DIVISION OF GENE EXPRESSION AND REGULATION I

*until April 30, 2001
**until Marchl 31, 2001
¹⁾from Chinese Academy of Sciences
²⁾from University of California
³⁾from Vrije University
⁴⁾from Yeungnam University
⁵⁾from Duke University
⁶⁾from Kasetsart University
⁷⁾Graduate University for Advanced Studies
⁸⁾from University of Shizuoka
⁹⁾from Toho University

The main interest of the group is in understanding the biology of the dynamic genome, namely, genome organization and reorganization and its impact on gene expression and regulation. Although there are many elements affecting organization and reorganization of the genomes, we are currently focused on mobile genetic elements in general and plant transposable controlling elements in particular. Since plant transposable elements are known to be associated with both genetic and epigenetic gene regulations, we are characterizing various aspects of genetic and epigenetic gene regulations. In addition, we are also undertaking reverse genetic approaches in order to elucidate the nature of dynamic genome in plants.

I. Spontaneous mutants in the Japanese morning glory.

The Japanese morning glory (Ipomoea nilor Pharbitis nil), displaying blue flowers, is believed to be originated from southeast Asia and has an extensive history of genetic and physiological studies. The plant had been introduced into Japan from China in about 8th century as a medicinal herb, seeds of which were utilized as a laxative, and has become a traditional horticultural plant in Japan since around 17th century. A number of its spontaneous mutants related to the color and shape of the flowers and leaves have been isolated, and about 10% of these mutants carry mutable alleles conferring variegated phenotypes. All of the mutants available are spontaneous mutants and most of them were isolated more than 60 years ago. Several lines of evidence indicate that an En/Spm-related transposable element Tpn1 and its relatives, which we termed Tpn1-family elements, are a common spontaneous mutagen in the plant. Indeed, we have succeeded to identify three of these mutable alleles for flower pigmentation, flecked,speckledand purple-mutable(pr-m), which are caused by integration of Tpn1-related elements, Tpn1, Tpn2and Tpn4, respectively. All of them are non-autonomous elements and their transposition is mediated by a Tpn1-related autonomous element which appears to be subjected to egigenetic regulations. Due to epigenetic inactivation of the autonomous element, rare excision of these non-autonomous elements could occur and such lines displayed apparently stable mutant flowers. In accordance with this notion, an apparent stable r-1allele conferring white flowers is also caused by insertion of a non-autonomous Tpn1-family element, Tpn3,into the CHS-Dgene encoding a chalcone synthase for anthocyanin biosynthesis.

II. Spontaneous mutants having Tpn1-related transposons inserted into the gene encoding anthocyanidin synthase in the Japanese morning glory.

The anthocyanidin synthase (ANS) catalyzes 2-oxoglutarate-dependent oxidation of leucoanthocyanidins to yield anthocyanidins in the anthocyanin biosynthesis pathway. The ANSgene was first isolated from maize and snapdragon, mutants of which are deficient in pigmentation: the A2mutations caused by insertion of transposons in maize control coloration in the aleurone layer of the kernel and a large deletion mutation including the candigene in snapdragon confers completely acyanic flowers. Out of 23 mutants of the Japanese morning glory displaying white flowers examined, three were found to carry an identical insertion of a 6.6-kb Tpn1-related transposon, Tpn8, at the promoter of the ANSgene (Fig. 1). In addition, we also characterized a mutable line Shibori-chidori exhibiting white flowers with red sectors, obtained from a red flower line Beni-chidori about 3 years ago, and found that the mutable allele was caused by integration of a 6.4-kb transposon, Tpn9, into the ANSgene. Interestingly, Tpn9is almost identical to the Tpn1sequence found at the mutable fleckedallele. To our knowledge, the ANS mutations characterized here are the first ANS insertion mutations, including the mutable allele, that affect flower pigmentation. Since the mutable line Shibori-chidori was isolated as a spontaneous mutant very recently, Tpn1-related elements still act as active spontaneous mutagens that would generate new interesting traits in the Japanese morning glory.

Fig. 1. Two mutant alleles in the ANSgene of the Japanese morning glory. The mutants r-3and Shibori-chidorihave Tpn8and Tpn9inserted into the ANSgene, respectively.

III. The transposable element Tip100found at the mutable flaked allele for flower variegation of the common morning glory is an autonomous element.

