- © 2016 American Society of Plant Biologists. All rights reserved.
Abstract
Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5′-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5′-phosphate (PMP), pyridoxine 5′-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism.
INTRODUCTION
Vitamin B6 is a water-soluble vitamin essential for all living organisms. The term vitamin B6 collectively refers to six different vitamers that have a pyridine ring in common but differ in their 4ʹ as well as their 5ʹ moieties, comprising the alcohol pyridoxine (PN), the amine pyridoxamine (PM), the aldehyde pyridoxal (PL), and their 5′ phosphorylated forms (pyridoxine 5′-phosphate [PNP], pyridoxamine 5′-phosphate [PMP], and pyridoxal 5′-phosphate [PLP]) (Fitzpatrick et al., 2007). The PLP vitamer acts as a cofactor in numerous (over 140) enzymatic reactions involved in a variety of processes (http://bioinformatics.unipr.it/cgi-bin/bioinformatics/B6db/home.pl). In addition, vitamin B6 has been shown to be an efficient reactive oxygen species quencher in vitro and is considered to act in the same capacity in vivo (Bilski et al., 2000; Havaux et al., 2009). Furthermore, it was recently suggested that PLP is important for proper folding of certain PLP-dependent enzymes, fulfilling a chaperone role (Cellini et al., 2014).
Biosynthesis de novo of PLP takes place in bacteria, plants, and fungi, but not in animals, making it an essential compound in the diet of the latter. Two different pathways have been described for biosynthesis de novo: the deoxyxylulose 5-phosphate (DXP)-dependent and the DXP-independent pathway (Fitzpatrick et al., 2007). The DXP-dependent pathway takes place in Escherichia coli as well as some other members of the γ-division of proteobacteria, uses DXP as an intermediate, and requires seven enzymes to form PLP (Fitzpatrick et al., 2007). By contrast, the DXP-independent pathway involves the action of only two enzymes, PDX1 and PDX2 (Tambasco-Studart et al., 2005), and takes place in the majority of bacteria, fungi, and plants (Ehrenshaft et al., 1999; Mittenhuber, 2001). PLP can also be generated from other B6 vitamers in salvage pathways, which are present in all kingdoms of life including those that do not produce vitamin B6 de novo, such as animals (Ruiz et al., 2008). In the salvage pathways of the plant model Arabidopsis thaliana, PN, PM, and PL can be phosphorylated by a kinase (named SALT OVERLY SENSITIVE4 [SOS4]; Shi et al., 2002; Shi and Zhu, 2002), and the phosphorylated forms, PNP and PMP, can be converted into PLP by the action of the oxidase PDX3 (González et al., 2007; Sang et al., 2007, 2011) (Figure 1). Interestingly, the PDX3 enzyme consists of two domains; the PMP/PNP oxidase domain is located at the C terminus and is fused to an N-terminal epimerase that has recently been implicated in nicotinamide nucleotide repair (annotated NNRE) (Marbaix et al., 2011; Colinas et al., 2014; Niehaus et al., 2014). In addition, a pyridoxal reductase converting PL into PN, found originally in yeast (Morita et al., 2004), has more recently been characterized in Arabidopsis (Herrero et al., 2011). Activities of (possibly unspecific) phosphatases involved in vitamin B6 interconversion have been suggested to occur in plants also, but genes encoding these enzymes have not been identified to date (Huang et al., 2011). The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood.
Scheme of B6 Vitamer Metabolism in Arabidopsis.
PLP can be biosynthesized de novo from ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P), and glutamine by the catalytic action of PDX1 and PDX2 (orange panel). A salvage pathway also operates (blue panel) in which PLP may also be produced from either PMP or PNP through the action of PDX3. The kinase SOS4 is also part of this pathway and can phosphorylate PM, PN, or PL. Phosphatases (probably unspecific) perform the reverse reactions to the latter. PL may also be reduced to PN through the action of pyridoxal reductase (PLR1). PN and PL may also be glycosylated, but the enzymes carrying out these reactions are not known in plants. In all cases, the cofactor form PLP may be transferred to confer catalytic activity to PLP-dependent enzymes such as transaminases and decarboxylases. Modified from González et al. (2007).
Several Arabidopsis mutants completely or partly impaired in vitamin B6 metabolism have been described. A complete loss of vitamin B6 biosynthesis de novo is embryo lethal (Tambasco-Studart et al., 2005; Titiz et al., 2006), while mutants in either one of the two catalytic PDX1 genes (PDX1.1 or PDX1.3) have short roots and are stress hypersensitive (Chen and Xiong, 2005; Titiz et al., 2006; Wagner et al., 2006; Boycheva et al., 2015). The latter phenotypes are remarkably similar to mutants in the salvage pathway kinase SOS4 (Rueschhoff et al., 2013), originally isolated due to their salt sensitivity (Shi et al., 2002). Recently, the phenotypes associated with pdx1.1 and pdx1.3 have been linked primarily to a perturbation in ethylene and auxin homeostasis (Boycheva et al., 2015), two phytohormones that require PLP as a cofactor for their biosynthesis. By contrast, mutants partially impaired in the expression of the salvage pathway enzyme PDX3 were reported to be morphologically indistinguishable from wild-type plants when grown on soil under normal conditions but hypersensitive to high sucrose levels, high light, and chilling (González et al., 2007). An independent study employing knockdown lines using small interfering RNA constructs also reported no significant visible phenotype but hypersensitivity to high light was confirmed (Sang et al., 2011). It was thus suggested that a minimum level of expression of PDX3 is sufficient for homeostasis, but the effect of its complete abolishment is not known. In eukaryotes, PLP is needed as a cofactor in numerous subcellular compartments most notably associated with enzymes involved in amino acid metabolism, as well as phytohormone biosynthesis, photorespiration, and others (Mooney and Hellmann, 2010). Biosynthesis of PLP de novo takes place in the cytosol, whereas the PLP salvage enzyme SOS4 has been reported to be plastidic (Rueschhoff et al., 2013). An early study reported a plastidic location for PDX3 (Sang et al., 2011), but it was more recently demonstrated to localize to mitochondria (Colinas et al., 2014; Niehaus et al., 2014). Taken together, the contribution of PDX3 in provisioning PLP within the subcellular pool and its role in growth and development remain to be elucidated.
To further our understanding, we report here on the functional relevance of PDX3 and demonstrate that it is essential for normal growth and development in Arabidopsis. Significantly, pdx3 knockout mutants have an imbalance in B6 vitamer levels and a severe aberrant leaf phenotype and show delayed development. Genetic complementation with PDX3 reverts the developmental phenotype of pdx3 mutants and restores vitamin B6 homeostasis. The B6 vitamer imbalance in pdx3 mutants results in gross changes in the metabolite profile, notably toward steady state amino acid levels. Most surprisingly, RNA sequencing reveals strong induction of stress-related genes, particularly those associated with biotic stress. This coincides with an increase in salicylic acid levels, implying that the pdx3 mutant is constitutively activated for defense. Intriguingly, exogenous ammonia rescues the pdx3 growth and developmental phenotype, as nitrate reductase activity is severely reduced. Overall, our analyses demonstrate an important link between B6 vitamer status and nitrogen metabolism. In particular, the effect of the ratio of PMP to PLP content on the perceived nitrogen status of the plant is discussed.
