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


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.


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.


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















Author contribution statement

TN and MN conceived and designed research. TN, MS and MN conducted experiments. TN and MN analyzed data. TN and MN wrote the manuscript. All authors read and approved the manuscript.


We thank Dr. Norihiro Otsubo and Dr. Katsutomo Sasaki, NARO Institute of Floricultural Science, for SEM observations of floral surface structure, and Dr. Akira Kanno, Tohoku University, for critical advice and suggestions. We thank Ms. Akiko Kubota for producing transgenic plants. This study was supported in part by the Japan Society for the Promotion of Science KAKENHI (grant numbers 21780028 and 24780029), and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.

Compliances with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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)


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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

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