olomouc biotech 2013 plant biotechnology: green for good ii

Centre of the Region Haná

for Biotechnological and Agricultural Research

Olomouc Biotech 2013

Plant Biotechnology: Green for Good II

Book of Abstracts

June 17 – 21, 2013, Olomouc, Czech Republic

CENTRE OF THE REGION HANÁ FOR BIOTECHNOLOGICAL AND AGRICULTURAL

RESEARCH

OLOMOUC BIOTECH 2013

PLANT BIOTECHNOLOGY: GREEN FOR GOOD II

BOOK OF ABSTRACTS

OLOMOUC, JUNE 17 – 21, 2013

WWW.CR-HANÁ.EU

http://www.cr-haná.eu/

1st edition

Eds © Eva Tomaštíková & Karolina Chvátalová

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CONTENT

PROGRAM OF THE CONFERENCE 4

SCIENTIFIC COMMITTEE: 8

LOCAL ORGANIZING COMMITTEE – CR HANÁ OLOMOUC: 8

KEYNOTE LECTURE 10

ABSTRACTS – ORAL PRESENTATIONS 12

SESSION I: CROP IMPROVEMENT 12

SESSION II: BIO-TECHNO: INDUSTRY MEETS ACADEMIA 23

SESSION III: PLANT STRESS RESPONSES AND TOLERANCE 32

SESSION IV: PLANT BIOENERGETICS 46

ABSTRACTS – POSTER SESSION 52

LIST OF PARTICIPANTS 116

NOTES 120

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PROGRAM OF THE CONFERENCE

Monday June 17, 2013 10:00 - 14:00 Formal opening of C.R. Haná and press konference

14:00 – 16:00 Registration, poster mounting

16:00 – 17:00 Opening of the conference

Jan Káš, President of the Biotechnology Society of the Czech Republic, ICT

Prague, Czech Republic

Ivo Frébort, Executive Director of C.R. Haná, Olomouc, Czech Republic

Jaroslav Doležel, Scientific Director of C.R. Haná, Olomouc, Czech

Republic

17:00 – 18:30 Keynote lecture

Marc van Montagu, President of EFB, University of Gent, Belgium

30 years of transgenic plants

19:00 – 21:00 Welcome reception

Tuesday June 18, 2013 Session I - Crop improvement (Chair Ivo Frébort)

09:00 – 09:30 Rodomiro Ortiz, Swedish University of Agricultural Sciences, Alnarp,

Sweden

From crossbreeding to biotechnology-facilitated improvement of banana and

plantain

09:30 – 10:00 Kristin D. Bilyeu, USDA, Columbia, MO, USA

A ‘conventional’ success story for improving the functionality of soybean oil

10:00 - 10:30 Jaroslav Doležel, C.R. Haná, Olomouc, Czech Republic

Advances in plant chromosome genomics

10:30 - 11:00 Coffee and tea break

11:00 - 11:30 Ivo Frébort, C.R. Haná, Olomouc, Czech Republic

GM barley with modulated cytokinin level: aiming at improved yield and

stress tolerance

11:30 - 11:50 Takashi R. Endo, Kyoto University, Japan

Promotion of homoeologous paring by the rye B chromosomal segments in

common wheat

11:50 - 12:10 Michael Abrouk, C.R. Haná, Olomouc, Czech Republic

Evolution of Triticae genomes

12:10 - 13:30 Lunch break

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13:30 - 15:30 Guided excursion to the research facilities and experimental fields of C.R. Haná

15:30 - 16:00 Coffee and tea break, poster viewing

Session I - Crop improvement (Chair Jaroslav Doležel) 16:00 – 16:30 Francisco Pérez-Alfocea, CEBAS-CSIC, Murcía, Spain

Regulation of source-sink relations to improve crop salt-tolerance: a role for root-to-shoot signaling

16:30 – 17:00 Karel Doležal, C.R. Haná, Olomouc, Czech Republic New cytokinin derivatives for plant biotechnology and agriculture

17:00 - 17:20 Hans Motte, University College Ghent, Belgium A chemical screen identifies a cytokinin degradation inhibitor as a strong promoter of shoot regeneration in plant tissue culture

17:20 - 17:40 Helena Hanosová, C.R. Haná, Olomouc, Czech Republic Claviceps purpurea as a factory producing ergot alkaloids

Wednesday June 19, 2013 Session II - BIO-TECHNO: Industry meets academia (Chair Marc Van Montagu)

09:00 – 09:30 Silvia Travella, Plants for the Future, European Technology Platform,

Brussels, Belgium

The European Technology Platform ‘Plants for the Future’ – A European

Partnership for Research and Innovation

09:30 – 10:00 Fabio Fiorani, Jülich Plant Phenotyping Centre, Germany

Traits and technologies for plant phenotyping

10:00 - 10:30 Katsuyuki Tanizawa, Osaka University, Japan

Development of versatile bionanocapsules displaying immunoglobulin-

binding domains for application to immunosensors

10:30 – 11:00 Coffee and tea break

11:00 - 11:30 Ralph Hückelhoven, Technische Universität München, Freising, Germany

Disease susceptibility as a target of biotechnology for durable resistance?

11:30 - 12:00 Tony Bacic, University of Melbourne, Australia

The role of non-starchy polysaccharides (“soluble” dietary fibre), in particular

(1,3;1,4)-β- glucans and arabinoxylans, in promoting bowel health

12:00 - 12:30 M. Tufan Öz, C.R. Haná, Olomouc, Czech Republic

Antimicrobial peptide expression in barley endosperm

12:30 – 14:00 Lunch break

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14:00 - 14:30 Yuri Gleba, Nomad Bioscience and Icon Genetics, Munich, Germany

Plant Biotechnology Based on Transient Agrobacterium Delivery

14:30 - 15:00 Martin Trtílek, Photon Systems Instruments, Brno, Czech Republic

PlantScan- New tool in high throughput Phenotyping

15:00 - 17:30 Poster session and discussion

17:30 - 18.00 Awards of student poster contest (Marc Van Montagu and Jaroslav

Doležel)

Thursday June 20, 2013

Session III - Plant stress responses and tolerance (Chair Jozef Šamaj)

09:00 - 09:30 Heribert Hirt, EPSO and URGV Plant Genomics, Evry, France

New insights into plant-microbe signalling

09:30 - 10:00 Claus Wasternack, University of Halle, Germany

Action of jasmonates in stress responses and development - advanced

aspects

10:00 - 10:30 Marek Šebela, C.R. Haná, Olomouc, Czech Republic

MALDI mass spectrometry of intact fungal microorganisms


11:00 - 11:30 Tibor Pechan, Mississippi State University, USA

Maize defense against insect and fungus attacks

11:30 - 11:50 Roman Hobza, C.R. Haná, Olomouc, Czech Republic

Heavy metal stress in plants – from weeds to crops

11:50 - 12:10 Pavel Vítámvás, Crop Research Institute, Prague, Czech Republic

Cereals proteome changes under cold stress

12:10 - 12:30 Palmiro Poltronieri, CNR-ISPA, Lecce, Italy

Exploiting specific isoforms of lipoxygenase, allene oxide synthase genes in

the jasmonic acid biosynthesis pathway and specific classes of small RNAs

in roots, to select varieties and hybrids better tolerating abiotic stresses in

legumes

12:30 - 14:00 Lunch break

Session III - Plant stress responses and tolerance (Chair Marek Šebela)

14:00 - 14:30 Thierry Gaude, École Normale Supérieure de Lyon, France

Nutrient deprivation in Arabidopsis reveals a function for the retromer

complex in autophagy

14:30 - 15:00 Irene Lichtscheidl, University of Vienna, Austria

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Support of plant growth by soil microbes for phytoremediation of heavy metal

soil

15:00 - 15:30 Jozef Šamaj, C.R. Haná, Olomouc, Czech Republic

Abiotic stress signaling by MAPKs in Arabidopsis thaliana


16:00 - 16:30 Anastasios Melis, University of California, Berkeley, CA, USA

Photosynthesis-to-fuels: maximizing solar-to-product efficiencies

16:30 - 16:50 Jens D. Berger, CSIRO Plant Industry, Australia

Mediterranean adaptive strategies: Phenology, productivity and water

relations in wild and domesticated Lupinus luteus L. from habitats with

contrasting terminal drought

16:50 - 17:10 A. Umut Tüyel, Marmara University, Istanbul, Turkey

Determination of morphological and molecular characteristics of Colchicum L.

species in the Turkish flora

18:30 – 21:00 Meeting of the Scientific Board of C.R. Haná and guests

Friday June 21, 2013

Session IV – Plant bioenergetics (Chair Jan Nauš and Petr Ilík)

09:00 - 09:30 Roman Kouřil, C.R. Haná, Olomouc, Czech Republic

Towards the structures of protein complexes involved in plant bioenergetics

using single particle electron microscopy

09:30 - 10:00 Javier Pozueta-Romero, Universidad Pública de Navarra, Nafarroa, Spain

Starch metabolism, its regulation and biotechnological approaches to

improve yields

10:00 - 10:30 Evelyn Lawrenz, Institute of Microbiology AS CR, Třeboň, Czech Republic

Ocean bioenergetics

10.30 - 11.00 Coffee and tea break

11:00 - 11:20 Sandeep Kumar Verma, Marmara University, Istanbul, Turkey

Sequencing of chloroplast trn-T-L-F intron for phylogenetic analysis of

Colchicum genus from flora of Turkey

11:20 - 11:40 Andrej Pavlovič, C.R. Haná, Olomouc, Czech Republic

Rapid changes of chlorophyll a fluorescence and photosynthesis in response

to action potentials generated by touch in carnivorous plant Dionaea

muscipula Ellis.

12:00 – 12:30 Concluding remarks

Marc van Montagu, President of European Federation of Biotechnology, University of Gent, Belgium

Ivo Frébort, C.R. Haná, Olomouc, Czech Republic

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SCIENTIFIC COMMITTEE:

Jaroslav Doležel, Scientific Director, C.R. Haná, Olomouc, Czech Republic

Marc van Montagu, President of European Federation of Biotechnology, University of

Gent, Belgium

Heribert Hirt, President of European Plant Science Organization, URGV, Evry France

Jan Káš, President of Biotechnology Society of the Czech Republic, Institute of Chemical

Technology, Prague

Ivo Frébort, Executive Director, C.R. Haná, Olomouc, Czech Republic

Jozef Šamaj, C.R. Haná, Olomouc, Czech Republic

Karel Doležal, C.R. Haná, Olomouc, Czech Republic

Marek Šebela, C.R. Haná, Olomouc, Czech Republic

Petr Ilík, C.R. Haná, Olomouc, Czech Republic

LOCAL ORGANIZING COMMITTEE – CR HANÁ OLOMOUC:

Karolina Chvátalová, Vladimíra Gužiaková, Michaela Holecová, Romana Čuprová, Martina

Špundová, Pavla Suchánková, Lucie Havlíková and others

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Dear Colleagues, It is our pleasure to welcome you to the conference “Olomouc Biotech 2013 Plant Biotechnology: Green for Good II”. The conference takes place in Olomouc, a town with a rich history and a vibrant presence. Its centre is the second largest urban preservation area in the Czech Republic with beautiful churches, fountains and civil buildings. The Holy Trinity Column is the biggest Baroque sculptural group in the country inscribed on the UNESCO World Heritage List. Olomouc has been a traditional business and transit centre and served as a strategically important fortress between 16th and 19th centuries. The town is an administrative centre of the Olomouc Region, the seat of the regional government, the Moravian archbishopric and Palacký University - the oldest university in Moravia. The University is one of the largest and most prestigious universities in the country and the second oldest after Charles University in Prague. With over 23 thousand students, Olomouc is the country’s largest student city by percentage of population. Olomouc lies in the heart of the Haná region, lowland with fertile soils and long agricultural tradition. Farmers in the area have profited from agricultural research and improvement programs in a number of institutions and breeding stations. Yet, the growing pressure to produce more and better quality food and a need to explore the potential of plant biotechnology called for multidisciplinary and more effective research and stimulated the establishment of the Centre of the Region Haná for Biotechnological and Agricultural Research. The Centre organized its first international conference in 2011 and as a response to its success, decided to organize a second international meeting in 2013. The research teams of the Centre cover a broad range of disciplines, including cell biology, physiology, genetics and biophysics. This is reflected by the topics of the conference, which as we hope, provides a representative picture of the current plant biotechnical research. The conference is held under auspices of the European Federation of Biotechnology and the Biotechnological Society of the Czech Republic and is commemorated with the grand official opening of the Centre. We hope that you enjoy the science, the spirit of the meeting, and your stay in one of the most beautiful cities in the Czech Republic.

Ivo Frébort Jaroslav Doležel

Executive Director of C.R. Haná Scientific Director of C.R. Haná

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30 YEARS OF TRANSGENIC PLANTS: DISCOVER, INNOVATE,

COMMUNICATE

Van Montagu M

Institute Plant Biotechnology Outreach (IPBO), Gent University, Belgium

The first transgenic plants expressing a foreign gene (Kanamycine resistance) were presented at the Miami Winter Symposium 1983. This new technology has been and still is at the base of the tremendous progress in plant science. However only some innovations in agriculture have been applied, be it on 170 Mi hectares a year (in 2012). Much disinformation was spread on possible dangers for health or environment. Major confusion focused on denying the social and economic value of these applications. Re-information on this technology is badly needed. Today, the major challenge for plant science is how to integrate the fast growing fundamental research and the more empirical knowledge built up by plant breeders and agronomists. Another important challenge is how to communicate to society the importance of this research. Trying to find out the molecular base of crown gall induction by the ubiquitous soil bacterium Agrobacterium tumefacians, we stumbled on the finding that this cell proliferation was induced through a natural event of gene engineering. This observation brought a lot of interesting information on plant bacteria interactions. It also led to the development of a universal gene transfer method for plants. The availability of such a genome modification technology made plant molecular genetics and developmental biology possible. The wealth of data that became available in the last decennia, should be transformed into knowledge on the molecular mechanisms that evolved and were selected during the evolution of today’s plants. The powerful "omics" technologies, the emerging understanding of epigenetics and the progress in IT, guarantee us that such knowledge creation will continue at an accelerated speed. Parallel with this fundamental progress in basic research, an innovative applied research developed which resulted in the construction of crop plants with new agronomical beneficial traits, the now called "GM-Crops". For the scientists, the economic advantages and the benefit for environment were so obvious that they proceeded rapidly with constructing a variety of food/feed and industrial crops as well as transgenic trees. By not communicating with society on the importance of this technology, plant scientists alienated themselves from the public at large. Organized disinformation created a lot of fear of transgenic crops, often extending to general fear of science applications. This attitude, particularly in Europe, made that the possible innovations remained blocked for more than 15 years. The action of Non Governmental Organizations against GM-Crops also resulted in a complex series of extremely expensive regulatory processes. This price tag makes that no SME or none of the developing countries can afford to commercialize their own GM crops, leaving a monopoly to six major multinationals. Some of the emerging countries like Brazil, China and India did develop novel GM-crops but large scale production is not always evident. Meanwhile the ever increasing world population, the necessity to replace fossil oil and coal by renewable plant derived raw materials for the chemical industry and the climate

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instability confront us with the need for a more intensive but sustainable agriculture. This year the UN declared at the Rio+20 meeting that our goals for the coming decennium should be to stop hunger, stop poverty and stop deforestation. Will scientists be able to develop in due time the novel crops needed and obtain their global commercialization ? Results presented at this meeting stress the potential of the research tools. If the public sector scientists would also collaborate in communicating their results to society, stressing that there is no danger to health of humans or animals and surely not for the environment, then there is hope that the ban on GM-crops can be lifted. If the political will is there to fight for the Rio+20 goals, then plant R&D should be able to construct the performing GM-crops requested. Literature: Van Montagu M. It Is a Long Way to GM Agriculture. Annual Review of Plant Biology June 2011 Vol. 62: 1-23. Grunewald W, Bury J. Comment on ‘‘Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize’’ by Séralini et al. Food and Chemical Toxicology 2012. in press.

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ABSTRACTS – ORAL PRESENTATIONS

SESSION I: CROP IMPROVEMENT

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FROM CROSSBREEDING TO BIOTECHNOLOGY-FACILITATED IMPROVEMENT OF

BANANA AND PLANTAIN

Ortiz R*, 1, Swennen R2

1 Swedish University of Agricultural Sciences (SLU), Department of Plant Breeding, Sundsvagen

14, Box 101, 23053 Alnarp, Sweden

2 Katholieke Universiteit Leuven – International Institute of Tropical Agriculture (IITA) – Bioversity

International, Willem de Croylaan 42, Box, 2455, 3001 Heverlee, Belgium

The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tonnes worldwide. About 85% of this global production comes from small plots and kitchen or backyard gardens from the developing world, and only 15% goes to the export trade. Musa acuminata and M. balbisiana are the ancestors of several hundreds of parthenocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hybridizations from these two wild diploid species. Generating hybrids combining host plant resistance to pathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired remains a challenge for Musa crossbreeding, which started about one century ago in Trinidad. The success of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels of female sterility, particularly among polyploidy cultivars. All banana export cultivars grown today are however selections from somatic mutants of the group Cavendish and have a very narrow genetic base, while some bred-hybrids (mostly cooking types) are used by smallholders in sub-Saharan Africa, tropical Asia and Latin America. Musa improvement goals need to shift to address emerging threats because of the changing climate. Innovative cell and molecular biology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micro-propagation has been successfully for high throughput production of clean planting materials while in vitro seed germination continues assisting to obtain seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are used for checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps based on them, and the recent sequencing of the banana genome offers means for gaining more insights in the genetics of the crops and to identify genes that could lead to accelerating Musa betterment. Likewise, DNA fingerprinting has been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musa breeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or enhance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on a few crossbreeding programmes (based in Brazil, Cameroon, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda) or a handful of genetic engineering endeavours (Australia, Belgium, India, Kenya, Malaysia and Uganda). Development investors (namely international aid and philanthropy) should therefore increase their funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world.

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A ‘CONVENTIONAL’ SUCCESS STORY FOR IMPROVING THE FUNCTIONALITY OF

SOYBEAN OIL

Bilyeu K

Plant Genetics Research Unit, USDA/ARS, Columbia, Missouri, USA

Soybean is the world's foremost oilseed crop. Soybean is valued for both the vegetable oil and high protein meal present in the seeds. The functionality of oil is dependent on the fatty acid profile present in the triacylglycerol fraction. Soybean oil typically consists of five major fatty acids: the two saturated fatty acids palmitic acid and stearic acid, the mono-unsaturated oleic acid, and the polyunsaturated fatty acids linoleic acid and linolenic acid. In order to meet the evolving market demands for oils with positive health attributes as well as superior qualities for flavor and cooking functionality, the target oil is very high in oleic acid and low in linolenic acid. A high oleic acid content improves the oxidative stability and thus the functionality of the oil. The biochemical understanding of the fatty acid desaturation pathway in model systems has been employed to alter the accumulation of oleic acid in soybean seed oil by conventional plant breeding through selection of variant alleles of genes encoding microsomal fatty acid desaturase enzymes. Mutant alleles of the soybean FAD2-1A gene were combined mutant alleles of the soybean FAD2-1B gene to produce soybean seeds with the high oleic acid trait in the seed oil. The objective of this research was to utilize a molecular breeding approach capturing induced and natural variation identified by forward and reverse genetics to develop and critically evaluate high oleic acid soybean lines. The high oleic acid soybean trait was backcrossed into an elite cultivar and the developed lines were analyzed for yield, fatty acid profile of the seed oil, and residual genome contributions from the non-recurrent parent allele donor.

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ADVANCES IN PLANT CHROMOSOME GENOMICS

Doležel J, Šimková H, Šafář J, Vrána J, Cápal P, Kopecký D, Burešová V, Číhalíková J,

Kubaláková M, Bartoš J

Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of

Experimental Botany, Šlechtitelů 31, CZ-783 71 Olomouc-Holice, Czech Republic

Genomics aims to sequence and analyze the structure and function of complete genomes. This may be insurmountable task in some plant species, including important agricultural crops, as their genomes are large and composed mostly of repetitive DNA. Moreover, many species are recent polyploids and interspecific hybrids. Sequence redundancy and presence of homoeologous sequences hampers gene mapping and cloning, construction of physical maps and sequence assembly. We have been pioneering chromosome genomics to overcome these difficulties. Chromosome genomics uses all tools of genomics, but applies them to individual chromosomes and sub-chromosome parts. This avoids problems due to polyploidy and significantly reduces sample complexity. Samples of mitotic chromosomes are prepared from synchronized root tips and chromosomes of interest are purified by flow cytometric sorting. Flow-sorted chromosomes have been used for physical mapping using PCR and FISH, construction of BAC libraries to facilitate construction of physical maps, and for targeted development of molecular markers. The International Wheat Genome Sequencing Consortium adopted chromosome genomics as main tool to develop ready to sequence physical map of hexaploid bread wheat. DNA amplified from flow-sorted chromosomes is suitable for next-generation sequencing, providing easy access to DNA composition of chromosomes. This approach enabled identification of most of genes in barley and ordering them along its chromosomes. In rye it clarified molecular structure and evolution of accessory B chromosomes. Until recently, only those chromosomes could be sorted that differed in DNA content. This restriction was overcome after Giorgi and colleagues developed a method called FISHIS (PLoS ONE 8: e57994, 2013) for fluorescent labeling of particular DNA sequences on chromosomes prior to flow cytometry. Considering the successful contributions to the analysis of complex plant genomes and given the recent methodological improvements, chromosome genomics is bound to continue playing important role in mapping and analysis of complex plant genomes.

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GM BARLEY WITH MODULATED CYTOKININ LEVEL: AIMING AT IMPROVED YIELD

AND STRESS TOLERANCE

Frébort I, Pospíšilová H, Mrízová K, Jiskrová E, Andrýsková E, Galuszka P

Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,

Palacký University, Šlechtitelů 11, CZ-78371 Olomouc-Holice, Czech Republic

Rapidly growing human population brings new challenges for plant breeders to increase and stabilize crop yield under various environmental stresses. Recent studies on quantitative trait loci have shown that limitations of conventional breeding set by natural variations can be overcome by plant biotechnology approaches. As shown for model plants, enhanced abiotic stress tolerance can be achieved by lowering the content of plant hormones cytokinins in the roots that leads to a selective expansion of the root system. In the present study, immature barley embryos were transformed with different genes coding for the hormone degrading enzyme cytokinin dehydrogenase (CKX) under root-specific promoters with vacuolar, plastidial, apoplastic or cytoplasmic targeting. Transgenic homozygous plants of T2 generation produced an expanded root system and in greenhouse experiments under reduced watering exhibited higher relative water content than control plants. Alternatively, nutrient transport into grain endosperm is positively regulated by cytokinins in the aleurone layer; therefore spatial silencing of the degradation enzyme can be used to increase grain filling and thus improve the yield. Transgenic barley plants with CKX silencing cassette were prepared and analyzed. To date, no commercial transgenic crop plant with altered cytokinin levels has been released. It will be discussed how these alterations influence various physiological processes during plant ontogenesis, focusing on possible applications in plant biotechnology and agriculture.

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PROMOTION OF HOMOEOLOGOUS PARING BY THE RYE B CHROMOSOMAL

SEGMENTS IN COMMON WHEAT

Takashi R. Endo

Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Japan

Rye B chromosomes are supernumeray chromosomes dispensable for the host but increase in number by non-disjunction after meiosis. The rye B chromosome has been reported to affect meiotic homoeologous chromosome pairing at meiosis in wheat-rye hybrids, but previous studies reported contradictory results. Using the gametocidal system, I dissected the rye B chromosome, which had been introduced into common wheat (2n=6x=42, genomes AABBDD), into segments. To study the effect of the rye B segments on homoeologous pairing in common wheat, I chose two dissected rye B chromosomes derived from a reciprocal translocation between the rye B chromosome and a wheat chromosome, i.e. one involving the rye B proximal segment including the pericentromeric region and the other involving the distal segment of the rye B long arm. I studied the effect of these rye B segments on meiotic homoeologous pairing in hybrids between a wild species Aegilops variabilis (2n=4x=28, UUSS) and common wheat. Both rye segments promoted homoeologous chromosome pairing, and the proximal segment had a greater effect than the distal segment; besides the effect was enhanced more in a monosomic addition than in a disomic addition. The mean chiasma frequency (10.23) in the hybrid with the monosomic addition of the rye B proximal segment was significantly higher than that (2.78) in the hybrid without the same segment, and was comparable with that (14.09) of the hybrid lacking chromosome 5B on which the Ph1 gene for the suppression of homoeologous pairing is located. These facts suggested that the rye B proximal segment can become a promising means, in addition to the Ph system, in order to introduce alien genes into common wheat through homoeologous pairing.