RESULTS
PDX3 Is Required for Plant Vegetative Growth and Development
To provide insight into the functional relevance of PDX3, two independent T-DNA insertion lines (SALK_054167C and GK-260E03) were obtained from the available collections isolated to homozygosity. PCR analysis established insertion of the T-DNA in intron 7 of SALK_054167C and in exon 4 of GK-260E03 (Figure 2A). Analysis of the transcript level of PDX3 expression by quantitative real-time RT-PCR (qPCR) with independent primer pairs confirmed severely reduced expression (Figure 2B), although residual expression downstream and upstream of the T-DNA insertion site can be observed in GK-260E03 and SALK_054167C, respectively. However, no or extremely low levels of protein could be detected in either mutant line by immunochemical analysis using an antibody specifically raised against Arabidopsis PDX3 (Figure 2C). By contrast, a strong protein band of ∼55 kD could be detected in wild-type protein extracts consistent with the expected molecular mass of mature PDX3 (52.2 kD) (Colinas et al., 2014; Niehaus et al., 2014). Therefore, these two lines can be considered as strong pdx3 mutants. Given that two partial knockout or weak mutants of pdx3 have been described previously (González et al., 2007) and annotated as pdx3-1 (SALK_060749C) and pdx3-2 (SALK_149382C), we annotate SALK_054167C as pdx3-3 and GK-260E03 as pdx3-4.
PDX3 Is Essential for Vegetative Growth and Development in Arabidopsis.
(A) Gene model of PDX3 with exons depicted as black bars, introns as lines, and the untranslated regions as gray bars. The regions corresponding to the NNRE and PMP/PNP oxidase (POX) domains are as indicated. The locations of the T-DNA insertion in SALK_054167C (pdx3-3) and GK-260E03 (pdx3-4) are as depicted and were confirmed by genotyping and sequencing. Primer pairs indicated (Supplemental Table 2) were used for qPCR.
(B) Quantitative analysis of PDX3 expression in mutant lines versus the wild type (Col-0). Two sets of primer pairs were used (see [A]). Some residual expression downstream of the insertion site can be observed in pdx3-4. The data are the average of three independent biological replicates with bars representing se.
(C) Immunochemical analysis of PDX3 levels indicates that the protein is not detectable in pdx3 mutant lines compared with the wild type. Expression is restored in transgenic pdx3 lines expressing PDX3 under the control of the CaMV 35S promoter. Immunochemical analysis of ACTIN-2 was used as a loading control.
(D) Photographs of 21-d-old pdx3 lines compared with the wild type (Col-0), showing gross morphological defects during leaf development. Leaves display reduced leaf blade area, enhanced serration at leaf margins, and asymmetric leaf expansion. Notably, the convex, slightly epinastic curvature typically observed in wild-type plants grown under the same conditions is lost. Complementation of the phenotype is achieved in transgenic pdx3-3 and pdx3-4 expressing PDX3.
(E) Photographs of the individual leaves of the pdx3 lines and wild-type plants shown in (D). The severe curling of the leaves and asymmetric leaf expansion can be observed in pdx3-3 in particular. Plants were grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C.
Interestingly, both pdx3-3 and pdx3-4 showed gross morphological defects during leaf development (Figures 2D and 2E). Leaves had reduced leaf blade area and enhanced serration at leaf margins and display asymmetric leaf expansion. Thus, the convex slightly epinastic curvature typically observed in wild-type plants grown under the same conditions was lost (Figure 2D). Moreover, severe curling of the leaves was observed at maturity (Figure 2E). These phenotypes were more pronounced in pdx3-3 compared with pdx3-4, consistent with a higher level of PDX3 transcript that would encode the PNP/PMP oxidase domain in the latter (Figure 2B) and were not observed in either pdx3-1 or pdx3-2 (Supplemental Figure 1A), corroborating only partial knockdown status. Notably, during early development (up to the emergence of the second pair of true leaves), pdx3-3 and pdx3-4 mutants were indistinguishable from the wild type. Photoperiod affected the severity of the developmental phenotype, which was most pronounced under continuous light or long days (16 h light) and less severe under a short photoperiod (8 h light) (Supplemental Figures 1B and 1C). To confirm that the phenotypic observations of pdx3-3 and pdx3-4 were a consequence of PDX3 impairment, we transformed the latter mutants with a construct designed to express PDX3 under the control of the CaMV 35S promoter. Several lines in which the expression of PDX3 was restored to wild-type protein levels were isolated, examples of which are shown (Figure 2C, pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3). In all cases, the aberrant vegetative growth and morphological defects in leaf development observed in both pdx3-3 and pdx3-4 were fully rescued (Figure 2D). We therefore conclude that the loss of PDX3 is responsible for the vegetative developmental defects observed in the pdx3-3 and pdx3-4 mutant lines.
PDX3 Is Also Required for Reproductive Development
In addition to the vegetative developmental phenotype, the transition to the reproductive stage was earlier in pdx3-3 in particular (Figure 3A), as judged from bolting time 23 d after germination (DAG) under long-day conditions, compared with wild-type plants (26 DAG) (Figure 3B). A weak early-bolting phenotype was also observed in pdx3-4 (25 DAG). Opening of the first flower in pdx3-3 (29 DAG) was 2 d earlier than in the wild type (31 DAG), while that of pdx3-4 was similar to the wild type under the same conditions (Figure 3C). Consequently, the number of leaves in pdx3-3 was less than in the wild type at the equivalent developmental stage (Figure 3D). Later during development, reduced apical dominance was observed in pdx3-3 in particular, compared with wild-type plants (Figure 3E), consistent with an increased number of primary stems (Figure 3F). There were fewer siliques overall; thus, there was a severe reduction in seed yield in pdx3-3 (by 60%) compared with the wild type (Figure 3G). The pdx3-4 mutant also had reduced seed yield, but this was less pronounced than that of pdx3-3 compared with the wild type. Furthermore, we performed reciprocal test crosses of pdx3-3 and pdx3-4, the T1 progeny of which displayed the weaker pdx3-4 phenotypes, demonstrating that the mutants are allelic and that the pdx3 phenotype is recessive (Supplemental Figure 2). Notably, these amalgamated pleiotropic phenotypes have not been described for a vitamin B6 mutant previously. Importantly, all of the reproductive phenotypes were fully restored to those of the wild type in the genetically complemented pdx3-3 and pdx3-4 lines (Figure 3). Taken together, it must therefore be concluded that PDX3 is needed for both vegetative and reproductive growth and development in Arabidopsis.
