National Institute for Basic Biology

Developmental Biology

LABORATORY OF PLANT ORGAN DEVELOPMENT

Director-General

OKADA, Kiyotaka

Assistant Professor:
Postdoctoral Fellows:

Visiting Scientists:

Technical Assistants:

Secretary:

TATEMATSU, Kiyoshi
IGARASHI, Hisako
TOYOKURA, Koichi
URAWA, Hiroko
YABE, Kimihiko
IKEUCHI, Momoko
IWASAKI, Akira
NAKATA, Miyuki
TAMESHIGE, Toshiaki
TOYOKURA, Koichi*
HARA, Reiko
MATSUMOTO, Miwako
NAKAMORI, Chihiro
SUGIMOTO, Nagisa
TSUZUKI, Yumiko
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.

likely to move cell to cell. We also analyzed the function
domain of miR165 when primary transcript of MIR165A wasexpressed by the FIL-promoter. this revealed that miR165
can act in the entirety of the leaf primordia, suggesting that
there is no physical barrier interfering with miRNA
movement between the adaxial- and the abaxial-side.
To examine the mechanisms of establishment of theadaxial-abaxial axis, we isolated novel mutants which show
altered patterns of FIL promoter::GFP expression, andnamed them enlarged fil-expression domain (enf). One ofthem, enf1, forms leaves with enlarged and reduced FILexpression domains, indicating that ENF1 is involved in thefixation or maintenance of the position of the adaxial-abaxial
boundary (Toyokura et al., 2011). We revealed that the ENF1
gene encodes SUCCINIC SEMALDEHYDE
DEHYDROGENASE, which catalyzes the conversion ofsuccinic semialdehyde (SSA) to succinate, and is strongly
expressed in leaf primordia although its expression was notfound in the SAM. Exogenous application of SSA at the sideof the SAM induced the adaxial-characters on the abaxial
side of newly formed leaves (Figure 1). These results
indicate that SSA and/or its derivatives affect the axisdependent cell fate in leaf primordia. We also isolated some
suppressor mutants of enf1, which show normal leaf shapes.
We determined the genes, which have the mutation for thesuppression of enf1 phenotype, using next-generation
sequence methods, and revealed that one of them has themutation in a gene which encodes a transaminase enzyme.

I. Genetic approach

Recent studies of Arabidopsis mutants show several genes
are involved in the axes-dependent control of lateral organdevelopment. 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 markergenes, FILAMENTOUS FLOWER (FIL) and YABBY. Using
the reporter gene-system, we visualized the function domainof microRNA165/166 (miR165/166), which targeted theHD-Zip III messenger RNA, and showed that miR165/166
act in the cells locating in the abaxial side and determine theadaxial-specific expression of PHB. One of the MIR165/166
genes, MIR165A, is expressed in the abaxial epidermal cells.
We revealed by the visualizing reporter genes that MIR165A
is enough to repress the PHB expression in the cells locatedin the abaxial side. These results suggested that miR165 is34

Figure 1. Effect of exogenous application of SSA on the adaxial-abaxial
polarity in leaf primorda. We applied SSA-containing lanolin paste on theside of the Arabidopsis SAM, and checked the newly formed leaves after
1-2 weeks. Yellow arrowheads indicate the application site, and red
arrows represent the newly formed leaves. (A) The lateral views ofextreme effect (class I) of SSA taken by stereomicroscopy (left) andscanning electron microscopy (right). Newly formed leaves have theadaxial-characteristics on the abaxial-side. (B) The abaxial view of strong
effect (class II) of SSA. Newly derived leaf developed with two laminas
with upper sides facing each other. (C) The sections of the leaf petiols of
the control leaf (left) and class II leaf (right). In class II leaves, theadaxial identity was observed not only on the side facing the SAM but
also on the abaxial side.

