  National Insitute for Basic Biology

DIVISION OF GENE EXPRESSION AND REGULATION I

Associate Professor:: Kiyotaka Okada

Research Associate:: Sumie Ishiguro

Graduate Students:: Azusa Yano; Yoichi Ono; Toshiro Ito¹⁾; Takuji Wada; Tokitaka Oyama¹⁾; Sinichiro Sawa²⁾

Technical Staff:: Hideko Nonaka; Akiko Kawai

(¹⁾ from Kyoto University)
(²⁾ from Nagoya University)

The principal interest of this laboratory is molecular genetic studies on the regulatory systems of organ development and on growth control by several environmental stimuli in higher plants. For these studies, we have mainly used a small crusifer, Arabidopsis thaliana. This plant is called "botanical Drosophila", because it has some remarkable features, such as a small genome size (1 x 10⁸ base pairs per haploid), short life-cycle (5-6 weeks), small size (20-30 cm in height), and ease of propagation. These features make the plant ideally suited for genetic and molecular biological studies. In addition, more than 360 loci and more than 650 RFLP and RAPD markers are mapped on 5 chromosomes. Experimental techniques such as transformation, regeneration of transgenic plants and gene tagging have been improved. Using this plant, we have isolated and characterized many mutants defective in flower development and morphogenesis or in root formation and responses toward physical stimuli such as gravity, light or touching.

I. Development and morphogenesis of flowers

The process of flower development can be divided into four major steps: phase transition from vegetative to reproductive growth, formation of inflorescence meristem, formation and identity determination of floral organs, and growth and maturation of floral organs. Several different types of signaling mechanism, between or within cells, must have important roles in each step of flower development, because each step requires cell division, cell growth and cell differentiation in a concerted fashion. In order to unveil the signaling mechanism and its genetic regulatory systems, we are carrying out genetic, biochemical, anatomical and physiological analyses on the processes of flower development using A. thaliana.

The filamentous flower (fil) mutant shows several structural defects in flowers and inflorescences. After flower initiation, fil mutant elongates an inflorescence axis normally, and forms about 10 flowers that show some structural abnormalities (phase 1). Then formation of floral buds is stopped at the inflorescence meristem, and instead, more than 10 filaments and more than 10 sepal-like structures are formed (phase 2). After forming the phase 2 cluster, a cluster of flowers is formed again (phase 3). One possible explanation of the phase shift is that the amount of the factor(s) or the amount of receptor molecules of the factor(s) changes to high-low-high as plants grow. In order to examine the genetic relationships of FIL gene with other known flower genes, we have constructed a series of double mutants. The results indicate that FIL gene is required for formation and maintenance of floral meristem in combination with APETALA 1, LEAFY and CAULIFLOWER.

The AGAMOUS (AG) gene of A. thaliana is a homeotic gene involved in the development of stamens and carpels. This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF. We confirmed that the AG protein is localized in the nuclei of the cell in stamen- and carpel-primodia. In order to identify AG target genes, we isolated the DNA fragments bound by AG protein in native chromatin by immunopurification, and then identified genes within or near the isolated fragments. Characterization of these genes is in progress.

II. Development and stimulus-response reactions in root

The root of A. thaliana has many advantages of simplicity, transparency and strict organization of cells to analyze the development of roots. One of the thick root mutant, RH32, has 10 cortical cells in the root, whereas wild type has 8 cortical cells. The epidermal cells of RH32 mutant in different tissues, such as root, hypocotyl, stem and sepal, are rounder than those of wild type. RFLP mapping has shown that RH32 gene is located on the lower portion of chromosome 1.

A T-DNA-tagged mutant K293 has a few root hairs of the primary root. Map position of K293 gene is on the lower portion of chromosome 2. We are now in progress of cloning of this gene.

Normal root hairs of A. thaliana are short and formed perpendicular to the root surface, when roots grow on agar plates stood vertical ("agar" form). The long and tilted root hairs are formed when roots grow in the air ("air" form). We showed that the "agar" form hairs are formed by the cells that have passed through the elongation zone, whereas the "air" form hairs are generated within the elongation zone. Mutants defective in these morphological alteration of root hairs are isolated and classified into three categories: namely, mutants generating root hairs of either the "agar" or the "air" form in both condition (class I); mutants defective in the "agar" form hair formation but normal in the "air" form hair formation (class II); and mutants defective in both hair formation (class III).

Roots alter their growth direction when their relative orientation against gravity is changed (gravitropic response), when they are illuminated from aside (phototropic response), or when they encounter obstacles (obstacle-escaping response). These responses were analyzed using agar plates. Young seedlings grown on vertical agar plates have roots which grow straight downward on the agar surface. When the plates are put aside, roots bend 90° and grow to the altered direction of gravity. If the plates are covered with black cloth and illuminated from a side, roots grow to the opposite direction of the incoming light. On the surface of agar plates which are set at an angle of 45° to the vertical, the roots exhibit a wavy growth pattern that is caused by periodic reversion of rotation of the root tip. Using these systems, mutants which show abnormal graviresponse, photoresponse, or obstacle-escaping responses were isolated. Several mutants with abnormal obstacle-escaping response also show abnormal gravitropism and/or phototropism. These results indicate that root gravitropic, phototropic and obstacle-escaping responses share at least in part a common genetic regulatory mechanism. Physiological characterization and molecular cloning of these mutants are in progress.

A mutant hy5 of A. thaliana, which was isolated as a long hypocotyl mutant, shows a variety of abnormalities in development and stimulus-response of roots. HY5 gene therefore plays a key function in signaling pathways of root morphogenesis as well as photomorphogenesis of hypocotyl. We have isolated a hy5 mutant allele from T-DNA inserted lines and cloned the putative HY5 gene. This gene encodes an open reading frame of 168 amino acid residues which include a bZIP motif at the C-terminus. Therefore, HY5 gene may function as a transcriptional regulator in the signaling pathways.

III. Technical improvement for molecular cloning

In order to isolate the genes responsible for the mutants, some technical improvements for gene tagging systems in A. thaliana were attempted. For such experiments, it is absolutely necessary to develop a good, efficient system of transformation mediated by Agrobacterium. We carried out so called in planta transformation, in which adult A. thaliana plants are directly infected with Agrobacterium and gene transfer occurs during floral development. In this system, it is expected that problems caused by somaclonal variation are avoided. About 1,100 lines of transformants were generated. Several interesting mutants have been identified and characterized.

Filamentous flower mutant forms clusters of deformed flowers and of filaments with a sepal-like structure at its top (left). In leafy mutant, flowers are homeotically transformed to shoots (middle). A double mutant carrying mutations in both FILAMENTOUS FLOWER and LEAFY genes fails to generate either flowers or shoots, and instead continues to form filaments and sepal-like structures (right).

Publication List:

Okamoto, H., Yano, A., Shiraishi, H., Okada, K. and Shimura, Y. (1994) Genetic complementation of a floral homeotic mutation, apetala 3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus. Plant Mol. Biol. 26, 465-472.

Ito, T., Shiraishi, H., Okada, K. and Shimura, Y. (1994) Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs. Plant Mol. Biol. 26, 529-533.

Okada, K. and Shimura, Y. (1994) Genetic analyses of signaling in flower development using Arabidopsis. Plant Mol. Biol. 26, 1357-1377.

Okada, K. and Shimura, Y. (1994) Modulation of root growth by physical stimuli. In Arabidopsis (E.M. Meyerowitz and C. Somerville, eds.). Cold Spring Harbor Lab. Press, pp.665-684.