Abstract
Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals2. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.
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Data availability
All data are available in the main text, extended data figures, Supplementary Information and supplementary data. In addition, the RNA-seq reads and the final transcript sequences have been uploaded to NCBI under BioProject ID PRJNA725986. Source data are provided with this paper.
Code availability
Supplementary Data 5 contains the R scripts used for obtaining the gene-to-metabolite correlations.
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Acknowledgements
We acknowledge the technical assistance of A.-M. Narvanto and L. Vottonen (University of Helsinki) as well as the bioinformatic support and analyses by J. Tanskanen (University of Helsinki). We also acknowledge the analytical support of R. Purves (University of Saskatchewan) as well as his kind gift of vicine and convicine standards. We thank NPZ Lembke KG for providing the F5 generation of one of their faba bean crosses. We thank G. Duc (INRA) for providing six seeds of the original, low-vicine faba bean line. We also thank F. Rook and S. Christensen (University of Copenhagen) for their contributions towards the funding of this work. This work was supported by Innovation Fund Denmark grant no. 5158-00004B, Academy of Finland decisions no. 298314 and no. 314961, UK Biotechnology and Biological Science Research Council award no. BB/P023509/1, VILLUM Foundation project no. 15476, Danish National Research Foundation grant no. DNRF99, Guangzhou Elite project no. JY201722, and German Federal Ministry of Food and Agriculture grant no. 2815EPS004.
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F.G.-F., S.U.A. and A.H.S. conceived the research plan. E.B., M.N., W.C., L.E.-H., D.M., D.A., X.X. and R.T. carried out the experiments and data analysis. H.K., C.C., D.M.O., F.L.S. and W.L. provided the instrumentation and resources. J.S., D.M.O., A.H.S., A.V., S.U.A., F.L.S. and F.G.-F. developed the project design and acquired the funding. J.S. coordinated the project. M.N. and S.U.A. prepared the figures. S.U.A. and F.G.-F. wrote the manuscript with input from all authors.
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Peer review information Nature Plants thanks Vincent Courdavault, John D’Auria and Benjamin Lichman for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Canonical function of the bifunctional enzyme 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II in plants.
(a) Domain structure. The protein is composed of a chloroplast targeting peptide (cTP) fused to two catalytic domains: the 3,4-dihydroxy-2-butanone-4-phosphate synthase domain, also called RibB, and the GTP cyclohydrolase II domain, also called RibA. (b) Biochemical function of each catalytic domain in the context of the riboflavin biosynthesis pathway.
Extended Data Fig. 2 Seed vicine and convicine phenotypes of Hedin/2 x Disco/1 pseudo-F2 recombinants within the vc- interval.
Recombinants are classified as Normal V&C (blue open circles) where vicine levels are >1.3 mg/g and convicine levels are >0.85 mg/g or as Low V&C (green open circles) where vicine levels are <1.05 mg/g and convicine levels are <0.2 mg/g. Parental means (n = 2) are shown as squares for Hedin/2 (green) and Disco/1 (blue).
Extended Data Fig. 3 Consequence of the additional AT dinucleotide on the predicted VC1 protein.
An alignment of Hedin/2 VC1 and Mélodie/2 vc1 amino acid sequences is shown. Predicted domains are shown underneath the alignment (RibB, 3,4-dihydroxy-2-butanone-4-phosphate synthase domain; RibA, GTP cyclohydrolase II domain). A measurement of residue conservation is shown underneath the predicted domains, distinguishing between identical residues and other scenarios (non-identical ones as well as gaps/insertions). The position of the AT insertion, which results in a frame shift, is marked with a black triangle underneath the conservation score (position 360). The following key residues in VC1 enzymatic domains are marked: blue arrows, substrate binding in RibB; red arrows, catalysis in RibB; green arrows, Zn binding in RibA; grey arrows, catalysis in RibA; orange arrows, GTP specificity. The prediction of key resides is based on Hiltunen et al.17 and Ren et al.41.
