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Interconversions of different forms of vitamin B6 in tobacco plants
Phytochemistry 72 (2011) 2124–2129
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Interconversions of different forms of vitamin B6 in tobacco plants ShuoHao Huang a,b, HaiBin Zeng a, JianYun Zhang c, Shu Wei a, LongQuan Huang a,⇑ a
Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People’s Republic of China Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan c College of Life Science, Anhui Agricultural University, Hefei 230036, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 18 September 2010 Received in revised form 18 July 2011 Available online 17 August 2011 Keywords: Nicotiana tabacum Solanaceae Vitamin B6 B6 vitamers
a b s t r a c t There are six different vitamin B6 (VB6) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 50 -phosphate (PLP), pyridoxamine 50 -phosphate (PMP), and pyridoxine 50 -phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB6 in the human diet, and VB6 plays an important role in plants, the mechanisms underlying the interconversions of different VB6 forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B6 vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB6 was about 3.9 lg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB6 by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B6 vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of nonphosphorylated forms. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Vitamin B6 (VB6) (1–6, Fig. 1) exists in several forms, which are termed ‘‘vitamers’’ and include pyridoxal (1) (PL), pyridoxamine (2) (PM), pyridoxine (3) (PN), and their phosphorylated derivatives pyridoxal 50 -phosphate (4) (PLP), pyridoxamine 50 -phosphate (5) (PMP), and pyridoxine 50 -phosphate (6) (PNP), respectively (Fig. 1). PLP (4) is an important coenzyme for numerous enzymes involved in different cellular reactions and processes, including metabolism of amino acids, fatty acids and carbohydrates, heme and chlorophyll biosynthesis, ethylene and auxin biosynthesis, and transcriptional regulation (Huq et al., 2007). Additionally, VB6 has been linked to stress responses in plants (Shi et al., 2002; Denslow et al., 2005, 2007; Sang et al., 2007) and is essential for root development (Shi and Zhu, 2002; Chen and Xiong, 2005; Titiz et al., 2006). VB6 is synthesized de novo by two different enzymatic pathways (Fig. 1). The first, referred to as the ‘‘DXP (10 -deoxy-D-xylulose-50 -phosphate)-dependent pathway’’, is found in Escherichia coli and a small number of other prokaryotes. In E. coli, PNP (6)
⇑ Corresponding author. Tel.: +86 551 5157862. E-mail address: [email protected] (L. Huang). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.07.019
after being synthesized is oxidized by a PNP/PMP oxidase to form PLP (4) (Hill et al., 1996; Laber et al., 1999; Drewke and Leistner, 2001). The other, referred to as the ‘‘DXP-independent pathway’’, is the predominant pathway in nature. This pathway involves a protein complex, consisting of two different proteins, encoded by the genes PDX1 and PDX2. This enzyme complex directly catalyzes PLP (4) biosynthesis (Burns et al., 2005; Raschle et al., 2005; Tambasco-Studart et al., 2005). Plants, a major source of VB6 in the human diet, synthesize VB6 via the DXP-independent pathway. PDX1 and PDX2 have been isolated and characterized in tobacco (Denslow et al., 2005), Arabidopsis thaliana (Tambasco-Studart et al., 2005; Titiz et al., 2006; Wagner et al., 2006; Denslow et al., 2007), and Ginkgo biloba (Leuendorf et al., 2008). There are other pathways of VB6 metabolism in nature. In addition to the de novo biosynthetic pathways of VB6, a salvage pathway is found in all organisms, and functions to inter-convert the six different vitamer forms (Fig. 1). The salvage pathway mainly involves an ATP-dependent PL kinase and a PNP/PMP oxidase (Tsuyoshi et al., 2004). The kinase catalyzes the phosphorylation of PL (1), PM (2) and PN (3) to form PLP (4), PMP (5) and PNP (6). The oxidase catalyzes PNP (6) and PMP (5) oxidation to form PLP (4). Alternatively, PMP (5) oxidation can also be catalyzed by a transaminase (Tsuyoshi et al., 2004). The phosphorylated forms are de-phosphorylated by phosphatase to restore the precursors
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“DXP-independent” de novo synthetic pathway
PNP/PMP oxidase
Salvage pathway
PNP/PMP oxidase
PMP (2) PL kinase
“DXP-dependent” de novo synthetic pathway
Transaminase PNP (3)
PLP (1)
Phosphatase
PL kinase
Phosphatase
PL reductase
PM-pyruvate aminotransferase PM (5)
PL kinase
Phosphatase
PL (4)
PN 4-oxidase
PN (6)
Fig. 1. Structures and interconversion pathways of vitamin B6 vitamers (1–6).
(McCormick and Chen, 1999; Mittenhuber, 2001). Additionally, a PL reductase that reduces PL (1) to PN (3) has been suggested to be involved in the salvage pathway (Nakano et al., 1999; Morita et al., 2004). Moreover, two degradation pathways of VB6 in bacteria have been reported (Burg et al., 1960). PM-pyruvate aminotransferase and PN 4-oxidase, respectively, use PM (2) and PN (3) as a substrate to form PL (1) in a VB6 degradation pathway (Yoshikan et al., 2006; Kaneda et al., 2002). The understanding of interconversions of the different VB6 forms in plants is very limited. Genes encoding PL kinases have been cloned and characterized in both A. thaliana and wheat (Lum et al., 2002; Wang et al., 2004), and that of PNP/PMP oxidase has been cloned from A. thaliana (Sang et al., 2007). González et al. (2007) further reported the effects on phenotype, vitamer levels, stress responses, enzyme activity, and regulation of salvage and de novo pathway genes in A. thaliana, resulting from mutations in the PNP/PMP oxidase (PDX3) and the PL kinase (SOS4) genes. In the sos4 mutant, significant shifts in vitamer concentration occurred compared to the wild type. Higher levels of PM (2), PLP (4), and PN (3), with a 2-fold increase in PM (2) and PN (3) and an almost 9-fold increase in PLP (4), were observed. Therefore, the major increase in total vitamer levels is due to a large increase in PLP (4). PLP (4) is a very reactive molecule forming complexes with amine groups of amino acid residues in proteins, and free PLP (4) will be promptly hydrolyzed by phosphatase to restore PL (1) and maintain PLP (4) homoeostasis, suggesting that the pool of free PLP (4) is maintained at a very low level (Di-Salvo et al., 2003; Lumeng et al., 1980; Merrill et al., 1984). Thus, the aim of the present study is to use a responsive analysis to investigate if there is an endogenous system to maintain PLP (4) homoeostasis and how the interconversions of different B6 vitamers take place in plants. The effect of supplementation of Murashige and Skoog (MS) basal medium with PM (2), PL (1) or PN (3), on the abundance of different B6 vitamers (1–6) in tobacco plants cultured on the medium was examined by using high performance liquid chromatography (HPLC). The results help us gain insights of VB6 metabolism taking place in plants.
determining all B6 vitamers (1–6) were established. The retention times of PMP (5), PM (2), PNP (6), PLP (4), PL (1) and PN (3) were 7.0, 7.6, 8.6, 9.0, 10.8 and 12.1 min, respectively, under the conditions described in the Experimental (Section 5.4). PMP (5) as well as PM (2) emits relatively strong fluorescence, while PLP (4) has poor fluorescence under the same conditions. The detection limit for PMP (5) as well as PM (2) was less than 30 pg, and that for PLP (4) was about 350 pg per injection. After treatment with KCN, the fluorescent signal of PLP (4) was enhanced over 10-fold due to the formation of pyridoxic acid 50 -phosphate (7) (PIC-P), and the retention time of PIC-P (7) was 9.3 min (data not shown). Thus, the detection limit of PLP (4) was increased to about 35 pg per injection, the same level as that of PNP (6). The recovery of each authentic B6 vitamer added to the extract from tobacco leaves was 95 ± 10%. 2.2. HPLC analysis of VB6 (1–6) in tobacco leaves, root and culture media The HPLC chromatogram of leaf B6 vitamers (1–6) in tobacco plants grown on MS basal media exhibited distinct peaks of PMP (5), PM (2), PL (1) and PN (3) (Fig. 2). The peak area or peak height was used for quantification. To obtain accurate results, KCNtreated leaf extracts were injected for quantitative analysis of PLP (4). No significant elution peak was found at the expected retention time for pure authentic PNP (6) during the whole
2. Results 2.1. HPLC analysis of standard B6 vitamers (1–6) Pure standard B6 vitamers (1–6) were well-separated using the HPLC analysis method used in this study and standard curves for
Fig. 2. HPLC pattern of leaf B6 vitamers from tobacco plants grown on MS basal media.
