European Frontiers of
Plant Reproduction Research
Final conference of the COST action FA0903 "Harnessing Plant Reproduction for Crop Improvement"
Bristol Hotel, OSLO, 2 – 4 OCTOBER 2013
www.plantreproduction.no
Program & Abstracts
Biosciences Department
Meiosis & Apomeiosis - Gametophyte Development & Function - Fertilization Mechanisms - Embryogenesis &
Parthenogenesis - Endosperm & Imprinting - Seed & Fruit Development - Polyploidy & Aneuploidy - Apomixis &
Emerging Technologies - Evolution of Reproductive Systems - Plant Reproduction & Environmental Change
*************************
local organization committee
Paul E. Grini
Katrine N. Bjerkan
Karina S. Hornslien
Barbara M. Glöckle
Ida M. Johannessen
Ingvild F. Ullmann
Maryam Kaletarian
Lene A. Kittelsen
Department of Biosciences,
University of Oslo, Norway
1
European Frontiers of
Plant Reproduction Research
Final conference of the COST action FA0903
"Harnessing Plant Reproduction for Crop Improvement"
2 – 4 OCTOBER 2013
Bristol Hotel, Oslo, Norway
www.plantreproduction.no
*************************
organised by
Paul E. Grini
Department of Biosciences,
University of Oslo, Norway
together with the COST action FA0903
Action Chair
Emidio Albertini
Faculty of Agriculture
University of Perugia, Italy
Workgroup Chairs
Thomas Dresselhaus
Cell Biology & Plant Biochemistry
University of Regensburg, Germany
Danny Geleen
Department of Plant production
University of Ghent, Belgium
Tim Sharbel
Leibnitz Institute of Plant Genetics and Crop Research(IPK)
Gatersleben, Germany
2
3
Welcome from the HAPRECI Chair
The
European
Network
the Netherlands, Belgium, and Italy as well as a
Crop
training school on metabolomics and plant breeding
Improvement (HAPRECI)) was established in 2009
that was organized in Italy this year. The aim of the
with financial support through the COST Action
schools was to train young investigators in state-of-
(Harnessing
Plant
Plant
Reproduction
Reproduction
for
program of the European Commission. Its major goal
the-art methodologies employed for understanding
was to stimulate research on plant reproduction in
plant reproduction and to improve crops. Therefore,
Europe
and
additionally, a number of short term scientific
multidisciplinary expertise of the various laboratories.
and
to
missions (STSMs) were facilitated to exchange PhD
Scientific goals aimed to understand the molecular
students
mechanisms
plant
laboratories for the purposes of training, collaborative
reproduction, and to facilitate the use of this
research, and to promote innovative interactions. The
knowledge in the development of new approaches in
HAPRECI network was subdivided into three working
biotechnology, agriculture, and food industry through
groups
improved crops. Further goals aimed to promote
development and embryogenesis
interaction
gametophyte
of
combine
sexual
between
and
interrelated
apomictic
fundamental
and
applied
and
with
young
a
focus
postdocs
on
development
among
female
and
partner
gametophyte
(WG1), male
pollen
viability
research areas, and to train young researchers in
(WG2) as well as apomixis technology development
various
Eighty-seven
(WG3). A special issue of Plant Reproduction Journal
laboratories from 28 countries participated in the
was dedicated to HAPRECI. Its editorial article by
network. In addition to annual meetings at Brussels
Dresselhaus and Albertini (DOI 10.1007/s00497-013-
(2009), Bristol (2010), Valencia (2011), and Porto
0232-9) gives an overview of the manuscripts and
(2012), a number of smaller focused workshops and
their relation to the Actions WGs. I hope you will
summer training schools were organized. These
enjoy the reading of those papers as well as of the
included a Hypericum workshop in Austria, five PhD
abstracts of this book. After four successful years,
schools on plant reproduction associated with flower
the Oslo meeting (to which this abstract book
and vegetative development, plant breeding, and
belongs) represents the final network meeting.
methods
and
disciplines.
apomixis, respectively, which took place in Germany,
Emidio A bertini
Chair COST FA0903
4
5
Sponsors
We thank our sponsors for their generous support:
The Molecular Life Science
initiative at the University of Oslo
http://www.uio.no/english/researc
h/interfaculty-research-areas/mls/
Departmenty of Biosciences,
University of Oslo
http://www.mn.uio.no/ibv/english/
COST (European Cooperation in
Science and Technology)
http://www.cost.eu/domains_acti
ons/fa/Actions/FA0903
Carl Zeiss AS
http://www.zeiss.no
Matriks AS,
the preferred supplier to
companies and research
institutes working with chemical,
biochemical and molecular
biology analysis.
http://www.matr ks.no/
Partec GmbH
www.partec.com
Activemotif
http://www.activemotif.com/
6
Practical information
Conference Venue
Hotel BRISTOL Conference Centre
Kristian IV's gate 7, 0164 Oslo, Norway
Hotel Bristol is located in the heart of Oslo city centre. It is only a short wa k from the hotel to Oslo's main street Karl
Johans gate, the Royal Palace, museums and the waterfront area Aker Brygge and most of Oslo's other attractions.
Hotel Bristol fronts one of Oslo’s most modern and complete congress halls with additional group rooms and
exhibition areas.
All Sessions are at the Maud Conference Hall.
Map
Coffe Breaks and Meals
All Meals (Lunches and Dinners) and Coffe Breaks are served in the Bristol Hotel Conference centre.
Included in the conference fee is:
Lunch, Dinner and Coffee breaks Wednesday
Lunch, Dinner and Coffee breaks Thursday
Lunch and Coffee breaks Friday
Tourist Information
Please see Visitors information included in the delagate folders. You will also find a Short guide to Sights below.
Exhibitions
Please visit our sponsors exhibitions in the Conferance hall Lobby
Emergency numbers
Medical- 113
Police- 112
Fire- 110
Conference organiser emergency number: +47 99390210
7
Oslo
A Short Guide...
Founded around 1048 by King Harald III (Hardråde), Oslo was elevated to a
capital under Haakon V (1299–1319), around 1300. He was the first king to reside
permanently in the city and also started the construction of the Akershus Castle.
During Personal Unions with Denmark from 1397 to 1523 and again from 1536 to
1814 Oslo's role was reduced to that of provincial administrative centre, with the
monarchs residing in Copenhagen.
After being destroyed by a great fire in 1624, the city was moved closer to
Akershus Castle during the reign of Danish King Christian IV and renamed
Christiania in his honour. The part of the city built starting in 1624 is now often
called Kvadraturen because of its orthogonal layout. Following a spelling reform, it
was known as Kristiania from 1877 to 1925, when its original Norwegian name –
Oslo - was restored.
Many of the main attractions of Oslo are situated within walking distance to the
Hotel Bristol: the National Gallery (Universitetsgata 13) and the main street Karl
Johan with the Parliament, the University Aula, the Royal Palace, the National
Theatre and the Grand Hotel.
The Town Hall, the fortress of Akershus, the Nobel Peace Center and the new
Astrup Fearnley Museum of Modern Art are all situated in the harbour area, not far
from the hotel.
Total area: 454 square kilometres
Population (2012): 613,000
Forest area: 242 square kilometres
Park and sports arena area: 8 square kilometres
Lakes within the city limits: 343
Islands within the city in the Oslofjord: 40
Length of the Oslofjord: 100 kilometres
The Town Hall
Oslo City Hall was completed in 1950 by the two architects Arnstein Arneberg and
Magnus Poulsson.
Every year on December 10, the Nobel Peace Prize is awarded during a
ceremony at the Oslo City Hall in Oslo, Norway. Located in the center of
downtown Oslo, the modern structure captures the history and culture of Norway.
The brick facade of Oslo City Hall is decorated with historical themes.
Two tall towers and an enormous clock echo the design of traditional northernEuropean town halls. The design and construction of Oslo City Hall spanned a dramatic thirty-year period in
Norway's history (The construction started in 1931, but was paused by the outbreak of World War II, before the
official inauguration in 1950.). Architectural fashions were shifting. The architects combined national romanticism with
modernist ideas. The elaborate carvings and ornaments showcase the talents of some of Norway's finest artists from
the first half of the twentieth century.
Oslo City Hall is the municipal of Oslo’s political center and the workplace for 450 municipal employees and
politicians. Opening hours: All days 9-16.
www.oslo.kommune.no/english
The Royal Palace
The Royal Palace is situated on a rise, the Bellevue, at one end of Oslo’s main
thoroughfare, Karl Johans gate. It is one of the Norway’s most important buildings,
and a concrete symbol of the course of Norwegian history since 1814. Building
activities commenced in 1824 (architect: Hans Ditlev Franciscus Linstow), and the
foundation stone was laid by Swedish King Carl Johan on 1 October 1825. The
Palace was officially taken into use on 26 July 1849 by his successor, King Oscar
I. The Royal Palace is where the daily work of the monarchy is conducted and
where the King and Queen live. It is where the King presides over the Council of
State, grants audiences and holds official dinners. Foreign heads of state who visit
Oslo stay at the Palace. Most of the members of the Royal Court have their workplace here.
The Norwegian monarchy dates back more than one thousand years. Harald Fairhair, regarded as the first
Norwegian king, united the petty kingships of Norway into a single realm in about 885. From the time of Harald
Fairhair until the present day, Norway has had more than 60 named sovereigns. The current King belongs to the
8
House of Glücksburg, which has ruled Norway since 1905. Although Norwegian history goes back many centuries,
modern Norway as an independent nation is relatively young. In 1380 Norway and Denmark were merged under a
single monarch, but Norway was given a subordinate role in the union and came increasingly under Danish control.
The union with Denmark was dissolved in 1814 in the aftermath of the Napoleonic Wars. For a brief time, Norway
once again became an independent nation, drawing up its own constitution. Just a few months later, however,
Norway was compelled to enter into a union with Sweden – this time as an independent nation, but with a common
king and joint foreign policy. On 7 June 1905 the norwegian parliament passed a resolution unilaterally dissolving the
union with Sweden. Prince Carl of Denmark ((1872-1957) was offered the throne and accepted after a referendum.
Upon assuming the throne he took the name of Haakon VII. His son, King Olav V (1903-1991) is the father of the
present King, Harald V.
Today`s members of the Norwegian Royal House are Their Majesties King Harald and Queen Sonja and Their Royal
Highnesses Crown Prince Haakon, Crown Princess Mette-Marit and Princess Ingrid Alexandra. www.kongehuset.no
th
Munch 150 anniversary
Edvard Munch was born in 1863. In 2013, the 150th anniversary of his birth is
celebrated with a whole year’s worth of events and activities that showcase Munch
and his artistry. The Anniversary Exh bition "Munch 150", taking place at Oslo's
National Gallery and the Munch museum, is the highlight of the celebrations.
Munch 150 is the most comprehensive presentation of Edvard Munch’s art ever
shown. In total, 200 paintings and approximately 50 works on paper are on
display. The selection is based on the two host museums' extensive collections,
and supplemented with borrowings from other museums and collectors around the
world.
Munch 150 presents an oeuvre that spans more than 60 years. Works from the period 1882-1903 can be seen in the
National Gallery, and works from 1904-1944 are shown at the Munch museum. Exhibition highlights include an
almost complete reconstruction of Munch's picture series The Frieze of Life from 1902, which comprises The Scream
and other well-known works, and the Reinhardt Frieze from 1906–07. The scope of the exhibition also invites the
exploration of central aspects of Munch's art. Among the themes the exhibition aims at highlighting are Munch's
many renderings of himself, the way he repeated and refined certain motifs, and his artistic use of landscapes he
knew well and cherished.
The National Gallery, in the centre of Oslo (close to Hotel Bristol) was the first public collection ever to buy a painting
from Munch, Night in Nizza, in 1891. Today the museum owns a notable set of paintings from Munch’s early career
and up until 1920. The museum has its own Munch room, with masterworks such as Puberty (1894-95), Ashes
(1895), The dance of life (1899), and the most well-known version of Scream, from 1893. In the period 2 June - 13
October 2013 the entire museum is devoted to the anniversary exhibition "Munch 150". Opening hours: All days: 10 –
17. Thursdays: 10 – 19.
When he died in 1944, Munch left all of his works still in his possession to the municipality of Oslo. These works got a
permanent home when the Munch Museum opened in 1963. The museum was after lengthy discussion built at
Tøyen in the Eastern part of Oslo, close to where Munch grew up. The Munch museum is one of the most
comprehensive one-man museums in the world. The museum owns more than half of Munch’s paintings, and all of
his graphic works. Opening hours: All days: 10 – 17. Thursdays: 10 – 19. Transport: All eastbound metro trains from
the city to Tøyen station/Munchmuset and bus no. 20 to the Munch Museum.
The Aula is an annex of Domus Media, the centre building of the University
of Oslo campus on Karl Johans gate is decorated by Edvard Munch and is
open to the public during the “Munch 150”. Munch commenced work on the
decoration in 1909. The decoration was not completed until September
2
1916. Munch’s 11 paintings cover a total of 223 m . Edvard Munch’s Aula
paintings are a major work within Norwegian monumental painting. The
main paintings are The Sun, History and Alma Mater. The central painting is
The Sun at the end of the hall. The sun as the source of life is
metaphorically an image of illumination, of learning and knowledge. In
History the old man passes on his experience and narratives to an attentive boy and Alma Mater gives nourishment
to a new generation. “She provides the milk of science” as Munch descr bed the woman. Both are placed in
recognizable Norwegian landscapes. Eight smaller paintings adorn the walls. Some of the motifs can be interpreted
as pictures of distinct sciences. All of them pay homage to light/enlightenment and the vital force of life. Edvard
Munch said about the Aula decorations: “My intention has been to make the decorations form a complete and
independent world of ideas, and its visual expression is to be intrinsically Norwegian as well as universally human”.
Opening hours: Friday - Sunday: 13:00-17:00. www.munch150.no
Vigeland Sculpturepark
Vigelandsparken is one of Norway's most visited attractions with more than 1
million visitors every year. The unique sculpture park is the life work of the
sculptor Gustav Vigeland (1869-1943) with more than 200 sculptures in bronze,
granite and cast iron. Don`t miss the angry little boy (on the bridge).
Vigeland was also responsible for the design and architectural outline of the park.
A monumental artistic creation with a human message that is well worth seeing.
9
The park is open all year at all times and is a popular recreation area. Distance City Centre 2 km, transport: tram no.
12 towards “Vigelandsparken”. www.vigeland.museum.no/en/vigeland-park
Nobel Peace Centre
In his testament from 1895 Alfred Nobel instructs the executors of the will to
give the Peace Prize “to the person who shall have done the most or the
best work for fraternity between nations, for the abolition or reduction of
standing armies and for the holding and promotion of peace congresses”.
Among those who have been awarded the Nobel Peace Prize since 1901,
are some of the most significant figures in our recent history. True to their
ideals, but at the same time focused on dialogue and collaboration, and of
doing mankind good. Laureates like Nelson Mandela, Martin Luther King,
Jr., Dalai Lama, Mother Teresa, Fridtjof Nansen, The International
Committee of the Red Cross (ICRC), Albert Schweitzer and Alva Myrdal all
contribute to making the Nobel Peace Prize the worlds most prestigious prize.
The Nobel Peace Center opened in the heart of Oslo in June 2005. It is a center where you can experience and learn
about the various Nobel Peace Prize Laureates and their activities as well as the remarkable history of Alfred Nobel.
The Center combines exhibits and films with digital communication and interactive installations, and has already
received attention for its modern design and use of state of the art technology.
The Nobel Peace Center is located in an old train station building from 1872, close to the Oslo City Hall and
overlooking the harbor. Opening hours: Tues–Sun 10–18. www.nobelpeacecenter.org/en/
The Astrup Fearnley Museum of Modern Art
The privately owned Astrup Fearnley Museum of Modern Art first opened in
1993, and was funded by two philanthropic foundations established by
descendants of the Fearnley shipping family and the Astrup family - the
Thomas Fearnley Foundation and the Heddy and Nils Astrup Foundation.
The two foundations merged in 1995 to become the Thomas Fearnley,
Heddy and Nils Astrup Foundation.
The Thomas Fearnley Foundation was established by shipping magnate
Thomas Fearnley (1880-1961) in 1939; he was the son of shipping magnate
Thomas Fearnley (1841-1927) and grandson of romantic painter Thomas Fearnley, whose works are exibited in the
National Gallery. The Heddy and Nils Astrup Foundation was named after Nils Ebbessøn Astrup, who was a
maternal grandson of Thomas Fearnley (1841-1927). Nicolai Astrup (1931-1990) is the most famous painter of his
family – some of his works are now part of the collection of the Norwegian National Gallery.
In October 2012 The Astrup Fearnley Museum moved into a new, spectacular building designed by Renzo Piano,
located in new-built neighborhood, Tjuvholmen, a former shipyard and mechanical industry area. Bordered by a
sculpture park situated along the water, Piano’s building is very sculptural itself. Ample glass canopies arch gently to
cover the three wings of the museum, creating a majestic silhouette that relates to the vastness of the fjord but also
to human scale. Facing the city with a height of six stories, the building is scaled down toward the park, gently
descending to almost touch the lawn.
The Astrup-Fearnley collection's main focus is the American appropriation artists from the 1980s, but it is currently
developing towards the international contemporary art scene, with artists like Jeff Koons, Richard Prince, Cindy
Sherman, Matthew Barney, Tom Sachs, Doug Aitken, Olafur Eliasson and Cai Guo-Qiang. The museum gives 6-7
temporary exh bitions each year. Astrup Fearnley Museum of Modern Art collaborates with international institutions,
and produces exhibitions that travels worldwide. The museum created a stir in the international art world in 2002
when it purchased the American artist Jeff Koons's monumental sculpture in gilt porcelain of the pop star Michael
Jackson with Bubbles, his favorite chimpanzee, for USD 5.1m
Opening hours: Tuesday, Wednesday, Friday: 12-17, Thursday 12-19, Saturday, Sunday 11-17. www.afmuseet.no/
The Oslo Grand Hotel
The Grand Hotel first opened its doors in 1874. Famous Norwegians such
as Henr k Ibsen (1828-1906) and Fridtjof Nansen (1861-1930) made the
Grand their second home. When he moved back to Oslo from southern
Europe in 1891, Henrik Ibsen brought with him the habit of visiting his
regular cafe every day. For nine years he made Norways' most famous
walk, from his home in Arbins Street, passing the University square where
he compared his watch with the larger one at the University, then down
Karl-Johans Street to the Grand Cafe. With his caractheristic look, he sat
down by his table - in his specially made armchair - "Reserved Dr. Ibsen".
Edvard Munch (1863-1944) is said once to have given a painting to Mr. Ulleberg, waiter at the Grand Café, which
entitled him to Chateaubriand with beer and a nip. Another well known head waiter, Olaf Olsen, was offered Munch's
now famous painting "The Sick Child" for a 100 steaks, but refused.
10
Today, the hotel is host to Nobel Peace Prize laureates, celebrities and heads of state, its guest book is a valuable
historical document. During the first years of its existence, the hotel expanded severeal times and on December 15,
1886, Speilsalen (the Mirror Room) opened its doors. When this room – The Rococo Room – was opened in 1894 in
the presence of Henrik Ibsen, it was the most magnificent reception room in Norway. It originally extended over two
floors and was elaborately decorated with murals by Wilhelm Krogh on the walls and ceiling. It provided an elegant
setting for countless balls, artistic exh bitions and shows. Last but not least, "The Ball Society", under the patronage
of Queen Maud and King Haakon, held their arrangements here. When Roald Amundsen (1872-1928) returned to
Oslo after his successful expedition to the South Pole in 1912, a banquet was held in his honor in this room. The
décor of the both the Mirror Room and the Rococo room was altered somewhat after the fire in 1957- they were
rebuilt and reopened i 1961 - but the crystal sconces and chandeliers from Venice, the tapestries and ornamental
mirrors still echo the historic sense of the room. www.rica.no/hoteller/oslo/grand-hotel
The Opera
Officially opened in April 2008, the Oslo Opera House was designed by
the acclaimed Norwegian architectural firm Snøhetta. The opera, which
took five years to complete, sits on the bank of the Bjørvika district,
near the stock exchange and the central station. From classics to world
premieres of renowned and new Norwegian works, as well as a
number of big concerts and one-off performances, the Oslo Opera
House aims to bring culture to a wider audience.
Oslo Opera has become a new landmark for the city and proved an
instant success with both locals and tourists. In its five years since
opening, over 8 million people have visited the house.
From the outside, the most striking feature is the white sloping marble roof which rises directly up from the Oslofjord,
allowing visitors to enjoy a stroll and take in views of the city. If you see the building from the fjord you will notice a
façade of solar panels. In fact, this is Norway’s biggest area of solar panels supplying the building with some of the
energy its needs.
The opera has also won an array of awards, both at home and abroad, including the prestigious Mies van der Rohe
Prize (2009) and the International Architecture Award 2010.
"The Oslo opera house is a powerful and beautiful statement, radiant with music and song, one that announces
Norway's arrival as a cultural centre. Most of all, it's a building to be shared: anyone who travels to Oslo will want to
see, and climb, Snøhetta's marble mountain," wrote Jonathan Glancey in the British newspaper The Guardian just
after the building’s inauguration in April 2008. Meanwhile The Times’ Richard Morrison declared: "I am in love. She's
Norwegian, gorgeous, full of fun, yet with surprising hidden depths. She's the new Oslo Opera House, an amazing
marble and granite vision that rises out of the fjord like a giant ice floe."
The floor area of the base of the building is equivalent to four international standard football fields and measures
more than 38,000 square metres. The building boasts three stages and a total of 1,100 rooms.
The foyer is a huge open room with a minimalist décor, using simple materials such as stone, concrete, glass and
wood. Here you find seating areas, bars and restaurants.
The main classical horseshoe shaped auditorium, which is one of the most technologically advanced in the world,
offers great scenographic flexibility and fantastic acoustics. The stage area measures several thousand square
metres and parts of it are as much as 16 metres below the surface of the water.
In contrast to the light foyer, the main auditorium is decorated in ammonia-treated Baltic oak. The seatbacks of the
1,350 seats contain individual screens with subtitles in eight different languages. Boat builders from the northwest
coast of Norway have carved the balconies, and hanging from the ceiling is Norway’s largest circular chandelier. It
is 7 metres in diameter, weighs 8 tons, has 5,800 crystal glass elements and was produced by the Norwegian
Hadeland Glassverk.
The 2013 season features over 330 performances and 11 new opera and ballet productions will be premiered.
www.operaen.no
Akershus Fortress
Akershus Fortress, located in the city centre by the Oslo Fjord, is a
great place to discover Oslo's history. The building of Akershus
Castle and Fortress was commenced in 1299 under king Håkon V.
The medieval castle, which was completed in the 1300s, had a
strategical location at the very end of the headland, and withstood a
number of sieges throughout the ages. King Christian IV (15881648) had the castle modernised and converted into a Renaisssance
castle and royal residence. During the 17th and 18th century the
castle fell into decay, and restoration work only started in 1899.
The fortress has never been successfully captured by a foreign
enemy. It surrendered without combat to Nazi Germany in 1940 when the Norwegian government evacuated the
capital in the face of the unprovoked German assault on Norway. During World War II, several people were executed
here by the German occupiers. The fortress was liberated on 11 May 1945. After the war, eight Norwegian traitors
11
who had been tried for war crimes and sentenced to death were also executed at the fortress. Among those executed
were Vidkun Quisling.
After the main building has undergone restoration, it has been used for official events and dinners for dignitaries and
foreign heads of state.
Akershus fortress is still a military area, but is open to the public daily until 21:00. In addition to the castle, the
Norwegian Armed Forces Museum and Norway`s Resistance Museum can be visited there. The Norwegian Ministry
of Defence and Defence Staff Norway (armed forces headquarters) have a joint modern headquarters in the eastern
part of Akershus Fortress.
Norwegian Royalty have been buried in the Royal Mausoleum in the castle. They include King Sigurd I, King Haakon
V, Queen Eufemia, Kong Haakon VII, Queen Maud, King Olav V and Crown Princess Märtha.
The Wikingship Museum
The Viking Ship Museum presents great V king ship discoveries from Gokstad, Oseberg and Tune as well as other
finds from V king tombs around the Oslo Fjord.
The museum displays the world's two best-preserved wooden V king
ships built in the 9th century, as well as small boats, sledges, a cart
with exceptional ornamentation, implements, tools, harness, textiles
and household utensils.
Opening hours: All days 10-16.
Distance city centre: 5 km.
Transport: Ferry nr. 91
(Vikingskipene).
(towards
Dronningen),
Bus
no.
30
http://www.khm.uio.no/english/visit-us/v king-ship-museum
Holmenkollen Ski Jump
At the very top of Oslo towers the new Holmenkollen Ski Jump - the
world's most modern ski jump. It is an imposing monument of steel
and concrete.
Holmenkollen national arena is one of the most visited sports arenas
in the world. The ski jump has been rebuilt or reconstructed 19
times, the last time for the World Ski Championships in 2011.
The Holmenkollen ski jump has been at the center of Norwegian
skiing for more than 100 years. There has been ski jumping
competitions in Holmenkollen every year since 1892. The first
competition here took place on January 31, 1892 with 12 000 spectators. The ski jump itself was built with twigs and
covered with snow. The longest jump reached 21,5 meters. Arne Ustvedt had the honors of setting the first record.
Today, the record in Holmenkollen is 136 meters, set by Tommy Ingebrigtsen, Norway, in 2006.
Located inside the base of the Holmenkollen Ski Jump tower, the Ski Museum presents the history of skiing over the
past 4,000 years, as well as the expeditions of Norwegian polar explorers Fridtjof Nansen and Roald Amundsen.
Here you will also get some historic glimpses from the Winter Olympics in Lillehammer in 1994 and Oslo in 1952. The
Ski Museum is the oldest museum of its kind in the world - it was opened in 1923.
The Holmenkollen Ski Jump is open 365 days a year. Here you will find two souvenir shops, a café and a ski
simulator.
From Oslo city centre, it takes around 20 - 30 minutes to get to Holmenkollen. By underground: This is the easiest
way to travel. Take the westbound underground marked Frognerseteren
www.holmenkollen.com
12
13
14
LSM 780
Obtain Quantitative Information About Your Cells
and Individual Molecules
The sensitivity of LSM 780 is quite simply
outstanding. The GaAsP detector achieves 45
percent quantum efficiency compared to 25 percent
typically by conventional PMT detectors. This results
in accurate details and contrast-rich images of the
challenging specimens you encounter in your live cell
imaging.
The system’s illumination and detection design
allows you to simultaneously acquire up to ten dyes.
You excite any common fluorophore with up to eight
different lasers, detecting the signals with the 32
channel GaAsP detector. LSM 780 is so sensitive,
the system even allows photon counting.
Awarded Quality and Sensitivity for Your Imaging
LSM 780 allows you to achieve images of
unparalleled detail, even from the most challenging
specimens. The superior quality and sensitivity has
been recognized by a distinguished R&D 100 Award
in 2011.