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COMPARATIVE AND EVOLUTIONARY GENOMICS IN GRASSES. PARTICULAR CASE

OF MICRO-RNA.

Abrouk M1, Zhang R2, Murat F2, Salse J2

1 Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of


2 INRA/UBP, UMR 1095, Génétique, Diversité et Ecophysiologie des Céréales, Laboratory

Paléogénomique des Plantes pour l’Amélioration Variétale, 63100 Clermont Ferrand, France.

Poaceae also called Grasses are an important botanical family consisting in nearly 12,000 species in over 700 genres including cereals. This family is of major economic interest because it comprises cereals that are among the most important crops for human and animal nutrition. This family has been extensively studied in comparative genomics since the 1990s and showed a high degree of gene conservation among species since they diverged from a common ancestor. With the complete sequencing of four grass species since 2005, we performed an analysis of its genome by the identification of twelve synteny blocks with the sequenced genomes of rice, sorghum and maize and seven duplications blocks shared with these last grass species. These data allowed us to suggest the five chromosomes of Brachypodium are from the common ancestor composed of twelve chromosomes and having undergone seven fusions during the evolution. Then, based on these comparative genomics results, we studied more particularly the evolution of different families of microRNAs (miRNAs). The comparison of non-coding RNA from rice, sorghum, maize and Brachypodium showed conservation into this family for the grass species with 50% of orthologs and 20% of paralogs. Based on the paleogenomics results, we proposed an evolutionary scenario of miRNA genes, which supports the hypothesis of an ancient origin of this gene silencing mechanism in plants.

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REGULATION OF SOURCE-SINK RELATIONS TO IMPROVE CROP SALT-TOLERANCE:

A ROLE FOR ROOT-TO-SHOOT SIGNALLING?

Moreno AAA, Martínez-Andujar C, Pérez-Alfocea F

Department of Plant Nutrition, CEBAS-CSIC, Campus de Espinardo, 25, E-30100 Murcia, Spain

Global food security for an expanding world population is increasingly threatened by environmental stresses that limit crop yield, thus maintaining crop productivity even under unfavourable environments is a major research topic in plant sciences. Abiotic stress conditions such as salinity and drought modify source-sink relations, thereby influencing plant growth, adaptive responses and consequently crop yield. Salinity decreases crop yield first by reducing growth of assimilate-consuming sink organs and, second, by decreasing assimilate production in photosynthetically active source tissues. One major area of crop improvement that has been comparatively neglected is the role of the plant root system in maximising water and nutrients capture and sensing and adjusting to environmental stresses in the soil to control plant growth and shoot physiology and ultimately agricultural productivity. This integrated plasticity probably involves long-distance communication between different organs with hormones, particularly differential changes in root and shoot hormone concentrations, playing an essential role. Hormonal signals, such as cytokinins, abscisic acid, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and the auxin indole-3-acetic acid may coordinate assimilate production and usage in competing sinks (biomass partitioning). For example, hormonal regulation of source-sink relations during the osmotic phase of salinity affects whole plant energy availability to prolong the maintenance of growth, root function and ion homeostasis, and could be critical to delay the accumulation of toxic ions. Growth maintenance probably requires coordinated regulation of stomatal conductance and source–sink relations to avoid premature stomatal and metabolic inhibitions of photosynthesis, and subsequent oxidative damage and senescence. Whether this coordination is achieved by hormonal signals integrating root and shoot physiologies remains an open question, but reciprocal grafting studies of mutants and transgenics with altered hormone biosynthesis (i.e. CKs) or metabolism (i.e. invertases) allows a structured approach to address this issue and its validity as an alternative approach to improve crop salt tolerance.

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NEW CYTOKININ DERIVATIVES FOR PLANT BIOTECHNOLOGY AND AGRICULTURE

Doležal K1,2, *, Malá J3, Spíchal L1 , Szűčová L1 , Mik V1 , Máchová P3, Cvrčková H3, Karady M2,

Novák O1,2, Mikulík J2 , Strnad M1, 2

1Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,

Palacký University, Šlechtitelů 11, CZ 783 71 Olomouc-Holice, Czech Republic

2Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany,

Šlechtitelů 31, 783 71 Olomouc-Holice, The Czech Republic

3 Reseach Institute of Forest and Game Management, Strnady 136, 252 02 Jíloviště, The Czech

Republic

Widely used 6-benzylaminopurine is an important and affordable cytokinin, routinely utilized for its effective stimulatory properties in micropropagation practice. On the other hand, it may negatively influence the growth, rooting and acclimatization processes in some crops (Werbrouck et al., 1996). It is known that hydroxylated aromatic cytokinins (topolins) are more resistant to cytokinin oxidase (CKX), more stable and in some biological systems active at lower concetrations than the isoprenoid CKs. In addition, meta - topolin does not inhibit root formation which is a typical inconvenient effect of high concentrations of BAP. These properties might help to enhance the future productivity of plant tissue culture industry (Werbrouck et al., 1996; Strnad, 1997). The development of other new CK derivatives exhibiting high morphogenetic activity might consequently be of a great practical importance in plant biotechnology. During our recent search for naturally occurring aromatic cytokinins (ARCKs) in plants, another new group of endogenous BAP derivatives –methoxytopolins – exhibiting high biological, especially anti-senescence (and surprisingly also anti-cancer) activity, was discovered. Based on these results, we synthesized several groups of their synthetic analogues that exhibited high activity in different CK bioassays and showed the ability to activate cytokinin receptors and/or to inhibit CKX. Best compounds so far, meta-topolin (mT) and the 6-(3-methoxybenzylamino)purine-9-β-D-ribofuranoside (MeomTR)) were used, together with some newly developed 9-substituted derivatives, for retardation of senescence during multiplication stage of micropropagation of selected tree species as well as for rooting support and acclimatization improvement. New cytokinin derivatives are also being tested in combination with inhibitors of cytokinin deactivation. Subsequently, wide range of endogenous plant hormones (isoprenoid cytokinins, IAA, ethylene) were quantified (using UPLC-MS/MS and/or GC/FID, respectively) in relation to cytokinin exogenously used as well as plant micropropagation and ex-vitro acclimatization efficiency. The results about optimal endogenous plant hormone concentrations and their dependence on different exogenous cytokinin used in the cultivation media, may improve in-vitro micropropagation efficiency as well as quality of ex vitro acclimatized plants, possibly also of other species. Work was supported by the Czech Ministry of Agriculture (QI 92A247) and Education ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research. References: Strnad M Physiol. Plant. 101: 674-688, 1997; Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC Physiol. Plant. 98: 291-298, 1996.

21

A CHEMICAL SCREEN IDENTIFIES A CYTOKININ DEGRADATION INHIBITOR AS A

STRONG PROMOTER OF SHOOT REGENERATION IN PLANT TISSUE CULTURE

Motte H1,2, Galuszka P3, Spíchal L3, Tarkowski P3, Plíhal O3, Šmehilová M3, Jaworek P3,

Vereecke D1,2, Werbrouck S1,2, Geelen D2

1Department of Plant Production, Associated Faculty of Applied Bioscience Engineering, University

College Ghent, Member of the Ghent University Association, BE-9000 Ghent, Belgium

2Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, BE-9000

Ghent, Belgium

1Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,


De novo shoot regeneration, or the development of shoots from non-meristematic tissue, is a widely applied technique for the genetic transformation of plants. For many plants, cultivars or tissues, efficient regeneration protocols are difficult to establish, also jeopardizing the isolation of transformed lines. To find new compounds that promote shoot regeneration and to identify target pathways important in the regeneration process, we followed a chemical genetic approach. A divers set of 10,000 small molecules was applied to a two-step regeneration protocol from Arabidopsis root explants. The chemical screen revealed a single compound, phenyl-adenine (Phe-Ade), as a potent inducer of adventitious shoots. Phe-Ade increased the regeneration capacity and was active at a very broad concentration range. Molecular and biochemical analyses showed that Phe-Ade is a strong competitive inhibitor of the cytokinin degrading CYTOKININ OXIDASE/DEHYDROGENASE (CKX) enzymes. This inhibition resulted in a moderate accumulation of endogenous cytokinins. Our results suggest that the application of Phe-Ade shifts the cytokinin homeostasis equilibrium in the explant and that this promotes de novo shoot organogenesis.

22

CLAVICEPS PURPUREA AS A FACTORY PRODUCING ERGOT ALKALOIDS

Hanosová H



Parasitic fungus Claviceps purpurea is a major producer of pharmaceutically active compounds, the ergot alkaloids. These alkaloids serve as a basic drug-stock (precursors) for semisynthetic production of various therapeutics, which are mainly used for migraine treatment, as antiparkinsonians, or as uterotonics and prolactin inhibitors in gynecology. The majority of characterized C. purpurea strains are able to synthesize ergot alkaloids only during parasitic interaction with plant species from Poaceae family. There are two ways how ergot is produced industrially – either by field production on hybrid lines of sterile rye, with yields depending on weather conditions and plant origin, or by submerged fermentation of mutated strains, with costs deriving from energy prices. Nowadays, the total worldwide ergot alkaloids production reaches 20 tons annually and is equally divided between these two technologies. Fifteen enzymes involved in ergot alkaloid synthesis are encoded by cluster of genes situated in unstable region of Claviceps genome. The last step is realized by non-ribosomal peptidyl synthetase leading to formation of D-lysergic acid amides and/or ergopeptines, main metabolites exploited pharmaceutically. Genetically modified C. purpurea strains with more stable or improved production of ergot alkaloids or strains producing only one specific ergopeptine without contamination by others are under preparation in our laboratory and would significantly reduce costs of therapeutics based on ergot alkaloids in future.

23


SESSION II: BIO-TECHNO: INDUSTRY MEETS ACADEMIA

24

THE EUROPEAN TECHNOLOGY PLATFORM ‘PLANTS FOR THE FUTURE’ – A

EUROPEAN PARTNERSHIP FOR RESEARCH AND INNOVATION

Travella S

Coordinator of the European Technology Platform ‘Plants for the Future’

Today, the European Technology Platform ‘Plants for the Future’ (Plant ETP) represents the most important stakeholders that provide plant-based raw materials for the (European) food and feed industry, the chemical, pharmaceutical and other processing industries, and the (bio)energy sector. This underpins that plants are one of the main pillars of the bioeconomy. Agriculture at the same time is one of the major drivers shaping the European rural landscape. The agri-culture dependent sector in its entirety is the largest sector of European industries illustrating its enormous role for employment and its socio-economic dimension. The sector faces short and mid-term major challenges, which include requirements for more yields, better and more tailored quality of biomass and more flexible production conditions to address reduced use of natural resources and climate change. In addition, there is a growing need for a European approach to the intensifying, global economic competition as well as to a balanced production that appropriately services existing markets and new ones such as energy. The European research, farming and agro-industry together are well positioned to solve these challenges. The best way forward is to develop plan(t)s for the future in its broadest sense. Representing the whole plant innovation chain from fundamental research to crop production and food processing, Plant ETP is committed to stimulating research and innovation in plant science and agriculture as a joint basis between industry, academia and the farming community, to the benefit of the growers and the final consumers. Developing plan(t)s for the future requires an integrated action including: research - generation of knowledge about plants, their production and their valorisation into products; innovation – transformation of knowledge into practice and products; education – development of skillful and knowledgeable people for research, industry and the farming community.

25

TRAITS AND TECHNOLOGIES FOR PLANT PHENOTYPING

Fiorani F1, Postma J1, Schurr U1

Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich

It is widely recognized that phenotyping plant traits at a faster pace is key to link genome to phenome, quantify genotypes x environment interactions, and identify useful germplasm with increased resource use and resource use efficiency (light, water, nutrients). These goals are crucial for both breeding programs and biotechnology applications. In this presentation we first focus on some of the key traits of interest in particular for water and nutrient use efficiency. Particular emphasis will be given to root systems architecture responses to sub-optimal water and nutrient limitation. Second, based on our portfolio of lab and field methods based on non- or minimally-invasive methodologies, we highlight the progress in the application of available optical and non-optical methods, from 2D RGB imaging to multi-spectral reflectance analyses and 3D reconstructions of shoot and root architecture. Based on knowledge generated at our Institute, these methods will be discussed according to the measurements principles, theoretical and practical limitations, and robustness for implementation of systematic phenotyping in Arabidopsis, Brassica napus, and cereals. Third, using specific examples focused on N and P acquisition by roots, we stress the importance of linking experimentation and mathematical modelling. Finally, we briefly present key national and international projects, such as the German Plant Phenotyping Network (DPPN: http://www.dppn.de) and the European Plant Phenotyping Network (EPPN: http://www.plant-phenotyping-network.eu) that support a development roadmap for new application of phenotyping systems and the sharing of existing European phenotyping infrastructure via user access to participating platforms, respectively.

26

DEVELOPMENT OF VERSATILE BIONANOCAPSULES DISPLAYING

IMMUNOGLOBULIN-BINDING DOMAINS FOR APPLICATION TO IMMUNOSENSORS

Tanizawa K1, Iijima M2, Kuroda S2

1Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan

2Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

To enhance the sensitivities and antigen-binding capacities of immunosensors, the oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We have developed an innovative method for adsorbing immunoglobulin proteins onto 30-nm bio-nanocapsules through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC), so that all the IgG Fv regions are displayed outward for effective binding of antigens. Although the binding capacities of ZZ-BNC for various IgGs differ depending on the animal species and IgG subclasses, the ZZ-BNC adsorbed on the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could maximally bind about 60 molecules of mouse total IgG, thereby markedly enhancing both the sensitivity and antigen-binding capacity of the chip. The ZZ-BNC-bound sensor chip is very stable and should prove useful for various immunosensor applications. In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNC in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNC with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNC. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNC and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNC showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays. We have also developed a novel method for simultaneous, rapid, and sensitive detection of multiple antigens using ZZ-BNC. When Cy2-labeled ZZ-BNC (Cy2-ZZ-BNC) was used instead of Cy2-labeled secondary antibody in western blot analysis, both sensitivity and signal intensity were significantly increased. The complex of Cy5-ZZ-BNC and mouse IgG2a (which shows moderate affinity to the Z domain) was not dissociated, even in the presence of 8-fold excess of free mouse IgG2a. In addition, crosslinking with bis-sulfosuccinimidyl suberate efficiently stabilized the interaction. The ZZ-BNCs labeled with various Cy dyes facilitated the simultaneous detection of multiple antigens using primary antibodies derived from the same host species, by western blot analysis, immunocytochemistry and flow cytometry, which could expand the possibility of bio-imaging probes in various immunofluorescence techniques.

27

DISEASE SUSCEPTIBILITY AS A TARGET OF BIOTECHNOLOGY FOR DURABLE

RESISTANCE?

Hückelhoven R, Hoefle C, Eichmann R, Pathuri I, Proels R

Lehrstuhl für Phytopathologie, Technische Universität München, 85350 Freising

Susceptibility of plants to microbial pathogens involves susceptibility factors, which can be targets of pathogen effectors. Additionally, successful pathogenesis requires host susceptibility factors, which meet general demands of pathogen development and nutrition. Plants also bear negative regulators of immune responses to avoid autoimmunity and unnecessary investment into defense in environments with little disease pressure. Consequently, disease susceptibility can get lost by loss of negative regulators of defense but also of host factors, that otherwise support the successful pathogen. Here, we used RNAi silencing cassettes against barley susceptibility factors of diverse modes of action in model barley cultivars for obtaining strong loss of susceptibility. We identified ADH1, BAX inhibitor-1 and RACB, a small RAC/ROP G-protein, as host susceptibility factors to powdery mildew of barley (1-3). Molecular dissection of RACB dependent susceptibility revealed a function of the host cytoskeleton in resistance and susceptibility to fungal penetration of the plant cell wall and to establishment of fungal infection structures in living cells of barley. In planta protein interaction studies and RNAi against host genes demonstrate the importance and dynamics of G-protein signalling in accessibility of living cells for parasitic entry. Future combination of RNAi silencing cassettes against susceptibility genes of diverse modes of action may create plants difficult to colonize by powdery mildew fungi. (1) Hoefle C, Huesmann C, Schultheiss H, Börnke F, Hensel G, Kumlehn J, Hückelhoven R (2011). Plant Cell 23:2422-2439 (2) Eichmann R, Bischof M, Weis C, Jane Shaw, Lacomme C, Schweizer P, Dimitar Duchkov, Hensel G, Kumlehn G, Hückelhoven R (2010). Mol Plant-Microbe Interact 23: 1217-1227. (3) Pathuri IP, Reitberger IE, Hückelhoven R, Proels RK (2011). J Exp Bot. 62:3449-3457

28

THE ROLE OF NON-STARCHY POLYSACCHARIDES (“SOLUBLE” DIETARY FIBRE),

IN PARTICULAR (1,3;1,4)-Β- GLUCANS AND ARABINOXYLANS, IN PROMOTING

BOWEL HEALTH

Bacic A1,2, Doblin MS1, Burton RA3, Fincher GB3, Jobling SA4 and Gidley MJ5

1ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Victoria,

Australia

2Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC, Australia

3ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of

Adelaide, SA, Australia

4CSIRO Division of Plant Industry & Food Futures Flagship, ACT, Australia

5ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition & Food Sciences, University of

Queensland, QLD, Australia

Total dietary fibre contains indigestible plant complex carbohydrates comprising two functionally distinct components, “soluble” and “insoluble” fibre. “Insoluble” fibre (cell walls) provides stool bulk thereby reducing the risk of constipation and diverticulitis. In contrast, high molecular weight (HMW) “soluble” fibre, also referred to as non-starchy polysaccharides (including 1,3;1,4-β-glucans (MLGs) and arabinoxylans (AXs)) lowers the risk of serious diet-related conditions of the developed world, such as Type II diabetes, cardiovascular diseases, colorectal cancer. The grains of cereals are an important source of caloric intake for a major part of the world’s population and are also a major source of HMW “soluble” dietary fibre in the form of MLGs and AXs. Recently oats and barley received FDA approval to be labelled as health promoting and we are working towards enhancing the soluble fibre content of wheat, barley and rice endosperm. The overall aim of our work is to determine the molecular mechanism of synthesis and assembly of AXs and MLGs and to use this information to enhance their content in grains through molecular breeding techniques. We are also studying their molecular mechanism(s) in promoting bowel health.

29

ANTIMICROBIAL PEPTIDE EXPRESSION IN BARLEY ENDOSPERM

Andrýsková E, Tufan Oz M, Galuszka P, Frébort I



Plant molecular farming is considered as a promising and competitive tool for the production of recombinant proteins, peptides and pharmaceuticals, owing to advantages of plants over other expression systems. Plants possess post-translational modification machinery, display no risk of contamination with mammalian viruses and pathogens, and require comparably lower costs for large scale production. Additionally, efficient transformation systems and simple downstream processing techniques are available for use in cereal expression systems. Grains of barley, a self pollinating cereal, display low protease activity, accommodate high levels of soluble proteins and low levels of phenolic compounds, and provide high product yields. The purposes of this study include expression of antimicrobial peptides in barley endosperm and evaluation of related parameters for molecular farming. Chosen parameters for evaluation cover expression levels and yield, purification techniques, and effects of various tissue specific promoters, purification tags, stabilization tags, and secretion signals. The selected antimicrobial peptides in this study are (1) LL-37 – a derivative from human CAP18, (2) pexiganan – an analog of frog magainins, and (3) hPAB-β – a mutant derivative from human beta-defensin 2. Barley grains might be used for large scale production of antimicrobial peptides which are attractive candidates for medicine.

30

PLANT BIOTECHNOLOGY BASED ON TRANSIENT AGROBACTERIUM DELIVERY

Gleba Y

Nomad Bioscience GmbH and Icon Genetics GmbH, Weinbergweg 22, Halle 06120 Germany

Transient expression technologies such as magnICON® developed by Icon genetics are new generation processes for biopharmaceutical and biomaterial production that are simple and indefinitely scalable protocols for heterologous protein expression in plants, which are devoid of stable genetic transformation of a plant, but instead rely on transient amplification of viral vectors delivered to multiple areas of a plant body (systemic delivery) by Agrobacterium. These eclectic technologies effectively address most of the major shortcomings of earlier plant-based technologies. The technologies have been brought to the GMP-compliant level and are currently being used to manufacture materials for clinical trials by Icon Genetics, KBP, Fraunhofer USA, Caliber, iBio and others. Transient technologies are also applicable for generating novel agronomic traits and biomaterials. The core process in development today is a technology that allows to rapidly temporarily re-program metabolism of green plants using agrobacteria sprays, thus generating valuable traits and bio-based materials. Because of its speed and versatility, the agrospray technology has a potential to become a disruptive new process that will redefine agriculture business as we know it. In particular, the time to market for traits delivered by transient process will be significantly shorter; the repertoire of traits and crops is expected to be much broader, and the genes encoding traits do not have to be built into the plant variety’s genome, thus allowing for ‘germplasm-independent’ business models like those of agrochemical manufacturers. Giritch, A., Marillonnet, S., Engler, C., van Eldik, G., Botterman, J., Klimyuk, V., and Gleba, Y. (2006). Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci U S A 103, 14701-14706. Gleba, Y., Klimyuk, V., and Marillonnet, S. (2007). Viral vectors for the expression of proteins in plants. Curr Opin Biotechnol 18, 134-141. Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V., and Gleba, Y. (2004). In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A 101, 6852-6857. Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V., and Gleba, Y. (2005). Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23, 718-723. Santi, L., Giritch, A., Roy, C.J., Marillonnet, S., Klimyuk, V., Gleba, Y., Webb, R., Arntzen, C.J., and Mason, H.S. (2006). Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system. Proc Natl Acad Sci U S A 103, 861-866. Werner, S., Marillonnet, S., Hause, G., Klimyuk, V., Gleba, Y. (2006) Immunoadsorbent nanoparticles based on a tobamovirus displaying protein A. Proc. Natl. Acad. Sci. USA, 103, 17678-17683. Werner, S., Breus, O., Symonenko, Y., Marillonnet, S., Gleba, Y. (2011) High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc. Natl. Acad. Sci. USA, 108, 14061-14066.

31

PLANTSCAN- NEW TOOL IN HIGH THROUGHPUT PHENOTYPING

Trtílek M

PSI (Photon Systems Instruments), spol. s r.o., Kolackova 39, 621 00 Brno, Czech Republic

Chlorophyll Fluorescence Imaging, Thermal Imaging, RGB Imaging, NIR Imaging, Hyperspectral Analysis, 3D reconstruction are methods used to characterize plant architecture and allow to speed up breeding progress, increase stress resistance, development of new fertilizers and others . Possibility to characterize one plant by such complex analytic methods, give us a solid view of the sample. New phenotyping approaches and methods of data integration should improve precision Agronomy, as well as basic Research. Biological objects have quite big complexity and putting all information together is necessary for understanding the system. New crops and farming practices are crucial to feed a warming world. Crops breeding required thousand years, but with advanced high throughput screening we may achieve our goal so much sooner.

