Skip to main content
Springer Nature Link
Log in

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5′ phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5′-phosphate, pyridoxamine, and pyridoxamine 5′-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5′-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Bilski P, Li MY, Ehrenshaft M, Daub ME, Chignell CF (2000) Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants. Photochem Photobiol 71:129–134

    Article  PubMed  CAS  Google Scholar 

  • Capitani G, Hohenester E, Feng L, Storici P, Kirsch JF, Jansonius JN (1999) Structure of 1-aminocyclopropane-1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene. J Mol Biol 294:745–756

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Xiong L (2005) Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stress. Plant J 44:396–408

    Article  PubMed  CAS  Google Scholar 

  • Denslow SA, Walls AA, Daub ME (2005) Regulation of biosynthetic genes and antioxidant properties of vitamin B6 vitamers during plant defense responses. Physiol Mol Plant Pathol 66:244–255

    Article  CAS  Google Scholar 

  • Denslow SA, Rueschhoff EE, Daub ME (2007) Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant Physiol Biochem 45:152–161

    Article  PubMed  CAS  Google Scholar 

  • Drewke C, Leistner E (2001) Biosynthesis of vitamin B6 and structurally related derivatives. In: Litwack G, Begley T (eds) Vitamins and hormones. Advances in research and applications, vol 61. Academic Press, San Diego, pp 121–155

    Google Scholar 

  • Ehrenshaft M, Bilski P, Li MY, Chignell CF, Daub ME (1999) A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis. Proc Natl Acad Sci USA 96:9374–9378

    Article  PubMed  CAS  Google Scholar 

  • Fitzpatrick TB, Amrhein N, Kappes B, Macheroux P, Tews I, Raschle T (2007) Two independent routes of de novo vitamin B6 biosynthesis: not that different after all. Biochem J 407:1–13

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez E, Danehower DA, Daub M (2007) 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. Plant Physiol 145:985–996

    Article  PubMed  CAS  Google Scholar 

  • Graham CM, Ehrenshaft M, Hausner G, Reid DM (2004) A highly conserved gene for vitamin B6 biosynthesis may have consequences for stress and hormone responses in plants. Physiol Plant 121:8–14

    Article  PubMed  CAS  Google Scholar 

  • Guirard BM, Snell EE (1988) Physical and kinetic properties of a pyridoxal reductase purified from baker’s yeast. Biofactors 1:187–192

    PubMed  CAS  Google Scholar 

  • Havaux M, Ksas B, Szewczyk A, Rumeau D, Franck F, Caffarri S, Triantaphylides C (2009) Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress. BMC Plant Biol 9:130

    Article  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Leuendorf JE, Genau A, Szewczyk A, Mooney S, Drewke C, Leistner E, Hellmann H (2008) The PDX1 family is structurally and functionally conserved between Arabidopsis thaliana and Ginkgo biloba. FEBS J 275:960–969

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Lum HK, Kwok F, Lo SCL (2002) Cloning and characterization of Arabidopsis thaliana pyridoxal kinase. Planta 215:870–879

    Article  PubMed  CAS  Google Scholar 

  • McCormick DB, Merrill AHJ (1980) Pyridoxamine (pyridoxine) 5′-phosphate oxidase. In: Tryfiates GP (ed) Vitamin B6 metabolism and role in growth. Food & Nutrition Press, Inc., Westport, pp 1–26

    Google Scholar 

  • Mittenhuber G (2001) Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and pyridoxal phosphate biosynthesis pathways. J Mol Microbiol Biotechnol 3:1–20

    PubMed  CAS  Google Scholar 

  • Mooney S, Leuendorf JE, Hendrickson C, Hellmann H (2009) Vitamin B6: a long known compound of surprising complexity. Molecules 14:329–351

    Article  PubMed  CAS  Google Scholar 

  • Morita T, Takegawa K, Yagi T (2004) Disruption of the plr1 + gene encoding pyridoxal reductase of Schizosaccharomyces pombe. J Biochem 135:225–230

    Article  PubMed  CAS  Google Scholar 

  • Nakano M, Morita T, Yamamoto T, Sano H, Ashiuchi M, Masui R, Kuramitsu S, Yagi T (1999) Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase—a possible additional family in the aldo-keto reductase superfamily. J Biol Chem 274:23185–23190

    Article  PubMed  CAS  Google Scholar 

  • Ollilainen V (1999) HPLC analysis of vitamin B6 in foods. Agric Food Sci Finland 8:515–618