15
17
18
19
Program in brief
Tuesday
Wednesday
Registration Bristol
Bristol Conference
Halls
Thursday
Friday
9:00
9:15
Welcome
Session I:
Evolution of
Reproductive
Systems
Session VI:
Meiosis &
Apomeiosis
Session IX:
Embryogenesis
&
Parthenogenesis
10:00
10:30
Session II:
Polyploidy &
Aneuploidy
11:00
Tea & Coffee break
11:30
Flash Poster
Presentations
8:00
12:15
12:30
Tea & Coffee
break
Tea & Coffee
break
Session VII:
Gametophyte
Development &
Function
Session X:
Seed & Fruit
Development
Lunch
COST member
meetings
Lunch
13:00
14:00
Session III:
Apomixis &
Emerging
Technologies
15:50
16:00
Tea & Coffee break
16:30
Session IV:
Endosperm &
Imprinting
Session VIII:
Fertilization
Mechanisms
18:00
Session V:
Plant Reproduction
& Environmental
Change
Poster Session odd
numbers
Poster Session
even numbers
20:00
20:30
Session VII:
Gametophyte
Development &
Function
Tea & Coffee
break
18:30
19:00
Concluding
remarks
Lunch
Registration
Bristol lobby
Dinner
Conference Dinner
20
21
Program in detail
WEDNESDAY 2. OCTOBER
08:00
Registration
Registration
09:00
Paul Grini & Emidio Albertini
Welcome
Session I: Evolution of Reproductive Systems
Chair: Anna Koltunow
09:15
John Carman
Utah State University, Logan,
USA
Apomictic life cycles: Ancient fair-weather alternatives to sexual life
cycles
S01
09:45
Elvira Hörandl
Georg-August-University of
Göttingen, Germany
The evolution of apomixis in angiosperms: a reappraisal
S02
Session II: Polyploidy & Aneuploidy
Chair: Anna Koltunow
10:00
Claudia Köhler
SLU, Uppsala, Sweeden
Epigenetic mechanisms establishing interploidy and interspecies
hybridization barriers in the endosperm
S03
10:30
Konstantinos Kritsas
University of Zürich,
Switzerland
ULEs, highly conserved non-coding sequences: Novel functional
elements hidden in the genome?
S04
10:45
Nico De Storme
University of Ghent, Belgium
Mild defects in cytokinesis form a basis for whole genome doubling in
plants
S05
Tea and Coffee Break
11:00-11:30
11:30
Flash Poster Presentations
Chairs; Paul Grini & Thomas Dresselhaus
(2-3 min presentations of selected posters without discussion)
Clément Lafon-Placette
SLU, Uppsala, Sweeden
Hua Jiang
SLU, Uppsala, Sweeden
Dorota Paczesniak
Eawag / ETH Zurich,
Switzerland
Philip Wolff
ETH Zurich, Switzerland
Barbara Glöckle
University of Oslo, Norway
Dieu Vo
University of Bremen,
Germany
Maura Cardarelli
IBPM-CNR Sapienza
University of Rome, Italy
Thomas Hackenberg
University of Regensburg,
Germany
Maria-Pilar Vallés
EEAD-CSIC, Zaragoza, Spain
Ewa Dubas
Institute of Plant Physiology
PAS, Kraków, Poland
Ida Myhrer Stø
University of Oslo, Norway
Tatyana Radoeva
Wageningen University,
Netherlands
12:30-14:00
The postzygotic hybridization barrier between diploid A. arenosa and
A. lyrata
Genetic analysis of postzygotic hybridization barriers
P01
Making bigger seeds to increase agricultural yield: the effects of
paternal contr bution and sexual conflict on variable endosperm
development in naturally-occurring asexual (apomictic) plants
Genomic Imprinting in Arabidopsis thaliana
P05
Dissecting cell-cycle and -differentiation in flowering plant gamete
formation
The Role of core-spliceosomal components during cell specification in
the female gametophyte
P20
Auxin and jasmonic acid interaction in the control of anther dehiscence
process in Arabidopsis
P27
Cell surface proteins mediating gamete interaction in Arabidopsis
P28
n-Butanol - induces bread wheat microspore embryogenesis by
microtubules depolymerization
Endogenous auxin and ABA in microspore embryogenesis of oilseed
rape (Brassica napus L.)
P31
IDA and IDL peptides in plant reproduction
P36
Auxin dependent patterning is mediated by distinct ARF/bHLH
modules
P40
Lunch
22
P03
P07
P25
P33
Session III: Apomixis & Emerging Technologies
Chair: Tim Sharbel
14:00
Anna Koltunow,
CSIRO, Adelaide, Australia
Control of apomixis in Hieracium (Asteraceae)
S06
14:30
Timothy Sharbel
IPK, Gatersleben, Germany
Evolutionary approaches to deciphering the functional switch from
sexual to asexual (apomictic) reproduction in natural plant populations
S07
15:00
Shai Lawit
DuPont Pioneer, Johnston,
USA
The agricultural frontier of plant female reproductive biology
S08
15:20
Sivina Pessino
Universidad Nacional de
Rosario, Zavalla, Argentina
Expression and functional analysis of candidate genes related to
aposporous apomixis in grass species
S09
15:35
Giulio Galla,
University of Padova, Italy
Exploring the coding and non-coding components of the H. Perforatum
flower transcriptome. Two sides of the same coin
S10
Tea and Coffee Break
15:50-16:30
Session IV: Endosperm & Imprinting
Chair: Paul Grini
16:30
Mary Gehring
Whitehead Institute MIT,
Cambridge, USA
Natural variation in DNA methylation leads to intraspecific variation in
gene imprinting
S11
17:00
Rod Scott
University of Bath, UK
Post-zygotic hybridisation barriers: paternal killer genes and maternal
modifiers
S12
17:30
Michael Raissig
University of Zürich,
Switzerland
Genomic imprinting in the Arabidopsis embryo is partly regulated by
PRC2
S13
Session V: Plant Reproduction & Environmental
Change
Chair: Paul Grini
18:00
Jose Gutierrez-Marcos
School of Life Sciences,
Warwick University, UK
Myths and facts about transgenerational epigenetic inheritance in
plants
18:30
Poster Session, odd numbers
(Tea, coffee and refreshments will be served during the session)
20:00
Dinner
S14
THURSDAY 3. OCTOBER
Session VI: Meiosis & Apomeiosis
Chair: Danny Geelen
9:00
Ravishankar Palanivelu
University of Arizona, Tucson,
USA
Molecular genetic analysis of expression of meiosis-related genes in
Arabidopsis
S15
09:30
Arp Schnittger
IBMP-CNRS, Strasbourg,
France
Control of germline entry in Arabidopsis
S16
10:00
Danny Geelen
Ghent University, Belgium
Multiple factors controlling maintenance of chromosome cohesion in
Arabidopsis thaliana meiosis
S17
S18
23
10:30
Martin Mau
IPK, Gatersleben, Germany
Genetic control of unreduced pollen formation in apomictic Boechera
(Brassicaceae)
10:45
Anja Schmidt
University of Zürich,
Switzerland
Transcriptome analysis of the Boechera gunnisoniana apomictic initial
cell and the female gametes reveals important differences in gene
regulatory pathways as compared to the sexual germline
S19
Tea and Coffee Break
11:00-11:30
Session VII: Gametophyte Development & Function
Chairs: Thomas Dresselhaus & Lucia Colombo
11:30
David Twell
University of Leicester, UK
Germline specification in male gametophyte development
S20
12:00
David Honys
ASCR, Prague, Czech
Republic
NTP303 - the story of translation regulation in pollen
S21
12:30
Diego Hojsgaard
Georg-August University of
Göttingen, Germany
Consequences of parallel development of meiotic and apomictic
pathways in aposporous plants
S22
12:45
Michael Borg
University of Leicester, UK
Gene expression profiling of regulatory mutants controlling sperm cell
formation
S23
Lunch
13:00-14:00
2+
14:00
José Feijó
University of Maryland,
College Park, USA
Coordination of pollen tube growth by Ca
regulated anion fluxes
S24
14:30
Lucia Colombo
University of Milan, Italy
Transcriptional control of the double fertilization process: the role of
REM family
S25
15:00
Rita Gross-Hardt
University of Tübingen,
Germany
Ethylene regulates seed composition and development
S26
15:30
Simona Masiero
University of Milan, Italy
Transcriptional regulation of egg cell specific genes in Arabidopsis
thaliana
S27
15:45
Mathieu Ingouff
University Montpellier II-IRD,
France
Real-time dynamics of DNA methylation during plant reproduction
S28
Tea and Coffee Break
16:00-16:30
Session VIII: Fertilization Mechanisms
Chair: Mary Gehring
16:30
Frederic Berger
Temasek Life Science
Laboratory, Singapore
Nuclear dynamics during fertilization in flowering plants
S29
17:00
Thomas Dresselhaus
University of Regensburg,
Germany
Peptide signaling during pollen tube perception
S30
17:30
Ana Marta Pereira
Universidade do Porto,
Portugal
Unravelling the function and expression pattern of Arabinogalactan
proteins in Arabidopsis thaliana reproductive tissues
S31
17:45
Cecile Bousquet-Antonelli
University of Perpignan
CNRS, France
The Arabidopsis LARP6c protein is an RNA binding factor specific to
mature pollen and required for proper male transmission
S32
18:00
Marta Adelina Mendes
University of Milan, Italy
The role of STK-ABS complex in the double fertilization process
S33
18:15
Satohiro Okuda
Nagoya University, Japan
Acquisition of LURE-reception ability at the pollen tube tip of Torenia
fournieri
S34
24
18:30
Poster Session even numbers
(Tea, coffee and refreshments will be served during the session)
20:30
Conference Dinner
FRIDAY 4. OCTOBER
Session IX: Embryogenesis & Parthenogenesis
Chair: Emidio Albertini
09:00
Emidio Albertini
University of Perugia, Italy
APOSTART: a candidate gene involved in embryo progression and
parthenogenesis
S35
09:30
Kim Boutilier
Plant Research International,
Wageningen, Netherlands
Epigenetic regulation of haploid embryo development
S36
09:45
Tatyana Radoeva
Wageningen University,
Netherlands
Auxin dependent patterning is mediated by distinct ARF/bHLH
modules
S37
10:00
Iwona Żur
Polish Academy of Sciences,
Kraków, Poland
Hormonal balance in triticale (×Triticosecale Wittm.) anthers and its
effect on androgenesis effectiveness
S38
10:15
Mercedes Soriano Kastan
Plant Research International,
Wageningen, Netherlands
A molecular framework for somatic embryogenesis induction in
Arabidopsis
S39
Tea and Coffee Break
10:30-11:00
Session X: Seed & Fruit Development
Chair: Claudia Köhler
11:00
Loïc Lepiniec
INRA-AgroParisTech,
Versailles, France
Dissecting gene regulatory networks that control seed development in
Arabidopsis
S40
11:30
Odd-Arne Olsen
Norwegian University of Life
Sciences, Ås, Norway
Alurone cell developmental programing is surface dependant and
relies on Dek1, a member of the ancient TML-calpain gene family
S41
11:45
Mara Cucinotta
University of Milan, Italy
The interaction between CUC genes and the hormonal network
controlling ovule numbers
S42
12:00
Adam Vivian-Smith
Bioforsk / Norw. Forest and
Landscape Inst., Ås, Norway
Rapid post-fertilization auxin responses, which activate fruit initiation,
are potentiated by a PINOID/AGC kinase to BDL/IAA12
phosphorylation pathway
S43
12:15
Albertini, Dresselhaus.
Geelen, Sharbel, Grini
Concluding remarks
12:30
Official program ends
12:30
Lunch
Session for cost MCs only:
For cost MCs only:
13:00
COST Management Committee (MC) final meeting.
13:30
COST evaluation meeting
15:00
COST session ends
25
26
27
Speaker Abstracts
28
Chair: Anna Koltunow
Session I: Evolution of Reproductive Systems
Abstract S01
Apomictic Life Cycles: Ancient Fair-weather
Alternatives to Sexual Life Cycles
John G. Carman
Plants, Soils and Climate Department, Utah State University, Logan, UT USA
A characteristic of eukaryotes is the production of discrete, epigenetically-reset cells that
re-initiate the life cycle, a process that generally occurs in association with meiosis and
syngamy. However, it also occurs in eukaryotes by the formation of discrete, epigeneticallyreset gametes or gamete-like cells that form without meiosis and re-initiate the life cycle
without syngamy. This is called apomixis, and it occurs with sex in all kingdoms of
eukaryotes, e.g., in single-celled protists and fungi, in primitive multicellular plants and
animals, and in more derived plants and animals. The most primitive apomicts today are
single-celled haplontic protists, and among them are those where apomixis cycles with sex (in
an organism) in response to environmental cues. Apomixis is the fair-weather mode. Sex
occurs in response to metabolic stress. Similar environment-regulated sex-apomixis switches
are found in organisms from all five eukaryote kingdoms. Consequently, we have
hypothesized that apomixis is an epigenetically-regulated life-cycle alternative that originated
with single-celled haplontic protozoa during eukaryogenesis where it cycled with sex in
response to the environment. If correct, two key implications emerge. First, apomeiosis is
not just unreduced gamete formation. It is “formation, without chromosome reduction, of
parthenogenesis-competent gametes or gamete-like cells.” This definition links all types of
apomeiosis together (by a conserved, epigenetics-based apomixis-life-cycle mandate),
including no meiosis, as occurs in primitive haplontic apomicts, and derived forms of
apomeiosis where meiosis is avoided or modified, as occurs in apospory and diplospory,
apogamy and adventitious embryony, and automictic parthenogenesis. Secondly, the
hypothesis implies an absence of de novo apomixis genes, the implication being that
abnormal levels of expression of normal genes, possibly stress-related genes and possibly
caused by hybridity or polyploidy-induced genetic or epigenetic modifications, could alter
perceptions of metabolic stress in germline and germline-associated tissues. Perceptions
might be weakened or strengthened thus increasing epigenetic shifts toward an apomictic
fate (if competencies have remained intact) or a sexual fate, respectively. Results of testing
for such switch-like mechanisms in transcriptomes of sexual and apomictic Boechera and
aposporous sorghum will be presented.
Abstract S02
The evolution of apomixis in angiosperms: a reappraisal
1
1
2
3
Hojsgaard, Diego ; Klatt Simone ; Baier, Roland ; Carman, John ; Hörandl, Elvira
1
1
Georg August University Göttingen, Albrecht-von-Haller Institute for Plant Sciences, Department of
2
Systematic Botany, Untere Karspüle 2, D37073 Göttingen, Germany Gesellschaft für wissenschaftliche
Datenverarbeitung mbH Göttingen (GWDG), Arbeitsgruppe Anwendungs- und Informationssysteme, Am
3
Fassberg 11, D37077 Göttingen, Germany Plants, Soils and Biometeorology Department, Utah State
University, Logan, UT 84322–4820, U.S.A.
Apomixis, the asexual reproduction via seed, has long been regarded a blind alley of
evolution. This hypothesis was based on the assumption that apomixis is an irreversible,
phylogenetically derived trait that would rapidly lead to extinction of the respective lineages.
However, recent updates of the taxonomic distribution of apomixis in angiosperms, which is
stored in an online database (http://www.apomixis.uni-goettingen.de) suggest an alternative
29
evolutionary scenario. Apomixis is taxonomically scattered and occurs in both early and late
branching lineages, with several reversals from apomixis to obligate sex along phylogeny.
The most frequent mode is adventitious embryony (148 genera), followed by apospory (110)
and diplospory (68), whereby these traits are not exclusive. Apomixis has a strong
association to larger families and genera-rich subfamilies, and orders with apomixis have
significantly more genera than sister taxa without apomixis. The majority of genera with
apomixis (62%) are distributed in more than one geographical zone, and apomixis enhances
range expansions. Genetic control of apomixis is based on altered expression patterns of the
same genes that control sexual development. Apomixis, therefore, could represent a
transition period in the evolution of polyploid complexes, with polyspory in paleopolyploids
being a remnant of lost apomixis. In neopolyploids, apomixis helps to overcome sterility and
allows for geographical range expansions of agamic polyploid complexes. The facultative
nature of apomixis allows for reversals to sexuality and further speciation of paleopolyploid
lineages. The positive correlation to biodiversity measures and to large geographical
distributions supports the hypothesis that apomixis may enhance diversification of genera.
30
Chair: Anna Koltunow
Session II: Polyploidy & Aneuploidy
Abstract S03
Epigenetic mechanisms establishing interploidy and interspecies
hybridization barriers in the endosperm
1,2
1,2
2
David Kradolfer , Philip Wolff , Hua Jiang , Carolin Rebernig, Claudia Köhler
2
1
2
Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland,
Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and
Linnean Center of Plant Biology, Uppsala, Sweden, claudia.kohler@slu.se
Polyploidization is a widespread phenomenon among plants and is considered a major
speciation mechanism. Polyploid plants have a high degree of immediate post-zygotic
reproductive isolation from their progenitors, as backcrossing to either parent will produce
mainly nonviable progeny. This reproductive barrier is called triploid block and it is caused by
malfunction of the endosperm. To elucidate the underlying mechanisms establishing the
triploid block we have performed a suppressor screen aiming to identify mutants that form
viable triploid seeds. One of the identified mutants is the paternally expressed imprinted gene
ADMETOS (ADM). We present evidence that increased dosage of ADM causes triploid seed
arrest. A large body of theoretical work predicted that deregulated imprinted genes establish
the barrier to interploidy hybridization. Our study thus provides evidence strongly supporting
this hypothesis and generates the molecular basis for our understanding of postzygotic
hybridization barriers in plants. We will furthermore discuss our results on interspecies
hybridization barriers in the genus Capsella that share striking similarities to interploidymediated seed failure in Arabidopsis.
Abstract S04
ULEs, highly conserved non-coding sequences: Novel functional
elements hidden in the genome?
1
1
2
3
1
1
K. Kritsas , C. Baroux , D. Hupalo , A.D. Kern , S. Wuest , T. Wicker & U. Grossniklaus
1
1
2
University of Zurich, Institute of Plant Biology & Zurich Plant Science Center, Zurich, SWITZERLAND
3
Dartmouth College, Department of Biological Sciences, New Hampshire, USA The State University of
New Jersey, Department of Genetics, New Jersey, USA
Ultraconserved elements (UCEs), DNA sequences which are 100% identical between animal
genomes are enigmatic features whose function is not well understood. Here, we report the
identification and characterization of UCE-like elements (ULEs). ULEs are highly conserved
non-coding sequences of unknown function in plant genomes. In addition to sequence
constraints, our data indicate that ULEs are functional elements. We have identified them
after whole genome comparison studies between Arabidopsis thaliana and Vitis vinifera (wine
grape). Arabidopsis and Vitis have diverged from their common ancestor ~115 MYA allowing
significant changes at the DNA sequence to occur. We found 36 ULEs, which are >55 bp long
and share at least 85% sequence identity. Further analysis showed that ULEs are not just
random sequences but are under strong purifying selection. They also have distinct structural
features. All of them have a sharp drop of their A+T content just at their borders, and they are
enriched next to genes involved in development. Intriguingly, the latter show preferential
expression in undifferentiated cells. Surprisingly, ULES are depleted from segmental
duplications, suggesting that the functions of ULEs or the regions that contain them are
dosage sensitive. Interestingly, ULEs have exactly the same properties as the animal UCEs
and ULEs that are only present in the monocots, suggesting that these elements appeared
independently several times. But why ULEs are so conserved and what would be their
function? Our hypothesis is that ULEs may serve as agents of chromosome copy counting.
31
The two parental ULEs may recognize each other perhaps through chromosome pairing to
ensure the exact number of chromosomes, which in a diploid cell should be exactly two. We
employed genetics and cytogenetics to address this question. Our latest results will be
presented.
Abstract S05
Mild defects in cytokinesis form a basis for whole genome doubling in
plants
1
2
2
3
3
N. De Storme , J. De Schrijver , W. Van Criekinge , V. Wewer , P. Dörmann and D. Geelen
1
1
Department of Plant Production, Faculty of Bioscience Engineering, University of Ghent, 9000 Ghent,
2
BELGIUM, Bioinformatics and Computational Genomics (BIOBIX), Department of Molecular
3
Biotechnology, University of Ghent, 9000 Ghent, BELGIUM, Institute of Molecular Physiology and
Biotechnology of Plants, University of Bonn, 53115 Bonn, GERMANY
Most if not all plants have undergone one or more polyploidization events throughout their
evolutionary history, which may have contributed to the emergence of new variation
(biodiversity) and plant speciation. It is generally considered that polyploidization in plants is
occurring through the production and fertilization of unreduced (2n) gametes, which
eventually generates triploid or tetraploid offspring. This process is referred to as sexual
polyploidization. Little is known about the cellular mechanisms through which 2n gametes are
formed and what conditions may provoke their production. In the last few years, several
genetic defects have been described that can lead to a restitution of the meiotic cell cycle and
the associated production of 2n gametes.
Here we present evidence that cellular defects in somatic cytokinesis also form an important
means for the generation of polyploid gametes. More specifically, we describe the
Arabidopsis mutant et2, which shows mild defects in cytokinesis due to alterations in the
expression of the callose synthase GLUCAN SYNTHASE LIKE 8 (GSL8). Due to the
incomplete cellular divisions primarily occurring in flower organs, multinuclear cells are
formed, which ultimately fuse to generate cells with an increased ploidy level. These
endomitotic cells can participate in male and female reproduction organ development and
then produce 5-20% of the male and female gametophytes, which are diploid. As a result, et2
produces a mixed population of diploid, triploid and tetraploid offspring. Similar ploidymaintenance defects (enlarged tetrads, frilled petals) also occur in the Arabidopsis mutants
frill1 and cvp1, which both have a mutation in the sterol biosynthesis STEROL METHYL
TRANSFERASE 2 (SMT2) enzyme. As SMT2 is a branching enzyme, positively regulating
the production of free phytosterols (sito- and stigmasterol), a putative link between free
sterols, GSL8-directed cell plate formation and reproductive ploidy maintenance is
established and discussed.
Altogether, these observations show that mild defects in cytokinesis can cause pre-meiotic
genome doubling (endomitosis), resulting in the formation of polyploid gametes and offspring.
The capacity of these cytokinesis mutants to produce a mix of diploids and polyploids
suggests that they may have contributed to plant evolution and diversity as they form a
sustained production vehicle driving polyploid plant induction.
32
Chair: Tim Sharbel
Session III: Apomixis & Emerging Technologies
Abstract S06
Control of apomixis in Hieracium (Asteraceae)
Susan Johnson, Melanie Hand, Steven Henderson, Dave Rabiger, Daisuke Ogawa and Anna
Koltunow
Commonwealth Scientific and industrial Research Organization (CSIRO), Plant Industry. Waite Campus.
PO Box 350 Glen Osmond, South Australia. 5064. anna.koltunow@csiro.au
Apomixis in Hieracium species occurs by the formation of meiotically unreduced embryo sacs
and both embryo and endosperm formation is fertilization-independent. In subgenus
Hieracium, meiotic avoidance during embryo sac formation occurs by diplospory while in
subgenus Pilosella, apospory is the mode of unreduced embryo sac formation. Our genetic
and molecular analyses of apomixis have been focused on aposporous subgenus Pilosella
species. We have previously shown that two dominant loci control apomixis in H. praealtum.
LOA is required for apospory and sexual suppression and LOP is required for fertilizationindependent seed formation. Using other genetic backgrounds we have recently separated
autonomous endosperm formation from both apospory and fertilization-independent
embryogenesis. Thus the three developmental components of apomixis in Hieracium appear
to have independent genetic control. Here we update our current knowledge of the control of
apomixis in subgenus Pilosella species, including information gained from recent
transcriptomic data and our progress towards isolating apomixis genes.
Abstract S07
Evolutionary approaches to deciphering the functional switch from
sexual to asexual (apomictic) reproduction in natural plant populations
Timothy F. Sharbel
Apomixis Research Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Corrensstrasse 3, D-06466 Gatersleben, Germany
An organism’s choice to reproduce with or without sex has long puzzled evolutionary
biologists. Apomixis, a natural form of reproduction in plants whereby seeds are produced
asexually, has evolved repeatedly from sexual ancestors in many taxa. Apomixis is of
interest on a number of levels, ranging from population genetics to evolution, but also from an
applied perspective, as it represents a disruptive technology which could significantly change
agricultural practices (e.g. fixing heterosis in hybrid crops). The switch from sex to apomixis
is hypothesized to result from deregulation of developmental pathways leading to sexual seed
development, and the trigger for deregulation involves the global genomic effects of
hybridization and polyploidy.
We study apomixis in wild plant populations, and use evolutionary theory to guide our
experimental approaches. High-throughput methods are employed to understand populationlevel phenotypic (seed production) and genetic (polyploidy, genetic structure) variability.
These data are then used to design targeted experiments, whereby candidate genes for
apomixis are identified using tissue-specific “omics” methods in particular genotypes. These
candidates are then used (1) in transformation experiments to attempt apomixis induction in
sexual plants, and (2) in population-level studies to understand the origin and evolution of
apomixis with respect to sexuality in natural populations.
33
Abstract S08
The Agricultural Frontier of Plant Female Reproductive Biology
1
1
S.J. Lawit , M.A. Chamberlin & M.C. Albertsen
1
1
DuPont Pioneer, Agricultural Biotechnology, Johnston, Iowa, UNITED STATES of AMERICA
Commercial seed businesses, like DuPont Pioneer, depend on controlled reproduction of the
crop plants they develop. Control of reproduction is critical in every stage of product
development from initial breeding stages through commercial seed production. Beginning with
the first commercial crosses for hybrid maize, innovative manipulations of plant reproductive
biology have led to increasing agricultural productivity in numerous crops. Some, such as
haploid induction technology have increased the rate of breeding. Others, such as
cytoplasmic male sterility and the harnessing of nuclear male sterility, have led to more
efficient production of hybrids and are enabling the next generation of hybrid crops. All these
technologies focus on male reproductive biology. However, studies have increasingly focused
on female reproductive biology and the megagametophyte. A major aim of this research is
synthetic apomixis resulting in seed derived wholly from the maternal plant. Such a
development could lead to fixation of important agronomic traits such as hybrid vigor, a key
benefit to agricultural productivity. Some of our work has focused on facilitating embryo sac
observations by fusing cell type specific promoters to different fluorescent proteins within a
single transcriptional unit and expressing them in Arabidopsis plants. We will discuss these
tools to facilitate female reproductive biology research, modifications of reproductive
mechanisms, and how reproductive biology can be further harnessed to benefit crop
production.
Abstract S09
Expression and functional analysis of candidate genes related to
aposporous apomixis in grass species
1
1
1
1
1
1
2
M. Podio , M. Mancini , H. Permingeat , L. Siena , L. Delgado , S. Felitti , M. Sartor , F.
2
3
3
3
4
5
Espinoza , L. Arrais-Guimaraes , D. Dusi , V. Carneiro , F. Pupilli , O. Leblanc , J.P.A.
