LABORATORY OF PLANT ORGAN DEVELOPMENTDirector-General

OKADA, Kiyotaka

Assistant Professor:
Postdoctoral Fellows:

Visiting Scientists:

Technical Assistants:
Secretary:

TATEMATSU, Kiyoshi
IGARASHI, Hisako
TSUCHIDA, Yuhei*
URAWA, Hiroko
YABE, Kimihiko
TOMINAGA, Rumi
WADA, Takuji
TSUCHIDA, Yuhei
IWASAKI, Akira
NAKATA, Miyuki
TAMESHIGE, Toshiaki
TOYOKURA, Koichi
HARA, Reiko
MATSUMOTO, Miwako
NAKAMORI, Chihiro
SAKAGAMI, Mari

Plant organs, leaves, flowers, and roots show impressive,
symmetrical shapes, based on an ordered arrangement ofdifferentiated cells. The organs are formed from a group ofundifferentiated cells located at the tip of the stem or theroot. In the case of leaves, the process of organogenesis starts
with the formation of a leaf primordium in the peripheral
zone of the shoot apical meristem (SAM) at a fixed position,
following an order called phyllotaxis. Cells in theprimordium then proliferate and differentiate according tothree spatially fixed axes: the apical-basal axis, the lateral(central-marginal) axis, and the adaxial-abaxial (foresidebackside) axis. In the course of proliferation anddifferentiation, plant cells are believed to exchange
information with neighboring or separated cells in order toregulate organ architecture. We are trying to understand themechanisms of information exchange between plant cells
during the development of lateral organs, such as leaves,sepals, petals, stamens and carpels by using genetic,biochemical, microsurgical and one-cell gene induction
approaches.

I. Genetic approach

Recent studies of Arabidopsis mutants show several genes
are involved in the axes-dependent control of lateral organ
development. The adaxial- and abaxial-specific tissue
differentiation in the leaf primordium is determined by theprecise expression of the adaxial marker genes, HD-Zip III
including PHABULOSA (PHB), and the abaxial marker
genes, FILAMENTOUS FLOWER (FIL) and YABBY. We
showed that the adaxial-specific expression of PHB isregulated by the action of microRNA165/166 (miR165/166)
which targeted the HD-Zip III messenger RNAs. We also
revealed that expression of MIR165/166 genes is observed
only in the abaxial side. One of the MIR165/166 genes,MIR165A, is expressed preferentially in the abaxialepidermal cells (Figure 1). We also found that MIR165A isenough to suppress the expression of PHB in the cells
located in the abaxial side. These results suggested thatmiR165/166 might suppress the expression of PHB in some
non-cell-autonomous manner.
To examine the mechanisms of establishment of theadaxial-abaxial axis, we isolated novel mutants which show
altered patterns of FIL promoter:GFP expression, and named
them enlarged fil-expression domain (enf). One of them,
enf1, forms leaves with enlarged and reduced FIL-expression
domains. In the extreme cases, leaves are filamentous. Thisphenotype indicates that ENF1 is involved in the fixation or
maintenance of the position of the adaxial-abaxial boundary.
We revealed that the ENF1 gene encodes an enzyme
associated with primary metabolism, and that ENF1 isstrongly expressed in leaf primordia although its expression
was not found in the SAM. This indicates that ENF1catalized metabolite and/or its derivatives affect the axisdependent cell fate in leaf primordia. In contrast, anothermutant, enf2, has leaves with an enlarged FIL-expression
domain, and the ENF2 gene encoded a plastid-localized
unknown protein. Chloroplast development was repressed ina severe allele of the enf2 mutant. Exogenous application ofchloroplast development inhibitors to Arabidopsis seedlings
mimics the defects of the FIL-expression pattern by enf2mutation. These results suggest that chloroplast development
is required for normal differentiation of leaf tissues.
A line of unique oblong cells is found at the marginal edge

Figure 1. Expression domain of the MIR165A promoter:GFPer in
Arabidopsis leaf primordia. Transverse section of the transgenic plant isshown. MIR165A is expressed mainly in the cells at the abaxial
epidermis. GFP signal and autofluorescence of chloroplasts are indicatedby green and red, respectively. White asterisk is the position of the SAM,and white dashed line outlines a leaf primordium. Scale bar is 100 µm.

of leaves. We noticed that a homeobox-related gene,
PRESSED FLOWER (PRS) and its homolog, WOX1, arerequired for forming the margin-specific cells. prs wox1
double mutants completely lack the margin-specific cells inleaves, and interestingly, the abaxial side-specific epidermalcells, which are smaller than the adaxial side-specificepidermal cells, “invade” the adaxial side surface. The results
indicate that the margin-specific cells act as a physicalbarrier separating the epidermal cells of the adaxial-side
surface from those of the abaxial-side surface.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2010. The former title isindicated by an asterisk (*).