PDX3 Is Essential for Timely Reproductive Development in Arabidopsis.
(A) Representative photographs of 27-d-old plants illustrating the early bolting phenotype of pdx3 mutant lines compared with the wild type (Col-0). Complementation of the phenotype is observed in transgenic pdx3 lines expressing PDX3 under control of the CaMV 35S promoter (pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3, respectively).
(B) Bolting time of the same lines as shown in (A). The bolting time of pdx3-3, in particular, is earlier than the wild type (Col-0), as indicated by the asterisk, but recovers in pdx3-3/35S-PDX3.
(C) and (D) Time of opening of the first flower and number of leaves at this stage in the same lines as shown in (A). The flowering time and number of leaves of pdx3-3, in particular, are statistically different from the wild type (Col-0), as indicated by the asterisks, but recover in pdx3-3/35S-PDX3.
(E) Reduced apical dominance is observed in pdx3-3, in particular, compared with the wild type (Col-0).
(F) Increased number of emerging primary stems (arrows) observed in pdx3-3 compared with the wild type (Col-0).
(G) Seed yield for all lines as in (A) measured as weight of total seeds per plant, shown as both fresh weight and dry weight. The yield is significantly decreased in pdx3-3.
Plants were grown under a 16-h photoperiod (120 µmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C. Statistical differences from the wild type were calculated by a two-tailed Student’s t test and indicated by an asterisk for P < 0.01. In all cases, error bars represent se.
The B6 Vitamer Profile Is Imbalanced in pdx3 Mutants
To obtain further insight into the molecular basis of the pdx3 mutant phenotypes, we determined the B6 vitamer profile of rosette leaves using HPLC. In this way, the six different B6 vitamers can be separated and quantified (Szydlowski et al., 2013) (Supplemental Figure 3). The analysis revealed that there was a considerable perturbation in the levels of certain B6 vitamers in pdx3-3 and pdx3-4 compared with the wild type (Figure 4A). Specifically, the levels of PMP and PNP were increased, while there was a tendency for a decrease in the levels of PLP. This is in agreement with the assumed in vitro biochemical function of PDX3, in that the enzyme oxidizes PNP and PMP to produce PLP (Sang et al., 2007). Thus, the results provide in vivo evidence for the function of PDX3 in Arabidopsis. Notably, the corresponding nonphosphorylated vitamers were also perturbed in a similar manner, i.e., PL contents were reduced, whereas PM contents were slightly increased (Figure 4A). This is likely due to the action of nonspecific cellular phosphatases. Therefore, the ratio of the PMP plus PM to PLP plus PL contents (PMP+PM:PLP+PL) was considerably higher in pdx3-3 (1.46) and pdx3-4 (1.28) compared with the wild type (0.57). The distribution of the B6 vitamer content was partially restored toward wild-type levels in the genetically complemented pdx3-3 and pdx3-4 mutant lines (Figure 4A); in turn, the PMP+PM to PLP+PL ratio was reduced (being 1.02 and 0.85 for pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3, respectively). The incomplete rescue in vitamer levels in the complemented lines to wild-type levels may be due to the slightly reduced expression of PDX3 in these lines compared with the wild type (Figure 2C). Supplementation with PM or PL did not serve to exaggerate or rescue, respectively, the developmental phenotypes in pdx3 but did rescue the pale leaf and stunted phenotype of the de novo biosynthesis mutant pdx1.3 (Supplemental Figure 4), as was shown previously with PL and PN (Chen and Xiong, 2005; Titiz et al., 2006). However, we noted that PM supplementation did not lead to an accumulation of PMP in the wild type but did in pdx3 (even though the corresponding kinase SOS4 is present in both). This suggests that PMP contents are kept in check through the action of PDX3. Significantly, we also found that the total vitamin B6 content (i.e., the sum of all vitamers) was not changed between the mutant lines and the wild type (Figure 4B). Taken together, the data demonstrate the in vivo function of PDX3 and that the growth and developmental phenotypes observed in pdx3-3 and pdx3-4 are not a consequence of a change in the total content of vitamin B6 but rather are due to an imbalance in the B6 vitamer profile.
PDX3 Is Required for Balancing B6 Vitamer Content.
(A) Individual B6 vitamer contents of lines as indicated, determined by HPLC from rosette leaves of 21-d-old plants. Contents of PMP and PNP are higher, whereas PLP contents are slightly lower in pdx3 mutant lines compared with the wild type (Col-0), confirming the enzymatic activity of PDX3 in vivo. Reversion of B6 vitamers toward wild-type levels is observed in the transgenic lines expressing PDX3 under control of the CaMV 35S promoter (pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3, respectively).
(B) Total B6 vitamer content of lines as indicated, demonstrating that the overall B6 content remains similar in all lines examined.
Different letters indicate statistically significant differences between means for each vitamer as determined by a two-way ANOVA (corrected with Tukey's multiple comparisons test) for P < 0.05. Plants were 21 d old grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C.
Loss of PDX3 Causes Major Alterations in Profiles of Metabolites, Particularly Amino Acids
Given the involvement of PLP in numerous metabolic pathways and due to the remarkable phenotype of the pdx3 mutants, we next determined the metabolic profile of pdx3-3 and pdx3-4 to provide insight into the predominant pathways affected. Rosette leaves of 21-d-old plants were chosen for the analysis, a developmental time point when the leaf phenotype of the pdx3 mutants is particularly pronounced. From this analysis, we were able to identify 66 compounds (Supplemental Table 1), out of which 41 were significantly changed compared with the wild type (Figure 5A). Among the changed compounds in the mutants, amino acids were particularly prominent. Seventeen of the detected compounds could be directly related to vitamin B6 metabolism because they are either substrates or products of PLP-dependent reactions (Figure 5B; Supplemental Data Set 1). Of these compounds, 13 were significantly changed in the mutants (Figures 5A and 5B). Thus, 76% of the compounds that could be detected and that are directly related to vitamin B6 metabolism based on known PLP-dependent reactions were altered in the pdx3-3 and pdx3-4 mutants. Interestingly, only 3 of the 41 altered compounds showed significantly lower levels, while the rest were all overaccumulated (Figure 5A).
Metabolite Profiling Reveals Perturbations in pdx3.
(A) List of changed metabolites in pdx3-3 (white) and pdx3-4 (gray) compared with the wild type (Col-0). Compounds underlined are directly related to PLP-dependent reactions.
(B) Proportional Venn diagram of metabolites detected as a function of vitamin B6 metabolism. Of the 66 compounds detected, 41 were changed in abundance in pdx3 mutants, 13 of which could be directly related to PLP-dependent enzymatic reactions, the remaining 28 being unrelated. Four of the detected compounds that could be associated with PLP-dependent reactions were unchanged in the pdx3 mutants relative to the wild type. Notably, another 71 compounds related to PLP-dependent reactions could be expected to be found in Arabidopsis (written in gray) but were not detected in our analysis.
