Plant Molecular Biology Reporter

, Volume 32, Issue 5, pp 957–970 | Cite as

Seed-Specific Expression of AINTEGUMENTA in Medicago truncatula Led to the Production of Larger Seeds and Improved Seed Germination

  • Massimo Confalonieri
  • Maria Carelli
  • Valentina Galimberti
  • Anca Macovei
  • Francesco Panara
  • Marco Biggiogera
  • Carla Scotti
  • Ornella Calderini
Original Paper


The increase of seed size is of great interest in Medicago spp., to improve germination, seedling vigour and, consequently, early forage yield as well as for optimizing seeding techniques and post-seeding management. This study evaluated the effects of the ectopic expression of the AINTEGUMENTA (ANT) cDNA from Arabidopsis thaliana, under the control of the seed-specific USP promoter from Vicia faba, on seed size, germination and seedling growth in barrel medic (Medicago truncatula Gaertn.). All the transgenic T2 barrel medic lines expressing ANT produced seeds significantly larger than those of control plants. Microscopic analysis on transgenic T3 mature seeds revealed that cotyledon storage parenchyma cells were significantly larger and contained larger storage vacuoles than those of the untransformed control. Moreover, the percentage of germination was significantly higher and germination was more rapid in transgenic than in control seeds. Our results indicate that the seed-specific expression of ANT in barrel medic led to larger seeds and improved seed germination, and revealed a regulatory role for ANT in controlling seed size development.


AINTEGUMENTA Cell expansion Medicago truncatula Seed germination Seed size 



The authors thank Dr. Stefania Barzaghi for the seed image analysis and Dr. Luciano Pecetti for the critical reading of the manuscript. We are grateful to Francesco Lascala, Massimo Sari and Annalisa Seminari (CRA-FLC, Lodi) and Giancarlo Carpinelli and Marco Guaragno (CNR-IGV, Perugia) for the excellent technical assistance. The scientific support of Dr. Efisio Piano (CRA-FLC, Lodi) and Dr. Sergio Arcioni (CNR-IGV, Perugia) during the completion of the project is greatly acknowledged. The research was supported by funds from “Programma di ricerca speciale: Incremento della Produzione di Proteine Vegetali per l’Alimentazione Zootecnica (legge 49/2001)”.

Supplementary material

11105_2014_706_MOESM1_ESM.doc (37 kb)
ESM 1 (DOC 37.0 kb)
11105_2014_706_MOESM2_ESM.doc (37 kb)
ESM 2 (DOC 37.0 kb)
11105_2014_706_MOESM3_ESM.doc (35 kb)
ESM 3 (DOC 35.0 kb)
11105_2014_706_MOESM4_ESM.doc (39 kb)
ESM 4 (DOC 39.0 kb)
11105_2014_706_MOESM5_ESM.doc (39 kb)
ESM 5 (DOC 39.0 kb)


