Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering

Abstract

Biofortification of staple crops could help to alleviate micronutrient deficiencies in humans. We show that folates in stored rice grains are unstable, which reduces the potential benefits of folate biofortification. We obtain folate concentrations that are up to 150 fold higher than those of wild-type rice by complexing folate to folate-binding proteins to improve folate stability, thereby enabling long-term storage of biofortified high-folate rice grains.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Folate stability in the 'first generation' folate rice and folate screening in the newly engineered 'second generation' lines.
Figure 2: Total folate concentrations in rice seeds engineered for greater folate content and stability (T3 and T4 generation) upon storage at 28 °C.

References

  1. Iyer, R. & Tomar, S.K. J. Food Sci. 74, R114–R122 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Díaz de la Garza, R.I., Gregory, J.F. III & Hanson, A.D. Proc. Natl. Acad. Sci. USA 104, 4218–4222 (2007).

    Article  PubMed  Google Scholar 

  3. Storozhenko, S. et al. Nat. Biotechnol. 25, 1277–1279 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Kiekens, F. et al. Mol. Nutr. Food Res. 59, 490–500 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. De Steur, H. et al. Nat. Biotechnol. 28, 554–556 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. De Steur, H. et al. New Biotechnol. 29, 432–442 (2012).

    Article  CAS  Google Scholar 

  7. De Steur, H. et al. Trends Food Sci. Technol. 39, 116–134 (2014).

    Article  CAS  Google Scholar 

  8. Fitzpatrick, T.B. et al. Plant Cell 24, 395–414 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Scott, J., Rébeillé, F. & Fletcher, J. J. Sci. Food Agric. 80, 795–824 (2000).

    Article  CAS  Google Scholar 

  10. De Brouwer, V. et al. Phytochem. Anal. 18, 496–508 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Blancquaert, D. et al. J. Exp. Bot. 65, 895–906 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Blancquaert, D. et al. Crit. Rev. Plant Sci. 29, 14–35 (2010).

    Article  CAS  Google Scholar 

  13. Yang, L. et al. Plant Biotechnol. J. 5, 815–826 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Takaiwa, F. et al. Plant Mol. Biol. 17, 875–885 (1991).

    Article  CAS  PubMed  Google Scholar 

  15. Nakase, M. et al. Gene 170, 223–226 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Orsomando, G. et al. J. Biol. Chem. 280, 28877–28884 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Sauberlich, H.E. et al. Am. J. Clin. Nutr. 46, 1016–1028 (1987).

    Article  CAS  PubMed  Google Scholar 

  18. Rueb, S. et al. Plant Cell Tiss. Org. Cult. 36, 259–264 (1994).

    Article  Google Scholar 

  19. Scarpella, E. et al. Development 127, 3655–3669 (2000).

    CAS  PubMed  Google Scholar 

  20. Counce, P.A., Keisling, T.C. & Mitchell, A.J. Crop Sci. 40, 436–443 (2000).

    Article  Google Scholar 

  21. Livak, K.J. & Schmittgen, T.D. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

  22. Hellemans, J. et al. Genome Biol. 8, R19 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  23. De Brouwer, V. et al. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878, 509–513 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Blancquaert, D. et al. Plant Mol. Biol. 83, 329–349 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Van Daele, J. et al. J. Agric. Food Chem. 62, 3092–3100 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank O. Saracutu for excellent rice transformation work. D.V.D.S. and W.L. gratefully acknowledge financial support from Ghent University, Belgium, (Bijzonder Onderzoeksfonds, BOF2004GOA012 and BOF2009G0A004), and the Research Foundation—Flanders (FWO, projects 3G012609 and 35963). D.B. is indebted to F.W.O. for a postdoctoral fellowship. Simon Strobbe is supported by a PhD fellowship from the Agency for Innovation by Science & Technology (IWT).

Author information

Authors and Affiliations

Authors

Contributions

D.B., experimental design, molecular cloning, analysis of transgenic lines, expression analysis, writing the manuscript; S. Strobbe, expression analysis; J.V.D., F.K., C.S. and W.L., development and application of chromatographic analyses; S. Storozhenko, experimental design, molecular cloning; H.D.S. and X.G., investigation of socio-economic impact; D.V.D.S., experimental design, data analysis, writing the manuscript, initiation and coordination of the research project.

Corresponding author

Correspondence to Dominique Van Der Straeten.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Folate stability in GA9.15

Total folate levels in GA9.15 until 7 months of storage at 28°C. Seeds of the sixth (T5) generation (upon harvest stored at -80°C) were used to confirm the results obtained in the pilot stability experiment (Fig. 1A). Values are means of two biological repeats (and four biological repeats for month 6 and 7); error bars indicate standard deviation. Dots represent the measured values.

Source data

Supplementary Figure 2 Folate content in GA lines over successive generations

Total folate levels in GA9.15 and GA26.5 of successive generations. Values are means of two biological repeats; error bars indicate standard deviation. Dots represent the measured values.