1
1
Ortiz & S. Pessino
1
2
Universidad Nacional de Rosario, Facultad de Ciencias Agrarias, Zavalla, ARGENTINA, Universidad
Nacional del Nordeste, Facultad de Ciencias Agrarias, Instituto de Botánica del Nordeste (IBONE3
4
CONICET), Corrientes, ARGENTINA, Embrapa, Cenargen, Brasilia, BRASIL, Istituto di Genetica
5
Vegetale, Perugia, ITALY, UMR DIADE, Institut de Recherche pour le Développement (IRD) &
Université de Montpellier 2, Montpellier, FRANCE
In the past decade, comparative transcriptome surveys carried out in Paspalum and
Brachiaria sp. provided a vast list of genes displaying differential expression in reproductive
organs of sexual and apomictic individuals. A candidates’s subset was then selected for
further characterisation using two main criteria: i) concurrent detection in comparative
transcriptome surveys from both species; and/or ii) occurrence of experimental or in silico
positional linkage to genomic regions associated with apospory control. Here, we report
detailed expression analyses for Pnserk (somatic embryogenesis receptor kinase), Pnexs
(exs domain-containing protein), Pn19 (unknown), Pn46 (MAP3K), Pn108 (far1 domaincontaining protein), Pn69 (DNA methyltransferase) and PnMTA-70 like (RNA
methyltransferase). Paspalum and Brachiaria candidates’ full sequences were obtained by
performing RACE (Rapid amplification of cDNA ends) or Illumina sequencing, respectively.
Chronological quantitative expression was analysed by real time PCR in reproductive organs
of sexual and apomictic genotypes at pre-meiosis, meiosis, post-meiosis and anthesis.
Moreover, in situ hybridization was used to determine more accurately the cell types where
expression was located. The selected candidates showed contrasting chronological and
spatial expression patterns in sexual and apomictic plants, sometimes involving both coding
and non-coding strands. Most of them displayed strong sense expression in the nucellus of
aposporous plants. A stable transformation platform is currently being used to explore their
34
potential functional role in reproductive development by altering timely or spatially their
expression pattern. Expression of candidate n46 (MAP3K) was down-regulated in a natural
aposporous genotype via the introduction of a hairpin construct. Transformed plants showed
a diminished capacity to form aposporous embryo sacs with respect to wild-type plants and
transformation controls. Collectively, our results suggest the existence of a molecular
pathway ectopically activated in the nucellus of aposporous genotypes, which regulates the
onset of apospory initials and their commitment to a gametophytic fate.
Abstract S10
EXPLORING THE CODING AND NON-CODING COMPONENTS OF THE H.
PERFORATUM FLOWER TRANSCRIPTOME. TWO SIDES OF THE SAME
COIN
1
2
Galla G. , Sharbel T.F. , Barcaccia G.
1
1
Laboratory of Plant Genetics, Department of Agriculture Food Natural resources Animals and
2
Environment (DAFNAE) - University of Padova, Viale dell’Università 16, 35020 Legnaro, Italy; Apomixis
Research Group, Department of Cytogenetics and Genome Analysis, Institut für Pflanzengenetik und
Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany.
St. John’s wort (Hypericum perforatum L.) is a medicinal plant that produces important
metabolites with antidepressant and anticancer activities. Beside the pharmaceutical interest,
recently gained information has shown that H. perforatum is also an attractive model system
for the study of apomixis. During the last decades the understanding of the molecular basis of
apomixis in this species has been complicated by the lack of biological data, e.g. genomic or
even transcriptomic sequences. The aim of our research project is the sequencing,
annotation and comparative investigation of the Hypericum flower transcriptome, as critical
steps toward a better understanding of the genetic control of aposporic and sexual
reproduction in the facultative apomict H. perforatum. We adopted the high-throughput Roche
454 technology to sequence the whole Hypericum flower transcriptome of single flower parts
collected from three apomictic and two sexual genotypes. Overall the sequencing approach
allowed us the investigation of the coding and non-coding RNA fractions of the flower
transcriptome and led us to the identification of RNAs that are likely exclusive of single flower
parts or reproductive strategies. The protein-coding sequences of the flower RNAs were
annotated according to the main vocabularies of Gene Ontology and Plant Ontology in order
to find out major biological processes and molecular functions belonging to the frame of cell
development, flower development and plant reproduction. Starting from this dataset, we could
discover and annotate a large number of transcripts related to meiosis, gametophyte and
gamete formation, in addition to genes exclusively expressed in sexual or apomictic libraries.
The annotation revealed that our dataset was also relatively rich in non-coding sequences.
Among these sequences some entries scored a high similarity with pre-miRNA precursors
that are expressed in apomictic plants by targeting transcription factors. A computational in
silico prediction of the structure, in combination with an in vitro validation, allowed us to
identify seven pre-miRNAs. We demonstrated that H. perforatum flowers share highly
conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide
range of molecular functions, including metabolism, response to stress, flower development
and plant reproduction. For both coding and non-coding RNA sequences, the expression
levels and patterns of the most promising ones were investigated in great details to verify the
tissue and stage specificity of genes potentially involved in gametophyte and gamete
formation. Overall data are presented and critically discussed. Our analyses aim to pave the
way toward a better understanding of the molecular basis of flower development and plant
reproduction, by discovering genes or RNAs that may differentiate and/or regulate the sexual
and apomictic reproductive pathways in H. perforatum.
35
Chair: Paul Grini
Session IV: Endosperm & Imprinting
Abstract S11
Natural variation in DNA methylation leads to intraspecific variation in
gene imprinting
Mary Gehring
Whitehead Institute for Biomedical Research, Dept. of Biology, Massachusetts Institute of Technology
DNA methylation is a heritable epigenetic mark important for genome stability, gene
imprinting, and transposable element silencing in diverse eukaryotes. During flowering plant
reproductive development the DNA methylation landscape is dramatically altered in the
central cell, the female gametophyte cell that gives rise to the seed endosperm tissue.
Previously we showed that short transposable elements are actively demethylated in the
central cell. Since the expression of some genes is tied to the epigenetic status of nearby
transposable elements, the resultant epigenetic asymmetry between maternal and paternal
alleles can create imprinted gene expression in the endosperm after fertilization. Both the
mechanisms that might create imprinted gene expression and the pressures that promote its
maintenance suggest that imprinting could be variable within and between species. Through
whole genome methylation and allele-specific expression profiling of reciprocal crosses
among three Arabidopsis ecotypes, we are using natural genetic and epigenetic variation
within Arabidopsis to test the hypothesis that transposable elements drive genomic imprinting.
We have defined all of the differentially methylated regions (DMRs) between embryo and
endosperm tissue and between different ecotypes. We discovered a set of approximately 100
genes that are imprinted in all crosses tested and a further 10-20 genes in which only certain
alleles are imprinted. Although ecotype DMRs represent a largely distinct set of sequences
from embryo-endosperm DMRs, we have found that several genes with allele-specific
imprinting are variably methylated among ecotypes. These genes might underlie variable
seed traits within the Arabidopsis thaliana species.
Abstract S12
Post-zygotic hybridisation barriers: paternal killer genes and maternal modifiers
1
2
1
1
R. J. Scott , A. Bolbol , M. Aljabri , J.L. Tratt , J. Doughty
1
1
2
Department of Biology and Biochemistry, University of Bath, Bath, UK Botany Department, Zagazig
University, Zagazig, Egypt
Many plants, including agronomically important species, exhibit post-zygotic barriers to
hybridization, in both interploidy crosses within a species and interspecific crosses between
related species, even at the same ploidy levels. These barriers prevent production of
potentially valuable new hybrids, but also provide opportunities to manipulate seed size and
potentially seed yield. In this talk, I will discuss genetic variation in the operation of the postzygotic hybridization barrier in Arabidopsis thaliana. Most Arabidopsis accessions tolerate
reciprocal diploid by tetraploid hybridizations (2xX4x and 4xX2x) to produce seed containing
viable triploid embryos, albeit of radically different weights. However, a small number,
including Columbia (Dilkes et al PlosB, 2008) whilst tolerating 4xX2x crosses are acutely
sensitive to paternal excess 2xX4x crosses, with most of the resulting seed dying sue to
severe disruption to endosperm development. These accessions therefore operate an
asymmetric hybridization barrier, where paternal excess causes lethality but maternal excess
does not. We have found that this Col-killer effect is associated with 2x sperm is independent
of the mechanism leading to chromosome doubling, and is under epigenetic control. Genetic
mapping indicates that the trait is multigenic but is likely to have the same genetic basis in at
least two accessions. There is also considerable variation among Arabidopsis accessions in
36
the ability to resist the killing effect of Col 2x sperm, with some diploid seed parents suffering
almost no seed abortion. I will describe progress toward identifying the gene(s) responsible
for this behaviour, and suggest how these and the Col-killer genes might be harnessed for
crop improvement.
Abstract S13
Genomic Imprinting in the Arabidopsis Embryo Is Partly Regulated by
PRC2
Michael T. Raissig, Marian Bemer, Célia Baroux and Ueli Grossniklaus
Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, CH-8008 Zürich,
Switzerland
Genomic imprinting results in monoallelic gene expression in a parent-of-origin-dependent
manner and is regulated by the differential epigenetic marking of the parental alleles. In plants,
genomic imprinting has been primarily described for genes expressed in the endosperm, a
tissue nourishing the developing embryo that does not contribute to the next generation. In
Arabidopsis, the genes MEDEA (MEA) and PHERES1 (PHE1), which are imprinted in the
endosperm, are also expressed in the embryo; whether their embryonic expression is
regulated by imprinting or not, however, remains controversial. In contrast, the maternally
expressed in embryo 1 (mee1) gene of maize is clearly imprinted in the embryo.
We identified several imprinted candidate genes in an allele-specific transcriptome of hybrid
Arabidopsis embryos and confirmed parent-of-origin-dependent, monoallelic expression for
eleven maternally expressed genes (MEGs) and one paternally expressed gene (PEG) in the
embryo, using allele-specific expression analyses and reporter gene assays. Genetic studies
indicate that the Polycomb Repressive Complex 2 (PRC2) but not the DNA
METHYLTRANSFERASE1 (MET1) is involved in regulating imprinted expression in the
embryo. In the seedling, all embryonic MEGs and the PEG are expressed from both parents,
suggesting that the imprint is erased during late embryogenesis or early vegetative
development.
Our finding that several genes are regulated by genomic imprinting in the Arabidopsis embryo
clearly demonstrates that this epigenetic phenomenon is not a unique feature of the
endosperm in both monocots and dicots.
37
Chair: Paul Grini
Session V: Plant Reproduction & Environmental Change
Abstract S14
Myths and facts about transgenerational epigenetic inheritance in plants
Jose Gutierrez-Marcos
School of Life Sciences, Warwick University, UK
Plants are sessile organisms that are known for their adaptive plasticity to the changing
environment. In addition to directly influencing plant growth, environmental changes also
influence gene expression patterns as well as affecting genome stability. Both these
responses are mediated by epigenetic mechanisms, which result in phenotypic changes
associated with adaptation to stress. Recent evidence suggests that some of these changes
can be transmitted to the offspring, and can remain stable for several generations. However,
the precise mechanisms regulating this epigenetic phenomenon and the impact that it might
have on plant adaptation remain unknown. My group has carried out genome-wide epigenetic
profiling in maize to understand the impact that abiotic environmental stress has on the
formation of new allelic variants. I will discuss our recent findings and propose a working
model to explain how the environment can direct discrete changes to the plant epigenome,
their mode of inheritance, and the wider significance for stress adaptation in plants.
38
Chair: Danny Geleen
Session VI: Meiosis & Apomeiosis
Abstract S15
Molecular Genetic Analysis of Expression of Meiosis-related Genes in
Arabidopsis
1,2
1
2
3
2
Yuan Qin, Lihua Zhao, Megan I. Skaggs, Sebastien Andreuzza, Tatsuya Tsukamoto,
3
2
4
3
5
Aneesh Panoli, Kirsten N. Wallace, Steven Smith, Imran Siddiqi, Zhenbiao Yang, Ramin
2
2
Yadegari, and Ravi Palanivelu
1
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology and
Shanghai Center for Plant Stress Biology, Shanghai Institutes for biological Sciences, Chinese
2
Academy of Sciences, Shanghai 200032, China School of Plant Sciences, University of Arizona,
3
Tucson, AZ 85721, USA Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007,
4
India School of Natural Resources and the Environment, University of Arizona, Tucson, AZ 85721,
5
USA Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of
California, Riverside, CA 92521, USA
In flowering plants, a series of coordinated developmental processes gives rise to meiocytes
from sub-epidermal cells of anthers and ovules. The gene-regulatory mechanisms controlling
the specification and maintenance of reproductive cell identities and function of meiocytes
before and after entering meiosis remain poorly characterized. Here, we show that before
entering meiosis, Arabidopsis ARP6, a subunit of the SWR1 complex, inhibits DMC1
expression in both the megasporocyte and surrounding non-sporogeneous ovule cells. After
entering meiosis, however, ARP6 activates DMC1 expression in the megasporocyte and
inhibits DMC1 expression in non-sporogenous ovule cells. The SWR1 complex remodels the
chromatin by depositing H2A.Z histone variant. Here, we demonstrate that H2A.Z deposition
at the DMC1 gene body requires ARP6. Consistent with these findings, arp6 mutants show
defects in prophase I of female meiosis, including defects reported in dmc1 mutants. Thus,
ARP6 controls female meiosis partly by modulating the spatial and temporal control of DMC1
expression.
Abstract S16
Control of germline entry in Arabidopsis
1
1
2,3
Xin’Ai Zhao , Daniel Bouyer, Jonathan Bramsiepe , Matthias van Durme , Moritz K.
2,3
Nowack , and
1,4
Arp Schnittger
1) Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des
Plantes du Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg, France,
2) Department of Plant Systems Biology, VIB, Gent, Belgium, 3) Department of Plant Biotechnology and
,
Bioinformatics, Ghent University, Gent, Belgium 4) Trinationales Institut für Pflanzenforschung
Establishment of the germline is a crucial step in the life cycle of multicellular organisms.
However, this developmental step is often challenging to study due to its limited accessibility
and the small number of cells involved. In contrast to animals, the germline is set up late in
the development of flowering plants and we are using Arabidopsis as a model system to
study entry into meiosis. Here we show that the mitosis-to-meiosis transition is controlled by
the cyclin-dependent kinase inhibitors of the KIP-RELATED PROTEIN (KRP) class. While
single and double krp mutants are indistinguishable from the wild type, meiocytes in triple krp
mutants underwent additional mitotic divisions before meiosis was executed. We reveal that
the function of KRP proteins in the germ line is the restriction of Cdk1/2-dependent control of
the Arabidopsis Retinoblastoma homolog RBR1 and rbr1 mutants also produced excessive
meiocytes. Reduction of Cdk1/2 activity rescued the multiple meiocyte phenotype of krp triple
39
mutants but remarkably not rbr1. Thus, RBR1 acts here independent of a cell proliferation
function. At the same time we show that RBR1 can directly bind to and represses genes
involved in cell differentiation and thus, directly links cell-cycle progression with differentiation.
To understand then the interconnection between differentiation and proliferation at a global
level, we have developed a new protocol to identify genome-wide Rb binding sites in
Arabidopsis and progress on the comparison between animal and plant Rb binding patterns
will be discussed here.
References:
Weimer, A.K., et al. (2012). Control of Asymmetric Cell Divisions by Retinoblastoma. Plant Cell, 24,
4083-95.
Zhao, X., et al. (2012). A general G1/S-phase cell-cycle module in Arabidopsis. PLoS Genetics,
8:e1002847
Nowack, et al. (2012). Genetic framework of cyclin-dependent kinase function in Arabidopsis. Dev Cell,
22, 1030-40.
Abstract S17
Multiple factors controlling maintenance of chromosome cohesion in
Arabidopsis thaliana meiosis
1
1
2
Linda Zamariola , Nico De Storme , Klaas Vandepoele , Danny Geelen
1
1
Department of Plant Production, Faculty of Bioscience Engineering, Gent University, Coupure Links
2
653 Gent, Belgium, Department of Bioinformatics and Systems Biology, VIB, Gent University,
Technologiepark 927, Belgium
Shugoshin is conserved protein protecting centromeric cohesion during meiosis. It was first
discovered in yeasts preventing REC8 cleavage by separase during meiosis I. Loss of
shugoshin leads to premature separation of sister chromatids. In fission yeast a paralogue of
Shugoshin, Sgo2, has been identified and it is required for faithful mitotic chromosome
division. In plants, homologous of Shugoshin1 have been identified in maize and rice and the
conserved function of the protein in meiosis has been demonstrated.
Within this research project, T-DNA insertion lines for the homologous genes of Shugoshin in
Arabidopsis thaliana were studied to investigate the chromosome cohesion. Meiotic spreads
on male meiocytes of one of the Shugoshin mutants (sgo1) showed that sister chromatids
separate before meiosis II leading to chromosome missegregation at the end of meiosis,
confirming the role of the protein as centromere protector. Furthermore, FISH analysis
performed with a centromeric probe on sgo1 meiocytes demonstrated loss of cohesion at the
end of meiosis I. However, in the sgo2 mutant no meiotic and mitotic defects were identified,
indicating that only sgo1 is required for maintaining apropriate chromosome dynamics.
sgo1/sgo2 double mutants are sterile whereas sgo1-/- mutants are not, suggesting that sgo2
may act redundantly to sgo1 during meiosis. In a search for additional mutants defective in
meiotic chromosome dynamics, we isolated a T-DNA line that showed defects in meiosis II in
which sister chromatids precociously separated, so that no normal metaphase II was
observed and chromatids were scattered on the equatorial plate. The observations imply that
chromosome cohesion is tightly regulated until the onset of metaphase II formation, and
implicates a novel factor, in addition to shugoshin, in protecting centromeric cohesion.
Abstract S18
Genetic control of unreduced pollen formation in apomictic Boechera
(Brassicaceae)
1
1
2
3
4
5
1
1
M. Mau , J. M. Corral , J. Lovell , H. Vogel , M. Koch , Ch. Kiefer , M. Melzer , J.Fuchs , M.
1
6
6
1
2
1
Kuhlmann , N. de Storme , D. Geelen , A. Olawale , J. McKay and T. F. Sharbel
1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Department of Cytogenetics and
2
Genome Analyses, Apomixis Group, Gatersleben, Germany, Colorado State University, Graduate
3
Degree Program in Ecology, Ft. Collins, USA, Max Planck Institute for Chemical Ecology, Department
4
of Entomology, Genomics Research Group, Jena, Germany, Heidelberg Institute of Plant Sciences,
40
5
Department of Biodiversity and Plant Systematics, Heidelberg, Germany, Max Planck Institute for Plant
6
Breeding Research, Department of Plant Developmental Biology, Cologne, Germany, Ghent University,
Plant Production, Faculty of Bioscience Engineering, Gent, Belgium
In sexual plants meiosis gives rise to recombined and reduced gametes. In diplosporous
apomictic accessions of the North American genus Boechera meiotic processes are
circumvented (i.e. apomeiosis) which lead to a supressed recombination and to production of
clonal unreduced male and female gametes. Unreduced male gametes in apomictic
Boechera are required to produce balanced endosperm. The objective of our study was to
identify the molecular genetic basis of unreduced pollen formation in Boechera. The
implementation of functional unreduced pollen formation from natural apomicts along other
apomixis elements in crops would hypothetically solve the “endosperm problem” by balancing
maternal and paternal genome ratios via central cell fertilization.
Using a Boechera-specific microarray, which was designed upon 454 cDNA libraries of
multiple sexual and apomictic accessions, this study identified and characterized a single
candidate genetic factor, UPGRADE-2, for unreduced pollen formation in apomictic Boechera.
Furthermore, its genus-wide dynamics was analyzed in order to contrast the hypotheses
whether apomeiosis expression was induced through interspecific hybridization or if it could
be an older characteristic of the genus (i.e. pre-Pleistocene).
Abstract S19
Transcriptome analysis of the Boechera gunnisoniana apomictic initial
cell and the female gametes reveals important differences in gene
regulatory pathways as compared to the sexual germline
1
1
2
3
1
Anja Schmidt , Marc W. Schmid , Ulrich C. Klostermeier , Weihong Qi , Daniela Guthörl ,
1
1
2
1
Christian Sailer , Manuel Waller , Philipp Rosenstiel , and Ueli Grossniklaus
1
University of Zürich, Institute of Plant Biology & Zürich-Basel Plant Science Center, Developmental
Genetics,
2
3
Switzerland, University of Kiel, Institute of Clinical Molecular Biology, Germany, University and ETH
Zürich, Functional Genomics Center Zürich, Switzerland
Apomixis (asexual reproduction through seeds) is a fascinating developmental process with a
great potential for agricultural applications, as it leads to the formation of clonal seeds. In
higher plants, both sexual reproduction and apomixis are common and closely interrelated.
During sexual reproduction, the megaspore mother cell (MMC) is the first cell of the germline
lineage formed in the female reproductive flower tissues, the ovule. The MMC is committed to
meiotic fate. After meiosis, typically only one reduced cell, the functional megaspore, survives
and ultimately gives rise to the mature female gametophyte harbouring the two female
gametes (egg cell, central cell). Subsequently embryo and endosperm development initiates
after double fertilization of the female gametes. In apomictic species at least two key
developmental steps are different: In the first cell of the apomictic germline, the apomictic
initial cell (AIC), meiosis is omitted or altered resulting in the development of an unreduced
gametophyte and the embryo develops by parthenogenesis from an unfertilized egg cell.
While the cell-type specific transcriptional basis underlying the development of the
Arabidopsis sexual MMC and female gametes has recently been described (1, 2, 3),
knowledge about the genetic basis and molecular mechanisms underlying apomictic germline
specification is still limiting.
To study the transcriptional basis underlying apomictic reproduction, we are using Boechera
gunnisoniana, a species closely related to Arabidopsis, as apomictic model plant. In this
species endosperm development requires fertilization of the central cell, while embryos are
formed from unreduced egg cells by parthenogenesis (98%) or after fertilization (2%, N = 84),
based on flow cytometry on individual seeds. As no reference genome is available for this
species to date, we generated a reference transcriptome based on RNA isolated from
microdissected ovule tissues using RNA-Seq. The assembled sequences were annotated by
Blast2GO and BLAT allowing functional categorization and identification of Arabidopsis
41
homologues. Transcriptome analysis of the Boechera g. germline cells was done by
microarray analysis and RNA-Seq. We identified 14’142, 15’156, and 12’860 genes with
evidence of expression in the apomictic initial cell, egg cell and central cell, respectively. For
selected genes, independent data confirmation was done by in situ hybridization, providing
good evidence for the accuracy of our dataset. Interestingly, gene ontology analysis of genes
expressed both in the sexual MMC and the apomictic initial cell suggests a conservation of a
number of functions, likely of general importance for initiation of the germline lineage. In
addition, functional classification of genes expressed only in the sexual Arabidopsis or
apomictic Boechera g. germline suggests a differential activity of a number of pathways,
including cell cycle, hormonal pathways, epigenetic regulation, and transcription factors.
Importantly, enriched activity of signal transduction pathways was identified as a feature of
the apomictic as compared to the sexual germline. In summary, our study gives important
new insights into the transcriptional basis underlying apomixis.
(1) Wuest SE, Vijverberg K, Schmidt A, Weiss M, Gheyselinck, J, Lohr M, Wellmer F,
Rahnenführer J, von Mering C, and Grossniklaus U (2010) Curr Biol 20: 506-512.
(2) Schmidt A, Wuest SE, Vijverberg K, Baroux C, Kleen D, and Grossniklaus U (2011)
PLoS Biol 9(9): e1001155.
(3)
Schmid MW , Schmidt A , Klostermeier UC , Barann M , Rosenstiel P , and
Grossniklaus U (2012) PLoS ONE 7(1): e29685.
42
Chair: Thomas Dresselhaus
Session VII: Gametophyte Development & Function
Abstract S20
Germline specification in male gametophyte development
1
D. Twell , M. Gherghiniou, M. Borg, N. Rutley, U. Sari
1
University of Leicester, Department of Biology, Leicester, UNITED KINGDOM
In recent years there have been major advances in understanding the ontogeny of the male
gametophyte generation of flowering plants which delivers the two sperm cells needed for
double fertilisation and seed development. A central outstanding question in this example of
‘micro-developmental patterning’ in plants is how cell polarity and asymmetric division of the
microspore are linked to the specification of the differential fate of the male germline and
vegetative cell. Mutants that disturb microspore division, together with the availability of new
cell-specific and chromatin markers have allowed the significance of cell isolation in the
establishment and maintenance of vegetative and germline fate to be examined. One
interesting finding is that elements of male germ cell fate do not strictly depend on cell
isolation, supporting the hypothesis that polarity associated ‘germ-plasm’ is operationally
linked to the initiation of germ cell fate. Once the discrete male germline is segregated at
asymmetric division, germ cell specification depends on the germline-specific transcription
factor DUO1 and downstream target genes that include zinc finger proteins (DAZ1 and DAZ2),
which ensure mitotic progression of the germ cell. We have started to explore the functional
conservation of DUO1 and DAZ1 and our results support a deep phylogenetic conservation of
the DUO1-DAZ regulatory network that determines the coordination of cell proliferation and
specification during angiosperm male germline development.
Abstract S21
NTP303 - the story of translation regulation in pollen
1,2
1
3,4
1
1
David Honys , Said Hafidh , David Potěšil , Katarína Breznenová , Pavel Bokvaj , Zbyněk
3,4
1
Zdráhal & Věra Čapková
1
Laboratory of Pollen Biology, Institute of experimental Botany ASCR, Prague, CZECH REPUBLIC
Department of Plant Experimental Biology, Charles University in Prague, Prague, CZECH REPUBLIC
3
CEITEC CEITEC - Central European Institute of Technology, Masaryk University, Brno, CZECH
4
REPUBLIC National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC
2
Male gametophyte, highly organized haploid flower organ, offers an unique chance to analyze
development and differentiation of single haploid cell, cell-cell interactions and recognition,
cellular polarity and pollen tube tip growth. Posttranscriptional control of gene expression
plays a vital role during tobacco pollen maturation and tube growth. NTP303 represents a
prominent example of translationally-regulated transcript encoding 69-kDa pollen tube cellwall glycoprotein. NTP303 and a number of other pollen genes showed apparent expression
discrepancy at mRNA and protein levels and their respective transcripts were shown to be
associated with long-term stored ribonucleoprotein particles annotated as EPP complexes.
Similarly to the role played in growing mammalian neurons, EPP particles represent preloaded complex machinery devoted to mRNA processing, transport, subcellular localization
and protein synthesis. Here we performed a detailed functional, transcriptomic and proteomic
characterisation of pollen storage RNP particles in tobacco (Nicotiana tabacum L.). In
particular, we aimed to integrate our knowledge on the categorization of translationally
regulated transcripts in developing pollen and to identify the mode of action of the
translational repression and derepression of mRNAs stored in developing pollen and
gradually activated during progamic phase.
43
Acknowledgement: The authors gratefully acknowledge the financial support from Czech
Science Foundation (P501/11/1462, P501/11/P321) and Czech Ministry of Education, Youth
and Sports (LD13049).