32


SESSION III: PLANT STRESS RESPONSES AND TOLERANCE

33

NEW INIGHTS INTO PLANT-MICROBE SIGNALLING

Hirt H

URGV Plant Genomics, INRA-CNRS-Univ. Evry, France

In their natural soil habitat, plants are immersed in a sea of microbes. Certain plants and microbes can form symbioses that profit both sides, but in many cases, the interaction is only beneficial to the microbial part and often results in desease and death of the host. Besides typical plant pathogens, a number of human pathogens such as Salmonella Typhimurium, can also infect plants thereby offering the unique possibility to compare the functioning of the human and plant defense system in response to the same pathogen (Schikora et al., 2012). We found that plants respond to S. Typhimurium with typical pathogen defenses and that the bacteria subvert the host immune system by a range of effectors that are injected into the host cells. Like many plant pathogens, S. Typhimurium recognizes and enters plants through their stomates. Although the ABA (abscisic acid) pathway was believed to mediate both environmental and pathogen signals, we uncovered a pathogen-specific pathway that triggers stomatal closure in response to pathogen contact. The identification of this pathway offers new approaches to enhance desease resistance in crops without interfering with the response of plants to environmental changes. Schikora A, Garcia AV, Hirt H. (2012) Plants as alternative hosts for Salmonella. Trends Plant Sci. 17:245-9. Montillet J.-L. et al. (2013) Identification of an ABA-independent oxylipin pathway that controls stomatal closure and immune defense in Arabidopsis. PLoS Biol. (in press).

http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/22513107

34

ACTION OF JASMONATES IN PLANT STRESS RESPONSES AND DEVELOPMENT –

APPLIED ASPECTS

Wasternack C

Leibniz Institute of Plant Biochemistry, Department of Molecular Signal Processing, Weinberg 3, D-

06120 Halle (Saale) Germany

Jasmonates (JAs) are lipid-derived compounds acting as key signalling components in plant stress responses and development. The JA receptor and several enzymes of JA biosynthesis have been crystallized, and various JA signal transduction pathways including cross-talk to most of the plant hormones have been intensively studied. Defence to herbivores and necrotrophic pathogens including systemic signalling are mediated by JA or JA compounds. Other environmental cues mediated by JA are light, seasonal and circadian rhythms, cold stress, desiccation stress, salt stress and UV stress. Regarding developmentally regulated processes, growth inhibition of roots, shoots and leaves occurs by JA, whereas seed germination and flower development are partially affected by its precursor 12-oxophytodienoic acid (OPDA). Based on these numerous JA mediated signal transduction pathways active in plant stress responses and development, there is an increasing interest in horticultural applications. Intercropping, the mixed growth of two or more crops, mycorrhization of plants, establishment of induced resistance, transgenerational priming of plants for enhanced insect resistance or pre- and post-harvest application of JA are few examples. Additional sources of horticultural improvement, where JAs might be involved, are defence against nematodes, altered carbon partitioning, biocontrol by plant growth promoting rhizobacteria, altered composition of rhizosphere bacterial community, sustained trade-off between photosynthesis and defence, and improved plant immunity by canopy light in cropping systems.

35

MALDI MASS SPECTROMETRY OF INTACT FUNGAL MICROORGANISMS

Chalupová J1,2, Chamrád I2, Helmel M5, Řehulka P1,4, Sedlářová M3, Marchetti-Deschmann M5,

Allmaier G5, Šebela M1,2

1Department of Biochemistry

2Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Protein

Biochemistry and Proteomics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ 783 71

Olomouc-Holice, Czech Republic

3Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71


4Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence,

Třebešská 1575, CZ-500 01 Hradec Králové, Czech Republic

5Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna,

Getreidemarkt 9, A-1060 Vienna, Austria

Biotrophic parasites such as downy and powdery mildews (DM and PM, respectively) cause damage on crop plants and their diagnostics is important to fight the diseases. Recently, intact spore MALDI-TOF mass spectrometry (MS) was optimized and applied for the identification and differentiation of DM and PM in our laboratory. Mass spectrometric profiles acquired using isolated spores or by a direct laser ablation from infected plant organs comprise peptide/protein peaks in the mass range of 1 to 20 kDa. Proteins extracted from spore surfaces of Bremia lactucae (DM), Oidium neolycopersici (PM) and Blumeria graminis (PM) were also analyzed. For separation of the extracted material, first 1-D electrophoresis using variable polyacrylamide gel concentration (10%, 12% and 15% T, all 3% C) and different gel composition (SDS-PAGE, tricine SDS-PAGE) was evaluated, which continued by two-dimensional gel electrophoresis experiments. Protein identification was achieved by in-gel digestion with trypsin followed by MALDI-TOF/TOF MS and tandem MS (MS/MS) or liquid chromatography (LC) coupled to offline to the MALDI instrument. In addition, in-solution tryptic digestion of the protein extracts was also done. Then LC separations were run coupled to MS and MS/MS analysis of peptides. An extraction procedure for surface peptides/proteins was developed, which relied on the application of matrix solution (ferulic acid diluted in ACN: 2.5% v/v, TFA, 7:3, v/v). The extraction process mimicked on-target conditions during MALDI-TOF MS. Proteins identified by similarity searches in protein databases included ribosomal proteins, histones, heat shock proteins, ATP synthases, ubiquitins, chaperonins and pathogenesis-related proteins. Optimizing extraction and separation methods for spore surface proteins is critical on the way to development of a methodology for the identification of protein biomarkers of fungal phytopathogens. We have accomplished a comparative screening of gel-based and gel-free separation protocols for the subsequent MS-based analysis of surface proteomes of phytopathogens from the category of mildews.

36

MAIZE DEFENSE AGAINST INSECT AND FUNGUS ATTACKS

Pechanova O1, Pechan T2, Luthe DS3, Williams WP4

1State Chem Lab, Mississippi State University

2Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University

3Department of Crop and Soil Sciences, Pennsylvania State University

4USDA ARS Mississippi State

Corn is not “only” a popular food, but globally, it became strategic commodity that is directly affecting supplies and prices of most food products found in both industrial and developing countries. Naturally, corn production and its sustainability is an object of scientific studies. At the Mississippi State University, number of researchers engaged in exploration of corn ability to withstand the attack of economically most important corn pests - insect and fungus. Results of multiyear and multifaceted research will be highlighted in the presentation by one of the team members. The journey will take the audience from the corn fields of US Deep South to high performance computing, via plant physiology, entomology, micro-surgery, electron microscopy, genetic engineering, proteomics and mass spectrometry. In particular, critical role of unique 33-kD cysteine proteinase in maize resistance against the insect infestation/damage will be presented in regards to both host and parasitic organism. The overview of complex rachis proteome changes in response to fungal infection will offer a base to conclusion that resistant maize cultivars rely on constitutive defences, while susceptible rachis is more dependent on inducible defences.

37

HEAVY METAL STRESS IN PLANTS – FROM WEEDS TO CROPS

Hobza R1,2, Hudzieczek V2, Horníková M1, Čegan R2



2Department of Plant Developmental Genetics, Institute of Biophysics ASCR, Brno, Czech

Republic

Almost all human activities influence the environment by heavy metals as a side effect of industry and mining. Plants growing in polluted sites exhibit altered expression of various stress responsive and metal homeostasis genes. These processes can finally lead to the lower biomass production and metal accumulation. On the other hand, past efforts focused on increasing crop yields is not followed by a tendency to control and/or modify concentration of metals in plants. Keeping sufficient micronutrients concentration has become an urgent task because about half of the world population suffers from the malnutrition of iron, zinc and selenium. In contrast, even low concentration of nonessential metals such as cadmium, arsenic and lead in food chain can negatively affect human population. Finding a balance in the plant metal uptake and keeping the metal content in crop plants under control has become an emerging task of plant breeding. Here we review how crop plants can learn from various weed species that have adapted to survive on suboptimal to extreme metal polluted niches. This work was supported by Czech Science Agency (grant 13-34962P)

38

CEREALS PROTEOME CHANGES UNDER COLD STRESS

Vítámvás P, Kosová K, Hlaváčková I, Prášil IT

Department of Genetics and Plant Breeding Methods, Crop Research Institute, Czech Republic

Winter cereals are able to survive long-term periods of cold during winter. Moreover, it requires a long-term cold treatment (vernalization) in order to gain flowering competence. However, within a winter cereal gene pool, there are large differences in the ability to acquire frost tolerance (FT) during cold acclimation (CA). The proteome composition and cold tolerance were analysed in winter barley Luxor and a set of two winter wheat cultivars Mironovskaya 808 and Bezostaya 1 and four reciprocal substitution lines with interchanged chromosomes 5A and 5B during a long-term cold-acclimation (CA) treatment (84 days). Changes in proteome were evaluated by two-dimensional difference gel electrophoresis (2D-DIGE) analysis, plant development was determined by analysis of morphological changes in shoot apex, and acquired frost tolerance (FT) was expressed as LT50 values determined by direct frost tests. Differently accumulated proteins were identified and divided into groups according to their functions (e.g., ROS, COR, chaperones, enzymes of carbohydrate metabolism, amino acid metabolism). The identification of differently accumulated proteins in differently tolerant genotypes was successful (e.g., legumin-like protein, malate dehydrogenase, proteins of ABA_WDS superfamily). Of these, WCOR615 protein and legumin-like protein revealed higher expression levels in genotypes harboring chromosome 5A from highly tolerant Mironovskaya 808 when compared to expression levels in genotypes carrying chromosome 5A from less tolerant Bezostaya 1. Proteomic analysis has thus confirmed previous findings of the prominent effect of chromosome 5A on expression of several COR/LEA proteins in wheat.Differently accumulated proteins could be tested as markers of tolerance. Comprehension of mechanism by which cereals cope with low temperatures is essential for breeding new resistant cultivars and thus for increasing yields in lower productive areas.

39

EXPLOITING SPECIFIC ISOFORMS OF LIPOXYGENASE, ALLENE OXIDE SYNTHASE

GENES IN THE JA BIOSYNTHESIS PATHWAY AND SPECIFIC CLASSES OF SMALL

RNAS IN ROOTS, TO SELECT VARIETIES AND HYBRIDS BETTER TOLERATING

ABIOTIC STRESSES IN LEGUMES

Poltronieri P, De Domenico S, Bonsegna S, Taurino M, Santino A

National Research Council of Italy, Agrofood Dept., Institute of Science of Food Productions CNR-

ISPA, via Monteroni km 7, 73100 Lecce, Italy

Legumes play an increasing role in mixed cropping systems, rotations and as target crops for food, livestock feeding, and in human diets. In our work we focused on improvement of response to abiotic stress in chickpea, and in studying the role of small RNAs in stress response in pea. Chickpea varieties with prolonged tolerance to drought were used to dissect the signaling pathway enabling to produce hormones and metabolites such as JA, OPDA, oxylipins, and ABA earlier than other non tolerant varieties. The lipoxygenase pathway seems to be responsible for the triggering of mechanisms letting the plant to adjust to the environment. Nowadays the chickpea genome draft has been made available, and we intend to individuate microRNAs such as miR-393 having a role in the control of TCP4 transcription factors, which activate many genes of the JA biosynthesis pathway. In pea, in the context of the FP7 project AB-STRESS, a library of small RNAs induced during biotic and abiotic stresses is under sequencing. We aim to identify legume specific and conserved microRNAs and other small RNA classes that may have a role in the response to environment and in the stress memory. A programme aiming to produce transformed tissues (roots) overexpressing and antisense constructs for individual genes in these pathways is under way. A series of Taqman probes able to differentiate between LOX, HPL, OAS, OAC and OPR isoforms involved in the synthesis of JA and expressed specifically in roots was developed together with GenXpro, Germany. These probes are suitable to be used in selection of hybrids obtained by crossing a high protein , high seed producing variety with a variety better responding to abiotic stress, to identify the hybrids with the desired characters.

40

NUTRIENT DEPRIVATION IN ARABIDOPSIS REVEALS A FUNCTION FOR THE

RETROMER COMPLEX IN AUTOPHAGY

Bayle V, Gaude T

Laboratoire Reproduction et Développement des Plantes UMR5667 CNRS-INRA-ENS Lyon-

Université Claude Bernard Lyon I, 46 allée d’Italie 69364 Lyon, France

Mechanisms controlling intracellular trafficking of molecules play a crucial role in cell function as they allow location of the right molecules at the right place and at the right time. This ensures that particular functions only occur at defined cellular sites. These mechanisms are not only important for cell fate but also for the proper development of whole multicellular organisms by positioning, maintaining or removing the tremendous variety of signaling molecules (hormone receptors, stress-sensing receptors, ion channels, immune related molecules, etc.) to/from their active sites so as to mediate cell communication. The mislocalization of molecules can cause dramatic cellular effects resulting in some cases to diseases, severe developmental defects or death. Retromer is a pentameric protein complex conserved from yeast to human that localizes to endosomal membranes and is involved in intracellular protein sorting. In plants, retromer components are required for the targeting of vacuolar storage proteins and phytohormone auxin receptor recycling. We previously showed that retromer loss-of-function mutants display altered cell polarity and organogenesis defects in Arabidopsis. Under certain stress conditions, such as starvation or nutrient restriction, organisms initiate an autophagy process that will provide novel nutrients for cell survival through organelle or cytosolic material degradation in lytic compartments. In the present study we show that the retromer loss-of-function mutant vps29 is able to initiate autophagy in response to sucrose or nitrogen deprivation, but contrary to other described autophagy deficient (atg) mutants, it fails to complete the process. Using confocal microscopy and intracellular compartment fluorescent markers, our results suggest that the retromer complex is required for the proper maturation of autophagosomes and further degradation in the lytic vacuole. Our goal is now to decipher the crosstalk between the retrograde endocytic pathway mediated by the retromer complex and the biogenesis and maturation of autophagosomes.

41

SUPPORT OF PLANT GROWTH BY SOIL MICROBES FOR PHYTOREMEDIATION OF

HEAVY METAL SOIL

Lichtscheidl I, Turnau K, Adlassnig W

Cell Imaging and Ultrastructure Research, University of Vienna, Althanstrasse 14, A-1090

Wien/Vienna, Austria

Land contaminated by mine wastes is characterized by extreme environmental conditions, not only because of high contents of toxic heavy metals (HMs), but also concerning nutrient supply, water holding capacity, stability of the matrix and microclimate. Accordingly, it often presents a barren desert-like landscape. For on-site soil remediation, first measures have to be the development of a cover of vegetation; suitable plants that endure the difficult ecological conditions stabilize the soil by their roots and prevent distribution of the fine sands by wind and water (“phytoremediation”). HMs can hence become either fixed in the rhizosphere and prevented from leaching into the ground water and from accumulating in the food chain (“phytostabilization”), or HM can be accumulated into above-soil parts of green plants and removed from the ecosystem by harvesting biomass (“phytoextraction”). Plant growth in nutrient-poor environment depends largely on interaction with symbiotic and mutualistic microbes in the rhizosphere. Soil microorganisms may influence plant growth by direct interaction with the plant as well as by their effects on soil chemistry, including synthesis of growth factors, release of nutrients and precipitation or dissolution of HM minerals. Phytoremediation promises cost-efficient, user-friendly and environmentally acceptable progress in cleaning up contaminated soil, but so far, we only start to understand the diversity of soil microorganisms and their interactions with plants. In this article, we discuss recent advances in bioremediation of HM polluted soil by promoting plant growth efficiently and sustainably through inoculation of plants with suitable microbes.

42

MITOGEN-ACTIVATED PROTEIN KINASES AND ABIOTIC STRESS IN PLANTS

Šamaj J

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell

Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech

Republic

Mitogen-activated protein kinases (MAPKs) are phosphorylating enzymes involved in stress signal transduction in plants. We are studying stress-induced MAPKs such as SIMK in Medicago sativa as well as MPK3, MPK4 and MPK6 in Arabidopsis thaliana. Our main interest is to dissect complex behaviour of these MAPKs including transcriptional regulation, post-translational activation as well as subcellular localization and compartmentalization under diverse stress and developmental conditions. For this purpose we are using complementary approaches such as qPCR, in situ hybridization, promoter-GUS histochemistry, phos-Tag, immunolocalization and recombinant GFP technology in wild type and mutant plants. Our previously published as well as recent results will be presented.

43

DRIVING THE PROCESS OF PHOTOSYNTHESIS TOWARD RENEWABLE FUELS AND

CHEMICALS

Melis A

University of California, Berkeley, CA, USA

The concept of Photosynthetic Biofuels entails the direct application of photosynthesis for the generation of fuel molecules, in a process where a single organism acts both as photocatalyst and processor, absorbing sunlight, photosynthesizing, and secreting ready to use product. An example of successful application of this concept is isoprene (C5H8) hydrocarbons photo-production by green microalgae and cyanobacteria, generated directly from sunlight, carbon dioxide, and water. The work will describe metabolic engineering approaches, whereby photosynthesis in microalgae and cyanobacteria is diverted toward terpenoid hydrocarbon synthesis and release. Green microalgae, e.g. Chlamydomonas reinhardtii, and cyanobacteria, e.g. Synechocystis sp., are model organisms amenable to transformation for bioenergetic and metabolic flux manipulation, leading to photosynthetic isoprene volatile hydrocarbons production. The work will further describe approaches of isoprene sequestration and trapping. Additional related issues include efforts to improve the solar-to-product energy conversion efficiency of photosynthesis, from a current max of 1-3% (solar-to-biomass energy conversion efficiency) up to a theoretical maximum of ~10%. The approach entails minimizing the chlorophyll antenna size of the photosystems, thereby preventing the over-absorption of bright sunlight and the wasteful dissipation of the extra energy. Such manipulation compromises the ability of individual cells to over-absorb sunlight and results in greater solar-to-biomass and solar-to-fuels conversion efficiencies in a mass culture. Genes that confer a “truncated chlorophyll antenna size” in photosynthesis will be discussed.

44

MEDITERRANEAN ADAPTIVE STRATEGIES: PHENOLOGY, PRODUCTIVITY AND

WATER RELATIONS IN WILD AND DOMESTICATED LUPINUS LUTEUS L. FROM

HABITATS WITH CONTRASTING TERMINAL DROUGHT

Berger JD1,2, Ludwig C1

1 CSIRO Plant Industry, Private Bag No. 5, Wembley WA 6913, Australia

2Centre for Legumes in Mediterranean Agriculture, Faculty of Natural and Agricultural Sciences,

The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

Our understanding of annual plant adaptation to rainfall gradients is rudimentary because C-S-R frameworks are often applied superficially, while detailed information on plant processes is scarce and contradictory. The Mediterranean legume Lupinus luteus was evaluated under contrasting terminal drought, using wild and domesticated material that had evolved under divergent drought stress. In wild germplasm, long-season, high rainfall habitats select for competitive traits: delayed phenology, high above- and below-ground biomass, greater productivity and fecundity, leading to high water-use, and early stress onset. Terminal drought-prone environments select for the opposite: ruderal traits that facilitate drought escape/avoidance but limit reproductive potential. While both ecotypes are similarly stress-sensitive in regulating water-use, the former reach a lower critical LWP, maintaining higher RWC than the latter, a tolerance capacity that may have evolved in response to intermittent self-imposed droughts driven by large biomass/water-use. Domesticated L. luteus is even more R-selected than low rainfall ecotypes, reflecting ongoing selection for timely ripening and drought escape. While this suits short-season environments, it forgoes potentially adaptive traits (eg. C-selected drought tolerance) and is inappropriate for productive, longer-season areas.

45

DETERMINATION OF MORPHOLOGICAL AND MOLECULAR CHARACTERISTICS OF

COLCHICUM L. SPECIES IN THE TURKISH FLORA

Tüyel U1, Verma SK1, Akfırat FS2, Şakiroğlu M3, Aydın Y4, Kaya E5, Uncuoğlu AA1

1Marmara University, Faculty of Engineering, Department of Bioengineering, Goztepe Campus,

34722, Istanbul -TURKEY

2Gebze Institute of Technology, Faculty of Science, Department of Molecular Biology and Genetics,

Cayirova Campus, Kocaeli – TURKEY

3Kafkas University, Faculty of Engineering and Architecture, Department of Bioengineering,36100,

Kars - TURKEY

4Marmara University, Faculty of Arts and Sciences, Department of Biology, Goztepe Campus,

34722, Istanbul -TURKEY

5Atatürk Central Horticultural Research Institute (AKMAE), 77102, Yalova – TURKEY

The genus Colchicum has a large number of species (ca.100) that are widely scattered throughout the different region of Anatolia and the region is considered as a major center of diversity and speciation. Colchicum L. is commonly known as autumn crocus and is a member of the Colchicaceae family. Colchicum L. is being represented by 49 taxa (47 species) in Turkey and 35 of those are endemic. So as to understand distinction among putative species, we aim to measure various morphological characters and deduce the degree of genetic differences via molecular markers. Initially, flower characteristics of 396 different plants from 128 taxa that are flowering in fall have been measured. We observed extensive morphological variations among taxa. For instance, C.micaceum K.Perss. showed the shortest vegetation by 8 days while the C.stevenii Kunth had the longest vegetation of 27 days. For molecular analysis, Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers are being employed. RAPD is one of the easiest and most commonly used molecular techniques for genetic variability analysis for the plants that have limited molecular tools. In the present study, the analysis of the genetic diversity of Colchicum L. collected from different geographical locations was investigated by RAPD-PCR analysis. Currently, a total of 206 populations are being genotyped by RAPD markers. A high level of polymorphism was observed in the RAPD profiles of plants. Hence, the results of this study indicate that there are significant morphological and molecular variations among the natural populations of Colchicum L. collected from various geographic and climatic regions of Turkey.

46


SESSION IV: PLANT BIOENERGETICS

47

TOWARDS THE STRUCTURES OF PROTEIN COMPLEXES INVOLVED IN PLANT

BIOENERGETICS USING SINGLE PARTICLE ELECTRON MICROSCOPY

Kouřil R

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of

Biophysics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ 783 71 Olomouc-Holice,

Czech Republic

Single particle electron microscopy is a powerful tool for structural characterization of biomolecular assemblies at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of electron microscopy. Image processing is performed by averaging large numbers of individual projections selected from electron micrographs. Average projection maps of large protein complexes/supercomplexes can be fitted with atomic x-ray structures of individual components and pseudo-atomic model of a supercomplex organization can be proposed. It can be applied for studies of unstable protein complexes, which cannot be purified to homogeneity, e.g. large, transient membrane complexes. Combination of this technique with biochemical characterization, separation methods and other biophysical techniques enables us to understand the structure and the function of protein complexes. Application of single particle electron microscopy and image analysis for structural characterization of protein complexes and supercomplexes involved in photosynthesis (Photosystem I and Photosystem II) and mitochondrial respiration (complex III and complex IV) will be presented.

48

STARCH METABOLISM, ITS REGULATION AND BIOTECHNOLOGICAL

APPROACHES TO IMPROVE YIELDS

Bahaji A1*, Li J1*, Sánchez-López A1*, Baroja-Fernández E1, Muñoz FJ1, Ovecka M1,2, Etxeberria

E3, Pozueta-Romero J1

1Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Mutiloako etorbidea z/g, 31192

Mutiloabeti, Nafarroa, Spain.

2Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell

Biology, Faculty of Science, Palacký University, Šlechtitelů11, CZ-783 71 Olomouc, Czech

Republic

3University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education

Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA

Starch is the main storage carbohydrate in vascular plants and structurally composed of the glucose homopolymers amylose and amylopectin. Synthesized in the plastids of both photosynthetic and non-photosynthetic cells, its abundance as a naturally occurring compound is surpassed only by cellulose, and represents both a cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including production of adhesives, biodegradable materials, and first-generation bioethanol. This talk will provide an update on different suggested pathways of starch metabolism in both autotrophic and heterotrophic organs, and emerging information about the networks regulating them and their interactions with the environment. Special emphasis will be given to recent findings showing that volatile compounds emitted by microorganisms promote the accumulation of exceptionally high levels of starch in both mono- and dicotyledonous plants. Finally, we will also review how plant biotechnologists have attempted to use basic knowledge on starch metabolism, mostly obtained using Arabidopsis as the reference system, for the rational design of genetic engineering traits aimed at increasing starch in annual crop species fitting both industrial needs and social demands.

49

TOWARDS AUTOMATED IN SITU PRIMARY PRODUCTIVITY MEASUREMENTS –

CONVERTING FLUORESCENCE BASED ELECTRON TRANSFER RATES TO

CARBON FIXATION

Lawrenz E1, Huete-Ortega M2, Suggett DJ.2, Geider RJ2, Prášil O1

1Microbiologický ústav AVČR, Opatovický mlýn, 37981 Třeboň

2School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK

Primary productivity is essential to describing structure and function of aquatic ecosystems. Development of bio-optical sensors, such as fast repetition rate (FRR) fluorometers, have opened up new avenues for deriving proxies of in situ primary productivity from measurements of electron transfer rates (ETR). However, FRR fluorometers determine productivity in a photosynthetic 'currency' of electrons produced by the splitting of water at PSII whereas most aquatic disciplines are interested in carbon-specific rates of primary productivity. Thus, measurements of ETRs must be converted to carbon-specific primary productivity rates via a conversion factor, that is the ratio of ETR:carbon fixation (units mol electrons/mol carbon fixed). This variable, however, is dependent upon the physiological state of phytoplankton. Here, we present results of a comprehensive laboratory study examining the effect of nitrogen availability on the variability of this conversion factor in model phytoplankton species from coastal and open ocean waters. Our results show a strong decline in carbon fixation in nitrogen limited phytoplankton, while ETR increased. This uncoupling of electron transfer and carbon fixation lead to large intraspecific variability in the conversion factor. Potential underlying cellular mechanisms driving this uncoupling and the resulting implications for field applications of FRR fluorometry will be discussed.

