Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

Abstract

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.

Figure 1.

Vitamin B₆ salvage pathway. PLP is produced both via the de novo pathway in organisms containing PDX1 and PDX2 as well as by conversion from PNP or PMP via the PNP/PMP oxidase. PMP oxidation can also be catalyzed by transaminases. PN, PM, and PL are phosphorylated by the action of a PL/PN/PM kinase or by an additional PL-specific kinase for PLP. Phosphorylated derivatives are dephosphorylated by phosphatases. PL can also be converted to PN by a PL reductase. This pathway is based on data from E. coli, yeast, B. subtilis, and mammals.

Figure 2.

Growth of E. coli pdxH mutants, mutants transformed with Arabidopsis PDX3, and the parental strain on minimal medium. NU887, NU1706, and NU1708, pdxH E. coli mutants; (T), pdxH E. coli mutants transformed with the Arabidopsis PDX3 gene; NU816, parental strain; (C), pdxH E. coli mutants transformed with vector control. Mutants were grown on minimal medium (liquid EM plus 0.01 mm FeSO₄).

Figure 3.

A, Diagram of the location of the T-DNA insertions in the pdx3 mutant lines (pdx3-1 and pdx3-2) within the PDX3 gene. FP and RP indicate forward and reverse primers used to amplify the 1,200-bp wild-type PDX3 segment. RB1 and RB2 are the T-DNA right-border primers used with RP to amplify the approximately 800-bp segments for the T-DNA insertion mutants. B, Screening of T-DNA pdx3 insertion lines by PCR. DNA from plants segregating for the T-DNA insertion was amplified with the FP and RP primers (top) to amplify the 1,200-bp wild-type sequence or with the RB1 or RB2 primers plus the RP primer (bottom) to amplify the approximately 800-bp sequence resulting from the T-DNA insertions. Plants lacking T-DNA insertions are identified by the amplification of only the 1,200-bp band; homozygous T-DNA mutants have only the 800-bp band and heterozygous plants have both bands. Lanes 1 and 14, 1-kb ladder; lanes 2, 5, 8, and 11, plants lacking the T-DNA insertion (wild type); lanes 3 and 6, pdx3-1 heterozygous plants; lanes 9 and 12, pdx3-2 heterozygous plants; lanes 4 and 7, pdx3-1 homozygous mutants; lines 10 and 13, pdx3-2 homozygous mutants. PCR products were visualized on 1% agarose gels. The difference in distance between the T-DNA insertion in the pdx3-1 and pdx3-2 mutant lines is only 38 bp, resulting in bands that are not distinguishable on the gel.

Figure 4.

PDX3 activity in Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT). The activity of PDX3 was measured in crude extracts of the mutant lines and WT by determining the amount of PLP formed with a colorimetric assay after a 1-h incubation of enzyme reactions containing increasing amounts of total protein. PDX3 activity data were analyzed with a regression line adjusted to the absorbance values obtained for the increasing amounts of total protein.

Figure 5.

In vitro root growth and foliar biomass of Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT). A and B, Root growth (A) and foliar biomass (B) of plants grown on Murashige and Skoog medium. Root length was measured every other day for 15 d. Biomass (wet weight) was determined at 15 d. Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

Figure 6.

Sensitivity of Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT) to Suc, NaCl, and mannitol. A and B, Relative root growth (A) and relative shoot weight (B) of plants grown on Murashige and Skoog medium amended with 100 mm of Suc, NaCl, or mannitol; values shown are relative to the growth of plants of the same line on Murashige and Skoog medium alone. Roots were measured every other day for 15 d. Shoot weight was determined at 15 d. Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

Figure 7.

Response of Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT) to high light and chilling conditions. A, Total dry weight of mutant and WT plants grown under control conditions (20°C, 8-h photoperiod, 200 μmol s⁻¹ m⁻² light) for 3 weeks. B, Relative dry weight of mutant and WT plants exposed to high light (20°C, 8-h photoperiod, 1,000 μmol s⁻¹ m⁻²) and chilling (5°C, 8-h photoperiod, 200 μmol s⁻¹ m⁻² light) conditions. Dry weight is relative to the dry weight of plants of the same line grown under control conditions. Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

Figure 8.

Levels of vitamin B₆ vitamers in Arabidopsis pdx3 and sos4 mutant plants and Arabidopsis ecotype Col-0 (WT) determined by HPLC analysis. Values represent the average concentration in plant extracts obtained from two independent sets of plants. Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

Figure 9.

Comparison of total vitamin B₆ in Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT) determined by yeast bioassay and HPLC analysis. HPLC values represent the sum of PMP, PM, PLP, PL, PNP, and PN. Total vitamin B₆ values represent the average concentration in plant extracts obtained from two independent sets of plants. Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

Figure 10.

Gene expression of de novo and salvage pathway genes in Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis Col-0 (WT). Lines are indicated across the top of the figure and fold changes in expression level for each gene are shown by the bars. Data represent the average of three replications. Genes were amplified by qRT-PCR using the primers listed in Table II and gene expression was normalized to UBQ10 expression.

Figure 11.

PDX1 activity in Arabidopsis pdx3 and sos4 mutant lines and Arabidopsis ecotype Col-0 (WT). The activity of PDX1 was measured in crude extracts of the mutant lines and WT by monitoring the formation of PLP in enzyme reactions containing the same amount of total protein for the mutant lines and WT. Before the addition of the substrate, a 1.5-min baseline was established. Absorbance values represent the average of three replications.