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experimental process (Fig. 2–5), suggesting that the level of PNP (6) in tobacco plants was different from those of the other vitamers and lower than the detection limit of our HPLC system. The levels of B6 vitamers (1–6) in tobacco leaves analyzed weekly during the experiments are given in Table 1. The mean level of total B6 vitamers (1–6) was about 3.9 lg/g fresh weight, and PL (1), PM (2), PN (3), PLP (4) and PMP (5) accounted for 23%, 14%, 37%, 20% and 6%, respectively. Thus, PN (3) was the major B6 vitamer in tobacco leaves. During the experiment for week 3, the levels of PL (1) and PLP (4) decreased, whereas that of PN (3) increased. There was no significant difference between tobacco leaves and roots in levels of B6 vitamers (1–6). Moreover, non-phosphorylated B6 vitamers of PL (1), PM (2) and PN (3) were all detected from media. The mean level of VB6 (1–3) was about 2.3 lg/g fresh weight, and PL (1), PM (2) and PN (3) accounted for 20%, 18% and 62%, respectively. PN (3) was also the major B6 vitamer in media. Since the medium (80 g) outweighed the plants (about 1.5 g with 4–5 tender leaves) grown on it greatly, a significant amount of non-phosphorylated VB6 (1– 3) was released from the tobacco plants.
Fig. 4. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PL (1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with non-phosphorylated B6 vitamers (1–3) VB6 compounds (1–6) are stable under acidic condition and unstable under neutral and especially under alkaline conditions. At room temperature for 3 weeks, B6 vitamers (1–3) were stable in the medium and no inter-conversion among the vitamers was detected. The recovery percentages for PM (2), PL (1) and PN (3) were 66.8%, 63.4% and 76.5%, respectively, detected by HPLC analysis. This was the basis for our further studies. Supplementation of medium with PM (2), PL (1), or PN (3) resulted in significant quantitative changes of the levels of the different B6 vitamers in the tobacco plants compared to those in control plants grown in medium without any supplementation. The levels of B6 vitamers (1–6) in leaves were determined weekly over the 3 week supplementation experiment. They did not change significantly after the 1st week of treatment, suggesting that they might attain new steady-state levels within this period. In addition, no difference in plant growth was observed between the plants supplemented with and without 100 mg/L of PM (2), PL (1) or PN (3) in the growth media (data not shown).
Fig. 5. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PN (3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3.1. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PM (2) Fig. 3 shows that supplementation of the medium with PM (2) resulted in concentration changes among B6 vitamers (1–6) in the tobacco plants compared to the control plants grown in the medium without the supplementation treatment. Following supplementation with PM (2), the total VB6 (1–6) level increased to 40 lg/g fresh weight, about 10-fold higher than that of the control plants. The mean contents for PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined as 4.40 ± 1.44, 24.23 ± 4.10, 9.35 ± 3.45, 1.45 ± 0.36 and 0.39 ± 0.02 lg/g fresh weight, respectively. An almost 45-fold increase in PM (2), an 8-fold increase in PN (3), and a 4-fold increase in PL (1), which accounted for about 61%, 23% and 11%, respectively of total VB6 (1–6), were observed. Both PLP (4) and PMP (5) levels changed slightly. Our data suggested that levels of the phosphorylated vitamers (4–6) were maintained steady in tobacco plants.
Fig. 3. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PM (2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3.2. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PL (1) Following supplementation with PL (1), the total VB6 (1–6) level was increased to 36 lg/g fresh weight, about 9 times as much as
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a b
Timeline
Sample
PL (1)
PM (2)
PN (3)
PLP (4)
1 Week
Leaf tissue Root Medium
1.11 ± 0.10 0.39 ± 0.05 0.45 ± 0.02
0.55 ± 0.05 0.67 ± 0.31 0.32 ± 0.01
1.18 ± 0.02 1.21 ± 0.01 0.95 ± 0.16
2 Week
Leaf tissue Medium
0.99 ± 0.11 0.50 ± 0.05
0.56 ± 0.01 0.60 ± 0.08
3 Week
Leaf tissue Medium
0.53 ± 0.18 0.47 ± 0.05
0.53 ± 0.04 0.38 ± 0.09
a
b
PMP (5)
PNP (6)
Total VB6
0.94 ± 0.15 0.81 ± 0.09 —
0.29 ± 0.03 0.17 ± 0.03 —
Trace Trace —
4.07 3.25 1.72
1.40 ± 0.06 2.17 ± 0.88
0.72 ± 0.05 —
0.25 ± 0.01 —
Trace —
3.92 3.27
1.81 ± 0.17 1.47 ± 0.59
0.63 ± 0.08 —
0.20 ± 0.01 —
Trace —
3.70 2.32
The amounts of PLP (4) were determined after KCN treatment. No significant elution peak can be used for quantitative analysis, the same in Tables 2 and 3.
lyzed after supplementation with PM (2), PL (1) or PN (3) for 1 week (Table 3).
that in control plants. The average levels for PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined as 4.58 ± 1.65, 3.11 ± 2.09, 25.93 ± 3.45, 1.38 ± 0.33 and 0.70 ± 0.64 lg/g fresh weight, respectively. The proportion of PN (3) to total VB6 (1–6) in leaves was increased to 73%, about 22-fold higher than in the control. PM (2) was also 6-fold higher than in the control. Whereas the ratio of PL (1) to total VB6 (1–6) was merely about 13%, a 4-fold increase compared to that of control plants. The changes in PLP (4) and PMP (5) levels due to the PL (1) supplementation were similar to those due to the PM (2) supplementation.
