Plant Cell Reports

, Volume 35, Issue 4, pp 895–904 | Cite as

Functional characterization of duplicated B-class MADS-box genes in Japanese gentian

  • Takashi Nakatsuka
  • Misa Saito
  • Masahiro Nishihara
Original Article

Abstract

Key message

The heterodimer formation between B-class MADS-box proteins of GsAP3a and GsPI2 proteins plays a core role for petal formation in Japanese gentian plants.

Abstract

We previously isolated six B-class MADS-box genes (GsAP3a, GsAP3b, GsTM6, GsPI1, GsPI2, and GsPI3) from Japanese gentian (Gentiana scabra). To study the roles of these MADS-box genes in determining floral organ identities, we investigated protein–protein interactions among them and produced transgenic Arabidopsis and gentian plants overexpressing GsPI2 alone or in combination with GsAP3a or GsTM6. Yeast two-hybrid and bimolecular fluorescence complementation analyses revealed that among the GsPI proteins, GsPI2 interacted with both GsAP3a and GsTM6, and that these heterodimers were localized to the nuclei. The heterologous expression of GsPI2 partially converted sepals into petaloid organs in transgenic Arabidopsis, and this petaloid conversion phenomenon was accelerated by combined expression with GsAP3a but not with GsTM6. In contrast, there were no differences in morphology between vector-control plants and transgenic Arabidopsis plants expressing GsAP3a or GsTM6 alone. Transgenic gentian ectopically expressing GsPI2 produced an elongated tubular structure that consisted of an elongated petaloid organ in the first whorl and stunted inner floral organs. These results imply that the heterodimer formation between GsPI2 and GsAP3a plays a core role in determining petal and stamen identities in Japanese gentian, but other B-function genes might be important for the complete development of petal organs.

Keywords

B-class MADS-box gene Japanese gentian Modified ABC model Petal identity Transgenic plant 

Abbreviations

AP1

APETALA1

AP3

APETALA3

DEF

DEFICIENS

GLO

GLOBOSA

PI

PISTILLATA

SEP

SEPALATA

Supplementary material

299_2015_1930_MOESM1_ESM.pdf (348 kb)
Figure S1. Alignment of the deduced amino acid sequences of three GsPI genes. Deduced amino acid sequences of GsPI1, GsPI2, GsPI3, GsAP3a,GsAP3b, and GsTM6 were aligned by ClustalW. Arrows indicate MADS-domain, I-region, and K-box. Open boxes indicate PI, PI-derived and euAP3 motifs. Gray boxes indicate conserved amino acids, Asn-98 and Glu-97, which are important for both the strength and specificity of AP3/PI heterodimer formation (Yang et al. 2003). Red font indicates specific amino acid sequence within MADS-domain and I-region of GsPI2. Figure S2. Anatomic observation of the ectopic GsPI2-overexpressing gentian flowers. Flower organs of vector control plant and AtACT2::GsPI2-transgenic plants are shown in upper (A to E) and lower panels (F to J), respectively. External appearance of floral buds (A and F), abaxial (B and G) and adaxial sides (C and H) of first-whorl organs, abaxial (D and I) and adaxial sides (E and J) of second- to fourth-whorl organs. Bar = 10 mm. Supplementary material 1 (PDF 347 kb)

