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ICES 2016 KYOTO
Sep 10th-14th, 2016
Location: Kyoto Prefectural Univ.
Registration & Welcome party: Sep 10th
Banquet: Sep 13th
Sponsors
Kyoto Prefectural University
Kyoto City and
the Kyoto Convention & Visitors Bureau
Leading Research Promotion Program
"Intercellular Chrono-communication" at
Chiba University
Life Science Foundation of Japan
Inoue Foundation for Science
Grant-in-Aid for Scientific Research
on Innovative Areas
Matryoshka-type Evolution
Kyoto Botanical Gardens
1
Congress(Venue
Inamori(Memorial(Hall,(Kyoto(Prefectural(University
稲盛記念会館 - Google Maps
2016/09/01 13:33
稲盛記念会館
Kitayama Station
Kyoto Botanical Garden
Kyoto Concert Hall
Inamori Memorial Hall
Kyoto Prefectural
University
Map data ©2016 Google, ZENRIN
■ 建物ゾーニング
100 m
From(JR(Kyoto(Sta?on(
Subway'(Karasuma-line)'to'Kitayama'Sta5on'(8th'stop.)'
and'7'min'walk'from'Exit'1.'
建物ゾーニング
1 階■ 府民利用・学生交流フロア
1 階府民利用・学生交流フロア
1st Floor
階段
出入口
WC
メインキッチン
出入口
メインキッチン
出入口
出入口
階段
階段
出入口
出入口
階段
自習室1
WC
WC
自習室1
事務室
事務室
101
講義室
(100人）
講師控室
WC
WC
WC
101
講義室
102
(100人）
講義室
WC
102
WC
WC
講義室
（170人）
講師控室
（170人）
EV
大廊下
大廊下
Poster Presentation
講義
・２０
EV
・１7
階段
・１０
EV
階段
Restaurant
稲盛記念
104
103
106
105105
103
104 104
稲盛記念
Welcome Reception
106
103
展示室
レストラン
(Room
講義室
講義室
講義室for
講義室
(Main
Hall)
出入口
Lunch
展示室
レストラン
講義室
講義室
講義室
講義室
出入口
(100人)
(100人)
（170人）
(200人）
Computer-Projecter
Coffee Break
メインホール
(100人)
(100人)
(200人）
test) （170人）
Light meal
https://www.google.co.jp/maps/place/〒606-0823+Kyōto-fu,+Kyōto-…6ad95:0x219732ffb4238511!8m2!3d35.0482354!4d135.7659457?hl=en
1/1 ページ
メインホール
(before ISE meeting)
メイン出入口
Entrance
メイン出入口
自習
稲盛
レス
事務
Entrance Hall
Registration desk (Sept. 10, 11)
Group Photo (Sept.12, Lunch Time)
Internet((
eduroam'Wi-Fi'is'available'in'the'Inamori'Memorial'Hall.'
稲盛記念展示室
レストラン
2 階学生講義室フロア
階段
稲盛記念展示室
2 階学生講義室フロア
104 講義室（
2
階段
201
講義室
(60人）
202
講義室
（60人）
レストラン
WC
自習室2
階段
大廊下
会議室
視聴覚室
講
(1
Time%Table%(Sept.%10%A%12)
Spt.%10
Saturday
Spt.%11
Sunday
Opening%Remarks
!Junichi!Obokata
9:00
Plenary%Lecture%3
Plenary%Lecture%1
John Archibald
(Chair: Yuji Inagaki)
10:00
Coffee Break
11:00
Mikio Nishimura
(Chair: Junichi Obokata)
Coffee Break
Session%1
Session%3
Gene transfer
Organellar gene expression
(Chair: Naoki Sato)
Group photo (Entrance Hall)
12:00
Spt.%12
Monday
Lunch
Lunch
13:00
Plenary%Lecture%2
Plenary%Lecture%4
Masamitsu Wada
Toshiya Endo
(Chair: Yoshiharu Yamamoto) (Chair: J rgen Soll)
14:00
15:00
Coffee Break
Coffee Break
Session%2
Poster%Presentation
Symbiosis and endosymbiosis
(Chairs: Ralf Oelmüller &
Ken Ishida)
16:00
Coffee Break
Session%2%(Continued)
17:00
18:00
(Chairs: Ralf Oelmüller &
Ken Ishida)
Registration
Welcome%Reception
19:00
(Restaurant of
the Inamori Memorial Hall)
Poster%Mounting
20:00
3
Light meal at the Restaurant
(Restaurant of the Inamori Memorial Hall)
ISE%Meeting
Time%Table%(Sept.%13%D%14)
Spt.%13
Tuesday
9:00
Spt.%14
Wednesday
Plenary%Lecture%5
Session%5
John F. Allen
(Chair: Mitsumasa Hanaoka)
Biogenesis of organelle
(Chair: Nobuyoshi Mochizuki)
10:00 Coffee Break
Session%4
Coffee Break
Nucleus-organelle interaction Session%6
Metabolisms and metabolite
and control
11:00
accumulation
(Chairs: John F. Allen &
(Chair: Uwe G. Maier)
Mitsumasa Hanaoka)
Lunch
12:00
Lunch
13:00
Excursion%(Guided%Tour) Session%7
14:00
Ryoanji Temple
|
Tojiin Temple
|
Kinkakuji Temple
15:00
Reductive evolution of
organelle
(Chair: Ryoma Kamikawa)
Coffee Break
Presentation%Ceremony%of%MiescherD
Ishida%Prize%and%other%awards
MiescherDIshida%Prize%Lecture
16:00
Uwe G. Maier
(Chair: Peter G. Kroth)
Closing%Remarks
Ralf%Oelmüller
17:00
18:00
19:00
Banquet
(The Sodoh Higashiyama
Kyoto)
(MC: Yoshiki Nishimura)
20:00
4
Excursion
Ryoanji Temple: This “Zen Buddhism temple”, a
UNESCO World Heritage Site, was established in
1450. The dry landscape garden, called
Karesannsui, consisting of fifteen rocks
surrounded by white gravel, is one of the most
beautiful gardens in Japan.
Toji-in Temple: Toji-in, founded in 1341, also has a
very beautiful garden. You can enjoy Japanese tea
and traditional sweets while viewing the garden.
Kinkakuji Temple: A UNESCO World Heritage Site,
Kinkakuji is one of the most famous temples in
Japan, the architecture which is also known as
“Golden Pavilion”. The wooden architecture is
covered in thin layers of pure gold and is
surrounded by a beautiful lake.
Itinerary
13:30
Departure from Kyoto Prefectural Univ. (20 minutes by bus)
13:50
Arrival at Ryoanji Temple
14:30
Departure (15 minutes-walk)
14:45
Arrival at Toji-in Temple
15:55
Departure (5 minutes by bus)
16:00
Arrival at Kinkakuji Temple
17:00
Departure
17:45
Arrival at a Parking area in Kodaiji Temple (Tour ends)
∗a few minutes-walk to the banquet venue
5
Banquet
Date & Time: 13 September 18:00- Venue: The Sodoh Higashiyama Tel: 075-541-3333 Access:
・ For those who attend the excursion, the bus will directly lead you to the restaurant after the tour. ・ For those who do NOT attend the excursion, a bus has been arranged that will leave Kyoto prefectural university (KPU) for the restaurant. Please come to the meeting room by 17:00. Otherwise, you can directly go to the restaurant for yourself, which is ~15 minutes’ walk from Yasaka shrine and close to Yasaka-no-tou. The room is “Pagoda”. (https://www.thesodoh.com/access/) *In case you have any problems, feel free to contact Yoshiki Nishimura (Cell phone: +81-80-5676-9327). 6
For oral presenters.
Please carry your computer to the conference hall, and connect it to the projector.
You can use VGA and HDMI cables.
The same projector and cables are set at the room next to the main hall, and you are
recommended to test your computer in the next room in advance of your talk.
Assigned time for presentation (20 ~ 30 min) includes discussions. Please leave ~3
minutes for discussions.
(bell: 15 min, 17 min and 20min, or 25 min, 27 min and 30 min)
（Projector information http://www.projectorcentral.com/TAXAN-PH-1001X.htm）
For poster presenters.
The size of the poster panel is 90 cm x150 cm.
Please put up your poster according to your poster number.
You are recommended to put up the poster in the evening of 10th or morning of 11th.
Posters are displayed throughout the conference.
Discussion time for posters is 14:50 - 17: 00 on 12th.
Young scientist awards.
The Local Organization Committee will give 6 awards.
・3 Best Presentation Awards for Young Scientists.
・3 Best Poster Awards for Young Scientists.
7
ICES 2016 Kyoto
2016. 9.10-14
The Program of Oral and Poster Presentations in ICES 2016 Kyoto
Saturday, September 10
18:00-21:00
Registration (Entrance Hall), Poster mounting (Hallway),
and Welcome Reception (Restaurant)
Sunday, September 11 9:00-9:05
Opening Remarks
Junichi Obokata
9:05-10:05
Plenary Lecture 1 (Chair: Yuji Inagaki)
John M. Archibald (Halifax): One plus one equals one: historical and modern
perspectives on endosymbiotic theory
10:05-10:25
Coffee Break (Restaurant)
10:25-
Session 1: Gene transfer 10:25-10:45
Ugo Cenci (Lille): Was the Chlamydial adaptive strategy to tryptophan starvation an
10:45-11:05
Shinichiro Maruyama (Sendai): The scope of the principle of parsimony in inferring
early determinant of plastid endosymbiosis?
the origins of evolutionary characters
11:05-11:25
Eriko Matsuo (Tsukuba): Differential impacts of plastid replacement on plastidal
biosynthetic pathways in dinoflagellates with non-canonical plastids, Karlodinium
veneficum and Lepidodinium chlorophorum
11:25-11:55
Junichi Obokata (Kyoto): How horizontal and endosymbiotic gene transfer occurs
from the time scale of minutes to million years
11:55-12:15
Group Photo (Entrance Hall)
12:15-13:30
Lunch
(Restaurant) 8
ICES 2016 Kyoto
2016. 9.10-14
13:30-14:30
Plenary Lecture 2
(Chair: Yoshiharu Yamamoto)
Masamitsu Wada (Tokyo): CHLOROPLAST PHOTO-RELOCATION MOVEMENT
The sophisticated and crucial phenomena for plant life
14:30-14:50
Coffee Break
(Restaurant)
14:50-
Session 2: Symbiosis and endosymbiosis (Chairs: Ralf Oelmüller& Ken Ishida)
14:50-15:10
Daniel Moog (Marburg): The role of peroxisomes in eukaryote-eukaryote
15:10-15:30
Mami Nomura (Shimoda): Fine-structural observations on engulfing behavior and
endosymbioses
enlargement of endosymbiont in Hatena arenicola
15:30-15:50
Toshinobu Suzaki (Kobe): Intracellular symbiosis of green algae in the ciliate
Paramecium bursaria with possible association with the host’s mitochondria
15:50-16:10
Przemyslaw Gagat (Wroclaw): Import of nuclear-encoded proteins into the
16:10-16:30
Shunichi
photosynthetic organelles of Paulinella chromatophora
Takahashi
(Okazaki):
Size-dependent
symbiont
specificity
in
cnidarian-dinoflagellate symbiosis
16:30-16:50
Coffee Break (Restaurant) 16:50-
Session 2: Continued 16:50-17:10
Cessa Rauch (Düsseldorf): Plastid retention in Elysia viridis is determined by algae
(Chairs: Ralf Oelmüller& Ken Ishida)
food source
17:10-17:40
Jürgen Steiner (Mautern): A c6-like cytochrome in the muroplast of Cyanophora
paradoxa
17:40-18:00
Maria Rosmarie Handrich (Düsseldorf): Comparative analysis of the response to high
18:00-18:20
Kai-Wun Yeh (Taipei): Isolation and characterization of effector genes during
light throughout plastid evolution
symbiotic interaction between Chinese cabbage and Piriformospora indica
18:20-18:50
Ralf Oelmüller (Jena): Fungi establishing mutualistic or pathogenic interactions with
roots
9
ICES 2016 Kyoto
2016. 9.10-14
Monday, September 12 9:00-10:00
Plenary Lecture 3 (Chair: Junichi Obokata)
Mikio Nishimura (Okazaki): Dynamics and the functional transition of plant
peroxisomes
10:00-10:20
Coffee Break
(Restaurant)
10:20-
Session 3: Organellar gene expression
10:20-10:40
Masayuki Nakamura (Nagoya): Coordination between the chloroplast psbA
(Chair: Naoki Sato)
5’-untranslated region and coding region activates the translation initiation
10:40-11:00
Marie-Kristin Sarah Lehniger (Berlin): Measuring the temperature-dependence of
cpRNP-RNA association in chloroplasts by a leaf-tissue based RIP-chip assay
11:00-11:20
Matthias Gordian Burger (Ulm): Elucidating target RNA editing sites of embryo
11:20-11:40
Anke-Christiane Hein (Bonn): Organelle RNA editing sites and their nuclear
lethal PLS class PPR proteins
specificity factors: Co-evolution among angiosperms and beyond
11:40-12:00
Bastian Oldenkott (Bonn): RNA editing in early land plants: the birds of paradise and
the workhorse
12:00-13:30
Lunch (Restaurant)
13:30-14:30
Plenary Lecture 4
(Chair: Jürgen Soll)
Toshiya Endo (Kyoto): Mitochondrial biogenesis through protein and lipid transport
14:30-14:50
Coffee Break
(Restaurant)
14:50-17:00
Poster Presentation
17:00- Light meal (sandwich & coffee) (Restaurant)
17:30
ISE Meeting
10
ICES 2016 Kyoto
2016. 9.10-14
Tuesday, September 13 9:00-10:00
Plenary Lecture 5 (Chair: Mitsumasa Hanaoka)
John F. Allen (London): Why chloroplasts and mitochondria retain their own genomes
and genetic systems: co-location for redox regulation of gene expression
10:00-10:20
Coffee Break (Restaurant) 10:20-
Session 4: Nucleus-organelle interaction and control
(Chair: John F. Allen & Mitsumasa Hanaoka)
10:20-10:40
Mitsumasa Hanaoka (Chiba): Chloroplast-dependent nuclear gene regulation:
10:40-11:10
Yoshiharu Y. Yamamoto (Gifu): Empirical identification of the transcriptional network
evolution of retrograde signaling
for H2O2 responses in Arabidopsis
11:10-11:30
Yoshiki Nishimura (Kyoto): Restructuring of chloroplast nucleoids by eukaryotic
factors during the green plant evolution
11:30-11:50
Stephan Greiner (Potsdam): Biparental inheritance of chloroplasts is controlled by
11:50-12:10
Takumi Arakawa (Sapporo): Post-translational regulation in nuclear-mitochondrial
lipid biosynthesis
interaction of cytoplasmic male sterility in sugar beet
12:10-13:30
Lunch
(Restaurant)
13:30-18:00
Excursion
(Ryoanji Temple ~ Tojiin Temple ~ Kinkakuji Temple / Guided Tour )
18:00-20:00
Banquet
(The Sodoh Higashiyama Kyoto) (MC: Yoshiki Nishimura )
11
ICES 2016 Kyoto
2016. 9.10-14
Wednesday, September 14 9:00-
Session 5: Biogenesis of organelle (Chair: Nobuyoshi Mochizuki)
9:00-9:30
Jürgen Soll (Munich): The plastid reticulum reloaded
9:30-9:50
Jasmine Theis (Kaiserslautern): Identification of factors involved in photosystem
9:50-10:10
Jörg Rolf Nickelsen (Munich): Biogenesis of thylakoid membranes
10:10-10:30
Coffee Break
10:30-
Session 6: Metabolisms and metabolite accumulation 10:30-11:00
Peter G. Kroth (Konstanz): Deciphering the chrysolaminarin biosynthetic pathway in
biogenesis using a forward genetics approach
(Restaurant)
(Chair: Uwe G. Maier)
the diatom Phaeodactylum tricornutum using molecular tools
11:00-11:20
Naoki Sato (Tokyo): Non-endosymbiotic origin of triacylglycerol accumulation: a
model of "chloroplast oil body" in Chlamydomonas reinhardtii
11:20-11:40
Md. Shafiqul Islam (Dhaka): New bioremediation technique for radioactive
cesium-contaminated soil using Paramecium bursaria
11:40-13:30
Lunch
(Restaurant)
13:30-
Session 7: Reductive evolution of organelle (Chair: Ryoma Kamikawa)
13:30-13:50
Goro Tanifuji (Tokyo): Plastid comparative genomics elucidates multiple independent
losses of photosynthesis in Cryptomonas (Cryptophyta)
13:50-14:10
Takuro Nakayama (Tsukuba): Phylogenetic positions of marine gregarines Selenidium
terebellae and Lecudina tuzetae, and molecular evidence of their remnant
nonphotosynthetic plastids (apicoplasts)
14:10-14:30
Herbert J. Santos (Tokyo): Screening and discovery of lineage-specific mitosomal
14:30-14:50
Ryoma
membrane proteins in Entamoeba histolytica
Kamikawa
(Kyoto):
Rare
loss
of
the
Calvin
Benson
non-photosynthetic plastids
14:50-15:10
Coffee Break
(Restaurant)
15:10-15:40
Presentation Ceremony of Miescher-Ishida Prize and other awards
15:40-16:40
Miescher-Ishida Prize Lecture
(Chair: Peter G. Kroth)
Uwe G. Maier (Marburg): Protein and metabolite transport in diatoms
16:40
Closing Remarks
Ralf Oelmüller
12
cycle
in
ICES 2016 Kyoto
2016. 9.10-14
Poster presentations
(*Asterisks indicate presentations also given by oral)
P1
Mamoru Sugita (Nagoya): On the expansion of the plastid RRM protein family in the Viridiplantae
P2 *(Oral, Session 2)
Cessa Rauch (Düsseldorf): Plastid retention in Elysia viridis is determined by algae food source
P3
Naoki Sato (Tokyo): Direct evidence for the presence of enigmatic peptidoglycan in the intermembrane
space of chloroplast envelope in the moss Physcomitrella patens
P4
Hidefumi Hamasaki (Yokohama): SnRK1 kinase and the NAC transcription factor SOG1 as possible
components of a mitochondrial retrograde signaling pathway mediating the low energy response triggered
by low ATP levels
P5
Junpei Fukumoto (Tokyo): Elucidating the mechanism of host mitochondrial recruitment of Toxoplasma
gondii
P6
Viktoria Schreiber (Marburg): The central vacuole of the diatom Phaeodactylum tricornutum:
Identification of new tonoplast proteins and a functional di-leucine motif
P7
Danijela Kozul (Potsdam): The plastome/nuclear-genome incompatibility in the evening primrose
Oenothera
P8
Euki Yazaki (Tsukuba): A phylogenomic study placed a previously undescribed eukaryote, strain
SRT308, at the base of the Euglenozoa clade.
