National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISMS

Professor

Associate Professor

NISHIMURA, Mikio

HAYASHI, Makoto

Assistant Professor:

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
GOTO-YAMADA, Shino
OIKAWA, Kazusato
KANAI, Masatake
WATANABE, Estuko
TANAKA, Mina
KAMIGAKI, Akane
NITO, Kazumasa
GOTO-YAMADA, Shino*
NAKAI, Atsushi*
CUI, Songkui*
SHIBATA, Michitaro
SAITO, Miyuki
NAKAYAMA, Tomomi
HIKINO, Kazumi
YOSHINORI, Yumi
YAMAGUCHI, Chinami
NAKAI, Atsushi
CUI, Songkui
UEDA, Chizuru

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

Graduate Student:
Technical Assistant:

Secretary:

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.

pumpkin cotyledons, the reverse transition of leaf
peroxisomes to glyoxysomes occurs during senescence. Gene
expression, alternative splicing, protein translocation andprotein degradation control the functional transformation
between glyoxysomes and leaf peroxisomes.

II. Transcriptomics, proteomics and phenomicsof plant peroxisomes
Enzymes localized in plant peroxisomes are synthesized inthe 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 by
inducing pectin degradation under the control of abscisicacid signaling. The overall results provide us with new
insights into plant peroxisomal functions.
Bioinformatic 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 theI. 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 constitutive
organelles. Etioplasts differentiate into chloroplasts while
mitochondria acquire the ability to oxidize glycine.
Glyoxysomes, which are peroxisomes engaged in thedegradation of reserve oil stored in oil body (Figure 1) via
β-oxidation and the glyoxylate cycle, are transformed intoleaf peroxisomes that function in several crucial steps ofphotorespiration. After the functional transition ofglyoxysomes to leaf peroxisomes during the greening of

8

Figure 1. Oil body visualized by oleosin-GFP. (A) Five-day-old darkgrown seedlings expressing oleosin-GFP. (B) Numerous oil bodies in thecell of dry seed. (C) High-resolution image of aggregated oil bodies. Bar= 5 μm.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

receptor-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
recently proposed that function of PEX7 is maintained by aquality control mechanism involving RabE1c.

III. Identification of novel components
essential for peroxisome biogenesis
To better understand peroxisome biogenesis and functions,
we isolated a number of Arabidopsis mutants having aberrant
peroxisome morphology (apem mutants) based on themhaving a different pattern of GFP fluorescence from theparent plant, GFP-PTS1, in which peroxisomes with normal
sizes and numbers 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, which is involved in division ofboth organelles. APEM2 and APEM4 (whose defects cause adecrease in the efficiency of protein transport) were revealed
to encode proteins homologous to PEROXIN 13 (PEX13)and PEX12, respectively, and both proteins are responsible
for protein translocation on peroxisomal membranes.
APEM9 is the plant-specific PEX that has a role in tethering
the PEX1-PEX6 complex on peroxisomal membranes. Inaddition, we found that APEM3 encodes Peroxisomal
membrane protein 38, and that its defect causes enlargementof peroxisomes (Figure 2).

We are currently characterizing other apem mutants. Fromthese analyses, we will be able to identify the components
responsible for peroxisome biogenesis and functions, and toaddress the mechanism at the molecular level.

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
β-glucosidase PYK10. When plant cells are damaged,
PYK10 forms large protein aggregates. The aggregate
formation increases glucosidase activity, possibly producing
toxic products. Arabidopsis nai1 mutants have no ER bodies
in the entire plant and do not accumulate PYK10. NAI1
encodes a transcription factor and regulates the expressionof PYK10 and NAI2. The Arabidopsis nai2 mutant has no ERbodies and reduced accumulation of PYK10. NAI2 encodes aunique protein that localizes to the ER body. We found thatthe membrane protein of ER body 1 (MEB1) and MEB2 areintegral membrane proteins of the ER body. MEB1 andMEB2 have homology to yeast iron/manganese transporter
CCC1. Heterologous expression of MEB1 and MEB2 inyeast ccc1 mutant suppresses the iron toxicity (Figure 3),indicating that MEB1 and MEB2 are iron/manganesetransporters. These results suggest that the ER body has
specific membrane proteins that are involved in defense
against metal stress as well as pathogens and herbivores.
NAI2 deficiency relocates MEB1 and MEB2 to the ERnetwork. These findings indicate that NAI2 is a key factor
that enables ER body formation. We are now investigating
the function of NAI2 on ER body formation byheterologously expressing it in onion and tobacco cells.

V. 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. We have found that HSP90 inhibitor
induces genes with heat shock response element (HSE)

Figure 2. PMP38/APEM3 has a role as a transporter on the peroxisomal
membrane. (A, B) Compared to the parent plant (A), peroxisomes areenlarged in the apem3 mutant (B). (C) Subcellular localization andfunction of PMP38. PMP38-GFP (green) and RFP-PTS1 (magenta) weretransiently expressed in Arabidopsis leaf cells (Inset). PMP38 is present
as a dimer, and has a role in transport of NAD+, CoA and ADP. Scalebars, 10 μm (A, B) and (B) 1 μm (C).

