As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B6 is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B6de novo, animals have to take up the vitamin from their diet. Plants are the major source of vitamin B6 for animals. The recent identification of vitamin B6 biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B6 in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B6 increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B6 content in seeds by metabolic engineering.
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Water-soluble pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) are collectively called vitamin B6. This B-complex vitamin is remarkably versatile. It can act as a cofactor for many enzymatic reactions, especially transaminations, decarboxylations and racemizations that are involved in amino acid, lipid and carbohydrate metabolism (Schneider et al., 2000; Percudani and Peracchi, 2003). Recently, PN has also emerged as a potent antioxidant equivalent to vitamins C and E and is particularly active in quenching singlet oxygen and the superoxide anion (Osmani et al., 1999; Daub and Ehrenshaft, 2000; Wrenger et al., 2005). Moreover, the vitamin B6 conjugation to a lysine residue in the nuclear repressor RIP140 is essential for its interaction with histone deacetylases, nuclear retention and subsequent transcriptional repression (Huq et al., 2007).
Vitamin B6 is essential for all cellular organisms and particularly renowned in the medical field as being involved in more bodily functions than any other single nutrient and required for the maintenance of mental as well as physical health (Gengenbacher et al., 2006). For example, vitamin B6 is essential for normal brain development and function, participating in the making of neurotransmitters (Malouf and Grimley Evans, 2003; Balk et al., 2007). On the other hand, it is an especially important vitamin for maintaining healthy muscle cells, for absorption of vitamin B12 and for the production of red blood cells and cells of the immune system (Cheng et al., 2006; Spinneker et al., 2007). Consequently, vitamin B6 deficiency will result in muscle weakness, nervousness, irritability, depression, difficulty in concentrating and short-term memory loss (Spinneker et al., 2007). As animals lack the ability to synthesize vitamin B6de novo, they have to take up vitamin B6 from their diet.
As plants are the major source of vitamin B6 for animals either directly or indirectly, it is of great interest to increase vitamin B6 levels in plants for improved nutrition value. So far, there has been only one recent report attempting to engineer vitamin B6 content in plants. Herrero and Daub (2007) overexpressed the PDX1 and PDX2 genes from the fungal Cercospora nicotianae in tobacco; however, yeast bioassays suggested that only one single transgenic line has about 21% increase in vitamin B6 level in such a heterologous expression system. In previous studies, we and others independently showed that the Arabidopsis PDX proteins function in vivo in vitamin B6 biosynthesis (Chen and Xiong, 2005). Here we further studied the localization of the PDX2 protein and found it has similar subcellular localization as the PDX1 protein, consistent with the notion that these two proteins interact to function in the biosynthesis of vitamin B6. To test whether the vitamin B6 biosynthesis pathway can be regulated, we generated transgenic Arabidopsis plants expressing the Arabidopsis PDX1 and PDX2 genes either individually or together under control of the CaMV 35S or Arabidopsis seed-specific 12S promoter. The levels of different types of vitamin B6 in leaves, roots and seeds were measured by using a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method. We found that transgenic plants with 12S driven PDX1 or PDX2 accumulated twofold more total vitamin B6 in seeds than the wild-type (WT). Our study suggests that it is feasible to engineer vitamin B6 contents in seeds.
Subcellular localization of the PDX2 protein is similar to that of the PDX1 protein
Sequence analysis suggested that the PDX1 protein does not have a clear signal peptide (Chen and Xiong, 2005), whereas PDX2 was predicted to be localized in the membrane or the lumen of the endoplasmic reticulum and in the peroxisomes (Denslow et al., 2007). Using transgenic Arabidopsis plants stably expressing GFP-tagged PDX1 fusion protein, we previously showed that the PDX1 protein is preferentially associated with the membrane system including plasma membrane, nuclear envelope and chloroplast outer membranes. Part of the PDX1 protein is also localized in the cytoplasm (Chen and Xiong, 2005). Similar subcellular localization of the PDX2 protein was recently reported by Denslow et al. (2007). These authors used epidermal peels from stable transformants and found that PDX2 is localized in the plasma membrane, the nucleus and to a less extent the cytoplasm (Denslow et al., 2007). However, transient expression assays in onion epidermal cells and Arabidopsis protoplasts found that both PDX1 and PDX2 are cytoplasmic (Tambasco-Studart et al., 2005). It is thus necessary to study, which of the above localization patterns of PDX2 may reflect the natural location of the protein.