Marie-Kristin Lehniger (Berlin): Measuring the temperature-dependence of cpRNP-RNA association in
chloroplasts by a leaf-tissue based RIP-chip assay
P10
Ryosuke Miyata (Tsukuba): Gregarine-like apicomplexan parasite isolated from the intestinal tract of a
centipede Scolopocryptops rubiginosus
13
ICES 2016 Kyoto
2016. 9.10-14
P11
Natsuki Hayami (Gifu): Metabolic dynamics and transcriptional regulation in temperature adaptation of
Arabidopsis
P12
Mayumi Kobayashi (Kobe): Molecular basis for β-1,3-glucan mediated self/nonself recognition in
unicellular protists
P13
Chinatsu Tsukakoshi (Tsukuba): Transcriptome analysis of Chrysochromulina parkeae, a haptophyte
with a cyanobacterial endosymbiont
P14
Chien-Hao Tseng (Taipei): The endophytic fungus Piriformospora indica enhances drought stress
tolerance in rice plants
P15
Rina Higuchi (Kobe): Changes in ultrastructure and chemical composition of the cell wall of Chlorella in
relation to endosymbiosis in Paramecium bursaria
Mami Nomura (Tsukuba): Fine-structural observations on engulfing behavior and enlargement of
endosymbiont in Hatena arenicola
P17
Jonny Gentil (Marburg): ER-subcompartmentalization allows the separation of the unfolded protein
response from protein transport into complex plastids
P18
Ayako Okuzaki (Machida): The acidic domain of Arabidopsis chloroplast CP31A is essential for
providing cold resistance
P19 *(Oral, Sesion3)
Matthias Burger (Ulm): Elucidating target RNA editing sites of embryo lethal PLS class PPR proteins
P20
Kumiko Kihara (Yatsushiro): Single-cell metagenomics of the termite-gut protozoa, Mixotricha
paradoxa
14
ICES 2016 Kyoto
2016. 9.10-14
P21
Keitaro Kume (Tsukuba): Prediction of Mitochondrial or Mitochondrion-related Organelle Proteins in
Non-model Organisms using Machine Learning
P22
Arisa Watanabe (Tsukuba): Evolution of organellar DNA polymerases in Chlorarachniophyte algae
P23
Yu Uchiumi (Hayama): Evolution of vertical transmission of symbionts by reducing the rate of cell
division
Herbert J. Santos (Tokyo): Screening and discovery of lineage-specific mitosomal membrane proteins in
Entamoeba histolytica
P25
Shigekatsu Suzuki (Tsukuba): Identification and transcriptome analysis of a non-photosynthetic
eukaryovorous amoeba phylogenetically related to chlorarachniophytes
P26
Yuki Hanadate (Tsukuba): Discovery of a mitosome-related compartment in Entamoeba histolytica
P27
Hirokazu Sakamoto (Tsukuba): Development of dual-transfection system in Perkinsus marinus
P28
Motomichi Matsuzaki (Tokyo): Conformation of mitochondrial DNA in the oyster parasite Perkinsus
marinus
P29
Soichirou
Satoh
(Kyoto):
DNA-barcode
technology
reveals
integration-dependent
stochastic
transcription activation of transgenes in the plant genome: its implication for the molecular basis of EGT
and HGT
P30
Mitsuhiro Matsuo (Kyoto): Evolutionary roles of SL-trans-splicing in the primary endosymbiosis
15
Plenary Lectures
16
PL1
One plus one equals one: historical and modern perspectives on endosymbiotic theory
John M. Archibald1,2,3
1
Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada;
Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, Nova
Scotia, Canada; 3Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity,
Toronto, Ontario, Canada
2
The evolution of the eukaryotic cell is a puzzle that has challenged biologists for over a century. The
prokaryote-eukaryote cellular divide is enormous and many fundamental questions about the origin of
eukaryotes and their endosymbiotically derived mitochondria and plastids remain unanswered. Comparative
genomics is a powerful tool with which to address such questions. Here I will place the modern-day study of
cellular evolution in a historical context, highlighting advances and lingering uncertainties in our
understanding of the role of endosymbiosis in the evolution of eukaryotic organelles.
17
PL2
CHLOROPLAST PHOTO-RELOCATION MOVEMENT
The sophisticated and crucial phenomena for plant life
Masamitsu Wada
Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan
University, Minamiosawa 1-1, Hachioji, Tokyo 192-0397, Japan. email: masamitsu.wada@gmail.com
Chloroplast photorelocation movement is a well-known phenomenon since late 19th century. However, the
precise mechanisms and chloroplast behaviors had not been analyzed until recently. Chloroplasts move to an
area irradiated with more light under weak light condition to perform efficient photosynthesis (accumulation
response), but move away from strong light to avoid photodamage of chloroplasts (avoidance response)
following the plants’ death. We analyzed chloroplast behaviors using the fern Adiantum capillus-veneris
gametophytes that have a long linear protonema cell and a young single-cell-layered prothallus cell. To find
out molecular factors for chloroplast movement, we screened mutants deficient in chloroplast movement
from Arabidopsis thaliana. I will talk on the importance of chloroplast movement for a plant life and the
mechanism of the phenomena.
Chloroplast movement is mediated by blue light receptor, phototropins, in general and a red and blue
light-absorbing neochrome in ferns. Chloroplasts are able to move to any direction by sliding, but not by
rolling or turning, using fine actin filaments specialized for chloroplast movement. The speed of movement
is about 1 µm min-1 or less. CHUP1 is the most important factor for polymerization and/or maintenance of
the actin filaments. The signal(s) connecting the photoreceptors and chloroplasts is not known, but some
characteristics are revealed. The signal for accumulation response has a longer life than that of avoidance
response, and can be transferred longer distance compering to that of avoidance response. The speed of
signal transfer is about 1 µm min-1, which is similar to the speed of chloroplast movement controlled by the
ratio of actin filament amounts in front and rear sides of chloroplasts.
Nuclei also show light-mediated movement like chloroplasts. Recently we found that nuclei do not move
by themselves but chloroplasts carry the nucleus. I will talk on this issue, if I have time.
18
PL3
Dynamics and the functional transition of plant peroxisomes
Mikio Nishimura1, Kazusato Oikawa1,2, Shino Goto-Yamada1,3, Cui Songkui1,4, Shoji Mano1
1
National Institute for Basic Biology, Okazaki 444-8585, Japan. email: mikosome@nibb.ac.jp; 2 Faculty of
Agriculture, Niigata University, Niigata 950-2181, Japan; 3Jagiellonian University, Krakow 30-387, Poland;
4
Nara Institute of Science and Technology (NAIST), Nara 630-0192, Japan
Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type by
expression of organ-specific genes. Glyoxysomes contain glyoxylate cycle enzymes and function in
gluconeogenesis in etiolated cotyledons. Leaf peroxisomes accumulated in glycolate cycle enzymes and
involve in photorespiration in leaves. The functional differentiation of glyoxysomes to leaf peroxisomes is
observed in greening process of etiolated cotyledon. To understand the functional differentiation of
peroxisomes, we isolated a number of Arabidopsis mutants having aberrant morphology (apem) and different
function of (ped) peroxisomes. From the analyses of these peroxisomal mutants, we revealed that proteolysis
of unnecessary protein by LON protease2 and autophagy are responsible for the functional differentiation of
peroxisomes(1-3). We also show dynamics and direct adhesion between peroxisomes and chloroplasts or oil
bodies by visualization of peroxisomes in the leaves(4-5).
References:
1) Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.
Shibata, M. et al. Plant Cell 25: 4967-4983 (2013)
2) Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation
with autophagy.
Goto-Yamada, S. et al. Plant Cell Physiol. 55(3): 482-496 (2014)
3) Dynamics of the light dependent transition of plant peroxisomes.
Goto-Yamada, S. et al. Plant Cell Physiol. 56(7): 1264-1271(2015)
4) Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis.
Oikawa K. et al. Nature Plants. 1: 15057 (2015)
5) Sucrose production mediated by lipid metabolism suppresses physical interaction of peroxisomes and oil
bodies during germination of Arabidopsis thaliana.
Cui S. et al. J. Biol. Chem. in press (2016)
19
PL4
Mitochondrial biogenesis through protein and lipid transport
Toshiya Endo1
1
Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan
Mitochondria perform central functions in cellular energetics, metabolism and signaling.
Since
mitochondria are only generated by growth and division of preexisting mitochondria, mitochondrial growth
relies on import of their resident proteins as well as transport and synthesis of phospholipids. Thus, protein
import and lipid transport constitute the central processes of mitochondrial biogenesis and maintenance.
Mitochondrial protein transport is mediated by the translocators in the outer and inner mitochondrial
membranes and by soluble factors in the cytosol, intermebrane space, and matrix. I will discuss how these
translocators and soluble factors cooperate to achieve precise as well as efficient transport of over 1,000
different mitochondrial proteins from the cytosol to different sub-compartments within mitochondria. How
hydrophobic phospholipid molecules can traverse aqueous compartments to shuttle between different
membranes is also a critical problem concerning the mechanism of organelle biogenesis. I will discuss how
different lipid transport machineries mediate transport of hydrophobic phospholipids between the ER and
mitochondria and between the outer and inner mitochondrial membranes by different mechanisms.
20
PL5
Why chloroplasts and mitochondria retain their own genomes and genetic systems: Co-location for
Redox Regulation of gene expression
John F. Allen
Research Department of Genetics, Evolution and Environment, Darwin Building, University College London,
Gower Street, London WC1E 6BT, United Kingdom. email: j.f.allen@ucl.ac.uk
Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic
systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of
characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of
chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their
cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and
precursor proteins that are synthesized in the cytosol for import into the organelle into which the
endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within
chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression
of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change
by means of a direct and unconditional regulatory control—control exerted by change in the redox state of
the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox
regulatory system has to be retained within its original membrane-bound compartment. Co-location of gene
and gene product for Redox Regulation of gene expression (CoRR) is an hypothesis in agreement with the
results of a variety of experiments designed to test it and that seem to have no other satisfactory explanation.
I present evidence relating to the CoRR hypothesis, and consider mechanisms of redox regulation in
chloroplasts. I discuss the development, conclusions, and implications of the CoRR hypothesis, and identify
predictions concerning the results of experiments that may yet prove it to be incorrect.
21
Miescher-Ishida
Prize Lecture
22
MIP
Protein and metabolite transport in diatoms
Uwe Maier
Philipps University Marburg, Cell Biology & LOEWE Center for Synthetic Microbiology (SynMikro)
Diatoms evolved via secondary endosymbiosis. Here, a red alga was engulfed by another eukaryote and
reduced to a complex plastid surrounded by four membranes. As most of the plastid proteome is encoded in
the cell nucleus, hundreds of nucleus-encoded proteins have to cross several plastid surrounding membrane
barriers to operate properly in the correspondent compartments of the plastid. The Marburg lab is interested
in protein transport mechanisms. Over the past years, we have investigated the protein and metabolite
translocators acting in the plastid surrounding membranes. Our findings highlight that no novel inventions
were made for protein import into complex plastids, instead pre-existing transport mechanisms were recycled
for novel tasks.
23
Oral Sessions
24
Session 1
Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid
Endosymbiosis?
Ugo Cenci1, Mathieu Ducatez1, Derifa Kadouche1, Christophe Colleoni1 and Steven G. Ball1
1
Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 Centre National de la Recherche
Scientifique, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France
Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a
common inclusion. The intracellular pathogens are thought to have donated those critical transporters that
triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also
suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for
photosynthetic carbon assimilation in the eukaryotic cytosol. We now turn our attention to the reasons
underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In
particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of
chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of
replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as
antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon
to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have
allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It
would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan
hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this
amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli
TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably
strengthening our proposal.
25
Session 1
The scope of the principle of parsimony in inferring the origins of evolutionary characters
Shinichiro Maruyama, Yukari Suzuki-Ohno, Masakado Kawata
Graduate School of Life
maruyama@tohoku.ac.jp
Sciences,
Tohoku
University,
Sendai
980-8578,
Japan.
email:
The principle of parsimony provides a useful guideline for inferring the origins of evolutionary characters
and/or events, including (endo)symbiosis, by suggesting that we should choose the simplest explanation
unless extra assumptions are necessary. However, when and in what conditions we should employ the
principle of parsimony are not evident a priori in many cases. Here, based on the Sober’s probabilistic model
on character evolution (Sober 1988), we implemented an additional parameter representing the
time-dependent decreasing rate of the probability of a character change, which determines how easily the
character state can be changed at a given time and makes it possible to fit the model to more realistic
situations. Simulation analyses using the model indicated that, compared to ancestral characters, derived
characters were more probably retained after a substantial number of generations in wider parameter ranges
than those shown in previous studies. Furthermore, even in the case where the initial probabilities of
character gain and loss were the same, the time-dependent decreasing rate of the character change probability
could be a factor to make it more probable that derived characters were more probably retained after
simulated generations. These results suggest that it is critical to understand biological processes
accompanying the evolutionary events and describe the model and the parameters more precisely in inferring
the origin and history of character evolution.
26
Session 1
Differential impacts of plastid replacement on plastidal biosynthetic pathways in dinoflagellates with
non-canonical plastids, Karlodinium veneficum and Lepidodinium chlorophorum.
Eriko Matsuo1, Yuji Inagaki1,2
1
Graduate School of Environmental and Biological Sciences, University of Tsukuba, Ibaraki, 305-8577,
Japan; 2Center for Computational Sciences, University of Tsukuba, Ibaraki, 305-8577, Japan; email:
yuji@ccs.tsukuba.ac.jp
The common ancestor of dinoflagellates most likely established the plastid through a red algal
endosymbiosis, and the vast majority of proteins work in the plastids (plastidal proteins) are encoded in their
nuclear genomes. Unlike any other eukaryotic algal groups, multiple independent lineages in dinoflagellates
replaced the original plastids by those acquired through the endosymbioses of phylogenetically diverse
eukaryotic algae. Previously published studies on the dinoflagellates with non-canonical plastids
demonstrated that a portion of the original set of plastidal proteins was replaced by the homologues acquired
from the endosymbionts. In this study, we focused on the evolutions of Chlorophyll a (Chl-a) biosynthetic
pathway and MEP/DOXP pathway for isopentenyl-diphosphate (IPP) biosynthesis in two dinoflagellates,
Karlodinium veneficum bearing a haptophyte-derive plastid and Lepidodinium chlorophorum bearing a green
alga-derive plastid. As the two pathways have been localized in the plastid beyond the plastid replacements,
it is intriguing to evaluate the impact of plastid replacement on the two metabolic pathways in the
dinoflagellates bearing non-canonical plastids. We surveyed the genes encoding the proteins of interest in the
transcriptomic data of the two dinoflagellates, and assessed their evolutionary origins individually by
maximum-likelihood phylogenetic analyses. Most or all of the proteins involved in MEP/DOXP pathway in
Karlodinium and Lepidodinium appeared to share the evolutionary ancestries with the homologues in
dinoflagellates bearing typical plastids, suggesting that plastid replacement gave no large impact on this
particular pathway. On the other hand, Chl-a biosynthetic pathway in Karlodinium was most likely
reorganized during plastid replacement. The Karlodinium pathway appeared to be occupied by ‘haptophyte
proteins,’ which were most likely acquired from the haptophyte endosymbiont that gave rise to the current
non-canonical plastid in this species. Similarly, the same pathway in Lepidodinium may have been also
reorganized, but in a different manner from the Karlodinium pathway. The Lepidodinium pathway seemingly
comprises the proteins acquired from phylogenetically diverse eukaryotes, rather than green algae that are
close relatives of the endosymbiont engulfed by the ancestral Lepidodinium. In this presentation, we will
propose a hypothesis to explain why plastid replacement reorganized Chl-a pathway intensely in both
Karlodinium and Lepidodinium, while little/weak impact on MEP/DOXP pathway was observed in either of
the two dinoflagellates.
27
Session 1
How horizontal and endosymbiotic gene transfer occurs from the time scale of minutes to million years
Junichi Obokata1
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8552, Japan.
email: obokata@kpu.ac.jp
Functional gene transfers between the organisms or organelles have greatly contributed to the evolutionary
innovation and diversification. However, we still have little knowledge about the molecular basis of these
phenomena. How do the transferred gene sequences become transcriptionally active and functional in the
alien genome environment? In this talk, I overview the recent studies of our research group with special
emphasis on two topics, and try to illustrate the HGT/EGT from the time scale of hours to million years.
The first topic concerns the mechanism by which newly translocated gene sequences acquire
transcriptional competence in the eukaryotic nuclear genome. To address this question, we became interested
in the similarity of the promoter-acquisition event between the gene-trap screening and HGT/EGT. Therefore,
we attempted a massive promoter trap experiment using a promoterless luciferase (LUC) gene in the plant
genome, assuming it as a model experiment of HGT/EGT. The obtained results were somewhat surprising.
Contrary to the widely accepted idea of promoter trap screenings, the great majority of the transcribed LUC
transgenes had nothing to do with the preexisting genes but did de novo transcription by itself. Further
analysis of this phenomenon suggests that this novel transcription should have occurred by the chromatin
remodeling accompanying the chromosomal integration of the LUC genes. Based on these findings, we
propose a new hypothesis for the initial transcription mechanism of the translocated genes in this meeting.
The second topic concerns the possible roles of the post-transcriptional regulation in establishing the
functional gene transfer, based on our analysis of a photosynthetic amoebae Paulinella chromatophora.
For the details of the first and second topics, please refer to the posters by Satoh et al. and by Matsuo et
al. in the poster session.
28
Session 2
The role of peroxisomes in eukaryote-eukaryote endosymbioses
Daniel Moog1,2, Bruce A. Curtis2, Goro Tanifuji3, Marlena Dlutek2, John M. Archibald2
1
Laboratory for Cell Biology, Philipps University Marburg, Karl-von-Frisch-Str. 8, 35043 Marburg,
Germany, 2Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper
Medical Building, 5850 College Street, Halifax, Nova Scotia, B3H 1X5 Canada, 3Department of Zoology,
National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba, Ibaraki, 305–0005 Japan
email: daniel.moog@biologie.uni-marburg.de
Peroxisomes are membrane-bound subcellular compartments present in most eukaryotic cells. Typical
peroxisomal functions include fatty acid beta-oxidation and detoxification of reactive oxygen species, but
peroxisomes are known to have an enormous metabolic versatility. Their abundance, shape and enzymatic
content can be highly dynamic and is rapidly adaptable to the requirements of cellular metabolism,
developmental stages and environmental signals. Whereas peroxisomal cell biology and the implication of
these organelles on metabolic networks are relatively well studied in model organisms such as fungi, higher
plants and animals, the presence and functions of peroxisomes in the majority of eukaryotes are enigmatic.
We are studying the role of peroxisomes and their derivatives in the evolution of metabolic
compartmentalization in eukaryotic cells with special focus on organisms that evolved via endosymbiotic
partnerships between two different eukaryotic cell types. Here we report molecular evidence for the presence
of two divergent, spatially separated peroxisomal compartments in Paramoeba spp., facultative-parasitic
amoebae, which are an unprecedented example for an endosymbiosis of two heterotrophic eukaryotic cells:
an amoebozoan host and a kinetoplastid endosymbiont. We also present preliminary data suggesting the
existence of peroxisomes in cryptophytes and chlorarachniophytes – evolutionarily and ecologically
significant organisms with complex plastids of red- and green-algal ancestry, respectively.
In this presentation we discuss the functional and metabolic diversity of peroxisomes and how these
remarkable organelles have contributed to the evolution of complex eukaryotes with endosymbiotic origin.
29
Session 2
Fine-structural observations on engulfing behavior and enlargement of endosymbiont in Hatena
arenicola.
Mami Nomura1, Ken-ichiro Ishida2
1
Shimoda Marine Research Center, University of Tsukuba, 415-0025, Japan; 2Faculty of Life and
Environmental Sciences, University of Tsukuba, 305-8571, Japan. Email: true82future@gmail.com
Hatena arenicola engulfs a prasinophyte alga, Nephroselmis spp. and maintain it as an
endosymbiont, which is distributed only to one of daughter cells during cell division. H. arenicola is a one of
key organisms for understanding endosymbiosis because H. arenicola is a one of kleptoplastidal protists and
suggested to be at an intermediate stage of secondary endosymbiosis. In this study, we observed the
Nephroselmis spp. cells that were being engulfed and the enlargement process of these as endosymbionts in
H. arenicola cells.
Usually Nephroselmis cells have extracellular organic scales, but the Nephroselmis endosymbionts
observed in H. arenicola cells lack the scales. Our fine structure observation showed that the feeding
apparatus of H. arenicola cell peeled off the scales of the Nephroselmis cell during engulfment. A pool of
peeled scales was observed in the feeding apparatus of H. arenicola cell and membrane-bounded cell
coverings were observed apart from Nephroselmis sp. cell body. It suggested that H. arenicola selectively
remove the cell coverings when it engulfs the Nephroselmis cell.