Abstract S22
Consequences of parallel development of meiotic and apomictic
pathways in aposporous plants
D. Hojsgaard
1
1
University of Göttingen, Department of Sytematic Botany, Göttingen, GERMANY
Gametophitic apomixis (apospory and diplospory) is characterized by formation of seeds via
an unreduced megagametophyte. In facultative and aposporous species, meiosis is not
avoided and aposporous embryo sacs arise from re-programmed nucellar cells. Hence, both
meiotic and apomictic reproductive pathways develop simultaneously, and compete to form a
seed. This developmental competition was evaluated in aposporous Paspalum
malacophyllum genotypes by using different methodological approaches and the relative
incidence of each meiotic or apomictic pathway along four different stages of the plant’s life
cycle –ie. beginning and end of gametogenesis, seed and offspring formation- was measured.
At embryological stages, frequencies of sexual and apomictic pathways show a high variation
as both reproductive pathways are unstable. By the end of gametogenesis apomixis is
stabilized; then in the last stages a clear decline in realized sexuality was observed and only
the apomictic pathway prevails. Results are discussed and bring new light on the functionality
and main factors modulating the penetrance of sexual reproduction and the low levels of
sexuality in natural populations of facultative apomictic species.
Abstract S23
Gene expression profiling of regulatory mutants controlling sperm cell
formation
1
2
Michael Borg , Jörg Becker & David Twell
1
1
Department of Biology, University of Leicester, Leicester, UK
2
Instituto Gulbenkian de Ciência, Oeiras, Portugal
In flowering plants, the male gametophyte plays a vital role in plant fertility through the
generation and delivery of two sperm cells to the embryo sac for double fertilisation. The male
germline is first established after division of the microspore and the small germ cell produced
goes on to differentiate and divide once again to produce two sperm cells. We have shown
previously that germ cell development is transcriptionally controlled by the male germlinespecific transcription factor DUO1. We have now shown that DUO1 directly activates a pair of
zinc finger proteins called DAZ1 and DAZ2, which together ensure progression of the germ
cell through mitosis. To explore the scale and specificity of the DUO1-DAZ1/2 network, we
used differentially expressed markers to isolate duo1 and daz1;daz2 mutant germ cells by
fluorescence-activated cell sorting (FACS) and profiled gene expression using microarrays.
This is the first report of the isolation and transcriptomic analysis of mutant germ cells in
plants. We will present our isolation strategy along with validation of our transcriptomic data,
which shows not only an abundance of down-regulated genes but significant numbers of upregulated genes. Deregulated chromatin-associated pathways offer a tantalising explanation
for the scale of observed gene expression changes. Our data provides compelling insight into
the importance of the DUO1-DAZ1/2 regulatory network in streamlining the male germline
transcriptome in flowering plants. This work was partially supported through a COST action
FA0903 Short Term Scientific Mission.
44
Abstract S24
Coordination of pollen tube growth by Ca2+ regulated anion fluxes
José A Feijó
Univ of Maryland, Cell Biol and Mol.Genetics Dept., 2134 Bioscience Research Building, College Park,
MD 20742-5815, USA, and Inst. Gulbenkian de Ciencia, Plant Biology, Oeiras, 2780-156, Portugal.
Pollen transcriptomics reveals the expression of of about 7.000 genes in pollen, but
theoretical modelling suggests that the cooperation of all of these into the processes of wall
surface and cytoplasmic volume production is a minimal condition to explain most of the
morphogenic events that characterize these cells. Spatial and temporal integration of
extended biochemical and biophysical processes is mandatory, and in the past we have
proposed that ion dynamics can be a common regulator of fundamental growth processes. I
will report on advances on the biology of Glutamate-Receptor Like Ca2+-channels. These
channels are hypothesized to participate on the generation of the Ca2+ focused gradient
characteristic of functional pollen tubes. I will also describe a new regulatory loop downstream
of the Ca2+ signal, based on the activation of specific Ca2+ dependent kinases (CPK) and
the regulation of the anion channel SLAH2. We have developed novel chloride (Cl-) sensing
genetic probes, and imaged for the first time the dynamics of the cytosolic concentration of
this ion. I will present data that allows the proposition of a feed-back between Cl- and Ca2+
as underlying the regulation of pollen tube growth.
Abstract S25
Transcriptional control of the double fertilization process: the role of
REM family
1
1
2
Marta Adelina Mendes , Rosalinda Guerra , Hugh Dickinson , Lucia Colombo
1
1
2
Dipartimento di Bioscienze, Università degli studi di Milano, Italy, Department of Plant Sciences,
University of Oxford, UK
In our lab we discover that the MADS-box protein domain complex formed by SEEDSTICK
(STK) and SEPPALATA3 (SEP3), known to control ovule identity, have as first direct target a
member of the REM family, VERDANDI.
VERDANDI (VDD, REM20) the first direct target of the MADS-box complex was shown to
have a very important role in the maintenance of the synergids function. Recently with the
combination of some Bioinformatical studies we were able to identify by ChIP-qPCR analysis
the second direct target of the MADS complex, REM11. Very interesting is the fact that this
gene belongs to the same family as the first target, but also that plays a similar role during the
development of the plant. We identify exactly the same defect in the REM11_RNAi mutant
line as shown before for vdd-1. The synergids in these mutants are still able to attract the
pollen tube but then are not able to degenerate and so the delivery of the sperm cells is
compromised. Additionally by backcrosses we saw that these mutants showed a maternal
defect. Plants in which REM11 was silenced by RNAi showed a phenotype similar to the one
described previously for the vdd mutant. Furthermore, by yeast two-hybrid assays we showed
that REM11 interact with VDD.
Using a RNA seq approach we have identified some putative target of the REM11-VDD
complex that might be involved in the last events of female-male gametophytic interaction.
45
Abstract S26
Ethylene regulates seed composition and development
Ronny Völz, Juliane Heydlauff, Thomas Nakel, Rita Gross-Hardt
Reproduction in flowering plants critically relies on a complex fertilization process involving
synergid-mediated pollen tube attraction, degeneration of the first synergid, and gamete
fusion. Successful double fertilization triggers programmed cell death of the second synergid,
thereby contributing to the establishment of a pollen tube block. We have recently shown that
the degeneration of the second synergid and the concomitant establishment of a pollen tube
block require a fertilization-dependent ethylene response cascade.
Here, the fate of the surviving synergid and potential developmental implications will be
discussed.
Abstract S27
Transcriptional regulation of egg cell specific genes in Arabidopsis
thaliana
1
3
1
3
Francesca Resentini , Philipp Cyprys , Piero Morandini , Stefanie Sprunck , Thomas
3
1-2
1
Dresselhaus , Lucia Colombo , Simona Masiero
1,
2,
Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano Italy, Istituto di Biofisica,
3,
CNR, Università di Milano, Italy, Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg,
Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany
In Angiosperms the gametophytes are composed of few haploid cells that develop within the
diploid sporophytic tissues of the flower sexual organs. The female gametophyte contains
seven cells among which the egg cell and the central cell (the two female gametes), which
respectively give rise to the embryo and the endosperm upon fertilization. Taking advantage
of the yeast one-hybrid strategy, a simple and fast protein-to-DNA interaction assay, we have
searched for transcription factors driving female gamete differentiation. We have chosen as
bait for our assay the EC1.1 gene, since it is exclusively expressed in the egg cell, the female
gamete, therefore the comprehension of its regulation can shed light into female gamete
differentiation. Our screenings indicated that EC1.1 promoter region is bound by SUF4
(SUPPRESSOR OF FRIGIDA 4), a C2H2 transcription factor. We have also been able to
show that SUF4 regulates EC1.1 also in in vivo since we could not detect pEC1.1:GUS
activity in suf4 egg cells. Coherently with its role, SUF4 is detected in mature embryo sacs
and real time PCR analyses pinpointed that all the 5 members of the EC1 family are
regulated by SUF4, since their expression level is strongly reduced in suf4 plants.
Using a bioinformatics approach we discovered that SUF4 is co-expressed with MOM1
(Morpheus’s Molecule1), a gene required for transcriptional gene silencing maintenance. RTPCR, real time PCR and promoter GUS line experiments indicate that MOM1 is also
expressed during ovule and embryo development. Interestingly in mom1 mutants, EC1.1
expression pattern is altered since it is also detected in developing carpel leaves.
The interactions among MOM1, SUF4 and EC1.1 will be presented and discussed.
46
Abstract S28
Real-Time Dynamics of DNA Methylation During Plant Reproduction
M. Ingouff, C. Michaud, T. Dumur, D. Grimanelli
Epigenetic regulations and Seed Development Group, University Montpellier II-IRD, France
DNA methylation is an key epigenetic mark in many organisms including plants, but remains
difficult to study dynamically with available tools, particularly for individual cells. This
generates important limitations when studying reproductive cells, which undergo very
dramatic and rapid epigenetic changes during gametogenesis, fertilization, and subsequently
early embryo development. We have developed transgenic lines in Arabidopsis that report
DNA methylation in living cells in a context-specific manner. The reporters take advantage of
methyl-binding domains specific to either CG or CHH methylation to target fluorescent
proteins to methylated cytosines on chromatin. They can be used to follow methylation
patterns in individual cells in real time, with very high temporal resolution. We used the
reporters to study reprogramming during male and female gamete formation in Arabidopsis.
The results suggest that DNA methylation patterns are much more dynamic that anticipated.
In addition, the reporters provide a powerful "readout" for genetic approaches. We used them
to screen for enzymes involved in either establishing or maintaining DNA methylation in the
germ cells.
47
Chair: Mary Gehring
Session VIII: Fertilization Mechanisms
Abstract S29
Nuclear Dynamics during Fertilization in Flowering Plants
Frederic Berger
Temasek Life Sciences Laboratory, Singapore (fred@tll.org.sg); Gregor Mendel Institute, Vienna,
Austria.
In most animal species, microtubules organized by centrosomes are essential for pronuclear
migration and subsequent fertilization events. By contrast, flowering plants lost genes
fundamental to centrosome generation during evolution.
To investigate cellular dynamics of female gametes during fertilization, we used fluorescent
markers to visualize cytoskeleton (F-actin and microtubules) in Arabidopsis female gametes.
We found that F-actin organizes as dynamic thick F-actin bundles that associate with the
sperm pronucleus during migration and we present additional mechanism important for this
process.
In addition, Genome wide profiling of H3 variants provides further insights in their impact on
transcriptional activity. From live observation of dynamics of chromatin during fertilization we
provide evidence for epigenetic reprogramming events linked to histone variants.
Abstract S30
Peptide signaling during pollen tube perception
1
1,2
3
2
3
Mayada Woriedh , Philipp Denninger , Christine Drübert , Guido Grossmann , Dirk Becker ,
1
Thomas Dresselhaus
1
Cell Biology and Plant Biochemistry, University of Regensburg, Germany
2
COS, University of Heidelberg, Germany
3
Molecular Plant Physiology, University of Würzburg, Germany
E-mail: thomas.dresselhaus@ur.de
Recent years have shown that proper cross-talk among gametophytic cells represents a key
to reproductive success in flowering plants. In addition to gametophytic interactions between
pollen tube and embryo sac cells, cell-cell-communication occurs inside both gametophytes
as well as between gametic cells during pollen tube burst. Here we will focus on
communication during pollen tube perception and discuss two classes of small cysteine-rich
proteins (CRPs) that are involved to induce pollen tube burst. Additionally, we will report on
possible roles of calcium signaling during this process. The defensin-like (DEFL) small protein
ZmES4 (Zea mays Embryo Sac4) was previously shown to induce pollen tube burst via
opening of the potassium channel KZM1 (Amien et al. 2010, PLoS Biol.). Here we show that
other members of the ZmES family are also capable to enhance the open probability of
potassium channels at physiological membrane potentials. Mutated proteins and short
peptides derived from various domains of ZmES4 were used to map active sites and channel
interaction domains. A second class of small CRPs secreted from the embryo sac encode
PME inhibitors (PMEI) that destabilize the pollen tube wall after external application
suggestion that they work in concert with DEFL-like toxins to induce pollen tube burst
(Woriedh et al. 2013, Plant Reprod.). Finally, we have studied the role of calcium during
pollen tube perception by monitoring its dynamics in the synergids, egg and central cell by
using a novel troponin-based biosensor.
48
Abstract S31
Unravelling the function and expression pattern of Arabinogalactan
Proteins in Arabidopsis thaliana reproductive tissues
1
2
1
1
3
Ana Marta Pereira , Simona Masiero , Sofia Nobre , Mário Costa , Stefanie Sprunck , Sílvia
1
Coimbra
1
2
Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Portugal, Dipartimento
3
di Bioscienze, Universitá degli Studi di Milano, Italy, Cell Biology & Plant Biochemistry, University of
Regensburg, Germany
Arabinogalactan Proteins (AGPs) undergoseveral post-translation modifications. The cDNAs
encoding the protein backbones of AGPs show a characteristic domain structure consisting of
an N-terminal secretion signal, absent from the mature protein, a central domain rich in
proline/hydroxyproline, alanine, serine and threonine, followed by a C-terminal signal
sequence for GPI-anchor attachment. Mature AGPs are therefore extensively glycosylated
and predicted to be attached by a GPI anchor to the plasma membrane (figure 1). The GPI
anchor can be cleaved by specific phospholipases, releasing the polypeptide into the
extracellular matrix in a regulated manner, suggesting that they might play signaling roles
(figure 1). Coimbra et al. in 2007 have shown that AGPs can be used as molecular markers
for reproductive development, showing that they are present in pistil tissues, especially along
the pathway followed by the pollen tube during its guidance to reach the egg cell inside the
embryo sac of the female gametophyte (figure 2). One of the purposes of this work is to study
the AGPs present in these tissues, as well as to unravel their roles along this path. Based on
microarray data available on-line (Genevestigator, Arabidopsis eFP Browser), and later, on
gametophytic cells microarray data (Stefanie Sprunck unpublished data, Wuest, S.E. et al.,
2010) AGP4 was one of the AGPs selected for further analysis and functional
characterization. Here we show some of the first results regarding the study of the agp4
mutant.
Coimbra, S. et al. (2007) J. Exp. Bot., 58-16, 4027-4035.
Wuest et al. (2010) Curr. Biol., 20, 506-512.
Abstract S32
The Arabidopsis LARP6c protein is an RNA binding factor specific to
mature pollen and required for proper male transmission
1,2
C.Bousquet-Antonelli ,E.Billey
1
1,2
1,2
1,2
, V.Jean , JJ Favory & JM Deragon
1,2
2
University of Perpignan, Laboratory of Plant Genome and Development, Perpignan, FRANCE CNRS,
Laboratory of Plant Genome and Development, Perpignan, FRANCE
RNA Binding Proteins (RBP) are key regulators of the mRNA metabolism involved in the
processing, nucleo-cytoplasmic shuttling, sub-cytoplasmic distribution, translation and stability
control of their targets. They can act as general regulatory factors such as the Poly(A) Binding
Protein (PABP) which by covering the mRNAs 3’-poly(A) tail stimulates their translation while
protecting them from decay. Alternatively RBPs can be specific factors devoted to the
regulation of few or a group of transcripts sharing specific cis elements. The La-Module is a
bipartite RNA binding domain composed of the highly conserved La-Motif (LAM) immediately
followed by an RNA Recognition Motif (RRM) of canonical or non canonical type. La-Modules
are found on several hundreds of eukaryotic proteins known as LaRPs (La-Related Proteins).
LaRPs can be classified into 4 sub-families (LARP1, 4, 6 and 7) which members share
evolutionary and structural features. In Arabidopsis thaliana we found that consistently with
previously published transcriptomic analyses, the LARP6c protein is exclusively expressed in
tricellular/mature pollen. Its loss of function specifically induces a decrease in male
transmission efficiency while female transmission is unaffected. Subcellular localization
studies show that the LARP6c protein forms aggregates in the vegetative cell cytoplasm, is
located in the vegetative nucleus and appears to label the periphery of the sperm cells and
49
the extension connecting the sperm cells to the vegetative nucleus. In vitro analyses allowed
to demonstrate the LARP6c has the ability to directly interact with the plant poly(A) Binding
Protein and displays RNA binding properties. We postulate that the LARP6c proteins is a
pollen specific mRNA binding factors involved in the translation and/or stability control of
transcripts sharing a cis LARP6c binding box.
Abstract S33
The role of STK-ABS complex in the double fertilization process
Marta Adelina Mendes and Lucia Colombo
Università degli Studi di Milano, Dipartimento di Bioscienze
The double fertilization process is one of the most essential processes for the development of
the next generation in plants. This process can be resumed to an interaction between the
male and female gametophytes. The male gametophyte, pollen grain, hydrates on the papillar
cells of the stigma, germinates, and forms a pollen tube in which the two male gametes are
transported to their destination, the female gametophyte. The female gametophyte on its turn
is composed by seven distinct cells: in the micropylar zone of the ovule there are two
synergids, close by the egg cell more in the center the central cell (diploid) and in the calazal
part three antipodals.
The synergids play a major role in the fertilization process, since they are responsible for the
attraction and reception of the pollen tube. When the pollen tube arrives one of the synergid
cells initiates to degenerate the pollen tube which arrests its growth, bursts, and releases the
two sperm cells to ensure double fertilization.
VERDANDI (VDD, REM20) the first direct target of the STK-SEP3 MADS-box complex was
shown to have a very important role in the maintenance of synergid function (identity).
Recently, by combining bioinformatics studies with ChIP-qPCR analysis, the second direct
target of the STK-SEP3 MADS complex, REM11 was identified. Very interesting is the fact
that this gene belongs to the same family as the first target, but also that it plays a very similar
role during the development of the plant. We observed exactly the same phenotype in
REM11_RNAi mutant lines as shown before for the vdd-1 mutant. The synergids in these
mutants are still able to attract the pollen tube but subsequently they are not able to
degenerate and therefore the delivery of the sperm cells is compromised. Additionally by
backcrosses we observed that these mutants showed only a maternal defect. Interestingly,
the genes responsible for the attraction of the pollen tube, like the transcription factor gene
MYB98, are correctly expressed in the mutants and another subset of genes are
missexpressed as we discover with an RNA-seq analysis. Two very different processes,
pollen tube attraction and reception of the gametes, are controlled by just one type of female
cells. VDD-REM11 regulate the reception of the gametes. Furthermore, yeast-2-hybrid
interaction studies demonstrated that these two REM factors are able to interact with other
REMs, which opens a new door to further studies.
Abstract S34
Acquisition of LURE-Reception Ability at the Pollen Tube Tip of Torenia
fournieri
1
S. Okuda , T. Suzuki
1
2, 3
4
3
3
, H. Mori , M. M. Kanaoka , N. Sasaki & T. Higashiyama
1, 2, 3
2
Nagoya University, Institute of Transformative Bio-Molecules, Nagoya, JAPAN JST, ERATO,
3
Higashiyama Live-Holonics Project, Nagoya, JAPAN Nagoya University, Division of Biological Science,
4
Nagoya, JAPAN Nagoya University, Department of Bioengineering Science, Nagoya, JAPAN
During fertilization in flowering plants, chemo-attractants from the synergid cells have been
thought to be key molecules in pollen tube guidance. We have identified defensin-like peptide
50
LUREs as attractant peptides (Okuda, Tsutsui et al., Nature, 2009). Here, we show that
pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent
manner to receive and respond to attractant LURE peptides (Okuda et al., Molecular Plant,
2013). We found that LURE peptides bound specifically to the tip of pollen tubes growing
through a cut style. The peptides also bound to pollen tubes growing through a shorter style,
which were not competent to respond to these peptides. These observations suggested a
possibility that acquisition of the LURE peptide reception ability and acquisition of full
competency are separable processes.
51
Chair: Emidio Albertini
Session V: Embryogenesis & Parthenogenesis
Abstract S35
APOSTART: a candidate gene involved in embryo progression and
parthenogenesis
1
2
3
2
2
3
G. Marconi , F. Resentini , A. Marrone , S. Masiero , L. Colombo , L. Storchi , E. Albertini
1
1
Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121
2
3
Perugia; Dipartimento di Bioscenze, Università di Milano, Via Celoria 26, 20133 Milano; Dipartimento
di Farmacia, Università “G d'Annunzio” di Chieti-Pescara, Via dei Vestini 31, 66100 Chieti
Seed is the key factor of crop productivity. The commercial success of a newly selected
cultivar depends not only on its vegetative attributes but also on its ability to produce seeds.
Breeding for seed yield and quality requires new sophisticated technologies, such as
apomixis, that will allow overcoming the conventional breeding limits. Apomixis is a naturally
occurring mode of asexual reproduction in flowering plants, resulting in embryo formation
without meiosis or fertilization of the egg. Seed derived progenies are genetically identical to
the maternal parent. In crop species, apomixis would enable the instantaneous fixation of the
complete genome of the best plants.
In Poa pratensis we have isolated a gene, termed APOSTART (Albertini et al. 2005, Plant
Phys 138:2185-2199). Our previous results demonstrate that some APOSTART members are
expressed exclusively in inflorescences and, overall, ore results suggest that APOSTART
may be related to the programmed cell death that is involved in the non-functional megaspore
and nucellar cell degeneration events that permit enlargement of maturing embryo sacs.
In addition, to strengthen the hypothesis of an involvement of APOSTART in apomixis, we
have isolated APOSTART members from two other aposporic species. Our results, showing
that at least one APOSTART member/allele is expressed differentially in all species, shed
light on the possible role of APOSTART in apomixis. To better understand this function we
are characterizing Arabidopsis thaliana APOSTART members. In particular, PpAPO1 shares
high homology with the Arabidopsis protein At5G45560, thus renamed AtAPOSTART1
(AtAPO1), and with with EDR2 (Enhanced Disease Resistance 2). Tang and co-workers
proved that EDR2 disruption enhances Arabidopsis capacities to resist to E. cichoracearum
infections (Tang et al. 2005; Vorwerk et al., 2010), edr2 homozygous plants does not show
any developmental defects and are indistinguishable from the wild type ones, whilst AtAPO1
down-regulation affects negatively seed germination. In order to verify if AtAPO1 and EDR2
have additive or redundant roles we generated and analyzed the atapo1-2/edr2 double
mutants. Double mutants plants appear smaller than the two parental lines, interestingly also
the double mutant developing siliques are smaller. Manual dissection of double mutant silique
show that also seed development is compromised in atapo1-2/edr2. Microscopic analyses
show that all the seeds contain embryos, 30% of which show a delayed or arrested
development. Moreover, by using a computational approach we have determined the tridimensional structure of the START domain with the aim of investigating the possible
interactions between the START domain and one or more phytosterols (i.e. stigmasterol,
campesterol, brassicasterol) which are believed to be important during the embryogenesis.
These data will showed and critically discussed.
52
Abstract S36
Epigenetic Regulation of Haploid Embryo Development
Hui Li, Mercedes Soriano, Gerco C. Angenent and Kim Boutilier
Plant Research International, e-mail: kim.boutilier@wur.nl.
The haploid multicellular male gametophyte of plants, the pollen grain, is a terminally
differentiated structure whose function ends at fertilization. Unlike pollen grains, the immature
gametophyte retains its capacity for totipotent growth when cultured in vitro. Haploid embryo
production from cultured immature male gametophytes is a widely used plant breeding and
propagation technique that was described nearly 50 years ago, but one that is poorly
understood at the mechanistic level. We have used a chemical genomics approach to
determine the role of epigenetic modifications in the establishment of cell totipotency from the
male gametophyte in Brassica napus and arabidopsis. The results of this screen will be
presented.
Abstract S37
Auxin dependent patterning is mediated by distinct ARF/bHLH modules
Tatyana Radoeva, Cristina Llavata-Peris, Annemarie Lokerse, Jos Wendrich & Dolf Weijers
Wageningen University, Laboratory of Biochemistry, Wageningen, The NETHERLANDS
The plant hormone auxin is required for many aspects of plant growth and development.
Recently, it was found that inhibition of auxin response in suspensor cells induces
transformation of suspensor cells to embryonic cells (1). Using a transcriptomics approach, a
small set of bHLH genes was identified that are: a) upregulated upon auxin response
inhibition and normally expressed in the proembryo or b) downregulated upon auxin inhibition
and normally expressed in the suspensor. Detailed investigation of gain-of-function and lossof-function mutants of these genes strongly suggests that their function is required for
controlling normal suspensor and hypophysis development. Strikingly, ectopic expression of
one of these is by itself sufficient for inducing embryo-like structures in suspensor cells.
Moreover, we show that these genes act downstream of auxin signaling, and proper auxin
signaling is indispensable for the regulation of these genes. Hence, this work identifies a
novel ARF-bHLH module that operates in parallel to the previously identified ARF5/MPTMO5/TMO7 module that operates in patterning the embryo (2,3). We conclude that auxinARF-bHLH modules are conserved elements that mediate auxin-dependent plant
development.
References:
(1) Rademacher, E. H., A. S. Lokerse, A. Schlereth, C. I. Llavata-Peris, M. Bayer, M. Kientz, A.
Freire Rios, J. W. Borst, W. Lukowitz, G. Jurgens and D. Weijers (2012). "Different auxin
response machineries control distinct cell fates in the early plant embryo." Dev Cell 22(1): 211222
(2) Schlereth, A., B. Moller, W. Liu, M. Kientz, J. Flipse, E. H. Rademacher, M. Schmid, G.
Jurgens and D. Weijers (2010). "MONOPTEROS controls embryonic root initiation by
regulating a mobile transcription factor." Nature 464(7290): 913-916.
(3) De Rybel, B., B. Moller, S. Yoshida, I. Grabowicz, P. Barbier de Reuille, S. Boeren, R. S. Smith,
J. W. Borst and D. Weijers (2013). "A bHLH complex controls embryonic vascular tissue
establishment and indeterminate growth in Arabidopsis." Dev Cell 24(4): 426-437.
53
Abstract S38
Hormonal balance in triticale (×Triticosecale Wittm.) anthers and its
effect on androgenesis effectiveness
I. Żur, E. Dubas, M. Krzewska, F. Janowiak, P. Waligórski, M. Dziurka
The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków, POLAND
Instant production of totally homozygous doubled-haploid (DH) plants through the process of
androgenesis is highly appreciated both from practical (breeding) and research perspective.
Unfortunately, in many plant species, including triticale, the efficiency of the process is still not
satisfactory. Many attempts have been undertaken to understand the molecular and
physiological background of androgenesis and improve the yield of DH plants . In order to
evaluate the effect of hormonal balance in triticale androgenesis, the present study examined
the endogenous content of plant hormones (auxins, cytokinins, and ABA) in anthers of ten DH
lines which differed significantly in androgenic responsiveness.
Five highly responsive (HR) and five recalcitrant (RC) genotypes were selected from the
population of ‘Saka 3006’ × cv. ‘Modus’ using the anther culture method described by
Wedzony (2003). The measurements were performed in anthers excised from freshly cut
tillers at the moment optimal for androgenesis induction and in anthers excised from lowtemperature (LT) treated tillers (3 weeks at 4°C), in which microspores switched toward
androgenic development. Standard HPLC protocols were used for the determination of
endogenous content of auxins and cytokinins (Dobrev and Kamínek 2002; Stefancic et al.
2007). ABA measurements were conducted by indirect enzyme-linked immunosorbent assay
(ELISA) according to Walker-Simmons and Abrams (1991).
