Yoshiro Shimura

Associate Professor:
Kiyotaka Okada

Research Associate:
Sumie Ishiguro
Nobuyoshi Mochizuki

Graduate Students:
Azusa Yano
Yoichi Ono
Toshiro Ito 1)
Takuji Wada
Tokitaka Oyama 1)

Technical Staff:
Hideko Nonaka
Akiko Kawai

(1) from Kyoto 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 10^8 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 Ioci and more than 500 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 responses toward physical stimuli such as gravity, light or touching.

I. Development and morphogenesis of flowers

Mutants with abnormal floral morphology could be divided into the following categories on the basis of the stages of floral development where the genetic defects were presumed to occur, namely, stage 1 : transition from vegetative to reproductive growth (mutants with delayed transition or earlier transition), stage 2: elongation of inflorescence axis (mutants with short inflorescence axis, dwarfs), stage 3: formation of floral meristem (mutants lacking floral meristem at the top of the inflorescence axis), stage 4: formation of floral organ primordia (mutants with increased or decreased numbers of floral organs, or aberrant positions), stage 5: fate determination of the floral organ primordia (homeotic mutants: mutants where some floral organs are replaced by other organs), and stage 6: development and morphogenesis of floral organs (mutants with organs of aberrant structure and function). Most of the mutants have been shown to have single, recessive, nuclear mutations (Komaki et at (1988) Development, 104, 195 203; Okada et al. (1989) Cell Differ Dev., 28, 27-38).

The AGAMOUS gene of Arabidopsis 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. Using the MADS domain of AGAMOUS protein overproduced in E. coli, we have shown that the consensus sequence of the high-affinity binding sites of the AGAMOUS MADS domain was 5'-TT(A/T/G)CC(A/T)6GG(A/T/C)AA- 3'. Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCMI binding-sites show similarities with the binding-site sequence of the AGAMOUS MADS domain (Shiraishi et al. (1993) Plant J, 4, 385-398).

The mutant apetala3 of A. thaliana and the mutant deficiens of Antirrhinum majus have a homeotic conversion of petals to sepals and stamens to carpels. We have isolated a homologous gene to the DEFICIENS from A. thaliana and shown complete complementation of apetala3 mutation by introducing the isolated gene. These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally. The 5'-upstream region of APETALA3 gene contains three SRE-Iike sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue- and stage-specific manner (Okamoto et al. (1994) Plant Mol. Biol., in press). Now we are trying to isolate the target genes, of which the expression is regulated by the MADS-box proteins, AGAMOUS, APETALA3 or PISTILLATA.

Attempts were also made to isolate cDNAs which are specifically expressed in floral organs. From a cDNA library of young floral buds of A. thaliana, we isolated two cDNA clones whose amino acid sequences are highly homologous with the known amidophosphoribosyltransferase cDNAs. Northern blot analysis showed that one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves (Ito et at, submitted).

II. Stimulus-response interactions in root

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). Using a newly devised system which provides a constant obstacletouching stimulus to root tips on agar plate, mutants which show abnormal responses to obstacle-touching stimulus were isolated (Okada & Shimura (1990) Science, 250, 274 276). Gravitropic and phototropic responses were also analyzed using agar plates. Young seedlings grown on vertical agar plates have roots which grow straight downward on the agar surface. When the plates were put aside, roots bend 90 degrees and grow to the new direction of gravity. If the plates were covered with black cloth and illuminated from the side, roots grow to the opposite direction of incoming light. Using these systems, mutants which show abnormal graviresponse or photoresponse were isolated (Okada & Shimura (1992) Aust. J. Plant Physiol., 19, 439-448). 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 (Okada & Shimura (1992) Cell, 70, 369-372).

Physiological and biochemical aspects of stimulus-response reaction in roots were also analyzed. Proteins newly synthesized in cells of root tips of Arabidopsis seedlings after gravistimulation and photo-induced tactile stimulation were separated by two-dimensional gel electrophoresis. Intensities of 14 protein spots were shown to increase after continuous rocking treatment for 24 hours. Analysis of [32P]-labeled proteins revealed that the continuous rocking enhanced the phosphorylation of proteins in two spots. When the seedlings in flasks were illuminated from the front, and the roots bent towards the back wall of the flasks, total of 12 spots were newly appeared or enhanced (Sakamoto et at (1993) Plant Cell Physiol., 34, 297-304).

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 and of transgenic plant regeneration. We have tested several combinations of A. thaliana ecotypes and Agrobacteria strains and established an efficient system (Akama el. al. (1992) Plant Cell Rep., 12, 7-11). We also 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 thought that problems caused by somaclonal variation are avoided. About four hundred of transformants were generated and screened.

Publication List:

Sakamoto, K., Shiraishi, H., Okada, K. and Shimura, Y. (1993) Proteins induced by physical stimuli in root tips of Arabidopsis thaliana seedlings. Plant Cell Physiol. 34, 297-304.

Shiraishi, H., Okada, K. and Shimura, Y. (1993) Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro. PlantJ. 4, 385-398.