At the middle of endosymbiont enlargement process, the eyespot of endosymbiont was observed
at the anterior end of H. arenicola cell where the feeding apparatus was usually present in colorless H.
arenicola cells. The nucleus of endosymbiont did not have heterochromatin which is present in the nucleus
of free living Nephroselmis sp. cell. The nucleus of endosymbiont in the middle of enlargement process was
not observed near the nucleus of H. arenicola cell, although it has been reported that the nucleus of
endosymbiont is associated to the nucleus of H. arenicola. These observations may imply that the gene
expression pattern of endosymbionts may change during the enlargement process in the H. arenicola cell.
30
Session 2
Intracellular symbiosis of green algae in the ciliate Paramecium bursaria with possible association with
the host’s mitochondria
Toshinobu Suzaki1, Chihong Song1,2, Masashi M. Hayakawa1, Kazuyoshi Murata2, Jun Makimoto1
1
Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; 2 National
Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan. email:
suzaki@kobe-u.ac.jp
Some eukaryotes accommodate endosymbiotic algae in their cytoplasm; however, the mechanisms involved
in the interaction between the host and the symbiont remain largely unexplained. Here, electron microscopy
analyses and three-dimensional reconstructions were used to examine the ultrastructures of symbiotic algal
cells and their interactions with organelles in the host ciliate, Paramecium bursaria. In cryofixed P. bursaria
specimens, the perialgal vacuole (PV) membrane, which surrounds the symbiotic algae, was found to be
apposed closely to the cell wall of the symbiotic algae. However, the distance between these structures was
markedly expanded in chemically fixed samples, suggesting that the procedures involved in chemical
fixation introduce artefacts. Structural connections between the host endoplasmic reticulum-mitochondria
network and the symbiotic algae or its surrounding perialgal vacuole membrane were also identified, thereby
suggesting additional mechanisms involved in the interaction between intracellular symbiotic algae and their
eukaryotic hosts. The PV membrane prevents digestion of the symbionts in the host's cytoplasm and controls
the mutual exchange of various substances between the two partners, indicating that the presence of the PV
membrane is essential for their successful mutual endosymbiosis. Combined analysis of transcriptome and
proteome data was employed to identify proteins that are associated with the PV membrane, and the results
were compared with those obtained from the digestive vacuole membrane. The PV membrane fraction was
found to contain various proteins, including amino acid and lipid transporters and a V-type H+-ATPase,
which are considered to be closely related to the functions of the PV membrane. Many mitochondria-specific
proteins were also identified as constituents of the PV membrane fraction, further strengthening the view that
the PV membrane is tightly associated with mitochondria.
31
Session 2
Import of nuclear-encoded proteins into the photosynthetic organelles of Paulinella chromatophora
Przemysław Gagat1, Andrzej Bodył2, Paweł Mackiewicz1
1
Faculty of Biotechnology, 2Faculty of Biological Sciences, University of Wroclaw, ul. Przybyszewskiego
63/77, 51-148 Wroclaw, Poland. e-mail: gagat@smorfland.uni.wroc.pl
Paulinella chromatopora is a thecate filose amoeba of the supergroup Rhizaria that thrives in sediments of
brackish and freshwater eutrophic ponds. It harbours two photosynthetically active endosymbionts of
cyanobacterial origin (chromatophores), acquired about 60–140 million years ago independently of ancient
primary plastids typical of Archaeplastida (glaucophyte, red algae and green plant). Similarly to primary
plastids, chromatophores have lost many essential genes, and experienced substantial endosymbiotic gene
transfer (EGT), including transfer of genes involved in photosynthesis. This indicates that, similarly to
primary plastids, Paulinella endosymbionts evolved a transport system to import their EGT-derived proteins.
Based on bioinformatics analyses of the chromatophore genomes, Paulinella EST database and presequences
of proteins imported to the chromatophores, we elaborated a model for protein import into Paulinella
endosymbionts and their thylakoid membranes. For comparative studies, we analysed genomes of primary
plastids, all sequenced cyanobacteria and some bacteria. Our model involves (i) vesicular trafficking to the
outer chromatophore membrane, (ii) a simplified Tic-like complex at the inner chromatophore membrane,
(iii) a molecular motor responsible for pulling imported proteins into the chromatophore matrix and (iv)
thylakoid trafficking based on Sec, Tat and Srp pathways. The model of protein import system into
Paulinella chromatophores seems to be an example of convergent evolution as it is largely conserved in
primary plastids. Since Paulinella endosymbionts indeed evolved a protein import system (partly proved
experimentally), the endosymbionts should be acknowledged as true call organelles, among primary plastids
and mitochondria.
32
Session 2
Size-dependent symbiont specificity in cnidarian-dinoflagellate symbiosis
Elise Biquand1, Nami Okubo2, Yusuke Aihara3, Vivien Rolland1, David C. Hayward1, Masayuki Hatta4, Jun
Minagawa3, Tadashi Maruyama5, and Shunichi Takahashi1,3
1
Research School of Biology, The Australian National University, Canberra, Australian Capital Territory,
2601 Australia; 2Department of Economics, Tokyo Keizai University, Kokubunji, Tokyo, 185-8502 Japan;
3
Division of Environmental Photobiology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji,
Okazaki 444-8585, Japan; 4Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo
112-8610, Japan; 5Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka,
Kanagawa 237-0061 Japan. email: shun@nibb.ac.jp
Many cnidarians, including reef-building corals and sea anemones, harbor endosymbiotic dinoflagellate
(genus Symbiodinium), and each host species typically associates with specific Symbiodinium phylotypes. It
has been assumed that Symbiodinium phylotypes express distinct cell-surface molecules that determine the
infectivity into each host species. Here we propose a new model to explain the symbiont specificity for the
host in cnidarian-dinoflagellate symbiosis. We examined the relationship between infectivity and cell size of
different cultured Symbiodinium strains using a model cnidarian host, sea anemone Aiptasia. Results showed
that, of the fifteen Symbiodinium strains tested, the largest four strains failed to infect. Further experiments
with fluorescent microspheres of different sizes showed inefficient uptake of larger microspheres into host
cells. Together these results lead us to conclude that in Symbiodinium-Aiptasia symbioses cell surface
proteins are not necessary for symbiont uptake, and that cell size is a major factor determining the difference
in infectivity among Symbiodinium phylotypes.
33
Session 2
Plastid retention in Elysia viridis is determined by algae food source
Cessa Rauch1, Aloysius G.M. Tielens2,3, Sven B. Gould1, Gregor Christa1.
1 Institute for Molecular Evolution, Heinrich-Heine-University Düsseldorf 40225, Germany.
2 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, The
Netherlands.
3 Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center,
Rotterdam, The Netherlands.
Photosynthetic symbioses are remarkably widespread among the animal kingdom. It occurs for instance in
sponges, corals, mollusks and even a vertebrate. One of the most curious photosynthetic symbioses is found
in some sacoglossan sea slugs: functional kleptoplastidy. Here, only the plastids of the algal prey are
sequestered and embedded photosynthetically functional inside the animals’ own cells. In some elysoid
Sacoglossa, the kleptoplasts are kept functional for several months of starvation, but the basics of how the
plastids are kept active remains unknown. In its natural habitat Elysia viridis feeds on different algae, which
include Bryopsis or Cladophora. We found that specimens collected from Cladophora and subsequently
solely fed on that species for 3 months, do not retain any functional kleptoplasts; all plastid are digested with
the remaining food. During starvation, however, the plastid-free animals shrink more rapidly compared to a
control group that was nurtured on Bryopsis before exposure to starvation. The latter supports a beneficial
role of the kleptoplasts during periods of food depletion. Contrary, in the presence of food algae, feeding on
the cytosol seems to be sufficient and slugs feeding on Cladophora do not rely on any sequestered plastids.
Elysia viridis appears able to switch between plastid retention and digestion, which, intriguingly, depends on
the algae it feeds on.
34
Session 2
A „c6-like“ cytochrome in the muroplast of Cyanophora paradoxa
Jürgen M. Steiner and Friedrich Kleiner
Institute of Biology-Plant Physiology, Martin Luther University Halle-Wittenberg, Weinbergweg 10, 06120
Halle (Saale), Germany. email: juergen_steiner@gmx.at
Cyanophora paradoxa (Glaucocystophyta), the „coelocanth“ of the algal world, is an obligatory
photoautotrophic biflagellated protist containing cyanelles (muroplasts), peculiar plastids surrounded by a
peptidoglycan wall, apparently a relict from the cyanobacterial endosymbiotic partner. Glaucocystophyte
algae serve as an ideal model system to study essential aspects of plastid evolution such as protein
translocation and assembly processes of supramolecular structures such as phycobilisomes and
carboxysomes. In Cyanophora, which does not contain plastocyanin, photosynthetic electron transport from
the cytochrome b6/f complex to photosystem I is mediated by cytochrome c6 (PetJ). As we could show earlier
(via homologous and heterologous import experiments), apocytochrome c6 is a Sec passenger in cyanelles. In
the course of the genome project we found a gene coding for a „c6-like“ cytochrome harboring a
twin-arginine (Tat) consensus motif in its signal peptide. Import experiments and localization studies will be
presented and the enigmatic nature of the „c6-like“ cytochromes in cyanobacteria and higher plants will be
discussed.
35
Session 2
Comparative analysis of the response to high light throughout plastid evolution
Maria R. Handrich, Cessa Rauch, Madeline C. Weiß, Jan de Vries, Sven B. Gould
Institute for Molecular Evolution, Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf,
Germany
Corresponding author: gould@hhu.de
Photosynthesis research in eukaryotes focuses to a large degree on a few species, almost always stemming
from the green lineage (Chloroplastida). Diversity among phototrophic eukaryotes, however, is immense and
the many plastids they harbour differ e.g. regarding their pigment compositions, non-photochemical
quenching mechanisms and genome coding capacities. We set out to perform a comparative analysis of the
response to highlight stress in representatives of all three primary algal lineages (Glaucophyta, Rhodophyta
and Chloroplastida), and two representatives of two lineages carrying plastids of secondary endosymbiotic
origin (Chlorarachniophyta, Cryptophyta). Our analysis combines (i) a large-scale RNA-seq approach,
probing in total 54 novel transcriptomes from 3 controlled and comparable conditions and 6 species, (ii) the
analysis of D1 turnover and pigment profiles, and (iii) simultaneous TEM-imaging to monitor changes in
morphology such as alterations in thylakoid stacking. The bird’s eye perspective of the RNA-seq data
uncovered an unexpected response in the glaucophyte Cyanophora paradoxa, while the zoom in on
individual pathways associated with plastid maintenance uncovered differences in particular between the
green and red lineage (and which reflects on the differences regarding their plastid genome coding
capacities). Our global comparison is the first of its kind and it sheds light on the different trajectories the
individual eukaryotic lineages have experienced with regard to housing a photosynthezing organelle.
36
Session 2
Isolation and characterization of effector genes during symbiotic interaction between Chinese cabbage
and Piriformospora indica
Kai-Wun Yeh1 and Fa-xing Chen2
1. Institute of Plant Biology, 106, National Taiwan University, Taiwan
2. College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002 PR China
Piriformospora indica is a mutualistic endophyte with a broad host range and great application potential in
agriculture. Recent studies indicate that, like pathogenic microbes, mutualistic microbes use effector proteins
to adjust plant physiology resulting a promotion of symbiosis with plant. The study of P.indica effector
proteins and their mechanism can be aadequate model for endophyte symbiosis. In this study, the objective is
to establish an examining system for identification and functional characterization of P.indica effector
proteins. From the double subtractive cDNA library and bioinformatics methods, we picked up nine genes to
investigate their expression pattern during symbiosis stage and the subcellular localization in plant cell.
Both PIIN_09643 and PIIN_11103 are induced to early express during symbiosis stage, and their encoded
proteins show the subcellular localization in nucleus. Using the hairy-root culture transformed by
PIIN_09643 and PIIN_11103 gene, we found PIIN_09643 and PIIN_11103 significantly suppress the
expression level of SA pathway marker gene, PR1; and cause a promotion of symbiotic colonization.
However, expression level of PDF1.2. in JA pathway was not affected by PIIN_09643 or PIIN_11103.
Moreover, the treatment of PIIN_09643 and PIIN_11103 recombinant proteins to Chinese cabbage leaves
showed the hypersensitive symptom of SA response. The results suggest that P.indica applies PIIN_09643
and PIIN_11103 to suppress SA response, and further facilitate the symbiosis process in planta. Moreover,
we found PIIN_09643 and PIIN_11103 can enhance the expression level of redox status relative
genes, DHAR5 and GSTU. It might be the indirect result of the down-regulation of SA pathway.
37
Session 2
Fungi establishing mutualistic or pathogenic interactions with roots
Ralf Oelmüller
Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich Schiller Universität, Jena, Dornburger
Str. 159, 07743 Jena; E-mail: b7oera@uni-jena.de
A huge number of beneficial and pathogenic fungi release biomolecules into the rhizosphere which initiate
signaling events in the roots leading to mutualistic or pathogenic plant/fungus interactions. The combination
and concentration of the individual biomolecules in the rhizosphere is critical for the plant´s decision to
invest in either growth or defense. These biomolecules activate receptors in the roots and induce cytoplasmic
Ca2+ ([Ca2+]cyt) elevation in a phosphorylation-dependent manner. Furthermore, they stimulate
phospholipid signaling which coordinate the balanced response between growth/development and defense,
by cross-talking to the Ca2+ signals. We use transgenic Arabidopsis and tobacco plants expressing the Ca2+
sensor aequorin to isolate and identify biomolecules from the exudates of the beneficial root-colonizing fungi
Piriformospora indica and Mortierella hyalina, and from the pathogenic fungi Alternaria brassicae and
Verticillium dahliae. The exudate components induce a rapid and transient increase in [Ca2+]cyt levels in the
roots. We isolated and characterized the fungal biomolecules responsible for the Ca2+ responses, and
Arabidopsis mutants, which do not respond to these biomolecules. While Ca2+ responses are induced by all
four fungi, and [Ca2+]cyt elevation is necessary for the proper plant responses to these fungi, production of
reactive oxygen species occurs only in response to pathogenic fungi, and in a Ca2+-dependent manner. We
study early signaling events in plants (e.g. reversible phosphorylation) in response to exudates from these
fungi, as well as systemic signaling, which informs the entire plant body about the presence of a particular
fungus.
Furthermore, the two beneficial fungi promote plant performance and root and shoot growth, and we identify
compounds (genes and metabolites), metabolic and signaling pathways which are involved in root growth
promotion and optimal root adaptation to environmental cues. These compounds are currently characterized
and tested for their agricultural applications. Finally, we want to know whether microbes are aware of other
microbes colonizing the same root, and how root colonization by multiple fungal species affects the
microbial community in the rhizosphere.
38
Session 3
Coordination between the chloroplast psbA 5’-untranslated region and coding region activates the
translation initiation.
Masayuki Nakamura, Masahiro Sugiura
Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan.
email: mnak@gene.nagoya-u.ac.jp
The 5’-UTR from psbA mRNA encoding the D1 protein of photosystem II complex has the highest
translation activity in in vitro translation system examined, and is the best choice to express recombinant
proteins, such as selection marker enzymes, reporter proteins, and therapeutic proteins in chloroplasts. Using
an in vitro translation system from tobacco chloroplasts, we detected no translation from a human
immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5’-UTR. The Tat
protein was translated using the E. coli phage T7 gene 10 5’-UTR, a highly active 5’-UTR, and the added tat
mRNAs and Tat protein synthesized were stable during translation reactions. These results suggest that the
translation of tat mRNA with psbA 5’-UTR was arrested at the translation initiation. When the tat coding
region was fused to the psbA 5’-UTR with a cognate 5’-coding segment, significant translation was detected
from the tat coding region. Therefore, cooperation between the 5’-UTR and its coding region is important for
translational initiation.
39
Session 3
Measuring the temperature-dependence of cpRNP-RNA association in chloroplasts by a leaf-tissue
based RIP-chip assay
Marie-Kristin Lehniger1, Stephanie Gathmann1, Christian Schmitz-Linneweber1
1
Molecular Genetics, Institute of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Philippstr.
11-13,
Bldg.
22
(Rhoda
Erdmann
Haus),
D-10115
Berlin,
Germany.
email:
marie-kristin.lampe@hu-berlin.de
The nuclear–encoded chloroplast ribonucleoproteins (cpRNPs) are a group of plastid hnRNP-like
RNA-binding proteins. They are highly abundant proteins that are regulated in a light- and
temperature-dependent manner. cpRNPs bind multiple RNA targets and are involved in RNA processing,
stabilization and editing. The cpRNPs CP29A and CP31A were shown to be important for mRNA
accumulation and processing at low temperatures. In this study, we wanted to analyze if the transcript pools
bound by CP29A change in quantity and/or quality under cold acclimation conditions. Therefore, a new
method for immunoprecipitation of RNA-protein complexes from plant leaf tissue was established.
An increased expression of CP29A under cold acclimation could be demonstrated by immunoblot analysis as
well as immunofluorescence assays of Arabidopsis thaliana protoplasts using a polyclonal antibody against
CP29A. Furthermore, the sub-organellar localization of CP29A changed after cold acclimation. Results on
RNA co-immunoprecipitation and microarray (RIP-chip) analysis of CP29A in leaf tissue of cold acclimated
versus normal grown Arabidopsis thaliana and Nicotiana tabacum will be presented.
40
Session 3
Elucidating target RNA editing sites of embryo lethal PLS class PPR proteins
Matthias Burger1, Anja Zehrmann1, Mizuki Takenaka1
1
Molecular Botany, Ulm University, 89069 Ulm, Germany
RNA editing in the organelles of Arabidopsis thaliana is converting selected cytidine residues to uridines.
More than 400 and 30 RNA editing events take place in mitochondria and chloroplasts, respectively, in
Arabidopsis. Most of them lead to amino acid exchanges in the translational products.
The cytidines to be edited are specifically chosen by PLS class PPR proteins, which have been characterized
as editing factors by reverse genetics of PLS class PPR genes. However, in many cases molecular functional
analyses including RNA editing in the PPR protein knock-out mutants are impossible, as they show severe
defects in organellar function which leads to seed abortion in early embryo stages.
To investigate the function of PLS class PPR proteins which show embryo lethality in the
knock-out lines, we undertook three approaches. At first, artificial microRNAs (amiRNAs) are employed to
establish knock-down lines of the PPR protein genes with milder effects on organelles. Secondly, embryos of
homozygous knock-out lines are cultivated on a MS-based embryo rescue medium with 8% sucrose content.
Finally, PPR protein genes are expressed in the respective mutant lines under control of the embryo specific
ABI3 promotor to overcome embryo lethality and obtain enough mature plant material with homozygous
mutant background. With the help of our target site prediction program based on the PPR code, it is possible
to concentrate the first RNA editing analysis on several candidate target sites of individual PPR proteins.
Here we show that our approaches successfully identified several novel RNA editing factors, of which
knock-out lines display embryo lethality at standard condition, as well as their target editing sites which must
be involved in embryo lethality.
41
Session 3
Organelle RNA editing sites and their nuclear specificity factors: Co-evolution among angiosperms
and beyond
Anke Hein, Monika Polsakiewicz and Volker Knoop
IZMB – Institute of Cellular and Molecular Botany, Department of Molecular Evolution, University of Bonn,
Germany
RNA editing converting cytidines into uridines is a common feature of plant organelle transcript maturation.
