracted in 25 mM phosphate buffer (pH. 4.0), homogenized, sonicated for 30 min, and incubated at 65 °C for 30 min. After centrifugation as described above, the supernatants were analyzed by high-performance liquid chromatography (HPLC), using the LC10 system (Shimadzu, Japan) equipped with a COSMOSIL Cholester column (4.6 mm id. 250 mm, Nacalai tesque, Japan). Pyridoxamine (5), pyridoxine (6), and pyridoxal (7) were eluted from the column with an isocratic mobile phase of 25 mM phosphate buffer (pH 4.0) at a flow rate of 1 ml min1 at 40 °C, and were detected with a fluorescence detector at an excitation of 328 nm and an emission of 393 nm. Adenine (8) was extracted from tobacco cells in 20 mM phosphate buffer (pH 7.0), homogenized, sonicated for 30 min, and incubated at 65 °C for 30 min. After centrifugation as described above, the supernatants were analyzed by HPLC as above. Adenine (8) was eluted from the column with an isocratic mobile phase of 20 mM phosphate buffer (pH. 7.0) at a flow rate of 1 ml min1 at 40 °C, and detected at UV 254 nm. Rutin (10) was extracted from yeast and tobacco cells with EtOH–H2O (1:1, v/v), and processed as described above. Supernatants from centrifugation were filtered using a COSMO Spin Filter H (Nacalai tesque, Japan) and analyzed by HPLC, using the MD-2018 system (Jasco, Japan), equipped with a COSMOSIL 5C18
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
8
K. Kato et al. / Phytochemistry xxx (2014) xxx–xxx
MS-II column (5 lm, 4.6 mm 150 mm, Nacalai tesque, Japan). Rutin (10) was eluted from the column with a linear gradient program from 12% to 100% buffer (B), relative to buffer (A), in 15 min at a flow rate of 1 ml min1 at 40 °C, and detected at UV 358 nm. Buffer (A) consisted of H2O:HCOOH (9:1), whereas buffer (B) was composed of H2O:HCOOH:MeOH:CH3CN (40:10:22.5:22.5). 5.6. Chemicals (20 R)(+)-Nicotine (1b) (TRC, Canada), (20 S)()(1a)-nicotine (Wako Chemicals, Japan), anatabine (Wako Chemicals, Japan), hyoscyamine (3) (Sigma, Germany), scopolamine (4) (TOCRIS bioscience, UK), pyridoxine (6) (Nacalai tesque, Japan), pyridoxamine (5) (Sigma, Germany), pyridoxal (7) (Wako Chemicals, Japan), rutin (10) (Nacalai tesque, Japan), and adenine (8) (Sigma, Germany) were purchased commercially. To prepare a racemic mixture of nicotine (1a/b), equal molar amounts of (20 S)-nicotine (1a) and (20 R)-nicotine (1b) were mixed. [methylene-3H]-pyridoxine (6) (37 GBq ml1) and [N-methyl-3H]-(20 S)-nicotine (1a) (37 GBq ml1) were obtained from ARC Inc. (Saint Louis, MO).
Acknowledgments We thank W. Frommer (Carnegie Institution), K. Yazaki (Kyoto University), N. Inada (Nara institute of Science and Technology), and A. Goffeau (Université Catholique de Louvain) for providing us with pDR196, pDR196GW, pUGW2m-tagRFP, and the yeast strain AD12345678, respectively. This study was supported in part by a Grant from the Japan Society for the Promotion of Science to T.S. (Grant-in-Aid for Scientific Research (C), No. 23570035) and to N.S. (Grant-in-Aid for Young Scientists (A), No. 25712012).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 05.011.
References An, G., 1985. High efficiency transformation of cultured tobacco cells. Plant Physiol. 79, 568–570. Bürkle, L., Cedzich, A., Döpke, C., Stransky, H., Okumoto, S., Gillissen, B., Kühn, C., Frommer, W.B., 2003. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J. 34, 13–26. Chintaparkorn, Y., Hamill, J.D., 2003. Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine. Plant Mol. Biol. 53, 87–105. Decottignies, A., Grant, A.M., Nichols, J.W., de Wet, H., McIntosh, D.B., Goffeau, A., 1998. ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J. Biol. Chem. 273, 12612–12622. Gillissen, B., Bürkle, L., André, B., Kühn, C., Rentsch, D., Brandl, B., Frommer, W.B., 2000. A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis. Plant Cell 12, 291–300. Hildreth, S.B., Gehman, E.A., Yang, H., Lu, R.-H., Rithish, K.C., Harich, K.C., Yu, S., Lin, J., Sandoe, J.L., Okumoto, S., Murphy, A.S., Jelesko, J.G., 2011. Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism. Proc. Natl. Acad. Sci. U.S.A. 108, 18179–18184. Kajikawa, M., Shoji, T., Kato, A., Hashimoto, T., 2011. Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco. Plant Physiol. 155, 2010–2022. Morita, M., Shitan, N., Sawada, K., Montagu, M.C.E.V., Inzé, D., Rischer, H., Goossens, A., Oksman-Caldentey, K.-M., Moriyama, Y., Yazaki, K., 2009. Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc. Natl. Acad. Sci. U.S.A. 106, 2447–2452. Shoji, T., Hashimoto, T., 2008. Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells? Plant Cell Physiol. 49, 1209– 1216. Shoji, T., Hashimoto, T., 2013. Smoking out the masters: transcriptional regulators for nicotine biosynthesis in tobacco. Plant Biotechnol. 30, 217–224. Shoji, T., Inai, K., Yazaki, Y., Sato, Y., Takase, H., Shitan, N., Yazaki, K., Goto, Y., Toyooka, K., Matsuoka, K., Hashimoto, T., 2009. Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots. Plant Physiol. 149, 708–718. Szydlowski, N., Bürkle, L., Pourcel, L., Moulin, M., Stolz, J., Fitzpatrick, T.B., 2013. Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis. Plant J. 75, 40–52. Tambasco-Studart, M., Tews, I., Amrhein, N., Fitzpatrick, T.B., 2007. Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiol. 144, 915–925. Xie, J., Song, W., Maksymowicz, W., Jin, W., Cheah, K., Chen, W., Carnes, C., Ke, J., Conkling, M.A., 2004. Biotechnology: A tool for reduced risk tobacco products. Recent Adv. Tobacco Sci. 30, 17–37. Yazaki, K., Kunihisa, M., Fujisaki, T., Sato, F., 2002. Geranyl diphosphate:4hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon: Cloning and characterization of a key enzyme in shikonin biosynthesis. J. Biol. Chem. 277, 6240–6246.
Please cite this article in press as: Kato, K., et al. Tobacco NUP1 transports both tobacco alkaloids and vitamin B6. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.05.011