    CAS  Google Scholar 

  • Penning TM (2004) Introduction and overview of the aldo-keto reductase superfamily. In: Penning TM, Petrash JM (eds) Aldo-keto reductases and toxicant metabolism. American Chemical Society Symposium Series, vol 865, pp 3–20

  • Preiss J, Ball K, Hutney J, Smith-White B, Li L, Okita TW (1991) Regulatory mechanisms involved in the biosynthesis of starch. Pure Appl Chem 63:535–544

    Article  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Sang Y, Barbosa JM, Wu H, Locy RD, Singh NK (2007) Identification of a pyridoxine (pyridoxamine) 5′-phosphate oxidase from Arabidopsis thaliana. FEBS Lett 581:344–348

    Article  PubMed  CAS  Google Scholar 

  • Sang Y, Locy RD, Goertzen LR, Rashotte AM, Si Y, Kang K, Singh NK (2011) Expression, in vivo localization and phylogenetic analysis of a pyridoxine 5′-phosphate oxidase in Arabidopsis thaliana. Plant Physiol Biochem 49:88–95

    Article  PubMed  CAS  Google Scholar 

  • Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37:501–506

    Article  PubMed  CAS  Google Scholar 

  • Shi H, Xiong L, Stevenson B, Lu T, Zhu JK (2002) The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance. Plant Cell 14:575–588

    Article  PubMed  CAS  Google Scholar 

  • Shimomura S, Emman K, Fukui T (1980) The role of pyridoxal 5′-phosphate in plant phosphorylase. J Biochem 87:1043–1052

    PubMed  CAS  Google Scholar 

  • Takagi M, Shimomura S, Fukui T (1981) Function of the phosphate group of pyridoxal 5′-phosphate in the glycogen phosphorylase reaction. J Biol Chem 256:728–730

    PubMed  CAS  Google Scholar 

  • Tambasco-Studart M, Titiz O, Raschle T, Forster G, Amrhein N, Fitzpatrick TB (2005) Vitamin B6 biosynthesis in higher plants. Proc Natl Acad Sci USA 102:13687–13692

    Article  PubMed  CAS  Google Scholar 

  • Tambasco-Studart M, Tews I, Amrhein N, Fitzpatrick TB (2007) Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiol 144:915–925

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Titiz O, Tambasco-Studart M, Warzych E, Apel K, Amrhein N, Laloi C, Fitzpatrick TB (2006) PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis. Plant J 48:933–946

    Article  PubMed  CAS  Google Scholar 

  • Tsang EWT, Hu Z, Chang Q, McGregor DI, Keller WA (2003) Expression of a Brassica napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein. Prot Expr Purif 29:193–201

    CAS  Google Scholar 

  • Valls F, Sancho MT, Fernando-Muino MA, Checa MA (2001) Determination of vitamin B6 in cooked sausages. J Agric Food Chem 49:38–41

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Liu D, Liu C, Zhang A (2004) The pyridoxal kinase gene TaPdxK from wheat complements vitamin B6 synthesis-defective Escherichia coli. J Plant Physiol 161:1053–1060

    Article  PubMed  CAS  Google Scholar 

  • Weigel D, Glazebrook J (2002) Arabidopsis: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Winkler ME (2000) Genetic and genomic approaches for delineating the pathway of pyridoxal 5′-phosphate coenzyme biosynthesis in Escherichia coli. In: Iriarte AJ, Kagan HM, Martinez-Carrion M (eds) Biochemistry and molecular biology of vitamin B6 and PQQ-dependent Proteins. Birkhauser Verlag, Basel, pp 3–10

    Google Scholar 

  • Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ (2008) Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS ONE 3:e1994

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by grant MCB-0322562 from the National Science Foundation and from the North Carolina Agricultural Research Service.

Author information

Author notes

  1. Eugenia González

    Present address: Pathology Research Department, Methodist Hospital Research Institute, Houston, TX, 77030, USA

  2. Heriberto Vélëz

    Present address: Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Authors and Affiliations

  1. Department of Plant Biology, North Carolina State University, Raleigh, NC, 27695-7612, USA

    Sonia Herrero, Eugenia González, Jeffrey W. Gillikin, Heriberto Vélëz & Margaret E. Daub

Authors
  1. Sonia Herrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eugenia González
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey W. Gillikin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Heriberto Vélëz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Margaret E. Daub
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Margaret E. Daub.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 27 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Herrero, S., González, E., Gillikin, J.W. et al. Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis. Plant Mol Biol 76, 157–169 (2011). https://doi.org/10.1007/s11103-011-9777-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-011-9777-x

Keywords

Search

Navigation