In contrast to low numbers of only 30-50 chloroplast RNA editing sites in model flowering plant chloroplasts,
we recently demonstrated ca. 4-fold amounts of RNA editing in basal angiosperm chloroplast transcriptomes,
e.g. 138 C-to-U editing events in Amborella trichopoda, the likely sister taxon to all other angiosperms.
More than 60 RNA-binding pentatricopeptide repeat (PPR) proteins have already been identified as
chloroplast or mitochondrial RNA editing factors in plants, mostly in the key model system Arabidopsis and
in the moss Physcomitrella patens. We currently test recombinant editing factor constructs in Physcomitrella
towards further refining the recently proposed PPR-RNA binding code.
Making use of the ever increasing amounts of plant genome sequence data we currently investigate the
evolutionary fates of those RNA editing sites and their cognate recognition factors, which can be traced back
for at least 120 Mio. years of angiosperm evolution. We will report on CLB19/PDE247 and AEF1/MPR25,
the latter being the hitherto only documented example for an RNA editing factor targeting both
endosymbiotic organelles at the same time. Whereas we observe strong sequence conservation of the two
editing factors in all functionally relevant parts, we found highly different scenarios of their rare vs. frequent
losses across angiosperm evolution, however, consistently connected with changes in the affected organelle
editomes. Complementation approaches to test the functionality of phylogenetically distant editing factor
homologues are currently underway with a focus on cases where we observe interesting changes in the
organelle editomes for the multi-target RNA recognition factors.
42
Session 3
RNA editing in Early Land Plants: The birds of paradise and the workhorse
Bastian Oldenkott1, Mareike Schallenberg-Rüdinger1,2, Nils Knie1, Simon Zumkeller1 and Volker Knoop1
1
IZMB – Institute of Cellular and Molecular Biology, University of Bonn, Germany
Department of Cell Biology, Faculty of Biology, University of Marburg, Germany
email: bastian.oldenkott@uni-bonn.de
2
Plant RNA editing converts cytidines to uridines in organelle transcripts, mostly restoring conserved codon
identities, thus indicating a repair mechanism. The base conversions are mediated by DYW-type
pentatricopeptide repeat (PPR) proteins.
The numbers of RNA editing sites in chloroplast and mitochondrial transcripts vary widely across land plant
phylogeny. Record amounts of RNA editing have been identified in the lycophyte genus Selaginella with
more than 2,100 RNA editing events in the mitochondrial transcripts of S. moellendorffii and with even more
than 3,400 in the chloroplast of S. uncinata. Extensive analyses of the eleven group II introns in the
chloroplast genome of S. uncinata has now likewise revealed frequent RNA editing. This will provide new
insights into RNA editing affecting highly structured RNA and its role in RNA processing including group II
intron splicing.
Assembly of the Selaginella peruviana chloroplast genome revealed a hitherto unique plastome featuring
frequent gene losses, recombination and retroprocessing events. Comparative studies show unparalleled
organelle genome diversity in the genus Selaginella, particularly regarding RNA editing patterns.
The model moss Physcomitrella patens in contrast is at the opposite end of editing complexity with only
eleven RNA editing sites in its mitochondrial and two sites in the chloroplast transcripts. Recently, it became
the first model system with all RNA editing sites assigned to their DYW-type PPR specificity factors. We
here report on complementation studies in knockout lines of PPR78 responsible for mitochondrial editing
sites cox1eU755SL and rps14eU137SL and PPR79 affecting nad5eU598RC, which both feature only mild
phenotypes. Recombinant PPR constructs with a special focus on the C-terminal domains, including the
DYW-domain with cytidine deaminase similarity, will help to elucidate the mechanisms of specific RNA
target recognition and the biochemistry responsible for the deamination reaction in RNA editing.
43
Session 4
Chloroplast-dependent nuclear gene regulation: evolution of retrograde signaling
Hikaru Ohara1, Hiroyuki Ando1, Atsushi Ikari1, Misato Anma1, Atsuko Era2, Shin-ya Miyagishima2, Yuki
Kobayashi3, Kan Tanaka3, Masayuki Igarashi4, Ryutaro Utsumi5, Mitsumasa Hanaoka1
1
Graduate School of Horticulture, Chiba University, Yayoi-cho, Inage-ku, Chiba, Chiba 263-8522, Japan;
Department of Cell Genetics, National Institute of Genetics, Yata, Mishima, Shizuoka 411-8540, Japan;
3
Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama
226-8503, Japan; 4Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan;
5
Faculty of Agriculture, Kinki University, Nakamachi, Nara, Nara 631-8505, Japan.
email: mhanaoka@faculty.chiba-u.jp
2
Chloroplasts are originated from the endosymbiosis of an ancient cyanobacterium. Just after symbiosis, gene
expression at the nucleus (host) and the chloroplast (symbiont) would be independent, and therefore light
sensing and light-dependent gene expression should have been completed inside the chloroplast. At the first
step of evolution, along with endosymbiotic gene transfer, light information in the chloroplast would have to
be somehow transduced into the nucleus, to coordinate gene expression of both genomes. We here used
Cyanidioschyzon merolae, a primitive, unicellular red alga showing many ancestral characteristics. This alga
does not have any typical plant-specific photoreceptors but still possesses the cyanobacterial two-component
system (TCS) in the chloroplast, which is speculated to be required for light-dependent transcriptional
regulation of chloroplast genes. Surprisingly, specific inhibition of the histidine kinase of TCS resulted in
change of several chloroplast-encoded genes, as well as a specific group of nuclear genes, suggesting that
this TCS can regulate nuclear gene expression as well, by light-dependent chloroplast-to-nucleus retrograde
signaling. We further analyzed this signal transduction and found that red light possibly influences this
regulation, and that heme, MAP kinase(s) and specific transcription factor(s) might be involved. Thus,
light-dependent signal(s) from the symbiont, and pre-existing cytosolic regulatory system of the host, might
be combined to establish initial retrograde signaling. After the long evolution, chloroplasts have lost their
autonomy, and light sensing and chloroplast gene expression have become under control of the nucleus.
Nevertheless, retrograde signaling still has a certain degree of roles on nuclear gene regulation, particularly
in the case of chloroplast dysfunction. Among them, we recently found in Arabidopsis thaliana that several
specific nuclear genes could be regulated by different light environments in chloroplasts, depending on CSK
(chloroplast sensor kinase), the orthologous protein of the histidine kinase of TCS. These results suggest that
retrograde light signaling has a significant role for coordination of gene expression beyond evolution.
44
Session 4
Empirical identification of the transcriptional network for H2O2 responses in Arabidopsis
Ayaka Hieno1, Naznin Hshuna Ara2, Keiko Hasegawa-Inaba2, Tomoko Yokogawa2, Daichi Obata2, Mika
Nomoto3, Yasuomi Tada3, Takashi Yokogawa2, Mieko Higuchi4, Kosuke Hanada5, Minami Matsui4, Takashi
Hirayama6, Nobutaka Mitsuda7, Yoshiharu Y. Yamamoto1,2,4,8,9
1
United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan;
Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan; 3Center
for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; 4RIKEN CSRS,
Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama 230-0045, Japan; 5Department of Bioscience and Bioinformatics,
Kyushu Insititute of Technology, Kawa zu 680-4, Iizuka, Fukuoka 820-8502, Japan; 6Institute of Plant
Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama 710-0046, Japan;
7
Bioproduction Research Institute, AIST, Chuo-dairoku, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan;
8
JST ALCA. email: 9yyy@gifu-u.ac.jp
2
Hydrogen peroxide (H2O2) is a molecule for retrograde signaling from plastids and mitochondria to the
nucleus, and also known to be a mediator involved in all the known biotic and abiotic stress responses
through transcriptional activation of a variety of genes. Now the first switch for the transcriptional responses
is thought to be translocation of a membrane-bound transcription factor, ANAC017, from ER to the nucleus1.
We tried to understand the transcriptional network for the responses.
Time course analysis of transcriptional response at shoots after H2O2 treatment to shoots revealed that
around 50 transcription factors (TFs) are activated within 24 h after the treatment, suggesting that they are
the nodes of the transcriptional network. Our microarray data, together with public microarray data of
knockout mutants and/or overexpressors of these TFs, which were available for 12 of them, were used for
determination of regulatory hierarchy in vivo among the TFs. These data was also used for prediction of their
target elements in the promoter region, and possible combination between regulator TF proteins and their
target promoter element of the regulated TFs were validated in vitro binding assays using regulator TF
proteins synthesized in vitro and biotinylated oligo DNA probes. In summary, we could empirically
determine 19 regulatory pairs of regulator TFs and regulated promoter of TFs, including 1 previously
reported2 and 18 novel pairs. The identified regulatory pairs served as directional connectors of the
potential transcriptional network for the H2O2 response, a part of which is expressed in specific physiological
conditions for biotic or abiotic stress responses, or for responses to the mitochondrial or plastid signal in
Arabidopsis.
1
2
Ng et al. Plant Cell 25:3450, 2013
Matsuo et al., Mol Plant 8: 1253, 2015.
45
Session 4
Reconstitution of chloroplast nucleoids by eukaryotic factors during the green plant evolution
Yusuke Kobayashi1, Mari Takusagawa1,6, Naomi Harada1, Yoichiro Fukao2,7, Shohei Yamaoka3, Takayuki
Kohchi3, Koichi Hori4, Hiroyuki Ohta4,5, Toshiharu Shikanai1, Yoshiki Nishimura1
1
Laboratory of Plant Molecular Genetics, Department of Botany, Kyoto University, Oiwake-cho,
Kita-Shirakawa, Kyoto 606-8502, Japan; 2Plant Global Educational Project, and Graduate School of
Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan; 3Graduate School of
Biostudies, Kyoto University, Kyoto 606-8502, Japan; 4Center for Biological Resources and Informatics,
Tokyo Institute of Technology, Yokohama City, Kanagawa 226-8501, Japan; 5Earth-Life Science Institute,
Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8551, Japan; 6Department of
Biological Science and Chemistry, Faculty of Science, Graduate School of Medicine, Yamaguchi University,
Yamaguchi 753-8512, Japan; 7Department of Bioinformatics, Ritsumeikan University, 1-1-1 Noji-higashi,
Kusatsu, Shiga 525-8577, Japan. e-mail: yoshiki@pmg.bot.kyoto-u.ac.jp
Chloroplast (cp) DNA is compacted to form nucleoprotein complexes, cp nucleoids. The structure of cp
nucleoids is ubiquitously observed in diverse plants. However, the protein compositions of cp nucleoids is
inconsistent among unicellular algae and flowering plants. In this research, we aimed to reveal the
evolutionary history of cp nucleoid organization by analyzing the key organisms representing four
evolutionary stages of eukaryotic phototrophs; Chlamydomonas reinhardtii, Klebsormidium flaccidum,
Marchantia polymorpha, Arabidopsis thaliana. In C. reinhardtii, we performed a proteomic analysis and
identified a novel SAP domain-containing protein as a constitutive core component of eukaryotic origin.
Furthermore, homologous genes for cp nucleoid proteins (SAP, HU, Whirly, and SWIB proteins) were
analyzed in the key organisms, and their intracellular localizations to cp nucleoids and DNA binding
activities were confirmed. Based on these analyses, we propose that recurrent modifications of cp nucleoid
organization by eukaryotic factors might have been the driving force for the diversification of cp nucleoids
since the early stage of green plant evolution.
46
Session 4
Biparental inheritance of chloroplasts is controlled by lipid biosynthesis
Johanna Sobanski1, Patrick Giavalisco1, Mark Aurel Schöttler1, Hieronim Golczyk1, Toshihiro Obata1, Ralph
Bock1, Barbara B. Sears2, and Stephan Greiner1
1
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam – Golm, Germany;
Department of Plant Biology, Michigan State University, East Lansing, MI 48824-1312, USA.
email: greiner@mpimp-golm.mpg.de
2
In most eukaryotes organelle genomes are transmitted preferentially by the mother, but the underlying
reasons for this fundamental biological principal are far from being understood. It is believed that biparental
inheritance favors competition between cytoplasmic genomes and thus allows the spread of selfish
cytoplasmic elements. Those can be, for example, chloroplasts which replicate faster, but are incompatible
with and maladaptive to the hybrid nuclear genome. This scenario is found in natural populations of our
model organism Oenothera (the evening primrose), which shows fairly high frequencies of biparental
chloroplast inheritance.
To identify the loci that make chloroplast genomes selfish we employed association mapping
using wild-type chloroplasts differing in their competitive ability and created chloroplast mutants from them
that have altered competitive abilities. In this material, we could identify two loci in the chloroplast genome
that change plastid lipid synthesis and consequently membrane lipid profile. We showed, that polymorphisms
in ycf2 (an ORF of unknown function) and in the regulatory region of accD (the chloroplast encoded subunit
of the acetyl-CoA carboxylase, catalyzing the first step of lipid biosynthesis) are responsible for the
differences in competitive behavior. These loci most likely determine chloroplast division rates and hence
competitiveness. Since chloroplast competition can result in uniparental inheritance (through the elimination
of the weak chloroplasts), this work uncovers for the first time a molecular mechanism for organelle
inheritance.
47
Session 4
Post-translational regulation in nuclear-mitochondrial interaction of cytoplasmic male sterility in
sugar beet
Takumi Arakawa, Kazuyoshi Kitazaki, Muneyuki Matsunaga, Hiroaki Matsuhira, Tetsuo Mikami and
Tomohiko Kubo
Graduate School of Agriculture,
takumia@abs.agr.hokudai.ac.jp
Hokkaido
University,
Sapporo,
060-8589,
Japan,
email:
Cytoplasmic male sterility (CMS) in plant is maternally inherited trait that confers inability to produce
functional pollen. CMS mitochondria bear sterility-inducing factor (perhaps) causing mitochondrial
dysfunction, whereas the trait is repressed by nuclear restorer-of-fertility gene (Rf). Sugar beet Rf resembles
Oma1 gene, which is known to encode mitochondrial chaperone-like protein in yeast. Because of the
similarity between sugar beet Rf and Oma1, we suspected that Rf regulates sterility-inducing factor in
post-translational manner. Immunoprecipitation using anti-RF antibody revealed that RF protein binds to
preSATP6, a protein unique to CMS mitochondria. Analysis of mitochondrial protein complex of sugar beet
plant and transgenic suspension cells using blue-native polyacrylamide gel electrophoresis suggested that the
higher-order structure of preSATP6 complex was altered by Rf but not by recessive rf. Thus, certain structure
of preSATP6 complex may be essential for the expression of CMS. The question what the effect of
preSATP6 complex on mitochondrial function should be investigated.
48
Session 5
The plastid reticulum reloaded
Jürgen Soll
Department Biologie I-Botanik, Ludwig-Maximilians-Universität, Großhaderner Straße 2-4, 82152
Planegg-Martinsried, Germany, e-mail: soll@lmu.de
Photosynthesis is an essential life-supporting process. All green parts of a plant perform photosynthesis in
small distinct compartments called chloroplasts. These organelles belong to a larger family known as plastids,
and while functionally and structurally distinct, at least one of its kind is present in every plant cell. The
progenitor of all plastids are proplastids, organelles which are present in non-differentiated stem cells
(meristematic tissue). During leave development proplastids differentiate into photosynthetically active
chloroplasts. This process is accompanied by the de novo formation of an extensive internal membrane
system called thylakoids, which house the photosystems. A vast literature exists on the assembly, turnover
and repair of the photosynthetic machinery in pre-existing thylakoids. In contrast, very little to nothing is
known about the generation and formation of thylakoid membranes in the physiological
proplastid-chloroplast transition phase. The genesis of thylakoids is fundamental for our understanding of
how a plant develops and functions. Early electron microscopy pictures from the 1950-1970s describe some
phenomena such as vesicle budding and membrane invagination from chloroplast envelopes which seem to
contribute to thylakoid biosynthesis. In addition, in C4 plants a dense layer of vesicles located between the
inner envelope and the thylakoids persists even after differentiation and is called the plastid reticulum.
49
Session 5
Identification of factors involved in photosystem biogenesis using a forward genetics approach
Jasmine Theis 1, Mark Rütgers 1, Ligia S. Muranaka 1, Stefan Geimer 2, Michael Schroda 1
1
Molecular Biotechnology & Systems Biology, Technische Universität Kaiserslautern, 67663 Kaiserslautern,
Germany; 2 Biologie/Elektronenmikroskopie, Universität Bayreuth, 95440 Bayreuth, Germany.
A plethora of different functions were assigned to the vesicle inducing protein in plastids (VIPP1), including
the formation/maintenance of inner envelope and thylakoid membranes, the biogenesis of photosystems, the
transfer of lipids, or the formation of lipid microdomains. Chlamydomonas VIPP2 is a paralog of VIPP1
which represents only a minor fraction of the cell’s VIPP pool under low light conditions. However VIPP2 is
induced by high light and when processes apparently involved in thylakoid/photosystem biogenesis are
impaired. To identify factors involved in photosystem biogenesis, we performed a forward genetics screen
based on a luciferase reporter construct driven by the VIPP2 promoter and insertional mutagenesis. This way
we identified 11 insertional mutants showing accumulation of VIPP2 under low light conditions. The
mutants exhibit a wide range of chloroplast-associated phenotypes. Some mutants display altered levels of
subunits of photosynthetic complexes as well as differences in complex composition, as demonstrated by
blue native PAGE and sucrose density gradient centrifugation. Our results suggest that VIPP2 is a sensitive
reporter for the identification of factors associated with photosystem formation and function.
50
Session 5
Biogenesis of thylakoid membranes
Jörg Nickelsen
Biozentrum der LMU München, AG Molekulare Pflanzenwissenschaften, Botanik, Großhaderner Str. 2-4,
82152 Planegg-Martinsried, Munich, Germany, e-mail: joerg.nickelsen@lrz.uni-muenchen.de
Cyanobacteria, algae and plants convert light energy into chemical energy by using a very similar type of
photosynthetic membrane system, named thylakoids. Current molecular analyses suggest that the biogenesis
of the cyanobacterial energy conversion system, in particular photosystem (PS) II, is initiated in dedicated
membrane sub-fractions at the cellular periphery. Here, these membranes form centres which are marked by
the manganese- transporting PS II assembly factor PratA. Center formation depends on the membrane
curving protein CurT, a homolog of the chloroplast grana-forming CURT1 protein family. By applying cryo
EM tomography, we dismantled the in situ ultrastructure of biogenesis centers.
51
Session 6
Deciphering the Chrysolaminarin Biosynthetic Pathway in the Diatom Phaeodactylum tricornutum
using Molecular Tools
Weichao Huang, Carolina Rio Bartulos, Bernard Lepetit, Peter G. Kroth
Fachbereich
Biologie,
University
Peter.Kroth@uni-konstanz.de
of
Konstanz,
78457
Konstanz,
Germany.
E-Mail:
Chrysolaminarin is the main storage compound in diatoms, a glucan consisting of linear 1,3-β-chains with
1,6- -branches. In diatoms, chrysolaminarin is stored in intracellular vacuoles in a non-crystalline form. The
biosynthetic pathway of chrysolaminarin in diatoms as well as the involved enzymes so far are poorly
investigated. Therefore, we aimed at studying this pathway in the diatom model system Phaeodactylum
tricornutum. We screened the respective genome and identified genes encoding enzymes that are potentially
involved in chrysolaminarin synthesis or modification, including UDP glucose pyrophosphorylases, a
-glucan synthase and -1,6-transglycosylases. By expression of GFP fusion proteins in P. tricornutum, we
determined the respective intracellular localizations of the proteins. We also investigated the functionality of
the glucan synthase and the putative transglycosylases from P. tricornutum, by applying gene silencing
techniques or by complementation of transglycosylase-deficient yeast strains. Silencing of the glucan
synthase yielded a number of phenotypic cellular changes including reduced growth, a higher NPQ and a
changed thylakoid morphology. Phylogenetic analyses finally revealed that these proteins are conserved
between the Stramenopiles, a taxonomic group including diatom, brown algae and non-photosynthetic
Oomycetes. Here, we will present model of the carbohydrate storage pathway in diatoms.
