Abstract
Genetic variation at the Tri locus, controlling seed trypsin inhibitor activity, is relevant to both food and feed uses of Pisum sativum L. (pea). Near-isogenic lines of Pisum sativum L. (pea) were developed previously to examine the impact on digestibility of variation at Tri on linkage group V. Further studies of these lines have now revealed a significant difference in seed nitrogen concentration between near-isolines having contrasting seed trypsin inhibitor activity. In order to investigate this apparent association, the multiple genes at a closely linked locus, Vc-2, encoding a set of major vicilin polypeptides, were analysed and shown to differ between the near-isolines. Characterisation of Vc-2 cDNAs revealed distinguishing features of the functional genes between the parents of the near-isolines, while analyses of gene structure showed that a disrupted variant Vc-2 gene was present in one, but not the second, parent of the near-isolines. The variant gene appeared to be non-functional, based both on its deduced truncated protein lacking part of one conserved cupin structural domain, and the fact that it did not correspond to any isolated cDNA. Recombinant near-isolines were generated between the closely linked Tri and Vc-2 loci to investigate the genetic association with seed nitrogen concentration. Seeds from near-isolines and recombinant inbred lines where the variant Vc-2 gene was present had lower seed nitrogen concentration than lines lacking the variant gene. Furthermore, the disrupted Vc-2 gene was absent from several pea genotypes with high seed protein content. Expression analyses suggested that gene expression at the Vc-2 locus was higher when the non-functional gene variant was absent. Markers based either on the element which disrupts the coding sequence within the variant gene at the Vc-2 locus, or on the closely linked Tri locus, may be exploited in the selection of haplotypes associated with genetic variation in seed protein composition and concentration. The gene content in the genomic region of Medicago truncatula chromosome 7 that is syntenic with the pea linkage group V has identified further candidates for functional analyses and marker assisted selection.
Similar content being viewed by others
References
Bown D, Levasseur M, Croy RRD, Boulter D, Gatehouse JA (1985) Sequence of a pseudogene in the legumin gene family of pea (Pisum sativum L.). Nucleic Acid Res 13:4527–4538
Burstin J, Marget P, Huart M, Moessner A, Mangin B, Duchene C, Desprez B, Munier-Jolain N, Duc G (2007) Developmental genes have pleiotropic effects on plant morphology and source capacity, eventually impacting on seed protein content and productivity in pea. Plant Physiol 144:768–781
Casey R, Domoney C (1999) Pea globulins. In: Shewry PR, Casey R (eds) Seed proteins. Kluwer Academic Publishers, The Netherlands, pp 171–208
Clemente A, Moreno FJ, Marín-Manzano MC, Jiménez E, Domoney C (2010) The cytotoxic effect of Bowman-Birk isoinhibitors from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases. Mol Nutr Food Res 54:396–405
Cooper LD, Doss RP, Price R, Peterson K, Oliver JE (2005) Application of Bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin. J Exp Bot 56:1229–1237
Crévieu I, Carré B, Chagneau AM, Quillien L, Guéguen J, Bérot S (1997) Identification of resistant pea (Pisum sativum L.) proteins in the digestive tract of chickens. J Agric Food Chem 45:1295–1300
Domoney C, Casey R (1985) Measurement of gene number for seed storage proteins in Pisum. Nucleic Acids Res 13:687–699
Domoney C, Casey R (1990) Another class of vicilin gene in Pisum. Planta 182:39–42
Domoney C, Welham T, Sidebottom C (1993) Purification and characterization of Pisum seed trypsin inhibitors. J Exp Bot 44:701–709
Domoney C, Welham T, Ellis N, Hellens R (1994) Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum. Theor Appl Genet 89:387–391
Domoney C, Welham T, Ellis N, Mozzanega P, Turner L (2002) Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants. Plant Mol Biol 48:319–329
Ellis THN (1993) The nuclear genome. In: Casey R, Davies DR (eds) Peas: genetics, molecular biology and biotechnology. CAB International, London, pp 13–47
Ellis THN, Domoney C, Castleton J, Cleary W, Davies DR (1986) Vicilin genes of Pisum. Mol Gen Genet 205:164–169
