Molecular Breeding

, Volume 30, Issue 1, pp 453–467 | Cite as

Ectopic expression of a truncated Pinus radiata AGAMOUS homolog (PrAG1) causes alteration of inflorescence architecture and male sterility in Nicotiana tabacum

  • Jun-Jun LiuEmail author


Plant MADS-box genes of the AG/PLE subfamily control the identity of sexual organs. An AG-homologous gene (PrAG1) was characterized in the Pinus radiata genome. PrAG1 transcript was detected only in the female and male strobili. To investigate its regulatory function during floral development, three binary vectors were constructed and transformed into tobacco plants for overexpression of the PrAG1 full-length protein (35S::MIKC), and truncated proteins with deletion of C domain (35S::MIK) or deletion of both C and K domains (35S::MI) under control of the CaMV 35S promoter. All transgenic tobacco lines with ectopic expression of 35S::MIKC and 35S::MIK showed no phenotypic effect on floral development. However, 23.8% of the 35S::MI transgenic lines displayed altered inflorescence architecture and variety of floral development changes, including complete male sterility, suggesting that PrAG1 may be the P. radiata AG-homologous gene with C-function and that it may play a role in the determination of meristem identities in both inflorescence and flowering. Expression of truncated AG MI genes could be useful in reducing plant pollen and seed formation, as well as increasing inflorescence branching and flower production, providing a novel engineered sterility strategy for transgenic plants with potential commercial application in molecular breeding of horticultural flowering plants.


Agamous Inflorescence architecture MADS-box gene Nicotiana tabacum Pinus radiata Transgenic sterility 



This research was supported in part by Canadian Forest Service. The author thanks the late Drs. D. F. Karnosky and G. K. Podila for their support and help in initiation of the project. This manuscript was prepared in memory of their great contribution to forest biotechnology.

Supplementary material

11032_2011_9635_MOESM1_ESM.doc (66 kb)
Fig. 1 Alignment of amino acid sequence of P. radiata PrAG1 protein with Arabidopsis AG and representative AG-homologous proteins from a variety of angiosperm and gymnosperm taxa. Four protein domains (MADS-box, I, K, and C) are marked with dashed arrows. The conserved amino acid residues are indicated by a black background; and other less conserved amino acids are indicated by grey shading. Dashes in the sequences represent single amino acid gaps for best alignment (DOC 66 kb)