52
Session 6
Non-endosymbiotic origin of triacylglycerol accumulation: a model of “chloroplast oil body” in
Chlamydomonas reinhardtii
Naoki Sato, Masakazu Toyoshima, Takashi Hirashima, Takashi Moriyama, Natsumi Mori, Masakazu Saitoh,
Hajime Wada
1
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902,
Japan; 2CREST, Japan Science and Technology Agency, Tokyo, 102-0076, Japan
Production of biofuel is a commercially active field of research, but the scientific background for these
researches remain weak with respect to the studies on the plant oil production. Unfortunately, discrepancy in
the subcellular localization of triacylglycerol (TAG) or 'oil' persists in the model alga Chlamydomonas
reinhardtii, making it difficult to construct a solid blue-print for the high-level production of microalgal oil.
Is TAG a constituent of the chloroplast? Is TAG present in the cyanobacterial ancestor? What is the
difference between the oil body and plastoglobule? Is the 'chloroplast oil body' (called 'cpst-LB' for
chloroplast lipid body) an enlarged plastoglobule? Does oil body really exist in the chloroplast? We have
been working on these problems over years, using comparative genomics, biochemical analysis, electron
microscopy and fluorescence microscopy. In the present report, we summarize all the results of observations
regarding these questions, as follows: Currently, there is no solid evidence for the presence or synthesis of
TAG in cyanobacteria. The presence of TAG in plastoglobule has been reported repeatedly in plants, but it is
important to assess the level of inevitable cytoplasmic contaminants. No enzyme for the synthesis of TAG
has been unambiguously demonstrated as being targeted to the chloroplast in C. reinhardtii. We examined by
electron microscopy the oil bodies apparently present within the chloroplast in C. reinhardtii. We focused on
the presence of the chloroplast envelope membranes that separate the inside and outside of chloroplast. All
what we found as resembling the 'cpst-LB' was the oil bodies within invaginations of the chloroplast. The
envelope membranes always surrounded the oil body, and therefore, a thin layer of cytosol was present
between them. Some of the oil bodies appeared nearly completely engulfed by the chloroplast, but a very
narrow cytosol extends to the oil body. Currently, we found no oil body without being surrounded by the
envelope membranes or completely isolated from the bulk cytosol compartment. Plastoglobules might
contain TAG, but at most as a minor component, and no plastoglobules could be as large as oil bodies (up to
1 µm in diameter). Based on these observations, we present a model of 'chloroplast oil body', which is in fact
the oil body present in the invagination of the chloroplast wrapped aroujd by a thin layer of cytosol.
53
Session 6
New bioremediation technique for radioactive cesium-contaminated soil using Paramecium bursaria
MD Shafiqul Islam1, Chisato YOSHIMURA2 and Toshinobu SUZAKI1
1
Dept. Biol., Grad. Sch. Sci., Kobe Univ., Japan, 2Ctr. Environ. Management, Kobe Univ., Japan, e-mail:
sshafiq1969@gmail.com
Physicochemical approaches for removal of radioactive cesium from contaminated soil proved to be
cost-ineffective than biological methods, and many biological techniques have been proposed. However,
none of the bioremediation techniques have been found suitable for fulfilling the purpose, especially for
removing cesium contamination from the siol. Paramecium bursaria has a mechanism of dissociating metal
elements that are strongly bound to the soil particles, and incorporating them into the cell. P. bursaria takes
up soil particles of up to 10 µm in size. In the digestive vacuole,pH decreases from ∼7 to 3 and dissociation
of metal element from the soil particles is facilitated. This unique character encouraged us to use
Paramecium for removal of radioactive cesium from the contaminated soil, and we found that P. bursaria
has a strong ability of cesium accumulation. After treatment with 1 mM CsCl for 24 hours, the average
concentration of cesium in P. bursaria cells(green P. bursaria) was increased to 8mM,while aposymbiotic
white P. bursaria (without symbiotic zoochlorellae)showed no accumulation. When cesium-adsorbed kaolin
particles (a model soil) were mixed with green P. bursaria for 4 days, a pronounced accumulation of cesium
was observed; the average concentration of cesium in green P. bursaria became ∼300 times higher than that
in the outer medium environment (0.1 mM). Paramecium cells were effectively recovered(∼95%)by
applying DC current to the soil suspension. This technique could be a promising cost-effective, eco-friendly
and time-saving method for bioremediation that can be operated on-site at individual farms.
54
Session 7
Plastid comparative genomics elucidates multiple independent losses of photosynthesis in Cryptomonas
(Cryptophyta)
Goro Tanifuji1, Ryoma Kamikawa2, Christa E. Moore3, Tyler Mills3, Kengo Kato4, Yuji Inagaki5, Tetsuo
Hashimoto6, John M. Archibald3
1
Department of Zoology, National Museum of Nature and Science, Tsukuba, 305-8577, Japan; 2Graduate
School of Global Environmental Studies, Kyoto University, Kyoto, 606-8501, Japan; 3Department of
Biochemistry and Molecular Biology, Dalhousie University, Halifax, B3H 4R2 Canada; 4Center for
Computational Sciences, University of Tsukuba, Tsukuba, 305-8577, Japan ; 4Pathogen Genomics Center,
National Institute of Infection Diseases, Tokyo, 162-8640, Japan; 5Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, 305-8577, Japan: email:gorot@kahaku.go.jp
In addition to photosynthesis, plastids are the site of a variety of significant biochemical processes such as
fatty acid, isoprenoid, and amino acid biosyntheses. This explains the persistence of non-photosynthetic
plastids in various eukaryotes, e.g., the malaria parasite Plasmodium. The unicellular algal genus
Cryptomonas (Cryptophyta) contains both photosynthetic and non-photosynthetic members, the latter having
recently evolved on at least three separate occasions. In order to elucidate the evolutionary mechanisms
underlying the loss of photosynthesis in Cryptomonas, we sequenced the plastid genomes of two
non-photosynthetic strains, Cryptomonas sp. M1634B and SAG977-2f, as well as the photosynthetic strain
Cryptomonas curvata CCAP979/52. Together with the previously sequenced plastid genome of the
non-photosynthetic C. paramecium CCAP977/2a, we carried out a four-way comparison of genome size,
coding capacity, pseudogene content, and genome synteny. Here we discuss how the non-photosynthetic
plastid evolved in these strains.
55
Session 7
Phylogenetic positions of marine gregarines Selenidium terebellae and Lecudina tuzetae, and molecular
evidence of their remnant nonphotosynthetic plastids (apicoplasts)
Kevin Wakeman1†, Takuro Nakayama2†, Goro Tanifuji3, Eriko Matsuo4, Brian S. Leander5 Yuji Inagaki2, 4
1
Department of Biological Sciences, Hokkaido University, 10-8 Kita-ku, Sapporo, Hokkaido 060-0810,
Japan; 2Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki
305-8577, Japan; 3Department of Zoology, National Museum of Nature and Science, Tsukuba, Ibaraki
305-0005, Japan; 4Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba,
Ibaraki 305-8577, Japan; 5Departments of Botany and Zoology, University of British Columbia, Vancouver,
BC V6T 1Z4, Canada; email: ntakuro@ccs.tsukuba.ac.jp †These authors contributed equally to this work
Gregarines are a diverse group of apicomplexan parasites that mostly infect the intestines of insects and
marine invertebrates. Because some marine gregarines have traits inferred to reflect the most recent
apicomplexan ancestor, improved knowledge of these parasites is important for understanding the early
evolution of apicomplexans as a whole. However, molecular data, with the exception of small subunit
ribosomal RNA (SSU rRNA) genes, has not been widely gathered for gregarines as a whole. The SSU rRNA
gene sequences that have been gathered for this group have been used to discriminate different species from
one another and in assessing the deeper phylogenetic relationship among gregarines, as well as the
relationship between these groups and non-gregarine apicomplexan parasites. Unfortunately, these data are
unable to resolve the deepest relationships among apicomplexans because of the divergent nature of
gregarine SSU rRNA genes. In the present study, we generated RNA-seq data from two species of marine
gregarines, Selenidium terebellae and Lecudina tuzetae, that belong to different subgroups of gregarines,
archigregarines and eugregarines, respectively. The results of a phylogenomic analysis using a dataset
comprised of 76 protein sequences indicated that S. terebellae and L. tuzetae formed a clade. This gregarine
clade formed the sister group to a clade of non-gregarine apicomplexan parasites. The monophyly of
gregarines and the Apicomplexa as a whole were robustly supported by maximum-likelihood analyses. Our
RNA-seq data also enabled us to establish evidence for the presence of nonphotosynthetic plastids in S.
terebellae and L. tuzetae. In both gregarine RNA-seq data, we found transcripts encoding putative proteins
bearing specific phylogenetic affinities to plastid proteins in other photosynthetic organisms. The potential
plastid proteins included enzymes for fatty acid metabolism, pyruvate metabolism, and transporters for
phosphorylated carbohydrates. Noteworthy, some of the potential plastid proteins were predicted to have
signal peptides, which have been established as a part of the apicoplast-targeting signal in non-gregarine
apicomplexans. Therefore, we conclude that both S. terebellae and L. tuzetae retain metabolically active, but
non-photosynthetic plastids. Based on the results presented here, we propose that the common ancestor of
archigregarines, eugregarines, and non-gregarine apicomplexan parasites evolved from a single ancestral cell
bearing a nonphotosynthetic plastid.
56
Session 7
Screening and discovery of lineage-specific mitosomal membrane proteins in Entamoeba histolytica
Herbert J. Santos1, Yuki Hanadate1,2, Kenichiro Imai3, Yoshinori Fukasawa3, Toshiyuki Oda3, Fumika
Mi-ichi4 and Tomoyoshi Nozaki1,2 1
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama,
Shinjuku-ku, Tokyo 162-8640, Japan. 2Graduate School of Life and Environmental Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. 3Biotechnology Research Institute for Drug
Discovery, National Institute of Advanced Industrial Science and
Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan. 4Division of Molecular and Cellular
Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan.
email: herbert@niid.go.jp
Entamoeba histolytica, an anaerobic intestinal parasite causing dysentery and extra-intestinal abscesses in
humans, possesses highly reduced and divergent mitochondrion-related organelles (MROs) called mitosomes.
This organelle lacks many features associated with canonical aerobic mitochondria and even other MROs
such as hydrogenosomes. The Entamoeba mitosome has been found to have a compartmentalized sulfate
activation pathway, which contributes to cellular differentiation, an essential process allowing for the
transmission of the parasite to its host. It also has other lineage-specific features such as a shuttle system via
Tom60, and a novel subclass of β-barrel outer membrane protein called MBOMP30. With the discoveries of
such unique characteristics of Entamoeba mitosomes, there still remain a number of significant unanswered
issues pertaining to this organelle. Particularly, the present understanding of the inner mitosomal membrane
is extremely limited. So far, only a few homologs of transporters for various substrates have been confirmed,
while the components of the protein translocation complexes appear to be absent or are yet to be discovered.
Employing a similar strategy as in our previous work, we collaborated to screen and discover mitosomal
membrane proteins. Using a specialized prediction pipeline, we searched for proteins possessing α-helical
transmembrane domains, which are unique to E. histolytica mitosomes. From the prediction algorithm, 25
proteins emerged as candidates, and three were observed to be localized to the mitosomal membranes.
Further analyses of the predicted proteins may provide clues to answer key questions on mitosomal evolution,
biogenesis, dynamics, and biochemical processes.
57
Session 7
Rare loss of the Calvin Benson cycle in non-photosynthetic plastids
Ryoma Kamikawa1,2, Stefan Zauner3, Daniel Moog3,4, Goro Tanifuji5, Ken-ichiro Ishida6, Shigeki Mayama7,
Tetsuo Hashimoto6,8, John M Archibald4, Uwe-G Maier3,9, Hideaki Miyashita1,2 Yuji Inagaki6,8
1
Graduate School of Global Environmental Studies, 2Graduate School of Human and Environmental Studies,
Kyoto University, Kyoto, Japan; 3Laboratory for Cell Biology, Philipps-Universität Marburg, Marburg,
Germany; 4Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada;
5
National Museum of Nature and Science, Tsukuba, Ibaraki, Japan; 6Faculty of Life and Environmental
Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 7Department of Biology, Tokyo Gakugei University,
Koganei, Tokyo, Japan; 8Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki,
Japan; 9LOEWE-Zentrum für Synthetische Mikrobiologie (SynMikro), Hans-Meerwein-Strasse, Marburg,
Germany. Email: kamikawa.ryoma.7v@kyoto-u.ac.jp
Although a number of eukaryotes have experienced secondary loss of photosynthesis, loss of photosynthesis
does not always result in loss of plastids. Non-photosynthetic plastids still retain some functions and are
indispensable for cell viability. However, how these non-photosynthetic organelles lose functions after loss
of photosynthesis remained to be fully understood. In this study, we characterized the non-photosynthetic
plastids of the apochlorotic diatom, Nitzschia sp. NIES-3581 by transcriptome analyses, cell biological
experiments, and phylogenetic analyses. We found that these plastids retained various pathways beyond
photosynthesis, which include amino acid biosynthesis and the Calvin Benson cycle lacking RuBisCO. By
comparative analyses, we discuss why the Calvin Benson cycle unable to fix CO2 is still retained and
difficult to be lost in non-photosynthetic plastids.
58
Posters
59
P1
On the expansion of the plastid RRM protein family in the Viridiplantae
Hiromichi Uchiyama1, Mizuho Ichinose1,2, Mamoru Sugita1
Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan; 2Institute of Transformative
Bio-Molecules (ITbM), Nagoya University, Nagoya, 464-8602, Japan; email:sugita@gene.nagoya-u.ac.jp
1
Many plastid and mitochondrial proteins are derived from their bacterial endosymbiotic ancestors, but most
of their genes now reside in the nucleus while others reside in the retained small ancestral genome. In
plastids, gene expression is strongly regulated at the posttranscriptional level that is mediated by numerous
nuclear-encoded RNA-binding proteins. Among them, RNA recognition motif (RRM)-containing proteins
are considered as a major regulator in various types of RNA metabolism but their function is not fully
understood. In this article, we surveyed the plastid RRM proteins encoded in Viridiplantae genomes. This
survey revealed that the plastid-localized RRM family constituted four types of members containing 1 to 4
RRMs. The plastid-targeted RRM protein gene family might have expanded during endosymbiosis and plant
evolution.
60
P2 (Oral, Session 2)
Plastid retention in Elysia viridis is determined by algae food source
Cessa Rauch1, Aloysius G.M. Tielens2,3, Sven B. Gould1, Gregor Christa1.
1 Institute for Molecular Evolution, Heinrich-Heine-University Düsseldorf 40225, Germany.
2 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, The
Netherlands.
3 Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center,
Rotterdam, The Netherlands.
Photosynthetic symbioses are remarkably widespread among the animal kingdom. It occurs for instance in
sponges, corals, mollusks and even a vertebrate. One of the most curious photosynthetic symbioses is found
in some sacoglossan sea slugs: functional kleptoplastidy. Here, only the plastids of the algal prey are
sequestered and embedded photosynthetically functional inside the animals’ own cells. In some elysoid
Sacoglossa, the kleptoplasts are kept functional for several months of starvation, but the basics of how the
plastids are kept active remains unknown. In its natural habitat Elysia viridis feeds on different algae, which
include Bryopsis or Cladophora. We found that specimens collected from Cladophora and subsequently
solely fed on that species for 3 months, do not retain any functional kleptoplasts; all plastid are digested with
the remaining food. During starvation, however, the plastid-free animals shrink more rapidly compared to a
control group that was nurtured on Bryopsis before exposure to starvation. The latter supports a beneficial
role of the kleptoplasts during periods of food depletion. Contrary, in the presence of food algae, feeding on
the cytosol seems to be sufficient and slugs feeding on Cladophora do not rely on any sequestered plastids.
Elysia viridis appears able to switch between plastid retention and digestion, which, intriguingly, depends on
the algae it feeds on.
61
P3
Direct evidence for the presence of enigmatic peptidoglycan in the intermembrane space of chloroplast
envelope in the moss Physcomitrella patens
Naoki Sato1,2, Masakazu Toyoshima1,2, Naoyuki Tajima1,3, Katsuaki Takechi4 and Hiroyoshi Takano4
1
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902,
Japan; 2CREST, Japan Science and Technology Agency, Tokyo, 102-0076, Japan; 3College of Bioresource
Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan; 4Graduate School of
Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan.
Chloroplasts are believed to be descendants of ancestral cyanobacteria that have peptidoglycan layer between
the outer and the inner membranes. Comparative genomic analysis has suggested that some land plants such
as the moss Physcomitrella patens have a complete set of genes involved in the synthesis of peptidoglycan,
but the presence of peptidoglycan layer has remained elusive. Recently, a technique using a labeled
derivative of the peptidoglycan precursor D-alanyl-D-alanine revealed that the incorporated label was
distributed at the periphery of the chloroplast (Hirano et al. 2016. Plant Cell, in press). Here we re-examined
if peptidoglycan is visible by conventional electron microscopy using the hot-osmium fixation intended to
stain densely any sugar-containing molecules. The chloroplast envelope membranes of P. patens appear
exceptionally straight and rigid, which is never the case in the chloroplasts of other plants or algae. This
gives us the first suggestion that there might be a solid support of the envelope membranes. We obtained
high-magnification images of the envelope membranes, and measured the density of the two membranes and
the inter-membrane space using the thylakoid membranes as a reference. We compared various moss
materials (mutants and ampicillin-treated) that are either expected or not expected to contain peptidoglycan.
We developed a new method for the analysis of the density of the digital images at a resolution of 1 pixel.
Statistical image analysis clearly indicated that the density of the inter-membrane space was reduced in the
moss treated with ampicillin that is known to block synthesis of peptidoglycan or in the mutants defective in
peptidoglycan synthesis. These results provide direct evidence for the presence of peptidoglycan between the
two membranes of the chloroplast envelope.
62
P4
SnRK1 kinase and the NAC transcription factor SOG1 as possible components of a mitochondrial
retrograde signaling pathway mediating the low energy response triggered by low ATP levels
Hidefumi Hamasaki1,2, Yukio Kurihara1, Takashi Kuromori3, Megumi Kobayashi4, Hiroaki Kusano2, Yuko
Imura3, Noriko Nagata4, Hiroaki Shimada2 , Yoshiharu Y. Yamamoto1,5, Minami Matsui1
1
Synthetic Genomics Research Group, Biomass Engineering Research Division, RIKEN Center for
Sustainable Resource Science, Yokohama 230-0045, Japan; 2Department of Biological Science and
Technology, Tokyo University of Science, Katsushika-ku, Tokyo 125-8585, Japan; 3Discovery Research Group,
RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan; 4Japan Woman’s University,
2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan; 5 Faculty of Applied Biological Sciences, Gifu
University, 1-1 Yanagido, Gifu, Gifu, 501-1193, Japan. email: gif193@gifu-u.ac.jp
Plant growth is achieved by cell division, elongation and differentiation, all of which consume ATP.
However, it is unclear how the plant growth is regulated by the level of intracellular ATP. To understand the
possible regulatory pathway from the mitochondria, we took a chemical and genetic analyses on the sd3,
which has abnormal mitochondria, decreased in ATP level, and seedling-lethal phenotype.