In contrast, another mutant, enf2, has leaves with anenlarged FIL-expression domain, and the ENF2 gene

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

encoded a plastid-localized unknown protein. Chloroplastdevelopment was repressed in a severe allele of the enf2
mutant. Exogenous application of inhibitors for the gene
expression of chloroplast genome-encoding genes toArabidopsis seedlings mimics the defects of the FILexpression pattern by enf2 mutation. These results suggestthat the expression of chloroplast genome-encoding genes isrequired for the determination of adaxial-abaxial polarity.
A line of unique oblong cells is found at the marginal edgeof leaves. We noticed that a homeobox-related gene,
PRESSED FLOWER (PRS) and its homolog, WOX1, arerequired for forming the margin-specific cells. The analysesof prs wox1 double mutants indicate that the margin-specificcells act as a physical barrier separating the epidermal cellsof the adaxial-side surface from those of the abaxial-sidesurface. Genetic analyses also showed that PRS and WOX1
function in blade outgrowth downstream of the adaxialabaxial polarity. The expression of PRS and WOX1 wasobserved in two middle mesophyll layers and the marginal
region of leaf primordia. We also revealed that the expressionof PRS and WOX1 are upregulated by the function of FIL
and YAB genes, and repressed by those of the abaxial-specificgenes, KANADI. We propose that the blade outgrowth andthe adaxial-abaxial patterning during leaf developments arecontrolled by the middle domain-specific function of PRS
and WOX1 genes.
To reveal how floral organs fix their forms throughdevelopment processes, we analyzed mutants named foldedpetals (fop). In the early stage of flower development, foppetals are similar to those of wild type, but the petals cannot
grow through the narrow space between the sepal and theanther in the flower buds. In fop mutants, petals growstraight when the sepals are removed in the early stage offlower development. We found that the petal epidermal cellsof fop petals bear epicuticular nanoridges like as those ofwild type. FOP proteins are related to wax/cutin synthesis ortransport. Thus, we proposed that wax/cutin components
secreted by FOP proteins on the surface of the petal
epidermis might act as a lubricant before the epicuticularnanoridge formation.

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 the curds ofcauliflower (Brassica oleracea L. var. botrytis). Through abiological assay using purified fractions, we obtained afraction, which increased the number of SAMs after
exogenous application of the fraction to Arabidopsis
seedlings (Figure 2). Then we analyzed the peptide
sequences included in the fraction by LC-MS/MS methods,
and a putative lipid transfer protein was identified. We arepreparing a recombinant protein of the candidate protein andexamining effects of the recombinant protein on thedevelopment and growth of Arabidopsis seedlings.

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 SAM
of young Arabidopsis seedlings, some of the newly generated
leaves changed to a filamentous structure lacking theadaxial-abaxial identity, suggesting that a flow of signal(s)from the SAM to the leaf primordia has a role of fixing theabaxial-adaxial polarity. We also examined the leaf serration
mechanism by ablating some cells at the margin in serrated
leave of E. californica, which form the leaflet acropetally.
When leaflet incipient site is ablated, a new leaflet initiatedat intact tissue near leaf tip, escaping from the ablated site,
suggesting that a regular space of the leaflet initiation site isactively kept from the leaf tip.

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 InfarRed Laser Evoked
Gene Operator (IR-LEGO) system, and showed gene
expression in only a single cell of the root. When WUSSEL(WUS) gene, which functions in maintenance of the SAM,
was ectopically expressed in the root, callus or a shoot-likestructure was generated at the root tip. We observed
induction of ectopic cell division when WUS was ectopicallyexpressed in lateral-root-cap cells.

Publication List

〔Original papers〕

K., Watanabe, K., Oikawa, A., Kusano, M., Tameshige, T.,• Toyokura,Tatematsu, K., Matsumoto, N., Tsugeki, R., Saito, K., and Okada, K.

Figure 2. A purified fraction increasing the number of the SAMs in
Arabidopsis seedlings. Purified fractions obtained from the apoplastic
region of the curds of cauliflower were applied to Arabidopsis CLV3
promoter:GUS transgenic lines. A purified fraction increased the numberof the SAMs (Right) compared to the control (Left). GUS expression
driven by the CLV3 promoter indicates the position of the SAM. White
dashed lines indicate the shape of the SAM. Bars indicate 200 µm.

•(2011) Succinic semialdehyde dehydrogenase is involved in the robust
patterning of Arabidopsis leaves along the adaxial-abaxial axis. Plant
Cell Physiol. 52, 1340-1353.
Ueda, M., Matsui, K., Ishiguro, S., Kato, T., Tabata, S., Kobayashi, M.,Seki, M., Shinozaki, K., and Okada, K. (2011) Arabidopsis RPT2aencoding the 26S proteasome subunit is required for various aspects ofroot meristem maintenance, and regulates gametogenesis redundantlywith its homolog, RPT2b. Plant Cell Physiol. 52, 1628-1640.

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