National Institute for Basic Biology

Cell Biology

LABORATORY OF ORGANELLE REGULATION

Associate Professor

MANO, Shoji

Specially Appointed Assistant Professor:
KANAI, Masatake
Visiting Scientist:
KAMIGAKI, Akane
OIKAWA, Kazusato
Technical Assistant:
HIKINO, Kazumi
HOSHI, Rie
Admin Support Staff:
UEDA, Chizuru
ASAI, Sanae

Because plants spread their roots in the ground, they must
survive in a given environment. To adapt to their environment, they utilized various signals generated from environmental changes as being necessary for their survival. Assuch, the flexibility of plant organelles is the basis for such
adaptation.
The aims of this laboratory are to clarify the molecularmechanisms underlying the induction, differentiation, andinteraction of organelles, especially peroxisomes and oilbodies, as well as to understand the integrated functions ofindividual plants through organelle dynamics.

Visual overview of this lab’s work.

26I. Molecular mechanisms of peroxisome dynamics
and functions in plant cells

Peroxisomes are single-membrane-bound organelles, which
are frequently present in eukaryotic cells, and are involvedin various biological processes such as lipid metabolism andphotorespiration. These functions change dramatically duringcertain developmental stages, and when they are confronted
with environmental changes. For example, light induces thetransformation of peroxisomes from glyoxysomes, which areperoxisomes engaged in the degradation of reserve oil stored
in the oil body via b-oxidation and the glyoxylate cycle, toanother type of peroxisome, leaf peroxisomes, that function
during several crucial steps of photorespiration. In addition
to functioning in vegetative tissues such as leaf and rootcells, it has been revealed that peroxisomes play essential
roles in reproductive processes. Studies using Arabidopsis
mutants defective in peroxisomal functions demonstratethat peroxisomes contribute to pollen fertility, pollen tube
elongation, and male–female gametophyte recognition. Gene
expression, alternative splicing, protein transport, protein
degradation and degradation of peroxisomes themselvescontrol these functions.
To better understand peroxisome biogenesis and functions, we isolated a number of Arabidopsis mutants that
displayed aberrant peroxisome morphology (apem mutants)
and unusual peroxisome positioning (peup mutants) basedon them having a different pattern of GFP fluorescencecompared to their parent plant, GFP-PTS1, in which peroxisomes with normal sizes, numbers and distribution
could be visualized with GFP (Figure 1). As of writing, wehave reported the functions of APEM1, APEM2, APEM3,APEM4, APEM9 and APEM10. Based on these results wewere able to update the model for protein transport, proliferation and quality control of peroxisomes via autophagy, using
these apem mutants in concert with the analyses of peup1,peup2, peup4, peup17 and peup22 mutants, which weredefective in Autophagy-related (ATG) genes.
We are also currently investigating other apem and peupmutants. Through these analyses, we will be able to identify
the components responsible for peroxisome biogenesis, functions and maintenance, and address the mechanism underlying this phenomenon at the molecular level.

Figure 1. Phenotype of Arabidopsis apem11 mutants. (A) GFP fluorescence was observed in the parent plant, GFP-PTS1, and apem11 mutants.
The apem11 mutant has enlarged peroxisomes in leaf cells, but not inroot cells. Bars, 20 µm. (B) The apem11 mutant showed dwarfism undernormal atmosphere, but grew normally under high CO2 atmosphere as
did the GFP-PTS1 plant.

II. Accumulation mechanism of seed storage
oils and proteins

Plant seeds accumulate huge amounts of storage reserves
such as oils, carbohydrates and proteins. Humans use
these storage reserves in food and industrial materials.
Storage reserves vary among different types of plantseeds. Wheat, maize and rice seeds mainly accumulate
starch, whereas rapeseed, pumpkin and sesame containlarge amounts of oils. Soybeans’ major reserve areproteins. Storage oils and proteins are synthesized in theendoplasmic reticulum (ER) and accumulated in oil andprotein bodies, respectively, during seed development.
We are currently analyzing the molecular mechanisms
controlling oil and protein contents in seeds (Figure 2).
Based on the analysis of the temporal sequence of oil andprotein synthesis during seed development in Arabidopsis
thaliana, which produces seeds containing approximately
30% oil and 30% protein, we revealed that the extensionof WRINKLED1 (WRI1), a transcription factor in fatty
acid biosynthesis, expression during the mid-phase of
seed development significantly enhanced seed oil content,
and caused seed sizes to enlarge.
We are also investigating the mechanisms of oil accumulation in other plant species. In the soybean (Glycine max.
L), we identified four lipases, GmSDP1s, on the oil bodymembrane. The analyses of GmSDP1s revealed that plantseeds have a mechanism required for the quality controlof fatty acids by degrading particular fatty acids in oilbodies.

Lipid synthesis

IV. C onstruction of The Plant Organelles
Database 3 (PODB3) and Plant Organelles
WorldThe Plant Organelles Database 3 (PODB3) was built
to promote a comprehensive understanding of organelle
dynamics (Figure 3), and, as a public database, it is open
to all researchers. PODB3 consists of six individual
sections: the electron micrograph database, the perceptive
organelles database, the organelles movie database, theorganellome database, the functional analysis database,and external links. The function of each database is asfollows:・The electron micrograph database provides information
concerning the ultrastructures in plant cells
・The perceptive organelles database shows organelles
dynamics responding to environmental stimuli
・The organelles movie database contains time-lapse
images and 3D structural rotations
・The organellome database is a compilation of static
image data of various tissues of several plant species atdifferent stages of development.
・The functional analysis database is a collection of protocols for plant organelle research
Through these databases, users can more easily comprehend plant organelle dynamics. Plant Organelles World,which is built based on PODB3, is an educational toolfor engaging members of the non-scientific community
to explore plant biology. We hope that both PODB3 andPlant Organelles World are of help to researchers as wellas the general public.

Lipid synthesis (µg/seed)Protein synthesis (µg/seed)Protein synthesis

Early

Mid

Late

Early

Mid

LateFigure 2. Biosynthesis of lipids and proteins during Arabidopsis seeddevelopment. Lipid synthesis and accumulation begins before those of
seed storage proteins during seed development.

III. Development of Gateway-technology vectors
for plant research

Gateway cloning is a useful and powerful technology whichallows the simultaneous generation of multiple constructs
containing a range of fusion genes. We have developed
various types of Gateway cloning-compatible vectors to
improve resources in the plant research field. Up until now,
we have provided vector sets to detect multiple proteinprotein interactions in vivo using multi-color bimolecularfluorescence complementation, and the binary vectors tofacilitate tripartite DNA assembly and promoter analysis withvarious reporters and tags in the liverwort Marchantia polymorpha. We will continue developing other useful Gateway
cloning-compatible vectors to contribute to the plant researchcommunity.

Figure 3. The graphical user interface of the PODB3 (http://podb.nibb.
ac.jp/Organellome).

27