  1. Bäumlein H, Boerjan W, Nagy I, Bassüner R, Van Montagu M, Inzé D, Wobus U (1991) A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants. Mol Gen Genet 225:459–467PubMedCrossRefGoogle Scholar
  2. Beaulieu JM, Moles AT, Leitch IJ, Bennett MD, Dickie JB, Knight CA (2007) Correlated evolution of genome size and seed mass. New Phytol 173:422–437PubMedCrossRefGoogle Scholar
  3. Beveridge JL, Wilsie CP (1959) Influence of depth of planting, seed size, and variety on emergence and seedling vigor in alfalfa. Agron J 51:731–734CrossRefGoogle Scholar
  4. Bingham E, Armour D, Irwin J, Jayaraman D, Ané JM (2009) Report on progress hybridizing herbaceous Medicago sativa and woody M. arborea. Medicago Genetic Reports. Accessed 17 Feb 2014
  5. Bögre L, Magyar Z, López-Juez E (2008) New clues to organ size control in plants. Genome Biol 9:226PubMedCentralPubMedCrossRefGoogle Scholar
  6. Borrás L, Otegui ME (2001) Maize kernel weight response to post-flowering source-sink ratio. Crop Sci 49:1816–1822CrossRefGoogle Scholar
  7. Butaye KMJ, Cammue BPA, Delaure SL, De Bolle MFC (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91CrossRefGoogle Scholar
  8. Carleton AE, Cooper CS (1972) Seed size effects upon seedling vigor of three forage legumes. Crop Sci 2:183–186CrossRefGoogle Scholar
  9. Clough RC, Casal JJ, Jordan ET, Christou P, Vierstra RD (1995) Expression of functional oat phytochrome A in transgenic rice. Plant Physiol 109:1039–1045PubMedCentralPubMedCrossRefGoogle Scholar
  10. Confalonieri M, Cammareri M, Biazzi E, Pecchia P, Fevereiro P, Balestrazzi A, Tava A, Conicella C (2009) Enhanced triterpene sapogenin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel ß–amyrin synthase (AsOXA1) gene. Plant Biotechnol J 7:172–182PubMedCrossRefGoogle Scholar
  11. Cook DR (1999) Medicago truncatula—a model in the making! Curr Opin Plant Biol 2:301–304PubMedCrossRefGoogle Scholar
  12. Cooper CS, Ditterline RL, Welty LE (1979) Seed size and seeding rate effects upon stand density and yield of alfalfa. Agron J 71:83–85CrossRefGoogle Scholar
  13. Cosson V, Durand P, d'Erfurth I, Kondorosi A, Ratet P (2006) Medicago truncatula transformation using leaf explants. Methods Mol Biol 343:115–127PubMedGoogle Scholar
  14. Djemel N, Guedon D, Lechevalier A, Salon C, Miquel M, Prosperi J-M, Rochat C, Boutin J-P (2005) Development and composition of the seeds of nine genotypes of the Medicago truncatula species complex. Plant Physiol Biochem 43:557–566PubMedCrossRefGoogle Scholar
  15. Dutta SK, Nema VK, Bhardwaj RK (1972) Physical properties of gram. J Agric Eng Res 12:128–137Google Scholar
  16. Easton LC, Kleindorfer S (2008) Germination in two Australian species of Frankenia L., F. serpyllifolia Lindl. and F. foliosa J. Black (Frankeniaceae). Effects of seed mass, seed age, light, and temperature. Trans R Soc S Aust 132:29–40Google Scholar
  17. Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WJ, Gerentes D, Perez P, Smyth DR (1996) AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8:155–168PubMedCentralPubMedCrossRefGoogle Scholar
  18. Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M (2005) Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res 14:703–712PubMedCrossRefGoogle Scholar
  19. Firnhaber C, Pühler A, Küster H (2005) EST sequencing and time course microarray hybridizations identify more than 700 Medicago truncatula genes with developmental expression regulation in flowers and pods. Planta 222:269–283PubMedCrossRefGoogle Scholar
  20. Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove JD (2002) Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth. Physiol Plant 116:238–247PubMedCrossRefGoogle Scholar