It was revealed that both the genotype of the donor plant and LT androgenesis-induction
treatment had significant effect on the content of endogenous hormones. Moreover,
significant differences were observed between the groups of HR and RC DH lines. In
addition, an almost 3-fold increase in ABA content, LT enhanced accumulation of IAA (at
28%) and IBA (at 62%) were detected, as well as an almost 2-fold increase in cis isomers of
zeatin (cisZ) and 57%-increase in zeatin ryboside (cisZR) levels. At the same time the
concentration of transZ isomers and its derivative transZR decreased significantly (86% and
20%, respectively). It was also revealed that after LT treatment, anthers of HR DH lines were
characterized by significantly lower IAA as well as higher IBA, transZ, and ABA content in
comparison to RC ones. It seems that such variation in endogenous hormones balance could
be at least one of the reasons of different androgenesis responsiveness.
Dobrev P, Kamínek M 2002. J Chromatogr A. 950(1-2): 21-9.
Stefancic M, Stampar F, Veberic R, Osterc G 2007. Sci. Hort. 112: 399–405
Walker-Simmons MK, Abrams SR 1991. In: Davies WJ, Jones HG (red) Abscisic acid, physiology and
biochemistry. Bios Scientific Publishers, Oxford, str. 53-63.
Wędzony M 2003. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled Haploid
Production in Crop Plants. A Manual. Kluwer Acad. Publ. Dordrecht/Boston/London, pp 123-128.
Abstract S39
A molecular framework for somatic embryogenesis induction in
Arabidopsis.
M. Soriano, M. Weemen, Cheryl Phillipsen, R. Offringa and Boutilier K.
Plant Research International. kim.boutilier@wur.nl
Differentiated somatic plant tissues can be induced to produce embryos in vitro. In the model
species Arabidopsis thaliana, this process can be induced in immature zygotic embryos (IZEs)
by the application of the synthetic auxin 2,4-D. The molecular mechanism that leads to the
initiation of somatic embryogenesis is still largely unknown. In order to characterize the
54
sequence of molecular events that lead to embryo induction we are using a collection of
fluorescent reporters that mark the onset of embryo identity (LEC1, WOX2), together with
reporters of auxin response (DR5, DII), cytokinin response (TCS), and root and shoot
meristem activities (WOX5, WUS). Our results show that root and shoot meristem-like
developmental programs are activated in internal tissue regions (procambium) in which there
is proliferative growth, and that expression of the shoot and root meristem programs precedes
the onset of embryo identity. These inner regions of the explant show a strong cytokinin
signalling response. Embryo identity is initiated later, in the outer cell layer(s) (L1/L2?) in the
proximity of the shoot meristem and on the adaxial side of the cotyledons. Auxin signalling is
restricted to the outer cell layers of the explant, but does not coincide with the sites of embryo
formation. We are investigating further the roles of the auxin/cytokinin signalling environment
and the establishment of meristem identity during the developmental switch towards
embryogenesis.
Chair: Claudia Köhler
Session IX: Seed & Fruit Development
Abstract S40
Dissecting Gene Regulatory Networks
That Control Seed Development in Arabidopsis
Kelemen Z., Berger N., Barthole G., Marchive C., Fiume E., Nikovics K., Boulard C., Thévenin
J., To A., Grain D., Dubos C., Miquel M., Baud S., Dubreucq B., and Lepiniec L.
IJPB, UMR 1318 INRA-AgroParisTech, ERL CNRS 3559, Saclay Plant Sciences (SPS), Route de SaintCyr, 78026 Versailles, France.
A network of transcriptional regulators that control seed maturation has been characterized
and named “LAFL” according to the first partners characterized (i.e. LEC1, ABI3, FUS3, and
LEC2). LEC2 (LEAFY COTYLEDON 2), FUS3 (FUSCA3), and ABI3 (ABSCISIC
INSENSITIVE 3) encode related transcriptional regulators of the “B3”-domain family. LEC1
belongs to a different class of proteins homologous to HAP3 subunits of the CAAT Boxbinding Factors (CBFs). Closely related genes have been described in the other plants
studied (dicots as well as monocots).
The key role of the LAFL genes in seed development is well established. Mutations of these
genes lead to partially overlapping and pleiotropic abnormal embryo phenotypes such as
abnormal suspensor, precocious cell cycle activation and growth of apical and root meristems,
“leafy” cotyledons (accumulating more chlorophyll and/or anthocyanins and lower levels of
storage compounds) and seeds are less tolerant to desiccation and/or display precocious
germination.
The AFL-B3 proteins (i.e. LEC2, FUS3 and ABI3) can directly trigger the expression of
structural genes encoding for instance seed storage proteins (SSP) and proteins of the oil
bodies (e.g. oleosins), by interacting with RY DNA-motives (CATGCA) of the target promoters.
Nevertheless, other important regulators have been identified including various bZIPs
proteins (e.g. ABI5 or EEL), and additional regulatory cis-elements like G boxes (CACGTG)
are usually required to confer the proper expression pattern of target genes. Transcriptional
activation of the fatty acid biosynthetic network appears to involve additional factors such as
WRINKLED 1 (WRI1), a member of the AP2-ethylene response element binding factor family.
Interestingly, WRI1 specifies the regulatory action of LEC2, and possibly other master
regulators, in this metabolic pathway.
The LAFL proteins are involved in an intricate self-regulated network of local and partially
redundant functions, interacting each other and with other factors (such as AGL15, a MADS-
55
box factor), as well as with metabolic and hormonal signalling. LEC1 and LEC2 are
expressed specifically, early and transiently during embryo development. Interestingly, the
ectopic expression of LEC1 or LEC2 is sufficient to trigger the abnormal formation of embryos
on vegetative tissues. Therefore, a tight control of their expression is of paramount
importance for plant life cycle. Consistent with this idea, LAFL genes expression is repressed
during vegetative development through a variety of pathways involving chromatin
modifications, Polycomb related proteins, and/or the VAL/HSI family of transcription factors
(that also contain B3 domain).
For instance, the expression of LEC2 is negatively regulated in vegetative tissues by
Polycomb Repressive Complex2 (PRC2) that catalyses histone H3 Lys 27 trimethylation
(H3K27me3) and plays a crucial role in developmental phase transitions. Two cis-activating
elements and a cis-repressing element (RLE) that is required for H3K27me3 marking were
characterized in the promoter of LEC2. Nevertheless, how LAFL genes are regulated and
control the expression of their targets genes in specific chromatin contexts, during the
different transitions of embryo development, remains to be elucidated.
Abstract S41
Alurone cell developmental programing is surface dependant and relies
on Dek1, a member of the ancient TML-calpain gene family.
1
2
3
1
4
Odd-Arne Olsen , Wenche Johansen , Pierre-Francois Perroud , Zhe Liang , Sen Zhao ,
2
4
1
Robert C. Wilson , Kamran Shalchian-Tabrizi and Viktor Demko .
1
2
Norwegian University of Life Sciences, As, N-1432, Norway, Department of Natural Science and
4
Technology, Hedmark University College, Hamar N-2318, Norway, Department of Biology, Washington
5
University in St Louis, Campus Box 1137, St Louis, MO 63130, USA, Microbial Evolution Research
Group, Department of Biology, University of Oslo, Oslo N-0136, Norway.
Insight into the cell biology and developmental programing of nuclear endosperm has
increased considerably over the past 20 years. Follwing the second event of double
fertilization, the central cell of maize develops through the syncytial stage before cellularizing
around 4 days after fertilization. Based on observations from maize endosperm in vitro organ
cultures, the endosperm developmental program has already been set, and is executed
largely without maternal tissue input. Tissue differentiation ensues according to a simple
program; default identity of the endosperm is the starchy endosperm cell fate. This fate is
overridden in cells that are at the surface of the endosperm, which become aleurone cells.
Lastly, in species with transfer cells, peripheral cells in the interface between the maternal
vascular tissue and the surface layers of the endosperm assumes the transfer cell fate via by
maternal signalling. We and others have shown earlier that surface dependant aleurone cell
fate determination depends on the Dek1 gene. Surprisingly, the DEK1 protein plays an
essential role in epidermis cell fate determination in all tissues of angiosperms, and may have
played an essential role in the evolutionary transition from single celled green algae to
multicellular land plants. Recent data on the function of DEK1 in the moss Physcomitrella
patens will be presented and discussed in the context of endosperm development.
Abstract S42
The interaction between CUC genes and the hormonal network
controlling ovule numbers.
1
1
2
2
Mara Cucinotta , Francesca Galbiati , Candela Cuesta , Eva Benkova and Lucia Colombo
1
1
2
Dipartimento di BioScienze, Università di Milano, Via Celoria 26, 20133 Milano, Institute of Science
and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria.
The pistil, the female reproductive structure of the flower, contains ovules that develop into
seeds after fertilization. The ovule primordia emerge as lateral organs from a meristematic
tissue within the carpel referred to as placenta. It is of great importance to understand the
56
mechanisms that control ovule numbers as they ultimately determine the final number of
seeds and, thereby, the yield in seed-crop plants.
In Arabidopsis thaliana the number of ovules that develop from the placenta is controlled by
CUP SHAPED COTILEDON1 (CUC1) and CUC2, two transcription factors belonging to the
NAM family. The cuc2 pSTK::RNAi-CUC1 lines in which both genes are silenced a significant
decrease in ovule numbers is observed. The plant hormones cytokinin and auxin have shown
to play fundamental roles during ovule development. In fact plants with a reduction in
cytokinin biosynthesis or perception show a drastic reduction in ovules numbers. A similar
phenotype is observed, when auxin biosynthesis or transport were compromised.
A connection between CUCs and auxin has recently been proposed for apical patterning
during embryogenesis and for the formation of leaf serration. In our lab we found that CUC
genes regulate PIN1 expression and localization in ovule primordia since in the cuc2
pSTK::RNAi-CUC1, PIN1 was down-regulated and the PIN1 protein was not correctly
localized at the cell membrane. The main goal of my project is to clarify how genetic and
hormonal pathways are related during ovule formation, focusing on the central role of the
CUC1 and CUC2 genes in this process. For this purpose a transcriptome analysis by RNAdeep-sequencing has been performed (comparing WT with cuc2 pSTK::RNAi-CUC1 RNA
extracted from pre-fertilization inflorescences) in order to identify genes that are putatively
regulated by CUC1 and CUC2 and that are connected to cytokinin and auxin metabolism and
signalling. Some interesting candidate genes have been selected and experiment will be
performed to confirm and study their involvement in the molecular and hormonal network
responsible for the determination of ovule numbers.
Abstract S43
Rapid post-fertilization auxin responses, which activate fruit initiation,
are
potentiated by a PINOID/AGC kinase to BDL/IAA12 phosphorylation
pathway
1,2
2
2
2
2
A. Vivian-Smith , F. Maraschin , M. Kemel-Zago , W. Helvensteyn , J. Memelink , & R.
2
Offringa
1
Present address: Norwegian Forest and Landscape Institute/Bioforsk, Høgskoleveien 8, 1430 Ås,
2
NORWAY, IBL, Molecular and Developmental Genetics, Leiden University, Leiden, THE
NETHERLANDS
Participation of the AUXIN RESPONSE FACTOR8 (ARF8) gene in the control of fruit initiation
implicates auxin as a crucial player in post-fertilization signaling. We demonstrate that
Arabidopsis has pre-fertilization stages where equilibriums exist in the ovule for both a
background but nominal auxin-related transcriptional activation and for polar auxin transport
(PAT) which prevents fruit set. Transcriptional activation thresholds are partially set by
communication with other floral tissues, like anthers which coordinate pollination, and now
several mutations which disrupt the pathway have been characterized. Under normal
circumstances fertilization activates concomitant ARF8 transcript clearance and an abrupt
ovule auxin response (AR) occurs in the integuments and chalaza 1-2 h postfertilization, in
tandem with the first nuclear endosperm division. By the next division, the AR strengthens
and fruit growth is observed at 5 h. There is a complete dependency for the degradation of
both BDL/IAA12 and SLR/IAA14 repressor proteins for the transmission of the AR to the
carpel and for the differentiation of the ovule-carpel vascular network. We show that the
potentiation of the post-fertilization auxin response, together with fruit initiation, occurs by
phosphorylation of a key motif on BDL/IAA12 directed by the PINOID protein kinase in L2 or
TIR1
interaction domain, and that the bdl-1/+
L3 cell layers. Proximity of the motif to the SCF
phenotype is ameliorated when the motif is removed, indicates this specific motif is a key
determinant of Aux/IAA protein activity and ARF activation threshold, and has special
relevance for angiosperm reproduction and the canalization of auxin responses.
57
Poster abstracts
58
Poster index:
Evolution of Reproductive Systems
Abstract P01
Clément Lafon-PlacetteThe postzygotic hybridization barrier between diploid A. arenosa and A. lyrata
Polyploidy & Aneuploidy
Abstract P02
Stefanie HilpertThe influence of ploidy variation on apomixis penetrance and gene expression in Poa
pratensis
Abstract P03
Hua JiangGenetic analysis of postzygotic hybridization barriers
Apomixis & Emerging Technologies
Abstract P04
Viviana EcheniqueExpression profiles of methyltransferases and RdDM proteins during sexual and apomictic
reproduction in Eragrostis curvula
Abstract P05
Dorota PaczesniakMaking bigger seeds to increase agricultural yield: the effects of paternal contribution and
sexual conflict on variable endosperm development in naturally-occurring asexual
(apomictic) plants
Abstract P06
Paride RizzoConnecting ploidy with female gametophyte development of the two apomicts Boechera spp.
and Hypericum perforatum
Endosperm & Imprinting
Abstract P07
Philip WolffGenomic Imprinting in Arabidopsis thaliana
Abstract P08
Aslıhan ÖzbilenIn silico and molecular characterization of some paternally expressed imprinted genes in
sexual and apomict boechera species
Abstract P09
Ida Marie JohannessenAGAMOUS-LIKE imprinting and regulation in Arabidopsis
Plant Reproduction & Environmental Change
Abstract P10
Åshild ErgonRelationships between induction of flowering, cold acclimation, deacclimation and
reacclimation in a perennial forage grass species
Abstract P11
Katalin JägerTissue-specific expression of the TaVRN1 transcription factor during vegetative-togenerative transition in wheat (Triticum aestivum L.)
Abstract P12
Georgina KosturkovaRegenerated In Vitro Soybean Plants With Improved Productivity
Abstract P13
Lu LiuFTIP1 Is an Essential Regulator Required for Florigen Transport
Abstract P14
Geeta PrasadInvestigating the relationship between substrates of the N-end rule Pathway and genes
regulated by EBP binding sites (GCCGCC) in Arabidopsis thaliana
Abstract P15
Oziniel RuzvidzoThe Arabidopsis thaliana Pentatricopeptide Repeat Protein (AtPPR) Contains a Domain that
Functions as an Adenylate Cyclase
Abstract P16
Anjar WibowoEpigenetic Adaptation to High Salinity in Plants
Meiosis & Apomeiosis
Abstract P17
Fatih SezerIsolation and characterization of dyad gene orthologs from boechera species
Gametophyte Development & Function
Abstract P18
Vicente BalanzàLooking for new insights in ovule primordia and femalegametophyte specification
Abstract P19
Antonia GibalováCharacterization of transcription regulators acting during male gametophyte development
Abstract P20
Barbara GlöckleDissecting cell-cycle and -differentiation in flowering plant gamete formation
Abstract P21
David KawadzaMolecular characterization and the elucidation of the physiological roles of a novel maternal
effect embryo arrest protein from Arabidopsis thaliana
Abstract P22
María Noel LuccaMolecular and functional characterization of POLLEN SPECIFIC KINASE1-4 genes (PSK1-4)
in Arabidopsis thaliana
Abstract P23
Nicholas RutleyFunctional Characterisation of a Novel Pair of Male Germline Proteins in Arabidopsis
Abstract P24
Francesca TedeschiNovel mutant alleles of the RKD transcription factor gene family of Arabidopsis
59
Abstract P25
Abstract P26
Abstract P27
Dieu VoThe role of core-spliceosomal components during cell specification in the female
gametophyte
Daniela Muñoz-StraleIdentification of pollen specific promoters from Arabidopsis thaliana and their use to
generate male sterility
Maura CardarelliAuxin and jasmonic acid interaction in the control of anther dehiscence process in
Arabidopsis
Fertilization Mechanisms
Abstract P28
Thomas HackenbergCell surface proteins mediating gamete interaction in Arabidopsis
Embryogenesis & Parthenogenesis
Abstract P29
Maria Antonietta GermanàAdvancement on isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. Monreal
Rosso and Nules
Marines Marli Gniech
Abstract P30
KarasawaStudy on anther culture in hazelnut (Corylus avellana L.)
Abstract P31
Maria-Pilar Vallésn-Butanol - induces bread wheat microspore embryogenesis by microtubules
depolymerization
Abstract P32
Attila FábiánEffects of n-Butanol on Maize Anther Culture - Androgenic Response and Cytological
Aspects
Abstract P33
Ewa DubasEndogenous auxin and ABA in microspore embryogenesis of oilseed rape (Brassica napus
L.)
Abstract P34
Anneke HorstmanAINTEGUMENTA-LIKE and HOMEODOMAIN GLABROUS transcription factors have
antagonistic functions in the control of cell proliferation
Seed & Fruit Development
Abstract P35
Katrine BjerkanWDR55 interacts with DDB1 and is required for apical patterning in the Arabidopsis embryo
Abstract P36
Ida Myhrer StøIDA and IDL peptides in plant reproduction
Abstract P37
Dario PaoloSeed size regulation in Arabidopsis thaliana
Abstract P38
Carolin Anna RebernigGenetic basis of postzygotic hybridization barriers in Capsell
Abstract P39
Bo SunTiming mechanism by cell cycle-dependent polycomb eviction in plant stem cells
Abstract P40
Tatyana Radoeva
Auxin dependent patterning is mediated by distinct ARF/bHLH modules
60
Abstract P01
The postzygotic hybridization barrier between diploid A. arenosa and A.
lyrata
1
1
C. Lafon-Placette , C. Rebernig & C. Köhler
1
1
Swedish University of Agricultural Sciences and Linnean Center of Plant Biology, Uppsala BioCenter,
Department of Plant Biology and Forest Genetics, Uppsala, SWEDEN
Arabidopsis lyrata and Arabidopsis arenosa diverged relatively recently (2 MYA). Both
species exist as diploid or tetraploid plants. There is a natural hybridization zone in central
Europe between both species that is thought to occur between tetraploid plants or even in an
interploidy manner, with a unidirectional gene flow from A. arenosa (♂) to A. lyrata (♀).
However, in the same geographical region, diploid plants of both species are strongly
genetically isolated. While a prezygotic hybridization barrier can’t be excluded, the occurrence
of gene flow between tetraploid plants in the same region suggests a postzygotic
hybridization barrier between diploid A. arenosa and A. lyrata. We will test this hypothesis by
characterizing hybrid seeds at the morphological level. For this purpose, we performed
reciprocal crosses between diploid A. arenosa and A. lyrata and compared the development
of hybrid seeds with intraspecies seeds. Our results suggest that there is a strong postzygotic
hybridization barrier between both species that depends on the direction of the cross. We will
present initial data on the characterization of this hybridization barrier.
Abstract P02
The influence of ploidy variation on apomixis penetrance and gene
expression in Poa pratensis
1
2
2
S. Hilpert , M. Bocchini , E. Albertini & T. Sharbel
1
1
2
IPK Gatersleben, Dept. of Cytogenetics and Genome Analyses, Gatersleben, GERMANY University of
Perugia, Department of Applied Biology, Perugia, ITALY
The important forage and turf grass Poa pratensis is characterized by versatile modes of
reproduction, varying from completely sexual to obligate aposporous apomictic. Because
apomixis appears most frequently in polyploids, P. pratensis flexibility in reproduction might
be correlated with ploidy variation.
123 Poa pratensis accessions from 29 different countries are being genotyped using
microsatellites to assess genetic diversity and Flow Cytometric Seed Screening is being used
to quantify apomixis penetrance. The ploidy levels of these accessions are additionally being
estimated using flow cytometry and verified by karyotyping of root tip cells. Ovules and
anthers in the post-meiotic stage of 12 Poa pratensis individuals with ploidy levels ranging
from 5x to 13x were live microdissected and a genome wide comparative gene expression
study will be performed using a Poa-specific custom designed expression profiling
microarrays.
Abstract P03
Genetic analysis of postzygotic hybridization barriers
1
Hua Jiang , Claudia Köhler
1
1
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of
Agricultural Sciences and Linnean Center of Plant Biology, 750 07 Uppsala, Sweden
Polyploidization is a widespread phenomenon among plants and is considered to be a major
speciation mechanism. Polyploids have a high degree of immediate post-zygotic reproductive
61
isolation from their progenitors, as backcrossing to either parent will produce mainly nonviable
progeny. The main determinant for interploidy lethality is the endosperm, a nutritious tissue
supporting embryo growth, similar to the functional role of the placenta in mammals. In
response to interploidy hybridizations the endosperm fails to cellularize, leading to embryo
arrest and seed abortion. To identify the genetic basis for this response we performed a
suppressor screen of triploid seed abortion. We made use of the jason mutant that generates
unreduced male gametes and triploid seeds at high frequency. We identified two suppressors
named akastos (aka) and hylos (hyl) that form viable triploid seeds. In jas;aka and jas;hyl
double mutants endosperm cellularization was restored and embryo development progressed
to the mature stage. Normalization of endosperm cellularization correlates with normalized
expression of AGL MADS-box transcription factor genes in both mutants. However, the
temporal regulation of AGL-gene expression in both mutants differs, suggesting they act in
different genetic pathways.
Abstract P04
Expression profiles of methyltransferases and RdDM proteins during
sexual and apomictic reproduction in Eragrostis curvula
1
2
1
3
1
J.P. Selva , S. Pessino , J. Romero , O. Leblanc , I Garbus & V. Echenique
1
1
Universidad Nacional del Sur, Departamento de Agronomía and CERZOS-CONICET, Bahía Blanca,
2
Argentina, Universidad Nacional de Rosario, Facultad de Ciencias Agrarias and CONICET, Zavalla,
3
Argentina. UMR DIADE, IRD and University of Montpellier 2, Montpellier, France.
Apomixis in plants refers to a diverse group of developmental behaviors resulting in asexual
reproduction through seeds. Apomictic individuals bypass both meiotic reduction and egg cell
fertilization to produce offspring that are exact genetic replicas of the maternal plant. Given
the established relationships between sexual and apomictic pathways, different models have
been proposed to explain the occurrence of apomixis at the molecular level involving genetic
and epigenetic mechanisms. Weeping lovegrass (Eragrostis curvula [Schrad.] Nees) is an
apomictic perennial grass native of Southern Africa. The type of apomixis present in the E.
curvula is pseudogamous diplospory. Meiotic stages are absent from the Eragrostis-type
apomixis, and the megasporocyte undergoes only two rounds of mitotic division to form a
non-reduced tetranucleated embryo sac with an egg, two synergids, and one polar nucleus.
The E. curvula complex includes cytotypes with different ploidy levels (from 2x to 8x) that may
undergo sexual reproduction, facultative apomixis or obligate apomixis. Diploid (2n = 2x = 20)
plants are sexual and rare. Polyploids reproduce mainly by obligate apomixis, but sexual and
facultative apomixis has also been reported. Since long time ago our group has been
studying different aspects of the genetics and physiology of weeping lovegrass. There are
recent evidences on the role played by certain proteins of the RdDM pathways involved in
gametophytic apomixis expression, suggesting an epigenetic regulation of the trait. It was
reported in maize that loss-of-function of the dmt103 and dmt102 methyltransferases genes
partially mimics apomictic developments. Similarly, loss-of-function in ago104, a maize
homolog of ago9, also results in apomixis-like traits, giving rise to up to 70% of functional
unreduced female gametes. These data suggest that methyltransferases deregulation plays a
role on the establishment of apomixis. The aim of this work was to study the expression of
genes associated to RdDM pathway in sexual and apomictic flowers and leaves of E. curvula
plants. The BlastX algorithm was used to find the corresponding Eragrostis curvula RdDM
genes using the ChromDB database. The phylogenetic analysis indicated that these genes
group together in the same clade with the corresponding ones in Arabidopsis and maize. The
dmt104, chr106, hdt104 and ago104 genes profiles were analysed using qRT-PCR in leaves
and in different stages of reproductive tissues. Primers were designed on EST sequences
from E. curvula and also on genes associated to RdDM pathway in Arabidopsis and maize.
The expression studies indicated a decreased activity of all these four genes in the sexual
genotypes, suggesting a chromatin remodeling pathway is active in the apomictic ones.
62
Abstract P05
Making bigger seeds to increase agricultural yield: the effects of
paternal contribution and sexual conflict on variable endosperm
development in naturally-occurring asexual (apomictic) plants
1
D.Paczesniak & T.F. Sharbel
2
1
Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Aquatic Ecology,
2
Dübendorf, and ETH-Zürich, Institute of Integrative Biology (IBZ), Zürich, SWITZERLAND, Leibniz
Institute of Plant Genetics and Crop Plant Research (IPK), Department of Cytogenetics and Genome
Analysis, Apomixis Research Group, Gatersleben, GERMANY
Human population growth, climate change and the environmental effects associated with
intensive agriculture are significant challenges which necessitate improvements to crop plant
productivity. Apomixis, a naturally-occurring form of asexual reproduction through seed, is
heralded as disruptive technology which could spur an agricultural revolution when
engineered into crops, as it would allow immediate fixation of any desired genotype and lead
to faster and simpler breeding schemes.
Naturally-occurring apomictic plants provide an excellent model system for studying seed size
variation (an agronomically important trait). The goal of our project is to undersdand factors
affecting seed size in apomictic lineages of Boechera holboellii species complex.
Endosperm development is essential for seed development, and has been shown to be
highly-influenced by genomic imprinting whereby either maternally- and paternally-inherited
alleles are differentially expressed at different loci. In plants reproducing by pseudogamous
apomixis, paternal (pollen) contribution is a potential factor which contributes to seed size
variation, as the embryo is a genetic clone of the mother plant. Thus, while there is no direct
paternal contribution to the offspring genotype, there is the capacity for paternal influence (an
echo of sexual conflict inherited from sexual ancestors) on offspring’s growth and survival
through its effects on endosperm development.
Using comparative genome-wide expression profiling and expression profile comparisons we
intend to identify specific candidate factors controlling endosperm size.
Abstract P06
Connecting ploidy with female gametophyte development of the two
apomicts Boechera spp. and Hypericum perforatum.
1,2
1,2
2
2
Paride Rizzo , Olga Kirioukhova , Helmut Bäumlein , Jörg Fuchs and Amal J. Johnston
1
1,2
University of Heidelberg, Centre for Organismal Studies (COS), Im Neuenheimer Feld 329, 69120
2
Heidelberg, Germany, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Corrensstrasse 3, Gatersleben, Germany
In order to decode the genetics of asexual female gametophyte development during apomixis,
several labs have established wild-apomicts such as Boechera spp. and Hypericum
perforatum (diplosporous and aposporous, respectively) as model plants.
Thus far, independent investigations suggest that there are many different genetic factors
regulating apomixis events nevertheless deeper investigation is currently needed to unveil the
role of every such single factor connected with the apomictic mode of reproduction.
The first goal of our research is to clarify how strong is the effect of ploidy level on the
development of the female gametophyte in Boechera spp. and Hypericum perforatum.
