Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis

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.

Figure 1.

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).

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.

Figure 8.

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.

Figure 9.

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⁺.