50

SEQUENCING OF CHLOROPLAST TRN-T-L-F INTRON FOR PHYLOGENETIC

ANALYSIS OF COLCHICUM GENUS FROM FLORA OF TURKEY

Aydemir M1, Verma SK1, Şakiroğlu M2, Aydın Y3, Kaya E4, Uncuoğlu AA1

1Marmara University, Faculty of Engineering, Department of Bioengineering, Goztepe Campus,

34722, Istanbul-TURKEY

2Kafkas University, Faculty of Engineering and Architecture, Department of Bioengineering, 36100,

Kars, TURKEY

3Marmara University, Faculty of Arts and Sciences, Department of Biology, Goztepe Campus,

34722, Istanbul-TURKEY

4Atatürk Central Horticultural Research Institute (AKMAE), Yalova, 77102, TURKEY

The number of studies aimed to tackle the taxonomic classification of Colchicum species via molecular tools is extremely limited. Occurrence of the high frequencies of Colchicum species in Turkey and the elevated number of the endemics could be a strong indication that Turkey is a major center of diversity for Colchicum. Phylogenetic relationships among 49 species of large Colchicum genus were investigated using DNA sequence data from the chloroplast intergenic region trnT-trnL-trnF. Plant material was obtained from Atatürk Central Horticultural Research Institute in Yalova and genomic DNA from 206 populations from a total of 49 species was isolated. In this study, noncoding trnT-trnL-trnF regions of chloroplast genome were amplified using six universal primers. PCR products were directly sequenced using Cycle Sequencing Ready Reaction Kit following the cycle sequencing procedure with BigDye terminator. The Sequences were aligned with the software program ClustalW. To infer the population structure of the entire set of species avoiding superimposing the preexisting classification, we used the software program STRUCTURE. In order to deduce the evolutionary relationship among the species, a Neighbor-joining tree was subsequently created using phylogenetic package Phylip. This study will help to reveal the relationship among the naturally growing 206 populations of Turkish Flora and help to deduce the structure and the evolution of the genome.

51

RAPID CHANGES OF CHLOROPHYLL A FLUORESCENCE AND PHOTOSYNTHESIS

IN RESPONSE TO ACTION POTENTIALS GENERATED BY TOUCH IN

CARNIVOROUS PLANT DIONAEA MUSCIPULA ELLIS.

Pavlovič P1,2, Libiaková M 2

1 Centre of the Region Haná for Biotechnological and Agricultural Research, Department of


Czech Republic

2 Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava,

Mlynská dolina B-2, SK-84215, Bratislava, Slovakia

The modified leaf called trap in carnivorous plant Venus flytrap (Dionaea musipula) catches prey by very rapid movement of its bilobed halves that shut when the trigger hairs protruding from the upper leaf epidermis are stimulated by touch. Two subsequent stimulations of trigger hairs generate two action potentials (APs) which are the immediate cause of the rapid trap movement. Repeating generation of APs by entrapped prey is responsible for releasing of digestive fluid. We found that generation of AP has significant impact on CO2 uptake: photosynthetic rate (AN) transiently decrease and respiration rate (RD) increase. In this study we investigate: i) the spatio-temporal dynamic of photosynthetic reactions using chlorophyll a fluorescence and gas exchange measurements in response to APs to find out the primary target of APs on photosynthetic reactions, ii) the enzymatic properties of digestive fluid in response to AP generation. We conclude that APs play an indispensable role in prey digestion and nutrient economy in carnivorous plant but inhibition of photosynthetic reactions is a negative and in view of carbon economy costly consequence of such type of signaling, which pay off after digestion; increase uptake of nitrogen from prey can later stimulate photosynthetic reactions. This study was supported by grant VEGA 1/0520/12 from the Ministry of Education of Slovak Republic and Ministry of Education, Youth and Sport of Czech Republic CZ.1.07/2.3.00/20.0057.

52

ABSTRACTS – POSTER SESSION

53

BIOLOGICAL ACTIVITIES OF NEW BRASSINOSTEROID DERIVATIVES

Bazgier V1, Oklešťková J2, Rárová L1, Kvasnica M3 , Berka K4 and Strnad M1,2

1Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth

Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc-Holice, Czech

Republic

2Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of


3Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,

Flemingovo nam. 2, 16610 Praha 6

4 Department of Physical Chemistry, Regional Center of Advanced Technologies and Materials,

Faculty of Science, Palacký University in Olomouc, tř. 17. listopadu 12, 771 46 Olomouc, Czech

Republic

Brassinosteroids are steroid plant hormones with important regulatory roles in various physiological processes, including growth, differentiation, root and stem elongation, disease resistance, stress responses, and vascular differentiation of many plants1. This group of natural plant steroids includes more than 70 compounds distributed from lower to higher plants. Brassinosteroids have been detected and isolated from seeds, fruits, leaves, galls and pollen. It was shown that natural BRs have inhibitory effects on the growth and viability of various normal and cancer cell lines2.

The aim of our study relates to synthesis of a series of new brassinosteroid monohydroxylated derivatives and to study of their biological and anticancer properties. Molecular modelling was used for design of new potentionally interesting derivatives. The modified versions of natural steroid brassinolide were docked using AutoDock Vina3. The most promising molecules selected from docking were synthesized and tested. Plant growth promoting activity of synthetic analogs was assayed using the bean second internode bioassay. The anticancer activity of the new BRs analogues was evaluated in vitro using cancer cell lines of different histopathological origins and normal human fibroblasts. Bajguz A.: Plant Physiol Bioch. 45, 95-107, 2007. Malíková, J., et al.: Phytochemistry, 69, 418-26, 2008. O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461

54

GENOMIC CONSTITUTION OF CEREALS WITH BLUE ALEURONE TRAIT

Burešová V1, Kopecký D1, Šafář J1, Vyhnánek T2, Martinek P3, Doležel J1



2Mendel University of Agriculture and Forestry in Brno, Zemědělská 1, 613 00 Brno, Czech

Republic

3Agrotest Fyto, Ltd., Havlíčkova 2787, 767 01 Kroměříž, Czech Republic

Anthocyanins are of great importance for human health due to their antioxidant potential. Their content is rather low in common varieties of wheat (Triticum aestivum L.). However elite lines with blue aleurone and introgressed chromatin from wild relatives exhibit significantly increased levels of anthocyanins. There is evidence that the donor of chromosome introgressions has been Thinopyrum ponticum (syn. Agropyron elongatum). The aim of our study was to characterize genomic constitution of selected cereals (wheat, barley and Triticale) with blue aleurone using genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). We used total genomic DNA of Th. ponticum and two repetitive DNA sequences (GAA repeat, Afa family) as probes to identify individual chromosomes. This enabled precise localization of introgressed chromatin. Our results revealed large variation in genomic constitution of blue aleurone wheat genotypes. Out of 14 analyzed lines, 11 lines carried an introgression from Th. ponticum. In the remaining three genotypes (cvs. Indigo, Skorpion, and line H83-952-1), we were unable to detect any introgressed chromosome segment. Six different types of introgressions were found, ranging from the addition of a telocentric chromosome pair (cv. Blue Norco) to substitution of one chromosome pair (cv. Blue Baart), substitution of complete (homologous) chromosome arms (line UC660-49) and various substitutions of distal parts of chromosome arm(s). Different types of introgressions observed in or work support a hypothesis that the introgressions activate the blue aleurone trait pathway, which is present, but deactivated in common wheat germplasm. This work was supported by the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01).

55

THE RESPONSE OF THE MULTIPLE HYPERACCUMULATORS THLASPI

CAERULESCENS, THLASPI GOESINGENSE, AND THE EXCLUDER PLANTAGO

MAJOR TOWARDS RADIONUCLIDE 238U

Burger A1,3, Baumann N2, Weidinger M1, Zöger N3, Arnold T2, Lichtscheidl I1

1Cell Imaging and Ultrastructure Research, University of Vienna, Althanstrasse 14, 1090 Vienna,

Austria

2Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstraße, 01328 Dresden, Germany

3Nuclear Engineering Seibersdorf GmbH, 2444 Seibersdorf, Austria

8 plant species were cultivated hydroponically in medium containing uranium (U) in concentrations between 1 µM and 10 mM.The effects of U were tested by describing plant growth parameters such as biomass, length of roots, stems and leaves, the degree of chlorosis and necrosis, and the rate of photosynthesis. Uptake of U into roots, stems and leaves was analyzed from extracts by inductively coupled plasma mass spectrometry (ICP-MS). Localization of U in plant organs on a tissue-specific level was determined by energy-dispersive X-ray spectroscopy (EDX) in the scanning electron microscope (SEM). The amount of accumulated U was strongly influenced by U concentration in the cultivation medium. Increase U contamination caused visible negative effects such as necrosis and chlorosis. Most sensitive was Thlaspi goesingense from Flatz, a non-contaminated habitat; it showed also the highest U uptake (17.200 mg U kg-1 in roots and 550 mg U kg-1 in leaves). Most tolerant up to 10 mM U in hydroponic culture was Plantago major, which occurs also naturally on U containing soil such as former uranium mining heap site in western Thuringia, close to Ronneburg. On an average, roots contained most of the U, less U was transported further up to the leaves. Element analysis by EDX-SEM revealed, however, that U is not distributed evenly within leaves and roots, but that it is accumulated in the cortex of the root and in the central tissues of the leaves; in this mesophyll, U concentration may even surpass the amount of U in the roots, similar as it is observed in hyperaccumulating plants.

56

EFFECT OF SILICON SUPPLY ON THE ANTIOXIDANT SYSTEM OF PERENNIAL

RYEGRASS PLANTS

Govinden B1, Pontigo S2, Ribera A3, de la Luz Mora M4, Cartes P4

1Doctoral Program in Science of Natural Resources, Universidad de La Frontera, P.O. Box 54-D,

Temuco, Chile

2Doctoral Program in Science of Natural Resources, Universidad de La Frontera, P.O. Box 54-D,

Temuco, Chile

3Center of Plant-Soil Interaction and Natural Resources Biotechnology, Scientific and

Technological (BIOREN-UFRO), Universidad de La Frontera, Avenida Francisco Salazar Nº 01145,

P.O. Box 54-D, Temuco, Chile

4Departamento de Ciencias Químicas y Recursos Naturales and Center of Plant-Soil Interaction

and Natural Resources Biotechnology (BIOREN-UFRO), Universidad de La Frontera, P.O. Box 54-

D, Temuco, Chile

Silicon (Si) exerts beneficial effects on higher plants, especially alleviating both biotic or abiotic stresses and the consequent oxidative damage. Earlier findings about the impact of Si on plant antioxidant system indicated that the capacity of this element to counteract oxidative stress may be closely related to plant species. In this context, we investigated the role of Si on the antioxidant capacity in perennial ryegrass (Lolium perenne L.), one of the most important forage species sown in Southern Chile. Plants were hydroponically cultivated at increasing Si doses (0, 2, 5 or 10 mM Si). At 21 days shoots and roots were harvested, and growth, lipid peroxidation, superoxide dismutase (SOD) activity and total phenolic (TPH) concentration were evaluated. As a result of Si applications no significant differences in growth of plants were observed. A decrease of lipid peroxidation and SOD activity in shoots at 2 mM Si occurred, whereas in roots, a slight increase of oxidative damage at Si supply up to 5 mM was found. This increment coincided with both, a rise of TPH concentration and a reduction of SOD activity. In shoots, phenolics increased in response to Si additions up to 5 mM, but above this dose, TPH levels were significantly decreased. This behaviour seems to be related to an increase of root TPH concentration when the greatest Si treatment was applied. Our results indicated that Si exerted a positive influence on the perennial ryegrass antioxidant system by enhancing phenolics, mainly in roots, that could be counteracting the oxidative stress in aboveground tissues. Acknowledgements. This research was supported by the FONDECYT project 1120901 and Doctoral Studies CONICYT Scholarships 21120704 and 21120779.

57

MOLECULAR AND CYTOGENETIC CHARACTERIZATION OF WILD MUSA SPECIES

NEWLY INTRODUCED TO ITC COLLECTION

Čížková J1, Hřibová E1, Christelová P1, Van den Houwe I2, Roux N3, Doležel J1



2Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Kasteelpark Arenberg

13, B-3001 Leuven, Belgium

3Bioversity International, Parc Scientifique Agropolis II, Montpellier Cedex 5, 34397, France

Bananas and plantains (Musa spp.) are a major staple food and an important export commodity in many countries of humid tropics. The production of bananas is, however, threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world's largest banana and plantain collection is maintained at the Biodiversity International Transit Centre (ITC) in Belgium and contains more than 1000 accessions. This reference collection is continuously extended by various edible cultivars and wild species. Detailed morphological and molecular characterization of the deposited as well as newly introduced accessions is of a great significance for efficient collection and protection of banana diversity. The aim of this work was to characterized a set of wild Musa species, which were newly introduced into the ITC collection. Altogether, 23 wild Musa accessions were described in terms of cytogenetic as well as molecular characters. Nuclear genome size, chromosome number and genomic distribution of rRNA genes were determined in individual accessions to shed light on their genome structure and evolution. The molecular characterization was performed using a set of 19 microsatellite markers. The SSR genotyping platform enabled us to identify species most closely related to the unknown accessions and thus clarify their phylogeny. In several cases, SSR genotyping was coupled with the sequence analysis of ITS DNA spacer region, to elucidate the putative hybrid character of some accessions. The ITS1-5.8S-ITS2 region was amplified using specific primers and PCR products were sequenced after cloning. Sanger sequencing of ITS region was also used to support incongruent results of SSR analysis.

58

BAC-POOL SEQUENCING OF THE PHYSICAL MAP FROM WHEAT CHROMOSOME-

ARM 3DS

Cviková K1, Bartoš J1, Šimková H1, Šafář J1, Stein N2, Cattonaro F3, Bergès H4, Mayer K5,

Doležel Jl



2Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstrasse 3, 06466

Gatersleben, Germany

3Instituto di Genomica Applicata, Via J. Linussio 51, 33100 Udine, Italy

4INRA, National Resources Centre for Plant Genomics, F-31326 Castanet Tolosan Cedex, France

5Institute for Bioinformatics and Systems Biology (MIPS), Helmholtz Center f. Health and

Environment, Ingolstadter Landstr. 1, D-85764 Neuherberg, Germany

Construction of a physical map using BAC library from individual chromosome or chromosome-arm after flow-sorting is an important step towards sequncing complex genome of hexaploid wheat. Overlaps of BAC clones are detected by High-Information Content Fingerprinting (HICF) technology on the basis of shared bands in restriction spectrum. Fingerpinted data are then elaborated by Finger Printed Contigs (FPC) or Linear Topological Contig (LTC) software to build contigs. For sequencing, an order and true orientation of contigs is necessary to avoid distortions on reference sequence. Usually, screening BAC library through pools with available and newly developed molecular markers is used for contig anchoring. We joined screening of pools from BAC library and next-generation sequencing to anchor and organize contigs of the physical map in an high-throughput manner. Minimum Tilling Path of the physical map of wheat chromosome-arm 3DS was used for preparation of pools that were sequenced by Illumina Hiseq2000. Reads gained with this sequencing technology were alignment to reference sequences representing in silico markers. First reference sequence for read alignment was obtained by sequencing of chromosome-arm 3DS with low coverage using Roche 454 GS-FLX followed by sequence ordering according to sequenced genomes relative to hexaploid wheat. Second reference represented SNPs sequences from Aegilops tauschii. BAC addresses of positive clones were de-convoluted from positive pools for each sequence and over 21,000 sequences of intra- and interspecific origin were anchored to the physical map. These sequences were used for contig ordering with help of genetic maps and collinearity with related grass species.

59

METABOLOME GRAIN ANALYSIS OF WHEAT TRITICUM AESTIVUM L. HYBRID

OBTAINED BY INTERSPECIFICAND INTERGENERIC HYBRIDIZATION WITH

POACEAE FAMILY SPECIES

Czaplicki AZ, Pilch J., Żebrowski J., Zimny J.

Plant Breeding & Acclimatization Institute – NRI, Radzików, 05-870 Błonie, Poland

Metabolome grain analysis of 49 wheat offspring (Triticum aestivum L.) obtained by interspecific and intergeneric hybridization with Poaceae family species was carried out. Six commercial varieties of wheat were used as a reference material. The analysis was performed using Fourier Transform spectroscopy (FTIR) in the mid-infrared range. This method provides information about the presence of functional groups in tested material, based on the spectral analysis of absorption/transmission of electromagnetic radiation in the wave range 4000 - 400 [1/cm]. It allows to determine the metabolome characteristics of material tested in terms of chemical compounds content (e.g. polysaccharides, fats, proteins, and the characteristics of the compounds within the carbohydrates, fats and proteins). Two random samples of 5-6 grains ground in a ball mill were analyzed. The analysis was performed using a Nicolet FTIR spectrometer iZ10 and the total internal absorption ATR technique. Collected data wwere analyzed using principal component analysis (PCA) and cluster analysis. The overall range of metabolome variation of seeds obtained in interspecific hybrids significantly exceeded that observed in the current available wheat varieties. A detailed spectral analysis was carried out in the ranges typical for: fatty compounds, proteins and carbohydrates. Grains of the hybrids (usually from the same crosses) were found to be characterized by much greater variability in the quality and the relative amounts of fat, proteins and carbohydrates than that observed in the commercial wheat varieties. Dendrogram for cluster analysis confirmed that the resulting variability in the fraction of carbohydrates in grains significantly exceeded that, which characterizes the current commercial varieties of wheat.

60

IN VITRO PROPAGATION OF OKRA, ABELMOSCHUS ESCULENTUS BY APICAL

MERISTEMS

Chika NE

Department of Biology, Faculty of Science, University Putr Malaysia, 43400 UPM Serdang,

Selangor Darul Ehsan, Malaysia

Prior to Abelmoschus esculentus economic, nutritional and medicinal uses, the need to provide a reliable pathogen free varieties, establish appropriate media/explants for regeneration, improved yield variety and availability in all year round prompted the study of Malaysian okra variety. Procedures for in vitro propagation of okra using meristematic culture were developed. Meristematic tissues (0.4-0.6mm) of Okra seedlings (at day 13) were excised from aseptically and non-aseptically grown seedlings. The shoot apical meristem (SAM) was cultured using MS medium (Murashige and Skoog) supplemented with different concentrations of 1-Naphthaleneacetic Acid (NAA) in combination with BAP (0.5+1.0mg/l , 0.5+2.0mg/l, 1.0+4mg/l, 1.5+4mg/l) and Indole-3-Acetic Acid (IAA) in combination with Zinetin(0.5+1.0mg/l , 0.5+2.0mg/l, 1.0+4mg/l, 1.5+4mg/l) While BAP and Zinetin(1.0mg/l, 2.0mg/l, 3.0mg/l and 4.0mg/l) were supplemented singly. The SAM from aseptically and non-aseptically grown seedlings results showed proliferations and shoots formation on semi-solid and solid Ms medium cultured in 2.0mg/l BAP with four shoots per culture and three shoots among NAA+BAP at 1.0+4mg/l, 1.5+4mg/l and Kinetin 3.0mg/l. The plantlets were transferred into pots containing soil for acclimatization. This research has harnessed the media for in-vitro culture of Malaysian okra variety.

61

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DROUGHT STRESS

RESPONSIVE DEHYDRIN GENES IN OIL SEED RAPE

Chikkaputtaiah C1, Havlickova L1, Urban M2, Vitamvas P2, Prasil IT2 and Curn V1

1Biotechnological Centre, Faculty of Agriculture, University of South Bohemia, Ceske Budejovice,

Czech Republic

2Crop Research Institute, Drnovska 507, CZ-16106, Prague, Czech Republic

Dehydrins have been identified as one of the major classes of Late embryogenesis-abundant (LEA) proteins from many plant species which play a fundamental role in plant response and adaptation to abiotic stresses. Dehydrins are primarily localized to the cytoplasm and nucleus, accumulate late in embryogenesis, and nearly in all the vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration such as drought, cold and salinity. Drought stress is one of the major abiotic stress factor induces the expression of dehydrin genes. Although the identification and functional analysis of different stress induced dehydrins are well studied in model plant systems and other crop plants, despite their importance in crop breeding, little is known about the genetic and functional roles of dehydrins in Brassica napus, one of the major oil seed crops in the world and prominent member of family Brassicaceae. Preliminary proteomic analysis results have shown different Brassica napus cultivars with contrast proteomic profiles of dehydrin proteins in response to drought stress. Using a range of genetic, transcriptomic and proteomic analysis, we aim to identify & quantify the expression pattern of potential dehydrin genes in Brassica napus and study their polymorphism in different cultivars in response to drought stress. The correlation between proteomic and genetic analysis would reveal whether the change in dehydrin pattern typical for resistant cultivars is induced either by dehydrin genes and their TFs or by post-transcriptional/ post-translational modifications. This will enable to develop drought-induced dehydrins as gene and allele specific markers for improvement of practical plant breeding in family Brassicaceae. This research was supported by the project Postdoc USB (reg.no. CZ.1.07/2.3.00/30.0006) realized through EU Education for Competitiveness Operational Programme. Project is funded by European Social Fund and Czech State Budget.

62

ROLE OF TOCOPHEROL AND PLASTOCHROMANOL IN SCAVENGING OF SINGLET

OXYGEN UNDER PHOTOOXIDATIVE STRESS IN ARABIDOPSIS THALIANA

Deepak Kumar Yadav1, Anshu Rastogi1, Szymańska R2, Kruk K2, Sedlářová M3 and Pospíšil P1

1Centre of the Region Haná for Biotechnological and Agricultural Research, Department of


Czech Republic

2Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and

Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.

3Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc,

Czech Republic

Plant has evolved different types of protective mechanism against the photooxidative stress, such as stimulation of synthesis of low molecular weight antioxidant in chloroplast. At the final step of biosynthesis process, tocopherol and plastochromanol is formed by the activity of tocopherol cyclase enzyme vte1 in plants. We studied the singlet oxygen scavenging activity of tocopherol and plastochromanol using vte1 mutant (lacking tocopherol and plastochromanol) in comparison with WT of Arabidopsis plant. High pressure liquid chromatography analysis confirmed the absence of tocopherol and plastochromanol in leaves of vte1 Arabidopsis plant. It is demonstrated here that the higher singlet oxygen formation in leaves and chloroplast of vte1 compared to WT Arabidopsis plant by using confocal laser scanning microscopy and electron paramagnetic resonance spin-trapping technique, respectively. Furthermore, it is shown that the two dimensional imaging of ultra-weak photon emission and malondialdehyde (secondary product of lipid peroxidation) content were higher in vte1 compared to WT Arabidopsis plant, using charge coupled device camera and high pressure liquid chromatography, respectively. Our data revealed that lipid soluble tocopherol and plastochromanol act as singlet oxygen scavenger in Arabidopsis plant and protect them against photooxidative stress.

63

DIVERSITY WITHIN COLLECTIONS OF THE GENUS LACTUCA L. AND

CUCURBITACEOUS GENETIC RESOURCES

Doležalová I, Dušek K

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Genetic

Resources for Vegetables, Medicinal and Special Plants, Crop Research Institute, Šlechtitelů 29,

Olomouc, CZ-78371, Czech Republic

The collections of the genus Lactuca L. and cucurbitaceous crops maintained at the Department of Genetic Resources for Vegetables, Medicinal and Special Plants of CRI in Olomouc are of a great importance regarding to their extent and species representation. Within the genus Lactuca 841 accessions (acc.) of L. sativa and 567 acc. of wild Lactuca spp. have been conserved. Cucurbitaceous genetic resources are represented by 670 acc. of Cucurbita spp., 754 acc. of Cucumis sativus, 103 acc. of Cucumis melo and 86 acc. of wild Cucumis spp. It includes also 33 acc. of Benincasa spp., Lagenaria spp., Luffa spp. and Momordica spp. The germplasm collections comprise especially landraces, historical and local cultivars and wild species originating from various ecogeographical areas (16 wild Lactuca spp. from Europe, Asia, Africa and North America and 12 wild Cucumis spp. mainly from African continent). Regeneration is carried out in an insect-free glasshouse (Lactuca spp.) and/or in isolation cages covered by glass or plastic net for controlled pollination (cucurbitaceous crops). The multiplication procedures follow international standards and respect regeneration protocols based on the biology of individual species. Extent of regenerations depends on technical facilities, recently allowing regeneration of about 75 accessions per year. The main task of the work with the collections of Cucurbita spp. and wild Lactuca spp. and Cucumis spp. is the regeneration (ca 70 % of accessions remains to be regenerated) followed by taxonomic verification and/or re-determination. Current research includes taxonomical, morphological and biochemical studies. The research was supported by the Ministry of Agriculture of the Czech Republic, Project No. 206553/2011-17253 and Project No. ED0007/01/01 from the Centre of the Region Haná for Biotechnological and Agricultural Research.