  21. Gallardo K, Lesignor C, Darmency M, Burstin J, Thompson R, Rochat C, Boutin J-P, Kuester H, Buitink J, Leprince O, Limami A, Grusak MA (2006) Seed biology of Medicago truncatula. In: Mathesius U, Journet EP, Sumner LW (eds) The Medicago truncatula handbook. Accessed 17 Feb 2014
  22. Garciarrubio A, Legaria JP, Covarrubias A (1997) Abscisic acid inhibits germination of mature Arabidopsis seeds by limiting the availability of energy and nutrients. Planta 203:182–187PubMedCrossRefGoogle Scholar
  23. Gardarin A, Dürr C, Colbach N (2011) Prediction of germination rates of weed species: relationships between germination speed parameters and species traits. Ecol Model 222:626–636CrossRefGoogle Scholar
  24. Gjuric R, Smith SR (1997) Inheritance in seed size of alfalfa: quantitative analysis and response to selection. Plant Breed 116:337–340CrossRefGoogle Scholar
  25. Glevarec G, Bouton S, Jaspard E, Riou M-T, Cliquet J-B, Suzuki A, Limami AM (2004) Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula. Planta 219:286–297PubMedCrossRefGoogle Scholar
  26. Grotkopp E, Rejmanek M, Sanderson MJ, Rost TL (2004) Evolution of genome size in Pines (Pinus) and its life-history correlates: supertree analyses. Evolution 58:1705–1729PubMedCrossRefGoogle Scholar
  27. Haas TJ, Thomas EM (2004) Survey of seeds per gram of individual alfalfa plants. Medicago Genetic Reports. Accessed 17 Feb 2014
  28. Hansen B (1989) Determination of nitrogen as elementary N, an alternative to Kjeldhal. Acta Agric Scand 39:113–118CrossRefGoogle Scholar
  29. Harper JL, Lovell PH, Moore KG (1970) The shapes and sizes of seeds. Annu Rev Ecol Syst 1:327–356CrossRefGoogle Scholar
  30. Hojjat SS (2011) Effects of seed size on germination and seedling growth of some Lentil genotypes (Lens culinaris Medik.). Int J Agric Crop Sci 3:1–5Google Scholar
  31. Jack T, Fox GL, Meyerowitz EM (1994) Arabidopsis homeotic gene APETALA3 ectopic expression: transcriptional and posttranscriptional regulation determine floral organ identity. Cell 76:703–716PubMedCrossRefGoogle Scholar
  32. Johnson K, Lenhard M (2011) Genetic control of plant organ growth. New Phytol 191:319–333PubMedCrossRefGoogle Scholar
  33. Jurado E, Westoby M (1992) Germination biology of selected central Australian plants. Aust J Ecol 17:341–348CrossRefGoogle Scholar
  34. Kiniry JR, Wood CA, Spanel DA, Bockholt AJ (1990) Seed weight response to decreased seed number in maize. Agron J 54:98–102CrossRefGoogle Scholar
  35. Klucher KM, Chow H, Reiser L, Fischer RL (1996) The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. Plant Cell 8:137–153PubMedCentralPubMedCrossRefGoogle Scholar
  36. Knight CA, Ackerly DD (2002) Variation in nuclear DNA content across environmental gradients: a quantile regression analysis. Ecol Lett 5:66–76CrossRefGoogle Scholar
  37. Knight CA, Molinari NA, Petrov DA (2005) The large genome constraint hypothesis: evolution, ecology, and phenotype. Ann Bot 95:177–190PubMedCrossRefGoogle Scholar
  38. Koelewijn HP, Van Damme JMM (2005) Effects of seed size, inbreeding and maternal sex on the offspring performance of gynodioecious Plantago coronopus. J Ecol 93:373–383CrossRefGoogle Scholar
  39. Krannitz PG, Aarssen LW, Dow JM (1991) The effect of genetically based differences in seed size on seedling survival in Arabidopsis thaliana (Brassicaceae). Am J Bot 78:446–450CrossRefGoogle Scholar
  40. Krizek BA (1999) Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs. Dev Genet 25:224–236PubMedCrossRefGoogle Scholar
  41. Krizek BA, Eaddy M (2012) AINTEGUMENTA-LIKE6 regulates cellular differentiation in flowers. Plant Mol Biol 78:199–209PubMedCrossRefGoogle Scholar
