National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISMS

Professor

Associate Professor

Assistant Professors:

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
OIKAWA, Kazusato
KANAI, Masatake
WATANABE, Etsuko
TANAKA, Mina
KAMIGAKI, Akane
GOTO-YAMADA, Shino
GOTO-YAMADA, Shino*
NAKAI, Atsushi
CUI, Songkui
SHIBATA, Michitaro
NISHINA, Momoko
ARAKI, Masami
SAITO, Miyuki
NAKAYAMA, Tomomi
HIKINO, Kazumi
MATSUDA, Azusa
YOSHINORI, Yumi
YAMAGUCHI, Chinami
UEDA, Chizuru

NISHIMURA, Mikio

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:Graduate Students:

Technical Assistants:

Secretary:

HAYASHI, Makoto

Since plants spread their roots in the ground, they must
survive in a given environment. To adapt to the environment,
they positively utilize environmental changes in the life cycle
as important signals that are necessary for their survival.
Plant cells can induce, degenerate and differentiate theirorganelles to adapt to environmental changes. The flexibilityof plant organelles is the basis of the strategy forenvironmental adaptation in plants.
The aim of this division is to clarify the molecularmechanisms underlying the induction, differentiation, andinteraction of organelles, and to understand the integratedfunction of individual plants through organelle dynamics.
The Scientific Research on Innovative Areas “Environmental
sensing of plants: Signal perception, processing and cellularresponses” was started to clarify the molecular mechanisms
underlying organelle differentiation and interaction.

I. R e v e r s i b l e t r a n s f o r m a t i o n o f p l a n t
peroxisomes

Dramatic metabolic changes that underlie the shift fromheterotrophic to autotrophic growth occur in the greening ofseed germination. Accompanying these metabolic changes
are the functional transformations of many constitutiveorganelles. Etioplasts differentiate into chloroplasts while
mitochondria acquire the ability to oxidize glycine.
Glyoxysomes, which are peroxisomes engaged in thedegradation of reserve oil via b-oxidation and the glyoxylate
cycle, are transformed into leaf peroxisomes that function inseveral crucial steps of photorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during thegreening of pumpkin cotyledons, the reverse transition of8leaf peroxisomes to glyoxysomes occurs during senescence.
Gene expression, alternative splicing, protein translocationand protein degradation control the functional transformation
between glyoxysomes and leaf peroxisomes.

II. Transcriptomics, proteomics and phenomicsof plant peroxisomes

Enzymes localized in plant peroxisomes are synthesized in
the cytosol and function after their post-translationaltransport into peroxisomes. Almost all of the peroxisomal
matrix proteins contain one of two targeting signals (PTS1and PTS2) within the molecules. PTS1 is a unique tripeptide
sequence found in the carboxyl terminus of the mature
proteins. In contrast, PTS2 is involved in a cleavable amino
terminal presequence of peroxisomal proteins that aresynthesized as a precursor protein with larger molecularmass.
We identified 256 gene candidates of PTS1- and PTS2containing proteins and another 30 genes of non-PTScontaining proteins from the Arabidopsis genome. Custommade DNA microarrays covering all these genes were used
to investigate expression profiles of the peroxisomal genes invarious organs. They revealed that peroxisomes in root cells
play a pivotal role in polyamine catabolism. We also made a
two-dimensional protein map of glyoxysomes and leaf
peroxisomes isolated from Arabidopsis and soybean. Peptide
MS fingerprinting analyses allowed us to identify novel
peroxisomal membrane proteins, i.e. voltage-dependentanion-selective channel and adenine nucleotide carrier 1(PNC1). We also found that peroxisomal membrane ATPbinding cassette transporter promotes seed germination byinducing pectin degradation under the control of abscisicacid signaling. The overall results provide us with new
insights into plant peroxisomal functions.

Bioinfomatic analysis of the Arabidopsis genome predicted
the presence of 15 kinds of genes, called PEX genes, forperoxisomal biogenesis factors. We demonstrated that PEX5and PEX7 form a cytosolic receptor complex and recognize
PTS1- and PTS2-containing proteins, respectively. PEX14 isa peroxisomal membrane docking protein that captures thereceptor-cargo complex. We also comprehensively
investigated whether or not these predicted PEX genes
function in peroxisome biogenesis by generating knockdown mutants that suppress PEX gene expression by RNAinterference. Phenotypes of these mutants allowed us toidentify the functional PEX genes, which can be classified
into two groups: PEX genes regulating for peroxisomal
protein import and PEX genes regulating for peroxisomal
morphology. We continue to investigate the detailed
molecular functions of other PEX genes. Of these, we
proposed that PEX10 is essential for the maintenance of ER
morphology and for biosynthesis of cuticular wax.