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National Institute for Basic Biology

Developmental Biology

To reveal how floral organs fix their forms through thedeveloping processes, we analyzed mutants named folded
petals (fop) (Figure 2). In the early stage of flower
development, fop petals are similar to those of wild type, but
the petals cannot grow through the narrow space between thesepal and anther in the flower buds. In fop mutants, petals
can grow straight when the sepals are removed in the earlystage of flower development. We also revealed that FOP
proteins are related to wax/cutin synthesis or transport. Thus,
we concluded that secreted wax/cutin on the petal epidermis
might be important for the precise development of petals.

This suggests a flow of signal(s) from the SAM to the leaf
primordia has a role of fixing the abaxial-adaxial regions inthat primordia. We are also examining the leaf serration
mechanism by ablating some cells at the margin in serrated
leaves of several plants.

IV. One-cell gene induction approach

As a new tool for examining the intercellularcommunication system, we are developing a one-cell geneinduction system in planta using the InfraRed Laser Evoked
Gene Operator (IR-LEGO) system, and showed gene
expression in only a single cell of the root. Currently, we areendeavoring to examine cell-to-cell communication in theSAM using this system.

Publication List

〔Original papers〕

S., Nishimori, Y., Yamada, M., Saito, H., Suzuki, T.,• Ishiguro,Nakagawa, T., Miyake, H., Okada, K., and Nakamura, K. (2010). The

Figure 2. fop flowers (right) have folded petals (white arrow heads),
while petals of wild type (left) elongate straightly.

II. Biochemical approach

We are taking a biochemical approach to study of theintercellular signaling system by analyzing small peptides ascandidates for intercellular signaling ligands, which arepresent in the apoplastic region of the SAM. Small peptides
were collected from apoplast fractions of floral buds ofArabidopsis apetala1 cauliflower double mutants and wereanalyzed by peptide sequence methods or LC-MS/MSmethods. We chose about 30 peptides as candidates, andprepared synthetic peptides based on the obtained sequences.
When applied to Arabidopsis seedlings, several peptides
caused morphological defects in the SAM, vascular tissue,
and root development. We are currently examining themechanisms involved. We are also isolating small peptides
from the apoplastic region in the heads of cauliflower(Brassica oleracea L. var. botrytis). Recently, we obtained
several purified fractions of the apoplastic region, which
affect axis-dependent leaf developments after exogenous
application to Arabidopsis seedlings, and are analyzing thepeptide sequences by LC-MS/MS methods.

III. Microsurgical approach

We are also carrying out microsurgical approaches usingnovel laser-ablating microscopy to investigate the cell-to-cellsignaling system working during leaf development. When weablated a small number of cells at the peripheral of the SAMof young Arabidopsis seedlings a few days after germination,some of the newly generated rosette leaves changed to afilamentous structure lacking the adaxial-abaxial identity.

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•••

Arabidopsis FLAKY POLLEN1 gene encodes a 3-hydroxy-3methylglutaryl-coenzyme A synthase required for development oftapetum-specific organelles and fertility of pollen grains. Plant Cell
Physiol. 51, 896-911.
Tsugeki, R., Ditengou, F.A., Palme, K., and Okada, K. (2010). NOVEIN facilitates auxin-mediated development in Arabidopsis. Plant
Signal. Behav. 5, 1249-1251.
Yuguchi, M., Yokouchi, T., Tominaga-Wada, R., Kuromori, T.,
Shinozaki, K., Okada, K., and Wada, T. (2010). Phenome analysis ofroot development in Arabidopsis. Plant Biotech. 27, 345-347.
Preston, J.*, Tatematsu, K.*, Kanno, Y., Hobo, T., Kimura, M.,
Jikumaru, Y., Yano, R., Kamiya, Y., and Nambara, E. (2009†). Temporal
expression patterns of hormone metabolism genes during imbibition ofArabidopsis thaliana Seeds: a comparative study on dormant and nondormant accessions. Plant Cell Physiol. 50, 1786-1800. (*: Equallycontributed)〔Review paper〕

K. (2010). Start of Arabidopsis research in Japan: a personal• Okada,memoir of the seedling stage 1985-2000. J. Plant Research 123, 267273.

† a paper that was not listed in the 2009 Annual Report