Figure 3. MEB1 and MEB2 suppress the toxicity of iron. Yeast ironsensitive ccc1 mutants harboring empty vector, vector for MEB1, orMEB2 were grown on synthetic minimal medium with (upper) or without(lower) 2 mM iron.

9National Institute for Basic Biology

Cell Biology

motifs in their promoters, suggesting that heat shocktranscription factor (HSF) is involved 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. These data
indicate that HSP90 regulates correct gene expression forheat acclimatization in plants. We also observed that HSP90
is involved in hormone responses in Arabidopsis. The
evolutional and functional characterizations are now being
investigated.

ultrastructures of organelles by electron microscopy, soon. Itis expected that PODB2 will contribute to systems biology
through the combination of the included data with other‘omics’ data and computational analyses. In addition, weupdated the website, Plant Organelles World, which is basedon PODB2 as an educational tool to engage members of thenon-scientific community. We expect that PODB2 and Plant
Organelles World will enhance the understanding of plantorganelles among researchers and the general public whowant to explore plant biology.

VI. Update of The Plant Organelles Database 2(PODB2) and release of Plant Organelles
WorldPublication List

The Plant Organelles Database 2 (PODB2) was built topromote a comprehensive understanding of organelledynamics. This public database is open to all researchers.
PODB2 consists of five individual units: the perceptive
organelles database, the organelles movie database, theorganellome database, the functional analysis database, andexternal links. The perceptive organelles database, which
was added as new content, shows organelles dynamics inresponse to environmental stimuli (Figure 4). 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 to〔Original papers〕
M., Nanba, C., Saito, M., Kondo, M., Takeda, A., Watanabe,• Hayashi,Y., and Nishimura, M. (2012). Loss of XRN4 function can trigger••••cosuppression in a sequence-dependent manner. Plant Cell Physiol. 53,1310-1321.
Nakano, R.T., Matsushima, R., Nagano, A.J., Fukao, Y., Fujiwara, M.,Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2012). ERMO3/MVP1/GOLD36 is involved in a cell type-specific mechanism formaintaining ER morphology in Arabidopsis thaliana. PLoS ONE 7,e49103.
Nakayama, M., Kaneko, Y., Miyazawa, Y., Fujii, N., Higashitani, N.,
Wada, S., Ishida, H., Yoshimoto, K., Yamada, K., Nishimura, M., andTakahashi, H. (2012). A possible involvement of autophagy inamyloplast degradation in columella cells during hydrotropic responseof Arabidopsis roots. Planta 236, 999-1012.
Negishi, T., Oshima, K., Hattori, M., Kanai, M., Mano, S., Nishimura,M., and Yoshida, K. (2012). Tonoplast- and plasma membrane-localized
aquaporin-family transporters in blue hydrangea sepals of aluminum
hyperaccumulating plant. PLoS ONE 7, e43189.
Toyokura, K., Hayashi, M., Nishimura, M., and Okada, K. (2012).
Adaxial-abaxial patterning: A novel function of the GABA shunt. Plant
Signal. Behav. 7, 705-707.

〔Original papers (E-publication ahead of print)〕
K., Narise, T., Sonoike, K., Hashimoto, H., Sato, N., Kondo,• Kobayashi,M., Nishimura, M., Sato, M., Toyooka, K., Sugimoto, K., Wada, H.

•

Masuda, T., and Ohta, H. Role of galactolipid biosynthesis incoordinated development of photosynthetic complexes and thylakoid
membranes during chloroplast biogenesis in Arabidopsis. Plant J. 2012
Nov. 26.
Yamada, K., Nagano, A.J., Nishina, M., Hara-Nishimura, I., andNishimura, M. Identification of two novel endoplasmic reticulum bodyspecific integral membrane proteins. Plant Physiol. 2012 Nov. 19.

〔Review articles〕
S., Kimori, Y., Takeda, T., Miwa, T., Nishikawa, S., Mimura, T.,• Mano,Nagatani, A., and Nishimura, M. (2012). Using image-based resources:•Figure 4. The graphical user interface of the perceptive organelles
database in PODB2 (http://podb.nibb.ac.jp/Orgenellome).

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Databases for plant organelle dynamics and applications based onimage information. In Introduction to Sequence and Genome Analysis.
iConcept Press Ltd. ed. (Brisbane, iConcept Press Ltd.), chapter 16, pp.
1-25.
Tanaka, Y., Kimura, T., Hikino, K., Goto, S., Nishimura, M., Mano, S.,and Nakagawa, T. (2012). Gateway vectors for plant genetic
engineering: Overview of plant vectors, application for bimolecularfluorescence complementation (BiFC) and multigene construction. In
Genetic Engineering – Basics, New Applications and Responsiblities.
Barrera-Saldaña, H. A ed. (Rijeka, INTECH), pp. 35-58.