To determine the subcellular localization of PDX2 protein, we generated stable transgenic Arabidopsis expressing GFP-PDX2 fusion protein under the control of the CaMV 35S promoter. As shown in Figure 1a–c, PDX2 was found to be mainly associated with the cell periphery and the endomembrane systems. Part of the protein was also localized in the cytoplasm. In root cells, the peripheral and cytoplasmic localization of PDX2 is particularly clear. In addition, some PDX2 may be associated with the nucleus (Figure 1d–i). To further demonstrate that the PDX2 protein is associated with the plasma membrane rather than cell wall, GFP-PDX2 transgenic seedlings were incubated in 30% sucrose to induce plasmolysis. Figure 1g–i shows that in plasmolysed root hairs, the PDX2 protein was not associated with cell wall. Thus, the PDX1 and PDX2 are similarly localized, being preferentially associated with membranes but also found in the cytoplasm.
Overexpression of PDX1 or PDX2 does not affect plant growth and development
To test whether we could increase vitamin B6 levels in plants, we generated transgenic plants overexpressing PDX1, PDX2 or both genes. We made constructs consisting of the PDX1 or PDX2 cDNA under control of the CaMV 35S promoter and transferred them separately into WT Arabidopsis plants. For the generation of transgenic plants overexpressing both PDX1 and PDX2, homozygous transgenic plants with a high level of PDX1 expression were transferred with the PDX2 overexpression construct and transformants were selected.
We randomly chose several transgenic lines to check for the expression levels of the PDX1 and PDX2 genes and presented data for two representative independent lines. Figure 2a shows that the PDX1 transcript level is significantly increased in the two 35S::PDX1 lines and the 35S::PDX1/35S::PDX2 line. Similarly, the PDX2 transcript level is significantly increased in the 35S::PDX2 lines (Figure 2b). However, the PDX2 transcript level was only slightly increased in the 35S::PDX1 background (Figure 2b). By checking more than 15 independent transformants, we could not find a single line that expressed both the PDX1 and PDX2 genes at high levels (data not shown).
Although all these transgenic plants have higher expression of either the PDX1 or PDX2 gene, the plants grow normally with no clear phenotypic alteration in growth or development (Figure 2c and data not shown). We also did not observe alterations in seed germination or stress sensitivity of seedlings in response to salt, abscisic acid (ABA), UV or heat-shock treatments (data not shown).
Overexpression of PDX1 or PDX2 increases pyridoxine content in seeds
Information about vitamin B6 contents in plants and particularly in different tissues of plants is rather limited, perhaps partly because of the difficulty in determining the various forms of the vitamin. To determine vitamin B6 in plants, we adopted a LC/MS/MS method recently developed to determine vitamin B6 in human plasma (Midttun et al., 2005) that we found can quantitatively and accurately determine vitamin B6 in plant samples (see Materials and methods section). Using this method, we measured PL, PM and PN (Figure 3) levels in the pdx1-1 null mutant (Chen and Xiong, 2005), Col-0 (WT) and PDX1 or PDX2 overexpressing lines respectively. We found that different plant parts have different compositions of vitamin B6. For example, leaves of 3-week-old plants tend to have more PM than roots and seeds, whereas seeds have higher PN contents than leaves and roots. Interestingly, PL levels are generally lower than those of PM or PN, particularly in seeds where it was nearly undetectable (Figure 3).
