NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF GENE EXPRESSION AND REGULATION I

Professor:
Shigeru Iida
Associate Professor (adjunct):
Yoshihiro Ozeki
Research Associate:
Rie Terada
Yoshiki Habu
Yoshishige Inagaki
Postdoctoral Fellow:
Yasuyo Hisatomi 2
Graduate Student:
Atsushi Hoshino
Technical Staff:
Sachiko Tanaka-Fukada
Tomoko Mori
Yoshiko Inoue
Visiting Fellows:
Ken-ichi Shiokawa
Yasumasa Morita
Fumiyoshi Myoga
Makiyo Yamagishi


The main interest of the group is in understanding the biology of dynamic genome, namely, genome organization and reorganization and their impact on gene expression and regulation. Although there are many elements affecting organization and reorganization of the genomes, we have currently focused on mobile genetic elements in general and plant transposable controlling elements in particular.


I. Identification and characterization of mutable alleles in the Japanese morning glory

The Japanese morning glory (Pharbitis nil or Ipomoea nil) is a traditional horticultural plant in Japan, and extensive physiological and genetical studies on the plant have been conducted. Among several mutable loci that condition variegated flower phenotypes, two mutable alleles, flecked and speckled have been chosen for molecular elucidation of the variegated phenotypes.

The flecked mutant bears white flowers with colored flecks and sectors (Fig. 1A). The flecking was regarded as recurrent somatic mutation from the recessive white to the dominant pigmented allele, accompanied by changes in genotypes from the homozygous recessive to the heterozygous condition. We have molecularly characterized the flecked mutation in one of the anthocyanin genes, A-3, and found that the mutable a-3flecked (flecked) allele carries the transposable element, Tpn1, in the anthocyanin biosynthesis gene for dihydroflavonol 4-reductase (DFR). Among the three copies of the DFR gene in the genome of the Japanese morning glory, Tpn1 resides within the second intron of the DFR-B gene. The flower variegation is due to somatic reversion of the DFR-B gene by Tpn1 excision. The 6.4 kb Tpn1 element belongs to the En/Spm family and is likely to be a non-autonomous element deficient to produce active transposases.

It is known that the frequency and the timing of the flecking are generally heritable by their progeny but sometimes conversion of these phenotypes is also observed. Thus the flecking caused by the excision of Tpn1 from the DFR-B gene may be determined either by trans-acting activities of its related autonomous element or by heritable modifications of Tpn1 itself. We are currently focusing on these lines of investigations.

Fig. 1B shows another flower variegation caused by the mutable speckled allele. Our genetic studies indicate that another element termed speckled-activator acting in trans on the speckled allele is necessary to confer the speckled variegation phenotype. Our current hypothesis is that the recessive speckled allele carries a non-autonomous transposable element and the dominant speckled-activator must be a related autonomous element. The plants carrying the speckled allele without active speckled-activator bear pale yellow flowers. Based on the chemical analysis of the pale yellow flowers, it was proposed that the mutation in the gene for flavanone 3-hydroxylase (F3H) is a likely candidate for the speckled allele. We have tested this hypothesis and shown that the speckled allele is neither F3H gene nor a regulatory gene acting on the F3H gene expression. Currently, we are characterizing the CHI gene which contains an insertion in the mutable speckled lines.


II. Identification of the mutable alleles in the common morning glory

The mutable aflaked line of the common morning glory (Pharbitis purpurea or Ipomoea purpurea) also bears white flowers with colored flakes and sectors (Fig. 1C). The mutable aflaked allele is known to exhibit incomplete dominance. Interestingly, not only intensely colored flakes but also white spots and sectors were often observed in lightly colored flowers of the morning glory with the heterozygous state A/aflaked (Fig. 1D). To identify the mutable aflaked allele, we have characterized DNA rearrangements found at the DFR and ANS (Anthocyanidin synthase) gene regions in the anthocyanin biosynthesis and identified several new mobile genetic elements. However, subsequent genetic studies revealed that these elements have not associated with the mutable aflaked allele.

Based on amplified restriction fragment length polymorphism (AFLP) technology, we have also developed a new protocol termed AMF (AFLP-based mRNA fingerprinting) for the fingerprinting of mRNA to detect differentially expressed genes. Using both AMF and the differential display (DD) method, we were able to identified new CHS (chalcone synthase) genes, CHS-D and CHS-E, in the anthocyanin biosynthesis. Both CHS-D and CHS-E are expressed in the flower buds of both the common and the Japanese morning glory, and CHS-D is more abundantly expressed than CHS-E. In the mutable aflaked line of the common morning glory, the CHS-D gene carries an about 4 kb insertion and its mRNA accumulation is drastically reduced. Further characterization of these newly identified CHS genes is in progress.


III. Transposition of the maize Ac/Ds transposable elements in transgenic rice plants.
To understand the behavior of the transposable elements, we are characterizing transposition in heterologous system using transgenic plants. A transgenic rice carrying a non-autonomous Ds element was crossed with another transgenic rice containing the active Ac transposase (T. Izawa, et al., (1997) Plant Mol. Biol. 35, 219-229), we are currently investigating somatic transposition of Ds and its target selectivity in the F1 plants.

Fig. 1
Flower variegation.
(A) a-3flecked; (B) speckled; (C) aflaked [aflaked/aflaked]; (D) aflaked [A/aflaked]


Publication List:
Abe, Y., Hoshino, A. and Iida, S. (1997) Appearance of flower variegation in the mutable speckled line of the Japanese morning glory is controlled by two genetic elements. Genes Genet. Syst. 72, 57-62.
Fukada-Tanaka, S., Hoshino, A., Hisatomi, Y., Habu, Y., Hasebe, M. and Iida, S. (1997) Identification of new chalcone synthase genes for flower pigmentation in the Japanese and common morning glories. Plant Cell Physiol. 38, 754-758.
Habu, Y., Fukada-Tanaka, S., Hisatomi, Y. and Iida, S. (1997) Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochem. Biophys. Res. Commun. 234, 516-521.
Hisatomi, Y., Hanada, K. and Iida, S. (1997) The retrotransposon RTip1 is integrated into a novel type of minisatellite MiniSip1 in the genome of the common morning glory, and carries another new type of minisatellite MiniSip2. Theor. Appl. Genet. 95, 1049-1056.
Hisatomi, Y., Yoneda, Y., Kasahara, K., Inagaki, Y. and Iida, S. (1997) DNA rearrangements at the region of the dihydroflavonol 4-reductase gene for flower pigmentation and incomplete dominance in morning glory carrying the mutable flaked mutation. Theor. Appl. Genet. 95, 509-515.
Hoshino, A., Abe, Y., Saito, N., Inagaki, Y. and Iida, S. (1997) The gene encoding flavanone 3-hydroxylase is expressed normally in the pale yellow flowers of the Japanese morning glory carrying the speckled mutation which produce neither flavonol nor anthocyanin but accumulate chalcone, aurone and flavanone. Plant Cell Physiol. 38, 970-974.


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Last Modified: 12:00, May 28, 1998