Statistically significant changes compared with the wild type were calculated by a two-tailed Student’s t test for P < 0.05 in (A) (Supplemental Table 1) and are indicated by an asterisk. In all cases, error bars represent se.
Defense Responses Are Activated in pdx3 Mutants
To further investigate and relate changes in the metabolite profile of pdx3 to global changes in gene expression, we performed high-throughput RNA-seq analysis. To generate RNA-seq data sets, cDNA libraries were prepared of three independent biological replicates of 21-d-old rosette leaves of pdx3-3, pdx3-4, and the wild type (Col-0) and subjected to Illumina sequencing. The resulting reads were mapped onto the reference Arabidopsis genome assembly (TAIR10 version) and evaluated for changes in gene expression. A global analysis of changes in gene expression in pdx3-3 and pdx3-4 showed that 1184 genes were induced at least 2.0-fold and 254 genes were repressed at least 0.5-fold in both pdx3 alleles (P < 0.05, see Methods for P value calculation), respectively (Figure 6A; Supplemental Data Set 2), out of a total of 17,721 expressed genes. Importantly, while there is misregulation of a unique set of transcripts in RNA-seq data sets of both pdx3-3 and pdx3-4, the vast majority of the misregulated transcripts overlap (Figure 6A). Given the biochemical function of PDX3 in supplying PLP, we first searched for alteration of genes associated with PLP-dependent pathways. However, our analysis revealed that of the 133 genes annotated as encoding PLP-dependent enzymes in the Arabidopsis genome (Supplemental Data Set 1), the majority was not significantly altered. Specifically, while the transcriptome data set covered 87% of the annotated PLP-dependent genes, only 11% of this gene category was affected (Figure 6B). Notably, a previous study reported on the upregulation of genes associated with vitamin B6 metabolism in the pdx3-1 and pdx3-2 mutant lines (González et al., 2007), specifically SOS4 of the salvage pathway and PDX1.1/PDX1.2/PDX1.3 of the biosynthesis de novo pathway. By contrast, the transcriptome analysis performed in this study with pdx3-3 and pdx3-4 showed that neither of the salvage pathway genes (SOS4 and PLR1) nor PDX2 of the biosynthesis de novo pathway were significantly changed and only slight changes (<2.0-fold) were observed for PDX1.1-1.3 (Figure 6C). Importantly, reads corresponding to expression of PDX3 were virtually absent after the location of the T-DNA insertion in pdx3-3; however, reads could be observed in pdx3-4 after the location of the T-DNA insertion, although at lower levels than those of the wild type (Supplemental Figure 5). Therefore, we also specifically examined the expression of these genes by qPCR in pdx3-3 and pdx3-4 compared with the wild type, which served to confirm these findings (Figure 6D). Only PDX1.2 was slightly upregulated in pdx3-3 in particular, compared with the wild type, whereas all other genes were not significantly altered in the mutants.
RNA-Seq Analysis Reveals That Defense Responses Are Activated in pdx3-3 and pdx3-4.
(A) Venn diagrams demonstrating that the majority of transcripts misregulated in pdx3-3 and pdx3-4 overlap, according to the RNA-seq transcriptome analysis.
(B) Pie charts demonstrating the percentage of genes altered in pdx3-3 and pdx3-4 that encode PLP-dependent enzymes. Note that the filtered transcriptome set contained 115 genes out of 133 genes that encode or are predicted to encode for PLP-dependent enzymes in the Arabidopsis genome.
(C) Expression of genes encoding enzymes involved in vitamin B6 metabolism as depicted by the RNA-seq transcriptome analysis of pdx3-3 and pdx3-4. Only slight changes (<2.0-fold) were observed for PDX1.1-1.3, whereas expression of the other genes was not statistically significantly altered compared with Col-0, with the exception of PDX3, which was reduced (P < 0.05 calculated as described for the transcriptome analysis).
(D) Expression of the same set of vitamin B6 metabolism genes as shown in (B) measured by qPCR with gene-specific primers. The data are representative of three biological replicates and two technical repeats with error bars representing se. Only the expression of PDX1.2 was statistically significantly upregulated (P < 0.05, two-tailed Student’s t test) in both pdx3 mutants.
(E) Gene Ontology terms related to pathogen defense and immune response are strongly overrepresented in the list of 2-fold upregulated genes in both pdx3-3 and pdx3-4. A list of Gene Ontology terms and the associated genes found in each list and taken into account for this graph can be found in Supplemental Data Set 2, as well as a GOslim analysis.
(F) Expression analysis of selected key genes involved in salicylic acid-mediated defense by qPCR analysis. The data are presented as means ± se for three biological and three technical replicates. Statistically significant changes compared with the wild type were calculated with a two-tailed Student’s t test for P < 0.05 and are indicated by an asterisk.
In all cases, the analyses were done on rosette leaves of 21-d-old plants grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C.
Intriguingly, an analysis by Gene Ontology (GOslim; https://www.arabidopsis.org/tools/bulk/go/index.jsp) indicated that among the induced or repressed genes the categories “response to stress” and “response to abiotic or biotic stimulus” were overrepresented (Supplemental Data Set 2). In particular, genes associated with pathogen defense were strongly enriched among the upregulated transcripts (Figure 6E). Moreover, the list of upregulated genes was considerably enriched (20%) for several process categories associated specifically with salicylic acid-mediated defense and immunity responses, as well as functional categories associated with the transmembrane receptor activities (Supplemental Data Set 2). This is interesting considering the overaccumulation of salicylic acid observed in the metabolite analysis of pdx3 mutants compared with the wild type (Figure 5A) and suggests that pdx3 is hyperactivated/primed for defense. Furthermore, we could demonstrate by qPCR that key genes in the salicylic acid-mediated defense pathway, such as PATHOGENESIS RELATED1 (PR1), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), and SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1; specific to Col), were upregulated in both pdx3-3 and pdx3-4 (Figure 6F). Thus, the data show that defense responses are activated in pdx3. Notably, among the misregulated genes there is enrichment for those encoding proteins localized to the plasma membrane or extracellular space according to a GOrilla analysis (http://cbl-gorilla.cs.technion.ac.il/) (Eden et al., 2009; Supplemental Data Set 2). This further supports the observation that pdx3 displays an activated defense response, as the latter is associated with major rearrangements of the cell wall and plasma membrane (Malinovsky et al., 2014).