3. Discussion VB6 exists, in at least six biologically-active forms (1–6) in biological materials. In order to quantify these derivatives in their simplest and most sensitive way, many analytical approaches have been proposed in which HPLC always figures prominently. A reversed-phase HPLC method in conjunction with highly sensitive fluorescence detection has been developed and it allows the analysis of six familiar VB6 derivatives (1–6) in an isocratic solvent within 30 min. By using fluorescence detection, many interfering peaks were eliminated; however some negative effect from such peaks still remained. In our previous study, many VB6 analyses on various plants using HPLC ended in failure. More interfering peaks appeared in plants grown in soil than those grown in a MS basal medium. As for PMP (5) and PM (2), the fluorescence detection method was over 50-fold more sensitive than the UV detection method. However, the fluorescent signal of PLP (4) was not sufficiently intense in this analytical system to obtain accurate results. To overcome this difficulty a pre-column treatment with KCN to enhance the fluorescence of PLP (4) was developed (Tsuge et al., 1988) and applied successfully in this study. In the mammalian system, PLP (4) is synthesized mainly in the liver, and then released to the bloodstream in association with
2.3.3. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PN (3) Supplementation with PN (3) for 1 week resulted in an increase in total VB6 (1–6) to 36 lg/g fresh weight, about 9 times as much as that of the control. The average levels of PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined to be 1.70 ± 0.18, 1.65 ± 0.52, 31.70 ± 6.66, 0.83 ± 0.10 and 0.28 ± 0.04 lg/g fresh weight, respectively. The increased PN (3) level accounted for about 88% of total VB6 (1–6) in leaves, an almost 26-fold increase compared to the control value. The other B6 vitamer levels were unchanged compared to those of control plants. Additionally, we analyzed the levels of B6 vitamers (1–6) in tobacco plants and media after supplementation with PM (2), PL (1) or PN (3) for 48 h (Table 2). The results show a similar trend of the concentration shifts of B6 vitamers (1–6) in the tobacco plants ana-
Table 2 Levels of leaf B6 vitamers in tobacco plants analyzed after supplementation with PM (2), PL (1) or PN (3) for 48 h. (lg/g fresh weight). Supplement
Sample
PLP (4)
PMP (5)
PNP (6)
Total VB6
PM (2)
Leaf tissue Medium
PL (1) 2.90 ± 1.37 1.58 ± 0.88
PM (2) 10.31 ± 5.17 85.64 ± 20.9
PN (3) 1.66 ± 0.45 1.18 ± 0.14
1.13 ± 0.06 —
0.53 ± 0.39 —
trace —
16.53 88.40
PL (1)
Leaf tissue Medium
2.21 ± 2.15 72.45 ± 26.8
0.93 ± 0.48 0.81 ± 0.09
6.94 ± 3.10 5.97 ± 5.28
1.20 ± 0.34 —
0.33 ± 0.06 —
Trace —
11.61 79.23
PN (3)
Leaf tissue Medium
1.20 ± 0.17 0.81 ± 0.19
0.80 ± 0.09 3.14 ± 3.96
8.67 ± 2.30 89.29 ± 8.32
1.12 ± 0.33 —
0.33 ± 0.10 —
Trace —
12.12 93.24
Table 3 Levels of leaf B6 vitamers in tobacco plants analyzed after supplementation with and without (control) PM (2), PL (1) or PN (3) for 1 week. (lg/g fresh weight). Supplement
Sample
PL (1)
PM (2)
PN (3)
PLP (4)
PMP (5)
PNP (6)
Total VB6
Without
Leaf tissue Medium
1.11 ± 0.10 0.45 ± 0.02
0.55 ± 0.05 0.32 ± 0.01
1.18 ± 0.02 0.95 ± 0.16
0.94 ± 0.15 —
0.29 ± 0.03 —
0.04 ± 0.02 —
4.07 1.72
PM (2)
Leaf tissue Medium
4.40 ± 1.44 3.21 ± 0.19
24.23 ± 4.10 74.68 ± 0.32
9.35 ± 3.45 3.08 ± 0.95
1.45 ± 0.36 —
0.39 ± 0.02 —
Trace —
39.82 80.97
PL (1)
Leaf tissue Medium
4.58 ± 1.65 43.06 ± 8.06
3.11 ± 2.09 2.40 ± 1.01
25.93 ± 3.45 11.16 ± 3.06
1.38 ± 0.33 —
0.70 ± 0.64 —
Trace —
35.70 56.62
PN (3)
Leaf tissue Medium
1.70 ± 0.18 0.75 ± 0.39
1.65 ± 0.52 2.24 ± 1.26
31.70 ± 6.66 70.56 ± 25.37
0.83 ± 0.10 —
0.28 ± 0.04 —
Trace —
36.16 73.55
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albumin (Lumeng et al., 1980; Merrill et al., 1984). During supplementation with PN (3) (100 mg/day orally for 1–3 weeks), the plasma content of PLP (4) increased 7-fold and that of PL (1) increased 12-fold, but the PN (3) level did not increase (Lumeng et al., 1980). In this study, administering tobacco plants with the non-phosphorylated vitamers (1–3) resulted in an increase in total VB6 (1–6) by around 10-fold with very little effect on the concentrations of the phosphorylated vitamers (4–6) including PLP (4). This suggests that a stable level of PLP (4), the coenzyme form of VB6, is maintained in tobacco even though the levels of the endogenous nonphosphorylated forms (1–3) changed significantly. Furthermore, exogenous addition of PM (2) or PN (3) to the plant growth medium increased its endogenous level in the plant more effectively than the levels of any other endogenous B6 vitamers, whereas PL (1) administration did not affect its endogenous level, instead, endogenous PN (3) abundance was increased significantly. These results indicate that the endogenous PL (1) level is also stringently controlled, and an efficient PL (1) to PN (3) conversion system may exist in plants. Therefore, we speculate that PN (3) may be downstream of VB6 metabolism in tobacco. This is consistent with a previous finding on the levels of VB6 (1–6) in plant-based foods where PN (3) was shown to be the predominant vitamer (Ollilainen, 1999). Additionally, the increase in level of endogenous PM (2) was also strikingly observed in the plants administered with PL (1) (Fig. 4), suggesting that an interconversion pathway between PL (1) and PM (2) also exists in tobacco. VB6 (1–6) is a group of water-soluble and small organic molecules. Transport of B6 vitamers across cellular membranes has been studied extensively. It has been known for many years that only the non-phosphorylated vitamers (1–3) move across cell membranes. In this study, tobacco plants were grown in sterile Magenta boxes; VB6 (1–3) detected in culture medium was derived solely from the plants (Table 1), confirming that the excretion of superfluous VB6 (1–3) from roots occurs in tobacco plants. Moreover, supplementation with any of the single non-phosphorylated vitamers (1–3) raised the total leaf levels of VB6 (1–6) by about 10-fold, suggesting that plants can take up the different non-phosphorylated B6 vitamers (1–3) with similar efficiency. In spite of the importance of this vitamin, the regulation of its metabolism is only beginning to be defined, and the consequences of altering VB6 content on plant growth and development is still unclear. The abundance of PLP (4), the functional form of VB6, was found to be quite stable in this study. That might be the reason why the greatly elevated level of VB6 in tobacco plants did not significantly affect plant growth and development. 4. Conclusions In conclusion, the present work reports that a stable level of the co-factor form PLP (4) is maintained in tobacco plants even though the levels of the endogenous non-phosphorylated forms (1–3) changed significantly due to supplementation of PM (2), PL (1) or PN (3) in the plant growth media. Efficient conversion pathways from PL (1) to PN (3) and PM (2) are present in the plant. To our knowledge, this is the first report of interconversions of B6 vitamers (1–6) in plants following administration experiments. These results raise some interesting questions about how, and why, the metabolism of VB6 is regulated in plants.
