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Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum

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We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation.

Abstract

In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.

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Acknowledgments

This experiment was performed at the Radioactive Ion Beam Factory, operated by RIKEN Nishina Center and CNS, University of Tokyo. We thank the RIKEN Research Resources Center of the Brain Science Institute for performing DNA sequencing analyses. This work was partially supported by grants from the Research Project Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries (Ministry of Agriculture, Forestry and Fisheries of Japan), the Social Infrastructure Technology Development Program (RIKEN), and the Funding Program for Next Generation World-Leading Researchers (Council for Science and Technology Policy from the Japan Society for the Promotion of Science), for TA. This work was also partially supported by Grants-in-Aid for Scientific Research (no. 20780009 and no. 23770070 to YK) from the Japan Society for the Promotion of Science, and the Special Postdoctoral Research Program of RIKEN (to YK).

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Authors and Affiliations

  1. RIKEN Innovation Center, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Yusuke Kazama & Tomoko Abe

  2. RIKEN Nishina Center, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Makoto T. Fujiwara, Hinako Takehisa, Sumie Ohbu, Hiroyuki Saito, Hiroyuki Ichida, Yoriko Hayashi & Tomoko Abe

  3. Department of Life and Material Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Tokyo, 102-8554, Japan

    Makoto T. Fujiwara

  4. Genome Resource Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan

    Hinako Takehisa

  5. Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA, 94305, USA

    Hiroyuki Ichida

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  1. Yusuke Kazama
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  2. Makoto T. Fujiwara
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  3. Hinako Takehisa
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Correspondence to Tomoko Abe.

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Communicated by K. Toriyama.

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Kazama, Y., Fujiwara, M.T., Takehisa, H. et al. Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum . Plant Cell Rep 32, 11–19 (2013). https://doi.org/10.1007/s00299-012-1336-7

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