Compared with the WT, the PDX1 or PDX2 overexpression lines had little or no increase in PL levels in leaves, roots, or dry seeds. Although no significant difference in PM content in leaves and roots was detected between WT and PDX1 or PDX2 overexpression lines (Figure 3b), dry seeds of PDX2 overexpression lines had 30.3%–70.8% increase of PM compared with WT seeds. Consistent with a defect in vitamin B6 biosynthesis, PN is barely detectable in all the tissues of the pdx1 mutant. In contrast, except for one PDX1-overexpressing line, the leaves of other transgenic lines had 28.7%–54.8% more PN than that of WT, although this increase in PN levels in leaves is statistically significant only for the 35S::PDX2#1 line (Fig. 3c). Importantly, 45.9%–105.3% increase in PN levels was found in the seeds of all transgenic lines (Figure 3c). In general, PDX2-overexpressing seeds have higher levels of PN and total vitamin B6 than PDX1-overexpressing seeds (Figures 3). These data suggest that seed might be a better target for engineering vitamin B6 levels than vegetative parts.
As the CaMV 35S promoter has a low activity in early embryogenesis (Higgins and Spencer, 1991; Sunilkumar et al., 2002), and its activity greatly decreased during seed development compared with the seed storage protein promoter phaseolin (Williamson et al., 1989), we decided to generate transgenic plants overexpressing the PDX genes specifically in seeds to see whether it is possible to further boost vitamin B6 accumulation. We made constructs consisting of the PDX1 or PDX2 cDNA under the control of the Arabidopsis 12S seed storage protein gene promoter individually or in tandem and transferred them separately into WT Arabidopsis plants. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed a great increase of either PDX1 transcript in 12S::PDX1 transgenic lines, or PDX2 transcript in 12S::PDX2 transgenic lines, or both PDX1 and PDX2 transcripts in 12S::PDX1/12S::PDX2 lines (Figure 4a). We then measured the vitamin B6 content in dry seeds of these transgenic lines. It was found that all these lines have at least over 40% increases in PN and PM content (Figure 4b). The total vitamin B6 level in some transgenic lines is three times that of WT (Figure 4c).
Although these transgenic seeds have increased total vitamin B6, no clear difference was observed between these lines and WT in seed germination and early seedling growth (data not shown). Given that vitamin B6 can act as a scavenger of reactive oxygen species, we examined the germination of these transgenic line seeds with different concentrations of H2O2. However, no enhanced tolerance to reactive oxygen species (ROS) in germination was found in these transgenic line seeds (data not shown), suggesting that vitamin B6 may not be the major ROS scavenger during seed germination.
Besides acting as a coenzyme for the metabolism of amino acids, glucose and lipids, vitamin B6 also play other important roles such as scavenging of ROS and even modulation of gene expression (Osmani et al., 1999; Daub and Ehrenshaft, 2000; Schneider et al., 2000; Wrenger et al., 2005; Huq et al., 2007; Spinneker et al., 2007). Vitamin B6 deficiency has been associated with impaired cognitive functions, Alzheimer’s disease, cardiovascular diseases and different types of cancer, especially in the elderly population (Spinneker et al., 2007). Since plants are the major source of vitamin B6 for animals, it is of great interest to engineer plants for high levels of vitamin B6. In this study, we report the achievement of increasing vitamin B6 content in Arabidopsis seeds by overexpressing Arabidopsis PDX1 or PDX2, two genes that were recently shown to encode committed enzymes in the vitamin B6 biosynthesis pathway.
We first examined the subcellular location of the PDX2 protein by generating transgenic plants stably expressing the PDX2-GFP fusion protein. Our data (Figure 1) are consistent with those recently reported (Chen and Xiong, 2005; Tambasco-Studart et al., 2005; Denslow et al., 2007) indicating that peripheral membrane association with cytoplasmic accumulation might be the most natural location for PDX1 and PDX2 proteins. This co-localization of the PDX1 and PDX2 proteins allows their interaction in the biosynthesis of vitamin B6.
We generated transgenic Arabidopsis plants overexpressing the PDX1 or PDX2 gene. Our study indicates that these transgenic plants had significantly increased transcript levels of the respective genes (Figure 2). Nonetheless, these higher levels of PDX1 or PDX2 gene expression did not impair plant growth or development (Figure 2c).