Through a deep morphological characterization of the female gametophyte in a set of lines of
the above mentioned species derived from distinct native geographic locations, we show a
correlation between ploidy levels and developmental speed over different reproductive modes.
63
Specifically, we observed a strong connection between ploidy and reproductive efficiency of
different sexual and apomictic lines of Boechera spp.
The evidence coming from our work is of crucial importance not only to better clarify the role
of ploidy in apomixis, but also to constitute the basis for further molecular dissection of this
phenomenon across these two model systems.
Abstract P07
Genomic Imprinting in Arabidopsis thaliana
P.Wolff
1,2
& C. Köhler
1
1
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of
2
Agricultural Sciences and Linnean Center of Plant Biology, 750 07 Uppsala, Sweden, Department of
Biology and Zurich-Basel Plant Science Center, Swiss Federal Institute of Technology, ETH
Centre, CH-8092 Zurich, Switzerland
Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific
differential expression of maternally and paternally inherited alleles. In plants, genomic
imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived
after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify
novel imprinted genes in Arabidopsis thaliana we generated deep sequencing RNA profiles of
F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions.
Using polymorphic sites to quantify allele-specific expression levels we could identify more
than 60 genes with potential parent-of-origin specific expression. Further experimental
confirmation of allele-specific expression of 30 genes greatly extends the number of imprinted
loci in plants. The distribution of DNA methylation and epigenetic marks established by
Polycomb group (PcG) proteins implicate that for maternally expressed genes (MEGs)
repression of the paternally inherited alleles largely depends on DNA methylation or PcGmediated repression whereas repression of the maternal alleles of paternally expressed
genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are
also targeted by PcG proteins, such targeting does not cause complete repression. Many
MEGs and PEGs encode for transcriptional regulators, implicating important functional roles
of imprinted genes for endosperm and seed development in Arabidopsis thaliana.
Abstract P08
In silico and molecular characterization of some paternally expressed
imprinted genes in sexual and apomict boechera species
1
A. Özbilen & K. M. Taskin
1
2
Çanakkale Onsekiz Mart University, Institute of Science, Department of Biology Çanakkale, TURKEY,
Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, Çanakkale,
TURKEY
2
Genomic imprinting as an epigenetic mechanism plays an important role on seed
development. In sexually reproducing angiosperms, genomic imprinting primary occurs in the
endosperm and causes parent-of-origin specific allelic gene expression. The ratios of parental
genomes within a diploid embryo and triploid endosperm have serious effects on seed
development. However, in some of flowering plants, there is an asexual seed formation which
clones embryos with mother, known as apomixis. Many apomicts are pseudogamous, the
endosperm still needs fertilization, and “the endosperm” problem circumvented by modified
pathways of female gametogenesis or fertilization. In our work, we would like to determine the
effects of genomic imprinting on seed development in sexual and apomict Boechera species.
Therefore, we have selected eight genes that previously reported as paternally expressed
imprinted genes (PEGs) in Arabidopsis thaliana L.. The genomic sequences of these PEGs
(AGL92, AT2G21930, AT2G36560, AT5G63740, HDG3, SUVH7, VIM1, YUC10) were BLAST
using the SRA databases for diploid sexual B. stricta and triploid apomict B. holboellii species.
64
Then, we selected the SRA sequences according their E-value and assembled. Thus,
genomic sequences for the PEGs similar genes in various Boechera species were revealed
for the first time. We designed Boechera specific primers by using these sequences. Until
now, we have investigated the structures and expression profiles of SUVH7 and YUC10
similar genes in Boechera with RT-PCR method in different development stages of flower and
silique tissues. We found that the expression level of SUVH7 was decreased 4 days after
pollination, while no expression were observed for YUC10. Moreover, expression of FIE gene,
a component of PRC2, was determined in these tissues and used as control. SUVH7
(SU(VAR)3-9 HOMOLOG 7) encodes a SET domain protein and involved in epigenetic
control of gene expression by acting as histone methyltransferases and YUC10 (YUCCA10)
encodes flavin monooxygenases involved in auxin transport.
Abstract P09
AGAMOUS-LIKE imprinting and regulation in Arabidopsis
Johannessen I.M. , Hornslien K.S. , Kalantarian M. , Kittelsen L.A. , Ullmann I.F. , Stø I.M. ,
Andersen E.D. , Bjerkan K.N. , Shirzadi R. and Grini P.E.
Department of Biosciences, University of Oslo, 0316 Oslo, Norway
Parent of origin dependent gene expression is crucial in the offspring of animals and flowering
plants and mutations in the imprinting machinery can lead to embryonic lethality. In order to
identify novel factors involved in parent of origin specific regulation, microarray transcriptional
profiles of seeds with only maternal endosperm was generated. We show that AGAMOUSLIKE36 (AGL36) is a novel imprinted gene, only expressed from the maternal genome after
fertilization. AGL36 imprinting requires MET1, the major Arabidopsis maintenance DNA
methyltransferase. In addition, maternal polycomb chromatin remodeling factors (PRC2) act
as repressors of maternal AGL36 expression during seed development. Recently the focus
has expanded to investigate the role of small RNAs in regulation of imprinting AGL genes.
Plants have two extra RNA polymerases which take part in making siRNA and directing them
to certain loci where de novo methylation can take place. RNA polymerase mutants were
used to affect the machinery producing siRINAs, and mutants of interacting AGLs was made
to gain mutant phenotypes. With these findings, we hope to gain new insight into the
interplay between maternal and paternal genomes in seeds and how imprinting is coordinated.
Abstract P10
Relationships between induction of flowering, cold acclimation,
deacclimation and reacclimation in a perennial forage grass species
1
2
Å. Ergon , M. Höglind & O.A. Rognli
1
1
Norwegian University of Life Sciences, Department of Plant and Environmental Sciences, Ås,
2
NORWAY, Bioforsk Særheim, Klepp, NORWAY
Winter cereals and many temperate perennial grasses have a vernalization requirement for
flowering which is gradually saturated by low temperatures during winter (typically many
weeks). At the same time low temperatures (<~10ºC) confer freezing tolerance through the
process of cold acclimation (typically a few weeks). Freezing tolerance can be lost rapidly
upon exposure to higher temperatures, but some freezing tolerance may be regained if plants
are again exposed to low temperatures. The tendency to deacclimate and/or the ability to
reacclimate have been suggested to depend on whether vernalization is saturated or not (e.g.
Laudencia-Chingcuanco et al. 2011). Understanding the genetic control of these processes
are important in relation to the global climate change and its effect on plant growth and
reproduction.
Individuals of a mapping family of meadow fescue (Festuca pratensis Huds.) (Alm et al. 2003)
vary in vernalization requirement (Ergon et al. 2006). Individuals with either a high or a low
vernalization requirement were crossed in two separate groups to create two divergently
selected F2 populations (Ergon et al. 2013). In order to study the relationship between
65
vernalization and the tendency to deacclimate and ability to reacclimate, individuals from the
two F2 populations were subjected to an intermediate length of vernalization (7 weeks at 6ºC
+ 2 weeks at 2ºC) (V-CA), followed by 1 week of deacclimation at 12ºC (DA) and 2 weeks of
reacclimation at 2ºC (RA), all at 8h photoperiod. Control plants were only cold acclimated for
2 weeks at 2ºC (CA). Plants sampled after CA, V-CA, DA and RA were characterized for
freezing tolerance and flowering response. The expression of VRN1 (controlling vernalization
response) and COR14b (involved in freezing tolerance) was also studied. The results will be
presented and discussed.
Alm et al. 2003. A linkage map of meadow fescue (Festuca pratensis Huds.) and comparative mapping
with other Poaceae species. Theor Appl Genet 108: 25–40.
Ergon et al. 2006. Quantitative trait loci controlling vernalisation requirement, heading time and number
of panicles in meadow fescue (Festuca pratensis Huds.). - Theor Appl Genet 112: 232-242.
Ergon et al. 2013. Differential expression of VRN1 and other MADS-box genes in Festuca pratensis
selections with different vernalization requirements. Biol Plantarum 47: 245-254.
Laudencia-Chingcuanco et al. 2011. Genome-wide gene expression analysis supports a developmental
model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.). BMC
Genomics 12: 299.
Abstract P11
Tissue-specific expression of the TaVRN1 transcription factor during
vegetative-to-generative transition in wheat (Triticum aestivum L.)
K. Jäger, Á. Boldizsár, G. Kocsy, B. Barnabás & G. Galiba
Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences,
Brunszvik u. 2, 2462 Martonvásár, Hungary, e-mail: jager.katalin@agrar.mta.hu
In the pooid subfamily of grasses wheat vernalization responsiveness is determined by allelic
differences in the MADS-box transcription factor VERNALIZATION1 (VRN1). Winter cereals
are tolerant to low temperatures. Their extended exposures to low temperatures during winter
induce the transition of the apical meristem to the reproductive phase. On the contrary, spring
cultivars are freezing-sensitive and flower later with prolonged periods of cold. In our
experiment plants of Chinese Spring wheat variety, Cheyenne winter wheat variety and two
Chinese Spring substitution lines carrying either T. spelta (spring wheat) 5A or T. aestivum
Cheyenne 5A chromosomes were grown at 20°C for 2 weeks under controlled environment.
Subsequently plants were subjected to 4°C and apical meristems were sampled after 14 days
and at both double ridge and generative stages. Specimens were fixed, dehydrated in ethanol
®
series, embedded in Unicryl resin, sectioned and chromogenic in situ hybridisation was
carried out to demonstrate the tissue-specific expression of the TaVRN1 transcription factor.
Our results demonstrate significant up-regulation of VRN1 expression in apical meristems of
the winter wheat genotype in response to vernalization; no treatment effect was found for the
spring wheat variety or substitution lines.
Abstract P12
Regenerated In Vitro Soybean Plants With Improved Productivity
G.Kosturkova, K .Tasheva & M. Dimitrova
Bulgarian Academy of Sciences, Institute of Plant Physiology and Genetics, Department of Regulation
of Plant Growth and Development, Sofia, BULGARIA
Soybean (Glycine max (L.) Merrill.) is the largest cultivated pulse crop in the world. As an oil
and protein rich plant it provides about 60% of the plant based protein in the world, and it is a
source of more than 200 industrial products. Soybean is receiving great global importance
due to its nutraceutical value but its cultivation suffers the problems of biotic and abiotic stress
[1, 2]. Due to the great importance of the problem and the complex nature of the tolerance
efforts and studies for improvement of plant performance are of different aspects. Classical
breeding methods have been complemented with new techniques of plant biotechnology and
66
molecular biology giving possibility for simulating the desired stress in vitro, providing
selection on cell level and manipulating genes for resistance. Bulgarian and foreign soybean
genotypes with various nutritional qualities and agronomic traits were used [2, 3]. To improve
soybean germplasm biotechnological approaches were applied in our previous studies. The
possibilities for establishment of in vitro cultures which can be used for genetic manipulations
and modelling of stress were studied bringing to development of schemes for callus induction,
adventitious shoot formation and regeneration of plants [4, 5, 6]. These systems were applied
to examine abiotic stress and the drought effects by simulation of osmotic stress by PEG
which caused alterations in growth and in vitro regeneration, as well as in the osmotic
potential and isozyme profiles of peroxidase [7, 8].
One of the objectives of the investigations was to obtain plants with better performance. Here
we report about in vitro plants which were regenerated under osmotic stress conditions,
rooted in pots with soil and after adaptation transferred to the experimental field. Their
development was studied making physiological and agronomical characterization. Obtained
lines differ in plant height, vegetative mass, number of stem branches, pods and seeds –
values reflecting to crop productivity. Some of the lines were superior compared to the initial
parent varieties.
Acknowledgements: The research was funded by the Bulgarian NSF under the
Intergovernmental Program for S&T between Bulgaria and India (projects B17-2006-2010 and
DNTS-05/2 2013-2016)
References:
[1] Kosturkova G., R. et al. 2011. Soybean in healthful human nutrition. Dietetika, 4, 17-18.
[2] Todorova R., G. Kosturkova 2010. Achievements, problems and perspectives in soybean breeding.
“Breeding & Technological Aspects in Production of Soybean” 2010, Pavlikeni, 27-36
[3] Sakthivelu et. Al. 2008. Isoflavone composition, phenol content, and antioxidant activity of soybean
seeds from India and Bulgaria. J. Agricultural and Food Chemistry, 56, 6, 2090-2095.
[4] Kosturkova G. 2005. In vitro development of various soybean explants from mature seeds. “Breeding
and Technological Aspects in Production and Processing of Soybean and Other Legume Crops”.
Pavlikeni, 94-99.
[5] Nedev Т., G. Kosturkova, et al. 2007. Variation in in vitro Morphogenic Response to Growth
Regulators in Soybean Genotypes from India and Bulgaria. BIOAUTOMATION, 8, S1, 193-200.
[6] Kosturkova G. et al. 2008. Response of Bulgarian and Indian soybean genotypes to drought and
water deficiency in field and laboratory conditions. GAPP, 34, 3-4, 239-250
[7] Sakthivelu G., et al. 2008. Drought induced alterations in growth, osmotic potential and in vitro
regeneration of soybean cultivars. GAPP, 34, 1-2, 103-112
[8] Kosturkova G. et al. 2011. Isoperoxidase profiles in soybean in vitro cultures under osmotic
stress. BIOTECHNOLOGY (Romania), 2011, p. 8 – 15. ISSN 1224-7774
Abstract P13
FTIP1 Is an Essential Regulator Required for Florigen Transport
Lu Liu, Chang Liu, Xingliang Hou, Wanyan Xi, Lisha Shen, Zhen Tao, Yue Wang & Hao Yu
Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of
Singapore, Singapore
The capacity to respond to day length, photoperiodism, is crucial for flowering plants to adapt
to seasonal change. The photoperiodic control of flowering in plants is mediated by a longdistance mobile floral stimulus called florigen that moves from leaves to the shoot apex.
Although the proteins encoded by FLOWERING LOCUS T (FT) in Arabidopsis and its
orthologs in other plants are identified as the long-sought florigen, whether their transport is a
simple diffusion process or under regulation remains elusive. Here we show that an
endoplasmic reticulum (ER) membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), is an
essential regulator required for FT protein transport in Arabidopsis. Loss of function of FTIP1
exhibits late flowering under long days, which is partly due to the compromised FT movement
to the shoot apex. FTIP1 and FT share similar mRNA expression patterns and subcellular
localization, and they interact specifically in phloem companion cells. FTIP1 is required for FT
export from companion cells to sieve elements, thus affecting FT transport through the
67
phloem to the SAM. Our results provide a mechanistic understanding of florigen transport,
demonstrating that FT moves in a regulated manner and that FTIP1 mediates FT transport to
induce flowering.
Abstract P14
Investigating the relationship between substrates of the N-end rule
Pathway and genes regulated by EBP binding sites (GCCGCC) in
Arabidopsis thaliana
1
1
1
G.Prasad , C. Sousa Correia , D. Gibbs , M. Holdsworth
1
1
University of Nottingham, Division of Plant and Crop Science, Sutton Bonington Campus,
Loughborough, LE12 5RD, UK
The N-end rule pathway is a ubiquitin-dependent proteolysis pathway that exists in all
eukaryotes. The proteins bearing primary destabilizing residues are recognized by Nrecognins, a class of E3 ligases that label N-end rule substrates through covalent linkage to
ubiquitin, that is later degraded by the downstream 26S proteasome complex. Recently, the
role for this pathway in seed germination (Holman et al. 2009,) and survival of plants under
low oxygen condition was established (Gibbs et al, 2011).
In Arabidopsis, the Group VII Ethylene Response Factor (ERF) transcription factors (that bind
the cis-element GCCGCC; EBP box) are substrates of the N-end rule pathway and begin with
the amino acids ‘Met-Cys’ (Gibbs et al. 2011). Their degradation takes place via the
arginylation branch of the N-end rule pathway. Under normal conditions, the N-terminal
cysteine is oxidized and can then be arginylated leading to recognition by the E3 ligase PRT6.
Previous studies demonstrated that ABA sensitivity during germination is regulated by the Nend rule pathway. Genetic analysis revealed that during seed germination the E3 ligase PRT6
mediates Abscisic Acid (ABA) sensitivity through ABI5 (Holman et al., 2009). Studies showed
that the ABI5 5’UTR region contains 2 EBP boxes that mediate positive regulation of the ABI5
via Group VII ERFs. Whereas the wild type 5’UTR region is ectopically activated in prt6,
deletion of one or both elements destroys expression in prt6. Hence, this suggests that genes
containing EBP elements in a similar configuration to that found in the 5’UTR region of ABI5
might be regulated by Group VII ERFs through the N-end rule pathway. In this project
fourteen genes containing two copies of GCCGCC in the 5’UTR/promoter were identified as
potential candidate genes to be regulated by Group VII ERFs, similarly to ABI5. Successful
amplification of the promoter region of five of these genes (AT4G01026, AT1G14810,
AT3G54510, At3G13440 and AT5G44420) was followed by cloning into a GUS-reporter
plasmid and transformation into Arabidopsis wild type (Col-0) and prt6-1 plants. Leaves and
flowers of transgenic plants were analysed by GUS staining to reveal promoter activity. The
present analysis has shown that these promoters up regulated GUS expression in prt6 in
comparison to the wild type. This suggests that these genes are regulated by substrates of
the N-end rule pathway. Future work will involve analysis of in-vitro DNA binding specificity of
Group VII ERFs through Electro Mobility Shift Assay. As well as also in-vivo binding
specificity of ERFs to selected promoters through ChIP. These experiments will confirm our
hypothesis that Group VII ERFs bind to GCCGCC binding sites and acts as substrates of Nend rule pathway. Further, analysis of selected promoters mutated in GCC elements will be
done. That will help to understand the role of Group VII ERFs in regulation of genes that
contain GCCGCC binding sites.
68
Abstract P15
The Arabidopsis thaliana Pentatricopeptide Repeat Protein (AtPPR)
Contains a Domain that Functions as an Adenylate Cyclase
1
1
1
1
1
O. Ruzvidzo , T.B. Dikobe , P. Chatukuta , D.T. Kawadza , J. Asong , & C.A. Gehring
1
2
2
North-West University, Department of Biological Sciences, Mmabatho, Republic of South Africa. King
Abdullah University of Science and Technology, Division of Chemistry, Life Science and Engineering,
Thuwal Kingdom of Saudi Arabia
Since climate changes and extreme environmental conditions are likely to continue, there is
therefore an urgent need to use rational and system-based approaches to develop crop
plants with increased tolerance to both biotic and abiotic stress factors. This need has to date,
led to an impressive body of work in the areas of plant genetics, plant physiology, plant
biochemistry and plant molecular biology, and a realization that only an integrated and
systems-based approach can possibly deliver effective biotechnological solutions. Part of this
approach involves the study of a special group of proteins termed adenylate cyclases (ACs)
that are thought to systemically affect homeostasis in plants. Adenylate cyclases are
enzymes capable of converting adenosine-5′- triphosphate to the second messenger, cyclic 3′,
5′-adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their
product cAMP have firmly been established as important signaling molecules with important
roles in several cellular signal transduction pathways. However, in higher plants and to date,
the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of
generating cAMP and is responsible for regulating pollen growth. Recently a number of
candidate AC-encoding genes in the Arabidopsis genome have been proposed based on
functionally assigned amino acids in the catalytic centre of annotated and/or experimentally
tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and
functional characterization of a candidate AC domain from Arabidopsis thaliana in the form of
a pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a series of
complementation tests and enzyme immunoassays, the recombinant AtPPR-AC was
thoroughly pre-screened and functionally evaluated for its possible in vitro and/or in vivo
adenylate cyclase activities. Findings from these tests and assays all indicated that the
recombinant AtPPR-AC indeed does possess some functional adenylate cyclase activities
and is therefore, a bona fide higher plant adenylate cyclase with potential roles in cell
signaling and transduction systems.
Abstract P16
Epigenetic Adaptation to High Salinity in Plants
Anjar Wibowo, Quentin Saintain, Christopher Barrington, Jose Gutierrez-Marcos
SLS, University of Warwick, Coventry, UK
Claude Becker, Detlef Weigel
Max Planck Institute for Developmental Biology, Spemannstrasse 35-39, 72076 Tübingen, Germany
High soil salinity is a major environmental stress that adversely affects plant growth and crop
production throughout the world. It is now estimated that half of the world’s cropland is
affected by salt stress. To cope with various environmental stresses, plants are able to
spatially and temporally regulate gene expression through changes in DNA methylation and
chromatin conformation, known as epigenetic modifications. Recent studies have shown that
epigenetic modifications induced by environmental stress can be inherited over several
generations, despite a genome-wide epigenetic resetting of epigenetic imprints, which is
thought to take place during gametogenesis. The aim our project is to evaluate the effect of
multi-generation salt stress treatments on the genome-wide dynamics of DNA methylation
and any acquired salinity tolerance in plants across multiple generations. Our results show
that progenies of Arabidopsis plants exposed to salt stress for four consecutive generations
display higher germination and survival rates under salinity stress, which is associated with
reduced salt intake. Our data suggest that salt stress induced epigenetic changes can be
69
transmitted to the offspring. However, the increased tolerance to high salinity did not persist in
subsequent generations when plants were grown under non-stress conditions, thus indicating
that plants developed efficient mechanisms to erase epigenetic information acquired as a
result of environmental stress.
Abstract P17
Isolation and characterization of dyad gene orthologs from boechera
species
1
1
F. Sezer , G. Nişli , K.M. Taşkın
1
1
Çanakkale Onsekiz Mart University, Department of Biology, Çanakkale, TURKEY
Apomixis leads to clonal reproduction via seeds in plants. Apomict plants form diploid female
gametes without meiosis (apomeiosis) followed by parthenogenetic development of embryo
and formation of functional endosperm. DYAD protein (At5g51330) is expressed in early
stages of meiosis I and involved in sister chromatid cohesion and meiotic chromosome
organisation. Characterized DYAD mutations were reported to show different phenotypes.
While dyad and swi1.1 effects only female meiosis, swi1.2 and dsy10 effects both female and
male. The dyad mutation leads to formation of 10 univalents at metaphase I instead of
expected 5 bivalents, leading to formation of functional diploid gametes. This phenotype of
dyad mutants resembles apomeiosis. However, there is no report about DYAD like genes in
natural apomicts. In order to identify the structures of DYAD like gene in natural apomicts, we
have isolated DYAD orthologs from Boechera species as close relatives of A. thaliana. The
Boechera DYAD cDNAs were obtained by RT-PCR from young flower buds and sequenced.
We found that the Boechera DYAD transcripts obtained from triploid apomict B. holboellii and
B. gunnisoniana and diploid sexual B. stricta showed high similarity to At5g51330. We also
amplified Bochera genomic sequences and compared with At5g51330 genomic sequences.
Transcript and protein sequences were predicted from these genomic sequences and used
for phylogenetic analyses. Bioinformatic analyses showed that Boechera DYAD orthologs
also contain phospholipase C domain and a nuclear localization signal. We analysed
Boechera DYAD expression with RT PCR in mature flowers, silique, leaves and very young
flower buds and found expression only in very young flower buds.
Abstract P18
Looking for new insights in ovule primordia specification
1,2
1
V. Balanzà , P. van Dijk and L. Colombo
1
2
2
KeyGene N.V., Wageningen, The Netherlands Università degli Studio de Milano, Dip. di Bioscienze,
Milan, Italy
In Arabidopsis, ovule primordia are initiated as outgrowths on the marginal meristematic
tissue of the carpel. After primordium emergence, three differents regions are recognizable:
the funiculus in the base that will comunicate the ovule with the fruit, the chalaza in the center,
where integuments will develop, and the nucella in the apical region, where the megaspore
mother cell (MMC) differentiates and will develop, after the meiosis, the female gametophyte.
Recent works have provided evidences that the hormonal action of auxins and citokinins are
crucial for the initials steps of ovule specification, as well as the biogenesis and control of
small RNAs for the MMC specification.
The aim of our project is to identify possible relationships between the morphogenic role of
hormones during the initial steps of ovule development and the action of small RNA that
contribute to the MMC specification.
70
Abstract P19
Characterization of transcription regulators acting during male
gametophyte development
1,2
1
Antonia Gibalová , David Reňák & David Honys
1,2
1
Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263
2
Praha 6, CZECH REPUBLIC, Department of Experimental Plant Biology, Faculty of Science, Charles
University in Prague, Viničná 5 Praha 2, CZECH REPUBLIC
Molecular mechanisms underlying many developmental processes, including the production
of both male and female gametes, remain largely unknown. To uncover such mechanisms,
our approach is to identify and characterize transcription factors (TFs) taking part in the
haploid regulatory networks that governs gamete production in flowering plants.
Candidate genes were chosen exploiting pollen developmental transcriptomic data (Honys
and Twell, 2004). Based on a wide screen of T-DNA mutant lines (Reňák and Dupľáková et
al. 2012) we have identified several promising candidates and subjected them to the
functional characterization. So far, genes from two TF families were found most interesting.
We have identified the atren1 mutation (restricted to nucleolus1) in early pollen gene
At1g77570, a member of the heat shock transcription factor (HSF) gene family. Complete
knock down of the AtREN1 transcript causes multiple defects in male gametophyte
development in both structure and function appearing from bicellular pollen stage to pollen
maturation and beyond, until progamic phase. In addition, atren1/- plants are defective in heat
stress (HS) response, causing aberration of pollen grains (Reňák et al. 2013).
Previously, we have also functionally characterized the late pollen-expressed gene from bZIP
TF family. Our results show that AtbZIP34 has multiple roles in the development of
gametophytic and sporophytic tissues. Characteristic phenotype and genetic transmission
defects demonstrate a requirement for AtbZIP34 for correct formation of pollen cell walls, lipid
metabolism, cellular transport and/or intine synthesis. This hypothesis is further supported by
the downregulation of distinct subsets of genes (Gibalová et al. 2009). As a next step we
have aimed to extend our knowledge to understand the bZIP networking during male
gametophyte development as these proteins exist as dimers as well as to uncover the
downstream targets of our candidate transcription factors.
Acknowledgements: Authors gratefully acknowledge the financial support from the Ministry of Education
of the Czech Republic (grant LD11018) and Grant Agency of the Czech Republic (grant P305/12/2611).
Abstract P20
Dissecting cell-cycle and -differentiation in flowering plant gamete
formation
1
1
1
2
1
Glöckle, B.M., Zhao, X.-A., Grini, P.E. and Schnittger, A.
2
Institut de Biologie Moléculaire des Plantes, UPR2357 du CNRS, Strasbourg, France University of
Oslo, Department of Molecular Biosciences, Oslo, Norway
In contrast to animals, plants do not generate gametes immediately after meiosis. Rather two
sex-specific haploid organisms are produced, the gametophytes, that undergo a distinct
series of cell divisions generating the gametes. In flowering plants, typically two gametes are
produced by each sex, both of which fuse during double fertilization. High precision control of
the gametophytic cell cycle is needed, not only to generate the exact number of gametes, but
also to arrest the gametes in the same cell-cycle phase allowing proper fusion at fertilization,
and preventing chromosomal disorders like aneuploidy. Furthermore, the development of
gametes involves a number of specialized cell divisions such as symmetric divisions and free
nuclear division that require a tight coordination and mutual control of cell proliferation with
differentiation. Despite its importance in the plant life cycle and for breeding, little is known
71
about the patterned control of cell proliferation and differentiation of the gametophytes. Based
on a unique set of mutants that disrupt the typical course of cell divisions during male pollen
development, we have set up an approach to dissect the interplay between cell proliferation
and cell differentiation of the gametes in flowering plants. A particular focus is given to the
dynamic changes of chromatin composition in gamete formation and an assessment of the
biological role of gamete chromatin regulation for the developing progeny.