Plant growth is strictly controlled by cell cycle for which adequate of intracellular ATP are required.
However, it is unclear how changes in the level of intracellular ATP affect the growth response. To reveal the
specific pathway that is induced at low ATP concentration, we analyzed a mutant of a mitochondrial protein
transporter gene, sd3, which shows decrease in ATP level, abnormal mitochondria, and sever growth arrest at
seedling stage.
Our genetic analysis revealed three genetic loci that can rescue the seedling lethal phenotype of sd3, that
are kin10 and kin11, both of which are mutants of SnRK genes, and sog1, a mutant of a NAC transcription
factor gene involved in regulation of cell cycle. These results strongly suggest that the growth arrest by
dysfunction of SD3 is not inevitable, but a signal for growth arrest is activated in the sd3 mutants. Our results
also suggest that the identified three gene products locates downstream of the signal generated by
dysfunction of SD3. The idea fits with an observation that SOG1 is phosphorylated by treatment of plants
with antimycin A blocking the mitochondrial electron transport and thus expected to suppress ATP synthesis
at mitochondria.
Relationship of our speculative "low ATP signal", so called mitochondrial retrograde signals, one of
which is activated by antimycin A, and ROS signals, which are also involved in the retrograde signals will be
discussed.
63
P5
Elucidating the mechanism of host mitochondrial recruitment of Toxoplasma gondii
Junpei Fukumoto1,2, Sakura Takaya1, Ryuma Matsubara1,2, Kisaburo Nagamune1,3
1
Department of Parasitology, National Institute of Infectious Disease, Shinjyuku-ku, Tokyo 162-8640, Japan;
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571,
Japan; 3Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571,
Japan. email: junpei@niid.go.jp
2
Toxoplasma gondii is an obligate intracellular protistic parasite and has a potential to infect virtually all
warm blooded animals. In the host cell, the parasite is confined to a membrane called parasitophorous
vacuole membrane (PVM). It is known that the parasite recruits some host organelles such as mitochnodria
and endoplasmic reticulum on the PVM, whereas some organelles like as lysosomes are excluded from there.
The mechanism of recruitment of host organelles has not been fully understood. Manifesting the mechanism
of host mitochondrial recruitment, we performed a quantitative mass spectrometry analysis of T. gondii
proteins on the host mitochondria in the host cell infected with the parasite and compared it with
non-infected ones, because there is a strong possibility that the recruitment factor(s) binds to the host
mitochondria. As a result, we could detect 777 proteins of T. gondii and set some criteria to narrow down the
candidates. Thirty proteins remained as the candidates and we further observed the localization of these
candidates by employing confocal microscopy to confirm whether they are truly transported to host
mitochondria. Six of 30 proteins colocalize, at least partially, with the host mitochondria on the PVM. We
determined the recruitment ability of 6 candidates and found that a functionally unknown protein could
enhance the mitochondrial recruitment.
64
P6
The central vacuole of the diatom Phaeodactylum tricornutum: Identification of new tonoplast proteins
and a functional di-leucine motif
Viktoria Schreiber1, Josefine Dersch1, Katharina Puzik1, Simone Stork2, Xiaojuan Liu1, Julian Schulz1,
Andreas Klingl3, Stefan Zauner1, Uwe-G. Maier1,4
1
Laboratory for Cell Biology, Philipp University of Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg;
Technische Hochschule Köln, Gustav-Heinemann-Ufer 54, 50968 Köln; 3Department Biology I – Botany,
Ludwig-Maximilians University Munich, Großhardener Str. 2-4, 82152 Planegg-Martinsried; 4LOEWE
Center for synthetic microbiology (SYNMIKRO), Hans-Meerwein-Str. 6, 35032 Marburg
2
The diatom Phaeodactylum tricornutum is a unicellular microalga that originated through secondary
endosymbiosis, the uptake of a red alga by a eukaryotic host. As a result their plastid possesses two
additional membranes in comparison to primary plastids. Besides the so-called complex plastid, P.
tricornutum holds vacuolar-like structures surrounded by only one membrane comparable to those present in
yeast or plants. One of these structures is a dominant compartment with a mabled phenotype, which is
supposed to represent a chrysolaminarin storage compartment. By now, little is known about the vacuolar
system and the vacuolar trafficking in phototropic protists. In order to study the functions of this
compartment as well as the trafficking of proteins to the surrounding membrane, we inspected putative
proteins in silico and performed in vivo studies. Here we present the vacuolar localization of several proteins
involved in carbohydrate metabolism and water homeostasis as well as proteins of unknown functions. In
addition, we could identify a di-leucine motif important for correct targeting of a protein to the vacuolar
membrane.
65
P7
The plastome/nuclear-genome incompatibility in the evening primrose Oenothera
Danijela Kozul, Arkadiusz Zupok, Mark Aurel Schöttler, Ralph Bock, Stephan Greiner
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
email: kozul@mpimp-golm.mpg.de
The model plant Oenothera (evening primrose) is perfectly suited to study chloroplast-mediated hybridizing
barriers. Hybrid offspring within Oenothera regularly displays incompatibility between the chloroplast and
the nuclear genome (plastome-genome incompatibility; PGI). In sum 18 incompatibilities of different
strength occur. Those genetically define the species and prevent colonization of different geographical
regions by some genotypes.
The aim of this work is to find molecular and evolutionary mechanism that causes
incompatibilities in Oenothera. Recently, by applying association mapping, physiological and molecular
analysis, we could demonstrate that one of the incompatibilities is due to the promoter region in front of the
psbB operon. This work is now extended to the nuclear factors, quite likely RNA polymerase sigma factors.
By employing our draft nuclear genome sequence, based on a homology search, suitable candidates have
been identified. The gene models are now verified and subsequently association mapping will reveal the
differences that might be responsible for the incompatibility. Further, to pinpoint the remaining
incompatibilities in Oenothera, a high-throughput screen is currently established. We aim to determine
translation efficiencies for the entire (incompatible) chloroplast genome using deep sequencing approaches
combined with ribosomal footprints (ribosome profiling).
66
P8
A phylogenomic study placed a previously undescribed eukaryote, strain SRT308, at the base of the
Euglenozoa clade.
Euki Yazaki1, Takashi Shiratori1, Tetsuo Hashimoto1,2, Ken-ichiro Ishida1, Yuji Inagaki1,2
Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki, 305-8577, Japan; 2Center for Computational Science, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki, 305-8577, Japan;
1
A novel unicellular flagellate, strain SRT308, was isolated from a marine sediment sample collected from the
Republic of Palau in 2013, and has been maintained in the laboratory. We firstly explored the position of this
flagellate using the maximum-likelihood (ML) phylogenetic analysis of small subunit ribosomal DNA (SSU
rDNA) sequences. In the SSU rDNA tree, SRT308 showed no strong affinity to any eukaryotes or eukaryotic
lineages, suggesting that this flagellate represents an unprecedented eukaryotic lineage. Therefore, to
determine the accurate phylogenetic position strain SRT308, we conducted a ML analysis based on 153
nucleus-encoded gene sequences, which included a part of the transcriptomic data of this flagellate. Our
phylogenomic analyses robustly placed SRT308 at the base of the clade of kinetoplastids, euglenids, and
diplonemids, collectively called the Euglenozoa. The intimate affinity between SRT308 and Euglenozoa is
consistent with the discoidal mitochondrial cristae observed in the electron microscopy of SRT308. The
clade of SRT308 + Euglenozoa further grouped with Heterolobosa, Jakobida, and Tsukubamonas globosa,
forming the Discoba clade. Finally, we will evaluate two possibilities — (1) this flagellate is a very
early-branching euglenozoan or (2) represents a novel lineage, which is closely related to, but distinct from
Euglenozoa — by comparing the ultrastructural characteristics between SRT308 and euglenozoans.
67
Measuring the temperature-dependence of cpRNP-RNA association in chloroplasts by a leaf-tissue
based RIP-chip assay
Marie-Kristin Lehniger1, Stephanie Gathmann1, Christian Schmitz-Linneweber1
1
Molecular Genetics, Institute of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Philippstr.
11-13,
Bldg.
22
(Rhoda
Erdmann
Haus),
D-10115
Berlin,
Germany.
email:
The nuclear–encoded chloroplast ribonucleoproteins (cpRNPs) are a group of plastid hnRNP-like
RNA-binding proteins. They are highly abundant proteins that are regulated in a light- and
temperature-dependent manner. cpRNPs bind multiple RNA targets and are involved in RNA processing,
stabilization and editing. The cpRNPs CP29A and CP31A were shown to be important for mRNA
accumulation and processing at low temperatures. In this study, we wanted to analyze if the transcript pools
bound by CP29A change in quantity and/or quality under cold acclimation conditions. Therefore, a new
method for immunoprecipitation of RNA-protein complexes from plant leaf tissue was established.
An increased expression of CP29A under cold acclimation could be demonstrated by immunoblot analysis as
well as immunofluorescence assays of Arabidopsis thaliana protoplasts using a polyclonal antibody against
CP29A. Furthermore, the sub-organellar localization of CP29A changed after cold acclimation. Results on
RNA co-immunoprecipitation and microarray (RIP-chip) analysis of CP29A in leaf tissue of cold acclimated
versus normal grown Arabidopsis thaliana and Nicotiana tabacum will be presented.
68
P10
Gregarine-like apicomplexan parasite isolated from the intestinal tract of a centipede Scolopocryptops
rubiginosus
Ryosuke Miyata1, Takuro Nakayama2, Goro Tanifuji3, Yasuhiko Chikami1, Kensuke Yahata4, Yuji Inagaki1,2
1
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki, 305-8577, Japan; 2Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki, 305-8577, Japan; 3Department of Zoology, National Museum of Nature and Science, 4–1–
1 Amakubo, Tsukuba, Ibaraki, 305–0005 Japan; 4Faculty of Life and Environmental Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; Email: yuji@ccs.tsukuba.ac.jp
Gregarines are a group of apicomplexan parasites that inhabit in the digestive tracts or body cavities of
diverse invertebrates. Because of their morphological characteristics and phylogenetic positions inferred
mainly from small subunit rRNA gene (SSU rDNA) sequences, gregarines are anticipated to provide novel
insights into the early evolution of the Apicomplexa. Some of us have already gathered RNA-seq data of two
gregarines parasitizing marine invertebrates (marine gregarines), and examined their phylogenetic positions
as well as the presence/absence of remnant nonphotosynthetic plastids (apicoplasts) (see the presentation of
Wakeman & Nakayama et al.). On the other hand, gregarines parasitizing terrestrial invertebrates (terrestrial
gregarines) have not been subjected to RNA-seq analysis. Due to the lack of large-scale transcriptomic data
of terrestrial gregarines, their phylogenetic positions amongst apicomplexan parasites (including marine
gregarines) or the presence/absence of apicoplasts remain controversial. In this study, we isolated
gregarine-like parasites from the digestive tract of a centipede Scolopocryptops rubiginosus, as the first step
toward large-scale transcriptomic analyses of terrestrial gregarines. We amplified and determined the SSU
rDNA sequence of the gregarine-like parasite, and the resultant sequence matched none of the known SSU
rDNA sequences in NCBI nr database with high nucleotide identity. In the maximum-likelihood tree inferred
from a SSU rDNA alignment, the parasite in S. rubiginosus grouped with non-gregarine apicomplexans,
Cryptosporidium spp., albeit this grouping received little statistical support. Combining the results described
above, we conclude that the gregarine-like parasite in S. rubiginosus is a novel, potentially early-branching
member of the Apicomplexa. We will also report preliminary results from the RNA-seq analysis of this
apicomplexan parasite currently underway.
69
P11
Metabolic dynamics and transcriptional regulation in temperature adaptation of Arabidopsis
Natsuki Hayami1#, Ayaka Hieno1, Miyako Kusano2,3, Kyonoshin Maruyama4, Mieko Higuchi2, Kousuke
Hanada5, Minami Matsui2, Yoshiharu Y. Yamamoto1,2,6
1
The United Graduate School of Agricultural Sciences, Gifu University, Yanagido 1-1, Gifu City, Gifu,
501-1193, Japan; 2RIKEN Center for Sustainable Resource Science, Suehiro-cho 1-7-22, Tsurumi-ku,
Yokohama City, Kanagawa, 230-0045, Japan; 3Faculty of Life and Environmental science, Tsukuba
University, Tennodai 1-1-1 Tsukuba City, Ibaraki, 305-8577, Japan; 4 Biological Resources Division, Japan
International Research Center for Agricultural Sciences, Ohwashi 1-1, Tsukuba City, Ibaraki, 305-8686,
Japan; 5Ftontier Research Academy for Young Researchers, Kyushu Institute of Technology, Sensui-cho 1-1,
Tobata-ku, Kitakyushu-shi, Fukuoka, 804-8550, Japan, 6JST ALCA. #email: v6103007@edu.gifu-u.ac.jp Temperature is an environmental factor that is largely fluctuating in the field. In higher plants, the fluctuation
is thought to disturb metabolic balance, and thus adaptation to temperature is considered to be a major
determinant of biomass production in higher plants. In order to understand disorder of metabolism by
temperature shift and also transcriptional responses which could recover the disorder, we decided to do
parallel analysis of metabolome and transcriptome during adaptation to rather moderate temperatures from 4
to 30 oC. In order to estimate effect of photosynthesis in the temperature adaptation, low and moderately
strong light conditions were compared at the same time. We investigated five temperature points (4℃, 8℃,
14℃, 22℃, 30℃) and two light intensities (limited and saturated light conditions) for the analysis. In our
presentation, we would like to show observed disorder of metabolism, possible metabolite-driven
transcriptional regulation, possible metabolite regulation by transcriptional responses during temperature
adaptation.
70
P12
Molecular basis for β-1,3-glucan mediated self/nonself recognition in unicellular protists
Mayumi Kobayashi, Mousumi Bhadra, Toshinobu Suzaki
Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe
657-8501, Japan. email: k_mayumi@people.kobe-u.ac.jp
In heterotrophic protists, self and nonself discrimination system is essential, because it is important not only
for correct targeting of predatory organisms to the desired prey, but also for establishment and maintenance
of the cellular interactions between host and endosymbiont. Heliozoans are unicellular protists that usually
feed on algae and protozoa, and occasionally harbor endosymbiotic green algae in the cytoplasm. In an
attempt to understand the most primitive self/nonself recognition mechanisms, identification of heliozoan proteins
that recognize the prey cell surface was carried out. In actinophryid heliozoan Actinophrys sol, prey recognition was
found to be initiated by the aid of a β-1,3-glucan binding protein (β-GBP) that is usually stored in vesicles
called extrusomes, and secreted in response to the prey signals. In a centrohelid heliozoan Raphidiophrys
contractilis, in spite of a similar behavior in food capturing, a protein besides β-GBP was identified that also
recognizes β-1,3-glucan. This protein showed a strong homology to the major vault protein (MVP) of
vertebrates, whose function is still not clear. The involvement of the heliozoan MVP homolog in prey
recognition suggests a possible novel function of this protein in self/nonself discrimination, at least in
unicellular organisms.
71
P13
Transcriptome analysis of Chrysochromulina parkeae, a haptophyte with a cyanobacterial
endosymbiont
Chinatsu Tsukakoshi1, Shigekatsu Suzuki2, Atsushi Nakamura1, Kyoko Hagino3, Ken-ichiro Ishida4
1
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1- Tennodai, Tsukuba,
Ibaraki 305-8577, Japan; 2Center for Environmental Biology and Ecosystem Studies, National Institute for
Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan; 3Center for Advanced Marine
Core Research, Kochi University, 200 Monobe-Otsu, Nankoku, Kochi 783-8502, Japan; 4Faculty of Life and
Environmental Sciences, University of Tsukuba, 1-1-1 Tennnodai, Tsukuba, Ibaraki 305-8577, Japan. email:
s1621008@u.tsukuba.ac.jp
Uncultivated unicellular N2-fixing cyanobacteria (UCYN-A) play an important role in oceanic nitrogen
fixation. The genome of UCYN-A lacks genes for major metabolic pathways including the tricarboxylic acid
cycle and photosystem II, and thus it is thought that UCYN-A is a symbiont. Phylogenetic analyses show that
its host is a haptophyte closely related to Chrysochromulina parkeae. However, the association of UCYN-A
and the host remained unclear because C. parkeae was unculturable. In this study, we isolated C. parkeae
with a cyanobacteria-like endosymbiont. Our phylogenetic analysis showed that this endosymbiont is one of
the members of UCYN-A. We sequenced the endosymbiont genome, and it was very similar to that of
UCYN-A in gene contents. Both of the genomes had nitrogen fixation genes, indicating that the
endosymbiont is probably an organelle for nitrogen fixation. To search genes transferred from the
endosymbiont to the host, we established stable cultures of C. parkeae, and performed a transcriptome
analysis. As a result, we found no candidates for endosymbiotic gene transfer. We consider that the
endosymbiont is at the very early stage in the process of becoming an organelle.
72
P14
The endophytic fungus Piriformospora indica enhances drought stress tolerance in rice plants
Ko-Hsuan Shao1, Hsuan-Ju Tsai1, Chien-Hao Tseng1, Shu-Jen Wang1
1
Department of Agronomy, National Taiwan University, Taipei, Taiwan.
Corresponding author: Shu-Jen Wang (email: shujen@ntu.edu.tw)
Presenting author: Chien-Hao Tseng
Piriformospora indica (P. indica), a culturable endophytic fungus, is known to play a crucial role in
promoting plant growth and enhancing environmental stress tolerance in several plant species. Drought is a
serious abiotic stress which leads to crop yield decreasing and enormous economic loss. In our study, it was
found that P. indica can promote rice plant growth and grain production. Furthermore, the activities of some
antioxidant enzymes were enhanced and drought damages were reduced in P. indica-colonized rice plants
under water stress conditions. To clarify the mechanisms involved with the P. indica-promoted drought
tolerance, RNA sequencing (RNA-seq) was employed to analyze transcript profiles of P. indica-colonized
rice plants. According to the RNA-seq results, several groups of genes (e.g. oxidation reduction and
antioxidant activity) were selected and considered to be involved with improving drought stress tolerance in
rice plants. Taken together, our study provides both physiological and molecular evidences to reveal the
mechanisms of P. indica-promoted drought tolerance in rice plants and furthers our understanding of how
plants defend environmental stresses.
Key words: Piriformospora indica, endophytic fungus, drought tolerance, RNA sequencing
73
P15
Changes in ultrastructure and chemical composition of the cell wall of Chlorella in relation to
endosymbiosis in Paramecium bursaria
Rina Higuchi1, Chihong Song1,2, Toshinobu Suzaki1
1
Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; 2National
Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan. email:
hig.m.rina@gmail.com
For successful establishment and stable maintenance of endosymbiosis in the Chlorella-Paramecium
bursaria system, interaction between the symbiotic Chlorella and the host’s cytoplasm is important, in which
the outer-most surface of the endosymbiotic Chlorella may play a pivotal role in mutual recognition between
the partner cells. Morphology and chemical properties of the cell wall of symbiotic Chlorella variabilis strain
Kb1 were examined in both free-living and endosymbiotic condition in the cytoplasm of Paramecium
bursaria. The cell wall of Kb1 was stained with Calcofluor white M2R, FITC-WGA, or FITC-LFA.