  1. Ahearn KP, Johnson HA, Weigel D, Wagner DR (2001) NFL1, a Nicotiana tabacum LEAFY-like gene, controls meristem initiation and floral structure. Plant Cell Physiol 42:1130–1139PubMedCrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  3. Alvarez-Buylla ER, Pélaz S, Liljegren SJ, Gold SE, Burgeff C, Ditta GS, Ribas de Pouplana L, Martínez-Castilla L, Yanofsky MF (2000) An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proc Natl Acad Sci USA 97:5328–5333PubMedCrossRefGoogle Scholar
  4. Becker A, Theißen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phylogenet Evol 29:464–489PubMedCrossRefGoogle Scholar
  5. Benedito VA, Visser PB, van Tuyl JM, Angenent GC, de Vries SC, Krens FA (2004) Ectopic expression of LLAG1 an AGAMOUS homologue from lily (Lilium longiflorum Thunb.) causes floral homeotic modifications in Arabidopsis. J Exp Bot 55:1391–1399PubMedCrossRefGoogle Scholar
  6. Bradley D, Carpenter R, Sommer H, Hartley N, Coen E (1993) Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of Antirrhinum. Cell 72:85–95PubMedCrossRefGoogle Scholar
  7. Carlsbecker A, Tandre K, Johanson U, Englund M, Engström P (2004) The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce (Picea abies). Plant J 40:546–557PubMedCrossRefGoogle Scholar
  8. Causier B, Bradley D, Cook H, Davies B (2009) Conserved intragenic elements were critical for the evolution of the floral C-function. Plant J 58:41–52PubMedCrossRefGoogle Scholar
  9. Chaidamsaria T, Samanhudi U, Sugiarti H, Santoso D, Angenent GC, de Maagd RA (2006) Isolation and characterization of an AGAMOUS homologue from cocoa. Plant Sci 170:968–975CrossRefGoogle Scholar
  10. Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interaction controlling flower development. Nature 353:31–37PubMedCrossRefGoogle Scholar
  11. Datla RSS, Bekkaoui F, Hammerlindl JK, Pilate G, Dunstan DI, Crosby WL (1993) Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci 94:139–149CrossRefGoogle Scholar
  12. De Bodt S, Raes J, Van de Peer Y, Theissen G (2003) And then there were many: MADS goes genomic. Trends Plant Sci 8:475–483PubMedCrossRefGoogle Scholar
  13. Dingwall C, Laskey RA (1991) Nuclear targeting sequences: a consensus? Trends Biochem Sci 16:481–487CrossRefGoogle Scholar
  14. Dinneny JR, Yanofsky MF (2004) Floral development: an ABC gene chips in downstream. Curr Biol 14:R840–R841PubMedCrossRefGoogle Scholar
  15. Dubois A, Raymond O, Maene M, Baudino S, Langlade NB, Boltz V, Vergne P, Bendahmane M (2010) Tinkering with the C-function: a molecular frame for the selection of double flowers in cultivated roses. PLoS One 5:e9288PubMedCrossRefGoogle Scholar
  16. Futamura N, Totoki Y, Toyoda A, Igasaki T, Nanjo T, Seki M, Sakaki Y, Mari A, Shinozaki K, Shinohara K (2008) Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili. BMC Genomics 9:383PubMedCrossRefGoogle Scholar
  17. Goetz M, Godt DE, Guivarc’h A, Kahmann U, Chriqui D, Roitsch T (2001) Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proc Natl Acad Sci USA 98:6522–6527PubMedCrossRefGoogle Scholar
  18. Gómez-Mena C, de Folter S, Costa MM, Angenent GC, Sablowski R (2005) Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development 132:429–438PubMedCrossRefGoogle Scholar
  19. Henschel K, Kofuji R, Hasebe M, Saedler H, Munster T, Theissen G (2002) Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens. Mol Biol Evol 19:801–814PubMedCrossRefGoogle Scholar
  20. Herskowitz I (1987) Functional inactivation of genes by dominant negative mutations. Nature 329:219–222PubMedCrossRefGoogle Scholar
  21. Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  22. Irish VF, Litt A (2005) Flower development and evolution: gene duplication, diversification and redeployment. Curr Opin Genet Dev 15:454–460PubMedCrossRefGoogle Scholar
  23. Ito T, Wellmer F, Yu H, Das P, Ito N, Alves-Ferreira M, Riechmann JL, Meyerowitz EM (2004) The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS. Nature 430:356–360PubMedCrossRefGoogle Scholar