ma Chemicals (Sigma, USA). Due to unavailability of a commercial standard, PNP (6) was prepared from PLP (4) by reduction with NaBH4, according to a previously reported method (Argoudelis, 1986) validated by HPLC. Standard VB6 (1–6) was dissolved in H2O to make a 1 mM stock solution which was then diluted with 25 mM NaClO4-25 mM KH2PO4 buffer (pH 2.5) to the final concentration used for HPLC analysis. To avoid photo-degradation of VB6 all procedures were conducted in a dark room and the H2O was distilled and filtered through a 0.45 lm membrane filter. HPLCgrade CH3CN, HClO4, NaClO4, KH2PO4, K2HPO4, and all other chemicals and reagents were purchased from Sangon (Shanghai, China). 5.2. Plants and treatment Fresh and young leaves of tobacco plants (Nicotiana tabacum) were used as explants in this study. A standard method for in vitro plant culture was adopted. Culture media for inducing callus, buds and rooting were MS + 6-BA 1.0 mg/L + 2,4-D 0.5 mg/L, MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L and MS + NAA 0.2 mg/L, respectively. The media were all complemented with sucrose 30.0 g/L and agar 8.0 g/L, and adjusted to pH 5.8. Under normal plant growth conditions (25 ± 2 °C and 14 h photoperiod at 2500 lux), culture periods were 2, 4 and 8 weeks for three different induction treatments, respectively. The generated plantlets were then transplanted into MS media plus sucrose 30.0 g/L and agar 8.0 g/L at pH 5.8, supplemented with PM (2), PL (1), or PN (3) 100 mg/L. Meanwhile, some plants transplanted into MS basal medium were used as control plants. The plants were grown for 3 weeks under the above-described conditions. To minimize the decomposition of VB6 induced by light, the lower part of a plant growth flask was covered with aluminum foil. The whole operation was performed under sterile conditions. 5.3. Extraction of VB6 (1–6) VB6 compounds (1–6) were extracted from leaf tissue as follows: freshly excised leaf tissue (0.5 g) was completely homogenized with liq. N2 in a mortar with a pestle, and then 3 M HClO4 (1.5 mL) was added and mixed by vigorous votexing. The mixture was next centrifuged at 12,840g for 20 min at 4 °C. To 1 mL of supernatant 1 M phosphate buffer (pH 5.5, 1 mL) was added and the solution was centrifuged again as above. The pH of the solution was adjusted to 2.5 with 0.1 M HCl, and then made up to 3 mL with distilled H2O. This solution was named as the leaf extract. The KCN treatment of leaf extract was carried out according to the method of Tsuge et al. (1988) with a slight modification: briefly leaf extract (2 mL) was placed in a centrifuge tube and the pH was adjusted to 7.5 with 5 M KOH. After addition of 0.1 M KCN (0.1 mL), the solution was incubated at 50 °C for 3 h with vigorous shaking under dark conditions in order to accomplish complete oxidation of PLP (4) to PIC-P (7). Then, the pH of the solution was again adjusted to 2.5 with 1 M HCl and allowed to stand for 24 h at 25 °C. Finally, the volume of this solution was adjusted to 3 mL with distilled water. Extracts were kept on ice in the dark. Prior to injection into an HPLC -line solutions were passed through a 0.22 lm membrane filter. Unless otherwise specified, all VB6 assays were performed three times. 5.4. HPLC analysis of B6 vitamers (1–6)
5. Experimental 5.1. Materials Reference standards of PL (1) (PL-HCl), PM (2) (PM-2HCl), PN (3) (PN-HCl), PLP (4), and PMP (5) (PMP-HCl) were obtained from Sig-
The levels of six B6 vitamers (1–6) in tobacco leaves were determined by HPLC analysis weekly during the 3 week supplementation experiments. A reversed-phase HPLC method described by Zhang et al. (2004) was used with some modifications. The HPLC system was a Waters 6000 with a 2475 fluorescence detector.
S. Huang et al. / Phytochemistry 72 (2011) 2124–2129
The B6 vitamers (1–6) were separated with COSMOSIL 5Ph column (4.6 mm i.d 250 mm, Waters type, Nacalai Tesque, Inc., Kyoto, Japan). The column was mounted in a column heater set at 30 °C. The detector excitation and emission wavelengths were set at 290 and 395 nm, and at 320 and 420 nm after KCN treatment, respectively. The sample injection volume was 5 lL. Separations were performed at a flow rate of 0.5 mL/min using an isocratic solvent of 25 mM NaClO4-25 mM KH2PO4 buffer (pH 2.5) containing 1% CH3CN. In order to elute slow-moving components, a solvent containing a higher concentration of CH3CN (usually 10%) in the buffer solution as above was flushed for 25 min after each analysis. The mobile phase was filtered through a 0.45 lm membrane filter and sufficiently degassed in an ultrasonicator with suction before adding CH3CN. Spiking of the leaf extracts with reference standards of PL (1), PM (2), PN (3), PLP (4), PMP (5) and PNP (6) was used to confirm peak identity. Six-point standard curves were prepared and run daily before running the leaf extract samples. B6 vitamer concentration was determined from the standard curves obtained by plotting peak areas or peak heights against the different known concentrations of each vitamer. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30870338) and Natural Science Foundation of Education Department of Anhui Province (No. KJ2010A116). References Argoudelis, C.J., 1986. Preparation of crystalline pyridoxine 50 -phosphate and some of its properties. J. Agric. Food Chem. 34, 995–998. Burns, K.E., Xiang, Y., Kinsland, C.L., McLafferty, F.W., Begley, T.P., 2005. Reconstitution and biochemical characterization of new pyridoxal-50 phosphate biosynthetic pathway. J. Am. Chem. Soc. 127, 3682–3683. Burg, R.W., Rodwell, V.W., Snell, E.E., 1960. Bacterial oxidation of vitamin B6. III. Metabolites of pyridoxamine. J. Biol. Chem. 235, 1164–1169. Chen, H., Xiong, L., 2005. Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses. Plant J. 44, 396–408. Denslow, S., Walls, A., Daub, M., 2005. Regulation of biosynthetic genes and antioxidant properties of vitamin B6 vitamers during plant defense responses. Physiol. Mol. Plant. 66, 244–255. Denslow, S.A., Rueschhoff, E.E., Daub, M.E., 2007. Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant Physiol. Biochem. 45, 152–161. Di-Salvo, M.L., Safo, M.K., Musayev, F.N., Bossa, F., Schirch, V., 2003. Structure and mechanism of Escherichia coli pyridoxine 50 -phosphate oxidase. Biochim. Biophys. Acta 1647, 76–82. Drewke, C., Leistner, E., 2001. Biosynthesis of vitamin B6 and structurally related derivatives. Vitam. Horm. 61, 121–155. González, E., Danehower, D., Daub, M.E., 2007. Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 50 -phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway. Plant Physiol. 145, 985–996. Hill, R.E., Himmeldirk, K., Kennedy, I.A., Pauloski, R.M., Sayer, B.G., Wolf, E., Spenser, I.D., 1996. The biogenetic anatomy of vitamin B6. A 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli. J. Biol. Chem. 271, 30426– 30435.
