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Tissue culture-induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3′, 5′ hydroxylase (F3′5′H) promoter

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Abstract

The variegated Saintpaulia cultivar Thamires (Saintpaulia sp.), which has pink petals with blue splotches, is generally maintained by leaf cuttings. In contrast, tissue culture-derived progeny of the cultivar showed not only a high percentage of mutants with solid-blue petals but also other solid-color variants, which have not been observed from leaf cuttings. Solid-color phenotypes were inherited stably by their progeny from tissue culture. Petals from each solid-color variant were analyzed by high-performance liquid chromatography and shown to contain different proportions of three main anthocyanin derivatives: malvidin, peonidin, and pelargonidin. Analysis of flavonoid 3′, 5′-hydroxylase (F3′5′H) sequences showed no differences in the coding region among the variants and variegated individuals. However, a transposon belonging to the hAT superfamily was found in the promoter region of variegated individuals, and the presence of transposon-related insertions or deletions correlated with the observed flower-color phenotypes. Solid-blue flower mutants contained 8-base pair (bp) insertions (transposon excision footprints), while solid-pink mutants had 58- to 70-bp insertions, and purple- and deep-purple mutants had 21- and 24-bp deletions, respectively. Real-time reverse transcription polymerase chain reaction (RT–PCR) analysis showed that F3′5′H expression levels correlated with insertions and deletions (indels) caused by hAT excision, resulting in flower-color differences. Our results showed that tissue culture of Saintpaulia ‘Thamires’ elicits transposon excision, which in turn alters F3′5′H expression levels and flower colors.

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Acknowledgments

We thank Professor A. Hoshino (National Institute for Basic Biology) for his meaningful advice. This work was supported by a Grant-in-Aid for Challenging Exploratory Research (21658011) from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

    Mitsuru Sato, Takashi Kawabe, Munetaka Hosokawa & Motoaki Doi

  2. Faculty of Agriculture, Iwate University, Morioka, 020-8550, Japan

    Fumi Tatsuzawa

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  1. Mitsuru Sato
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  2. Takashi Kawabe
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  3. Munetaka Hosokawa
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Correspondence to Munetaka Hosokawa.

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Communicated by K. Kamo.

A contribution to the Special Issue: Plant Biotechnology in Support of the Millennium Development Goals.

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Electronic Supplemental Fig. 1

Pictures of Saintpaulia used in this study. (A) Full image of ‘Thamires’. (B) Full image of ‘Akemi’ (1) and a closeup of the flower (2). (C) Original species of the genus Saintpaulia: S. intermedia (1), S. ionantha (2), and S. rupicola (3) (EPS 6422 kb)

Electronic Supplemental Fig. 2

Schematic representation of type 1 and type 2 F3′5′H found in Saintpaulia ‘Thamires’. Type 1 has intact coding region, but type 2 has undergone retrotransposon insertion and its coding region is impaired (414 bp of the first exon is lost). The retrotransposon appeared to be inserted in the antisense direction. LTR: long terminal repeat; RVT: reverse transcriptase. Accession numbers (GenBank): type 1 F3′5′H, AB596830; type 2 F3′5′H, AB596832 (EPS 604 kb)

Electronic Supplemental Fig. 3

The results of RT–PCR for F3′H expression at each bud developmental stage of solid-blue and pink mutants. Stages 1-3 correspond to Fig. 3. F3′H sequence is obtained using the degenerate primers SP-F3′H deg1F-K (5′-YTGTKCVSTCCACCTYTTCTC-3′) and SP-F3′H deg1R-K (5′-DGCCCAHRCATTTGGATCRCG-3′). Nucleotides of degenerate positions are represented by single letters: Y is C and T; K is G and T; V is A, C, and G; S is C and G; D is A, G, and T; H is A, C, and T; R is A and G. RT–PCR is performed using Blend Taq polymerase (TOYOBO, Japan). Primers used are F3′H1 entire1F (5′-TGTTAAGCTCCACCGTAACA-3′), F3′H1 entire1R (5′-AACAAGTATGCAACATGGAG-3′), saintActin 1F (5′-GGCTGGAACAAGACTTCAGG-3′) and saintActin 1R (5′-GAGCCACACTGTTCCCATTT-3′) (EPS 5976 kb)

Electronic Supplemental Table 1

Primers used for determining the F3′5′H and its upstream sequences (XLS 21 kb)

Electronic Supplemental Table 2

Protein homology suggested by a BLASTp search of the putative pol gene of the retrotransposon (XLS 19 kb)

Electronic Supplemental Table 3

Protein homology suggested by a BLASTp search of the putative transposase coding region (XLS 20 kb)

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Sato, M., Kawabe, T., Hosokawa, M. et al. Tissue culture-induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3′, 5′ hydroxylase (F3′5′H) promoter. Plant Cell Rep 30, 929–939 (2011). https://doi.org/10.1007/s00299-011-1016-z

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