Although the PDX1 or PDX2 transcript levels are greatly increased in these 35S promoter driven overexpression lines, no dramatic increase in vitamin B6 content was detected in either shoots or roots. This observation is consistent with the result found with tobacco plants overexpressing fungal PDX genes. Herrero and Daub (2007) were unable to detect any significant increase in the vitamin level except for one single line (out of total six lines) which had only ∼21% increase in total vitamin B6 level. It seems that vitamin B6 homeostasis is more tightly regulated in vegetative parts. In contrast to shoots and roots, a 45.9%–105.3% increase in PN levels was found in the seeds of all transgenic plants in our study (Figure 3c).
To test the possibility to further increase vitamin B6 accumulation in seeds, we generated Arabidopsis plants expressing the PDX genes under control of the Arabidopsis 12S seed storage protein promoter. Using this seed-specific promoter, both PDX1 and PDX2 transcript levels could be elevated in the developing seeds (Figure 4a). As a consequence, the accumulation of vitamin B6 in seeds is more significant in these transgenic plants (Figure 4). Compared with the WT seeds, PN and PM were increased in the transgenic seeds (Figure 4b), and the total vitamin B6 could be increased more than twofold (Figure 4c). Although information concerning vitamin B6 contents in different plant parts are very limited, the level of vitamin B6 in seeds detected in our study is comparable with that reported in wheat flours (Sampson et al., 1996). Our results indicate that seed might be a suitable target organ for further engineering of higher levels of vitamin B6. This finding is particularly valuable for crop plants whose seeds are the major source of food and feed.
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in all experiments. The pdx1-1 T-DNA allele (SALK_086418) in At5g01410 was described previously (Chen and Xiong, 2005). Seeds were surface-sterilized with bleach plus 0.01% triton and planted onto a half-strength Murashige-Skoog (MS) medium supplemented with 3% sucrose and 0.6% agar (Sigma Aldrich, St Louis, MO, USA). After 2 days of cold treatment, plates were incubated at 22 °C under constant white light for seed germination and seedling growth.
The PDX1 cDNA was amplified with the primers 5′-CACCATGGAAGGAACCGGCGTTGT-3′ and 5′-CTCGGAGCGATTAGCGAAC-3′. The PDX2 cDNA was amplified with the primer pair: 5′-CACCATGACCGTCGGAGTTTTAGC-3′ and 5′-GAAATATAGGAAGATCAGGCTTAG-3′. The PCR products were ligated with pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA). After sequencing confirmation, it was cloned into pMDC Gateway vector (Curtis and Grossniklaus, 2003) through LR clonase recombination. Constructs of the CaMV 35S promoter driven PDX1 or PDX2 cDNA alone or GFP-PDX2 fusion were made with the respective Gateway vectors. Agrobacterium tumefaciens GV3101 were transformed by electroporation with these constructs. Four-week-old Arabidopsis plants were then vacuum-infiltrated with the transformed Agrobacterium strains (Bechtold et al., 1993) and transformants were selected using hygromycin resistance. For overexpression of both PDX1 and PDX2, the 35S promoter-PDX2-nos terminator was released from pMDC32-PDX2 with HindIII and EcoRI and the insert was put into HindIII and EcoRI digested pMDC100 vector, which harbours kanamycin resistant gene NPTII, and designated pMDC100-PDX2. One high PDX1 overexpression line was transformed with GV3101 harbouring pMDC100-PDX2 and screened with both hygromycin and kanamycin.