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Huq, M.D., Tsai, N.P., Lin, Y.P., Higgins, L., Wei, L.N., 2007. Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation. Nat. Chem. Biol. 3, 161–165. Kaneda, Y., Ohnishi, K., Yagi, T., 2002. Purification, molecular cloning, and characterization of pyridoxine 4-oxidase from Microbacterium luteolum. Biosci. Biotechnol. Biochem. 66, 1022–1031. Laber, B., Maurer, W., Scharf, S., Stepusin, K., Schmidt, F., 1999. Vitamin B6 biosynthesis: formation of pyridoxine 50 -phosphate from 4-(phosphohydroxy)l-threonine and 1-deoxy-d-xylulose-5-phosphate by pdxa and pdxj protein. FEBS Lett. 449, 45–48. Leuendorf, J.E., Genau, A., Szewczyk, A., Mooney, S., Drewke, C., Leistner, E., Hellmann, H., 2008. The Pdx1 family is structurally and functionally conserved between Arabidopsis thaliana and Ginkgo biloba. FEBS J. 275, 960–969. Lumeng, L., Lui, A., Li, T.K., 1980. Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. J. Clin. Invest. 66, 688–695. Lum, H.K., Kwok, F., Lo, S., 2002. Cloning and characterization of Arabidopsis thaliana pyridoxal kinase. Planta 215, 870–879. McCormick, D.B., Chen, H., 1999. Update on interconversions of vitamin B6 with its coenzyme. J. Nutr. 129, 325–327. Merrill, A.H., Henderson, J.M., Wang, E., Mcdonald, B.W., Millikan, W.J., 1984. Metabolism of vitamin B6 by human liver. J. Nutr. 114, 1664–1674. Mittenhuber, G., 2001. Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and pyridoxal phosphate biosynthesis pathways. J. Mol. Microbiol. Biotechnol. 3, 1–20. Morita, T., Takegawa, K., Yagi, T., 2004. Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe. J. Biochem. 135, 225–230. Nakano, M., Morita, T., Yamamoto, T., Sano, H., Ashiuchi, M., Masui, R., Kuramitsu, S., Yagi, T., 1999. Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily. J. Biol. Chem. 274, 23185–23190. Ollilainen, V., 1999. HPLC analysis of vitamin B6 in foods. Agric. Food Sci. Finland 8, 515–618. Raschle, T., Amrhein, N., Fitzpatrick, T.B., 2005. On the two components of pyridoxal 50 -phosphate synthase from Bacillus subtilis. J. Biol. Chem. 280, 32291–32300. Sang, Y., Barbosa, J.M., Wu, H., Locy, R.D., Singh, N.K., 2007. Identification of a pyridoxine (pyridoxamine) 50 -phosphate oxidase from Arabidopsis thaliana. FEBS Lett. 581, 344–348. Shi, H., Xiong, L., Stevenson, B., Lu, T., Zhu, J.K., 2002. The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance. Plant Cell. 14, 575–588. Shi, H., Zhu, J.K., 2002. SOS4, a pyridoxal kinase gene, is required for root hair development in Arabidopsis. Plant Physiol. 129, 585–593. Tambasco-Studart, M., Titiz, O., Raschle, T., Forster, G., Amruein, N., Fitzpatrick, T.B., 2005. Vitamin B6 biosynthesis in higher plants. PNAS 102, 13687–13692. Titiz, O., Tambasco-Studart, M., Warzych, E., Apel, K., Amrhein, N., Laloi, C., Fitzpatrick, T.B., 2006. PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis. Plant J. 48, 933–946. Tsuyoshi, T., Yoshio, T., Takashi, G., 2004. Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes. Mol. Biol. Evol. 22, 243–250. Tsuge, H., Toukairin-Oda, T., Shoji, T., Sakamoto, E., Mori, M., Suda, H., 1988. Fluorescence enhancement of PLP for application to HPLC. Agric. Biol. Chem. 52, 1083–1086. Wagner, S., Bernhardt, A., Leuendorf, J.E., Drewke, C., Lytovchenko, A., Mujahed, N., Gurgui, C., Frommer, W.B., Leistner, E., Fernie, A.R., Hellmann, H., 2006. Analysis of the Arabidopsis rsr4–1/pdx1–3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis. The Plant Cell. 18, 1722–1735. Wang, H., Liu, D., Liu, C., Zhang, A., 2004. The pyridoxal kinase gene tapdxk from wheat complements vitamin B6 synthesis-defective Escherichia coli. J. Plant Physiol. 161, 1053–1060. Yoshikan, Y., Yokochi, N., Ohnicji, K., Hayashi, H., Yagi, T., 2006. Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase. Biochem. J. 396, 499–507. Zhang, J.Y., Huang, L.Q., Hayakawa, T., Tsuge, H., 2004. Analysis of vitamin B6 derivatives in biological samples with high performance liquid chromatography. Chem. J. Chin. Univ. 26, 638–640.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Interconversions of different forms of vitamin B6 in tobacco plants ShuoHao Huang a,b, HaiBin Zeng a, JianYun Zhang c, Shu Wei a, LongQuan Huang a,⇑ a
Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People’s Republic of China Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan c College of Life Science, Anhui Agricultural University, Hefei 230036, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 18 September 2010 Received in revised form 18 July 2011 Available online 17 August 2011 Keywords: Nicotiana tabacum Solanaceae Vitamin B6 B6 vitamers
a b s t r a c t There are six different vitamin B6 (VB6) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 50 -phosphate (PLP), pyridoxamine 50 -phosphate (PMP), and pyridoxine 50 -phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB6 in the human diet, and VB6 plays an important role in plants, the mechanisms underlying the interconversions of different VB6 forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B6 vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB6 was about 3.9 lg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB6 by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B6 vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of nonphosphorylated forms. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Vitamin B6 (VB6) (1–6, Fig. 1) exists in several forms, which are termed ‘‘vitamers’’ and include pyridoxal (1) (PL), pyridoxamine (2) (PM), pyridoxine (3) (PN), and their phosphorylated derivatives pyridoxal 50 -phosphate (4) (PLP), pyridoxamine 50 -phosphate (5) (PMP), and pyridoxine 50 -phosphate (6) (PNP), respectively (Fig. 1). PLP (4) is an important coenzyme for numerous enzymes involved in different cellular reactions and processes, including metabolism of amino acids, fatty acids and carbohydrates, heme and chlorophyll biosynthesis, ethylene and auxin biosynthesis, and transcriptional regulation (Huq et al., 2007). Additionally, VB6 has been linked to stress responses in plants (Shi et al., 2002; Denslow et al., 2005, 2007; Sang et al., 2007) and is essential for root development (Shi and Zhu, 2002; Chen and Xiong, 2005; Titiz et al., 2006). VB6 is synthesized de novo by two different enzymatic pathways (Fig. 1). The first, referred to as the ‘‘DXP (10 -deoxy-D-xylulose-50 -phosphate)-dependent pathway’’, is found in Escherichia coli and a small number of other prokaryotes. In E. coli, PNP (6)
⇑ Corresponding author. Tel.: +86 551 5157862. E-mail address: [email protected] (L. Huang). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.07.019
after being synthesized is oxidized by a PNP/PMP oxidase to form PLP (4) (Hill et al., 1996; Laber et al., 1999; Drewke and Leistner, 2001). The other, referred to as the ‘‘DXP-independent pathway’’, is the predominant pathway in nature. This pathway involves a protein complex, consisting of two different proteins, encoded by the genes PDX1 and PDX2. This enzyme complex directly catalyzes PLP (4) biosynthesis (Burns et al., 2005; Raschle et al., 2005; Tambasco-Studart et al., 2005). Plants, a major source of VB6 in the human diet, synthesize VB6 via the DXP-independent pathway. PDX1 and PDX2 have been isolated and characterized in tobacco (Denslow et al., 2005), Arabidopsis thaliana (Tambasco-Studart et al., 2005; Titiz et al., 2006; Wagner et al., 2006; Denslow et al., 2007), and Ginkgo biloba (Leuendorf et al., 2008). There are other pathways of VB6 metabolism in nature. In addition to the de novo biosynthetic pathways of VB6, a salvage pathway is found in all organisms, and functions to inter-convert the six different vitamer forms (Fig. 1). The salvage pathway mainly involves an ATP-dependent PL kinase and a PNP/PMP oxidase (Tsuyoshi et al., 2004). The kinase catalyzes the phosphorylation of PL (1), PM (2) and PN (3) to form PLP (4), PMP (5) and PNP (6). The oxidase catalyzes PNP (6) and PMP (5) oxidation to form PLP (4). Alternatively, PMP (5) oxidation can also be catalyzed by a transaminase (Tsuyoshi et al., 2004). The phosphorylated forms are de-phosphorylated by phosphatase to restore the precursors
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“DXP-independent” de novo synthetic pathway
PNP/PMP oxidase
Salvage pathway
PNP/PMP oxidase
PMP (2) PL kinase
“DXP-dependent” de novo synthetic pathway
Transaminase PNP (3)
PLP (1)
Phosphatase
PL kinase
Phosphatase
PL reductase
PM-pyruvate aminotransferase PM (5)
PL kinase
Phosphatase
PL (4)
PN 4-oxidase
PN (6)
Fig. 1. Structures and interconversion pathways of vitamin B6 vitamers (1–6).