Abstract P21
Molecular characterization and the elucidation of the physiological roles
of a novel maternal effect embryo arrest protein from Arabidopsis
thaliana
1
D. Kawadza , O. Ruzvidzo
1
1
North-West University-Mafikeng, Department of Biological Sciences, Mafikeng, North-West Province,
SOUTH AFRICA, 2745
Over the years, the presence and functional role of adenylyl cyclases (AC) in plants has been
a contentious issue. In an attempt to characterize a possible adenylate cyclase we undertook
to test a putative plant AC. A bioinformatic search by a colleague of the Arabidopsis genome
using an amino acid motif specific for Adenosine cyclase binding, returned nine genes with
possible/ likely AC activity which included the Maternal-Effect-Embryo-arrest-22 (MEE)
protein encoded by the At2g34780 gene. The MEE protein is a developmental protein derived
from the maternal (gametophytic) Arabidopsis plant. In order to determine whether MEE
possesses adenylate cyclase activity, the At2g34780 gene was obtained from leaf material of
Arabidopsis thaliana plants, cloned into a vector then transformed into a prokaryotic host. The
prokaryotic host was induced to over-express the At2g34780 gene. The expressed protein
was then tested for endogenous, in vitro and in vivo adenylate cyclase activity. The results of
the assays indicate that the MEE protein from Arabidopsis thaliana possesses endogenous,
in vitro and in vivo adenylate cyclase activity. We further indicate that cAMP has a role in
MEE developmental signalling. In order to understand the possible role of MEE we used coexpression analysis tools to understand the possible biological role of the Arabidopsis
thaliana MEE. The analysis of MEE and its co-expressed genes revealed that genes
annotated as part of the developmental group were over-represented, thus suggesting that
MEE functions as a component of plant developmental strategies which are induced in the
presence of biotic and abiotic perturbations as well as inducer hormones. The results further
show that MEE shares many characteristics with development related genes in that its
transcription is strongly induced in response to pathogen challenges. We conclude that MEE
together with other genes plays a key role in signal transduction which may involve a phytohormone.
Abstract P22
Molecular and functional characterization of POLLEN SPECIFIC
KINASE1-4 genes (PSK1-4) in Arabidopsis thaliana
1
1
1
M. N. Lucca , M.A. Ibeas , S. Parra & G. León
1
1
Laboratory of Plant Reproduction and Development, Center of Plant Biotechnology, Universidad
Andres Bello, Santiago. CHILE
Pollen grains are the male gametophyte of plants and thus are essential for plant
reproduction and productivity. However, despite their biological and agronomical importance,
little is known about the molecular mechanisms that regulate its development and function. In
Arabidopsis, three cells compose mature pollen grains: a large vegetative cell and two small
sperm cells engulfed in the cytoplasm of the vegetative cell. During fertilization, the vegetative
cell must germinate and produce a pollen tube, a growing tip structure that directionally
transports the sperm cells to the ovule to produce the double fertilization event. Currently,
72
little is known about signal transduction pathways and molecular components involved in
these processes. Using publicly available microarray data we have previously identified 4
genes encoding kinase proteins (PSK1 to 4, for POLLEN SPECIFIC KINASE) that are
expressed exclusively during the last stages of pollen development, germination and tube
elongation. Promoter-GUS fusions suggest that these genes are regulated in pollen at the
transcriptional level and GFP-PSKs constructions were used to determine its sub cellular
distribution. To analyze the physiological relevance of these genes, we have generated
transgenic plants expressing specific amiRNAs under the transcriptional control of a pollenspecific promoter (LAT52) and we have analyzed pollen development and tube elongation in
these plants. Our preliminary data suggest that these genes may have an important role for
male gametophyte development and function. Funded by Fondecyt 1120766 and UNAB DI74-12/R.
Abstract P23
Functional Characterisation of a Novel Pair of Male Germline Proteins in
Arabidopsis
1
1
1
N. Rutley , A. Yim , M. Borg & D. Twell
1
1
University of Leicester, Department of Biology, Leicester, UK
During the male gametophytic life cycle of flowering plants two sperm cells are produced
which go on to fuse with the egg cell and central cell. The sperm cells result from a mitotic
division of the germ cell, and previous work has described duo1 in which germ cells fail to
divide (1). DUO1 is a male germline-specific R2R3 MYB transcription factor and following
ectopic expression of DUO1 in seedlings the expression of numerous DUO1 regulated genes
was induced including two paralogous C2H2 zinc finger proteins, DAZ1 and DAZ2 (2). Like
DUO1, DAZ1 and DAZ2 are putative transcription factors specific to the male germline and
when both genes are mutated, progression of germ cell division fails. Moreover, daz1-1
daz2-1 germ cells are rescued by a transgene coding for DAZ1 using in planta
complementation assays. DAZ1 and DAZ2 are members of the C1-3i subgroup of C2H2 zinc
finger proteins and have three dispersed zinc finger domains (3). Zinc finger domain one has
an unusual ‘KALFGH’ alpha helix, while the second and third domains have ‘QALGGH’ alphahelices. Phylogenetic alignment of amino acid sequences of DAZ1 and DAZ2 homologues
show that zinc finger domain one is well conserved in flowering plants, however monocots
(including rice, sorghum and maize) lack domain two. In a phylogenetic context, our work has
characterised the role of zinc finger domains one and two of DAZ1 in rescuing daz1-1 daz2-1
germ cells.
(1) Durbarry et al 2005 Plant Physiol 137:297-307
(2) Borg et al 2011 Plant Cell 23:534-49
(3) Englbrecht et al 2004 BMC Genomics 5:39
Abstract P24
Novel mutant alleles of the RKD transcription factor gene family of
Arabidopsis
F. Tedeschi, D. Koszegi, M. Rosso, T. Altmann and H. Bӓumlein
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) 06466 Gatersleben, Germany
The life cycle of higher plants alternates between a haploid gamete-producing gametophyte
and a diploid spore-producing sporophyte, with the transitions marked by fertilisation and
meiosis. The Arabidopsis female gametophyte consists of four different cell types including
three antipodal cells, two synergid cells, a central cell and the egg cell. Double fertilisation
includes the fusion between one sperm cell and the egg cell resulting in the diploid embryo
and between a second sperm cell and the central cell to initiate endosperm differentiation.
The Arabidopsis genome encodes a family of plant specific transcription factors called RKD,
73
based on their highly conserved RWP_RK domain. Members of this gene family are
specifically expressed in the egg cell. RKD factors are similar to MINUS DOMINACE (MID) of
green algae, where MID is required for gamete differentiation, indicating the evolutionary
conservation of RKD functions. Previous experiments have shown that RKD factors
reprogram sporophytic cells towards an egg cell-like transcriptome, suggesting that RKD
factors are involved in the control of gamete development. A further understanding of RKD
functions might provide tools to manipulate parthenogenetic processes as a component of
apomictic reproduction (Koszegi et al. Plant J., 67, 280, 2011). For a deeper understanding of
developmental functions of the RKD gene family we here describe the isolation and
characterisation of 12 mutant alleles based on T-DNA insertions and a novel allele of RKD2
created by a TILLING approach.
Abstract P25
The role of core-spliceosomal components during cell specification in
the female gametophyte
Nicola Nielsen, Sebastian Tiedemann, Dieu Vo, Rita Gross-Hardt
University of Bremen, Plant Molecular Genetics, Bremen, Germany
In contrast to animals, plant germ cells are formed along with accessory cells in specialized
haploid gametophytes. The female gametophyte of flowering plants comprises four different
cell types, which exert distinct functions in the reproductive process. For successful
fertilization, the development of the four cell types has to be tightly coordinated, however the
underlying mechanisms are not yet understood.
We have previously shown that lachesis (lis) and clotho (clo) mutants form supernumerary
gametes at the expense of accessory cells. LIS and CLO code for the Arabidopsis
homologues of the pre-mRNA splicing factors PRP4 and Snu114, respectively, which are
considered core spliceosomal components in yeast. The specific defects observed in lis and
clo mutants are difficult to reconcile with the general function of housekeeping genes. Here,
we discuss results addressing whether LIS and CLO act in a substrate specific manner.
Abstract P26
Identification of pollen specific promoters from Arabidopsis thaliana and
their use to generate male sterility
1
D. Muñoz-Strale & G. León
1
1
Laboratory of Plant Reproduction and Development, Center of Plant Biotechnology, Universidad
Andres Bello, Santiago. CHILE
The mature pollen grain displays a highly specialized function in angiosperms, accordingly,
the male gametophyte development involves many cell activities and mechanisms, making it
a complex and unique process in plants. In order to accomplish this, during the pollen
development, a whole new transcription program starts indicating the switch from a
sporophytic to a gametophytic tissue, involving the expression of many pollen specific genes.
With the search in microarrays databases we selected, according to their expression profile,
five candidates for pollen specific genes (PSG), and confirmed this through RT-PCR. A
transcriptional fusion where performed between the putative promoters of the PSG genes and
the GUS reporter gene, in order to determinate the spatial and temporal expression pattern,
this showed GUS activity exclusively confined to the pollen grain in Arabidopsis transgenic
lines. The expression of a cytotoxin under the transcriptional control of the PSG promoters
generated pollen specific ablation. Through the selection of genes that shown a pollen
specific expression profile in microarrays experiments, we where able to identify promoter
regions that confers pollen specific expression, providing a powerful tool for the expression of
74
genes exclusively in pollen during microgametogenesis, like the generation of male sterility.
Funded by Fondecyt 1120766 and UNAB DI-74-12/R.
Abstract P27
Auxin and jasmonic acid interaction in the control of anther dehiscence
process in Arabidopsis
1,2
3
2
3
Valentina Cecchetti , Maria Maddalena Altamura , Patrizia Brunetti , Giuseppina Falasca ,
1
2
Paolo Costantino and Maura Cardarelli
1
2
3
Dipartimento di Biologia e Biotecnologie, Istituto di Biologia e Patologia Molecolari CNR, Dipartimento
di Biologia Ambientale, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Anther dehiscence is a multistep-process occurring during late stamen development.
Endothecium lignification is an early event in this process that allows at anthesis, anther
opening in the stomium region. It has been shown that in Arabidopsis the anther dehiscence
process is controlled by two hormones: auxin and jasmonic acid (JA). We have shown that
auxin peaks before the start of the process and subsequently declines when endothecium
lignification occurs. By analysing auxin perception mutants afb1 and tir1afb2 afb3 and auxintreated wild type flower buds, we showed that auxin controls the timing of anther dehiscence
by negatively regulating endothecium lignification. The transcription factor MYB26, required
for endothecium lignification, is overexpressed at early stages in auxin perception mutants
whereas its transcript level is reduced in auxin-treated anthers, indicating that auxin acts
through MYB26. In contrast JA does not control endothecium lignification as opr3 mutants,
defective in JA biosynthesis, has a normal timing of endothecium lignification and MYB26
transcript levels comparable to wild type. By analysing OPR3 and DAD1 transcript levels in
afb1-3 and tir1afb2afb3 flower buds and in auxin-treated flower buds, and by measuring JA
content in auxin perception mutants, we showed that auxin negatively regulates JA
production at specific late stages of flower development. Furthermore the double mutant
afb1opr3 shows premature endothecium lignification, like in afb1-3, and indehiscent anthers
due to lack of JA, which is required for stomium opening. A model of auxin and JA interaction
in controlling the anther dehiscence process has been proposed (Cecchetti et al. Plant
Journal 2013). To determine which genes mediate the control of auxin on the expression of
MYB26 and on the synthesis of JA we are currently analysing the effects of the candidate
genes ARF8 and ARF6. Preliminary data obtained by the analysis of arf6 arf8 mutant lines
and ARF8ox and ARF6ox overexpressing lines will be presented.
Abstract P28
Cell surface proteins mediating gamete interaction in Arabidopsis
1
2
1
2
Thomas Hackenberg , Christine Glaesser , Lucija Soljic , Klaus F.X. Mayer , Stefanie
1
Sprunck
1
2
Cell Biology and Plant Biochemistry, University of Regensburg, Germany, Bioinformatics and Systems
Biology, Helmholtz Zentrum Muenchen, Germany
Contrary to animals, flowering plant sexual reproduction encompasses two distinct gamete
fusion events. After the sperm cell pair is delivered into the embryo sac, one sperm cell fuses
with the egg cell, while the second sperm cell fuses with the central cell. Microscopically,
three major events can be distinguished during this double fertilization process: (i) rapid
sperm cell delivery to the fusion site, (ii) a short phase of sperm cell immobility at the fusion
site (spanning a couple of minutes), and (iii) movement of the sperm nuclei, indicating
ongoing plasmo- and karyogamy. However, the underlying molecular mechanisms of
flowering plant gamete fusions remain almost elusive.
We aim to identify membrane-associated proteins involved in gamete adhesion, recognition,
or fusion. By analysing transcriptome data from isolated female gametes we discovered
genes encoding putative cell surface-localized proteins. Some of these proteins exhibit
features of protein classes known to be involved in sperm-egg interaction in other species.
75
Currently, we are analysing a set of promising and preferentially female gamete-specific
expressed candidate genes regarding their expression, subcellular localization, and impact on
gamete fusion. These include receptor(-like) kinases, glucosidases, and proteins of unknown
function.
Abstract P29
Advancement on isolated microspore culture in Citrus clementina Hort.
ex Tan., cvs. Monreal Rosso and Nules
Marines Marli Gniech Karasawa, Ahmed Abdelgalel, Benedetta Chiancone, Maria Antonietta
Germanà
Università degli Studi di Palermo, Dipartimento di Scienze Agrarie e Forestali Viale delle Scienze, 11.
90128 Palermo, Italy.
Haploid (Hs) and douled-haploid (DHs) technology represents a valuable and powerful tool
for breeding programs; in fact, it allows, in one step, the obtaining of complete homozygous
lines from heterozygous parents, through gametic embryogenesis (Germanà 2011b). This
technology is particularly useful for woody species, like Citrus, characterized by a long
juvenile stage, high degree of heterozygosity and often by self-incompatibility (Germanà 2006,
2009, 2011).
In vitro anther or isolated microspore culture are the most effective and widely used methods
to induce gametic embryogenesis. Even if isolated microspore culture is more time
consuming and requires better equipment than anther culture, it permits a better
understanding of the pollen embryogenesis process from cellular, physiological, biochemical,
and molecular point of view, without the influence of the anther somatic tissue.
The efficiency of gametic embryogenesis is influenced by several factors, among others, by
the culture medium composition and in particular, by the type and the concentration of plant
growth regulators. Meta-topolin, a naturally occurring aromatic cytokinins, is considered an
alternative to benzyladenine (BA), zeatin (ZEA) and kinetin (KIN) in plant tissue culture
(Aremu et al., 2012). It has been used, mainly, to increase the efficiency in in vitro plant
propagation of several species, among them also Citrus (Niedz and Evens, 2011), but never,
in our knowledge, to induce gametic embryogenesis from isolated microspores.
In this study, carried out on Citrus clementina Hort. ex Tan., cultivars Monreal Rosso and
Nules, the effect of the presence in the culture media of meta-topolin in substitution of BA or
ZEA, on pollen embryogenesis induction, through isolated microspore culture, was
investigated.
After five months of culture, for both genotypes and for all the media tested, different
structural features have been observed and registered: microspores uninucleated, with no
development, binucleated with two asymmetrical nuclei (normal gametophytic pathway: one
vegetative and one generative nucleus), binucleated with two equal-size vegetative-type
nuclei that had just started their sporophytic pathway, trinucleated, tetranucleated and
multinucleated. The sporophytic development of isolated microspores in culture has been
confirmed by the presence, in the culture, of calli and embryos at different stage.
The results presented represent an advancement in the knowledge of pollen embryogenesis
in Citrus clementina Hort. ex Tan., in fact, this is the first time that the regeneration of
embryos from Monreal Rosso and Nules isolated microspores is reported.
References
Aremu A.O., Bairu M.W., Doležal K., Finnie J.F., Van Staden J. (2012). Tissue and Organ Culture
108:1-16.
Germanà M.A. (2011) Special issue Plant Cell Reports: “Plant Biotechnology in support of the Millenium
Development Goals” 30, Issue: 5, pp. 839 - 857.
Germana` MA (2006). Plant Cell Tiss Org 86: 131-146.
76
Germanà M.A. (2009). In: Touraev A, Forster B, Jain M (eds) Advances in haploid production in higher
plants, Springer-Verlag, Heidelberg, Berlin, pp 241-263.
Germanà M.A. (2011). Special issue Plant Cell Reports: “Plant Biotechnology in support of the
Millenium Development Goals” 30, Issue: 5, pp. 839 - 857.
Niedz, R.P., Evens, T.J. (2011). ARPN Journal of Agricultural and Biological Science. 6: 64-73.
ACKNOWLEDGMENTS
This study has been partially supported by the projects: PON01_01623IT Citrus Genomics and thanks
are due to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarship
to Marines Marli Gniech Karasawa.
Abstract P30
Study on anther culture in hazelnut (Corylus avellana L.)
Benedetta Chiancone, Marines Marli Gniech Karasawa, Ahmed Abdelgalel, Maria Antonietta
Germanà.
Università degli Studi di Palermo, Dipartimento di Scienze Agrarie e Forestali Viale delle Scienze, 11.
90128 Palermo, Italy.
The European hazelnut (Corylus avellana L.), diploid species (2 n= 2x= 22) with a small
genome size (estimated to be 0.48 pg per 1C nucleus), is a monoecious, dichogamous, selfincompatible and wind pollinated plant (Rovira et al. 1993). Although hazelnut is the fourth
tree nut produced worldwide (FAOSTAT 2013), behind cashew (Anacardium occidentale L.),
almond (Prunus dulcis (Miller) D.A. Webb), and Persian walnut (Juglans regia L.), genetic
improvement efforts were started only recently (Mehlenbacher et al., 2006).
Biotechnologies and particularly haploid (H) and doubled haploid (DH) technology can
support the traditional breeding of this high value crop, because the production of
homozygosity in one step (rather than through several generations of selfing), accelerates the
breeding times. In woody species, such as hazelnut, generally characterized by a long
juvenile periods, a high degree of heterozygosity, large size and, often, self-incompatibility,
there is not a different way than the gametic embryogenesis to obtain homozygous breeding
lines (Germanà 2011a; 2011b).
This research was carried out to study the gametic embryogenesis in hazelnut, evaluating
some of the factors that influence this process: genotype and temperature stresses.
Particularly, after a morphological characterization of flower buds, anthers of six hazelnut
cultivars (Carrello, Gentile romana, Imperatrice Eugenia, Meraviglia de Bollwiller, Nostrana
and Tonda romana) were put in culture and, just after, they were subjected to two different
thermal stresses: 60 min at 35°C and 30 min at -20°C.
Microscopical observations have been carried out during the culture by fluorescence
microscope, after 4',6-diamidino-2-phenylindole staining. It was possible to observe both the
gametophytic pathway with the presence of bicellular pollen, but also, the sporophytic
pathway with the presence of bicellular (with symmetrical nucleus division), tricellular and
multicellular structures.
After seven months of culture, different features of anthers were observed: stopped, swollen
and with callus. The analysis of data recorded demonstrated that there is an important
interaction between the cultivar and the type of thermal stress; in fact, each cultivar
responded differently in dependence of the considered temperature.
This research, in our knowledge, represents the first study reported on anther culture in
hazelnut. Further studies are, however, necessary to better understand the process of
gametic embryogenesis in this species.
References
Germanà M.A. (2011a). Anther culture for haploid and doubled haploid production. Special issue: "In
Vitro Ploidy Manipulation in the Genomics Era". Plant Cell, Tissue and Organ Culture Volume 104,
Number 3, 283-300, DOI: 10.1007/s11240-010-9852-z
77
Germanà M.A. (2011b) Gametic embryogenesis and haploid technology as valuable support to plant
breeding. Special issue Plant Cell Reports: “Plant Biotechnology in support of the Millenium
Development Goals” 30, Issue: 5, pp. 839 - 857.
Mehlenbacher, S.A., R.N. Brown, E.R. Nouhra, T. Gokirmak, N.V. Bassil and T.L. Kubisiak. 2006. A
genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers. Genome
49:122–133.
Rovira, M., N. Aleta, E. Germain, and P. Arus. 1993. Inheritance and linkage relationships of ten
isozyme genes in hazelnut. Theor. Appl. Genet. 86:322–328
ACKNOWLEDGMENTS
Thanks are due to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the
scholarship to Marines Marli Gniech Karasawa.
Abstract P31
n-Butanol - induces bread wheat microspore embryogenesis by
microtubules depolymerization
1
2
2
A.M. Castillo , E. Dubas , I. Zur , & M.P. Vallés
1
1
Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, EEAD-CSIC, Av.
2
Montañana 1005, 50059 Zaragoza, SPAIN, The Franciszek Górski Institute of Plant Physiology,
Polish Academy of Sciences, Niezapominajek 21, 30-239 Kraków, POLAND
Microspore embryogenesis is the process in which stress-induced immature pollen develop to
embryo-like structures with ability to regenerate green sporophytes. One of the first
indications of microspore reprogramming is the cytoskeletal reorganization, where
microtubules (MTs) play a central role in the nuclear migration and the first sporophytic
division. To induce microspore embryogenesis MTs depolymerising substances have been
assayed. The application of the buthyl alcohol n-butanol, a cortical MT-depolymerizing agent,
to mannitol stressed anthers triggered microspore embryogenesis leading to a significant
increase in the number of bread wheat green DH plants (Soriano et al 2008; Broughton et al
2011).
In order to examine precisely the effect of n-butanol on MTs during microspore
embryogenesis induction in bread wheat (Triticum aestivum L.), a ‘whole mount’
immunolocalization protocol with confocal laser scanning microscopy were applied (Dubas et
al. 2011, 2013). Mannitol stress-treated anthers were incubated for 4h with 0.2 % n-butanol. For
the first time, 3-D visualization showed the MTs reorganization in the n-butanol treated
microspores induced to embryogenesis. Endoplasmic and cortical MTs depolymerization and
re-polymerization were observed.
ACKNOWLEDGMENTS
This work was supported by Project AGL2010-17509, from ‘Plan Nacional de Recursos y Tecnologías
Agroalimentarias’ of Spain, the Bilateral Project CSIC (Spain)-PAS (Poland) 2010PL0006 and by COST
Action FAO0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI).
REFERENCES
Broughton S (2011) The application of n-butanol improves embryo and green plant production in anther
culture of Australian wheat (Triticum aestivum L.) genotypes. Crop Pasture Sci 62(10) 813-822
Dubas E, Custers J, Kieft H, Wędzony M, van Lammeren AAM (2011) Microtubule configurations and
nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas
microspores. Plant Cell Rep 30:2105–2116
Dubas E, Custers J, Kieft H, Wędzony M, van Lammeren AAM (2013) Characterization of polarity
development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in
Brassica napus L. Protoplasma. Doi:10.1007/s00709-013-0530-y
Soriano M, Cistué L, Castillo AM (2008) Enhanced induction of microspore embryogenesis after nbutanol treatment in wheat (Triticum aestivum L.) anther culture. Plant Cell Rep 27:805-811
78
Abstract P32
Effects of n-Butanol on Maize Anther Culture - Androgenic Response
and Cytological Aspects
1
1
1
1
2
A. Fábián , P. K. Földesiné Füredi , H. Ambrus , K. Jäger , L. Szabó & B. Barnabás
1
1
Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences,
2
Martonvásár, HUNGARY Institute of Materials and Environmental Chemistry, Research
Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, HUNGARY
Production of double haploid (DH) plants is a valuable tool in conventional breeding. This
technique reduces the time needed for the development of new, improved varieties.
Application of androgenic anther or microspore culture is the most widely used method for
double haploid production, yielding diploid homozygous plants in large number and with high
genetic variability. Improvement of androgenic cultures can be achieved by the enhancement
of embryogenic induction frequency, which can be carried out by various pretreatments (e.g.:
heat, cold, starvation). Application of a biogenic alcohol, n-butanol was reported previously to
elevate the proportion of embryogenic microspores in wheat anther culture. N-butanol
inhibited the production of a signaling phospholipid, phosphatidic acid (PA), catalysed by
phospholipase D (PLD), and hence caused reversible microtubule depolymerisation and/or
the release of cortical microtubules from the plasma membrane in Arabidopsis BY-2 cell
culture. Besides this, high levels of PA was reported to enhance the amount of filamentous Factin in Arabidopsis epidermal cells.
In order to test the effect of n-butanol on maize anther cultures, anthers were treated with 0.2%
n-butanol for 6 and 18 hours. Treatments reduced the ratio of viable microspores, and in
parallel, increased the proportion of symmetric microspore divisions, which is thought to be a
relevant marker of embryonic induction. Embryo yield was significantly increased by n-butanol
treatments as well. To examine the effects of cold pretreatment and n-butanol
supplementation of culture media on cytoskeletal structure, microtubules and actin filaments
were visualized by confocal laser scanning microscopy. Ultrastructural changes were studied
by transmission electron microscopy. Cold pretreatment resulted in an elevated amount of
actin filaments, whereas microtubule network remained unaffected. N-butanol treatment
caused depolymerisation of microtubules to various extents, but had no effect on actin
cytoskeleton. Transmission electron microscopy revealed that n-butanol caused toxic effects
and elevated the amount of lipid bodies in treated microspores. Our results support the
concept that switching of gametophytic development of microspores to embryogenic pathway
in androgenic cultures is assisted by cytoskeletal rearrangements. Alterations of cytoskeleton
network induced by cold pretreatment and n-butanol may alter the polarized state of the
cytoplasm in maize microspores and facilitate embryogenic response by the promotion of
symmetric divisions.
Acknowledgements
Authors wish to thank for the support of Hungarian Scientific Research Fund grant No. OTKA 80260
Abstract P33
Endogenous auxin and ABA in microspore embryogenesis of
oilseed rape (Brassica napus L.)
1
2
1
1
1
1
E. Dubas , E. Benkova , F. Janowiak , P. Waligorski , M. Dziurka , M. Krzewska , I. Żur
1
1
The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21,
2
30-239 Kraków, POLAND, Plant Systems Biology Department (VIB) Institute of Ghent University in
Ghent, Technologiepark 927, 9052 Zwijnaarde – Gent, BELGIUM
* Corresponding author; e-mail: dubas@ifr-pan.krakow.pl
Under stress-inducible in vitro conditions, the phytohormones auxin and abscisic acid (ABA)
are involved in the developmental transition from the microspore into the embryo. However,
79
little is known about endogenous levels of these regulators at the initial stages of
embryogenesis. A better understanding of auxin/ABA role and the mechanism of their action
in the process of microspore embryogenesis (ME) could bring significant progress to the
utilization of doubled haploid (DH) technology. Brassica napus microspore suspensions are
considered to be a perfect model for such a study.