Extended Data Fig. 4 Control reactions for VC1 and vc1 primer pairs.
1 ng of pGEM-T plasmids carrying VC1 cloned from V. faba cv. Hedin/2 (Hed) or vc1 from Mélodie/2 (Mel) were subjected to PCR with primer pairs for either VC1 or vc1. The reaction mixtures and temperature program are the same as described in Methods for ‘specific amplification of VC1 and vc1 from seed coat cDNA of Hedin/2 and Mélodie/2’, except 25 cycles were used for the vc1 primer pair and 35 for the VC1 primer pair to account for their different efficiencies. This control experiment was carried out twice with similar results.
Extended Data Fig. 5 Predicted subcellular localization of VC1 by TargetP 2.0.
The protein sequence of VC1 from Hedin/2 was run through the prediction server TargetP 2.0 (http://www.cbs.dtu.dk/services/TargetP/) giving the output shown above. The protein is predicted to have an N-terminal chloroplast transfer peptide of 52 amino acids with a likelihood of 0.9933.
Extended Data Fig. 6 VC1 expression, purification, and assays.
(a) Polyacrylamide gel electrophoresis (PAGE) showing the affinity chromatography of His-tagged VC1 on a Ni-NTA matrix. Protein was visualized on a Stain-FreeTM pre-cast gel using the ChemiDocTM gel imaging system (BioRad). L, lysate; P, pellet; FT, flow-through; W1-3, three consecutive wash fractions; E1-4, elutions with increasing concentration of imidazole (20, 50, 100, and 250 mM, respectively); M, molecular weight marker (given in kDa). The expected molecular weight of His-tagged VC1 was 51.3 kDa. After buffer exchange to remove the imidazole, fraction E4 was used for the subsequent assays. The expression and purification of VC1 was carried out several times with similar results. (b) Representative result of the GTP cyclohydrolase II assays measuring the appearance of DARPP, which presents an absorption maximum at 310 nm. The graph shows the increase in absorbance at 310 nm (∆A310) against time for a control (no enzyme) and for an assay with purified VC1.
Extended Data Fig. 7 Co-elution of labeled vicine and convicine with their respective unlabeled forms.
The top panels show two of the chromatograms shown in Fig. 3d for labeled vicine and convicine, respectively. The bottom panels show two chromatograms obtained when analysing a mixture of unlabeled vicine and convicine standards. The panels on the left show the result of multiple reaction monitoring (MRM) for vicine, while chromatograms on the right show the result of MRM for convicine.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Supplementary Data 1
Transcriptome coding sequences in FASTA format.
Supplementary Data 2
Gene expression counts in transcripts per million.
Supplementary Data 3
List of metabolic features, their grouping into clusters and their abundances across tissue samples.
Supplementary Data 4
List of the top 20 genes correlated with vicine accumulation levels in all tissues except mid-maturation embryos.
Supplementary Data 5
R scripts used to analyse gene-to-metabolite correlations.
Supplementary Data 6
VC1 and vc1 cDNA sequences and predicted amino acid sequences.
Supplementary Data 7
Design of the expression constructs used in the study.
Source data
Source Data Fig. 3
Uncropped and unprocessed scan of the agarose gel presented in Fig. 3e,f.
Source Data Extended Data Fig. 4
Uncropped and unprocessed scan of the agarose gel presented in Extended Data Fig. 4.
Source Data Extended Data Fig. 6
Uncropped and unprocessed scan of the polyacrylamide gel presented in Extended Data Fig. 6a.
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Björnsdotter, E., Nadzieja, M., Chang, W. et al. VC1 catalyses a key step in the biosynthesis of vicine in faba bean. Nat. Plants 7, 923–931 (2021). https://doi.org/10.1038/s41477-021-00950-w
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DOI: https://doi.org/10.1038/s41477-021-00950-w
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