(McCormick and Chen, 1999; Mittenhuber, 2001). Additionally, a PL reductase that reduces PL (1) to PN (3) has been suggested to be involved in the salvage pathway (Nakano et al., 1999; Morita et al., 2004). Moreover, two degradation pathways of VB6 in bacteria have been reported (Burg et al., 1960). PM-pyruvate aminotransferase and PN 4-oxidase, respectively, use PM (2) and PN (3) as a substrate to form PL (1) in a VB6 degradation pathway (Yoshikan et al., 2006; Kaneda et al., 2002). The understanding of interconversions of the different VB6 forms in plants is very limited. Genes encoding PL kinases have been cloned and characterized in both A. thaliana and wheat (Lum et al., 2002; Wang et al., 2004), and that of PNP/PMP oxidase has been cloned from A. thaliana (Sang et al., 2007). González et al. (2007) further reported the effects on phenotype, vitamer levels, stress responses, enzyme activity, and regulation of salvage and de novo pathway genes in A. thaliana, resulting from mutations in the PNP/PMP oxidase (PDX3) and the PL kinase (SOS4) genes. In the sos4 mutant, significant shifts in vitamer concentration occurred compared to the wild type. Higher levels of PM (2), PLP (4), and PN (3), with a 2-fold increase in PM (2) and PN (3) and an almost 9-fold increase in PLP (4), were observed. Therefore, the major increase in total vitamer levels is due to a large increase in PLP (4). PLP (4) is a very reactive molecule forming complexes with amine groups of amino acid residues in proteins, and free PLP (4) will be promptly hydrolyzed by phosphatase to restore PL (1) and maintain PLP (4) homoeostasis, suggesting that the pool of free PLP (4) is maintained at a very low level (Di-Salvo et al., 2003; Lumeng et al., 1980; Merrill et al., 1984). Thus, the aim of the present study is to use a responsive analysis to investigate if there is an endogenous system to maintain PLP (4) homoeostasis and how the interconversions of different B6 vitamers take place in plants. The effect of supplementation of Murashige and Skoog (MS) basal medium with PM (2), PL (1) or PN (3), on the abundance of different B6 vitamers (1–6) in tobacco plants cultured on the medium was examined by using high performance liquid chromatography (HPLC). The results help us gain insights of VB6 metabolism taking place in plants.
determining all B6 vitamers (1–6) were established. The retention times of PMP (5), PM (2), PNP (6), PLP (4), PL (1) and PN (3) were 7.0, 7.6, 8.6, 9.0, 10.8 and 12.1 min, respectively, under the conditions described in the Experimental (Section 5.4). PMP (5) as well as PM (2) emits relatively strong fluorescence, while PLP (4) has poor fluorescence under the same conditions. The detection limit for PMP (5) as well as PM (2) was less than 30 pg, and that for PLP (4) was about 350 pg per injection. After treatment with KCN, the fluorescent signal of PLP (4) was enhanced over 10-fold due to the formation of pyridoxic acid 50 -phosphate (7) (PIC-P), and the retention time of PIC-P (7) was 9.3 min (data not shown). Thus, the detection limit of PLP (4) was increased to about 35 pg per injection, the same level as that of PNP (6). The recovery of each authentic B6 vitamer added to the extract from tobacco leaves was 95 ± 10%. 2.2. HPLC analysis of VB6 (1–6) in tobacco leaves, root and culture media The HPLC chromatogram of leaf B6 vitamers (1–6) in tobacco plants grown on MS basal media exhibited distinct peaks of PMP (5), PM (2), PL (1) and PN (3) (Fig. 2). The peak area or peak height was used for quantification. To obtain accurate results, KCNtreated leaf extracts were injected for quantitative analysis of PLP (4). No significant elution peak was found at the expected retention time for pure authentic PNP (6) during the whole
2. Results 2.1. HPLC analysis of standard B6 vitamers (1–6) Pure standard B6 vitamers (1–6) were well-separated using the HPLC analysis method used in this study and standard curves for
Fig. 2. HPLC pattern of leaf B6 vitamers from tobacco plants grown on MS basal media.
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experimental process (Fig. 2–5), suggesting that the level of PNP (6) in tobacco plants was different from those of the other vitamers and lower than the detection limit of our HPLC system. The levels of B6 vitamers (1–6) in tobacco leaves analyzed weekly during the experiments are given in Table 1. The mean level of total B6 vitamers (1–6) was about 3.9 lg/g fresh weight, and PL (1), PM (2), PN (3), PLP (4) and PMP (5) accounted for 23%, 14%, 37%, 20% and 6%, respectively. Thus, PN (3) was the major B6 vitamer in tobacco leaves. During the experiment for week 3, the levels of PL (1) and PLP (4) decreased, whereas that of PN (3) increased. There was no significant difference between tobacco leaves and roots in levels of B6 vitamers (1–6). Moreover, non-phosphorylated B6 vitamers of PL (1), PM (2) and PN (3) were all detected from media. The mean level of VB6 (1–3) was about 2.3 lg/g fresh weight, and PL (1), PM (2) and PN (3) accounted for 20%, 18% and 62%, respectively. PN (3) was also the major B6 vitamer in media. Since the medium (80 g) outweighed the plants (about 1.5 g with 4–5 tender leaves) grown on it greatly, a significant amount of non-phosphorylated VB6 (1– 3) was released from the tobacco plants.
Fig. 4. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PL (1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with non-phosphorylated B6 vitamers (1–3) VB6 compounds (1–6) are stable under acidic condition and unstable under neutral and especially under alkaline conditions. At room temperature for 3 weeks, B6 vitamers (1–3) were stable in the medium and no inter-conversion among the vitamers was detected. The recovery percentages for PM (2), PL (1) and PN (3) were 66.8%, 63.4% and 76.5%, respectively, detected by HPLC analysis. This was the basis for our further studies. Supplementation of medium with PM (2), PL (1), or PN (3) resulted in significant quantitative changes of the levels of the different B6 vitamers in the tobacco plants compared to those in control plants grown in medium without any supplementation. The levels of B6 vitamers (1–6) in leaves were determined weekly over the 3 week supplementation experiment. They did not change significantly after the 1st week of treatment, suggesting that they might attain new steady-state levels within this period. In addition, no difference in plant growth was observed between the plants supplemented with and without 100 mg/L of PM (2), PL (1) or PN (3) in the growth media (data not shown).
Fig. 5. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PN (3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3.1. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PM (2) Fig. 3 shows that supplementation of the medium with PM (2) resulted in concentration changes among B6 vitamers (1–6) in the tobacco plants compared to the control plants grown in the medium without the supplementation treatment. Following supplementation with PM (2), the total VB6 (1–6) level increased to 40 lg/g fresh weight, about 10-fold higher than that of the control plants. The mean contents for PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined as 4.40 ± 1.44, 24.23 ± 4.10, 9.35 ± 3.45, 1.45 ± 0.36 and 0.39 ± 0.02 lg/g fresh weight, respectively. An almost 45-fold increase in PM (2), an 8-fold increase in PN (3), and a 4-fold increase in PL (1), which accounted for about 61%, 23% and 11%, respectively of total VB6 (1–6), were observed. Both PLP (4) and PMP (5) levels changed slightly. Our data suggested that levels of the phosphorylated vitamers (4–6) were maintained steady in tobacco plants.
Fig. 3. HPLC patterns of leaf B6 vitamers from tobacco plants with (black curve) and without (red curve) supplementation by PM (2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3.2. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PL (1) Following supplementation with PL (1), the total VB6 (1–6) level was increased to 36 lg/g fresh weight, about 9 times as much as
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a b
Timeline
Sample
PL (1)
PM (2)
PN (3)
PLP (4)
1 Week
Leaf tissue Root Medium
1.11 ± 0.10 0.39 ± 0.05 0.45 ± 0.02
0.55 ± 0.05 0.67 ± 0.31 0.32 ± 0.01
1.18 ± 0.02 1.21 ± 0.01 0.95 ± 0.16
2 Week
Leaf tissue Medium
0.99 ± 0.11 0.50 ± 0.05
0.56 ± 0.01 0.60 ± 0.08
3 Week
Leaf tissue Medium
0.53 ± 0.18 0.47 ± 0.05
0.53 ± 0.04 0.38 ± 0.09
a
b
PMP (5)
PNP (6)
Total VB6
0.94 ± 0.15 0.81 ± 0.09 —
0.29 ± 0.03 0.17 ± 0.03 —
Trace Trace —
4.07 3.25 1.72
1.40 ± 0.06 2.17 ± 0.88
0.72 ± 0.05 —
0.25 ± 0.01 —
Trace —
3.92 3.27
1.81 ± 0.17 1.47 ± 0.59
0.63 ± 0.08 —
0.20 ± 0.01 —
Trace —
3.70 2.32
The amounts of PLP (4) were determined after KCN treatment. No significant elution peak can be used for quantitative analysis, the same in Tables 2 and 3.
lyzed after supplementation with PM (2), PL (1) or PN (3) for 1 week (Table 3).
that in control plants. The average levels for PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined as 4.58 ± 1.65, 3.11 ± 2.09, 25.93 ± 3.45, 1.38 ± 0.33 and 0.70 ± 0.64 lg/g fresh weight, respectively. The proportion of PN (3) to total VB6 (1–6) in leaves was increased to 73%, about 22-fold higher than in the control. PM (2) was also 6-fold higher than in the control. Whereas the ratio of PL (1) to total VB6 (1–6) was merely about 13%, a 4-fold increase compared to that of control plants. The changes in PLP (4) and PMP (5) levels due to the PL (1) supplementation were similar to those due to the PM (2) supplementation.