The Lack of PDX3 Renders the Plant with an Ammonium-Dependent Phenotype
We noted that pdx3 was indistinguishable from the wild type when grown in culture on MS salt medium (Murashige and Skoog, 1962) and that the strong developmental phenotypes (Figures 2 and 3) became apparent only when pdx3 was grown on soil, suggesting a nutrient-dependent response that was alleviated on MS salt medium. Notably, MS salt medium contains both ammonium and nitrate as nitrogen sources, whereas nitrate is the predominant source of nitrogen in soil due to microbial nitrification (Bloom, 2015). In this context, it has previously been shown that there is an interaction between defense responses and nitrogen metabolism (Park et al., 2011; Wang et al., 2013). Specifically, the developmental phenotypes observed in these aforementioned studies were caused by a low ammonium-to-nitrate ratio and could be repressed by simple ammonium supplementation. We therefore grew pdx3 on modified MS salt medium in which ammonium was omitted and indeed observed the aberrant leaf phenotype, in particular with pdx3-3, previously seen only on soil (Figure 7A). As this suggested that a low ammonium-to-nitrate ratio was responsible for the pdx3 phenotype on soil, we then watered soil-grown plants with different salts (ammonium nitrate, potassium nitrate, or ammonium chloride). When ammonium was part of the salt mixture, pdx3 could not be distinguished from the wild type, whereas in the absence of ammonium supplementation, the developmental phenotypes could be clearly observed (Figure 7B). That the response is specific to ammonium and not nitrate is highlighted by exclusive rescue of the pdx3 phenotype upon watering with ammonium chloride or ammonium nitrate but not with potassium nitrate (Figure 7B).
The pdx3 Mutants Are Dependent on Ammonium.
(A) On standard MS salt medium (Murashige and Skoog 1962), pdx3 mutant leaves are phenotypically indistinguishable from the wild type. When grown on a version of this medium with ammonium omitted, the narrow curly leaf developmental phenotype becomes visible. Pictures were captured of 21-d-old plants grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C.
(B) Plants were germinated and grown on soil with water supplemented with 50 mM of the indicated compounds or with water only as a control every nine days. Pictures were captured of 21-day-old plants grown under a 16-h photoperiod (120 μmol photons.m−2 s−1) at 22°C and 8 h of darkness at 18°C. Note that supplementation with either ammonium chloride or ammonium nitrate complements the phenotype, whereas supplementation with potassium nitrate does not.
To investigate a potential interaction between nitrogen metabolism and vitamin B6, the profile of B6 vitamers was analyzed in the ammonium-supplemented rescued lines. We found that ammonium supplementation led to considerable accumulation of the PMP vitamer, in both the wild type and the pdx3 mutant lines either when grown in culture or on soil (Figures 8A and 8B). This suggests that elevated PMP contents signify ammonium availability. As a corollary, ammonium availability may be constitutively perceived in pdx3 mutant lines (due to the abundance of PMP in these mutants even in the absence of ammonium). This would serve to explain the dependence of pdx3 mutants on ammonium when grown on soil. Furthermore, PMP has been reported to inhibit nitrate reductase activity in vitro (Sims et al., 1968). Therefore, we measured this activity in the pdx3 lines and the wild type. Indeed, there was a considerable decrease in nitrate reductase activity in both pdx3-3 and pdx3-4 (reduced by 60 to 80% of that in the wild type) (Figure 8C). This would serve to compound the dependence of pdx3 on ammonium. Taken together, it can be concluded that pdx3 is an ammonium-dependent mutant, which possibly results from its constitutive overaccumulation of PMP.
Interaction between Nitrogen Metabolism and Vitamin B6 Homeostasis.
(A) B6 vitamer profiles of plants grown on soil watered with either ammonium chloride (NH4Cl), ammonium nitrate (NH4NO3), or potassium nitrate (KNO3), or in the absence of supplementation (H2O) as indicated. The analysis was performed on rosette leaves of 21-d-old plants grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C. A considerable increase in PMP levels, in particular, is observed in both the wild type and pdx3 mutants upon ammonium supplementation. Different letters indicate statistically significant differences between means for each vitamer as determined by a two-way ANOVA (corrected with Tukey's multiple comparisons test) for P < 0.05.
(B) B6 vitamer profiles of plants grown on MS medium modified to be in the absence or presence of ammonium (NH4+). The analysis was performed on rosette leaves of 10-d-old plants grown under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C. Different letters indicate statistically significant differences between means for each vitamer as determined by a two-way ANOVA (corrected with Tukey's multiple comparisons test) for P < 0.05.
(C) Nitrate reductase activity in extracts of rosette leaves of 19-d-old plants grown on soil under a 16-h photoperiod (120 μmol photons m−2 s−1) at 22°C and 8 h of darkness at 18°C. Error bars represent se of 18 biological replicates. Activity was strongly reduced in pdx3 mutants, as indicated by the asterisks, which represent statistically significant changes compared with the wild type calculated by a two-tailed Student’s t test for P < 0.01.
DISCUSSION
In nature, the success of plants is determined by their ability to withstand adverse environmental conditions. Consequently, plants have developed an arsenal of mechanisms to sense unfavorable situations and respond through a variety of signaling pathways that mediate a response. Inevitably, the launch of such responses requires resources and the plant often needs to make a trade-off that can affect yield (Todesco et al., 2010; Kerwin et al., 2015). In this study, our initial observations suggested that pdx3 mutants were activated for defense and suffered a yield penalty. However, further investigation allowed us to demonstrate that these mutants were ammonium dependent and provided an unanticipated link between vitamin B6 homeostasis and nitrogen metabolism. Specifically, perturbation of the balance of B6 vitamers in the cell, in this case through disruption of the salvage pathway enzyme PDX3, leads to accumulation of PMP and compromises nitrogen metabolism, such that the plant becomes dependent on ammonium. In the absence of the latter nutrient, the plant suffers strong developmental impairments.