Arabidopsis seed-specific promoter 12S was amplified by PCR using the following primer pair: 5′-ACAAGCTTTACTTTTATTTATGC-3′ and 5′-TTGGTACCTCTTTATTGATTTACTTT-3′. The HindIII and KpnI digested 12S promoter fragment was used to replace the 35S promoter in the pMDC43-PDX1 or pMDC32-PDX2 plasmid to make the 12S::PDX1 or 12S::PDX2 constructs. To make the 12S driven both PDX1 and PDX2 construct, the 12S::PDX1 insert was released from the 12S::PDX1 plasmid with HindIII and EcoRI, blunted with klenow fragment and ligated into Klenow blunted EcoRI-cut 12S::PDX2 plasmid. Agrobacterium tumefaciens GV3101 was transformed by electroporation with these constructs. Four-week-old Arabidopsis were then vacuum infiltrated with the transformed Agrobacterium strains (Bechtold et al., 1993) and transformants were screened using hygromycin.
For expression analysis of the CaMV 35S promoter driven PDX1 and PDX2 overexpression lines, total RNA was extracted from seedlings grown under a long-day photoperiod by using the TRIZOL Reagent according to the manufacturer’s instruction (Invitrogen, Carlsbad, CA, USA). For expression analysis of the 12S promoter driven PDX1 and PDX2 overexpression lines, total RNA was extracted from young siliques as described (Downing et al., 1992). cDNA was synthesized using an oligo dT primer and moloney murine leukaemia virus reverse transcriptase (New England Biolabs, Boston, MA, USA). The primer pair for tubulin amplification was 5′-CGTGGATCACAGCAATACAGAGCC-3′ and 5′-CCTCCTGCACTTCCACTTCGTCTTC-3′. All PCR were performed with 30 cycles except for tubulin amplification with silique cDNA, which was conducted with 35 cycles. No fragments were amplified in the RT-PCR using control template prepared without reverse transcriptase (data not shown).
Vitamin B6 quantification
Because the glycosidic conjugates of vitamin B6 found in many plants have low bioavailability for animals (Bognar and Ollilainen, 1997), we only measured the level of free vitamin B6. Vitamin B6 was extracted with 0.1 m HCl at 120 °C for 30 min without subsequent enzymatic treatment. Under these conditions, nearly all PL-5-phosphate (PLP) or PM-5-phosphate (PMP) are hydrolysed into PL or PM and PN-5-phosphate (PNP) is also partially hydrolysed to PN (Bognar and Ollilainen, 1997). Determination of vitamin B6 content was performed similarly as described for human plasma (Midttun et al., 2005) using an API 4000 triple-quadrupole tandem mass spectrometer (Applied Biosystems, Foster City, CA, USA). In brief, either dry seeds or shoots or roots of 3-week-old Arabidopsis plants grown on MS agar plate were ground in liquid nitrogen and extracted in 0.1 m HCl at 120 °C for 30 min. After centrifugation at 15 000 g for 30 min, the supernatant was filtered through a 0.2 μm Millipore filter unit before injecting to LC/MS/MS. The mobile phase consisted of two components, solution A (100 mm heptafluorobutyric acid in 650 m acetic acid) and solution B (900 mL/L acetonitrile in water). The column was developed with gradient elution according to the following timetable: 0–0.2 min (A), 0.3–6.6 min (2.5% A and 97.5% B) and 6.7–8 min (A). Vitamin B6 was detected in the multiple reaction monitoring mode. The ion transitions are: parent ion 168.1, product ion 150.1 for PL, parent ion 169.3, product ion 134.1 for PM and parent ion 170.1, product ion 134.1 for PN. PN-hydrochloride, PL-hydrochloride and PM-dihydrochloride (Sigma, St Louis, MO, USA) were served as standards. There are three biological replicates for each line and the data were analysed using Student’s t-test.
Confocal microscopy for subcellular localization
Whole seedlings were mounted in water under glass coverslips for GFP fluorescent signal visualization using a confocal laser scanning microscope (Nikon Eclipse E-800 C1 confocal microscope, Nikon Instrument Inc., Melville, NY, USA) equipped with a kryptonargon laser. For plasmolysis, seedlings were treated with 30% sucrose for one hour before observation.
The authors thank Dr Leslie Hicks at the Proteomics and Mass Spectrometry Facility of Donald Danforth Plant Science Center and Dr Xiangqing Pan for the help with LC/MS/MS. This study was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (grant #2004-02111) (to LX).