Source data

Supplementary Figure 3 Rice T-DNA vectors

T-DNA vectors used for rice transformation. Orange arrows represent the different promoters.; blue arrows, hygromycin resistance gene (HPTII). Blue bars indicate transcriptional terminators. Abbreviations: LB and RB, left and right T-DNA borders; T35S, 35S transcriptional terminator; Tnos, nopaline synthase transcriptional terminator; CaMV35S, core cauliflower mosaic virus 35S promoter; HPTII, hygromycin phosphotransferase II; GluB1, rice glutelin B1 promoter; GluB4, rice glutelin B4 promoter; Glob, rice globulin promoter; GTPCHI, Arabidopsis thaliana cDNA encoding GTP cyclohydrolase I (green arrow); ADCS, Arabidopsis thaliana cDNA encoding aminodeoxychorismate synthase (pale brown arrow); mtFPGS, Arabidopsis thaliana coding cDNA encoding mitochondrial folylpolyglutamate synthetase (FPGS) (black arrows); ctFPGS, Arabidopsis thaliana cDNA encoding cytosolic FPGS (red arrows); sFBP, soluble folate binding protein (FBP) (dark brown arrows); CAFBP, fusion between coding sequence of β- carbonic anhydrase 2 from Arabidopsis thaliana and sFBP (lilac arrows); GluB4FBP, fusion between rice glutelin B4 coding sequence and sFBP (yellow arrows).

Supplementary Figure 4 Rice polishing experiment

Total folate levels in unpolished and polished seeds of GA9.15 (T4) and two lines engineered for a higher folate content and stability (T3). Upon harvest, seeds were stored at -80°C for 3 years. Values are means of four biological repeats; error bars indicate standard deviation. Dots represent the measured values. Abbreviations: A, aminodeoxychorismate synthase; CAFBP, fusion of β-carbonic anhydrase 2 from Arabidopsis thaliana with soluble synthetic folate binding protein (sFBP); ctF, cytosolic folylpolyglutamate synthetase (FPGS); G, GTP cyclohydrolase I; mtF, mitochondrial FPGS.

Source data

Supplementary Figure 5 Transgene expression

Expression levels of GTPCHI, ADCS, FPGS and FBP transgenes in green rice seeds of all lines in the stability experiment (T3 and T4 generation) presented in Figure 2 (except line GA-mtF-CAFBP 2, due to loss of RNA during extraction). Expression analyses were performed by real-time quantitative PCR. Rice tumor protein homologue (LOC_Os11g43900.1) and expressed protein (LOC_OS07g02340.1) were used as reference genes for normalization. Values are means of a sample and two technical replicates; error bars indicate standard error. Abbreviations: A, aminodeoxychorismate synthase; CAFBP, fusion of β-carbonic anhydrase 2 from Arabidopsis thaliana with soluble synthetic folate binding protein (sFBP); ctF, cytosolic folylpolyglutamate synthetase (FPGS); G, GTP cyclohydrolase I; GluB4FBP, fusion of rice glutelin B4 with sFBP; mtF, mitochondrial FPGS. Line GA-ctF-CAFBP 1 had a high expression of FBP (panel e), in combination with a high GTPCHI (panel a) and ADCS transgene expression (panel b), contributing to a high and stable folate content (Figure 2).

Supplementary Figure 6 Folate mono/polyglutamate content

Total folate levels in lines engineered for a higher folate content and stability (T3 and T4 generation), participating in the folate stability experiment (Fig. 2). Values are means of four biological repeats; error bars indicate standard deviation. Dots represent the measured values. The folate monoglutamate fraction is represented by green bars, the polyglutamate fraction by orange bars. Lines with an enhanced folate polyglutamylation (> 20%) and a high folate content (> 500 µg per 100 g FW) are indicated in bold. Abbreviations: A, aminodeoxychorismate synthase; CAFBP, fusion of β-carbonic anhydrase 2 from Arabidopsis thaliana with soluble synthetic folate binding protein (sFBP); ctF, cytosolic folylpolyglutamate synthetase (FPGS); G, GTP cyclohydrolase I; GluB4FBP, fusion of rice glutelin B4 with sFBP; mtF, mitochondrial FPGS; WT, wild type.

Source data

Supplementary Figure 7 GGH and FPGS expression

Expression levels of endogenous rice gamma-glutamyl hydrolase (GGH) (panel A and B), mitochondrial (mtFPGS) (panel C) and cytosolic (ctFPGS) (panel D) FPGS transgenes in seeds of the sixth (T5) generation of lines engineered for a higher folate stability through polyglutamylation. Expression analyses were performed by real-time quantitative PCR. Rice tumor protein homologue (LOC_Os11g43900.1) and expressed protein (LOC_OS07g02340.1) were used as reference genes for normalization. Values are means of a sample and two technical replicates; error bars indicate standard error. Abbreviations: A, aminodeoxychorismate synthase; CAFBP, fusion of β-carbonic anhydrase 2 from Arabidopsis thaliana with soluble synthetic folate binding protein (sFBP); ctF, cytosolic folylpolyglutamate synthetase (FPGS); G, GTP cyclohydrolase I; GluB4FBP, fusion of rice glutelin B4 with sFBP; mtF, mitochondrial FPGS; WT,wild type.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Note and Supplementary Table 1 (PDF 1443 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Blancquaert, D., Van Daele, J., Strobbe, S. et al. Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering. Nat Biotechnol 33, 1076–1078 (2015). https://doi.org/10.1038/nbt.3358

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3358

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing