Null mutations for genes encoding a major seed storage protein in pea, vicilin, were sought through screening a fast-neutron mutant population. Deletion mutations at four or five vicilin loci, where all vicilin genes within each locus were deleted, were combined to address the question of how removal or reduction of a major storage protein and potential allergen might impact the final concentration of protein per unit of mature seed weight, seed yield, and viability. While the concentration of seed protein was not reduced in mature seeds of mutant lines, indicative of a re-balancing of the proteome, notable differences were apparent in the metabolite, proteomic, and amino acid profiles of the seeds, as well as in some functional properties. Major effects of the deletions on the proteome were documented. The genomic regions which were deleted were defined by whole-genome sequencing of the parental line, JI2822, and its quintuple vicilin null derivative, providing a comprehensive description of each vicilin locus and its genic arrangement. An annotated reference genome has been generated for JI2822, which will serve as a very valuable resource for the research community and support further study of the associated deletion mutant population.

Biology Centre of the Czech Academy of Sciences Institute of Plant Molecular Biology 37005 České Budějovice Czech Republic

Department of Biochemistry and Metabolism John Innes Centre Norwich Research Park Norwich NR4 7UH UK

Department of Computational and Systems Biology John Innes Centre Norwich Research Park Norwich NR4 7UH UK

Department of Crop Genetics John Innes Centre Norwich Research Park Norwich NR4 7UH UK

IDna Genetics Ltd Centrum Norwich Research Park Norwich NR4 7UG UK

Quadram Institute Norwich Research Park Norwich NR4 7UQ UK

Zobrazit více v PubMed

Antonets  KS, Belousov  MV, Sulatskaya  AI, et al.  2020. Accumulation of storage proteins in plant seeds is mediated by amyloid formation. PLoS Biology  18, e3000564. PubMed PMC

AOAC. 1995. Official Method 994.12. Amino acids in feeds. Official Methods of Analysis of AOAC International. Vol. 1, 16th edn.

Barac  M, Cabrilo  S, Pesic  M, Stanojevic  S, Zilic  S, Macej  O, Ristic  N.  2010. Profile and functional properties of seed proteins from six pea ( PubMed PMC

Bäumlein  H, Braun  H, Kakhovskaya  IA, Shutov  AD.  1995. Seed storage proteins of spermatophytes share a common ancestor with desiccation proteins of fungi. Journal of Molecular Evolution  41, 1070–1075. PubMed

Beach  LR, Spencer  D, Randall  PJ, Higgins  TJV.  1985. Transcriptional and post-transcriptional regulation of storage protein gene expression in sulfur-deficient pea seeds. Nucleic Acids Research  13, 999–1013. PubMed PMC

Bourgeois  M, Francoise  J, Savois  V, Sommerer  N, Labas  V, Henry  C, Burstin  J.  2009. Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition. Proteomics  9, 254–271. PubMed

Braun  H, Cziha  A, Shutov  AD, Bäumlein  H.  1996. A vicilin-like seed protein of cycads: similarity to sucrose-binding proteins. Plant Molecular Biology  31, 35–44. PubMed

Campion  B, Perrone  D, Galasso  I, Bollini  R.  2009. Common bean (

Cantalapiedra  CP, Hernandez-Plaza  A, Letunic  I, Bork  P, Huerta-Cepas  J.  2021. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Molecular Biology and Evolution  38, 5825–5829. PubMed PMC

Cartelier  K, Aimé  D, Ly Vu  J, Combes-Soia  L, Labas  V, Jean-Marie Prosperi  J-M, Buitink  J, Gallardo  K, Le Signor  C.  2021. Genetic determinants of seed protein plasticity in response to the environment in PubMed

Casey  R, Domoney  C.  1999. Pea globulins. In: Shewry  PR, Casey  R, eds. Seed proteins. Dordrecht, The Netherlands: Kluwer Academic Publishers, 171–208.

Casey  R, Domoney  C, Forster  C, Hedley  C, Hitchin  E, Wang  T.  1998. The effect of modifying carbohydrate metabolism on seed protein gene expression in peas. Journal of Plant Physiology  152, 636–640.

Castillo  J, Genovés  A, Franco  L, Rodrigo  MI.  2005. A multifunctional bicupin serves as precursor for a chromosomal protein of PubMed

Castillo  J, Rodrigo  MI, Márquez  JA, Zúñiga  A, Franco  L.  2000. A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily. European Journal of Biochemistry  267, 2156–2165. PubMed

Cheng  H, Concepcion  GT, Feng  X, Zhang  H, Li  H.  2021. Haplotype-resolved PubMed PMC

Chinoy  C, Welham  T, Turner  L, Moreau  C, Domoney  C.  2011. The genetic control of seed quality traits: effects of allelic variation at the

Chung  RP-T, Neumann  GM, Polya  GM.  1997. Purification and characterization of basic proteins with

Clemente  A, Arques  MC, Dalmais  M, et al.  2015. Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea. PLoS One  10, e0134634. PubMed PMC

Deutsch  EW, Bandeira  N, Perez-Riverol  Y, et al.  2023. The ProteomeXchange Consortium at 10 years: 2023 update. Nucleic Acids Research  51, D1539–D1548. PubMed PMC

Dodt  G, Braverman  N, Wong  C, Moser  A, Moser  HW, Watkins  P, Valle  D, Gould  SJ.  1995. Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nature Genetics  9, 115–125. PubMed

Domoney  C, Casey  R.  1985. Measurement of gene number for seed storage proteins in PubMed PMC

Domoney  C, Casey  R.  1990. Another class of vicilin gene in PubMed

Domoney  C, Knox  M, Moreau  C, et al.  2013. Exploiting a fast neutron mutant genetic resource in PubMed

Dudchenko  O, Batra  SS, Omer  AD, et al.  2017. PubMed PMC

Dunwell  JM, Khuri  S, Gane  PJ.  2000. Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily. Microbiology and Molecular Biology Reviews  64, 153–179. PubMed PMC

Dunwell  JM, Purvis  A, Khuri  S.  2004. Cupins: the most functionally diverse protein superfamily? Phytochemistry  65, 7–17. PubMed

Ellis  N, Hofer  J, Sizer-Coverdale  E, et al.  2023. Recombinant inbred lines derived from wide crosses in PubMed PMC

Ellis  THN, Domoney  C, Castleton  J, Cleary  W, Davies  DR.  1986. Vicilin genes of

European Commission. 2009. Regulation (EC) No.152/2009. Annex III. Determination of amino acids (except tryptophan). Official Journal of European Union  L54/1, 23–32.

Eyraud  V, Karaki  L, Rahioui  I, Sivignon  C, Da Silva  P, Rahbé  Y, Royer  C, Gressent  F.  2013. Expression and biological activity of the cystine knot bioinsecticide PA1b (pea albumin 1 subunit b). PLoS One  8, e81619. PubMed PMC

Freitas  RL, Teixeira  AR, Ferreira  RB.  2007. Vicilin-type globulins follow distinct patterns of degradation in different species of germinating legume seeds. Food Chemistry  102, 323–329.

Gabriel  L, Bruna  T, Hoff  KJ, Ebel  M, Lomsadze  A, Borodovsky  M, Stanke  M.  2024. BRAKER3: fully automated genome annotation using RNA-seq and protein evidence with GeneMark-ETP, AUGUSTUS and TSEBRA. Genome Research  34, 769–777. PubMed PMC

Gagete  AP, Franco  L, Rodrigo  MI.  2011. The PubMed

Gao  Y, Liu  B, Wang  Y, Xing  Y.  2019. TideHunter: efficient and sensitive tandem repeat detection from noisy long-reads using seed-and-chain. Bioinformatics  35, i200–i207. PubMed PMC

Gell  G, Kovács  K, Veres  G, Korponay-Szabó  IR, Juhász  A.  2017. Characterization of globulin storage proteins of a low prolamin cereal species in relation to celiac disease. Scientific Reports  7, 39876. PubMed PMC

Gomes  VM, Mosqueda  MI, Blanco-Labra  A, Sales  MP, Fernandes  KVS, Cordeiro  RA, Xavier-Filho  J.  2002. Vicilin storage proteins from

Gravel  A, Dubois-Laurin  F, Turgeon  SL, Doyen  A.  2024. The role of the 7S/11S globulin ratio in the gelling properties of mixed β-lactoglobulin/pea proteins systems. Food Hydrocolloids  156, 110273.

Heim  U, Wang  Q, Kurz  T, et al.  2001. Expression patterns and subcelular localization of a 52 kDa sucrose-binding protein homologue of PubMed

Hellens  RP, Moreau  C, Lin-Wang  K, et al.  2010. Identification of Mendel’s white flower character. PLoS One  5, e13230. PubMed PMC

Higashi  Y, Hirai  MY, Fujiwara  T, Naito  S, Noji  M, Saito  K.  2006. Proteomic and transcriptomic analysis of PubMed

Huerta-Cepas  J, Szklarczyk  D, Heller  D, et al.  2019. eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Research  47, D309–D314. PubMed PMC

Hurkman  WJ, Tanaka  CK.  1986. Solubilisation of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiology  81, 802–806. PubMed PMC

Husband  H, Ferreira  S, Bu  F, Feyzi  S, Ismail  BP.  2024. Pea protein globulins: does their relative ratio matter? Food Hydrocolloids  148, 109429.

Kim  D, Paggi  JM, Park  C, Bennett  C, Salzberg  SL.  2019. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nature Biotechnology  37, 907–915. PubMed PMC

Kreplak  J, Madoui  M-A, Cápal  P, et al.  2019. A reference genome for pea provides insight into legume genome evolution. Nature Genetics  51, 1411–1422. PubMed

Krochko  JE, Bewley  D.  2000. Seed storage proteins in cultivars and subspecies of alfalfa (

Laetsch  DR, Blaxter  ML.  2017. BlobTools: interrogation of genome assemblies. F1000Research  6, 1287.

Lamont  BB, He  T, Milne  LA, Cowling  RM.  2024. Out of Africa: linked continents, overland migration and differential survival explain abundance of Proteaceae in Australia. Perspectives in Plant Ecology, Evolution and Systematics  62, 125778.

Lan  T, Xiong  W, Chen  X, Mo  B, Tang  G.  2022. Plant cytoplasmic ribosomal proteins: an update on classification, nomenclature, evolution and resources. The Plant Journal  110, 292–318. PubMed

Le Signor  C, Aimé  D, Bordat  A, et al.  2017. Genome-wide association studies with proteomics data reveal genes important for synthesis, transport and packaging of globulins in legume seeds. New Phytologist  214, 1597–1613. PubMed

Li  H, Durbin  R.  2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics  25, 1754–1760. PubMed PMC

Li  H, Handsaker  B, Wysoker  A, Fennell  T, Ruan  J, Homer  N, Marth  G, Abecasis  G, Durbin  R; 1000 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map (SAM) format and SAMtools. Bioinformatics  25, 2078–2079. PubMed PMC

Liu  N, Lyu  X, Zhang  X, et al.  2024. Reference genome sequence and population genomic analysis of peas provide insights into the genetic basis of Mendelian and other agronomic traits. Nature Genetics  56, 1964–1974. PubMed

Livak  KV, Schmittgen  TD.  2001.  Analysis of relative gene expression data using real-time quantitative PCR and the 2 PubMed

Lopez-Torrejon  G, Salcedo  G, Martin-Esteban  M, Diaz-Perales  A, Pascual  CY, Sanchez-Monge  R.  2003. Len c 1, a major allergen and vicilin from lentil seeds: protein and cDNA cloning. The Journal of Allergy and Clinical Immunology  112, 1208–1215. PubMed

Manni  M, Berkeley  MR, Seppey  M, Simão  FA, Zdobnov  EM.  2021. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Molecular Biology and Evolution  38, 4647–4654. PubMed PMC

Marcus  JP, Green  JL, Goulter  KC, Manners  JM.  1999. A family of antimicrobial peptides is produced by processing of a 7S globulin protein in PubMed

Martinez-Seidel  F, Beine-Golovchuk  O, Hsieh  Y-C, Kopka  J.  2020. Systematic review of plant ribosome heterogeneity and specialization. Frontiers in Plant Science  11, 948. PubMed PMC

Miele  A, Dekker  J.  2009. Mapping cis- and trans-chromatin interaction networks using chromosome conformation capture (3C). Methods in Molecular Biology  464, 105–121. PubMed PMC

Miranda  MRA, Uchôa  AF, Ferreira  SR, Ventury  KE, Costa  EP, Carmo  PRL, Olga  LTM, Fernandes  KVS, Oliveira  AEA.  2020. Chemical modifications of vicilins interfere with chitin-binding affinity and toxicity to PubMed

Moreau  C, Warren  FJ, Rayner  T, Perez-Moral  N, Lawson  DM, Wang  TL, Domoney  C.  2022. An allelic series of starch-branching enzyme mutants in pea ( PubMed

Neumann  P, Novák  P, Hoštáková  N, Macas  J.  2019. Systematic survey of plant LTR-retrotransposons elucidates phylogenetic relationships of their polyprotein domains and provides a reference for element classification. Mobile DNA  10, 1. PubMed PMC

Nguyen  T-P, Cueff  G, Hegedus  DD, Rajjou  L, Bentsink  L.  2015. A role for seed storage proteins in Arabidopsis seed longevity. Journal of Experimental Botany  66, 6399–6413. PubMed PMC

Novák  P, Hoštáková  N, Neumann  P, Macas  J.  2024. DANTE and DANTE_LTR: lineage-centric annotation pipelines for long terminal repeat retrotransposons in plant genomes. NAR Genomics and Bioinformatics  6, 113. PubMed PMC

Olías  R, Rayner  T, Clemente  A, Domoney  C.  2023. Combination of three null mutations affecting seed protein accumulation in pea ( PubMed

Pavlicevic  MZ, Tomic  MD, Djonlagic  JA, Stanojevic  SP, Vucelic Radovic  BV.  2018. Evaluation of variation in protein composition on solubility, emulsifying and gelling properties of soybean genotypes synthesizing β subunit. Journal of the American Oil Chemists Society  95, 123–134.

Perez-Riverol  Y, Bai  J, Bandla  C, et al.  2022. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Research  50, D543–D552. PubMed PMC

Perez-Riverol  Y, Xu  QW, Wang  R, et al.  2016. PRIDE Inspector Toolsuite: moving towards a universal visualization tool for proteomics data standard formats and quality assessment of ProteomeXchange datasets. Molecular & Cellular Proteomics  15, 30517. PubMed PMC

Pertea  M, Pertea  GM, Antonescu  CM, Chang  TC, Mendell  JT, Salzberg  SL.  2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology  33, 290–295. PubMed PMC

Quinlan  AR, Hall  IM.  2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics  26, 841–842. PubMed PMC

Rayner  T, Moreau  C, Ambrose  M, Isaac  PG, Ellis  N, Domoney  C.  2017. Genetic variation controlling wrinkled seed phenotypes in PubMed PMC

Ribeiro  AC, Monteiro  SV, Carrapico  BM, Ferreira  RB.  2014. Are vicilins another major class of legume lectins? Molecules  19, 20350–20373. PubMed PMC

Robinson  KA, St-Jacques  AD, Bakestani  ID, Beavington  BAG, Loewen  MC.  2022. Pea and lentil 7S globulin crystal structures with comparative immunoglobulin epitope mapping. Food Chemistry: Molecular Sciences  5, 100146. PubMed PMC

Rogers  H, Dora  M, Tsolakis  N, Kumar  M.  2024. Plant-based food supply chains: recognising market opportunities and industry challenges of pea protein. Applied Food Research  4, 100440.

Rose  TL, Gomes  VM, Da Cunha  M, Fernandes  KVS, Xavier-Filho  J.  2003. Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells. Biocell  27, 173–179. PubMed

Sales  MP, Gerhardt  IR, Grossi-de-Sa´  MF, Xavier-Filho  J.  2000. Do legume storage proteins play a role in defending seeds against bruchids? Plant Physiology  124, 515–522. PubMed PMC

Sales  MP, Pereira  ST, Gomes  VM, Fernandes  KVS, Pimenta  PPP, Grossi-de-Sá  F, Xavier-Filho  J.  1996. Localization of vicilins (7S) from cowpea (

Salgado  P, Freire  JB, Ferreira  RB, Seabra  M, Teixeira  AR, Toullec  R, Lallès  JP.  2002. Legume proteins of the vicilin family are more immunogenic than those of the legumin family in weaned piglets. Food and Agricultural Immunology  14, 51–63.

Sanchez-Monge  R, Lopez-Torrejon  G, Pascual  CY, Varela  J, Martin-Esteban  M, Salcedo  G.  2004. Vicilin and convicilin are potential major allergens from pea. Clinical and Experimental Allergy  34, 1747–1753. PubMed

Schmidt  MA, Barbazuk  WB, Sandford  M, May  G, Song  Z, Zhou  W, Nikolau  BJ, Herman  EM.  2011. Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome. Plant Physiology  156, 330–345. PubMed PMC

Seabra  M, Carvalho  S, Freire  J, Ferreira  R, Mourato  M, Cunha  L, Cabral  F, Teixeira  A, Aumaitre  A.  2001.

Shewry  PR, Casey  R.  1999. Seed proteins. In: Shewry  PR, Casey  R, eds. Seed proteins. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1–10.