Calcofluor is a fluorescent dye that stains β-D-glucopyranose polysaccharides. WGA is a lectin that binds
N-acetylglucosamine and sialic acid residues, and LFA is a sialic acid-specific lectin. The results suggest a
possible decrease of the amount of glycosaminoglycans at the surface of the cell wall under the
endosymbiotic condition. Transmission electron microscopy with quick-freezing and freeze-substitution
techniques showed that thickness of the cell wall of free-living Kb1 was 20-30 nm, while that of symbiotic
Kb1 was 7-12 nm. A mutant strain that lacks the ability of infection to P. bursaria was produced by heavy
ion beam irradiation from C. variabilis NC64A. As with Kb1 strain, the cell wall of NC64A was stained well
with Calcofluor, while that of the mutant strain (NC64A#48) showed a decreased stainability (~50%).
Transmission electron microscopy showed that the thickness of the cell wall of NC64A was 23-35 nm, while
that of NC64A#48 was 15-25 nm. These findings suggest that the cell wall of symbiotic Chlorella changes in
both structure and chemical properties with the establishment of Chlorella-Paramecium symbiosis.
74
Fine-structural observations on engulfing behavior and enlargement of endosymbiont in Hatena
arenicola.
Mami Nomura1, Ken-ichiro Ishida2
1
Shimoda Marine Research Center, University of Tsukuba, 415-0025, Japan; 2Faculty of Life and
Environmental Sciences, University of Tsukuba, 305-8571, Japan. Email: true82future@gmail.com
Hatena arenicola engulfs a prasinophyte alga, Nephroselmis spp. and maintain it as an
endosymbiont, which is distributed only to one of daughter cells during cell division. H. arenicola is a one of
key organisms for understanding endosymbiosis because H. arenicola is a one of kleptoplastidal protists and
suggested to be at an intermediate stage of secondary endosymbiosis. In this study, we observed the
Nephroselmis spp. cells that were being engulfed and the enlargement process of these as endosymbionts in
H. arenicola cells.
Usually Nephroselmis cells have extracellular organic scales, but the Nephroselmis endosymbionts
observed in H. arenicola cells lack the scales. Our fine structure observation showed that the feeding
apparatus of H. arenicola cell peeled off the scales of the Nephroselmis cell during engulfment. A pool of
peeled scales was observed in the feeding apparatus of H. arenicola cell and membrane-bounded cell
coverings were observed apart from Nephroselmis sp. cell body. It suggested that H. arenicola selectively
remove the cell coverings when it engulfs the Nephroselmis cell.
At the middle of endosymbiont enlargement process, the eyespot of endosymbiont was observed
at the anterior end of H. arenicola cell where the feeding apparatus was usually present in colorless H.
arenicola cells. The nucleus of endosymbiont did not have heterochromatin which is present in the nucleus
of free living Nephroselmis sp. cell. The nucleus of endosymbiont in the middle of enlargement process was
not observed near the nucleus of H. arenicola cell, although it has been reported that the nucleus of
endosymbiont is associated to the nucleus of H. arenicola. These observations may imply that the gene
expression pattern of endosymbionts may change during the enlargement process in the H. arenicola cell.
75
P17
ER-subcompartmentalization allows the separation of the unfolded protein response from protein
transport into complex plastids
Jonny Gentil1, Simone Stork1, Julia Lau1, Uwe-G. Maier1, 2
1
Laboratory for Cell Biology, Philipp University of Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg,
Germany; 2LOEWE Center for synthetic microbiology (SYNMIKRO), Hans-Meerwein-Str. 6, 35032 Marburg,
Germany
Diatoms are unicellular algae evolved by secondary endosymbiosis harboring a complex plastid of red algal
origin surrounded by four membranes. The outer plastid membrane (cER) is in continuum with the outer
nuclear envelope and thus part of the endoplasmatic reticulum (ER). Hence, the ER of diatoms can be
divided into several functionally and morphologically different regions, the host ER (hER), the nuclear
envelope (NE) and the cER. Nucleus-encoded plastidal proteins have to cross the cER membrane via the
Sec61 translocon to reach their final localizations. One can presume that the number of unfolded proteins
crossing the cER lumen varies between day and night according to the physiological needs in the plastid, a
situation which is not expected for the hER. Therefore, the different ER sub-compartments have to manage
different concentrations of unfolded proteins all over the day. In order to study this hypothesis we searched
the genome sequence of one diatom for Ire1 and Perk, which are responsible for sensing unfolded proteins in
the ER lumen. By localization experiments we could observe localization of the UPR proteins mainly in the
hER but not in the cER. On the contrary, the ER associated degradation (ERAD) machinery is not limited to
one specific ER sub-compartment. Our findings indicate that the functionally sub-compartmentalization of
the ER permits protein quality control, stress sensing and transport of unfolded proteins into the plastid
simultaneously.
76
P18
The acidic domain of Arabidopsis chloroplast CP31A is essential for providing cold resistance
Ayako Okuzaki1,2, Marie-Kristin Lehniger1, Christian Schmitz-Linneweber1
1
Humboldt University Berlin, Berlin D-10115, Germany; Tamagawa University, Machida 194-8610, Japan.
email:okuzaki8@agr.tamagawa.ac.jp
Chloroplast gene expression is controlled by a large number of nuclear-encoded RNA binding proteins
which play a role for individual steps in post-transcriptional RNA processing events. However, none of them
has been shown to be a true regulator of chloroplast gene expression. As previously reported, one of the
chloroplast RNA binding proteins, CP31A, has multiple roles in chloroplast RNAs stabilization and RNA
editing. CP31A has two RRM domains as well as an unstructured acidic domain, which is known to be
phosphorylated. It was reported that CP31A is phosphorylated in a light-dependent fashion. In sum, CP31A
is a prime candidate for mediating light-activated global regulation of chloroplast RNA processing. In
addition, we reported that cold treatment induced bleaching of emerging tissue paralleled by a reduction of
mRNAs accumulation and defects of RNAs processing in Arabidopsis cp31a knock-out lines.
In this study, we aimed to investigate the role of CP31A domains for RNA processing events in chloroplast.
We complemented the cp31a knock-out line with a CP31A version devoid of the acidic domain
(CP31AΔAD) controlled by the native CP31A promoter and the CAMV 35S promoter, respectively.
Overexpression of CP31AΔAD was able to complement the loss of ndhF transcripts and several RNAs
editing defects under normal growth conditions. However, CP31AΔAD levels comparable to wild type
CP31A protein amounts were only leading to partial complementation of RNA accumulation and RNA
processing. These results suggested that the RRM domains are sufficient for these RNAs processing events,
with the acidic domain only having a supportive role. By contrast, the bleaching phenotypic observed after
cold stress treatment in cp31a knock-out line was not complemented by overexpression of CP31AΔAD.
Thus, the acidic domain of chloroplast CP31A is essential for providing cold resistance.
77
Elucidating target RNA editing sites of embryo lethal PLS class PPR proteins
Matthias Burger1, Anja Zehrmann1, Mizuki Takenaka1
1
Molecular Botany, Ulm University, 89069 Ulm, Germany
RNA editing in the organelles of Arabidopsis thaliana is converting selected cytidine residues to uridines.
More than 400 and 30 RNA editing events take place in mitochondria and chloroplasts, respectively, in
Arabidopsis. Most of them lead to amino acid exchanges in the translational products.
The cytidines to be edited are specifically chosen by PLS class PPR proteins, which have been characterized
as editing factors by reverse genetics of PLS class PPR genes. However, in many cases molecular functional
analyses including RNA editing in the PPR protein knock-out mutants are impossible, as they show severe
defects in organellar function which leads to seed abortion in early embryo stages.
To investigate the function of PLS class PPR proteins which show embryo lethality in the
knock-out lines, we undertook three approaches. At first, artificial microRNAs (amiRNAs) are employed to
establish knock-down lines of the PPR protein genes with milder effects on organelles. Secondly, embryos of
homozygous knock-out lines are cultivated on a MS-based embryo rescue medium with 8% sucrose content.
Finally, PPR protein genes are expressed in the respective mutant lines under control of the embryo specific
ABI3 promotor to overcome embryo lethality and obtain enough mature plant material with homozygous
mutant background. With the help of our target site prediction program based on the PPR code, it is possible
to concentrate the first RNA editing analysis on several candidate target sites of individual PPR proteins.
Here we show that our approaches successfully identified several novel RNA editing factors, of which
knock-out lines display embryo lethality at standard condition, as well as their target editing sites which must
be involved in embryo lethality.
78
P20
Single-cell metagenomics of the termite-gut protozoa, Mixotricha paradoxa
Kumiko Kihara 1, 2, 5, Akinori Yamada3, Nathan Lo4, Shigeharu Moriya2, Yuichi Hongho5
1
Department of Biological and Chemical Systems Engineering, National Institute of Technology Kumamoto
College, 2627 Yatsushiro Hirayama-shinmachi, Kumamoto 866-8501, Japan; 2RIKEN Center for Sustainable
Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama city, Kanagawa, 230-0045, Japan; 3Graduate
School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi
Nagasaki-shi, Nagasaki, 852-8521; 4The University of Sydney, Edgeworth David Building A11, Sydney, NSW
2006, Australia; 5Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
2-12-1-W3-48 O-okayama, Meguro-ku, Tokyo, 152-8550, Japan. email: kihara@kumamoto-nct.ac.jp
Mixotricha paradoxa is one of the gut symbiont of lower termite Mastotermes darwiniensis living in
Australia. This huge protozoan is belonging to the phylum Parabasalia. It is noted that a unique symbiosis,
which involves multiple bacterial species adhere on its surface, have been thought to be driving force of the
huge protozoa-swimming. Those bacterial symbionts consist of spirochetes and bacteroidales. So far, the
phenomenon has attracted a lot of researchers, and the following questions have been posed: How do the
three organisms live together? Have they developed the symbiosis as is the case of Lynn Margulis‘s theory of
symbiosis evolution? These questions, however, still remains to be answered mainly due to the difficulty in
cultivation of the protist as well as the bacteria, while genome- coupled with transcriptome-approaches could
improve our understanding of the attractive symbiosis. Here we conducted comprehensive genome analysis
from a single cell, “single-cell metagenomics”, of Mixotricha paradoxa.
79
P21
Prediction of Mitochondrial or Mitochondrion-related Organelle Proteins in Non-model Organisms
using Machine Learning
Keitaro Kume1,2, Toshiyuki Amagasa2,3, Tetsuo Hashimoto1, Hiroyuki Kitagawa2,3
1
Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572, Japan;
Graduate School of Systems and Information Engineering, University of Tsukuba, Ibaraki, 305-8572,
Japan; 3Center for Computational Sciences, University of Tsukuba, Ibaraki, 305-8577, Japan.
2
Mitochondria or mitochondrion-related organelles (MROs) are almost commonly found organelles in
eukaryotic cells. These organelles provide essential functions such as, energy metabolism, oxidative stress
response, amino acid synthesis, iron-sulfer cluster assembly machinery, and others. To exactly understand the
organelle functions, it is necessary to directly identify the organelle proteins from experimental analyses of
the organelle proteome. Unless such proteome data are available, organelle proteins are predicted by
computer programs, such as Mitofates and SignalP, using sequence-based prediction algorithms. However,
previous predictors focused only on mitochondrial proteins of model organisms and their relatives. Since no
experimental proteome data from non-model organisms were used as machine learning data in such
predictors, these were not generally applicable to mitochondrial/MRO proteins from non-model organisms.
Here we developed a new predictor for mitochondrial/MRO proteins of non-model organisms. Our predictor
shows a better performance than existing ones on the prediction of such proteins.
80
P22
Evolution of organellar DNA polymerases in Chlorarachniophyte algae
Arisa Watanabe1, Shigekatsu Suzuki2, Yoshihisa Hirakawa3, Ken-ichiro Ishida3
1
Graduate school of Life and Environmental Sciences, Tsukuba University, Ibaraki 305-8572, Japan; 2
National Institute for Environmental Studies, Ibaraki 305-0053, Japan; 3Faculty of Life and Environmental
Science, Tsukuba University, Ibaraki 305-8572, Japan
Plastids and mitochondria, generally contain their own genomes of bacterial origins. These organellar
genomes lack essential genes for DNA replication proteins (e.g., DNA polymerases), and organellar DNA
replication is regulated by nucleus-encoded proteins. Plant organellar DNA polymerases, so-called the POPs,
were identified in land plants, and POP homologs were found in diverse algae and protists. Interestingly,
POPs are phylogenetically distinct from bacterial DNA polymerases, and their origins remain unknown.
Moreover, POPs were demonstrated to be dually targeted to plastids and mitochondria in Arabidopsis and the
red alga Cyanidioschyzon. Although organellar DNA replication has been investigated in Archaeplastida,
there are only a few reports from algal groups with complex plastids acquired by secondary endosymbioses.
In this study, we investigated organellar DNA replication of chlorarachniophytes that are marine unicellular
algae possessing complex plastids originated by the uptake of a green alga. The nuclear genome of the
chlorarachniophyte, Bigelowiella natans, was found to encode two POP homologs, BnPOP1 and BnPOP2.
Our phylogenetic analysis suggests that these two sequences have difference origins. Fluorescent protein
tagging experiments revealed that BnPOP1 and BnPOP2 were targeted into the plastid and mitochondrion,
respectively. We also performed quantitative PCR assay to reveal the timing of POP gene expression and
organellar DNA replication. Our data suggest that two phylogenetically distinct POPs function in respective
organelles in chlorarachniophytes, unlike plants.
81
P23
Evolution of vertical transmission of symbionts by reducing the rate of cell division
Yu Uchiumi1,2, Hisashi Ohtsuki1, Akira Sasaki1
1
Department of Evolutionary Studies of Biosystems, SOKENDAI (The Graduate University for Advanced
Studies), Hayama, Kanagawa 240-0193, Japan; 2email: uchiumi_yu@soken.ac.jp
Chloroplasts and mitochondria evolved from free-living bacteria via intracellular symbiosis. Through the
evolutionary transition from symbionts to organelles, symbionts has become transmitted vertically and
remained persistently in a host cell. In intracellular symbiosis, such as the relationship between protists and
algae, vertical transmission is ensured by synchronized cell divisions of symbionts and hosts. If symbiont
division is faster or slower than the host, symbionts accumulate in or are lost from their host cell. In
particular, excessive accumulation makes symbionts a burden to their host and will induce host cell death.
Indeed, the regulation of symbiont division rate is well observed in various symbiotic systems, for example
in the symbiosis between ciliates (Paramecium bursaria) and green algae (Chlorella spp.). There are two
hypotheses for the maintenance of synchronized cell divisions. The first hypothesis is that a host suppresses
excessive cell divisions of symbionts. The second hypothesis is that a symbiont restrains its own cell division
for the synchronization. The latter may seem paradoxical because symbionts’ growth rate may increase by
virtue of symbiotic interaction, and it is actually unclear whether symbionts self-restrain their own division
evolutionarily. Therefore, we theoretically explored the condition for the evolution of self-restraints in
symbiont cell division. We analyzed a mathematical model, in which symbiont dynamics within a host cell is
explicitly taken into account. We assumed that excessive accumulation of symbionts in a host cell leads to
the host death and its burst. We also assumed that the host may acquire free-living symbionts, and, once in
host cell, symbionts can be inherited randomly in daughter cells during host cell division. These assumptions
lead the tradeoff between horizontal and vertical transmission as in many protist-algae symbiotic systems. If
the division rate is higher in symbiont than in host, symbionts tend to accumulate in a host cell, leading to the
host burst and their release into the environment. As a result, such symbionts tend to be transmitted
horizontally and lose the opportunity of vertical transmission. Additionally, we also assumed that both host’s
and symbiont’s symbiotic benefits are to reduce their death rates. An analysis of our model reveals that a low
cell division rate evolves if symbiotic benefit is large in both host and symbiont. This is because a larger
symbiotic benefit given to hosts makes infected hosts advantageous over uninfected ones, leading to lower
density of uninfected hosts, with which released symbionts from dead host become difficult to find next
hosts. In contrast, sybmiont cell division rate evolves to a large value or increase without limit if either
symbiont’s or host’s benefit is not sufficiently large. Thus, our results suggest that if symbiotic benefit is
large both in host and symbiont, synchronized cell division can result from symbionts’ self-restraint of their
own division even if host’s suppression of symbiont’s division is absent.
82
Screening and discovery of lineage-specific mitosomal membrane proteins in Entamoeba histolytica
Herbert J. Santos1, Yuki Hanadate1,2, Kenichiro Imai3, Yoshinori Fukasawa3, Toshiyuki Oda3, Fumika
Mi-ichi4 and Tomoyoshi Nozaki1,2 1
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama,
Shinjuku-ku, Tokyo 162-8640, Japan. 2Graduate School of Life and Environmental Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. 3Biotechnology Research Institute for Drug
Discovery, National Institute of Advanced Industrial Science and
Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan. 4Division of Molecular and Cellular
Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan.
email: herbert@niid.go.jp
Entamoeba histolytica, an anaerobic intestinal parasite causing dysentery and extra-intestinal abscesses in
humans, possesses highly reduced and divergent mitochondrion-related organelles (MROs) called mitosomes.
This organelle lacks many features associated with canonical aerobic mitochondria and even other MROs
such as hydrogenosomes. The Entamoeba mitosome has been found to have a compartmentalized sulfate
activation pathway, which contributes to cellular differentiation, an essential process allowing for the
transmission of the parasite to its host. It also has other lineage-specific features such as a shuttle system via
Tom60, and a novel subclass of β-barrel outer membrane protein called MBOMP30. With the discoveries of
such unique characteristics of Entamoeba mitosomes, there still remain a number of significant unanswered
issues pertaining to this organelle. Particularly, the present understanding of the inner mitosomal membrane
is extremely limited. So far, only a few homologs of transporters for various substrates have been confirmed,
while the components of the protein translocation complexes appear to be absent or are yet to be discovered.
Employing a similar strategy as in our previous work, we collaborated to screen and discover mitosomal
membrane proteins. Using a specialized prediction pipeline, we searched for proteins possessing α-helical
transmembrane domains, which are unique to E. histolytica mitosomes. From the prediction algorithm, 25
proteins emerged as candidates, and three were observed to be localized to the mitosomal membranes.
Further analyses of the predicted proteins may provide clues to answer key questions on mitosomal evolution,
biogenesis, dynamics, and biochemical processes.
83
P25
Identification and transcriptome analysis of a non-photosynthetic eukaryovorous amoeba
phylogenetically related to chlorarachniophytes
Shigekatsu Suzuki1, Takashi Shiratori2, Ken-ichiro Ishida2
1
Center for environmental biology and ecosystem studies, National institute of environmental sciences,
Onogawa 16-2, Tsukuba, Ibaraki, 305-0053, Japan; 2 Faculty of Life and Environmental Sciences, University
of Tsukuba, Ten-nodai 1-1-1, Tsukuba, Ibaraki, 305-8571, Japan. email: suzuki.shigekatsu@nies.go.jp
Chlorarachniophyte is a cercozoan group, and has complex plastids acquired form a green alga in the
secondary endosymbiosis. Chlorarachniophytes retain relict nuclei of the symbiont, so-called nucleomorph,
and thus they are important to understand the process of the secondary endosymbiosis. Recently a
bacteriovorous relative of chlorarachniophytes, Minorisa minuta has been found, however few studies
focused on the host species have been performed. Here, we report a non-photosynthetic eukaryovorous
amoeba, Rhabdamoeba marina SRT404, which has been isolated from seawater sample collected in Tomari
port, Tottori, Japan, and maintained with Chrysochromulina sp. strain NIES-1333 as prey. Our molecular
phylogenetic analysis using 18S rRNA gene sequences showed that it is a cercozoan species related to
chlorarachniophytes. Multi-gene phylogeny also supported the monophyly of R. marina and
chlorarachniophytes. Observation of the ultrastructure of R. marina showed that there is no plastid-like
structure in the cell. By transcriptome analysis, we could not detect any protein-coding genes, which are
clearly homologous to plastid-targeted proteins of chlorarachniophytes. These suggest that R. marina can be
at the stage before acquiring plastids in chlorarachniophytes.