  42. Kuluev BR, Knyazev AV, Iljassowa AA, Chemeris AV (2012) Ectopic expression of the PnANTL1 and PnANTL2 black poplar genes in transgenic tobacco plants. Russ J Genet 48(10):993–1000CrossRefGoogle Scholar
  43. Le BH, Wagmaister JA, Kawashima T, Bui AQ, Harada JJ, Goldberg RB (2007) Using genomics to study legume seed development. Plant Physiol 144:562–574PubMedCentralPubMedCrossRefGoogle Scholar
  44. Leishman MR (2001) Does the seed size⁄number trade-off model determine plant community structure? An assessment of the model mechanisms and their generality. Oikos 93:294–302CrossRefGoogle Scholar
  45. Leishman MR, Wright IJ, Moles AT, Westoby M (2000) The evolutionary ecology of seed size. In: Fenner M (ed) Seeds—the ecology of regeneration in plant communities. CAB International, Wallingford, pp 31–57CrossRefGoogle Scholar
  46. Linkies A, Graeber K, Knight C, Leubner-Metzger G (2010) The evolution of seeds. New Phytol 186:817–831PubMedCrossRefGoogle Scholar
  47. Mason G, Provero P, Vaira AM, Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2:1–10CrossRefGoogle Scholar
  48. Mizukami Y, Fischer RL (2000) Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proc Natl Acad Sci U S A 97:942–947PubMedCentralPubMedCrossRefGoogle Scholar
  49. Nole-Wilson S, Tranby TL, Krizek BA (2005) AINTEGUMENTA-like (AIL) genes are expressed in young tissues and may specify meristematic or division-competent states. Plant Mol Biol 57:613–628PubMedCrossRefGoogle Scholar
  50. Orsi CH, Tanksley SD (2009) Natural variation in an ABC transporter gene associated with seed size evolution in tomato species. PLoS Genet. doi:  10.1371/journal.pgen.1000347
  51. Pay A, Heberle-Bors E, Hirt H (1992) An alfalfa cDNA encodes a protein with homology to translationally controlled human tumor protein. Plant Mol Biol 19:501–503PubMedCrossRefGoogle Scholar
  52. Porceddu A, Panara F, Calderini O, Molinari L, Taviani P, Lanfaloni L, Scotti C, Carelli M, Scaramelli L, Bruschi G, Cosson V, Ratet P, de Larambergue H, Duc G, Piano E, Arcioni S (2008) An Italian functional genomic resource for Medicago truncatula. BMC Res Notes 1:129PubMedCentralPubMedCrossRefGoogle Scholar
  53. Ramakers C, Ruijter JM, Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66PubMedCrossRefGoogle Scholar
  54. Rao SK (1981) Influence of seed size on field germination, seedling vigour, yield and quality in self pollinated crops—a review. Agric Rev 2:95–101Google Scholar
  55. Rogers C, Wen J, Chen R, Oldroyd G (2009) Deletion-based reverse genetics in Medicago truncatula. Plant Physiol 151:1077–1086PubMedCentralPubMedCrossRefGoogle Scholar
  56. Rotili P, Gnocchi G, Scotti C, Zannone L (1999) Some aspects of breeding methodology in alfalfa. In: Proceedings of The Alfalfa Genome Conference, Madison, WI, USA, Accessed 17 Feb 2014
  57. Saalbach I, Giersberg M, Conrad U (2001) High-level expression of a single chain Fv fragment (scFv) antibody in transgenic pea seeds. J Plant Physiol 158:529–533CrossRefGoogle Scholar
  58. Scaramelli L, Balestrazzi A, Bonadei M, Piano E, Carbonera D, Confalonieri M (2009) Production of transgenic barrel medic (Medicago truncatula Gaertn.) using the ipt-type MAT vector system and impairment of recombinase-mediated excision events. Plant Cell Rep 2:197–211CrossRefGoogle Scholar
  59. Scheller J, Leps M, Conrad U (2006) Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol J 4:243–249PubMedCrossRefGoogle Scholar
  60. Schruff MC, Spielman M, Tiwari S, Adams S, Fenby N, Scott RJ (2006) The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs. Development 133:251–261PubMedCrossRefGoogle Scholar