Shimada  T, Fuji  K, Tamura  K, Kondo  M, Nishimura  M, Hara-Nishimura  I.  2003. Vacuolar sorting receptor for seed storage proteins in PubMed PMC

Shutov  AD, Bäumlein  H, Blattner  FR, Müntz  K.  2003. Storage and mobilization as antagonistic functional constraints on seed storage globulin evolution. Journal of Experimental Botany  54, 1645–1654. PubMed

Shutov  AD, Braun  H, Chesnokov  YV, Bäumlein  H.  1998. A gene encoding a vicilin-like protein is specifically expressed in fern spores. European Journal of Biochemistry  252, 79–89. PubMed

Smit  AFA, Hubley  R, Green  P.  2013–2015. RepeatMasker Open-4.0. http://www.repeatmasker.org

Tabe  L, Hagan  N, Higgins  TJV.  2002. Plasticity of seed protein composition in response to nitrogen and sulfur availability. Current Opinion in Plant Biology  5, 212–217. PubMed

Tayeh  N, Hofer  JMI, Aubert  G, et al.  2024. PubMed

Taylor  M, Chapman  R, Beyaert  R, Hernandez-Sebastia  C, Marsolais  F.  2008. Seed storage protein deficiency improves sulfur amino acid content in common bean ( PubMed

Taylor  SL, Marsh  JT, Koppelman  SJ, Kabourek  JL, Johnson  PE, Baumert  JL.  2021. A perspective on pea allergy and pea allergens. Trends in Food Science &. Technology  116, 186–198.

Troya  M, Kurawadwala  H, Byrne  B, Dowdy  R, Weston  Z.  2021. Plant-based meat: anticipating 2030 production requirements. GFI (Good Food Institute) Publication. https://gfi.org/wp-content/uploads/2021/11/Plant-based-meat_-anticipating-2030-production-requirements.pdf

van Berkum  NL, Lieberman-Aiden  E, Williams  L, Imakaev  M, Gnirke  A, Mirny  LA, Dekker  J, Lander  ES.  2010. Hi-C: a method to study the three-dimensional architecture of genomes. Journal of Visualized Experiments  6, 1869. PubMed PMC

van Zanten  M, Carles  A, Li  Y, Soppe  WJ.  2012. Control and consequences of chromatin compaction during seed maturation in PubMed PMC

Vigeolas  H, Chinoy  C, Zuther  E, Blessington  B, Geigenberger  P, Domoney  C.  2008. Combined metabolomic and genetic approaches reveal a link between the polyamine pathway and albumin 2 in developing pea seeds. Plant Physiology  146, 74–82. PubMed PMC

Wang  Z, Luo  Y, Li  X, et al.  2008. Genetic control of floral zygomorphy in pea ( PubMed PMC

Xiong  W, Lan  T, Mo  B.  2021. Extraribosomal functions of cytosolic ribosomal proteins in plants. Frontiers in Plant Science  12, 607157. PubMed PMC

Yamada  K, Shimada  T, Kondo  M, Nishimura  M, Hara-Nishimura  I.  1999. Multiple functional proteins are produced by cleaving Asn–Gln bonds of a single precursor by vacuolar processing enzyme. Journal of Biological Chemistry  274, 2563–2570. PubMed

Yan  H, Chen  D, Wang  Y, Sun  Y, Zhao  J, Sun  M, Peng  X.  2016. Ribosomal protein L18aB is required for both male gametophyte function and embryo development in Arabidopsis. Scientific Reports  6, 31195. PubMed PMC

Yang  T, Liu  R, Luo  Y, et al.  2022. Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics. Nature Genetics  54, 1553–1563. PubMed PMC

Young  J, Johnston  A, Brewin  N.  1982. A search for peas (

Zhang  Y, Park  C, Bennett  C, Thornton  M, Kim  D.  2021. Rapid and accurate alignment of nucleotide conversion sequencing reads with HISAT-3N. Genome Research  31, 1290–1295. PubMed PMC

Zhang  Z, Schwartz  S, Wagner  L, Miller  W.  2000. A greedy algorithm for aligning DNA sequences. Journal of Computational Biology  7, 203–214. PubMed

Zinsmeister  J, Lalanne  D, Terrasson  E, et al.  2016. ABI5 is a regulator of seed maturation and longevity in legumes. The Plant Cell  28, 2735–2754. PubMed PMC