PDX3 Is Vital for Balancing B6 Vitamer Content
The functional significance of PDX3 becomes apparent in this study, as loss-of-function mutants display severe impairments in vegetative and reproductive development in Arabidopsis. These impairments are more pronounced in the stronger allele pdx3-3 than in the weaker pdx3-4 allele. Complementation of the aberrant phenotypes upon introduction of the PDX3 transgene validates the dependence of these phenotypes in the pdx3 mutant alleles. From a molecular point of view, there is perturbation of vitamin B6 homeostasis in the pdx3 alleles examined. In particular, the PMP-to-PLP ratio is imbalanced in these pdx3 mutants (with PMP contents markedly increased and PLP contents decreased), as are the ratios of the corresponding nonphosphorylated derivatives, although the total vitamin B6 content remains unchanged. Notably, this observation validates the presumed in vivo biochemical function of PDX3, which is the oxidation of PMP (or PNP) to the cofactor form PLP (Sang et al., 2007). In line with this, metabolite profiling revealed that a high proportion of detected compounds altered in the pdx3 mutants examined here could be directly related to vitamin B6 metabolism. The perturbation of amino acids was particularly prominent and may be due to the prevalence of PLP-dependent enzymes for their metabolism, suggesting that PDX3 is important for the supply of PLP to certain enzymes. Intriguingly, most of the metabolite compounds detected were increased rather than decreased. This is similar to what has been observed in other vitamin B6 biosynthesis mutants with the exception of the amino acid glycine, which has been observed to decrease in other mutants of this pathway (Wagner et al., 2006; Raschke et al., 2011), although glycine accumulation has been reported upon overexpression of the de novo biosynthesis gene PDX1.3 (Leuendorf et al., 2010). Interestingly, we did not observe substantial changes in the expression of the majority of the other genes involved in the biosynthesis of PLP, such as PDX1.1/PDX1.3 and PDX2 involved in biosynthesis de novo (Tambasco-Studart et al., 2005) or the salvage pathway kinase SOS4 (Shi et al., 2002; Sang et al., 2007). Thus, in contrast to a previous study based on partial knockdown mutants of pdx3 (pdx3-1 and pdx3-2), we did not see evidence of a compensatory mechanism for the lack of PLP provision by PDX3. In other words, PDX3 plays a specific role in Arabidopsis that cannot be fulfilled by other genes involved in vitamin B6 metabolism. Notably in this context, PDX3 is the only enzyme known in vitamin B6 metabolism that is able to recycle PLP from PMP or PNP. Significantly, it must be taken into account that the PDX3 protein was below detection in the pdx3-3 and pdx3-4 mutants described in this study, although pdx3-4 carries a transcript that would include the PNP/PMP oxidase domain, which despite not being detected at the protein level may account for the weaker phenotype in this allele. Nonetheless, we conclude that pdx3-3 and pdx3-4 represent stronger mutant alleles than the promoter T-DNA insertion mutants pdx3-1 and pdx3-2, which were shown to still express PDX3 and have PNP/PMP oxidase activity (González et al., 2007). This may suggest that even reduced amounts of PDX3 in pdx3-1 and pdx3-2 can sense PLP levels (by an as yet unknown mechanism) and transmit imbalances in homeostasis to the other vitamin B6 metabolic genes allowing them to compensate for a decrease in PDX3 activity and thereby maintain homeostasis. Thus, the functional significance of the gene only becomes apparent upon analyzing loss-of-function alleles and is exemplified in this study through the growth and developmental consequences described in the absence of PDX3. Despite the fact that PDX3 is necessary for vitamin B6 homeostasis, we did not observe changes in the expression of the vast majority of PLP-dependent enzymes at the transcriptional level, suggesting that PDX3 effects are mediated at the posttranscriptional level. In this context, a recent study has highlighted that the stability and abundance of certain PLP-dependent enzymes may be directly affected by a low PLP content (de la Torre et al., 2006; Cellini et al., 2014).
PDX3 Plays an Important Role in Nitrogen Metabolism
Although vitamin B6 has been previously implicated in defense (Denslow et al., 2005), the upregulation of specific pathogen response genes and increased salicylic acid content of pdx3 was unanticipated and initially suggested that PDX3 contributes to repression of pathogen responses. In fact, the stunted shoot growth, early flowering, dwarfism, and reduced seed yield of pdx3 are hallmarks of mutants that show constitutive defense responses in the absence of pathogen infection, i.e., autoimmune mutants (Todesco et al., 2010; Alcázar and Parker, 2011). However, a key finding in this study was the demonstration that whereas the pdx3 mutants show strong developmental impairments on soil, they are phenotypically normal when grown in culture on MS salt medium (Murashige and Skoog, 1962). An important difference between these two growth conditions is that the essential macronutrient nitrogen is provided in the form of nitrate and ammonium in MS salt medium but is present mainly as nitrate in soil due to microbial nitrification (Bloom, 2015). As ammonium supplementation completely abrogated the developmental impairments of pdx3 on soil, we therefore conclude that the primary defect in this mutant is due to ammonium insufficiency triggered by the abnormal accumulation of PMP and that the morphological irregularities and constitutive defense response are consequential to this defect.
Given the biochemical function of PDX3 as a PMP oxidase, it is interesting that several decades ago the ratio of PMP to PLP was hypothesized to reflect the nitrogen state of a plant (Teixeira and Davies, 1974; Matsumoto et al., 1975). These studies have been largely overlooked since, but the ammonium transporter mutant amt1.1 has recently been reported to have alterations in vitamin B6 content, although it was not clear which vitamer(s) were changing (Pastor et al., 2014). In a normal cell, the equilibrium between PMP and PLP levels must be maintained. An excess of PMP may interfere with PLP-dependent transaminase activity (the former being an intermediate in such reactions), and excess PLP can be toxic as it is a very reactive aldehyde that readily interacts with amines and thiols in the cell. From the data in this study, we can conclude that PDX3 plays an important role in this process; in its absence, PMP accumulates and PLP diminishes. In the vitamer profiles, part of this misregulation is likely to be reflected in the levels of the corresponding nonphosphorylated vitamers produced through the action of nonspecific phosphatases, such that the ratio of PMP+PN contents to PLP+PL contents is considerably perturbed (i.e., elevated) in pdx3 compared with the wild type. In this study, we also show that exogenous ammonium, but not nitrate, leads to accumulation of PMP (Figure 8), which allows us to propose a working model (Figure 9) on the possible mechanism underlying our observations. First, we suggest that the level of PMP is a chemical sign (either directly or indirectly) of the cellular ammonium status. Accumulation of PMP indicates that ammonium availability is sufficient and concomitantly inhibits nitrate reductase activity, preventing further reduction of nitrate to ammonium, which is energetically wasteful and could in turn lead to ammonia toxicity. Under standard soil conditions, ammonium is derived from nitrate, as the predominant nitrogen source due to microbial activity. Any imbalance in PMP contents is offset by the activity of PDX3, whose job is to maintain the PMP-to-PLP equilibrium, thus ensuring metabolic processes such as amino acid metabolism do not get perturbed. This is corroborated by the increase in PL contents (possibly derived from PLP) in the wild type but not in pdx3 in the presence of an ammonium source (Figures 8A and 8B). However, in the pdx3 mutants, the overaccumulation of PMP may inadvertently signal ammonium availability constitutively to the plant, leading to consistent inhibition of nitrate reductase activity. Therefore, pdx3 mutants grown on soil suffer ammonium insufficiency with the consequential negative effect on growth and development and the inappropriate defense response. These defects can be alleviated by ammonium supplementation. Taken together, our data indicate that PMP levels reflect the ammonium status of the plant and that PDX3 is important for maintaining PMP and PLP homeostasis.
Proposed Working Model for the Interaction of Vitamin B6 Homeostasis and Nitrogen Metabolism.
Growth on standard soil conditions, where nitrate (NO3−) is the predominant nitrogen source and ammonium (NH4+) is usually not abundant and freely available. Left panel: NO3− is taken up and reduced to NH4+ through the actions of nitrate/nitrite (NO2−) reductase (NR and NIR, respectively). The accumulation of NH4+ causes PMP levels to rise (as is observed in this study by NH4+ supplementation), which in turn inhibits NR activity regulating nitrate reduction and thus prevents accumulation of toxic levels of NH4+. PMP and PLP levels are in equilibrium during vitamin B6 metabolism and constantly turning over through the action of PLP-dependent enzymes that are predominantly involved in amino acid metabolism. Maintenance of this B6 vitamer homeostasis includes the action of PDX3, which ensures the PMP and PLP equilibrium such that amino acid metabolism is not perturbed. Right panel: In the absence of PDX3, the PMP and PLP equilibrium is perturbed, manifested as the accumulation of PMP and diminishing PLP. The excess PMP inhibits NR, which inadvertently leads to NH4+ insufficiency in pdx3 disturbing metabolic homeostasis, in particular amino acid metabolism. Thus, these defects can be bypassed by feeding with NH4+.