64

MITOGEN-ACTIVATED PROTEIN KINASE-DEPENDENT PHOSPHORYLATION AND

FUNCTIONAL REGULATION OF CYTOSKELETAL END BINDING 1C PROTEIN

Doskočilová A, Novák D, Komis G, Šamaj J


Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc-Holice, Czech

Republic

Microtubule cytoskeleton is crucial for plant development, morphogenesis, and cell cycle progression. Precise coordination of MT reorganization is necessary for proper cell division and cell expansion. Microtubule associated proteins (MAPs) and their post-translational regulation, especially phosphorylation, throughout the cell cycle remarkably influence microtubule dynamics and reorganization. End binding 1c (EB1c) is a member of EB1 protein family encompassing typical microtubule plus-end MAPs. EB1c plays a role in microtubule plus-end properties and it is proposed to be important for cell-cycle progression. Our recent in silico prediction studies using publicly available databases revealed putative mitogen-activated protein kinases (MAPKs) phosphorylation sites on several cytoskeletal proteins. One of these proteins is EB1c in which we identified two putative and unique MAPK phosphorylation sites. We started to clone EB1c protein from genomic DNA and cDNA library for both C-terminal and N-terminal fusions with selected

tags. For cloning strategies, we use both Gateway and Multisite Gateway technologies. We already prepared GFP-EB1c constructs under the EB1c native promoter and expressed them transiently in Nicotiana bentamiana leaves. We would like to elucidate the function of nuclear localization of EB1c protein in relation to MAPKs nuclear localization. We plan to address the role of MAPKs in EB1c phosphorylation-dependent regulation. This work was supported by structural research grants from EU and the Czech Republic to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic (grant no. ED0007/01/01) and by

grant no. P501/11/1764 from the Czech Science Foundation GAC R.

65

GENETIC RESOURCES OF VEGETABLES AND SPECIAL CROPS

Dušek K, Cenklová V, Doležalová I, Dušková E, Hýbl M, Kopecký P, Jelínková M, Peč J,

Petrželová I, Stavělíková H

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Genetic



Plant genetic resources (germplasm) maintained in the gene banks represent an irreplaceable natural source of biodiversity for the future generations (Dotlačil et al. 2002). In the Czech Republic, the systematic collecting, study and conservation of plant germplasms is supported by the Ministry of Agriculture within the National Programme on Conservation and Utilisation of Plant Genetic Resources and Agrobiodiversity, and is coordinated by the Crop Research Institute in Prague (CRI). In the Department of Genetic Resources for Vegetables, Medicinal and Special Plants of CRI in Olomouc, broad collections of genetic resources of vegetables (9,245 accessions), medicinal, aromatic and culinary plants (MAPs, 828 accessions) traditionally grown in the Central Europe, and a collection of fungi (mainly morel) are kept (EVIGEZ 2013). The main concept of research activities is to continue in a long-lasting work with genetic resources and to obtain detailed information on vegetable and MAPs germplasms via the comprehensive research. The main objectives are: I. Conservation and evaluation of genetic diversity of vegetables, medicinal, aromatic and culinary plants and fungi genetic resources; II. Optimization of cultivation technologies of selected vegetables and special crops; III. Resistance studies of selected vegetables and special crops to diseases and pests; IV. Development of new bioanalytical methods for natural products analysis; V. Molecular biological analyses of selected genetic resources. This multidisciplinary approach will yield more efficient work with genetic resources, facilitate selection of cultivars and genotypes applicable for further breeding, cultivation and utilization by the commercial subjects and enable the broadening of international cooperation and increase of competitiveness of the Czech agriculture. The research was supported by the Ministry of Agriculture of the Czech Republic, Project No. 206553/2011-17253 and Project No. ED0007/01/01 from the Centre of the Region Haná for Biotechnological and Agricultural Research. References: Dotlačil et al. (2002) Czech J Genet Plant Breed 38, 3–15 EVIGEZ (2013) http://genbank.vurv.cz/genetic/resources/

66

VACCINIUM CORYMBOSUM CULTIVARS EXHIBIT DIFFERENT STRATEGIES IN

RESPONSE TO UV-B RADIATION

Luengo-Escobar A1, Reyes-Díaz M2, 3, Ribera AE3, Cartes P2,3 , Acevedo P4,5, Inostroza-

Blancheteau C6 and Alberdi M2,3*

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera,

Temuco, Chile

2Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco,

Chile

3Center of Plant- Soil Interaction and Natural Resources Biotechnology, Scientific and

Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile

4Departamento de Ciencias Físicas, Universidad de la Frontera, Temuco, Chile

5Center for Optics and Photonics, Universidad de Concepción, Casilla 4016, Concepción, Chile

6Núcleo de Investigación en Producción Alimentaría, Escuela de Agronomía, Facultad de Recursos

Naturales, Universidad Católica de Temuco, P.O. Box 56-D, Temuco, Chile.

*Corresponding author: [email protected]

Plants can eventually prevent, mitigate and repair UV-B damage, due to plants possess a range of acclimation responses. UV-B induces different phenolic compounds in plants as photoprotection (avoidance) and/or as antioxidant mechanism (tolerance) depending on their own strategies. Currently, Vaccinium corymbosum L. is an economically important crop in South Central Chile, but studies about the effect of UV-B on this species are scarce. We evaluated oxidative damage as total lipid peroxidation and chloroplast ROS production. To assess the protective strategies, we evaluate antioxidant activity, phenolic, flavonoids, anthocyanins, total UV-B absorbing compounds and phenolic profiles. This experiment was carried out with two blueberry cultivars with contrasting resistance to other abiotic stresses (Legacy, resistant and Bluegold, sensitive). Plants (two years-old) were grown in an Andisol under controlled greenhouse conditions during 0 to 21 days with different irradiation treatments: i) PAR without UV-B; ii) PAR plus low UV-B (2.5 Wm-2, which represent winter daily course); and iii) PAR plus high UV-B (5.2 Wm-2). The last irradiance represents a summer daily course with 30% increase over the maximum irradiance for South of Chile. Our results indicated that Legacy was less affected in growth, with a higher induction in total phenols and lower levels of chloroplasts ROS. Also, Legacy showed a wider variety of compounds induced by UV-B, most of them flavonol glycosides. Moreover, Legacy presented a higher induction of UV-B photoprotective compounds, which could avoid the UV-B stress. In contrast, an increment in the antioxidant activity and anthocyanins was induced in Bluegold, but despite this behavior, seems to be sensitive to UV-B radiation, being more affected in growth, higher induction of ROS and more oxidative damage by lipid peroxidation. Therefore, the response of this cultivar seems to result insufficient to counteract the stress produced by UV-B radiation. Acknowledgments: FONDECYT 1110726; Berries San Luis and CONICYT fellowships.

67

LIGAND-BINDING CHARACTERIZATION OF HISTIDINE KINASES CYTOKININ

RECEPTORS FROM BRASSICA NAPUS

Gallová L1, Kuderová A2, Plíhal O3 and Spíchal L1,4

1 Centre of the Region Haná for Biotechnological and Agricultural Research, Department of

Chemical Biology and Genetics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71


2 Laboratory of Growth Regulators, Faculty of Science, Palacký University & Institute of

Experimental Botany AS CR, Šlechtitelů 11, 783 71 Olomouc – Holice, Czech Republic


Molecular Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc-

Holice, Czech Republic

4Laboratory of Plant Molecular Physiology, Department of Functional Genomics and Proteomics,

Institute of Experimental Biology, Masaryk University, Kamenice 5, CZ-625 00, Brno, Czech

Republic

Cytokinins, essential plant hormones, have impact on growth, development and physiological aspects of plants. They initiate signalling pathway due to activation of membrane-located receptors, eukaryotic histidin kinases. In the N-terminal part receptors contain an extracellular ligand-binding domain with a characteristic receptor-like architecture, called CHASE domain. Characterisation of interaction of receptor-ligand is basic step for better understanding of hormone-signalling. Various receptors exhibit differential affinities for different cytokinins. For comprehension of hormone-binding characteristics in Brassica napus, two cytokinin receptors, which are orthologs of AHK2 and AHK3, were expressed in E.coli transgenic clones. For both receptors, KD and a sequence of cytokinin preferences for different cytokinins were defined.

68

DEFENSE RESPONSES OF WHEAT UNDER WATER DEPRIVATION

Gálusová T1, Gregorová Z1, Maglovski M2, Mészáros P1, Kuna R1, Matušíková I2, Hauptvogel P3

1 The Constantine Philosopher University, Faculty of Natural Sciences, Department of Botany and

Genetics, Nábrežie mládeže 91, 949 74 Nitra, Slovak Republic

2 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, 950

07 Nitra, Slovak Republic

3 Plant Production Research Centre Piešťany, Bratislavská cesta 122, 921 68 Piešťany, Slovak

Republic

Despite of a number of studies on the genetic nature of plant adaptation to drought stress there is still no conclusion on the core mechanism of defense. Previously, aquaporin genes have been demonstrated to play important role in controlling the water homeostasis in plants. In this work the commercial winter wheat Triticum aestivum L. var. lutescens (ALEF.) MANSF. cv. Genoveva was studied under deficiency of water. This cultivar has a good agronomical qualities and is considered as relatively resistant to various biotic factors as well as to drought. We study the impact of water shortage on this cultivar and monitor the activity of individual aquaporin genes. We observe the signs of stress such as altered rate of membrane lipid peroxidation and proline accumulation. Furthermore, we study the accumulation of pathogenesis-related proteins, namely chitinases, to reveal their role during water stress in wheat. We expect to clarify the possible interplay between chitinases that modulate the cell wall flexibility and the aquaporins that are membrane water channels under drought.

69

ASSESSMENT OF GENETIC DIVERSITY IN OILSEED RAPE USING ISSR MARKERS

Havlíčková L, Jozová E, Čurn V

Biotechnological Centre, Faculty of Agriculture, University of South Bohemia, České Budějovice,

Czech Republic

Decrease of genetic variation and narrowing of the genetic pool of modern cultivars as a result of intensive breeding is a crucial problem both for creating new cultivars and estimating genetic diversity and managing genetic resources. This phenomenon causes troubles with selection of suitable genotypes in running breeding programmes. Genetic diversity among 156 different accessions of Brassica napus were characterized using ISSR markers. Five ISSR primers produced 126 scorable bands in which 121 were polymorphic. The average rate of polymorphic bands (96%) indicates high genetic diversity among the accessions. Genetics similarities (GS) between each pairs of accessions were estimated. All accessions were found to be genetically distinct from each other and molecular analyses based on ISSR markers proved to be useable for characterization of genetic resources and their identification (ID). Based on the genetic similarity matrix, the set of the most distinct varieties was determined and also UPGMA and PCO analyses were performed. This research was supported by the project University of South Bohemia GAJU 063/2013/Z and Postdoc USB (reg.no. CZ.1.07/2.3.00/30.0006) realized through EU Education for Competitiveness Operational Programme. Project is funded by European Social Fund and Czech State Budget.

70

THE POTENCIAL OF LOW-COPY FISH IN PHYSICAL MAPPING

Havránková M1, Bartoš J1, Kopecký D1, Lu M2, Sato K3, Houben A2, Stein N2, Doležel J1



2Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Gatersleben, Germany

3Barley Germplasm Center, Research Institute for Bioresources, Okayama University, Kurashiki,

710-0046, Japan

Recent genomic trends tend towards modern and high throughput techniques like sequencing. During the last decade, sequencing of the different genome portions or whole genomes of various species became more available and experience a boom. But as the every other technique, also sequencing has its weakness. Correct assembly of a genome sequence is difficult, especially in species with large and complex genomes like cereals have. The sequence orientation and the knowledge of the physical position are essential to integration of genetic and physical maps and to assembly genome sequence correctly. We introduce fluorescence in situ hybridization (FISH) with single-copy probes shorter than 4kb as an attractive alternative and useful approach for physical mapping. We chose the centromeric region of barley chromosome 7H and we concentrated on the ordering of the set of full-length cDNA originating in this non-recombining region using multi-color FISH. We successfully establish the order 13 out of 15 cDNA clones and show that region with highly reduced recombination frequency representing genetic centromere constitutes more than 30 % of the barley chromosome 7H. This finding underlines the necessity of combining the different mapping approaches to reach high resolution physical map and we proved that FISH could be one of them. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01 and Internal Grant Agency of Palacky University (Grant award No. IGA PrF/2012/001).

71

METABOLISM OF ZEATIN TYPE CYTOKININS IN MAIZE

Galuszka P1, Hluska T1, Motyka V2, Dobrev P2, Lenobel R3, Zatloukal M4, Šebela M3, Frébort I1


Molecular Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc-


2Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany of Czech

Academy of Science

3Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Protein

Biochemistry and Proteomics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71



Chemical Biology and Genetics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71


There are four major types of isoprenoid cytokinin determined in plant tissues: trans-zeatin, cis-zeatin, dihydrozeatin and isopentenyladenine. Their homeostasis is kept by various metabolic conversions. Genes coding for degradation, biosynthesis and glycosylation enzymes have been explored just after the Arabidopsis genome sequencing. In the past, the metabolism of trans-zeatin and isopentenyladenine has been predominantly investigated. Information concerning origin of cis-zeatin and dihydrozeatin remains rather scarce, even though they are prevalent cytokinin forms in many plant species, e.g. cereals. Enzyme activities for zeatin cis-trans isomerase and zeatin reductase has been described in the past, but the enzymes or respective genes have not been identified yet. We followed and purified activity of zeatin cis-trans isomerase and identified protein encoded in maize genome responsible for the conversion in vitro. Ability of novel protein to contribute to cis/trans-zeatin homeostasis in planta will be discussed. When searching for zeatin reductase activity, we identified enzymatic activity converting trans-zeatin to novel metabolic product. Structure of the compound was resolved and the compound was synthesized and tested in bioassays for cytokinin-like activity. Thus, a novel cytokinin deactivation pathway existing in specific organs is hypothesized.

72

EVALUATION OF PEA (PISUM SATIVUM L.) GERMPLASM FROM CENTRES OF

GENETIC DIVERSITY FOR BREEDING PURPOSES

Hýbl M1,3, Smýkal P2,3, Pavelková M3

1Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Genetic



2Department of Botany, Faculty of Science, Palacky University, Olomouc, Czech Republic

3Agritec, Plant Research s.r.o. Šumperk, Czech Republic

Study Czech collection of pea showed very low representation of germplasm from Armenia, Georgia, Mongolia, Tajikistan, Morocco and Algeria, the areas designated as centres of genetic diversity of pea with the occurrence of economic characteristics that are of current cultivars due to intensive breeding completely disappeared. 68 pea accessions were obtained from gene bank VIR St Petersburg. As a control cultivars were chosen cultivars of field peas (Pisum sativum L. ssp sativum var. sativum) - Sponsor and Zekon and fodder pea (P. sat. L. ssp arvense) - Arvika. Statistically significant positive correlation (r = 0.78) between leaflet margin shape in the second real leaves and leaflet margin shape in the first flowering node, character of antocyanin spot and wings (r = 0,93) and vexillium colour (r = 0,91) was confirmed. The Euclidean distance formula for calculate the coefficient of similarity was elected. The minimum value (1.41) was found between Kamyshlovskyj mramornyj myelkyj 1584 and Untitled 999 accessions. The maximum value of the coefficient of similarity (17.07) was found between Primavil and K 38_2314 accessions. High coefficient of similarity was found between the control cultivar Sponsor and 12 accessions. In control cultivar Arvika differing from cultivar Sponsor by flower colour and type of growth was found 12 accessions with high coefficient phenotypic similarity. With our developed method for the molecular analysis of eIF4E resistance gene PSbMV P1 virus were performed PCR analysis of length polymorphism of intron 3 using primers 750F + 586 g-R. Any accessions containing a known resistance allele to PSbMV were detected. At the sample no. 1589 was amplified PCR product suggesting the presence of a novel allele. This was confirmed by sequencing analysis of the product. Direct virological testing confirmed that this is new allele resistance PSbMV. This work was supported by grants no.: LH12227 and ED0007/01/01.

73

FAST ESTIMATION OF HEAT STABILITY OF PLANTS USING LINEAR HEATING OF

PLANT SEGMENTS

Ilík P1, Špundová M1, Šicner M2, Trtílek M2



Czech Republic

2PSI (Photon Systems Instruments) Ltd., Drásov 470, CZ-66424 Drásov, Czech Republic

Current techniques for the estimation of heat stability of plants are based on heating of plant segments for a defined time at given temperatures. The heated segments are soaked in a water bath for tens of minutes or even hours. The heat-induced damage of cell membranes leads to leakage of ions from plant tissues, resulting in an increase in electrical conductivity of the water in the bath (Levitt 1980). A critical point of this method is the choice of optimal thermal dose (time and temperature), which would enable to detect the differences in heat stability between the studied plant species. Therefore, preliminary measurements are usually required, which make the method even more time-consuming. We have developed an improved method for the estimation of heat stability of plant segments, which does not require any preliminary measurements. This method is based on gradual (linear) heating of plant segments from room temperature to about 70°C and simultaneous measurement of the electrical conductance of the water in the bath, in which the segments are immersed. For the heating rate of 2°C/min, the bath conductance steeply increases in the range of 55–65°C. Each sample is unequivocally characterized by the critical temperature of the steep conductivity rise, which is the measure of heat stability of plants and differs among species. Here, we present a comparison of the heat stability data obtained for different plant species using the traditional and the new method and we discuss their application for the estimation of constitutive and inducible heat stability of plants. First commercial version of the instrument for the estimation of heat stability of plants based on the new method will be available in August 2013. Supported by TACR (grant TA01010254) and MSMT (grant ED0007/01/01).

74

PHOTOSYNTHETIC AND LEAF ANATOMICAL CHARACTERISTICS IN HIGHBUSH

BLUEBERRY (VACCINIUM CORYMBOSUM L.) UNDER UV-B STRESS

Inostroza-Blancheteau C1, Arellano A2, Acevedo P3, Latsague M4, Reyes-Díaz M5, Alberdi M6

1Núcleo de Investigación en Producción Alimentaría, Escuela de Agronomía, Facultad de

Recursos Naturales, Universidad Católica de Temuco. P.O. Box 56-D, Temuco, Chile

2Escuela de Agronomía, Facultad de Recursos Naturales, Universidad Católica de Temuco. P.O.

Box 56-D, Temuco, Chile

3Departamento de Física, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.

4Escuela de Ciencias Ambientales, Facultad de Recursos Naturales, Universidad Católica de

Temuco, P.O. Box 56-D, Temuco, Chile


and Natural Resources Biotechnology ((BIOREN-UFRO), Universidad de La Frontera, P.O. Box

54-D, Temuco, Chile


and Natural Resources Biotechnology ((BIOREN-UFRO), Universidad de La Frontera, P.O. Box

54-D, Temuco, Chile

Chile is the main exporter of blueberries in the southern hemisphere, where high levels of UV-B radiation are reported in spring and summer. This radiation represents a serious risk for the biological and biochemical processes, affecting the yield and quality of fruits. Therefore, the aim of this work was to study the effect of high UV-B doses on the photosynthetic and leaf anatomical features of two blueberry cultivars (Brigitta and Bluegold) frequently cultivated in Chile. They were subjected to three levels of UV-B radiation (2.5; 4.0 and 5.2 Wm-2) for 0 to 72 h. Thereafter the net photosynthesis (Pn), maximum (Fv/Fm) and effective quantum yield ФPSII, of PSII electron transport rate (ETR) and non-photochemical quenching (NPQ) were determined and anatomical features of leaves (adaxial epidermis (ADE), palisade layer (P), mesophyll layer (M), intercellular cavities (IC) and abaxial epidermis (ABE) were measured in an optical microscope (OM). The results showed that both cultivars have a differential response to UV-B. Fv/Fm showed values (~0.8) for healthy plants. The Pn, ФPSII and ETR decreased significantly in all times and treatments in Bluegold compared to Brigitta. NPQ increased from the start of treatment with UV-B in Brigitta. Anatomical significant changes were observed in both cultivars by the effect of UV-B radiation. ADE cells were strongly affected, decreasing its size 4.3-fold in Brigitta and 2.0-fold in Bluegold. By contrast, IC increased in all treatments, this probably by decreasing the cells size of all cell layers, generating more IC and finally generating the decrease of the thickness of leaf in both cultivars. These changes are reported as adaptive mechanisms for high doses of UV-B radiation in plants. According to the results, Brigitta appear to be the most UV-B resistant cultivar due to the better photochemical performance indicated principally by Pn, ФPSII and ETR. Acknowledgments: FONDECYT-Postdoctoral N°3120248 and FONDECYT N°1120917.

75

DIFFERENT DAYLENGTH – DIFFERENT EFFECT

Ivaničová Z1, Milec Z 1, Trávníčková M2, Valárik M 1, Prášil IT 2, Pánková K2, Snape JW 3 and

Šafář J1



2 Crop Research Institute, Drnovská 507/73, Prague, Czech Republic

3 John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom

Accelerating and delaying of flowering time was identified in Czech wheat landrace Česká přesívka under long (LD) or short days (SD) conditions, respectively. The one day (LD) or two days (SD) difference indicates a possible role of earliness per se gene (QFt.cri-3B.1 locus) in determination this phenotypic trait. The presumed gene was localized within 30 cM near-centromeric region of the long arm of chromosome 3B. This locus is delineated by barc164 and cfa2170 microsatellites markers. Additional phenotyping and genotyping of nearly isogenic lines mapping population is currently in progress. Phenotypic effect was identified (i) in controlled conditions as well as (ii) in field experiment. The differences in apex development (i) or in heading time (ii) proved bipolar effect of assumed gene. The map around the gene was saturated with DArT, SSR, STS markers. Up to now we identified 30 new polymorphic markers which have been used to delimitate region. Fine mapping followed by positional cloning and sequencing will enable identification gene of our interest. Candidate gene analysis and its detailed characterization will provide novel information which could lead to deeper understanding of fine tuning flowering time in wheat. Furthermore, determined effect could be useful in creating new varieties, finding out new growth areas in different climate conditions. This work has been supported by the Czech Science Foundation (P501/10/1778) and MSMT CR and EU (Operational Programme Research and Development for Innovations No. ED0007/01/01).

76

PARAFAC - DECOMPOSITION METHOD FOR PLANT GENETIC RESOURCES

EVALUATION

Jelínková M1, Doležalová I1, Hron K2, Kalivodová A2, Dušek K1

1Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Genetic



2Department of Mathematical Analysis and Applications of Mathematics, Palacký University, 17.

listopadu 12, 771 46 Olomouc, Czech Republic

Plant genetic resources (germplasm) represent a unique natural source of biodiversity for future generations. A preserved genetic resource without accompanying information on its passport data (data of origin) and descriptive ones (morphological and phenological characteristics, yield parameters, data on resistance, biochemical and molecular data) is of limited value. Plants are, in general, complex organisms where intraspecific connections among various types of data are expected and as such must be considered. Standard (univariate) statistical methods, that are most common techniques for analyzing chemical or biological data, are not able to cope with complex data structures, where also relations between compounds (factors) are of interest. Processing of data using appropriate multivariate statistical methods thus provides a deeper understanding of plant genetic resources variability. Rocket (Eruca sativa L.), belonging to the Brassicaceae family, has been reported as a significant green food with a high content of vitamin C and various medicinal properties. It has a characteristic pungent taste and aroma that depend on its genotype, and environmental conditions. The individual yield parameters and content of important nutrients varying within species, cultivars and even genotypes were used to construct multi-way data for statistical evaluation. Parallel factor analysis (PARAFAC) is a decomposition method that represents a generalization of the well-known principal component analysis for multi-way data. This method enables to decompose multivariate observations in different layers into sets of scores and loadings that describe the data in a more condensed form than the original data array. The outputs are plots of the individual modes (individual plants - observations, their characteristics – variables, and time - layers) that enable to observe, e.g. significant changes of the plant parameters in time. The research was supported by Projects No. ED0007/01/01, No. 206553/2011-17253 and No. CZ.1.07/2.3.00/20.0170.