3. Discussion VB6 exists, in at least six biologically-active forms (1–6) in biological materials. In order to quantify these derivatives in their simplest and most sensitive way, many analytical approaches have been proposed in which HPLC always figures prominently. A reversed-phase HPLC method in conjunction with highly sensitive fluorescence detection has been developed and it allows the analysis of six familiar VB6 derivatives (1–6) in an isocratic solvent within 30 min. By using fluorescence detection, many interfering peaks were eliminated; however some negative effect from such peaks still remained. In our previous study, many VB6 analyses on various plants using HPLC ended in failure. More interfering peaks appeared in plants grown in soil than those grown in a MS basal medium. As for PMP (5) and PM (2), the fluorescence detection method was over 50-fold more sensitive than the UV detection method. However, the fluorescent signal of PLP (4) was not sufficiently intense in this analytical system to obtain accurate results. To overcome this difficulty a pre-column treatment with KCN to enhance the fluorescence of PLP (4) was developed (Tsuge et al., 1988) and applied successfully in this study. In the mammalian system, PLP (4) is synthesized mainly in the liver, and then released to the bloodstream in association with
2.3.3. Levels of B6 vitamers (1–6) in tobacco plants grown in media supplemented with PN (3) Supplementation with PN (3) for 1 week resulted in an increase in total VB6 (1–6) to 36 lg/g fresh weight, about 9 times as much as that of the control. The average levels of PL (1), PM (2), PN (3), PLP (4) and PMP (5) were determined to be 1.70 ± 0.18, 1.65 ± 0.52, 31.70 ± 6.66, 0.83 ± 0.10 and 0.28 ± 0.04 lg/g fresh weight, respectively. The increased PN (3) level accounted for about 88% of total VB6 (1–6) in leaves, an almost 26-fold increase compared to the control value. The other B6 vitamer levels were unchanged compared to those of control plants. Additionally, we analyzed the levels of B6 vitamers (1–6) in tobacco plants and media after supplementation with PM (2), PL (1) or PN (3) for 48 h (Table 2). The results show a similar trend of the concentration shifts of B6 vitamers (1–6) in the tobacco plants ana-
Table 2 Levels of leaf B6 vitamers in tobacco plants analyzed after supplementation with PM (2), PL (1) or PN (3) for 48 h. (lg/g fresh weight). Supplement
Sample
PLP (4)
PMP (5)
PNP (6)
Total VB6
PM (2)
Leaf tissue Medium
PL (1) 2.90 ± 1.37 1.58 ± 0.88
PM (2) 10.31 ± 5.17 85.64 ± 20.9
PN (3) 1.66 ± 0.45 1.18 ± 0.14
1.13 ± 0.06 —
0.53 ± 0.39 —
trace —
16.53 88.40
PL (1)
Leaf tissue Medium
2.21 ± 2.15 72.45 ± 26.8
0.93 ± 0.48 0.81 ± 0.09
6.94 ± 3.10 5.97 ± 5.28
1.20 ± 0.34 —
0.33 ± 0.06 —
Trace —
11.61 79.23
PN (3)
Leaf tissue Medium
1.20 ± 0.17 0.81 ± 0.19
0.80 ± 0.09 3.14 ± 3.96
8.67 ± 2.30 89.29 ± 8.32
1.12 ± 0.33 —
0.33 ± 0.10 —
Trace —
12.12 93.24
Table 3 Levels of leaf B6 vitamers in tobacco plants analyzed after supplementation with and without (control) PM (2), PL (1) or PN (3) for 1 week. (lg/g fresh weight). Supplement
Sample
PL (1)
PM (2)
PN (3)
PLP (4)
PMP (5)
PNP (6)
Total VB6
Without
Leaf tissue Medium
1.11 ± 0.10 0.45 ± 0.02
0.55 ± 0.05 0.32 ± 0.01
1.18 ± 0.02 0.95 ± 0.16
0.94 ± 0.15 —
0.29 ± 0.03 —
0.04 ± 0.02 —
4.07 1.72
PM (2)
Leaf tissue Medium
4.40 ± 1.44 3.21 ± 0.19
24.23 ± 4.10 74.68 ± 0.32
9.35 ± 3.45 3.08 ± 0.95
1.45 ± 0.36 —
0.39 ± 0.02 —
Trace —
39.82 80.97
PL (1)
Leaf tissue Medium
4.58 ± 1.65 43.06 ± 8.06
3.11 ± 2.09 2.40 ± 1.01
25.93 ± 3.45 11.16 ± 3.06
1.38 ± 0.33 —
0.70 ± 0.64 —
Trace —
35.70 56.62
PN (3)
Leaf tissue Medium
1.70 ± 0.18 0.75 ± 0.39
1.65 ± 0.52 2.24 ± 1.26
31.70 ± 6.66 70.56 ± 25.37
0.83 ± 0.10 —
0.28 ± 0.04 —
Trace —
36.16 73.55
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albumin (Lumeng et al., 1980; Merrill et al., 1984). During supplementation with PN (3) (100 mg/day orally for 1–3 weeks), the plasma content of PLP (4) increased 7-fold and that of PL (1) increased 12-fold, but the PN (3) level did not increase (Lumeng et al., 1980). In this study, administering tobacco plants with the non-phosphorylated vitamers (1–3) resulted in an increase in total VB6 (1–6) by around 10-fold with very little effect on the concentrations of the phosphorylated vitamers (4–6) including PLP (4). This suggests that a stable level of PLP (4), the coenzyme form of VB6, is maintained in tobacco even though the levels of the endogenous nonphosphorylated forms (1–3) changed significantly. Furthermore, exogenous addition of PM (2) or PN (3) to the plant growth medium increased its endogenous level in the plant more effectively than the levels of any other endogenous B6 vitamers, whereas PL (1) administration did not affect its endogenous level, instead, endogenous PN (3) abundance was increased significantly. These results indicate that the endogenous PL (1) level is also stringently controlled, and an efficient PL (1) to PN (3) conversion system may exist in plants. Therefore, we speculate that PN (3) may be downstream of VB6 metabolism in tobacco. This is consistent with a previous finding on the levels of VB6 (1–6) in plant-based foods where PN (3) was shown to be the predominant vitamer (Ollilainen, 1999). Additionally, the increase in level of endogenous PM (2) was also strikingly observed in the plants administered with PL (1) (Fig. 4), suggesting that an interconversion pathway between PL (1) and PM (2) also exists in tobacco. VB6 (1–6) is a group of water-soluble and small organic molecules. Transport of B6 vitamers across cellular membranes has been studied extensively. It has been known for many years that only the non-phosphorylated vitamers (1–3) move across cell membranes. In this study, tobacco plants were grown in sterile Magenta boxes; VB6 (1–3) detected in culture medium was derived solely from the plants (Table 1), confirming that the excretion of superfluous VB6 (1–3) from roots occurs in tobacco plants. Moreover, supplementation with any of the single non-phosphorylated vitamers (1–3) raised the total leaf levels of VB6 (1–6) by about 10-fold, suggesting that plants can take up the different non-phosphorylated B6 vitamers (1–3) with similar efficiency. In spite of the importance of this vitamin, the regulation of its metabolism is only beginning to be defined, and the consequences of altering VB6 content on plant growth and development is still unclear. The abundance of PLP (4), the functional form of VB6, was found to be quite stable in this study. That might be the reason why the greatly elevated level of VB6 in tobacco plants did not significantly affect plant growth and development. 4. Conclusions In conclusion, the present work reports that a stable level of the co-factor form PLP (4) is maintained in tobacco plants even though the levels of the endogenous non-phosphorylated forms (1–3) changed significantly due to supplementation of PM (2), PL (1) or PN (3) in the plant growth media. Efficient conversion pathways from PL (1) to PN (3) and PM (2) are present in the plant. To our knowledge, this is the first report of interconversions of B6 vitamers (1–6) in plants following administration experiments. These results raise some interesting questions about how, and why, the metabolism of VB6 is regulated in plants.
