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