III. Identification of novel components
essential for peroxisome biogenesis

To better understand peroxisome biogenesis, we isolated anumber of Arabidopsis mutants having aberrant peroxisome
morphology (apem mutants) based on them having aNote: 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 (*).

different pattern of GFP fluorescence from the parent plant,
GFP-PTS1, in which peroxisomes with normal sizes andnumbers can be visualized with GFP.
Of these apem mutants, APEM1 gene (whose defect causes
the elongation of peroxisomes and mitochondria) encodes
dynamin-related protein 3A, one member of the dynaminfamily. APEM2 and APEM4 (whose defects cause a decreasein the efficiency of protein transport) were revealed toencode proteins homologous to PEX13 and PEX12,respectively, and both proteins are components of theprotein-translocation machinery on peroxisomal membranes.
In addition, we reported on characterization of other apem
mutants, apem3 and apem9. APEM3 encodes Peroxisomal
membrane protein 38, and its defect causes enlargement ofperoxisomes. APEM9 is the plant-specific PEX that has arole in tethering the PEX1-PEX6 complex on peroxisomal
membranes (Figure 1).

IV. ER derived organelles for protein storingand defense strategy

Plant cells develop various types of endoplasmic reticulum(ER)-derived structures with specific functions. ER bodies
are ER-derived compartments observed in Arabidopsis. They
are rod-shaped structures surrounded by ribosomes, and arewidely distributed in the epidermal cells of whole seedlings.
Undamaged rosette leaves have no ER bodies, but
accumulate ER bodies after wounding or jasmonic acidtreatment. This suggests that ER bodies function in thedefense against herbivores. ER bodies in seedlings include
PYK10, a b-glucosidase with an ER retention signal. Whenplant cells are damaged, PYK10 forms large protein
aggregates that include other b-glucosidases (BGLUs),GDSL lipase-like proteins (GLLs) and cytosolic jacalinrelated lectins (JALs). The aggregate formation increases
glucosidase activity, possibly producing toxic products(Figure 2). Arabidopsis nai1 mutants have no ER bodies inthe entire plant and do not accumulate PYK10. NAI1 encodes
a transcription factor that has a basic-helix-loop-helix(bHLH) domain and regulates the expression of PYK10 andNAI2. The Arabidopsis nai2 mutant has no ER bodies andreduced accumulation of PYK10. NAI2 encodes a uniqueprotein that localizes to the ER body. We found that themembrane protein of ER body 1 (MEB1) and MEB2 areintegral membrane proteins of the ER body. NAI2 deficiency
relocates MEB1 and MEB2 to the ER network. Thesefindings indicate that NAI2 is a key factor that enables ERbody formation. We are now investigating the function ofNAI2 on ER body formation by heterologously expressing itin onion and tobacco cells.

V. Vacuoles responsible for programmed celldeath in plants

The vacuolar processing enzyme (VPE) belongs to thecysteine protease family found in higher plants and animals.
VPE is responsible for the maturation of various types ofvacuolar proteins. We revealed a novel function of VPE invarious instances of programmed cell death (PCD) in plants.
VPE is identified as the proteinase that exhibits caspase-1

Figure 1. APEM9 is required for peroxisomal protein transport. (A)APEM9 knockdown mutants (apem9i) show growth defects. (B)Peroxisomal localization of GFP-PTS1 protein in wild type plants. (C)GFP-PTS1 protein is accumulated in the cytosol in apem9i mutants. (D)Working model of peroxisomal protein transport in plants, in which
APEM9 tethers PEX1-PEX6 complex to the peroxisomal membrane.

We are currently analyzing the functions of other APEM
proteins such as APEM10 and APEM11. From theseanalyses, we will be able to identify the components
responsible for peroxisome biogenesis, and to address themechanism at the molecular level.

Figure 2. Activation model of β-glucosidases in ER bodies ofArabidopsis. (A) Healthy plants accumulate β-glucosidases (BGLUs) in
the ER body, GDSL lipase-like proteins (GLLs) and glucosides in thevacuole, and jacalin-related lectins (JALs) in the cytosol. (B) When cellcollapse occurs following herbivore or pathogen attack, β-glucosidases
leak from the ER body and bind to JALs, which then form a large
aggregate with increased glucosidase activity. This complex hydrolyzes
glucosides to produce toxic compounds.