Forster C, North H, Afzal N, Domoney C, Hornostaj A, Robinson DS, Casey R (1999) Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2. Plant Mol Biol 39:1209–1220
Hedemann MS, Welham T, Boisen S, Canibe N, Bilham L, Domoney C (1999) Studies on the biological responses of rats to seed trypsin inhibitors using near-isogenic lines of Pisum sativum L. (pea). J Sci Food Agric 79:1647–1653
Hofer J, Turner L, Moreau C, Ambrose M, Isaac P, Butcher S, Weller J, Dupin A, Dalmais M, Le Signor C, Bendahmane A, Ellis N (2009) Tendril-less regulates tendril formation in pea leaves. Plant Cell 21:420–428
Irzykowska L, Wolko B (2004) Interval mapping of QTLs controlling yield-related traits and seed protein content in Pisum sativum. J Appl Genet 45:297–306
Kaló P, Seres A, Taylor SA, Jakab J, Kevei Z, Kereszt A, Endre G, Ellis THN, Kiss GB (2004) Comparative mapping between Medicago sativa and Pisum sativum. Mol Gen Genomics 272:235–246
Khuri S, Bakker FT, Dunwell JM (2001) Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins. Mol Biol Evol 18:593–605
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Le Gall M, Quillien L, Sève B, Guéguen J, Lallès JP (2007) Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and albumin pea albumin 2. J Anim Sci 85:2972–2981
Marcus JP, Goulter KC, Manners JM (2008) Peptide fragments from plant vicilins expressed in Escherichia coli exhibit antimicrobial activity in vitro. Plant Mol Biol Rep 26:75–87
Mossé J (1990) Nitrogen to protein conversion factor for ten cereals and six legumes or oilseeds. A reappraisal of its definition and determination. Variation according to species and to seed protein content. J Agric Food Chem 38:18–24
Muel F, Carrouée B, Grosjean F (1998) Trypsin inhibitor activity of pea cultivars: new data and a proposal strategy for breeding programmes. In: AEP (Association Européenne de recherche sur les Protéagineux) (ed) Proceedings of the Third European Conference on Grain Legumes (Valladolid, Spain), AEP, Paris, pp 164–165
Page D, Aubert G, Duc G, Welham T, Domoney C (2002) Combinatorial variation in coding and promoter sequences of genes at the Tri locus in Pisum sativum accounts for variation in trypsin inhibitor activity in seeds. Mol Genet Genomics 267:359–369
Page D, Duc G, Lejeune-Hénaut I, Domoney C (2003) Marker-assisted selection of genetic variants for seed trypsin inhibitor content in peas. Pisum Genet 35:19–21
Poerio E, Carrano L, Garzillo AM, Buonocore V (1989) A trypsin inhibitor from the water-soluble protein fraction of wheat kernel. Phytochem 28:1307–1311
Richardson M (1991) Seed storage proteins: the enzyme inhibitors. Methods Plant Biochem 5:259–305
Riechman JL, Ratcliffe OJ (2000) A genomic perspective on plant transcription factors. Curr Opin Plant Biol 3:423–434
Rolletschek H, Borisjuk L, Radchuk R, Miranda M, Heim U, Wobus U, Weber H (2004) Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy. Plant Biotech J 2:211–219
Rolletschek H, Nguyen TH, Häusler RE, Rutten T, Göbel C, Feussner I, Radchuk R, Tewes A, Claus B, Klukas C, Linemann U, Weber H, Wobus U, Borisjuk L (2007) Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage. Plant J 51:468–484
Salgado P, Freire JPB, Ferreira RB, Teixera A, Bento O, Abreu MC, Toullec R, Lallès JP (2003) Immunodetection of legume proteins resistant to small intestinal digestion in weaned piglets. J Sci Food Agric 83:1571–1580
Sanders A, Collier R, Trethewy A, Gould G, Sieker R, Tegeder M (2009) AAP1 regulates import of amino acids into developing Arabidopsis embryos. Plant J 59:540–552
Tar’an B, Warkentin T, Somers DJ, Miranda D, Vandenberg A, Blade S, Bing D (2004) Identification of quantitative trait loci for grain yield, seed protein concentration and maturity in field pea (Pisum sativum L.). Euphytica 136:297–306
Tsubokura Y, Hajika M, Harada K (2006) Molecular characterization of a β-conglycinin deficient soybean. Euphytica 150:249–255
Turner SR, Barratt DHP, Casey R (1990) The effect of different alleles at the r locus on the synthesis of seed storage proteins in Pisum sativum. Plant Mol Biol 14:793–803
Verdier J, Kakar K, Gallardo K, Le Signor C, Aubert G, Schlereth A, Town CD, Udvardi MK, Thompson RD (2008) Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling. Plant Mol Biol 67:567–580
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 Physiol 146:74–82