77

FORMATION OF ECTOPIC MICROTUBULAR FIBRES WITHIN NUCLEI AND WITH

NUCLEAR ENVELOPE REQUIRES OVERPRODUCTION OF ARABIDOPSIS TPX2

PROTEIN

Jeřábková H1, Petrovská B1, Cenklová V2, Pochylová Ž2, Gelová Z2, Váchová L2, Kurejová M1,

Tomaštíková E, and Binarová P3



2Institute of Experimental Botany, AS CR, v.v.i., Sokolovská 6, 772 00, Olomouc, Czech Republic

3Institute of Microbiology, AS CR, v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic

The targeting protein for Xklp2 (TPX2) is a microtubule associated coiled-coil protein with multiple functions in microtubule nucleation and spindle organization. The plant TPX2 molecule contains all the functional domains of vertebrate counterpart, TPX2 signature motif is present once in vertebrate sequences but twice in plants. Compared to animals, in plant systems, proteins with coiled-coil signature are less understood. Previously we showed that AtAurora1 kinase and AtTPX2 localize in plant microtubules in a cell cycle specific manner from preprophase to early telophase. In addition, AtTPX2 protein is intranuclear and although important mitotic functions for plant TPX2 protein have already been well documented, any functional role for its accumulation in interphase nuclei is far from understood. Here we present data on specific arrays of microtubules decorated with AtTPX2 formed in the vicinity of the nuclear envelope and in nuclei. Microscopic analysis of cells overproducing AtTPX2 showed the “dots” of overexpressed AtTPX2-GFP signal in interphase nuclei. Later AtTPX2-GFP dots were rebuilt into the thick fibrilar structures or new ectopic sites for microtubule formation positive for AtTPX2-GFP. AtTPX2-GFP signal decorated filamentous structures reminiscent of microtubules. Fibrils were arranged into cage-like structures surrounding nuclei. We proved that AtTPX2 fibres were positive for α-tubulin immunolabeling, while they were not recognized by anti-actin antibody. The interconnection between the cage-like structures surrounding the nuclear envelope and intranuclear arrays was visible and fibres often extended to the cell periphery as well. The rearrangement of AtTPX2 from the dots through the fibres might be caused by co-assembly of the AtTPX2-GFP with endogenous microtubule-nucleating units comprised γ-tubulin and TPX2 protein.Acknowledgements: This work was supported by the Grant Agency of the Czech Republic [204/09/P155, 204/07/1169, P501/12/2333, P501/12/G090], IGA UP Prf/2013/003 for H.J., E.T. and the Centre of the Region Haná for Biotechnological and Agricultural Research.

78

CHEMICALLY DIRECTED EXPRESSION OF ATCKX2 IN TRANSGENIC BARLEY

PLANTS

Jiskrová E, Šmehilová M, Pospíšilová H, Galuzska P, Frébort I

Centre of the Region Haná for Biotechnological and Agricultural research, Department of Molecular

Biology, Palacký University, Šlechtitelů 11, 783 71 Olomouc-Holice, Czech Republic

The possibility to control the expression of the gene of interest in a transgenic plant brings a promising tool for determination of the gene’s function. As such, chemically inducible expression is based on special promoters that are responsive to chemical solutions. Induced promoters then direct the transcription of a transgene. Our approach is aimed at manipulating cytokinins, plant hormones, which play an important role in plant development. They influence many key processes including the cell division, seed germination and delay of senescence. Interferences with the cytokinin metabolism may cause dramatic changes in the plant development and can help better understanding to the hormone functions. Cytokinin oxidase (CKX, EC 1.5.99.12) catalyses the degradation of cytokinins so it may be used to decrease their endogenous level. Increased expression of CKX genes causes significant changes in the plant growth, which lead for instance to dwarfism and enlarged root system. In this work, the AtCKX2 gene from Arabidopsis thaliana was used for the transformation of barley plants. The transgene was placed under the control of a chemically inducible promoter responsive to the glucocorticoid analogue dexamethasone. The construct was incorporated into the barley genome by Agrobacterium tumefaciens infection. Assessment of the expression level of AtCKX2 gene and related phenotypic changes in barley plants depending on dexamethasone application is in progress.

79

EFFECT OF DROUGHT STRESS ON ROOT SYSTEM SIZE AND ABOVEGROUND

BIOMASS OF SPRING BARLEY (HORDEUM VULGARE L.)

Klimešová J, Středa T, Hajzler M

Mendelova univerzita v Brně

The aim of the study is evaluation of influence of abiotic stress caused by drought on aboveground and underground biomass production of spring barley (Hordeum vulgare L). Four varieties of spring barley with different root system size (RSS) were tested by pot experiment. Irrigation control of four variants of soil moisture conditions (unstressed variation, moderate stress variation at the level 65 % available water holding capacity, strong stress variation at wilting point and the variation with the natural rainfall conditions) were started at stem elongation phase (BBCH 30). Two types of methods were used for root system evaluation. For RSS measuring by its electric capacity was used the capacitance meter. The second (soil-core) method allows the subsequent digital image analysis of collected root system samples (software WinRHIZO). The root and aboveground biomass were taken at the end of the experiment (BBCH 70). Depending on moisture conditions the RSS was compared with aboveground biomass production. The results of RSS measurement by mentioned methods were compared.

80

CONSTRUCTION AND CHARACTERIZATION OF WHEAT 4A CHROMOSOME

SPECIFIC PHYSICAL MAP AS A BASE STEP FOR THE CHROMOSOME

SEQUENCING

Klocová B1, Kladivová M1, Frenkel Z3, Kumar A4, Kianian SF4, Šimková H1, Šafář J1, Hu Y2, Zhang

Y2, You FM2, Luo M2, Korol A3, Doležel J1, Valárik M1



2Department of Plant Sciences, University of California, Davis, CA 95616, USA

3Institute of Evolution, University of Haifa, Haifa 31905, Israel

4 Department of Plant Sciences, North Dakota State University, Fargo, Loftsgard Hall 470G,ND

58108, USA

Triticum astivum L. (bread wheat) has large and complex genome. The genome size is approximately 17 Gb with 80% of repetition and is composed of three homeologous genomes A, B and D. All of this makes sequencing, mapping, and marker assisted breeding a difficult task. To facilitate sequencing of wheat 4A chromosome we are developing sequence-ready chromosome arm specific physical maps. Physical maps for both arms were assembled from fingerprinted arm-specific BAC libraries. The physical maps were assembled using both, LTC (Linear Topology Contig) and FPC (FingerPrint Contigs) softwares. The 4AS library was assembled into 415 contigs and MTP consist of 4 422 clones. After superclustering, the advanced tools of LTC, there were 67 super-contigs with more than 100 clones in one contig. The longest supercontig comprised 582 clones. Also, 4AL physical map was assembled into 1127 contigs and MTP contains of 8 369 clones. After additional superclustering there were 74 supercontigs with more than 100 clones in one contig. Moreover, the supercontigs of both physical maps contains more than half of informative clones. 3D pools of MTP clones were prepared to simplify marker anchoring to the maps. To the physical map of 4AL were anchored 20 markers, four of this markers were found in one supercontig. After sequencing of the MTP BAC clones high-capacity physical map anchoring will be performed in silico. Endosperm radiation hybrid panel comprising 1 100 lines will be used for contig ordering and orientation. This work has been supported by the Czech Science Foundation (P501/10/1740), MSMT CR and EU (Operational Programme Research and Development for Innovations No. ED0007/01/01) and Internal Grant Agency PrF-2012-003.

81

INVOLVEMENT OF MITOGEN ACTIVATED PROTEIN KINASES IN PLANT CELL

DIVISION, GROWTH AND ROOT TISSUE PATTERNING UPSTREAM OF

MICROTUBULE REGULATION

Komis G1, Smékalová V1, Šamajova O1, Beck M2, Mueller J3, Doskočilová A1, Ovečka M, Šamaj J1

1 Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell


Republic

2The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK

3Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Weinberg 3,

06120 Halle (Saale), Germany

Hereby we report roles of Arabidopsis thaliana mitogen activated protein kinases MPK4 and MPK6 in the organization of microtubular arrays in cycling root cells. Both mpk4 and mpk6 knock-out mutants show defects in root growth and tissue patterning at variable degrees. Deficiency of MPK4 signalling in mpk4 knock-out mutant, results in gross mitotic spindle progression delays, cytokinetic aberrations, incomplete cell plate formation and the generation of multinucleate cells, while mpk6 knock-out, results in delayed root formation, excessive adventitious roots, and disturbance of cell division plane determination leading to the observation of oblique cell plate formation. These results are consistent with relevant microtubule organization disturbances and the colocalization of the respective MPKs with distinct microtubular arrays including the cortical interphase array, the preprophase microtubule band and the cytokinetic phragmoplast. As suggested from previous work, a major target of both MPKs is the microtubule associated protein MAP65-1, which is under phosphorylated and overexpressed in at least mpk4 mutant while it was recently shown to drive periclinal divisions in the epidermal root layer downstream of auxin signalling. In light of recent results, it is likely that Arabidopsis MPK4 and MPK6, act upon root cell fate decisions, both as transcriptional transactivators of cell plane determinants in the long term, but also as transient regulators via MAP65 protein phosphorylation. Funding: GAČR P501/11/1764, ED0007/01/01 (CR Hana/EU Structural Grants) and OP

VK CZ.1.07/2.3.00/20.0165 (Project INTERHANA).

82

EFFECTS OF DIVERSE ABIOTIC STRESSES ON ROOT GROWTH AND PHENOTYPES

OF MPK4, MPK6 AND ANP3ANP2 MUTANTS

Košútová P, Šamajová O, Šamaj J



Republic

Plants developed mechanisms for rapid sensing of signals from a changing environment and for transmitting them to adaptive or defensive responses. Mitogen activated protein kinase (MAPK) pathways play an essential role in signal transduction involved in the regulation of cell growth, differentiation, proliferation, death and stress responses. MAPK pathways are known to be activated by diverse abiotic stresses. We studied effects of osmotic, oxidative and cold stresses on root growth and phenotypes of mpk4, mpk6 and anp3anp2 mutants. All used stresses inhibited root elongation growth and affected root and root hair phenotypes in these mutants. Mutant mpk6 was more resistant to osmotic stresses in comparison to mpk4 and anp2anp3 mutants. However, anp2anp3 mutant showed the highest resistance to oxidative stress. Distribution of superoxide was monitored by histochemical staining with nitroblue tetrazolium while SOD activity was measured by biochemical assay in this mutant. This work was supported by grant No. P501/11/1764 from the Czech Science Foundation GAČR and by grant No. ED0007/01/01 to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.

83

QTL ANALYSIS OF SUGARS AND ORGANIC ACIDS IN PEACH

Kuznetsova K1 , Gennari F1, Pachesco-Cruz I2, Forschi S3 , Bassi D4, Cattonaro F5, Verde I6,

Dondini L1, Tartarini S1

1Dipartimento di Scienze Agrarie,Universita di Bologna

2Parco Technologico Padano,Lodi

3Centro Ricerche Produzioni Vegetali, Cesena

4Dipartimento di Produzione Vegetale, Università di Milano

5Istituto di Genomica Applicata Udine

6Centro di Ricerche in Frutticoltura CRA, Roma

Peach and nectarine (prunus persica l. Batsch) are the second most important fruit crop in the european union (approx. 4.3 million tons) after apple (faostat 2010), and the most important within the prunus genus. Italy is the second world biggest producer of peaches after china, followed by usa (faostat 2010). Fruit quality improvement is one of the main objectives in peach breeding. The recent delivery of the whole peach genome sequence by the international peach genome initiative (ipgi) together with the italian project “drupomics” has opened new horizons in peach genomics and development of molecular markers. The availability of snp markers linked to quality trait in peach could be very useful in marker assisted selection to assist future breeding programs. The concept of fruit quality of prunus fruit crops includes sugar and organic acid content, which highly contribute to peach taste. The objectives of the given project are to identify qtls controlling main sugars and organic acid contents in a peach f2 population and to develop snp markers on selected candidate genes within the qtl regions. A phenotypic analysis of the main fruit quality traits was done for qtl analysis. In this work malic, citric, quinic acids as well as fructose, glucose and sucrose contents were analysed as they are the main sugars and organic acids present in the peach flesh. Qtl analysis of sugar and organic acids was performed through kruskal wallis and interval mapping tests. Thanks to already available peach map, six interesting qtls were identified on the eight linkage groups of peach. Snps tightly linked to qtl regions were developed on the 20 x resequencing data of the parent of this f2 progeny (f1_cxa, conteder x ambra).

84

PRESENCE OF MELATONIN IN CHARACEAE CHARA AUSTRALIS AND EFFECT OF

EXOGENOUS MELATONIN ON CHARA´S PHOTOSYNTHESIS

Lazár D1, Murch SJ2, Beilby MJ3, Al Khazaaly S3



Czech Republic

2Department of Chemistry, University of British Columbia, Okanagan Campus, Kelowna,

BC,Canada

3School of Physics, University of NSW, Sydney, NSW, Australia

Melatonin, known to provide also a protection against reactive oxygen species, was found in the fresh water characeae Chara australis. The concentrations (~4 μg/g of tissue) were similar in photosynthesizing cells, independent of their position on the plant and rhizoids (roots) without chloroplasts. Exogenous melatonin, added at 10 μM to the artificial pond water, increased quantum yield of photochemistry of photosystem II by 34%. The increased efficiency appears to be due to the increased amount of open reaction centers of photosystem II, rather than increased efficiency of each reaction center. More open reaction centers reflect a better functionality of all photosynthetic transport chain constituents. We suggest that melatonin protection against reactive oxygen species covers not only chlorophyll, but also photosynthetic proteins in general.

85

THE PREPARATION OF ARABIDOPSIS THALIANA MPK6 MUTANT WITH GFP-FUSED

MPK6 PROTEIN UNDER ITS OWN PROMOTER

Luptovčiak I, Doskočilová A, Šamaj J



Republic

Mitogen activated protein kinases (MAPKs) regulate numerous processes in eukaryotic cells. In plants, it was discovered that MAPKs are important for stress responses, hormonal regulation, innate immunity and plant development. The MPK6 protein plays a key role in plant responses to stress stimuli in A. thaliana. To study in vivo localization of MPK6 in plant cells under various stress conditions, the GFP-fusion of MPK6 protein under 35S promoter was prepared and published before. Now we are focusing on the preparation of the A. thaliana mpk6 mutant lines expressing GFP-fused MPK6 protein under its own promoter to visualize in vivo native pool and localization of MPK6 in living cells under control and stress conditions. We prepared constructs with C-terminal and N-terminal fusions of genomic and cDNA sequences of the MPK6 gene under its own promoter and with GFP tag by using Multisite Gateway technology. We will proceed with floral dip stable transformation to get rescue of phenotype in already genotyped mpk6-2 mutants in order to prove functionality of MPK6-GFP fusion constructs. This work was supported by structural research grants from EU and the Czech Republic to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic (grant no. ED0007/01/01), by grant

no. P501/11/1764 from the Czech Science Foundation GAC R.

86

CHITINASE ENZYME-ACTIVITIES REVEAL SOME DIFFERENCES IN RESPONSE TO

VARIOUS STRESSORS

Mészáros P1, Piršelová B1, Kuna R1, Boleček P1, Matušíková I2

1Constantine the Philosopher University in Nitra, Department of Botany and Genetics, Nábrežie

mládeže 91, 949 74 Nitra, Slovak republic

2 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, 950

07 Nitra, Slovak republic

This work monitors the sensitivity of 10 soybean (Glycine max L.) cultivars to different types of abiotic stressors. Based on the sensitivity of roots to the applied stressors there were two cultivars selected and subsequently subjected to further analyses of activated defense. Chitinase enzymes, belonging to so called pathogenesis-related proteins, act as possible second line defense compounds in plants exposed to metals. In this work their activity was studied after the exposure to different metal(loid)s (cadmium, arsenic and aluminium) that are expected to cause toxicity in different ways. For comparison, NaCl was applied as well. Impacts of the applied stressors on a set of soybean varieties revealed a pattern that coincided with the behaviour of total chitinases. After further analyses of the acidic and neutral proteins there was one additional chitinase isoform detected that distinguishes between a tolerant and sensitive soybean cultivar. Simultaneously, this isoform was stress-responsive pointing to possible evolutional adaptation to the environment. Activities of the individual chitinases were dependent on the type of stress as well as the cultivar indicating their apparently more complex role in plant defense during this type of stress.

87

EFFECTS INFLUENCING READINGS OF CHLOROPHYLL-METERS

Nauš J, Hlavinka J, Špundová M



Czech Republic

The chlorophyll amount or concentration in leaves belong to the most important information about nutritional and stress state of plants. Chlorophyll may be estimated analytically or nondestructively using portable instruments – chlorophyll-meters. Recently, application of chlorophyll-meters is preferred due to much easier and faster gathering a large amount of information. However the readings of the chlorophyll-meters are influenced to a desirable degree not only by the amount of chlorophylls in the leaf but also by other effects. Some of these effects are analyzed theoretically and supported experimentally in this contribution. There are two principles of the chlorophyll-meter performance. They use detection of leaf transmittance (e.g. SPAD 502) or reflectance (e.g. detecting the NDVI index). Both these methods are analyzed. We concentrate on the effects of leaf structure, chloroplast configuration in cells, chloroplast movement, sieve effect, surface reflection, optical field below the leaf and contribution of other pigments. Several theoretical concepts are introduced to describe the optical effects in the leaves. Among them the specific absorbance and leaf spectral polarity should be mentioned. It is shown that the chloroplast arrangement in the cells may change the readings of SPAD up to 30 %. A consideration of surface reflections leads to changes in pigment estimation up to 40%. It is also analyzed theoretically and shown experimentally that the presence of blue pigments, mostly anthocyanins, leads to distortion of chlorophyll estimation. A procedure for the correction of the readings for the presence of blue pigments is suggested. As model plants, mostly tomato and tobacco plants have been used. A special cultivation conditions have been selected to obtain the blue colored leaves. The spectra of diffusive transmittance and reflectance have been measured with LI-1800 spectral radiometer. Portable chlorophyll-meters SPAD-502L for the transmittance mode and NDVI-300 (P.S.I. Brno) for the reflectance mode have been used.

88

INCREASED ALTERNATIVE OXIDASE (AOX) EXPRESSION EARLY DURING

ADVENTITIOUS ROOT INDUCTION IN SHOOT CUTTINGS OF OLEA EUROPAEA L.

Noceda C1,2, Santos-Macedo E1,2, Peixe A2, Arnholdt-Schmitt B1

1EU Marie Curie Chair, ICAAM –Instituto de Ciências Agrárias e Ambientais Mediterrânicas–,

University of Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal

2Laboratory of Biotechnology and Plant Breeding, ICAAM –Instituto de Ciências Agrárias e

Ambientais Mediterrânicas–, University of Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora,

Portugal

Alternative oxidase (AOX) has a central role in stress acclimation. It is involved in cell-reprogramming upon various abiotic and biotic stresses that can lead also to adventitious behaviours of commercial interest, such as the adventitious rooting (AR) for plant propagation. In shoot cuttings of olive tree (Olea europaea L.), AR is induced by exogenous indol-3-butyric acid (IBA). However, induction of AR is blocked when IBA is supplied together with the AOX inhibitor salicylhydroxamic acid (SHAM). To study the involvement of AOX in AR, oxygraphic measurements of AOX enzyme capacity were taken and semi-quantitative RT-PCR was performed for four recently identified OeAOX genes: AOX1a, AOX1b, AOX1c and AOX2. The influence of IBA-SHAM treatments and genotype were explored. Before treatments, AOX1a and AOX2 transcript levels were similar and the more abundant. Few hours after IBA treatments (both without and with SHAM), peaks in transcript accumulation were observed for AOX1a, AOX1c, and AOX2, corresponding the highest increase to AOX1a. Compared with only-IBA treatment, application of IBA+SHAM led to a minor capacity of AOX. Further, lower levels of AOX1c transcripts and AOX2 transcripts with the longest 3’-UTR were observed after the treatment with additional SHAM. Also, kinetics of recovering to original transcript levels was modified by SHAM treatment. These results and those obtained from comparison between difficult and easy-to-root genotypes suggest an involvement of AOX expression levels in the early induction phase with effects on later root manifestation.

89

HIGH TEMPERATURE STRESS AND ITS IMPACT ON THE STRUCTURE OF

PHOTOSYSTEM II

Nosek L



Czech Republic

Photosynthesis is one of the most important processes on the Earth, because it is responsible for sustaining of life. Because plants in nature are exposed to many changing conditions, it is important to understand how they influence plant photosynthetic activity. Elevated temperature is one of these natural conditions. Since it is known, that photosynthetic apparatus, mainly photosystem II, is sensitive to high temperature, thus it is important to understand the impact of temperature on its structure. In this study, we studied the impact of high temperature treatment on the structure of photosystem II. We used thylakoid membranes or photosystem II membranes isolated from barley leaves. Membranes were exposed to elevated temperature (25, 42, 47 and 55°C) and these membranes were then subjected to solubilization by mild, nonionic detergents α- or β-dodecylmaltoside. Obtained solubilized membranes were subsequently separated by clear-native PAGE or ultracentrifugation, respectively. Separated photosynthetic complexes were then subjected to denaturing SDS-PAGE in order to assess their subunit composition. Preliminary analysis reveals that elevated temperature induces degradation of high molecular mass-complexes of photosystem II, which results in a decrease in band density with these high molecular-mass complexes at temperature of 42°C. This decay is caused mainly by dissociation of light-harvesting complexes (LHCII) from the core complex of photosystem II. At 47°C new bands of lower molecular-mass complexes appeared. These bands are likely composed of degraded photosystem II complexes. However, the exact subunit composition of these bands still remains unclear and it is subject of further study.

90

TRANSFORMATION AND EXPRESSION OF A GENE BPHC FOR

PHYTOREMEDIATION OF PCB

Lišková V1, Vašková J2, Ohnoutková L2

1Research Centre for Toxic Compounds in the Environment, Faculty of Science Masaryk University

Brno, Czech Republic


Molecular Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ 783 71 Olomouc-


Polychlorinated biphenyls (PCBs) are very stable toxic compounds that persist in the environment and accumulate in the food chain. Decontamination of PCB-polluted areas is problematic due to hydrophobic properties of the PCB molecules. Phytoremediation can serve as an alternative to physical and chemical methods which are very expensive and greatly contribute to the environmental destruction. Spring barley (Hordeum vulgare L.) cv. Golden Promise and switchgrass (Panicum virgatum L.) cv. Alamo 2 were transformed with the gene bphC which was isolated from an aerobic bacterium Pandorea pnomenusa B-356. The gene bphC encodes an enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase, responsible for breaking the biphenyl ring. Agrobacterium strains AGL1 was used for the transformation of vector pBract214 which contains the bphC gen under the control of the Ubi promoter from maize and nos terminator.The presence of the transgene was verified by PCR and RT-PCR in transgenic plants. By performing these methods, the possibility of effective transformation of monocotyledons with bphC gene was confirmed.

91

CONFOCAL FLUORESCENCE MICROSCOPIC VISUALIZATION OF STARCH

GRANULES IN PLANT SAMPLES

Ovečka M1,2, Bahaji A1, Muñoz FJ1, Almagro G1, Ezquer I1, Baroja-Fernández E1, Li J1, Pozueta-

Romero J1

1Instituto de Agrobiotecnología (Consejo Superior de Investigaciones Científicas, Gobierno de

Navarra, Universidad Pública de Navarra) Nafarroa, Spain

2Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell


Republic

Starch represents a predominant storage carbohydrate in plants. It is synthesized by starch synthases using ADP-glucose as the sugar donor molecule. Biosynthesis of transitory starch in leaves is a complex, light-dependent process. We demonstrated that the so-called “starch-less” Arabidopsis thaliana adg1-1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Using Arabidopsis WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV starch synthases, gbss plants lacking the granule-bound starch synthases, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, we characterized the presence, size and number of starch granules in leaves. Our data were obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules in leaf chloroplasts. This method was valid not only for starch granules occurring in plant cells, but also for pure, isolated polymers, like amylose and amylopectin. Furthermore, we show that this method can be used to visualize glycogen granules occurring in bacteria. By presenting preparation methods for starch macro- and microscopic examination, we show how microscopic analysis may help in plant carbohydrate research. This work was partially supported by the grants BIO2007–63915 and BIO2010–18239 from the Comisión Interministeria de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain), Iden Biotechnology S.L., the grant IIM010491.RI1 of the Government of Navarra, and by structural research grants from EU and the Czech Republic to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic (grant no. ED0007/01/01).

92

CANNABIS SATIVA L. AS A SOURCE OF IMPORTANT BIOACTIVE SECONDARY

METABOLITES: RESEARCH AND HEALTH CARE APPLICATION NEEDS IN

TERMS OF THE CZECH REPUBLIC.