The mutable flakedline of the common morning glory (Ipomoea purpurea or Pharbitis purpurea) displays white flowers with colored flakes, and the flaked mutation is caused by the insertion of a transposable element Tip100into the CHS-D gene for anthocyanin biosynthesis. The 3.9 kb Tip100 element belonging to the Ac/Ds family contains an open reading frame encoding a polypeptide of 808 amino acids. The frequency and timing of the flower variegation vary in different flakedlines and a genetic element termed Modulator has been postulated to affect the variegation pattern. Since the pattern of the flower variegation is determined by the frequency and timing of the excision of Tip100 from the CHS-Dgene, we examined whether Tip100 is an autonomous element capable of transposition in heterologous tobacco plants. The intact Tip100element was able to excise from its original position in an introduced vector and to reinsert into new sites in the tobacco genome, whereas its internal deletion derivative was not. Based on these results, we concluded that Tip100 is an autonomous element.

IV. Targeted gene disruption by homologous recombination in rice.

The modification of targeted chromosomal genes through homologous recombination is a powerful tool of reverse genetics for characterizing gene functions. In higher plants, however, a transgene is integrated randomly into the genome by illegitimate recombination even the introduced sequence contains targeted homologous region. Although generation of a single transgenic Arabidopsisline having a targeted gene disrupted was reported, the procedure was far from a common practice in higher flowering plants. Rice is an important staple food for more than half of the world?fs population and has become a model monocotyledonous plant because of accumulating information from rice genome projects as well as efficient transformation. To develop a reproducible and reliable procedure for targeted gene disruption by homologous recombination in rice, we tried to improve the following parameters: efficiency of Agrobacterium-mediated transformation, utilization of a strong positive-negative selection, and PCR amplification for screening and detecting a long junction fragment produced by homologous recombination. Subsequently, we conducted detailed Southern blot analysis to confirm the occurrence of the expected precise homologous recombination. We chose the single locus gene Waxy encoding granule-bound starch synthase, a key enzyme in amylose synthesis, in rice as a model gene to be targeted, because its mutant is of agronomic importance and because its phenotype in endosperm and pollen can be easily monitored by simple iodine staining. A transgenic rice plant having a targeted gene disrupted by a transgene (Fig. 2) was isolated, which appears to be a heterozygote with only one copy of the integrated transgene in its geneome.

Fig. 2. Regeneration of a targeted callus through multiple shoots, a plantlet and a fertile transgenic plant from the targeted callus. More than 50 shoots can be usually obtained from a targeted callus.

Publication List:

Fukada-Tanaka, S., Inagaki, Y., Yamaguchi T. and Iida, S. (2001) Simplified transposon display (STD): a new procedure for isolation of a gene tagged by a transposable element belonging to the Tpn1 family in the Japanese morning glory. Plant Biotech.18,143-149.

Hoshino, A., Johzuka-Hisatomi Y. and Iida, S. (2001) Gene duplication and mobile genetic elements in the morning glories. Gene265,1-10.

Ishikawa, N., Johzuka-Hisatomi, Y., Sugita, K., Ebinuma H. and Iida, S. The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants. Mol. Gen. Genet. (in press).

Kamiunten, H., Inoue, S., Yakabe Y. and Iida, S. Characterization of ISPsy2 and ISPsy3, newly identified insertion sequences inPseudomonas syringaepv. Eriobotryae. Gen. Plant Pathol. (in press).

Terada, R., Ignacimuthu, S., Bauer, P., Schultze, M., Kondorosi, A., Potrykus I. and Sautter, C. (2001) Expression of early nodulin promoter gene in transgenic rice. Curr. Sci.81, 270-276.

Toki, K., Saito, N., Iida, S., Hoshino, A., Shigihara A. and Honda, T. (2001) Acylated pelargonidin 3-sophoroside-5-glucosides from the flowers of the Japanese morning glory cultivar 'Violet'. Heterocycles55, 1241-1248.

Toki, K., Saito, N., Iida, S., Hoshino, A., Shigihara A. and Honda, T. A novel acylated pelargonidin 3-sophoroside-5-glucosides from greyish-purple flowers of the Japanese morning glory. Heterocycles55, 2261-2267.

Yamaguchi, T., Fukada-Tanaka, S., Inagaki, Y., Saito, N., Yonekura-Sakakibara, K., Tanaka, Y., Kusumi T. and Iida, S. (2001) Genes encoding the vacuolar Na⁺/H⁺ exchanger and flower coloration. Plant Cell Physiol.42,451-461.