Wang TL, Hedley CL (1993) Genetic and developmental analysis of the seed. In: Casey R, Davies DR (eds) Peas: genetics, molecular biology and biotechnology. CAB International, London, pp 83–120
Weber H, Rolletschek H, Heim U, Golombek S, Gubatz S, Wobus U (2000) Antisense-inhibition of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation. Plant J 24:33–43
Weigelt K, Küster H, Radchuk R, Müller M, Weichert H, Fait A, Fernie AR, Saalbach I, Weber H (2008) Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism. Plant J 55:909–926
Weigelt K, Küster H, Rutten T, Fait A, Fernie AR, Miersch O, Wasternack C, Emery RJN, Desel C, Hosein F, Müller M, Saalbach I, Weber H (2009) ADP-glucose pyrophosphorylase-deficient pea embryos reveal specific transcriptional and metabolic changes of carbon-nitrogen metabolism and stress responses. Plant Physiol 149:395–411
Welham T, Domoney C (2000) Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement. Plant Sci 159:289–299
Welham T, O’Neill M, Johnson S, Wang T, Domoney C (1998) Expression patterns of genes encoding seed trypsin inhibitors in Pisum sativum. Plant Sci 131:13–24
Wiseman J, Al-Mazooqi W, Welham T, Domoney C (2003) The apparent ileal digestibility, determined with young broilers, of amino acids in near-isogenic lines of peas (Pisum sativum L.) differing in trypsin inhibitor activity. J Sci Food Agric 83:644–651
Wiseman J, Al-Marzooqi W, Hedley C, Wang TL, Welham T, Domoney C (2006) The effects of genetic variation at r, rb and Tri loci in Pisum sativum L. on apparent ileal digestibility of amino acids in young broilers. J Sci Food Agric 86:436–444
Zhukov VA, Kuznetsova EV, Ovchinnikova ES, Rychagova TS, Titov VS, Pinaev AG, Borisov AY, Moffet M, Domoney C, Ellis THN, Ratet P, Weeden NF, Tikhonovich IA (2007) Gene-based markers of pea linkage group V for mapping genes related to symbioses. Pisum Genet 39:19–25
Acknowledgments
We acknowledge financial support for this work from the Department for Environment Food and Rural Affairs (Defra) projects AR0105 and AR0711, the Pulse Crop Genetic Improvement Network, UK. The JIC is supported by competitive grants from the Biotechnology and Biological Sciences Research Council (BBSRC), and from Defra. We are very grateful to Dr. Hans Weber, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben (Germany), and to Sigurd Boisen, Danish Institute of Agricultural Sciences, Department of Animal Nutrition and Physiology Research, Centre Foulum, P.O. Box 39 DK-8830 Tjele Denmark for carrying out N analyses of seed meals in their laboratories. We thank Jevneet Kular and Dominic Conquest, John Innes Centre, for their assistance with seed harvest, meal preparation and assays.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
10681_2011_363_MOESM3_ESM.ppt
Fig. S1: Seed phenotypes of near-isolines of pea, compared with those of the parent lines, JI 516 and JI 868. L and H refer to lines within a pair that have different alleles at the Tri locus (PPT 4806 kb)
10681_2011_363_MOESM4_ESM.ppt
Fig. S2: Mean seed weight of near-isolines of pea, based on triplicate measurements, ± SE. L and H refer to lines within a pair that have different alleles at the Tri locus. The mean seed weight of the parent lines, JI 516 and JI 868 were 0.391 and 0.355 g, respectively (PPT 67 kb)
10681_2011_363_MOESM5_ESM.doc
Fig. S3: Protein sequences deduced from a Vc-2 cDNA, vicK (X67429, top), and vicJ gene (X67428, lower) DNA sequences. The sequence shown in red for the deduced vicK protein is missing from that deduced for vicJ, with the latter showing divergence after amino acid 354 and a premature stop codon (*) after a further 14 amino acids (in red, underlined). Additional amino acid differences between the two sequences are coloured blue (DOC 29 kb)
10681_2011_363_MOESM6_ESM.ppt
Fig. S4: Seed nitrogen content determined for a range of Pisum round and wrinkled-seeded lines that are parents of mapping populations, based on triplicate measurements, ± SE. White bars indicate round-seeded lines (R), whereas black bars indicate wrinkled-seeded (r) lines (PPT 117 kb)
Rights and permissions
About this article
Cite this article
Chinoy, C., Welham, T., Turner, L. et al. The genetic control of seed quality traits: effects of allelic variation at the Tri and Vc-2 genetic loci in Pisum sativum L.. Euphytica 180, 107–122 (2011). https://doi.org/10.1007/s10681-011-0363-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10681-011-0363-8