In the present study, two oilseed rape genotypes with different embryogenic capability were
used. Endogenous auxin was purified and measured by common chromatographic technique.
ABA content was evaluated by ELISA. Both hormones were extracted from microspores (mcs)
isolated from plants growing at different temperatures (18°C or 10°C) and collected from the
subsequent in vitro culture conditions (isolation day, 1d and 5d at 18°C, 1d and 5d at 32°C).
-1
4
-1
Auxin concentration (ng mg protein) and ABA content (fmol per 10 mcs or pmol g FW)
were measured in mcs for the first time. In general, the level of hormones depends
significantly on the genotype and the treatment. Auxin derivate IBA (indole-3-butyric acid) was
the main auxin form that prevailed in isolated mcs. 24 h of heat shock significantly increased
IBA concentration (5-fold) and ABA content (2-fold) in mcs of the highly embryogenic line.
Assuming a mean mcs radius of 10 µm, ABA content corresponded to ABA concentration of
2.1 µM. Extended treatment of mcs at high temperature (32°C) was required for IBA increase
in mcs of the non-embryogenic cultivar. Prolonged heat shock significantly improved mcs
embryogenesis in the non-responsive genotype. Based on these observations, we found a
positive correlation between auxin level and efficient induction of embryogenesis. Heat
induced increase of ABA content in mcs had no clear-cut impact on androgenesis.
Our findings point to possible importance of endogenous auxin and ABA in microspore
embryogenesis induction. Presented results suggest a more complex mechanism of
embryogenesis process initiation.
ACKNOWLEDGMENTS
This work was supported by an agreement with the Polish Academy of Sciences (PAS) – The Research
Foundation-Flanders (FWO) in frame of the joint Polish-Belgian project: ‘Auxin as a trigger in double
haploids (DHs) production of oilseed rape’ and the national project 2011/01/D/NZ9/02547.
REFERENCES
Dubas E, Janowiak F, Krzewska M, Hura T, Żur I (2013) Endogenous ABA concentration and
cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica napus L.) genotypes differing
in responsiveness to androgenesis induction. Plant Cell Rep. doi:10.1007/s00299-013-1458-6
Abstract P34
AINTEGUMENTA-LIKE and HOMEODOMAIN GLABROUS transcription
factors have antagonistic functions in the control of cell proliferation
A. Horstman, H. Fukuoka, P. Passarinho, M. Weemen, G. Sanchez-Perez, L. Nitsch, G.
Angenent, R. Immink, K. Boutilier
Embryogenesis in plants is normally restricted to zygotic embryo development, which takes place in the
seed after fertilization. Embryogenesis can also be induced in vitro from both gametophytic and somatic
cells. Somatic embryogenesis (SE) is induced by stress conditions and/or exogenous growth regulators,
however ectopic expression of the AP2/ERF transcription factor BABY BOOM (BBM) is also sufficient to
induce SE from cotyledons and leaves of Arabidopsis seedlings in the absence of growth regulators. We
show that also other members of the AINTEGUMENTA-like (AIL) gene family to which BBM belongs are
able to induce SE.
To gain more insight into the BBM-mediated SE, we have studied BBM-interacting proteins. We have
shown that BBM interacts with several HOMEODOMAIN GLABROUS (HDG) transcription factors.
These HDG proteins are expressed in the L1 layer throughout the plant and have redundant functions in
establishing the epidermis and its specialized structures.
We have observed that ectopic overexpression of HDG proteins leads to root and shoot meristem
arrest, similar to what has been reported for double/triple ail mutants. On the other hand, downregulation of multiple HDG genes leads to ectopic meristems and somatic embryo formation, similar to
80
BBM (AIL) overexpression. These results suggest opposite roles for AIL and HDG proteins, with AILs
promote meristem activity, while HDGs stimulate meristem differentiation.
Abstract P35
WDR55 interacts with DDB1 and is required for apical patterning in the
Arabidopsis embryo
1
2
3
4
3
Katrine N. Bjerkan , Sabrina Jung , Veronica Gregis , Gerd Jürgens , Martin M. Kater ,
2
1
Pascal Genschik and Paul E. Grini
1
2
Department of Biosciences, University of Oslo, 0316 Oslo, Norway, Institut de Biologie Moléculaire
des Plantes, Centre National de la Recherche Scientifique UPR2357, 67084 Strasbourg, France,
3
Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, 20133
4
Milan, Italy. Department of Cell Biology, Max Planck Institute for Developmental Biology, 72076
Tübingen, Germany.
Plant reproduction and the development of a seed require coordinated regulation of genes
and gene products in gametophytes as well as in the different organisms of the seed. Protein
ubiquitination by cullin (CUL)-RING E3 ligases (CRLs) regulates an extensive range of
biological processes by attachment of ubiquitin to substrate proteins to either promote their
degradation by the UBIQUTIN-26S proteasome pathway, or by changing their function or
chromatin context. CRL4 ligases were recently shown to exert their specificity through the
binding of various substrate receptors, which bind the CUL4 interactor DDB1 through a DWD
or a WDxR motif. In a segregation-based mutagenesis screen we identified a WDxR motifcontaining protein (WDR55) required for male and female gametogenesis and seed
development. We demonstrate that WDR55 physically interact with DDB1A in planta,
suggesting WDR55 to be a novel substrate recruiter in CRL4 ubiquitin ligase complexes.
Examination of the mutant allele wdr55-1 revealed a delay in the fusion of the polar nuclei in
embryo sac development, in addition to embryo and endosperm developmental arrest.
Interestingly, the observed embryo and endosperm phenotype is reminiscent to CUL4 and
WDR55
ligase
DDB1A/B loss of function, in support of a regulatory role of a putative CUL4
complex. wdr55-2 embryos suggest a defect in the transition to bilateral symmetry in the
apical embryo domain. Auxin distribution in the wdr55-2 embryo by means of the synthetic
DR5 reporter appears not to be affected. However, the lack of bilateral symmetry and further
localization failure of DORNROESCHEN, a direct target of the auxin response factor protein
MONOPTEROS, may suggest a WDR55 function in targeting genetic components regulated
by auxin. Currently, we have isolated a homozygous WDR55 knockout, and here we report
that the adult plants display pleiotropic phenotype characteristics of which many are
reminiscent of mutants in auxin regulated pathways.
Abstract P36
IDA and IDL peptides in plant reproduction
1
1
1
Ida Myhrer Stø , Ullrich Herrmann , Paul E. Grini , Reidunn Aalen
1
1
Department of Biosciences, University of Oslo, Norway
Cell-separation events that break down the matrix between adjacent cells are important
throughout the life cycle of plants. It is needed for germination, the shaping of the plants form,
for pollen and seed dispersal, and it is also a means to get rid of organs that are no longer of
use. In Arabidopsis floral organs abscise after pollination, and this cell separation event is
controlled by the peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) which
signals through the leucine-rich receptor-like kinases HAESA (HAE) and HAESA-LIKE2
(HSL2), and a downstream MAP kinase cascade .IDA and its receptors are also involved in
separation of cells overlying emerging lateral roots.
81
IDA belongs to a small gene family of IDA-LIKE genes. We have hypothesized that they also
are involved in cell separation processes and have therefore wanted to generate double
mutants. Investigations of single mutants show no visible phenotype in the seed, but
surprisingly the cross between IDA and IDL4, did not generate any homozygous plants.
Reciprocal crosses of the double hemizygote to wildtype indicated developmental problems in
both the male and female gametophyte.
We are currently working on finding the mechanisms giving rise to these developmental
problems, and also the role of IDA and IDLs in reproduction.
Abstract P37
Seed size regulation in Arabidopsis thaliana
1
1
1
1
2
D. Paolo , G. Orozco-Arroyo , Ignacio Ezquer , C. Mizzotti , B. Ambrose & L. Colombo
1
1
2
Università degli Studi di Milano, Dipartimento di BioScienze, Milano, ITALIA The New York Botanical
Garden, Department of Plant Genomics, New York City, U.S.A.
Seed yield and seed size regulation represents one of the major goals in the study of plant
development, given the fundamental role that seeds have in plant reproduction and in food
production. We use Arabidopsis thaliana as model to investigate on the transcriptional and
hormonal regulation of the development of the seed coat, that is the part that mostly
influences the final seed size. We propose novel roles for MADS-domain transcription factors
that were previously reported to act very early in organ identity determination. We show that
the ovule identity gene SEEDSTICK (STK) is involved in the maternal control of seed
development, specifically promoting cell expansion in the seed coat, as we revealed by the
analysis of the stk mutant and through other genetic data. Moreover, RNA-seq data suggest
an involvement of STK in the regulation of different metabolic pathways including
carbohydrate metabolism, the synthesis of the cell wall as well as in cell cycle regulation. We
propose that another MADS-box gene, GORDITA (GOA) acts independently of STK in the
repression of seed coat cell expansion, similarly to its previously reported function in fruit
development. Protein-protein interaction experiments, expression data and analysis of seed
morphology for different combination of mutants further suggest that the expansion
mechanism that we propose could be triggered by hormonal signaling pathways downstream
of brassinosteroids and auxin, mainly through the regulation of another transcription factor,
AUXIN RESPONSE FACTOR 2 (ARF2).
Abstract P38
Genetic basis of postzygotic hybridization barriers in Capsella
Carolin Anna Rebernig, Claudia Köhler
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural
Sciences and Linnean Center for Plant Biology, PO-Box 7080, SE-75007 Uppsala, Sweden
Reproductive isolation among most plant species is established by different mechanisms that
can act either pre- or postzygotically. One important postzygotic barrier is caused by
malfunction of the endosperm. The endosperm is a terminal nutritive tissue supporting
embryo growth that is consumed by the embryo during seed development or after
germination. The endosperm is essential for viable seed formation and therefore,
mechanisms disrupting endosperm development play a major role in reproductive isolation.
Similar reciprocal effects on endosperm development have been reported in response to
interploidy crosses, suggesting that unbalanced parent-of-origin specific genes are the
underlying cause for endosperm failure in response to interspecies hybridizations.
A well-known phenomenon observed in interspecies hybridization is that the degree of seed
failure due to endosperm breakdown is depending on the direction of the cross. We show that
in the genus Capsella reciprocal crosses between outcrossing C. grandiflora and selfing C.
82
rubella result in clearly different seed abortion rates. We characterized seed development in
the hybrid crosses and aim for mapping the locus/loci that causes hybrid seed failure in
Capsella.
Abstract P39
Timing mechanism by cell cycle-dependent polycomb eviction in plant
stem cells
1
1,2
1
1,2
1,2
1,2
Bo Sun , Liang-Sheng Looi , Siyi Guo , Zemiao He , Eng-Seng Gan , Jiangbo Huang ,
1
1
1,2
Yifeng Xu , Wanyi Wee and Toshiro Ito *
1
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604,
2
Republic of Singapore. Department of Biological Sciences, National University of Singapore.
*Correspondence to: itot@tll.org.sg
Plant floral stem cells and most animal precursor cells divide a limited number of times before
they stop and terminally differentiate, but the mechanisms that control when the cells stop
dividing and differentiate remain unclear. The precise temporal induction of the Arabidopsis
zinc finger repressor KNUCKLES (KNU) is essential for the coordinated growth and
differentiation of floral stem cells. Herein, we identify an intrinsic epigenetic timer by showing
that the floral homeotic protein AGAMOUS causes the eviction of the Polycomb group
proteins from KNU, leading to delayed KNU activation, which is associated with cell division.
Furthermore, a synthetic promoter simulating PcG eviction recapitulated the timed gene
activation. These analyses demonstrate that floral stem cells measure time by a divisioncounting epigenetic timer triggered by Polycomb eviction.
Abstract P40
Auxin dependent patterning is mediated by distinct ARF/bHLH modules
Tatyana Radoeva, Cristina Llavata-Peris, Annemarie Lokerse, Jos Wendrich & Dolf Weijers
Wageningen University, Laboratory of Biochemistry, Wageningen, The NETHERLANDS
The plant hormone auxin is required for many aspects of plant growth and development.
Recently, it was found that inhibition of auxin response in suspensor cells induces
transformation of suspensor cells to embryonic cells (1). Using a transcriptomics approach, a
small set of bHLH genes was identified that are: a) upregulated upon auxin response
inhibition and normally expressed in the proembryo or b) downregulated upon auxin inhibition
and normally expressed in the suspensor. Detailed investigation of gain-of-function and lossof-function mutants of these genes strongly suggests that their function is required for
controlling normal suspensor and hypophysis development. Strikingly, ectopic expression of
one of these is by itself sufficient for inducing embryo-like structures in suspensor cells.
Moreover, we show that these genes act downstream of auxin signaling, and proper auxin
signaling is indispensable for the regulation of these genes. Hence, this work identifies a
novel ARF-bHLH module that operates in parallel to the previously identified ARF5/MPTMO5/TMO7 module that operates in patterning the embryo (2,3). We conclude that auxinARF-bHLH modules are conserved elements that mediate auxin-dependent plant
development.
References:
(1) Rademacher, E. H., A. S. Lokerse, A. Schlereth, C. I. Llavata-Peris, M. Bayer, M. Kientz, A.
Freire Rios, J. W. Borst, W. Lukowitz, G. Jurgens and D. Weijers (2012). "Different auxin
response machineries control distinct cell fates in the early plant embryo." Dev Cell 22(1): 211222
(2) Schlereth, A., B. Moller, W. Liu, M. Kientz, J. Flipse, E. H. Rademacher, M. Schmid, G.
Jurgens and D. Weijers (2010). "MONOPTEROS controls embryonic root initiation by
regulating a mobile transcription factor." Nature 464(7290): 913-916.
83
(3) De Rybel, B., B. Moller, S. Yoshida, I. Grabowicz, P. Barbier de Reuille, S. Boeren, R. S. Smith,
J. W. Borst and D. Weijers (2013). "A bHLH complex controls embryonic vascular tissue
establishment and indeterminate growth in Arabidopsis." Dev Cell 24(4): 426-437.
84
List of Participants
Aalen Reidunn Birgitta
University of Oslo
Oslo, NORWAY
reidunn.aalen@ibv.uio.no
Bousquet-Antonelli Cecile
Laboratory of Plant and Genome
Development-CNRS
Perpignan, FRANCE
cecile.antonelli@univ-perp.fr
Adriaanse Marcel Bejo
Zaden BV
Warmenhuizen, NETHERLANDS
m.adriaanse@bejo.nl
Boutilier Kim
Plant Research International
Wageningen, NETHERLANDS
kim.boutilier@wur.nl
Albertini Emidio
University of Perugia
Perugia, ITALY
emidio.albertini@unipg.it
Brysting Anne
University of Oslo
Oslo, NORWAY
a.k.brysting@ibv.uio.no
Andersen Ellen D.
University of Oslo
Oslo, NORWAY
ellen.d.andersen@gmail.com
Cardarelli Maura
Sapienza University of Rome
Rome, ITALY
maura.cardarelli@uniroma1.it
Balanza Vicente
KeyGene NV
Wageningen, NETHERLANDS
vtebalan@gmail.com
Carman John
Utah State University
Logan, Utah, UNITED STATES
john.carman@usu.edu
Barcaccia Gianni
University of Padova
Padova, ITALY
gianni.barcaccia@unipd.it
Coimbra Sílvia
University of Porto
Porto, PORTUGAL
scoimbra@fc.up.pt
Beata Barnabas
Hungarian Academy of Sciences
Martonvasar, HUNGARY
barnabas.beata@gmail.com
Colombo Lucia
Dipartimento di Bioscienze
Milano, ITALY
lucia.colombo@unimi.it
Berger Frederic
Temasek Life Sciences Laboratory
Singapore, SINGAPORE
fred@tll.org.sg
Cucinotta Mara
Università degli Studi di Milano
Milano, ITALY
mara.cucinotta@unimi.it
Bjerkan Katrine Nergård
University of Oslo
Oslo, NORWAY
katrine.bjerkan@ibv.uio.no
De Storme Nico
University of Ghent
Ghent, BELGIUM
nico.destorme@ugent.be
Bocchini Marika
University of Perugia
Santa Maria degli Angeli, ITALY
maryk87@hotmail.it
De Vries Sacco
Wageningen University
Wageningen, NETHERLANDS
sacco.devries@wur.nl
Borg Michael
University of Leicester
Leicester, UNITED KINGDOM
mb307@le.ac.uk
Demko Viktor
Norwegian University of Life Sciences
Ås, NORWAY
viktor.demko@umb.no
85
Dresselhaus Thomas
University of Regensburg
Regensburg, GERMANY
thomas.dresselhaus@ur.de
Grini Paul E.
University of Oslo
Oslo, NORWAY
p.e.grini@ibv.uio.no
Dubas Ewa
Polish Academy of Sciences
Kraków, POLAND
edubas@o2.pl
Groß-Hardt Rita
University of Bremen
Tübingen, GERMANY
rita.gross-hardt@zmbp.uni-tuebingen.de
Echenique Carmen V.
UNS/CONICET
Bahía Blanca, Bs. As., ARGENTINA
echeniq@criba.edu.ar
Grossniklaus Ueli
University of Zürich
Zürich, SWITZERLAND
grossnik@botinstuzh.ch
Ergon Åshild
Norwegian University of Life Sciences
Ås, NORWAY
ashild.ergon@umb.no
Gutierrez-Marcos Jose
University of Warwick
Warwickshire, UNITED KINGDOM
j.f.gutierrez-marcos@warwick.ac.uk
Fábián Attila
Hungarian Academy of Sciences
Martonvasar, HUNGARY
fabian.attila@agrar.mta.hu
Hackenberg Thomas
University of Regensburg
Regensburg, GERMANY
thomas.hackenberg@biologie.uniregensburg.de
Feijo Jose
University of Maryland
Oeiras, PORTUGAL
jfeijo@igc.pt
Galla Giulio
University of Padova
Padova, ITALY
giulio.galla@unipd.it
Geelen Danny
Ghent University
Ghent, BELGIUM
danny.geelen@ugent.be
Gehring Mary
Whitehead Institute
Cambridge, MA, UNITED STATES
mgehring@wi.mit.edu
Germanà Maria Antonietta
University of Palermo
Palermo, ITALY
agermana@unipa.it
Gibalova Antonia
Institute of Experimental Botany ASCR
Prague, CZECH REPUBLIC
gibalova@ueb.cas.cz
Glöckle Barbara
University of Oslo
Oslo, NORWAY
b.gloeckle@gmx.de
Hilpert Stefanie
IPK Gatersleben
Gatersleben, GERMANY
hilpert@ipk-gatersleben.de
Hojsgaard Diego
Georg-August University of Göttingen
Göttingen, GERMANY
diego.hojsgaard@biologie.unigoettingen.de
Honys David
Institute of Experimental Botany ASCR
Prague, CZECH REPUBLIC
david@ueb.cas.cz
Hornslien Karina
University of Oslo
Oslo, NORWAY
k.s.hornslien@ibv.uio.no
Horstman Anneke
Plant Research International
Wageningen, NETHERLANDS
anneke.horstman@wur.nl
Hörandl Elvira
Georg-August-University of Göttingen
Göttingen, GERMANY
elvira.hoerandl@biologie.unigoettingen.de
86
Ingouff Mathieu
University Montpellier II-IRD
Montpellier cedex 5, FRANCE
mathieu.ingouff@ird.fr
Jiang Hua
Swedish University of Agricultural
Sciences
Uppsala, SWEDEN
hua.jiang@slu.se
Johannessen Ida Marie
University of Oslo
Oslo, NORWAY
i.m.johannessen@ibv.uio.no
Johnston Amal
University of Heidelberg
Heidelberg, GERMANY
amal.johnston@cos.uni-heidelberg.de
Jäger Katalin
Hungarian Academy of Sciences
Martonvasar, HUNGARY
katajager@gmail.com
Kalantarian Maryam
University of Oslo
Oslo, NORWAY
maryam.kalantarian@ibv.uio.no
Karasawa Marines Marli Gniech
University of Palermo
Palermo, ITALY
mgniechk@hotmail.com
Kawadza Dave
North-West University
Northwest, SOUTH AFRICA
dave.kawadza@nwu.ac.za
Kirioukhova Olga
University of Heidelberg
Heidelberg, GERMANY
olga.kirioukhova@cos.uni-heidelberg.de
Kittelsen Lene A.
University of Oslo
Oslo, NORWAY
l.a.kittelsen@usit.uio.no
Koltunow Anna
CSIRO
South Australia, AUSTRALIA
anna.koltunow@csiro.au
Kosturkova Georgina
Bulgarian Academy of Sciences
Sofia, BULGARIA
georgina_kosturkova@abv.bg
Kritsas Konstantinos
University of Zürich
Zürich, SWITZERLAND
k_kritsas@access.uzh.ch
Köhler Claudia
Swedish University of Agricultural
Sciences
Uppsala, SWEDEN
claudia.kohler@slu.se
Lafon Placette Clément
Swedish University of Agricultural
Sciences
Uppsala, SWEDEN
clement.lafon.placette@slu.se
Lawit Shai
DuPont Pioneer
Johnston, Iowa, UNITED STATES
shai.lawit@pioneer.com
Leon Gabriel
Universidad Andres Bello
Santiago, CHILE
gleon@unab.cl
Lepiniec Loïc
INRA
Versailles, France
loic.lepiniec@versailles.inra.fr
Lillo Cathrine
University of Stavanger
Stavanger, NORWAY
cathrine.lillo@uis.no
Lu Liu
Temasek Life Sciences Laboratory
Singapore, SINGAPORE
liulu@tll.org.sg
Lucca Maria Noel
Universidad Andres Bello
Santiago, CHILE
noel.lucca@gmail.com
Masiero Simona
Universita' degli Studi di Milano
Milano, ITALY
simona.masiero@unimi.it
Mau Martin
IPK Gatersleben
Gatersleben, GERMANY
mau@ipk-gatersleben.de
87
Mendes Marta Adelina
University of Milan
Milano, ITALY
marta.miranda@unimi.it
Muñoz-Strale Daniela
Universidad Andres Bello
Santiago, CHILE
danimunozs@gmail.com
Nielsen Anders
University of Oslo
Oslo, NORWAY
anders.nielsen@ibv.uio.no
Okuda Satohiro
Nagoya University
Nagoya, JAPAN
okuda@bio.nagoya-u.ac.jp
Olsen Odd-Arne
Norwegian University of Life Sciences
Ås, NORWAY
odd-arne.olsen@mb.no
Paczesniak Dorota
ETH Zurich
Duebendorf, SWITZERLAND
dorota.paczesniak@eawag.ch
Palanivelu Ravishankar
University of Arizona
Tucson, UNITED STATES
rpalaniv@ag.arizona.edu
Paolo Dario
Università degli Studi di Milano
Milano, ITALY
dario.paolo@unimi.it
Pereira Ana Marta
Faculdade de Ciências da Universidade
do Porto
Porto, PORTUGAL
ambacpereira@fc.up.pt
Perroud Pierre-Francois
Washington University in St. Louis
MO, UNITED STATES
perroud@biology2.wustl.edu
Pessino Silvina
Universidad Nacional de Rosario
Zavalla/Provincia de Santa Fe,
ARGENTINA
pessino@arnet.com.ar
stxgp6@nottingham.ac.uk
Radoeva Tatyana
Wageningen University
Wageningen, NETHERLANDS
tatyana.radoeva@wur.nl
Raissig Michael
University of Zürich
Zürich, SWITZERLAND
mraissig@access.uzh.ch
Rashal Isaak
University of Latvia
Salaspils, LATVIA
izaks@email.lubi.edu.lv
Rebernig Carolin Anna
Swedish University of Agricultural
Sciences
Uppsala, SWEDEN
carolin.rebernig@slu.se
Rizzo Paride
IPK Gatersleben
Stadt Seeland, GERMANY
rizzo@ipk-gatersleben.de
Rutley Nicholas
University of Leicester
Leicester, UNITED KINGDOM
njr24@le.ac.uk
Ruzvidzo Oziniel
North-West University
Northwest, SOUTH AFRICA
oziniel.ruzvidzo@nwu.ac.za
Sangwan Rajbir Singh
Université de Picardie Jules Verne
Amiens, FRANCE
rajbir.sangwan@u-picardie.fr
Schmidt Anja
University of Zürich
Zürich, SWITZERLAND
aschmidt@botinst.uzh.ch
Schnittger Arp
IBMP-CNRS
Strasbourg, FRANCE
arp.schnittger@ibmp-cnrs.unistra.fr
Scott Rod
University of Bath
Bath, UNITED KINGDOM
bssrjs@bath.ac.uk
Prasad Geeta
University of Nottingham
Nottingham, UNITED KINGDOM
88
Sezer Fatih
Çanakkale Onsekiz Mart University
Çanakkale, TURKEY
fsezerfatih@gmail.com
van Dijk Peter
Keygene
Wageningen, NETHERLANDS
peter.van-dijk@keygene.com
Sharbel Tim
IPK Gatersleben
Gatersleben, GERMANY
sharbel@ipk-gatersleben.de
Vivian-Smith Adam
Norwegian Forest and Landscape
Institute/Bioforsk
Ås, NORWAY
adam.vivian-smith@skogoglandskap.no
Soriano Castan Mercedes
Plant Research International
Wageningen, NETHERLANDS
mersorica@yahoo.es
Streibig Jens C.
University of Copenhagen
Copenhagen, DENMARK
jcs@life.ku.dk
Vo Dieu
University of Bremen
Tübingen, GERMANY
dieu.vo@zmbp.uni-tuebingen.de
Voloudakis Andreas
Agricultural University of Athens
Athens, GREECE
avoloud@aua.gr
Stø Ida Myhrer
University of Oslo
Oslo, NORWAY
i.m.sto@ibv.uio.no
Wibowo Anjar
University of Warwick
Coventry, UNITED KINGDOM
a.t.wibowo@warwick.ac.uk
Sun Bo
Temasek Life Sciences Laboratory
Singapore, SINGAPORE
sunbo@tll.org.sg
Wolff Philip
ETH Zürich / SLU Uppsala
Uppsala, SWEDEN
pwolff@ethz.ch
Taskin Kemal Melih
Çanakkale Onsekiz Mart University
Çanakkale, TURKEY
kmtaskin@gmail.com
Zur Iwona
Polish Academy of Sciences
Kraków, POLAND
zur@ifr-pan.krakow.pl
Tedeschi Francesca
IPK Gatersleben
Saxony-Anhalt, GERMANY
tedeschi@ipk-gatersleben.de
Özbilen Aslihan
Çanakkale Onsekiz Mart University
Çanakkale, TURKEY
aslihanozbilen@hotmail.com
Thorstensen Tage
Bioforsk
Ås, NORWAY
tage.thorstensen@bioforsk.no
Twell David
University of Leicester
Leicester, UNITED KINGDOM
twe@le.ac.uk
Ullmann Ingvild Falkum
University of Oslo
Oslo, NORWAY
i.f.ullmann@ibv.uio.no
Valles Maria Pilar
EEAD-CSIC
Zaragoza, SPAIN
valles@eead.csic.es
89
Notes
91
92
93