9National Institute for Basic Biology

Cell Biology

activity in plants. The plant hypersensitive response (HR), atype of defense strategy, constitutes well-organized PCD. NoHR occurs on the tobacco mosaic virus-infected leaves ofVPE-deficient tobacco plants. These results suggest that VPE
is involved in vacuolar collapse, which triggers PCD.
Using inhibitors for caspase-3 and the proteasome (alsoknown to affect animal cell death), we found that theactivities of both are required for bacterium-induced cell
death in plants. RNA interference-mediated silencing
confirmed that one of the three Arabidopsis proteasome
catalytic subunits, PBA1, is required for the fusion of thevacuolar and plasma membranes, which triggers PCD.
Plants evolve a death strategy mediated by vacuolar
systems, which are not seen in animals. Interestingly,
vacuoles are the key players in the plant-specific cell deathsystem.

Organelles World, which is based on PODB2 as an
educational tool to engage members of the non-scientificcommunity. We expect that PODB2 and Plant Organelles
World will enhance the understanding of plant organelles
among researchers and the general public who want to
explore plant biology.

VI. Roles of molecular chaperones on celldifferentiation

Molecular chaperones are cellular proteins that function in
the folding and assembly of certain other polypeptides into
oligomeric structures. To clarify the roles of molecularchaperones on cell differentiation, we have purified andcharacterized chaperonin and HSP70s and analyzed theirroles in the translocation of proteins into chloroplasts.
We found that HSP90 inhibitor induced genes with heatshock response element (HSE) motifs in their promoters,suggesting that heat shock transcription factor (HSF) isinvolved in the response. Arabidopsis HSFs interacted with
HSP90.2. Thus, it appears that in the absence of heat shock,
HSP90 actively suppresses HSF function. During heat shock,
HSP90 is transiently inactivated, which leads to HSF
activation. This data indicates that HSP90 regulates correct
gene expression for heat acclimatization in plants. We also
observed that HSP90 is involved in hormone responses inArabidopsis. The evolutional and functional characterizations
are now being investigated.

VII. Update of The Plant Organelles Database
2 ( P O D B 2 ) a n d re l e a s e o f P l a n t
Organelles WorldThe Plant Organelles Database 2 (PODB2) was built topromote a comprehensive understanding of organelle
dynamics. This public database is open to all researchers.
PODB2 consists of four individual units: the organelles
movie database, the organellome database, the functional
analysis database, and external links. The organelles movie
database contains time-lapse images and 3D structure
rotations. The organellome database is a compilation of static
image data of various tissues of several plant species atdifferent developmental stages. The functional analysis
database is a collection of protocols for plant organelle
research. The amount of included data is increasing day byday. We will add new content, which is dedicated toorganelle movement and morphology in response toenvironmental stimuli, soon. It is expected that PODB2 willcontribute to systems biology through the combination of theincluded data with other ‘omics’ data and computational
analyses. In addition, we released a new website, Plant

10Figure 3. The graphical user interface of the organelles movie database inPODB2 (http://podb.nibb.ac.jp/Orgenellome).

Publication List

〔Original papers〕

S., Mano, S., Nakamori C., and Nishimura, M. (2011).
• Goto,Arabidopsis ABERRANT PEROXISOME MORPHOLOGY 9 is a••••peroxin that recruits the PEX1-PEX6 complex to peroxisomes. Plant
Cell 23, 1573-1587.
Hino, T., Tanaka, Y., Kawamukai, M., Nishimura, K., Mano, S., andNakagawa, T. (2011). Two Sec13p homologs, AtSec13A and AtSec13B,
redundantly contribute to formation of COPII transport vesicles inArabidopsis thaliana. Biosci. Biotechnol. Biochem. 75, 1848-1852.
Mano, S., Miwa, T., Nishikawa, S., Mimura, T., and Nishimura, M.
(2011). The Plant Organelles Database 2 (PODB2): An updated
resource containing movie data of plant organelle dynamics. Plant Cell
Physiol. 52, 244-253.
Mano, S., Nakamori, C., Fukao, Y., Araki, M., Matsuda, A., Kondo, M.,
and Nishimura, M. (2011). A defect of peroxisomal membrane protein
38 causes enlargement of peroxisomes. Plant Cell Physiol. 52, 21572172.
Masuda, S., Harada, J., Yokono, M., Yuzawa, Y., Shimojima, M.,
Murofushi, K., Tanaka, H., Masuda, H., Murakawa, M., Haraguchi, T.,
Kondo, M., Nishimura, M., Yuasa, H., Noguchi, M., Oh-oka, H.,Tanaka, A., Tamiaki, H., and Ohta, H. (2011). A
monogalactosyldiacylglycerol synthase found in the green sulfur
bacterium Chlorobaculum tepidum reveals important roles forgalactolipids in photosynthesis. Plant Cell 23, 2644-2658.

〔Review Article〕

K., Hara-Nishimura, I., and Nishimura, M. (2011). Unique• Yamada,
defense strategy by the endoplasmic reticulum body in plants. Plant
Cell Physiol. 52, 2039-2049.