METHODS
General Plant Material and Growth Conditions
Arabidopsis thaliana (Columbia ecotype), pdx3-1 (SALK_060749C, N681169), pdx3-2 (SALK_149382C, N669245), and pdx3-3 (SALK_054167C, N659883) were obtained from the European Arabidopsis Stock Centre, whereas the pdx3-4 mutant (GK-260E03) was obtained from the GABI-KAT collection (Kleinboelting et al., 2012). The mutant lines pdx3-3 and pdx3-4 were backcrossed to wild-type Columbia and reisolated based on cosegregation of genotype and phenotype, while ensuring the presence of a single insertion and used for all experiments described. Plants homozygous for pdx3-3 and pdx3-4 were reciprocally crossed to generate the double pdx3-3 pdx3-4 insertion mutants. The T1 progeny heterozygous for each of the pdx3 insertions were analyzed for the purpose of a test cross. Unless stated otherwise, plants were grown under long-day growth conditions (60% relative humidity, 100 to 150 μmol photons m−2 s−1 generated by fluorescent lamps [Philips Master T-D Super 80 18W/180] and 22°C for 16 h followed by 8 h of darkness at 18°C) and ambient CO2. Plant lines carrying the pdx3-3 and pdx3-4 T-DNA insertions were verified by PCR analysis of genomic DNA (see Supplemental Table 2 for oligonucleotides used). The absence of expression of PDX3 in the respective lines was verified by qPCR and immunochemical analyses (see below).
Construction of Transgenic Plant Lines
Full-length PDX3 including the region encoding the transit peptide but excluding the stop codon was amplified from cDNA of 10-d-old seedlings using a proofreading polymerase (Stratagene) and specific oligonucleotides (Supplemental Table 2). The amplified products were cloned into the pENTR/D-TOPO vector using the pENTR/D-TOPO cloning kit (Life Technologies) according to the manufacturer’s instructions and sequenced. A stop codon was introduced by site-directed mutagenesis using specific oligonucleotides (Supplemental Table 2). Subsequently, the PDX3 insert was cloned into the Gateway destination vector pB7YWG2 (Karimi et al., 2002) using LR clonase enzyme mix II (Life Technologies). The construct was introduced into Agrobacterium tumefaciens strain C58 and used to transform pdx3-3 and pdx3-4 Arabidopsis mutant plants by the floral dip method (Clough and Bent, 1998). As the respective constructs contain the BAR gene, transformants were selected by resistance to BASTA and phenotypic complementation. Resistant plants were allowed to self-fertilize, and homozygous lines were selected from the T3 generation according to their segregation ratio for BASTA resistance.
Metabolite Profiling
Ten experimental replicates of Col-0, pdx3-3, and pdx3-4 were grown under long-day conditions and harvested when they were 21 d old. The material of four to six plants was pooled and ground in liquid nitrogen using a mortar and pestle. The resulting powder was split for standard gas chromatography-mass spectrometry and fatty acid analysis; the exact weight was recorded and the material was kept at −80°C until extraction. Gas chromatography-mass spectrometry was performed exactly as described (Lisec et al., 2006) with peak annotation based on libraries of authentic standards (Kopka et al., 2005), while fatty acid analysis was performed as detailed by Raschke et al. (2011).
Transcriptome Analysis by RNA Sequencing
RNA was extracted from 21-d-old Col-0, pdx3-3, and pdx3-4 soil-grown plants under long-day conditions (three biological replicates) using the PureLink RNA Mini kit (Life Technologies) and poly(A)-selected using the TruSeq Stranded mRNA sample preparation LS kit (Illumina). The nine samples were pooled and sequenced on an Illumina HiSeq 2000 sequencing platform. The quality control of the resulting reads was done with FastQC and the reads mapped to the Arabidopsis TAIR10 Ensembl genome with the TopHat v.2 software. The number of reads and mapped reads per sample is given in Supplemental Data Set 2. For differential expression analysis, the gene features were counted with HTSeq v0.6p1 (htseq-count) on the Ensembl TAIR 10 gene annotation. The statistical analysis was performed using the R/Bioconductor package EdgeR v3.4.2. The counts were normalized according to the library size and genes with less than one count per million were filtered out. The filtered data set contained 17,721 genes. The differentially expressed gene tests were done with a general linear model using a negative binomial distribution. The P values of the differentially expressed genes were corrected for multiple testing with a 5% false discovery rate using Benjamini-Hochberg correction. Gene Ontology terms of the resulting gene set were retrieved from TAIR. The GOrilla analysis was done using the whole transcriptome as a background set.
Gene Expression Analysis by qPCR
Tissue samples were collected from 21-d-old soil grown plants under long-day conditions. RNA was extracted using the RNA NucleoSpin Plant kit (Machery-Nagel) according to the manufacturer’s instructions. DNA was removed by an on-column DNase digest during the RNA extraction and by an additional in-solution digest using DNase RQ1 (Promega) for 20 min according to the manufacturer’s instructions. Reverse transcription was performed using 1 μg total RNA as a template and Superscript II (Invitrogen) according to the instructions with the following modifications: oligo(dT)20 primers stock concentration was 50 ng/μL and 0.5 μL Superscript II enzyme was used per reaction. qPCR was performed in 384-well plates on an 7900HT Fast real-time PCR system (Applied Biosystems) using Power SYBR Green master mix (Applied Biosystems) and the following amplification program: 10 min denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The data were analyzed using the comparative cycle threshold method (2−ΔCT) normalized to three reference genes, UBC21, SAND, and PP2A, as described (Vandesompele et al., 2002). Primers used are listed in Supplemental Table 2. Each experiment was performed with three biological and three technical replicates.
