NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF GENE EXPRESSION AND REGULATION I
- Yoshiro Shimura
- Associate Professor:
- Kiyotaka Okada
- Research Associate:
- Sumie Ishiguro
- Graduate Students:
- Azusa Yano
Toshiro Ito 1)
Tokitaka Oyama 1)
- Technical Staff:
- Hideko Nonaka
- (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.
- 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,
- 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.
- 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.