84
P26
Discovery of a mitosome-related compartment in Entamoeba histolytica
Yuki Hanadate1,2, Herbert J. Santos2, Kenichiro Imai3, and Tomoyoshi Nozaki1,2 1
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8572, Japan. 2 Department of Parasitology, National Institute of Infectious Diseases, 1-23-1
Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. 3Biotechnology Research Institute for Drug Discovery,
National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo
135-0064, Japan. email: yuki@nih.go.jp
Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis. 50 million people are
infected worldwide, and 40,000 to 70,000 die of amoebiasis every year. Entamoeba possesses a markedly
reduced and highly divergent form of mitochondrion-related organelle, called mitosome. The mitosome,
typically demonstrated in parasitic and free-living protists such as E. histolytica, Giardia intestinalis, and
diverse microsporidian species, generally does not produce hydrogen or ATP and possesses a double
membrane, and mitochondrial chaperonin 60 (Cpn60). The E. histolytica mitosome is involved in the sulfate
activation pathway, which was recently implicated to have a role in stage conversion from trophozoite to cyst.
In this study, we are characterizing a novel mitosomal protein EHI_099350, which is localized to a
potentially new compartment that morphologically resembles the mitosome. Immunoelectron microscopy
revealed that the organelle is bound by a double membrane. However, the size of the unknown compartment
appears to be consistently smaller and its electron density seems to be lower compared to typical mitosomes.
In addition, it clearly lacks mitosomal matrix markers Cpn60 and/or adenosine-5’-phosphokinase (APSK),
an enzyme involved in sulfate activation. Exploring the significance and physiological function of
subpopulations of mitosomes may unveil some interesting aspects associated with biogenesis and dynamics.
85
P27
Development of dual-transfection system in Perkinsus marinus
Hirokazu Sakamoto1, Yoshihisa Hirakawa1, Ken-ichiro Ishida1, Kiyoshi Kita2, Motomichi Matsuzaki3
1
Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan; 2School of
Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8521, Japan; 3Department of
Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan.
e-mail: hzsakamoto@hotmail.co.jp
Plastids in apicomplexan parasites, such as malaria parasites, are highly degenerated and have lost the
photosynthetic ability. Interestingly, an oyster parasite Perkinsus marinus, which shares a phototroph
ancestor with dinoflagellates and apicomplexans, has a cryptic plastid that lacks DNA. There is not predicted
any functions other than isoprenoid biosynthesis pathway (MEP pathway) in this plastid. Recently, we
developed a stable transfection system by bleomycin selection for the parasite and established a stable cell
line expressing plastid-targeted gfp that is fused with ispC gene for MEP pathway. The cell line visualized
the cryptic plastid very easily. To identify new plastid proteins using the cell line, we newly developed a
dual-transfection system with two different sets of fluorescent protein genes and drug resistance genes for
co-localization analyses. Firstly we screened antibiotics and determined that blasticidin S and puromycin
fully inhibit parasite growth. Then, gfp-fused resistance genes for the drugs were transfected into
bleomycin-selected parasites expressing mCherry. After two months, puromycin treatment selected cells
having both of GFP and mCherry signals, but blasticidin S treatment did not. This result shows that the
combination of bleomycin and puromycin can select cells that co-express the two fluorescent protein genes,
which should be available for co-localization analyses. We believe that this co-expression system contributes
to identifying novel plastid-targeted proteins and to understanding functions of the cryptic plastid in
Perkinsus marinus.
86
P28
Conformation of mitochondrial DNA in the oyster parasite Perkinsus marinus
Motomichi Matsuzaki1,2, Hirokazu Sakamoto1,3, Masayuki Hata1, Isao Masuda4, Kisaburo Nagamune2,
Kiyoshi Kita1,5
1
Graduate School of Medicine, the University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; 2Department of
Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan; 3Faculty of Life
and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; 4Department of
Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA; 5School of
Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8521, Japan. E-mail:
mzaki@niid.go.jp
Mitochondrial genome is essential for oxidative phosphorylation and thus conserved throughout eukaryotes
living in the aerobic environment. However, those of protists heavily vary in their size, topology, and gene
content; sometimes how to encode proteins is modified. Perkinsus spp. are notorious unicellular marine
protists, which parasitize commercially important bivalve species like clams and oysters worldwide. In their
lifecycle, propagating and thus pathogenic trophozoite develops into an enlarged dormant hypnospore in an
anaerobic condition, and then into small dispersal stage zoospore when returned to an aerobic condition. This
is considered as an adaptation to the periodic anaerobic condition caused by tidal cycles, and the
mitochondrial respiratory chain is therefore supposed to have a major role in parasites’ physiology. We have
recently sequenced the mitochondrial DNA of Perkinsus marinus by IonTorrent technology to see the genetic
basis for the adaptation. Reconstituted 67-kb genome consisted of 2 protein-coding genes cox1 and cob as
well as numerous short transcribed regions possibly functioning as ribosomal RNA fragments. However,
experimentally determined physical structure of mitochondrial DNA extracted from P. marinus was an
assemblage of 1- to 10-kb linear molecules without telomeric repeats. Here, we show the in vivo
conformation of the mitochondrial DNA by a combination of TdT-mediated dUTP nick end labeling
(TUNEL) and quantitative PCR after exonuclease treatment. TUNEL assay indicates free 3’-OH groups of
DNA termini under a microscope, and the result showed abundant DNA termini in the mitochondrion of P.
marinus. Exonuclease digests linear DNA molecules from their termini, but keeps circular or nicked DNA
intact. Quantitative PCR showed that there is no master circle DNA in P. marinus mitochondria. Taken
together, mitochondrial DNA of P. marinus is truly composed of randomly fragmented linear DNA
molecules without telomeres. This conclusion raises “End replication problem,” and we have currently no
idea to solve it. Comments are welcome.
87
P29
DNA-barcode technology reveals integration-dependent stochastic transcription activation of
transgenes in the plant genome: its implication for the molecular basis of EGT and HGT
Soichirou Satoh1, Takayuki Hata1, Naoto Takada1, Makoto Tachikawa1, Mitsuhiro Matsuo1, Sergei Kushnir2,
Junichi Obokata1
1
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8552, Japan.
Vale Institute of Technology Sustainable Development, 66055-090, Belém, Pará, Brazil. email:
s-satoh@kpu.ac.jp
2
We still have little knowledge of the mechanism by which newly introduced alien protein-coding sequences
by EGT (endosymbiotic gene transfer) and HGT (horizontal gene transfer) become transcriptionally active,
in the eukaryotic nuclear genome. To address this question, we attempted an artificial evolutionary
experiment in the plant genome: thousands of promoterless luciferase coding sequences (LUCs) each labeled
by 12-base barcode (random nucleotide) were randomly introduced into the nuclear genome of Arabidopsis
thaliana suspension culture cells, and the transcriptional fate of the whole LUC transgenes was traced with
the aid of barcode. We identified 4,504 LUC integration sites, and one-third of them were transcribed.
Unexpectedly, it was only 10% of the transcribed LUCs that were explained by the transcriptional fusions
with the annotated genes, and the great majority of them indicated the occurrence of de novo transcription
that was unable to be explained by the conventional promoter-trap models. The degree of transcriptional
activity or chromatin silencing code at the respective genomic sites before LUC integration had little impact
on the expression of the LUC transgenes. We also found that this novel transcription activation occurs
stochastically for 30% of the chromosomal integration events, but independently of the inherent properties of
the respective genomic integration sites. Based on these novel findings, we propose that the initial
transcription of the horizontally or endosymbiotically transferred genes should occur by the stochastic
chromatin remodeling that accompanies the DNA double strand break repair following the chromosomal
integration of alien DNA fragments.
88
P30
Evolutionary roles of SL-trans-splicing in the primary endosymbiosis
Mitsuhiro Matsuo1, Atsushi Katahata1, Soichirou Satoh1, Motomichi Matsuzaki2, Mami Nomura3, Ken-ichiro
Ishida3, Yuji Inagaki4 and Junichi Obokata1
1
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8552, Japan;
Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan; 3 Graduate School of Life
and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572, Japan; Center for Computational
Sciences, University of Tsukuba, Tsukuba 305-8577, Japan; email:mmat@kpu.ac.jp
2
Spliced leader (SL) trans-splicing, which adds short non-coding RNA sequences (20-30 bases) to the 5’ end
of mRNAs by splicing in trans, is sporadically observed in the diverse eukaryotic lineages, including
metazoa, kinetoplastida and dinoflagellata. Although this peculiar RNA maturation process is essential in
those trans-splicing organisms, it remains largely unknown how those splicing systems have evolved in their
lineages. Here we report a novel finding that SL trans-splicing occurs in a photosynthetic rhizarian organism,
Paulinella chromatophora. This organism has a unique photosynthetic organelle termed cyanelle, which was
derived from the cyanobacterial endosymbiosis that had occurred about 0.1 billion years ago. Considering
that the cyanobacterial endosymbiosis that led the evolution of chloroplasts occurred 1.0-1.5 billion yeas
before, P. chromatophora nuclear-organelle genome system is thought to be still young, thus suitable for
studying the genome-transcriptome features in the initial stage of the primary endosymbiosis. In this
presentation, we demonstrate the characteristics of the SL-trans-splicing in this unique organism, and discuss
its possible roles and advantages for endosymbiotic evolution.
89
Participants
Affiliation
E-mail address
John F. Allen
University College London
j.f.allen@ucl.ac.uk
Takumi Arakawa
Hokkaido University
takumia@abs.agr.hokudai.ac.jp
Honoka Araki
Gifu University
s8022005@edu.gifu-u.ac.jp
John M. Archibald
Dalhousie University
jmarchib@dal.ca
Matthias Gordian Burger
Ulm University
matthias.burger@uni-ulm.de
Ugo Pierre Cenci
Université des Sciences et Technologies de Lille
ugocenci@hotmail.com
Ming-Tsair Chan
Academia Sinica
mbmtchan@gate.sinica.edu.tw
Toshiya Endo
Kyoto Sangyo University
tendo@cc.kyoto-su.ac.jp
Hiroki Fukuizumi
obokata@kpu.ac.jp
Junpei Fukumoto
National Institute of Infectious Disease
junpei@niid.go.jp
Przemyslaw Gagat
University of Wroclaw
gagat@smorfland.uni.wroc.pl
Jonny Michael Gentil
Philipp University of Marburg
jonny.gentil@biologie.uni-marburg.de
Stephan Greiner
Max Planck Institute of Molecular Plant Physiology
Greiner@mpimp-golm.mpg.de
Hidefumi Hamasaki
RIKEN Center for Sustainable Resource Science
gif193@gifu-u.ac.jp
Yuki Hanadate
National Institute of Infectious Diseases
yuki@nih.go.jp
Mitsumasa Hanaoka
Chiba University
mhanaoka@faculty.chiba-u.jp
Maria Rosmarie Handrich
Heinrich-Heine-University
maria.handrich@hhu.de
Tetsuo Hashimoto
University of Tsukuba
hashimoto.tetsuo.gm@u.tsukuba.ac.jp
Takayuki Hata
h811320039@gmail.com
Natsuki Hayami
Gifu University
natsuki-hayami@hotmail.co.jp
Anke-Christiane Hein
University of Bonn
ahein@uni-bonn.de
Ayaka Hieno
Gifu University
ayakatwb@gmail.com
Rina Higuchi
Kobe University
hig.m.rina@gmail.com
Yoshihisa Hirakawa
hirakawa.yoshi.fp@u.tsukuba.ac.jp
Yuji Inagaki
yuji@ccs.tsukuba.ac.jp
90
Ken-ichiro Ishida
ken@biol.tsukuba.ac.jp
Yu Ishii
Tohoku University
yu.ishii.a1@tohoku.ac.jp
Md Shafiqul Islam
Kobe University
sshafiq1969@gmail.com
Ryoma Kamikawa
Kyoto University
kamikawa.ryoma.7v@kyoto-u.ac.jp
Atsushi Katahata
s814631008@kpu.ac.jp
Kumiko Kihara
National Institute of Technology Kumamoto College
kihara@kumamoto-nct.ac.jp
Mayumi Kobayashi
Kobe University
k_mayumi@people.kobe-u.ac.jp
Danijela Kozul
Max Planck Institute of Molecular Plant Physiology
Kozul@mpimp-golm.mpg.de
Peter Georg Kroth
University of Konstanz
Peter.Kroth@uni-konstanz.de
Keitaro Kume
kkei444@gmail.com
Marie-Kristin Sarah
Lehniger
Humboldt University of Berlin
Uwe G. Maier
University of Marburg
maier@biologie.uni-marburg.de
Shinichiro Maruyama
Tohoku University
maruyama@tohoku.ac.jp
Eriko Matsuo
abbey.ohayo@gmail.com
Mitsuhiro Matsuo
mmat@kpu.ac.jp
Motomichi Matsuzaki
mzaki@niid.go.jp
Mikio Nishimura
National Institute for Basic Biology
mikosome@nibb.ac.jp
Ryosuke Miyata
haclogic.ssute@gmail.com
Nobuyoshi Mochizuki
Kyoto University
mochizuki@physiol.bot.kyoto-u.ac.jp
Daniel Moog
Philipps University Marburg
daniel.moog@biologie.uni-marburg.de
Shigeharu Moriya
RIKEN Center for Sustainable Resource Science
smoriya@riken.jp
Norbert Eugen Müller
norbertmueller@mac.com
Kisaburo Nagamune
nagamune@niid.go.jp
Masayuki Nakamura
Nagoya University
mnak@gene.nagoya-u.ac.jp
Takuro Nakayama
ntakuro@ccs.tsukuba.ac.jp
Jörg Rolf Nickelsen
Ludwig-Maximilians-Universität München
joerg.nickelsen@lrz.uni-muenchen.de
91
Yoshiki Nishimura
Kyoto University
yoshiki@pmg.bot.kyoto-u.ac.jp
Mami Nomura
true82future@gmail.com
Junichi Obokata
obokata@kpu.ac.jp
Ralf Oelmüller
Friedrich Schiller Universität
b7oera@uni-jena.de
Ayako Okuzaki
Tamagawa University
okuzaki8@agr.tamagawa.ac.jp
Bastian Oldenkott
University of Bonn
bastian.oldenkott@gmx.net
Cessa Rauch
Heinrich-Heine-University
cessa.rauch@hhu.de
Hirokazu Sakamoto
hzsakamoto@hotmail.co.jp
Herbert Jimenez Santos
herbert@nih.go.jp
Naoki Sato
University of Tokyo
naokisat@bio.c.u-tokyo.ac.jp
Soichirou Satoh
s-satoh@kpu.ac.jp
Viktoria Schreiber
Philipp University of Marburg
victoria.schreiber@biologie.uni-marburg.de
Jürgen Soll
Ludwig-Maximilians-Universität München
soll@lmu.de
Jürgen Steiner
Martin Luther University Halle-Wittenberg
juergen_steiner@gmx.at
Atsushi Sugioka
obokata@kpu.ac.jp
Mamoru Sugita
Nagoya University
sugita@gene.nagoya-u.ac.jp
Masahiro Sugiura
Nagoya University
sugiura@gene.nagoya-u.ac.jp
Makoto Sugizaki
University of Tsukuba?
rankweed.sgzk392@gmail.com
Toshinobu Suzaki
Kobe University
suzaki@kobe-u.ac.jp
Shigekatsu Suzuki
National Institute for Environmental Studies
shigekatsu.szk@gmail.com
Naoto Takada
maikeeeerumuuuua@yahoo.co.jp
Shunichi Takahashi
National Institute for Basic Biology
shun@nibb.ac.jp
Goro Tanifuji
National Museum of Nature and Science
gorot@kahaku.go.jp
Jasmine Theis
Technische Universität Kaiserslautern
theisj@rhrk.uni-kl.de
Keita Torazawa
Aichi Gakuin University
torakay@dpc.agu.ac.jp
Chien-Hao Tseng
National Taiwan University
B98612037@ntu.edu.tw
92
Chinatsu Tsukakoshi
every90362@gmail.com
Tomohiro Uchikoba
obokata@kpu.ac.jp
Yu Uchiumi
The Graduate University for Advanced Studies
uchiumi_yu@soken.ac.jp
Masamitsu Wada
Tokyo Metropolitan University
masamitsu.wada@gmail.com
Arisa Watanabe
arisa321w@gmail.com
Tetsushi Yada
Kyushu Institute of Technology
ytetsu@bio.kyutech.ac.jp
Yoshiharu Y. Yamamoto
Gifu University
yyy@gifu-u.ac.jp
Euki Yazaki
euki87@gmail.com
Fanf-Fei Yeh
Kai-Wun Yeh
mbmtchan@gate.sinica.edu.tw
National Taiwan University
ykwbppp@ntu.edu.tw
93
ORGANIZATION
President of the Congress
Obokata, Junichi (Kyoto)
Local Organizing Committee
Hanaoka, Mitsumasa (Chiba)
Inagaki, Yuji (Tsukuba)
Ishida, Kenichiro (Tsukuba)
Kamikawa, Ryoma (Kyoto) Excursion
Matsuzaki, Motomichi (Tokyo)
Nakamura, Masayuki (Nagoya) Treasurer
Nishimura, Yoshiki (Kyoto) Banquet
Obokata, Junichi (Kyoto)
Terachi, Toru (Kyoto)
Yamamoto, Yoshiharu (Gifu) Program
Advisory Board
Sugiura, Masahiro (Nagoya)
Löffelhardt, Wolfgang (Vienna)
Oelmüller, Ralf (Jena)
Kroth, Peter G. (Konstanz)
Contact
E-mail : secretary@ices2016.com
Office : Kyoto Prefectural University, Laboratory of Plant Genome Biology
94
ISE
International Society of Endocytobiology
http://www.endocytobiology.org
Endocytobiosis and Cell Research
http://ecb.thulb.uni-jena.de/submit.html
AIMS AND SCOPE
Endocytobiosis and Cell Research (online journal), an international research journal and organ of
the “International Society of Endocytobiology” (ISE), launched the first issue in 1984. The journal
was founded to publish information on evolutionary biology, symbiosis research and cell biology.
The journal offers a communication forum for (micro)biologists, bioinformatics, biophysicists,
biochemists, geneticists, medical scientists, veterinary researchers, and molecular biologists that
are concerned with the origin, development, differentiation, evolution and phylogeny of
endosymbioses and cell research (including endocytoparasitism) in the context of the eukaryotic
cell.
The editorial board ensures an efficient, fast and transparent review process for all submitted
manuscripts. Over the last year the manuscript has adapted a modern publication layout and new
sections have been launched in order to make the journal more attractive for a broader range of
readers. The web portal has been restructured and now emphasizes on the new sections.
Editor-in-Chief
Prof. Dr. Ralf Oelmüller,
Friedrich Schiller University Jena
Institute of General Botany and Plant Physiology
Dornburger Str. 159, 07743 Jena
Germany
Section editors
Research Articles - Ralf Oelmüller, Jena, Germany
Communication - Ralf Oelmüller, Jena, Germany
Mini and Full Reviews - Jörg Nickelsen, München, Germany
Technical Notes - Enrico Schleiff, Frankfurt, Germany
Addendum - Peter Kroth, Konstanz, Germany
Hypothesis Papers - Junichi Obokata, Kyoto, Japan
News and Views - Sven B. Gould, Düsseldorf, Germany
Editorial Office
Dr. Irena Sherameti
Friedrich Schiller University Jena
Institute of General Botany and Plant Physiology
Dornburger Str. 159, 07743 Jena Germany
e-mail: irena.sherameti@uni-jena.de

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