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VC1 catalyses a key step in the biosynthesis of vicine in faba bean

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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Fig. 1: Gene expression analysis and metabolite profiling of eight faba bean tissues.
Fig. 2: Identification of VC1.
Fig. 3: Characterization of VC1 as the vc gene.
Fig. 4: Characterization of VC1 as a GTP cyclohydrolase II and establishment of GTP as a precursor for vicine and convicine.

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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.

References

  1. Duc, G. et al. in Handbook of Plant Breeding: Grain Legumes Vol. 10 (ed. De Ron, A.) Ch. 5 (Springer, 2015).

  2. Luzzatto, L. & Arese, P. Favism and glucose-6-phosphate dehydrogenase deficiency. N. Engl. J. Med. 378, 1068–1069 (2018).

    Article  Google Scholar 

  3. IPCC Summary for Policymakers. In Special Report on Climate Change and Land (eds Shukla, P. R. et al.) (IPCC, 2019).

  4. FAOSTAT (Food and Agriculture Organization of the United Nations, accessed 27 February 2020); http://www.fao.org/faostat/en/#home

  5. Feedipedia—Animal Feed Resources Information System (INRA, CIRAD, AFZ and FAO, accessed 27 February 2020); https://www.feedipedia.org

  6. Stoddard, F. L. in Legumes in Cropping Systems (eds Murphy-Bokern, D. et al.) 70–87 (CAB International, 2017).

  7. Meletis, J. & Konstantopoulos, K. Favism—from the ‘avoid fava beans’ of Pythagoras to the present. Haema 7, 17–21 (2004).

    Google Scholar 

  8. Duc, G., Sixdenier, G., Lila, M. & Furstoss, V. Search of genetic variability for vicine and convicine content in Vicia faba L.: a first report of a gene which codes for nearly zero-vicine and zero-convicine contents. In Proc. 1st International Workshop on Antinutritional Factors (ANF) in Legume Seeds (eds Huisman, A. J. et al.) 305–313 (Pudoc, 1989).

  9. Ramsay, G. & Griffiths, D. W. Accumulation of vicine and convicine in Vicia faba and V. narbonensis. Phytochemistry 42, 63–67 (1996).

    Article  CAS  Google Scholar 

  10. Lin, J. Y. et al. Similarity between soybean and Arabidopsis seed methylomes and loss of non-CG methylation does not affect seed development. Proc. Natl Acad. Sci. USA 114, E9730–E9739 (2017).

    Article  CAS  Google Scholar 

  11. Ray, H., Bock, C. & Georges, F. Faba bean: transcriptome analysis from etiolated seedling and developing seed coat of key cultivars for synthesis of proanthocyanidins, phytate, raffinose family oligosaccharides, vicine, and convicine. Plant Genome 8, plantgenome2014.07.0028 (2015).

  12. Khazaei, H. et al. Development and validation of a robust, breeder-friendly molecular marker for the vc locus in faba bean. Mol. Breed. 37, 140 (2017).

    Article  Google Scholar 

  13. Khazaei, H. et al. Flanking SNP markers for vicine–convicine concentration in faba bean (Vicia faba L.). Mol. Breed. 35, 38 (2015).

    Article  Google Scholar 

  14. O’Sullivan, D. M. & Angra, D. Advances in faba bean genetics and genomics. Front. Genet. 7, 150 (2016).

    PubMed  PubMed Central  Google Scholar 

  15. Kereszt, A. et al. Agrobacterium rhizogenes–mediated transformation of soybean to study root biology. Nat. Protoc. 2, 948–952 (2007).

    Article  CAS  Google Scholar 

  16. Reid, D. et al. Cytokinin biosynthesis promotes cortical cell responses during nodule development. Plant Physiol. 175, 361–375 (2017).

    Article  CAS  Google Scholar 

  17. Hiltunen, H.-M., Illarionov, B., Hedtke, B., Fischer, M. & Grimm, B. Arabidopsis RIBA proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis. Int. J. Mol. Sci. 13, 14086–14105 (2012).