References

  1. An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10:107–121CrossRefPubMedGoogle Scholar
  2. Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427CrossRefPubMedGoogle Scholar
  3. Chang YY, Kao NH, Li JY, Hsu WH, Liang YL, Wu JW, Yang CH (2010) Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiol 152:837–853CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330CrossRefPubMedGoogle Scholar
  5. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  6. Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower development. Nature 353:31–37CrossRefPubMedGoogle Scholar
  7. Davies B, Di Rosa A, Eneva T, Saedler H, Sommer H (1996) Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes. Plant J 10:663–677CrossRefPubMedGoogle Scholar
  8. Egea-Cortines M, Saedler H, Sommer H (1999) Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J 18:5370–5379CrossRefPubMedPubMedCentralGoogle Scholar
  9. Ferrario S, Immink RG, Shchennikova A, Busscher-Lange J, Angenent GC (2003) The MADS box gene FBP2 is required for SEPALLATA function in petunia. Plant Cell 15:914–925CrossRefPubMedPubMedCentralGoogle Scholar
  10. Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409:525–529CrossRefPubMedGoogle Scholar
  11. Hsu HF, Hsu WH, Lee YI, Mao WT, Yang JY, Li JY, Yang CH (2015) Model for perianth formation in orchids. Nat Plants 1:15046CrossRefGoogle Scholar
  12. Kanno A, Nakada M, Akita Y, Hirai M (2007) Class B gene expression and the modified ABC model in nongrass monocots. ScientificWorldJournal 7:268–279CrossRefPubMedGoogle Scholar
  13. Kramer EM, Dorit RL, Irish VF (1998) Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics 149:765–783PubMedPubMedCentralGoogle Scholar
  14. Krizek BA, Meyerowitz EM (1996) The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development 122:11–22PubMedGoogle Scholar
  15. McGonigle B, Bouhidel K, Irish VF (1996) Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression. Genes Dev 10:1812–1821CrossRefPubMedGoogle Scholar
  16. Mishiba K, Nishihara M, Nakatsuka T, Abe Y, Hirano H, Yokoi T, Kikuchi A, Yamamura S (2005) Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J 44:541–556CrossRefPubMedGoogle Scholar
  17. Mondragon-Palomino M, Theissen G (2011) Conserved differential expression of paralogous DEFICIENS- and GLOBOSA-like MADS-box genes in the flowers of Orchidaceae: refining the ‘orchid code’. Plant J 66:1008–1019CrossRefPubMedGoogle Scholar
  18. Mondragón-Palomino M, Theißen G (2008) MADS about the evolution of orchid flowers. Trends Plant Sci 13:51–59CrossRefPubMedGoogle Scholar
  19. Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant Cell Physiol 49:1818–1829CrossRefPubMedGoogle Scholar
  20. Nakatsuka T, Saito M, Yamada E, Nishihara M (2011) Production of picotee-type flowers in Japanese gentian by CRES-T. Plant Biotechnol 28:173–180CrossRefGoogle Scholar
  21. Nakatsuka T, Saito M, Yamada E, Fujita K, Yamagishi N, Yoshikawa N, Nishihara M (2015) Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian. BMC Plant Biol 15:182CrossRefPubMedPubMedCentralGoogle Scholar
  22. Nishihara M, Mishiba K, Imamura T, Takahashi H, Nakatsuka T (2015) Molecular breeding of Japanese gentians—applications of genetic transformation, metabolome analyses, and genetic markers. In: Rybczyński JJ, Davey MR, Mikula A (eds) The Gentianaceae, vol 2., Biotechnology and ApplicationsSpringer, New York, pp 239–265Google Scholar
  23. Rijpkema AS, Royaert S, Zethof J, van der Weerden G, Gerats T, Vandenbussche M (2006) Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage. Plant Cell 18:1819–1832CrossRefPubMedPubMedCentralGoogle Scholar
  24. Sasaki K, Aida R, Yamaguchi H, Shikata M, Niki T, Nishijima T, Ohtsubo N (2010) Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind. Mol Genet Genomics 284:399–414CrossRefPubMedPubMedCentralGoogle Scholar
  25. Sasaki K, Yamaguchi H, Nakayama M, Aida R, Ohtsubo N (2014) Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture. Plant Mol Biol 86:319–334CrossRefPubMedGoogle Scholar
  26. Tsai WC, Kuoh CS, Chuang MH, Chen WH, Chen HH (2004) Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant Cell Physiol 45:831–844CrossRefPubMedGoogle Scholar
  27. Tsai WC, Lee PF, Chen HI, Hsiao YY, Wei WJ, Pan ZJ, Chuang MH, Kuoh CS, Chen WH, Chen HH (2005) PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant Cell Physiol 46:1125–1139CrossRefPubMedGoogle Scholar
  28. Tsai WC, Pan ZJ, Hsiao YY, Jeng MF, Wu TF, Chen WH, Chen HH (2008) Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid. Plant Cell Physiol 49:814–824CrossRefPubMedGoogle Scholar
  29. van Tunen AJ, Eikelboom W, Angenent GC (1993) Floral organogenesis in Tulipa. In: Flow Newsl, p 33–37Google Scholar
  30. Vandenbussche M, Zethof J, Royaert S, Weterings K, Gerats T (2004) The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development. Plant Cell 16:741–754CrossRefPubMedPubMedCentralGoogle Scholar
  31. West AG, Causier BE, Davies B, Sharrocks AD (1998) DNA binding and dimerisation determinants of Antirrhinum majus MADS-box transcription factors. Nucleic Acids Res 26:5277–5287CrossRefPubMedPubMedCentralGoogle Scholar
  32. Whipple CJ, Ciceri P, Padilla CM, Ambrose BA, Bandong SL, Schmidt RJ (2004) Conservation of B-class floral homeotic gene function between maize and Arabidopsis. Development 131:6083–6091CrossRefPubMedGoogle Scholar
  33. Xu Y, Teo LL, Zhou J, Kumar PP, Yu H (2006) Floral organ identity genes in the orchid Dendrobium crumenatum. Plant J 46:54–68CrossRefPubMedGoogle Scholar
  34. Yang Y, Fanning L, Jack T (2003) The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins, APETALA3 and PISTILLATA. Plant J 33:47–59CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Takashi Nakatsuka
    • 1
  • Misa Saito
    • 2
  • Masahiro Nishihara
    • 2
  1. 1.Graduated School of AgricultureShizuoka UniversityShizuokaJapan
  2. 2.Iwate Biotechnology Research CenterKitakami, IwateJapan

Personalised recommendations