ma Chemicals (Sigma, USA). Due to unavailability of a commercial standard, PNP (6) was prepared from PLP (4) by reduction with NaBH4, according to a previously reported method (Argoudelis, 1986) validated by HPLC. Standard VB6 (1–6) was dissolved in H2O to make a 1 mM stock solution which was then diluted with 25 mM NaClO4-25 mM KH2PO4 buffer (pH 2.5) to the final concentration used for HPLC analysis. To avoid photo-degradation of VB6 all procedures were conducted in a dark room and the H2O was distilled and filtered through a 0.45 lm membrane filter. HPLCgrade CH3CN, HClO4, NaClO4, KH2PO4, K2HPO4, and all other chemicals and reagents were purchased from Sangon (Shanghai, China). 5.2. Plants and treatment Fresh and young leaves of tobacco plants (Nicotiana tabacum) were used as explants in this study. A standard method for in vitro plant culture was adopted. Culture media for inducing callus, buds and rooting were MS + 6-BA 1.0 mg/L + 2,4-D 0.5 mg/L, MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L and MS + NAA 0.2 mg/L, respectively. The media were all complemented with sucrose 30.0 g/L and agar 8.0 g/L, and adjusted to pH 5.8. Under normal plant growth conditions (25 ± 2 °C and 14 h photoperiod at 2500 lux), culture periods were 2, 4 and 8 weeks for three different induction treatments, respectively. The generated plantlets were then transplanted into MS media plus sucrose 30.0 g/L and agar 8.0 g/L at pH 5.8, supplemented with PM (2), PL (1), or PN (3) 100 mg/L. Meanwhile, some plants transplanted into MS basal medium were used as control plants. The plants were grown for 3 weeks under the above-described conditions. To minimize the decomposition of VB6 induced by light, the lower part of a plant growth flask was covered with aluminum foil. The whole operation was performed under sterile conditions. 5.3. Extraction of VB6 (1–6) VB6 compounds (1–6) were extracted from leaf tissue as follows: freshly excised leaf tissue (0.5 g) was completely homogenized with liq. N2 in a mortar with a pestle, and then 3 M HClO4 (1.5 mL) was added and mixed by vigorous votexing. The mixture was next centrifuged at 12,840g for 20 min at 4 °C. To 1 mL of supernatant 1 M phosphate buffer (pH 5.5, 1 mL) was added and the solution was centrifuged again as above. The pH of the solution was adjusted to 2.5 with 0.1 M HCl, and then made up to 3 mL with distilled H2O. This solution was named as the leaf extract. The KCN treatment of leaf extract was carried out according to the method of Tsuge et al. (1988) with a slight modification: briefly leaf extract (2 mL) was placed in a centrifuge tube and the pH was adjusted to 7.5 with 5 M KOH. After addition of 0.1 M KCN (0.1 mL), the solution was incubated at 50 °C for 3 h with vigorous shaking under dark conditions in order to accomplish complete oxidation of PLP (4) to PIC-P (7). Then, the pH of the solution was again adjusted to 2.5 with 1 M HCl and allowed to stand for 24 h at 25 °C. Finally, the volume of this solution was adjusted to 3 mL with distilled water. Extracts were kept on ice in the dark. Prior to injection into an HPLC -line solutions were passed through a 0.22 lm membrane filter. Unless otherwise specified, all VB6 assays were performed three times. 5.4. HPLC analysis of B6 vitamers (1–6)
5. Experimental 5.1. Materials Reference standards of PL (1) (PL-HCl), PM (2) (PM-2HCl), PN (3) (PN-HCl), PLP (4), and PMP (5) (PMP-HCl) were obtained from Sig-
The levels of six B6 vitamers (1–6) in tobacco leaves were determined by HPLC analysis weekly during the 3 week supplementation experiments. A reversed-phase HPLC method described by Zhang et al. (2004) was used with some modifications. The HPLC system was a Waters 6000 with a 2475 fluorescence detector.
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The B6 vitamers (1–6) were separated with COSMOSIL 5Ph column (4.6 mm i.d 250 mm, Waters type, Nacalai Tesque, Inc., Kyoto, Japan). The column was mounted in a column heater set at 30 °C. The detector excitation and emission wavelengths were set at 290 and 395 nm, and at 320 and 420 nm after KCN treatment, respectively. The sample injection volume was 5 lL. Separations were performed at a flow rate of 0.5 mL/min using an isocratic solvent of 25 mM NaClO4-25 mM KH2PO4 buffer (pH 2.5) containing 1% CH3CN. In order to elute slow-moving components, a solvent containing a higher concentration of CH3CN (usually 10%) in the buffer solution as above was flushed for 25 min after each analysis. The mobile phase was filtered through a 0.45 lm membrane filter and sufficiently degassed in an ultrasonicator with suction before adding CH3CN. Spiking of the leaf extracts with reference standards of PL (1), PM (2), PN (3), PLP (4), PMP (5) and PNP (6) was used to confirm peak identity. Six-point standard curves were prepared and run daily before running the leaf extract samples. B6 vitamer concentration was determined from the standard curves obtained by plotting peak areas or peak heights against the different known concentrations of each vitamer. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30870338) and Natural Science Foundation of Education Department of Anhui Province (No. KJ2010A116). References Argoudelis, C.J., 1986. Preparation of crystalline pyridoxine 50 -phosphate and some of its properties. J. Agric. Food Chem. 34, 995–998. Burns, K.E., Xiang, Y., Kinsland, C.L., McLafferty, F.W., Begley, T.P., 2005. Reconstitution and biochemical characterization of new pyridoxal-50 phosphate biosynthetic pathway. J. Am. Chem. Soc. 127, 3682–3683. Burg, R.W., Rodwell, V.W., Snell, E.E., 1960. Bacterial oxidation of vitamin B6. III. Metabolites of pyridoxamine. J. Biol. Chem. 235, 1164–1169. Chen, H., Xiong, L., 2005. Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses. Plant J. 44, 396–408. Denslow, S., Walls, A., Daub, M., 2005. Regulation of biosynthetic genes and antioxidant properties of vitamin B6 vitamers during plant defense responses. Physiol. Mol. Plant. 66, 244–255. Denslow, S.A., Rueschhoff, E.E., Daub, M.E., 2007. Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant Physiol. Biochem. 45, 152–161. Di-Salvo, M.L., Safo, M.K., Musayev, F.N., Bossa, F., Schirch, V., 2003. Structure and mechanism of Escherichia coli pyridoxine 50 -phosphate oxidase. Biochim. Biophys. Acta 1647, 76–82. Drewke, C., Leistner, E., 2001. Biosynthesis of vitamin B6 and structurally related derivatives. Vitam. Horm. 61, 121–155. González, E., Danehower, D., Daub, M.E., 2007. Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 50 -phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway. Plant Physiol. 145, 985–996. Hill, R.E., Himmeldirk, K., Kennedy, I.A., Pauloski, R.M., Sayer, B.G., Wolf, E., Spenser, I.D., 1996. The biogenetic anatomy of vitamin B6. A 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli. J. Biol. Chem. 271, 30426– 30435.
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