Immunochemical Analyses
Plant material was ground either using a tissue lyser (Qiagen) or a micropestle on liquid nitrogen. One volume of extraction buffer (50 mM sodium phosphate buffer, pH 7.0, containing 5 mM β-mercaptoethanol, 10 mM EDTA, 0.5% Triton X-100 [v/v], 0.1 mM PMSF, and 1% [v/v] complete plant protease inhibitor cocktail [Sigma-Aldrich]) was added immediately to the ground material and homogenized briefly, and samples were then kept on ice. After centrifugation for 15 min at 16,000g at 4°C, the supernatant was decanted and the protein concentration determined by the Bradford assay kit (Bio-Rad) (Bradford, 1976). The samples were then separated by 10% SDS-PAGE loading 30 μg of total protein per lane. Immunoblot analyses for detection of PDX3 were performed with α-PDX3 (Colinas et al., 2014) (custom antibody raised against recombinant PDX3 in rabbits) at a 1:5000 dilution as well as a peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad) at a 1:5000 dilution, using the iBlot system (Invitrogen) and the SNAP i.d. 2.0 system (Millipore) as described previously (Colinas et al., 2014). As a loading control, a monoclonal antibody against ACTIN-2 (Sigma-Aldrich; catalog number A0480, lot 065K4793) was used in combination with the peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad; catalog number 1721019, lot 350003011), both at a 1:10,000 dilution. Chemiluminescence was detected using Western Bright ECL (Advansta) and captured using an ImageQuant LAS 4000 system (GE Healthcare).
Vitamin B6 Analysis by HPLC
Vitamin B6 was extracted from 21-d-old soil-grown plants under long-day conditions. Six rosettes from each line and experimental replicate were harvested separately and ground using glass beads and a tissue lyser. Two volumes of 50 mM ammonium-acetate (pH 4) were added, the samples were vortexed vigorously for 10 min, centrifuged at 20,238g for 15 min, heated to 99°C for 3 min, and centrifuged as before. The resulting supernatant was decanted and used for analysis in two runs, using 10- and 50-μL injection volumes. Separation and detection of B6 vitamers by HPLC was performed as described previously (Szydlowski et al., 2013). All standards except PNP were purchased from Sigma-Aldrich. PNP is not commercially available and was produced enzymatically using PN as a substrate and recombinant Escherichia coli PdxK (Park et al., 2004).
Growth under Different Ammonium Conditions
For culture in vitro, seeds were surface-sterilized with ethanol and sown on plates containing modified MS medium (Murashige and Skoog 1962) (Sigma-Aldrich; containing no ammonium nitrate) and 0.55% agar (Duchefa). For growth in vitro including ammonium, modified MS supplemented with 10.3 mM ammonium chloride was used. For growth on soil, seeds were sown on soil soaked with water or water supplemented with either ammonium chloride, ammonium nitrate, or potassium nitrate (50 mM) and subsequently watered with the described solutions every 10 d.
Measurement of Nitrate Reductase Activity
Nitrate reductase assays were performed as described previously (Yu et al., 1998; Park et al., 2011). Whole shoots from 19-d-old plants grown on soil under long-day conditions were harvested in liquid nitrogen and ground with glass beads on a tissue lyser (Qiagen). Then, 50 μL extraction buffer (250 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 5 µM FAD, 1 µM sodium molybdate, 3 mM DTT, 0.1% BSA, 12 mM β-mercaptoethanol, and 1% plant protease inhibitor cocktail [Sigma-Aldrich]) was added per 25 mg tissue. The samples were kept on ice and centrifuged at 13,000g for 15 min. Fifteen microliters of the supernatant was mixed with 85 μL reaction buffer (100 mM sodium phosphate buffer, pH 7.4, containing 40 mM sodium nitrate and 10 mM NADH) and incubated in the dark at 25°C. After 2 h, the reaction was stopped by the addition of 25 μL 1% (w/v) sulfanilamide and 25 μL 0.05% (w/v) napthylethylenediamine. As a background control, samples were mixed as above but the reaction was stopped immediately. The absorbance at 540 nm was then measured in a 96-well plate reader (BioTek Synergy 2).
B6 Vitamer Supplementation Experiments
Supplementation experiments with PM and PL were performed by watering plants on soil with a solution containing 500 μM of the respective vitamers.
Accession Numbers
Sequence data from this article can be found in the EMBL/GenBank/TAIR data libraries under the following Arabidopsis Genome Initiative locus identifiers: PDX3, At5g49970; SOS4, At5g37850; PLR1, At5g53580; PDX1.1, At2g38230; PDX1.2, At3g16050; PDX1.3, At5g01410; PDX2, At5g60540; PR1, At2g14610; EDS1, At3g48090; SNC1, At4g16890; UBC21, At5g25760; SAND, At2g28390; and PP2AA3, At1g13320. All RNA-seq data generated in this study were deposited in the Gene Expression Omnibus under accession number GSE77428.
Supplemental Data
Supplemental Figure 1. Photoperiod affects the severity of the pdx3 developmental phenotype.
Supplemental Figure 2. Reciprocal test crosses of pdx3-3 and pdx3-4.
Supplemental Figure 3. B6 vitamer profile of Arabidopsis extracts.
Supplemental Figure 4. Supplementation experiments with PM or PL.
Supplemental Figure 5. RNA sequencing reads corresponding to PDX3 expression in pdx3-3 and pdx3-4.
Supplemental Table 1. List of metabolites detected in Arabidopsis lines.
Supplemental Table 2. Oligonucleotide primers used in this study.
Supplemental Data Set 1. Arabidopsis genes predicted to encode PLP-dependent enzymes.
Supplemental Data Set 2. RNA sequencing analysis of pdx3-3 and pdx3-4 compared with the wild type.
Acknowledgments
We thank Mylène Docquier and Natacha Civic from the iGE3 Genomics Platform (University of Geneva) for performing the RNA-seq. Financial support is gratefully acknowledged from the Swiss National Science Foundation (Grant 31003A-141117/1) to T.B.F. as well as the University of Geneva. We also thank the Ernest Boninchi Foundation and the Ernst and Lucie Schmidheiny Foundation for financial contributions. Additional financial support is gratefully acknowledged from the European Molecular Biology Organization (ASTF 485-2014) to M.C. M.E. and A.P.M.W. appreciate support by the Deutsche Forschungsgemeinschaft (WE2231/8-2 and EXC 1028). We thank the European Arabidopsis Stock Centre for seeds of SALK_060749C (pdx3-1), SALK_149382C (pdx3-2), and SALK_054167C (pdx3-3), GABI-KAT for GK-260E03 (pdx3-4), and Svetlana Boycheva for critical reading of the article.
AUTHOR CONTRIBUTIONS
T.B.F. and M.C. designed the research. M.C., M.E., T.T., and M.P. performed the research. M.C., M.E., T.T., M.P., A.R.F., A.P.M.W., and T.B.F. analyzed data. M.C. and T.B.F. wrote the article with input from all other authors.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Teresa B. Fitzpatrick (teresa.fitzpatrick{at}unige.ch).
Glossary
- PN
- pyridoxine
- PM
- pyridoxamine
- PL
- pyridoxal
- PNP
- pyridoxine 5′-phosphate
- PMP
- pyridoxamine 5′-phosphate
- PLP
- pyridoxal 5′-phosphate
- DXP
- deoxyxylulose 5-phosphate
- qPCR
- quantitative real-time RT-PCR
- DAG
- days after germination
- Received December 10, 2015.
- Revised January 19, 2016.
- Accepted February 2, 2016.
- Published February 8, 2016.
References