    Article  CAS  Google Scholar 

  18. Brown, E. G. & Roberts, F. M. Formation of vicine and convicine by Vicia faba. Phytochemistry 11, 3203–3206 (1972).

    Article  CAS  Google Scholar 

  19. Frelin, O. et al. A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis. Biochem. J. 466, 137–145 (2015).

    Article  CAS  Google Scholar 

  20. Lehmann, M. et al. Biosynthesis of riboflavin: screening for an improved GTP cyclohydrolase II mutant. FEBS J. 276, 4119–4129 (2009).

    Article  CAS  Google Scholar 

  21. Yadav, S. & Karthikeyan, S. Structural and biochemical characterization of GTP cyclohydrolase II from Helicobacter pylori reveals its redox dependent catalytic activity. J. Struct. Biol. 192, 100–115 (2015).

    Article  CAS  Google Scholar 

  22. Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

    Article  CAS  Google Scholar 

  23. Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152 (2012).

    Article  CAS  Google Scholar 

  24. Gilbert, D. Gene-omes built from mRNA-seq not genome DNA. Poster presented at the 7th Annual Arthropod Genomics Symposium in Notre Dame, Indiana (2013).

  25. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013).

  26. Waterhouse, R. M. et al. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol. Biol. Evol. 35, 543–548 (2018).

    Article  CAS  Google Scholar 

  27. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  28. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinform. 12, 323 (2011).

    Article  CAS  Google Scholar 

  29. Tautenhahn, R., Patti, G. J., Rinehart, D. & Siuzdak, G. XCMS Online: a web-based platform to process untargeted metabolomic data. Anal. Chem. 84, 5035–5039 (2012).

    Article  CAS  Google Scholar 

  30. Saeed, A. I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003).

    Article  CAS  Google Scholar 

  31. Li, J., Witten, D. M., Johnstone, I. M. & Tibshirani, R. Normalization, testing, and false discovery rate estimation for RNA-sequencing data. Biostatistics 13, 523–538 (2012).

    Article  Google Scholar 

  32. R Core Team R: A language and environment for statistical computing v.3.4.3 (R Foundation for Statistical Computing, 2018); https://www.R-project.org/

  33. Purves, R. W., Khazaei, H. & Vandenberg, A. Toward a high-throughput method for determining vicine and convicine levels in faba bean seeds using flow injection analysis combined with tandem mass spectrometry. Food Chem. 256, 219–227 (2018).

    Article  CAS  Google Scholar 

  34. Webb, A. et al. A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.). Plant Biotechnol. J. 14, 177–185 (2016).

    Article  CAS  Google Scholar 

  35. Broman, K. W., Wu, H., Sen, S. & Churchill, G. A. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19, 889–890 (2003).

    Article  CAS  Google Scholar 

  36. Chang, W., Jääskeläinen, M., Li, S. P. & Schulman, A. H. BARE retrotransposons are translated and replicated via distinct RNA pools. PLoS ONE 8, e72270 (2013).

    Article  CAS  Google Scholar 

  37. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6, e16765 (2011).

    Article  CAS  Google Scholar 

  38. Nadzieja, M., Stougaard, J. & Reid, D. A toolkit for high resolution imaging of cell division and phytohormone signaling in legume roots and root nodules. Front. Plant Sci. 10, 1000 (2019).

    Article  Google Scholar 

  39. Stougaard, J. Agrobacterium rhizogenes as a vector for transforming higher plants: application in Lotus corniculatus transformation. Methods Mol. Biol. 49, 49–61 (1995).

    CAS  PubMed  Google Scholar 

  40. Armenteros, J. J. A. et al. Detecting sequence signals in targeting peptides using deep learning. Life Sci. Alliance 2, e201900429 (2019).

    Article  Google Scholar 

  41. Ren, J. et al. GTP cyclohydrolase II structure and mechanism. J. Biol. Chem. 280, 36912–36919 (2005).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Alan H. Schulman, Stig U. Andersen or Fernando Geu-Flores.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Vincent Courdavault, John D’Auria and Benjamin Lichman for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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.

Source data

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.

Reporting Summary

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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