Peč J, Dušková E, Dušek K, Jelínková M, Cenklová V


Genetic Resources for Vegetables, Medicinal and Special Plants, Crop Research Institute,

Šlechtitelů 29, Olomouc, CZ-78371, Czech Republic

Human health in 21st century is highly regulated area due to minimizing risk of possible harmful effect in coherence with newly developed remedies or introduced medical techniques. Also good quality of life of individual person is highly important. However some diseases are not possible to cure sufficiently with our best knowledge. Cannabis derived remedies are such the case, where long term positive effects observed on selected patients groups, leads to resumption of cannabis as a medicine. Due to long period of restricted possibility of growing and research of cannabis in the Czech Republic, the information gap has been created. Some cannabinoid remedies with pure substance (mainly ∆9-tetrahydrocannabinol) were already established as the registered drugs (Marinol®, Cesamet™), however plant material with the complex mixture of cannabinoids and other secondary metabolites shows also nowadays much better profile of pharmacological effects and also adverse effects. Even if the plant derived remedy Sativex® or new pharmaceutical dosage form like Namisol® are developed, plant material in the form of pharmacopeia defined substance is necessary. With the novelization of Addictive Substances and Remedy Act (No. 50/2013 Sb.) the possibility of prescription of cannabis as a medicine is available. Nevertheless in the Czech Republic is necessary to set up the quality, production or prescription standards, where interdisciplinary approach is essential. Our department is starting to establish collection of medicinal cannabis with support of analytical and molecular biology laboratories, with emphasis to characterize collection utilizable in the area of our region. Preliminary results concerning inflorescence and leaf yield or inflorescence compactness with seven registered technical varieties shows beneficial characteristics for Finola and Carmagnola representatives. More analytical work and field or greenhouse experiments will continue with extended number of varieties. Acknowledgement: grant No. ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research.

93

MONITORING OF PESTS AND DISEASES IN THE FIELD COLLECTIONS OF

MEDICINAL, AROMATIC AND CULINARY PLANTS (MAPS) - PRELIMINARY

RESULTS

Petrželová I, Dušek K, Dušková




Medicinal, aromatic and culinary plants (MAPs) can be attacked by various pests and diseases degrading herb quality. However, unlike the broadly grown crop species there is relatively little information available on pests and diseases of MAPs. Germplasm collection of MAPs maintained at the Centre of Applied Research of Vegetables and Special Crops of CRI in Olomouc comprises about 833 accessions, and another 1100 accessions have a status of working collection. During the vegetation season field collections were surveyed for occurrence of pests and diseases. Most frequently recorded diseases were rusts (caused by members of Uredinales: found on Agrimonia, Balsamita, Inula, Levisticum, Malva, Potentilla, Salvia, Sanguisorba, Verbascum) and powdery mildews (Erysiphales: on Agrimonia, Calendula, Carum, Monarda, Potentilla, Salvia, Silybum, Verbascum). Infection by Peronosporales occurred sparsely (Armoracia, Ocimum). Species of Agrimonia, Alchemilla, Betonica, Digitalis, Echinacea, Filipendula, Foeniculum, Lavandula, Leuzea, Levisticum, Melissa, Monarda, Origanum, Potentilla, Rhodiola, Saponaria, Silybum suffered from leaf spots which mostly had not big devastating effect. Symptoms of infection by Aster yellows phytoplasma were observed on Echinacea. A broad diversity of insect species was recorded feeding on MAPs, some of them causing extensive damage (by sucking or chewing). Most herbs suffered from various species of leafhoppers (Cicadellidae) and plant bugs (Miridae). Carum, Gentiana and Verbascum were badly damaged by caterpillars (Lepidoptera). Carum and Salvia were attacked by gall mites (genus Aceria - Eriophyidae). Strong attacks by aphids (Aphididae) were observed on some Althaea, Artemisia, Borago, Calendula, Carum, Gentiana, Malva, Plantago, Salvia and Verbascum plants. Frequent were also spider mites (Tetranychidae: on Betonica, Carum, Dictamnus, Filipendula, Gentiana, Hyssopus, Lavandula, Levisticum, Malva, Ocimum, Plantago, Polemonium, Ruta, Salvia, Sanguisorba), leafminers (Agromyzidae, Tephritidae: on Agrimonia, Levisticum, Linaria, Polemonium, Potentilla, Salvia, Saponaria) and flea beetles (Chrysomelidae: most damaging on Althaea, Armoracia, Bellamcanda, Hyssopus, Ruta). Other insects were in minority or did not represent bigger threat. The research was supported by the Ministry of Agriculture of the Czech Republic, Project No. 206553/2011-17253, and Project No. ED0007/01/01 from the Centre of the Region Haná for Biotechnological and Agricultural Research.

94

N9-SUBSTITUTED AROMATIC CYTOKININS WITH NEGLIGIBLE SIDE EFFECTS

ON ROOT DEVELOPMENT ARE AN EMERGING TOOL FOR IN VITRO

CULTURING

Plíhal O, Szűčová L, Galuszka P

Palacky University in Olomouc, Centre of the Region Haná for Biotechnological and

Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, 78371 Olomouc-


Natural cytokinins as well as the majority of their synthetic derivatives show negative effects on root growth and development. Changes in morphology, primarily linked to the inhibition of the cell division in the meristematic zone, are manifested as thickening and shortening of the primary root and impaired lateral root branching. Rational design of cytokinin derivatives can partially overcome these drawbacks and reduce the negative effects. Using our database of cytokinin derivatives, we selected several aromatic cytokinin analogues with modifications at the N9 atom of the adenine moiety. We found that tetrahydropyranyl and tetrahydrofuranyl substitutions at the N9 atom led to enhanced acropetal transport of the modified cytokinin, and both derivatives also showed potential anticytokinin activity. Consequently, changes in the distribution of the active cytokinin pool together with gradual metabolic conversion of the modified cytokinin to its free form prevented root growth inhibition that is associated with cytokinin use. Thus, these novel N9-substituted derivatives of the aromatic CKs have a great potential as alternative compounds to improve plant in vitro regeneration techniques.

95

PREPARATIONS OF TRANSGENIC BARLEY WITH MODIFIED CYTOKININ

LEVEL

Pospíšilová H1, Jiskrová E1, Mrízová K1, Husičková A2, Ježilová E2, Klimešová J3, Galuzska

P1, Frébort I1

1Palacky University in Olomouc, Centre of the Region Haná for Biotechnological and

Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, 78371 Olomouc-



Biophysics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ 783 71 Olomouc-


3MENDELU Brno, Department of Crop Science, Breeding and Plant Medicine,

Zemědělská 1, 61300 Brno. Czech Republic

Barley, Hordeum vulgare, is an agriculturally important crop. New techniques of molecular cloning and plant transformation accelerate classical breeding techniques and produce barley plants with enhanced traits. Cytokinins are plant hormones controlling many physiological processes including stress tolerance and root formation1. Cytokinins are irreversibly degraded by cytokinin dehydrogenase (EC 1.5.99.12; CKX), which is a principal factor to control cytokinin levels in plants2. For modulation of the cytokinin level in barley, constructs with cytokinin dehydrogenase with altered targeting under the control of potentially root specific promoter were prepared. All intended transgenic barley lines were prepared, confirmed and then analyzed in T2 generation of homozygous plants. Three unique transgenic barley lines with differently localized CKX showed distinct phenotypes.

96

CORRELATION OF SINGLET OXYGEN PRODUCTION AND ULTRA-WEAK

PHOTON EMISSION IN GREEN ALGAE CLAMYDOMONAS REANHARDTII

Prasad A1, Sedlářová M2, Pospíšil P1




2Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, Olomouc,

Czech Republic

To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring the oxidation of biomolecules, the production of singlet oxygen during heat treatment (40 °C) for a duration ranging from 0-30 min was studied in the unicellular green alga Chlamydomonas reinhardtii. The oxidation of biomolecules, predominantly of lipids and proteins was monitored by ultra-weak photon emission measured with the employment of highly sensitive charge coupled device camera and photomultiplier tube. The enhancement in ultra-weak photon emission by heat treatment correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. The enhancement in production of singlet oxygen with increasing duration of heat treatment was monitored utilizing Electron paramagnetic resonance spin-trapping spectroscopy. These observations revealed that the oxidation of biomolecules results in generation of singlet oxygen which upon recombination can emit ultra-weak photons at the wavelength of 634 nm and 703 nm.

97

DEHYDRIN RELATIVE ACCUMULATION IN CEREALS EXPOSED TO ABIOTIC

STRESS FACTORS

Kosová K, Vítámvás P, Vlasáková E, Prášil IT

Department of Genetics and Plant Breeding, Crop Research Institute, Prague, Czech

Republic

Dehydrins (DHNs) are highly hydrophilic late embryogenesis abundant (LEA) II proteins which accumulate in plant cells upon stress conditions displaying a dehydrative component. Accumulation of dehydrins represents an important part of the complex process of plant acclimation to stresses and induction of protective system. It is becoming evident that in cereals, abundance of some dehydrins can also be related to the level of acquired tolerance to various abiotic stresses indicating their potential use in plant biotechnology research. Our research has been focused on dehydrin protein relative accumulation in wheat and barley plants exposed to a broad range of growth temperatures, drought or salinity. In cold-treated common wheat, the major cold-inducible protein was WCS120, while in barley, the major cold-inducible protein was DHN5. The WCS120 proteins and DHN5 protein are orthologues and belong to high-molecular-weight DHNs. Both WCS120 and DHN5 relative accumulation increased exponentially along with a temperature decrease in the range from optimum (20 °C) to cold (5 °C) temperatures. Both WCS120 and DHN5 relative accumulation revealed a significant correlation with plant maximum acquired frost tolerance expressed as lethal temperature values (LT50s) at cold temperatures (5 °C, 10 °C), and, moreover, WCS120 relative accumulation revealed a significant correlation with maximum LT50 also at mild temperatures (15 °C, 20 °C). In drought-treated or salt-treated barleys, besides the accumulation of DHN5 protein, accumulation of several low-molecular dehydrins (29 kDa, 26 kDa, 21 kDa, 19 kDa) has been found. The highest levels of accumulated dehydrin proteins have been found in plant crowns which are crucial organs for the whole plant survival. Considering low-molecular-weight DHNs accumulated upon drought and salt stresses, both quantitative and qualitative differences have been observedd between the relatively susceptible Czech cultivar Amulet and the relatively tolerant Syrian landrace Tadmor.

98

APPLICATION OF METHYL JASMONATE AFFECTS THE ANTIOXIDANT

ACTIVITY AND OXIDATIVE RESPONSES IN VACCINIUM CORYMBOSUM L.

Reyes-Díaz M1, Ribera-Fonseca A2, Lobos T3, Alberdi M1

1Departamento de Ciencias Químicas y Recursos Naturales and Center of Plant-Soil

Interaction and Natural Resources Biotechnology (BIOREN-UFRO), Universidad de La

Frontera, Avenida Francisco Salazar Nº 01145, P.O. Box 54-D, Temuco, Chile.


Technological (BIOREN-UFRO), Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.


Casilla 54-D, Temuco, Chile.

Vaccinium corymbosum L. represents an economically important crop, being a good antioxidant source. A beneficial effect on the induction of antioxidant activity is manifested by exogenous application of methyl jasmonate (MeJA). Studies about the effect of MeJA applications on this crop are scarce. In this study we determine the effect of MeJA exogenously sprayed on antioxidant activity and lipid peroxidation of highbush blueberry cv. Bluegold. Two-years old blueberry plants grown under greenhouse conditions were sprayed with different MeJA concentrations (0, 0.01, 0.05 and 0.1 mM) using a 2L hand pump. One group of plants was sprayed for one opportunity at the same concentrations mentioned above. Another group was subjected to the same MeJA treatments for two times. Leaves were harvested after 0, 7, 14 and 21 days of treatments. Then antioxidant activity (DPPH method), total phenols (spectrophotometrically) and lipid peroxidation (TBARS method) were determined. It was found that the antioxidant activity increased by one MeJA application in all treatments, remaining over the time. Two MeJA applications augmented strongly the antioxidant activity at 7 and 14 days, decreasing thereafter. On the contrary, total phenols decreased with one MeJA application at all treatments, showing a slight increase with two MeJA applications at the highest treatment. Lipid peroxidation exhibited an increase when two MeJA applications at the highest treatment were applied. The antioxidant activity increases with one application, being constant through the time. Differentially, two MeJA applications increased this parameter only initially, decaying significantly compared to control. Despite the increase in antioxidant activity with one application, phenols decreased, suggesting that non-phenolic compounds would be responsible for this activity. Thus, we conclude that one MeJA application could be more suitable to avoid oxidative stress, which appeared with two applications of MeJA. Acknowledgements: FONDECYT Nº1120917, CONICYT fellowship and Berries San Luis farm.

99

ROLE OF SUPEROXIDE DISMUTASE ISOFORMS IN ALLEVIATE MN-INDUCED

OXIDATIVE STRESS IN PERENNIAL RYEGRASS

Ribera-Fonseca A1, Cartes P2, Inostroza-Blancheteau C3, Álvarez D4, de la Luz Mora M5


Technological (BIOREN-UFRO), Universidad de La Frontera, Avenida Francisco Salazar Nº

01145, P.O. Box 54-D, Temuco, Chile.



Frontera, P.O. Box 54-D, Temuco, Chile.

3Núcleo de Investigación en Producción Alimentaria, Escuela de Agronomía, Facultad de

Recursos Naturales, Universidad Católica de Temuco. P.O. Box 56-D, Temuco, Chile.

4Laboratory of Soil and Plant Analyses; all affiliations belong to Universidad de La Frontera,

Avenida Francisco Salazar 01145, Temuco, Chile.



Frontera, P.O. Box 54-D, Temuco, Chile.

In Southern Chile, ryegrass (Lolium perenne) is mainly cultivated in Andisols, in which manganese (Mn)-excess limits plant growth. Earlier we showed an increase of superoxide dismutase (SOD) activity in response to Mn-induced oxidative stress, mainly in Mn-tolerant cultivars. Here we evaluated Mn uptake, lipid peroxidation (LPO), SOD activity and the gene expression profiles of SOD isoforms in shoots of two ryegrass cultivars differing in their Mn-tolerance. Seedlings of Nui (Mn-sensitive) and Kingston (Mn-tolerant) cultivars were hydroponically grown at 2.4 (control) or 750 μM Mn, and harvested at 2, 6, 12, 24 and 48 h. During the experiment, Kingston contained higher shoot Mn than Nui at 2.4 μM Mn added. When plants grown under Mn-excess, shoot Mn gradually increased in both cultivars. Even though a strong increase in Mn uptake was detected from 24 h in both cultivars, Kingston had higher shoot Mn than Nui at the end of the assay. A significant increase in LPO under Mn excess was observed at 2 h. Nevertheless, only in Nui this increment was also found at 6 h and 48 h. Kingston exhibited higher SOD activity than Nui during the experiment. In fact, SOD was greatly activated by Mn only in this cultivar at 24 and 48 h (up to 200%). In general, basal and Mn-induced gene expression of SOD isoforms were higher in Nui (Mn-inductions: 50% for Mn-SOD, 40% for Cu/Zn-SOD and 270% for Fe-SOD respect to the control) than Kingston. However, we found a strong up-regulation of Fe-SOD expression (~540%) triggered by Mn-excess in Kingston at 12 and 24 h. Thus, under Mn-excess, the activation of SOD and the induction of the gene expression of Fe-SOD seem to contribute to the greater ability of Kingston to counteract the Mn-induced oxidative stress compared to Nui. Acknowledgements: FONDECYT projects 11100494, 1120901 and 3120248.

100

WORTMANNIN AND LY294002 INDUCE SUBCELLULAR CHANGES IN

VESICULAR TRAFFICKING OF ARABIDOPSIS ROOT CELLS

Šamajová O, Takáč T, Ovečka M, Šamaj J


Cell Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc-


Endocytosis is fundamental, dynamic and complex process of intracellular uptake of plasma membrane and extracellular cargos which is controlled by network of regulatory proteins. In plants, more specific and targeted inhibitors are necessary to study vesicular trafficking routes including the endocytosis. We studied effects of wortmannin and LY294002 on vesicular trafficking routes of Arabidopsis root cells. Wortmannin is widely used pharmaceutical compound employed to define vesicular

trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinases (PI4Ks) in a dose-dependent manner. Wortmannin causes the inhibition of protein vacuolar sorting and endocytosis. Wortmannin at low concentrations (up to 1 μM) specifically inhibits PI3K, but at high concentrations it inhibits both PI3K and PI4Ks. On the subcellular level, wortmannin (33 μM) caused clustering, fusion and swelling of multivesicular bodies (MVBs). All these changes lead to the formation of wortmannin-induced multivesicular compartments and depletion of TGN as revealed by electron microscopy. This indicates a possible consumption of TGN vesicles by MVBs during wortmannin treatment. Second inhibitor used in our studies was LY294002 which is a synthetic quercetin-like compound. In comparison to wortmannin LY294002 is a more specific inhibitor of phosphatidylinositol 3-kinase. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. Such affects are at least partially similar to those induced by wortmannin. This work was supported by grant No. ED0007/01/01 to the Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic and by grant No. P501/11/1764 from the Czech Science Foundation GAČR.

101

INACTIVATION OF CYTOKININS – DIFFERENT ROLES OF VARIOUS CYTOKININ

UDP-GLYCOSYLTRANSFERASE ISOFORMS IN A. THALIANA?

Šmehilová M, Dobrůšková J, Mičaníková R, Novák O, Galuszka P

Centre of the Region Hana for Biotechnological and Agricultural Research, Department of

Molecular Biology, Faculty of Science, Palacky University Olomouc, Slechtitelu 11, 783 71


Cytokinin UDP-glycosyltransferases-mediated deactivation of cytokinins plays an important role in proper maintenance of the hormones’ levels. Our recent results together with previously published data suggest different roles of distinct UGTs isoforms in the cytokinin metabolism and different mechanism of their regulation. Varying expression profiles of the cytokinin specific UGTs were observed during A. thaliana natural senescence and after exogenous cytokinin treatment. In leaves, majority of cytokinins decrease rapidly during senescence, including glucosides. That corresponds well with activation of cytokinin degradation enzymes - cytokinin dehydrogenases (CKXs), as well as with strong up-regulation of their genes expression. The cytokinin glucosides represent major forms of a cytokinin pool within plant tissues. Plants with depleted cytokinin glucosides content would comprise a powerful tool for their physiological role exploration. Since no mutants with silenced genes for the UGTs were characterized before, characterization of T-DNA insertional mutant lines of A. thaliana UGTs aims to shed more light into the process of UGTs-mediated regulation of active cytokinin levels. Here, knock-down mutants of A. thaliana UGTs will be characterized for morphological changes, cytokinin content and for expression profile of cytokinin metabolism genes. Further, physiological response to endogenous cytokinin treatment will be shown.

102

EFFECT OF ABIOTIC STRESS ON MAPK ACTIVATION

Smékalová V1, Šamajová O1, Petrovská B2, Šamaj J1


Cell Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc-


2Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of

Science, Palacký University, Centre of Plant Structural and Functional Genomics, Šlechtitelů

31, CZ-783 71 Olomouc, Czech Republic

Mitogen-activated protein kinases are signaling enzymes which are involved in regulation of gene expression, enzymatic activities, stress responses, cell growth and differentiation. It is known that MAPK signaling cascade is triggered by diverse environmental and stress stimuli. Therefore, we have studied effect of different types of abiotic stresses on three best characterized MAPKs in Arabidopsis thaliana, namely MPK3, MPK4 and MPK6. Transcriptional activation was studied by using promoter GUS histochemistry and quantitative Real-Time PCR (qPCR). Hyperosmotic (NaCl, KCl, sucrose and sorbitol), hypoosmotic (distilled water) and oxidative (H2O2) stress were used for stimulation of MAPK activation. According to results of GUS histochemistry, MPK3 promoter was significantly activated in roots and cotyledons by all types of abiotic stress. Activation of MPK4 promoter was primarily triggered by salt hyperosmotic stress, especially in stomata. Finally, MPK6 promoter was activated mainly after salt and sugar stress in roots, especially in root tips and in central cylinder. Results of qPCR show that transcriptional activation of MPK3 gene in roots and leaves was triggered mainly by oxidative and hyperosmotic stress. Activation of MPK4 gene in roots and leaves was not so pronounced. MPK6 gene was activated by hydrogen peroxide and sucrose in roots and also in leaves. These data indicate that activation of the MPK3 is induced by all types of stresses used. The MPK4 and MPK6 genes are activated by hyperosmotic stress.

103

PHOTOSYNTHETIC CHANGES INDUCED BY HIGH ENDOGENOUS LEVEL OF

CYTOKININS IN TOBACCO

Novák J1,2, Pavlů J 1,2, Novák O3, Nožková-Hlaváčková V 4, Špundová M4, Hlavinka J4,

Koukalová Š 1,2, Skalák J1,2, Černý M 1,2, Brzobohatý B 1,2

1Department of Molecular Biology and Radiobiology, Mendel University in Brno, Zemědělská

1, CZ-61300 Brno, Czech Republic

2CEITEC – Central European Institute of Technology, Mendel University in Brno,

Zemědělská 1, CZ-61300 Brno, Czech Republic

3Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany

AS CR, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic




Photosynthetic changes induced by an increase in endogenous level of cytokinins were investigated in transgenic tobacco CaMV35S > GR > ipt (pOp6-ipt/LhGR-N, line 303). The ipt expression was activated in the 5-week-old plants by an application of 20 µM dexamethasone (DEX) to the growth substrate. Photosynthetic gas exchange and chlorophyll fluorescence parameters were measured during the first 3 days after the DEX application. The levels of cytokinin bases as well as their ribosides and ribotides increased dramatically within 19 h after the DEX application and this increase levelled off over the following 24 h, reaching values about four orders of magnitude higher than in non-activated plants. The increase in cytokinin levels was followed by an accumulation of hydrogen peroxide, increased lipid peroxidation and by downregulation of CAB, FNR1 and VDE genes. Photosynthetic activity decreased, especially on the 3rd day after the DEX application. The analysis of photosynthetic gas exchange (measured by an LI-6400 open system) revealed an inhibition of light-induced stomatal opening and light-induced increase in a rate of CO2 assimilation (A) indicating down-regulation of Rubisco activation. The carboxylation efficiency of Rubisco was inhibited, too, as demonstrated by a decrease in the slope of the linear region of the A/Ci dependence. Changes in chlorophyll fluorescence parameters indicated an inhibition of electron transport through both photosystem II and I and a pronouncedly up-regulated dissipation of excitation energy. The need to dissipate excess chlorophyll excitation energy reflects a decrease in electron consumption by CO2 assimilating processes resulting from the down-regulation of Rubisco activity, together with a decrease in CO2 availability due to reduced stomatal opening. The overall response of tobacco plants to the dramatic increase in endogenous cytokinin level closely resembled a hypersensitive response to pathogen attack. This work was supported by grants Nos. 1M06030 and LK21306 and by projects CZ1.05/1.1.00/02.0068 and ED0007/01/01 from the European Regional Developmental Fund.

104

PHYSICAL MAP AND SEQUENCING OF WHEAT CHROMOSOME ARM 7DS

Staňková H1, Luo MC2, Visendi P3, Tulpová Z1, Batley J4, Bartoš J1, Doležel J1, Edward Ds3,

Šimková H1



2Department of Plant Sciences, University of California, Davis, CA 95616

3School of Agriculture and Food Sciences and Australian Centre for Plant Functional

Genomics, University of Queensland, Brisbane, QLD 4072, Australia

4Centre for Integrative Legume Research, University of Queensland, School of Agriculture

and Food Sciences, Brisbane, QLD 4072, Australia

Bread wheat (Triticum aestivum) is one of the most important crop species in the world. It provides the staple food for 35% of world’s population and its annual production can be challenged only by rice. Wheat huge genome size (1C = 17 Gb), presence of three homeologous subgenomes (A, B and D) and prevalence of repetitive sequences (>80%) hamper any genomic research in wheat including physical map construction and sequencing. The possibility to divide the wheat genome into individual chromosome arms by flow cytometric sorting enables coping with polyploidy and studying the wheat genome in a stepwise manner. In our project, we focused on sequencing the 7DS chromosome arm based on physical map of the 7DS. First, a 7DS-specific BAC library comprising 49,152 BAC clones representing 12.2 equivalents of the chromosome arm was constructed and all BAC clones were fingerprinted using SNaPshot-based HICF technology. Clones were automatically assembled into contigs based on fingerprint overlaps using FPC software. Integration of the 7DS physical map with that of Aegilops tauschii (D genome ancestor) provided a clue for further merging of contigs. Reliability of the assembly was verified through LTC software. Final BAC contig assembly resulted in 931 BAC contigs and a minimum tiling path (MTP) of 4,608 BAC clones. The physical map has been anchored to 7DS genetic maps by 583 markers, which located 307 contigs on the chromosome arm. MTP BAC clones were pooled in fours and the pools were sequenced by Illumina MiSeq platform. Using Sassy software developped to assemble short complex sequences from Illumina paired read data, we succeeded in assembling the reads into 1 - 7 sequence contigs per BAC. Optimisation of the procedure aiming to further reduce the number of contigs per BAC is in progress. Sequences of BAC clones from the 7DS MTP will be used for anchoring of the 7DS physical map. The anchored 7DS physical map as well as the sequence of the 7DS arm will become valuable tool for genetic mapping and positional cloning of genes located in this part of the wheat genome. This work has been supported by the Czech Science Foundation (P501/12/2554), MSMT CR and EU (Operational Programme Research and Development for Innovations No. ED0007/01/01) and Internal Grant Agency PrF-2012-003.