  24. Ito T, Ng KH, Lim TS, Yu H, Meyerowitz EM (2007) The homeotic protein AGAMOUS controls late stamen development by regulating a jasmonate biosynthetic gene in Arabidopsis. Plant Cell 19:3516–3529PubMedCrossRefGoogle Scholar
  25. Jager M, Hassanin A, Manuel M, Le Guyader H, Deutsch J (2003) MADS-box genes in Ginkgo biloba and the evolution of the AGAMOUS family. Mol Biol Evol 20:842–854PubMedCrossRefGoogle Scholar
  26. Kang H-G, Noh Y-S, Chung Y–Y, Costa MA, An KS, An GH (1995) Phenotypic alterations of petal and sepal by ectopic expression of a rice MADS-box gene in tobacco. Plant Mol Biol 29:1–10PubMedCrossRefGoogle Scholar
  27. Kater MM, Colombo L, Franken J, Busscher M, Masiero S, Van Lookeren-Campagne MM, Angenent GC (1998) Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate. Plant Cell 10:171–182PubMedCrossRefGoogle Scholar
  28. Kaufmann K, Melzer R, Theissen G (2005) MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene 347:183–198PubMedCrossRefGoogle Scholar
  29. Kelly AJ, Bonnlander MB, Meeks-Wagner DR (1995) NFL, the tobacco homolog of FLORICAULA and LEAFY, is transcriptionally expressed in both vegetative and floral meristems. Plant Cell 7:225–234PubMedCrossRefGoogle Scholar
  30. Kempin SA, Mandel MA, Yanofsky MF (1993) Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1. Plant Physiol 103:1041–1046PubMedCrossRefGoogle Scholar
  31. Khan MS (2005) Plant biology: engineered male sterility. Nature 436:783–785PubMedCrossRefGoogle Scholar
  32. Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224PubMedCrossRefGoogle Scholar
  33. Kramer EM, Jaramillo MA, Di Stilio VS (2004) Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS Box genes in angiosperms. Genetics 166:1011–1023PubMedCrossRefGoogle Scholar
  34. Krizek BA, Fletcher JC (2005) Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet 6:688–698PubMedCrossRefGoogle Scholar
  35. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinf 5:150–163CrossRefGoogle Scholar
  36. Liu J–J, Ekramoddoullah AKM (2003) Root-specific expression of a western white pine PR10 gene is mediated by different promoter regions in transgenic tobacco. Plant Mol Biol 52:103–120PubMedCrossRefGoogle Scholar
  37. Liu J-J, Podila GK (1997) Characterization of a MADS box gene (Y09611) from immature female cone of red pine (PGR97–032). Plant Physiol 113:665Google Scholar
  38. Liu J–J, Ekramoddoullah AKM, Podila GK (2003) A MADS-Box gene specifically expressed in the reproductive tissues of red pine (Pinus resinosa) is a homologue to floral homeotic genes with C-function in angiosperms. Physiol Mol Biol Plants 9:197–206Google Scholar
  39. Liu J–J, Ekramoddoullah AKM, Piggott N, Zamani A (2005) Molecular cloning of a pathogen/wound-inducible PR10 promoter from Pinus monticola and characterization in transgenic Arabidopsis plants. Planta 221:159–169PubMedCrossRefGoogle Scholar
  40. Mandel MA, Bowman JL, Kempin SA, Ma H, Meyerowitz EM, Yanofsky MF (1992) Manipulation of flower structure in transgenic tobacco. Cell 71:133–143PubMedCrossRefGoogle Scholar
  41. Mizukami Y, Ma H (1992) Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell 71:119–131PubMedCrossRefGoogle Scholar
  42. Mizukami Y, Ma H (1995) Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA. Plant Mol Biol 28:767–784PubMedCrossRefGoogle Scholar
  43. Mizukami Y, Ma H (1997) Determination of Arabodopsis floral meristem identity by AGAMOUS. Plant Cell 9:393–408PubMedCrossRefGoogle Scholar
  44. Mizukami Y, Huang H, Tudor M, Hu Y, Ma H (1996) Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations. Plant Cell 8:831–845PubMedCrossRefGoogle Scholar
  45. Mouradov A, Glassick TV, Hamdorf BA, Murphy LC, Marla SS, Yang Y, Teasdale RD (1998) Family of MADS-box genes expressed early in male and female reproductive structures of Monterey pine. Plant Physiol 117:55–62PubMedCrossRefGoogle Scholar
  46. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  47. Ng KH, Yu H, Ito T (2009) AGAMOUS controls GIANT KILLER, a multifunctional chromatin modifier in reproductive organ patterning and differentiation. PLoS Biol 7:e1000251PubMedCrossRefGoogle Scholar