  61. Scotti C, Gnocchi G (2004) Seed size and fertility relationships of WI643 alfalfa grown at Lodi, Italy. Medicago Genetic Reports. Accessed 17 Feb 2014
  62. Simons AM, Johnston MO (2000) Variation in seed traits of Lobelia inflata (Campanulaceae): sources and fitness consequences. Am J Bot 87:124–132PubMedCrossRefGoogle Scholar
  63. Singer SD, Liu Z, Cox KD (2012) Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators. Plant Cell Rep 31:13–25PubMedCrossRefGoogle Scholar
  64. Sun X, Shantharaj D, Kang X, Ni M (2010) Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr Opin Plant Biol 13:611–620PubMedCrossRefGoogle Scholar
  65. Tadege M, Wen J, He J, Tu H, Kwak Y, Eschstruth A, Cayrel A, Endre G, Zhao PX, Chabaud M, Ratet P, Mysore KS (2008) Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J 54:335–347PubMedCrossRefGoogle Scholar
  66. Tanska M, Konopka M, Rotkiewicz D (2008) Relationships of rapeseed strength properties to seed size, colour and coat fibre composition. J Sci Food Agric 88:2186–2193CrossRefGoogle Scholar
  67. Tan-Wilson AL, Wilson KA (2012) Mobilization of seed protein reserves. Physiol Plant 145:140–153PubMedCrossRefGoogle Scholar
  68. Thompson R, Burstin J, Gallardo K (2009) Post-genomics studies of developmental processes in legume seeds. Plant Physiol 151:1023–1029PubMedCentralPubMedCrossRefGoogle Scholar
  69. Trinh TH, Ratet P, Kondorosi E, Durand P, Kamaté K, Bauer P, Kondorosi A (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355CrossRefGoogle Scholar
  70. Van Daele I, Gonzalez N, Vercauteren I, de Smet L, Inzé D, Roldán-Ruiz I, Vuylsteke M (2012) A comparative study of seed yield parameters in Arabidopsis thaliana mutants and transgenics. Plant Biotechnol J 10:488–500PubMedCrossRefGoogle Scholar
  71. Van Son L, Tiedemann J, Rutten T, Hillmer S, Hinz G, Zank T, Manteuffel R, Bäumlein H (2009) The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development. Plant Mol Biol 71:319–329PubMedCrossRefGoogle Scholar
  72. Venable DL (1992) Size-number trade-offs and the variation of seed size with plant resource status. Am Nat 140:287–304CrossRefGoogle Scholar
  73. Westoby M, Falster DS, Moles AT, Vesk PA, Wright IJ (2002) Plant ecological strategies: some leading dimensions of variation between species. Annu Rev Ecol Syst 33:125–159CrossRefGoogle Scholar
  74. Wu GL, Du GZ (2007) Germination is related to seed mass in grasses (Poaceae) of the eastern Qinghai-Tibetan Plateau, China. Nord J Bot 25:361–365CrossRefGoogle Scholar
  75. Yi CX, Zhang J, Chan KM, Liu XK, Hong Y (2008) Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum). Anal Biochem 375:150–152PubMedCrossRefGoogle Scholar
  76. Young ND, Debellé F, Oldroyd GE et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524PubMedCentralPubMedCrossRefGoogle Scholar
  77. Zakharov A, Giersberg M, Hosein F, Melzer M, Müntz K, Saalbach I (2004) Seed-specific promoters direct gene expression in non-seed tissue. J Exp Bot 55:1463–1471PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Massimo Confalonieri
    • 1
  • Maria Carelli
    • 1
  • Valentina Galimberti
    • 2
  • Anca Macovei
    • 2
    • 4
  • Francesco Panara
    • 3
    • 5
  • Marco Biggiogera
    • 2
  • Carla Scotti
    • 1
  • Ornella Calderini
    • 3
  1. 1.Consiglio per la Ricerca e Sperimentazione in AgricolturaCentro di Ricerca per le Produzioni Foraggere e Lattiero-CasearieLodiItaly
  2. 2.Department of Biology and Biotechnology “L. Spallanzani”University of PaviaPaviaItaly
  3. 3.CNR, Istituto di Genetica VegetalePerugiaItaly
  4. 4.International Center for Genetic Engineering and Biotechnology (ICGEB)New DelhiIndia
  5. 5.ENEA Centro Ricerche TRISAIARotondellaItaly

Personalised recommendations