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

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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

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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-

methods

and

disciplines.

(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.

apomixis, respectively, which took place in Germany,

Emidio A bertini Chair COST FA0903

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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/

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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

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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

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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.

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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.

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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

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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.

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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

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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.

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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

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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

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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

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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

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26

27

Speaker Abstracts

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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, [email protected]

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.

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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. [email protected]

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.

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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.

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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.

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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

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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,

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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

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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.

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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.

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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.

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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.

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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.

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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.

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Chair: Mary Gehring

Session VIII: Fertilization Mechanisms Abstract S29

Nuclear Dynamics during Fertilization in Flowering Plants Frederic Berger Temasek Life Sciences Laboratory, Singapore ([email protected]); 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: [email protected]

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.

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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

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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

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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.

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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.

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Abstract S36

Epigenetic Regulation of Haploid Embryo Development Hui Li, Mercedes Soriano, Gerco C. Angenent and Kim Boutilier Plant Research International, e-mail: [email protected].

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.

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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. [email protected]

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

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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-

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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

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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.

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Poster abstracts

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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

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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

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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

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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.

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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.

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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.

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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 (