105

THE PEPPER (CAPSICUM ANNUUM L.) IN THE CZECH REPUBLIC

Stavělíková H




The pepper is very popular, widespread in the world, annual vegetable. The pepper is good source of many essential nutrients to produce high amounts of vitamin C, provitamin A, E, P (citrin), B1 (thiamine), B2 (riboflavin) and B3 (niacin). The pepper has been grown very long time. The oldest known records of pepper come from the desert valley of Tehuacán, in Southern Mexico. It is known that the indigenes were eating peppers as early 7000 B.C. Christopher Columbus brought the pepper to the Europe. The collections of pepper (Capsicum annuum L.) germplasm have very long and rich tradition since 1951. In this time the holder of this collection is Centre of Applied Research of Vegetables and Special Crops of the Crop Research Institute. The collection of pepper has 507 accessions. Management of the germplasm collection comprises conservation, evaluation and multiplication of pepper accessions, including their description according to international Descriptors for Capsicum (Capsisum spp.). We use for the pepper 27 characters. In this time the pepper is grown on 531 725 ha on the world and on 244 ha in the Czech Republic. Now, 175 varieties (59 hybrid and 116 open pollinated) of the pepper are in Recommended List of Varieties. These varieties were bred by nineteen breeding companies: twelve from the Czech Republic, two from Netherlands and Slovakia and one from Poland, Romania and Italy. The research was supported by the Ministry of Agriculture of the Czech Republic, Project No. 0002700604 and Project No. ED0007/01/01 from the Centre of the Region Haná for Biotechnological and Agricultural Research.

106

THE DEVELOPMENT OF SNP PLATFORM FOR ANALYSIS OF GENOMIC

CONSTITUTION AND GENE EXPRESSION IN XFESTULOLIUM HYBRIDS

Stočes Š1, Ruttink T2, Kopecký D1, Bartoš J1, Doležel J1



2Institute for Agricultural and Fisheries Research, Caritasstraat 21, 9090, Melle, Belgium

Grasses are one of the most important and widely cultivated crops. Italian and perennial ryegrasses (L. multiflorum Lam. and L. perenne L.) predominate in temperate regions as suitable species for intensive farming systems due to their yield, palatability, digestibility and nutritional characteristics. However, they are susceptible to biotic and abiotic stresses and tend to fail in less favorable conditions. On the other hand, tall fescue (F. arundinacea Schreb.) and meadow fescue (F. pratensis Huds.) perform better on poor soils and excel in drought tolerance, winter hardiness and persistence. Festulolium has been developed by intergeneric crossing of ryegrass and fescue species and combine complementary agronomic attributes of both genera. Despite the increasing popularity of these hybrids among seed companies and farmers, there is generally lack of knowledge on the genomic constitution and gene expression. The advent of Next Generation Sequencing technologies enables the production of large amount sequence datasets. These can be used for molecular marker development, phylogenetic and ecological studies and analysis of transcriptomes using RNA-sequencing. We developed a SNP platform from RNAseq data, which enables the precise reconstruction of genomic composition of hybrid plants and to analyze the gene expression of parental alleles including the information on particular allele loss or silencing. We used sequencing data from 12 parental genotypes (6 of F. pratensis, 6 of L. multiflorum) and for the first time we identified 30,000 to 90,000 highly species-specific SNPs based on the combination of F. pratensis × L. multiflorum genotypes used for F1 hybrids development. These SNPs are localized on 6,000 to 9,000 genes distributed more or less evenly over all chromosomes. The analysis of RNA sequence data from reciprocal F1 and F2 hybrids using this platform is in progress. This work was supported by grants P501/11/0504 and CZ.1.05/2.1.00/01.0007.

107

PROTEOMIC ANALYSIS SHOWS IMPORTANT ROLE OF ANTIOXIDANT

ENZYMES DURING MAIZE ANDROGENESIS

Takáč T1, Uváčková Ľ2, Šamaj J1


Cell Biology, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc,

Czech Republic

2Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2,

P. O. Box 39A, 950 07, Nitra, Slovak Republic

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenesis represents an important tool for the investigation of embryogenesis, as well as for breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. This study aimed to investigate the changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis. This work was supported by grant Nr. P501/11/1764 from the Czech Science Foundation GAČR and by grant Nr. ED0007/01/01 to Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.

108

IDENTIFICATION AND QUANTIFICATION OF CYTOKININS IN TWO

PHYTOPATHOGENIC ACTINOMYCETES, RHODOCOCCUS FASCIANS AND

STREPTOMYCES TURGIDISCABIES

Tarkowski T1, Vereecke D2, Francis I3, Loria R3, Werbrouck S2


Protein Biochemistry and Proteomics, Faculty of Science, Palacký University, Šlechtitelů 11,

CZ 783 71 Olomouc-Holice, Czech Republic

2Department of Plant Production, University College Ghent, Ghent, Belgium

3Department of Plant Pathology and Plant-Microbe Biology, Cornell University, USA

Although the dark side of plant pathogenic microbes is logically studied intensively, the application of their elicitors for beneficial purposes is often not considered. Rhodococcus fascians, the causative agent of the leafy gall syndrome, and Streptomyces turgidiscabies, one of the scab inducing streptomycetes, not only share the characteristic of having a very broad host range, but they are the only two organisms described to date to carry a fas operon involved in the biosynthesis of cytokinins (Joshi and Loria, 2007; Pertry et al., 2010). Because of the significant impact of these pathogens on plant development, especially on shoot formation, we propose that the use of the morphogens produced by these bacteria in tissue culture practices could be valuable, particularly for plants that are recalcitrant to the currently used compounds. We performed mass-spectrometric identification and quantification of isoprenoid cytokinins isolated from cultivation media by solid-phase extraction. Interestingly, despite the strong similarity between the fas operons of R. fascians and S. turgidiscabies, both bacteria secrete a different set and different levels of cytokinins. In addition, whereas R. fascians machinery mainly produces cZ-type cytokinins and iP-type cytokinins, S. turgidiscabies almost exclusively produces tZ.

M. Joshi and R. Loria (2007) MPMI 20, 751-758; I. Pertry et al (2010) MPMI 23,1164-74

109

DIFFERENTIAL ACTIVITY OF ARABIDOPSIS AURORA KINASE MEMBERS

TOWARDS HISTONE H3

Tomaštíková E1*, Doležel J1, Houben A2, Demidov D2§



2Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466

Gatersleben, Germany

Histones are subjected to a variety of post-translational modifications (PTM). Such modifications are required for interactions with specific protein domains and regulate many aspects of chromosome activity. Phosphorylation of histone H3 is crucial for cell-cycle dependent chromosome condensation and segregation. Various H3 kinases have been identified in plant, such as Aurora and Haspin. Members of the Aurora kinase family are responsible for cell cycle-dependent phosphorylation of serine 10 and serine 28 of H3 in both plant and non-plant organisms. In non-plant species cross-talk occurs between phosphorylation of H3S10 and acetylation or methylation of H3. For plants, information on the cross-regulation between H3S10 phosphorylation and PTMs of neighboring amino acids is limited. Data for the cross-talk between phosphorylation of H3S10 by AtAurora3 and other neighboring PTMs or around H3S28 by AtAurora1 and 3 are missing. To elucidate whether the phosphorylation activity of the Aurora kinase family members of Arabidopsis towards H3S10 and H3S28 is influenced by neighboring PTMs, an in vitro kinase assay was performed. As substrates, N-terminal peptides of H3 with various modifications close to S10 and S28, were used. Recombinant AtAurora1 and AtAurora3 were expressed in E. coli. Our kinase assay confirmed that both serine positions are phosphorylated by both kinases in vitro, although phosphorylation of H3S28 is much weaker than of H3S10. However, H3S28 phosphorylation is increased by dimethylation and acetylation of neighboring K27. H3S10 phosphorylation by AtAurora1 is increased by dimethylation of H3K4 and phosphorylation of H3T6. Decrease in kinase activity was observed for acetylated H3K4. Taken together our data suggest that a cross-talk between different H3 modifications occurs also in plants, although in similar rather than the same manner as in non-plant species. *Supported by ED0007/01/01 and IGA UP Prf/2013/003. §Supported by DFG Germany (SFB 648).

110

CHANGES OF BIOCHEMICAL AND PHYSIOLOGICAL FEATURES OF V.

CORYMBOSUM CULTIVARS INDUCED BY DIFFERENT MEJA DOSES

EXOGENOUSLY APPLIED UNDER ALUMINIUM TOXICITY

Ulloa-Inostroza E1, Alberdi M2, Reyes-Diaz M2


Avda. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.

2Departamento de Ciencias Químicas y Recursos Naturales and Center of Plant, Soil

Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource

Nucleus (BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D, Temuco, Chile.

Blueberry (Vaccinium corymbosum L.) is important crop and has a high antioxidant capacity, which can be stimulated under some stress conditions as aluminum (Al) toxicity. A low dose of methyl jasmonate (MeJA) exogenously applied may counteract the Al-toxic effects and can maintain a high antioxidant capacity. The objective of this study was to determine the dose effect of an exogenous MeJA application on biochemical and physiological features in Legacy (Al-resistant) and Bluegold (Al-sensitive) cultivars. Blueberry plants of two year-old grown in Hoagland solution with toxic Al (pH=4.5) under greenhouse conditions with five treatments: Control (1): without Al and MeJA; Control (2): Al (100 µM); Combined treatments with Al (100 µM) and MeJA (0.005; 0.01; 0.05 mM) were used by 48 hours. The MeJA was applied simultaneously with Al toxic. The parameters measured were: antioxidant activity (AA) by the DPPH method, lipid peroxidation (LP) by the TBARS method, and

fluorescence parameters of PSII (Fv/Fm, ɸPSII, ETR and NPQ) were measured with a portable modulated fluorescence system FMS 2. The LP decreased and AA increased with the application of low MeJA doses in both cultivars and in leaves and roots. The Fv/Fm remained within the range for healthy plants in both cultivars in all

treatments. The ɸPSII, ETR and NPQ in Legacy remained constants. However, ɸPSII and ETR in Bluegold was affected by the presence of Al in the solution, but this decrease was less with MeJA application. These results suggest that the application of low MeJA doses in blueberry plants under Al stress could be a good alternative to reduce the imbalances and biochemical alterations induced by aluminum toxicity in blueberry plants. Acknowledgements: FONDECYT Nº1120917, CONICYT fellowship and Berries San Luis Farm.

111

FOUNDATION OF ARTIFICIAL ASSOCIATIONS OF CUCUMBER WITH

BENEFICIAL MICROORGANISM

Vershinina ZR, Nigmatullina LR, Baymiev AK, Chemeris AV

Institute of Biochemistry and Genetics, Ufa Scientific Center, Russian Academy of Sciences

Creation of artificial symbiotic associations of cultivated plants with endophytic microorganism is a new long-range direction in bioengineering of symbiotic systems. Nodule bacteria a kind of Rhizobium, which fix nitrogen in symbiosis with leguminous plants, can act as associative microsymbiont for many economically valuable nonleguminous plants, including cucumber, encouraging growth of plants and protecting from phytopathogen. But associative processes in nature are difficult to control, and in the natural conditions beneficial bacteria can’t stand competition. The goal of researchers is to make plants to control microflora of their rhizosphere, and in case of use as beneficial microsymbiont of nodule bacteria, one of the tools are lectins of legume. Accumulated data in the past decades shows that lectins can connect with symbiotic bacteria and help to recognize and attach microsymbiont to macrosymbiont. In such a way transgenic cucumber synthesizing lectin of a definite leguminous plant, will be mainly colonized by the microsymbiont isolated from this legume. Using such a strategy gives a clue to formation of “artificial rhizosphere” of cucumber, specifically colonized only by those bacteria which fulfil useful for plants trophic, growth initiating and/or protective functions. The aim of our work was examination of R. leguminosarum (pea microsymbiont) colonization on cucumber roots transformed the pea lectin gene. Complete genomic pea lectin psl was cloned in the binary vector pCambia 1301. Then we used A. rhizogenes A4 carrying pCambia 1301-psl to induce hairy roots in Cucumis sativus L. by wounding the region of the axillary bud. Аpproximately 90% inoculated plants did have at least three gus-psl-activity hairy roots 10 days after infection. The number of adhered bacteria onto the roots transformed with lectin gene was 10 higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of cucumber.

http://www.multitran.ru/c/m.exe?a=118&t=3071138_1_2

http://www.multitran.ru/c/m.exe?t=4313787_1_2







http://www.multitran.ru/c/m.exe?a=118&t=3071138_1_2

112

PHYTASE ACTIVITY AND CONTENT OF DIGESTIBLE PHOSPHORUS IN

TRANSGENIC LINES OF SPRING BARLEY SCLW-GP-PHYA

Vlčko T, Vašková J, Ohnoutková L




Phosphorus is bound within molecules of phytic acid in plant cells, especially in plant seeds. The phytic acid also binds cations thus being considered as an antinutritional factor, which decrease the nutrition value of foodstuffs. The phytase is an enzyme which catalyze dephosphorylation of phytic acid molecules thus releasing tightly bound cations. Non-ruminants do not produce an adequate amount of this enzyme in their gastro-intestinal tract so they are not able to utilize completely nutrient potential of feedstuff. Hence, the transgenic lines of spring barleySCLW-GP-PHYA with an inserted microbial phytase enzyme (PhyA) were developed. The transgenic spring barley SCLW-GP-PHYA was launched in conformity with permission of the Ministry of Environment into environment in 2011. At present, transgenic lines SCLW-GP-PHYA are grown at two locations Olomouc and Lukavec in the Czech Republic. The activity of phytase was determined colorimetrically using molybdate-vanadate method. The content of digestible phosphorus was determined colorimetrically.

113

AFFECTING OF ERGOT ALKALOID PRODUCTION IN CLAVICEPS PURPUREA

BY GENETIC MANIPULATION

Vrabka J, Hanosová H, Čudejková M, Vojta P, Valík J, Galuszka P




Ergot alkaloids produced by the parasitic fungus Claviceps purpurea are pharmaceutically importantant substances widely used for production of anti-migraine drugs, uterotonics, anti-Parkinson agents, etc. Improving of ergot alkaloid production of this fungus could significantly reduce the costs of ergot alkaloid based drugs. To improve the alkaloid production we selected and overexpressed three genes from ergot alkaloid gene cluster, that could lead to increased production, however this was not confirmed yet. Using the Next Generation Sequencing techniques we are analyzing the transcriptomes of four commercial strains which differ in the spectrum of the produced alkaloids. We suppose that comparison of differential expression of genes directly or indirectly involved in ergot alkaloid biosythetic pathway between these strains might help us to find new candidate genes which could be manipulated to increase the production. Since the fungus strains which we are using for genetic manipulations can produce alkaloids only when infecting a host, we are studying the process of infection as well. Interestingly, we found that two selected strains differ not only in the spectrum of produced alkaloids, but also in the progress of infection.

114

CHARACTERISATION OF MAIZE CYTOKININ DEHYDROGENASE FAMILY

Zalabák D1, Frébortová J2, Galuszka P1

1 Centre of the Region Hana for Biotechnological and Agricultural Research, Department of



2 Centre of the Region Hana for Biotechnological and Agricultural Research, Department of

Chemical Biology and Genetics, Faculty of Science, Palacky University Olomouc,

Slechtitelu 11, 783 71 Olomouc-Holice, Czech Republic

Cytokinins are plant hormones responsible for many physiological processes in plants. The cytokinin homeostasis is crucial for normal plant growth and development and thus cytokinin level has to be finely controlled. One of possible mechanisms is the cytokinin irreversible degradation by cytokinin dehydrogenases. Here we focused on cytokinin dehydrogenase gene family in maize as a useful monocot model plant with the goal to characterize respective CKX isoforms in detail, especially their substrate preferences. Thus we have selected nine out of 13 isoforms based on the gene expression profiling. Open reading frames of CKX genes were subcloned in pTYB12 vector and recombinant proteins were expressed in E. coli. Recombinant proteins were then purified using affinity as well as ion exchange chromatography. Substrate preferences were studied using two different methods: continuous decolorisation of an electron acceptor dichlorophenol indophenol (Laskey et al., 2003) and direct measurement of cytokinin degradation using HPLC method. Here the decay of respective cytokinin in a cytokinin mixture was measured during a two hour period (Pertry et al. 2009). We expect that obtained results will help to understand the complexity of cytokinin metabolism in monocot plants.

115

THE RELATIONSHIP BETWEEN POLLEN FLOW AND GENE FLOW RANGE, AS

A FACTOR IN GM AND OTHER CROPPING SYSTEMS COEXISTENCE

Zimny J1, Otręba P1, Kozdój J1, Zimny A1, Jędryczka M2, Kaczmarek J2, Oleszczuk S1,

Czaplicki A1, Makowska K1, Sowa S1

1Plant Breeding and Acclimatization Institute – National Research Institute, Radzików, 05-870

Błonie, Poland, Department of Plant Biotechnology and Cytogenetics

2Institute of Plant Genetics of the Polish Academy of Sciences, Strzeszyńska 34, 60-479

Poznań, Poland, Laboratory of Resistance Genetics

Since GM plants appeared on the market, the admixture of GM products in non-GM products cannot be excluded. Therefore suitable measures during cultivation, harvest, transport, storage and processing of GM crops, should be applied. In view of the frequently used argument against genetically modified plants that their pollen can migrate for long distance and contaminate non transgenic crops and wild relatives, it is necessary to verify such hypotheses and find out what the truth is. It is known from the literature and from the experience of breeders that triticale is a partially open-pollinated plant, but only at a short distance from pollinator. The exact rates of different genotypes’ of triticale outcrossing under field conditions are not known and should be examined. Here we present a comparison of results of two field experiments performed on pollen and gene flow. Transgenic triticale plants expressing the uidA and the bar gene were used to estimate the outcrossing rates between genetically modified and conventional genotypes. Histochemical detection of β-glucoronidase is a very precise and convenient test for quantification of gene and pollen flow and a perfect model for coexistence studies. In our studies we applied histochemical detection on a large scale. Here we present a comparison of gene flow and pollen flow examination that was performed in Polish climatic conditions. While single pollen grains were found at a distance of 85 m, the outcrossing, albeit at a very low level of 0,03%, was reported at a maximum distance of 28 – 30 m, where no physical barrier was used and at 10 – 11 m, where non GM buffer planting was applied. The established method can be used for general studies of plant crossability and pollen flow.

116

LIST OF PARTICIPANTS

117

Surname, name e-mail address abstract on page nr.

Abrouk Michaël [email protected] 18

Bacic Tony [email protected] 28

Bazgier Václav [email protected] 53

Berger Jens [email protected] 44

Bilyeu Kristin D. [email protected] 14

Burešová Veronika [email protected] 54

Burger Anna [email protected] 55

Cartes Paula [email protected] 56

Clark Brian F.C. [email protected]

Cviková Kateřina [email protected] 58

Czaplicki Andrzej [email protected] 59

Čížková Jana [email protected] 57

Deepak Kumar [email protected] 62

Doležal Karel [email protected] 20

Doležalová Ivana [email protected] 63

Doležel Jaroslav [email protected] 15

Doskočilová Anna [email protected] 64

Dušek Karel [email protected] 65

Endo Takashi [email protected] 17

Escobar Luengo Ana Luisa [email protected] 66

Fiorani Fabio [email protected] 25

Frébort Ivo [email protected] 16

Gallová Lucia [email protected] 67

Gaude Thierry [email protected] 40

Gleba Yuri [email protected] 30

Gregorová Zuzana [email protected] 68

Hanosová Helena [email protected] 22

Havlíčková Lenka [email protected] 69

Havránková Miroslava [email protected] 70

Hirt Heribert [email protected] 33

Hluska Tomáš [email protected] 71

Hobza Roman [email protected] 37

Hückelhoven Ralf [email protected] 27

Hýbl Miroslav [email protected] 72

Chika Nwachukwu Emmanuel [email protected] 60

Chikkaputtaiah Channakeshavaiah [email protected] 61

Ilík Petr [email protected] 73

Inostroza-Blancheteau Claudio [email protected] 74

Ivaničová Zuzana [email protected] 75

Jelínková Marcela [email protected] 76

Jeřábková Hana [email protected] 77

mailto:[email protected]







































118


Jiskrová Eva [email protected] 78

Káš Jan [email protected]

Klimešová Jana [email protected] 79

Klocová Barbora [email protected] 80

Komis Georgios [email protected] 81

Košútová Petra [email protected] 82

Kouřil Roman [email protected] 49

Kuznetsova Kristina [email protected] 83

Lazár Dušan [email protected] 84

Lawrenz Evelyn [email protected] 47

Lichtscheidl Irene [email protected] 41

Luptovčiak Ivan [email protected] 85

Melis Anastasios [email protected] 43

Mészáros Patrik [email protected] 86

Motte Hans [email protected] 21

Nauš Jan [email protected] 87

Noceda Carlos [email protected] 88

Nosek Lukáš [email protected] 89

Ohnoutková Ludmila [email protected] 90

Ortiz Rodomiro [email protected] 13

Ovečka Miroslav [email protected] 91 Öz Tufan [email protected] 29

Pavlovič Andrej [email protected] 51

Pechan Tibor [email protected] 36

Pérez- Alfocea Francisco [email protected] 19

Peč Jaroslav [email protected] 92

Petrželová Irena [email protected] 93

Plíhal Ondřej [email protected] 94

Poltronieri Palmiro [email protected] 39

Pospíšilová Hana [email protected] 95

Pozueta-Romero Javier [email protected] 48

Prasad Ankush [email protected] 96

Prášil Ilja [email protected] 97

Reyes Díaz Marjorie [email protected] 98

Ribera Fonseca Alejandra [email protected] 99

Smékalová Veronika [email protected] 102

Staňková Helena [email protected] 104

Stavělíková Helena [email protected] 105

Stočes Štěpán [email protected] 106

Šamaj Jozef [email protected] 42

Šamajová Olga [email protected] 100




















mailto:Miroslav%[email protected]





















119


Šebela Marek [email protected] 35

Šmehilová Mária [email protected] 101

Špundová Martina [email protected] 103

Takáč Tomáš [email protected] 107

Tanizawa Katsuyuki [email protected] 26

Tarkowski Petr [email protected] 108

Tomaštíková Eva [email protected] 109

Travella Silvia [email protected] 24

Trtílek Martin [email protected] 31

Tüyel Umut [email protected] 45

Ulloa Elizabeth [email protected] 110

Van Montagu Marc [email protected] 10

Verma Sandeep Kumar [email protected] 50

Vershinina Zilya Rifovna [email protected] 111

Vítámvás Pavel [email protected] 38

Vlčko Tomáš [email protected] 112

Vrabka Josef [email protected] 113

Wasternack Claus [email protected] 34

Zalabák David [email protected] 114

Zimny Janusz [email protected] 115





















120

NOTES

121

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NOTES

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NOTES

125

Olomouc Biotech 2013 – Plant Biotechnology: Green for Good II

Book of Abstracts, Olomouc, June 17 – 21, 2013

Editors: Eva Tomaštíková & Karolina Chvátalová

Publisher: Centre of the Region Haná for the Biotechnological and Agricultural

Research

Printed at: Tiskárna TWIN, Olomouc, Czech Republic

Olomouc 2013

1st edition, impression 150 pcs.

Not for sale

126

This Book of abstracts has been funded by the Operational Programme Research

and development for Innovation, grant nr. ED0007/01/01.

olomouc biotech 2013 plant biotechnology: green for good ii

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