  48. Nishimura T, Yokota E, Wada T, Shimmen T, Okada K (2003) An Arabidopsis ACT2 dominant-negative mutation, which disturbs F-actin polymerization, reveals its distinctive function in root development. Plant Cell Physiol 44:1131–1140PubMedCrossRefGoogle Scholar
  49. Pan IL, McQuinn R, Giovannoni JJ, Irish VF (2010) Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development. J Exp Bot 61:1795–1806PubMedCrossRefGoogle Scholar
  50. Parenicová L, de Folter S, Kieffer M, Horner DS, Favalli C, Busscher J, Cook HE, Ingram RM, Kater MM, Davies B (2003) Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell 15:1538–1551PubMedCrossRefGoogle Scholar
  51. Perez-Prat E, van Lookeren Campagne MM (2002) Hybrid seed production and the challenge of propagating male-sterile plants. Trends Plant Sci 7:199–203PubMedCrossRefGoogle Scholar
  52. Pnueli L, Hareven AD, Rounsley SD, Yanofsky MF, Lifschitz E (1994) Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell 6:163–173PubMedCrossRefGoogle Scholar
  53. Rutledge R, Regan S, Nicolas O, Fobert P, Coté C, Bosnich W, Kauffeldt C, Sunohara G, Séguin A, Stewart D (1998) Characterzation of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversion when expressed in Arabidopsis. Plant J 15:625–634PubMedCrossRefGoogle Scholar
  54. Sablowski R (2007) Flowering and determinacy in Arabidopsis. J Exp Bot 58:899–907PubMedCrossRefGoogle Scholar
  55. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  56. Sather DN, Jovanovic M, Golenberg EM (2010) Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism. BMC Plant Biol 10:46PubMedCrossRefGoogle Scholar
  57. Straus SH, Rottmann WH, Brunner AM, Sheppard LA (1995) Genetic engineering of reproductive sterility in forest trees. Mol Breed 1:5–26CrossRefGoogle Scholar
  58. Tandre K, Albert VA, Sundas A, Engstrom P (1995) Conifer homoloques to genes that control flower development in angiosperms. Plant Mol Biol 27:69–78PubMedCrossRefGoogle Scholar
  59. Tandre K, Svenson M, Svenson ME, Engström P (1998) Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms. Plant J 15:615–623PubMedCrossRefGoogle Scholar
  60. Velten J, Cakir C, Cazzonelli CI (2010) A spontaneous dominant-negative mutation within a 35S:AtMYB90 transgene inhibits flower pigment production in tobacco. PLoS One 5:e9917PubMedCrossRefGoogle Scholar
  61. Villarreal F, Martín V, Colaneri A, González-Schain N, Perales M, Martín M, Lombardo C, Braun H-P, Bartoli C, Zabaleta E (2009) Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence. Plant Mol Biol 70:471–485PubMedCrossRefGoogle Scholar
  62. Wellmer F, Riechmann JL, Alves-Ferreira M, Meyerowitz EM (2004) Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 16:1314–1326PubMedCrossRefGoogle Scholar
  63. Winter K-U, Becker A, Münster T, Kim JT, Saedler H, Theißen G (1999) MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants. Proc Natl Acad Sci USA 96:7342–7347PubMedCrossRefGoogle Scholar
  64. Yamaguchi T, Lee DY, Miyao A, Hirochika H, An G, Hirano H-Y (2006) Functional diversification of the two C-class MADS box genes OsMADS3 and OsMADS58 in Oryza sativa. Plant Cell 18:15–28PubMedCrossRefGoogle Scholar
  65. Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldman KA, Meyerowitz EM (1990) The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346:35–39PubMedCrossRefGoogle Scholar
  66. Zhang P, Tan HT, Pwee KH, Kumar PP (2004) Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms. Plant J 37:566–577PubMedCrossRefGoogle Scholar
  67. Zobell O, Faigl W, Saedler H, Münster T (2010) MIKC* MADS-box proteins: conserved regulators of the gametophytic generation of land plants. Mol Biol Evol 27:1201–1211PubMedCrossRefGoogle Scholar

Copyright information

© Crown Copyright  2011

Authors and Affiliations

  1. 1.Pacific Forestry Centre, Canadian Forest Service, Natural Resources CanadaVictoriaCanada

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