Following Text Represents The Selection Of Materials Compiled

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IRP identification code AV0Z50380511 Following text represents the selection of materials compiled for the international evaluation of the Institute of Experimental Botany AS CR in 2004. 1 IRP identification code AV0Z50380511 Institutional Research Plan (IRP) proposal Provider‘s code IRP identification code Research plan title Applicant 1 Institution 2 Principal investigator 3 A A1. AV0 Z50380511 Mechanisms of regulation of plant growth and development on the level of cells, organs and whole organisms: physiological, genetic and molecular bases Institute of Experimental Botany AS CR RNDr. Ivana Macháčková, CSc. General information on the applicant Organisational scheme of the applicant, including the number of employees in the applicant’s units Governing bodies of the apllicant are listed in part A2. Number of employees at the IEB (including part-time jobs): in total 196 employees (consisting of 63 researchers, 34 research assistants, 42 technicians, and 57 other employees). IEB is located in six centres in Prague and Olomouc. There are 16 laboratories in IEB (names and heads of the laboratories are listed below). CENTRE 1: Rozvojová 135 Lysolaje 165 00 Prague 6 RNDr. Radomíra Vaňková, CSc.  (420) 220 390 427 fax: (420) 220 390 446 e-mail: [email protected] RNDr. Ivana Macháčková, CSc. Laboratory of Plant Morphogenesis (since 2004 RNDr. Jan Martinec, CSc.)  (420) 220 390 453 fax: (420) 220 390 456 e-mail: [email protected] Ing. Miroslav Kamínek, CSc. Laboratory of Hormonal Regulations in Plants (since 2004 RNDr. Eva Zažímalová, CSc.)  (420) 220 390 445 fax: (420) 220 390 446 e-mail: [email protected] RNDr. Milena Cvikrová  (420) 220 390 409 fax: (420) 220 390 419 Laboratory of Biologically Active Compounds 1 2 3 Legal name of the applying organisation, legal entity Name of the department or applicant’s principal organisational unit, which will carry out the research according to the proposal, if different from “applicant”. Applies only if more than one proposal of IRP was submitted by the “applicant”. Person in charge, who is responsible, on behalf of the applicant/institution, in scientific and financial matters of IRP 2 IRP identification code AV0Z50380511 e-mail: [email protected] RNDr. Věra Čapková, CSc.  (420) 220 390 452 fax: (420) 220 390 461 e-mail: [email protected] RNDr. Viktor Žárský, CSc.  (420) 220 390 457, 220 390 458 fax: (420) 220 390 461 e-mail: [email protected] Laboratory of Pollen Biology Laboratory of Cell Biology CENTRE 2: Na Karlovce 1a Dejvice 160 00 Prague 6 RNDr. Milada Šindelářová, CSc.  secretariat: (420) 224 310 108 fax: (420) 224 310 113 e-mail: [email protected] RNDr. Milada Šindelářová, CSc. : (420) 224 310 109 fax: (420) 224 310 113 e-mail: [email protected] RNDr. Karel J. Angelis, CSc.  (420) 224 322 603 fax: (420) 224 310 113 e-mail: [email protected] RNDr. Jana Pospíšilová, CSc.  (420) 224 320 198, (420) 233 331 032 fax: (420) 224 310 113 e-mail: [email protected] RNDr. Tomáš Gichner, DrSc.  (420) 224 310 109 fax: (420) 224 310 113 e-mail: [email protected] RNDr. Noemi Čeřovská, CSc.  (420) 224 320 338 fax: (420) 224 310 113 e-mail: [email protected] Ing. Jaroslav Tupý, DrSc. Laboratory of Pathological Plant Physiology Laboratory of DNA Repair Laboratory of Stress Physiology Laboratory of Mutational Genetics Laboratory of Virology CENTRE 3: Na Perníkářce 15 Dejvice 160 00 Prague 6 (since 2005 RNDr. Miloslav Juříček, CSc.)  (420) 233 336 791 fax: (420) 233 339 412 e-mail: [email protected] Ing. Jaroslav Tupý, DrSc. Laboratory of Pollen Embryogenesis (since 2005 RNDr. Miloslav Juříček, CSc.)  (420) 233 336 791 fax: (420) 233 339 412 e-mail: [email protected] Ing. Jaroslav Tupý, DrSc.  (420) 485 177 430 fax: (420) 233 339 412 Experimental Station Střížovice 3 IRP identification code AV0Z50380511 e-mail: [email protected] Josef Holík  (420) 241 032 381, (420) 241 062 728 fax: (420) 241 062 150 e-mail: [email protected] Josef Holík  (420) 241 032 381, (420) 241 062 728 fax: (420) 241 062 150 e-mail: [email protected] CENTRE 4: Vídeňská 1083 Krč 142 00 Prague 4 Isotope Laboratory CENTRE 5: Sokolovská 6 772 00 Olomouc Doc. Ing. Jaroslav Doležel, DrSc.  secretariat: (420) 585 228 521-2 fax: (420) 585 228 523 e-mail: [email protected] Doc. Ing. Jaroslav Doležel, DrSc.  secretariat: (420) 585 228 521-2 fax: (420) 585 228 523 e-mail: [email protected] Doc. Dr. Jiří Vagera, CSc.  secretariat: (420) 585 228 521-2 fax: (420) 585 228 523 e-mail: [email protected] Laboratory of Molecular Cytogenetics and Cytometry Laboratory of Genetic Manipulations in vitro Laboratory of Plant Cytoskeleton and Cell Cycle Doc. RNDr. Pavla Binarová, CSc.  (420) 585 228 521-2, (420) 241 062 130 fax: (420) 585 228 523 e-mail: [email protected], [email protected] CENTRE 6: Joint Laboratory of IEB ASCR and Faculty of Life Sciences Palacký University in Olomouc Šlechtitelů 11 783 71 Olomouc Prof. Ing. Miroslav Strnad, CSc.  (420) 585 634 850 sekretariat: (420) 585 634 851 fax: (420) 585 634 870 e-mail: [email protected] Laboratory of Growth Regulators Prof. Ing. Miroslav Strnad, CSc.  (420) 585 634 850 secretariat: (420) 585 634 851 fax: (420) 585 634 870 e-mail: [email protected] 4 IRP identification code AV0Z50380511 A2. Governing bodies of the applicant and names of their personnel DIRECTOR: Rozvojová 135 Lysolaje 165 00 Prague 6 RNDr. Ivana Macháčková, CSc.  (4202) 220 390 453  - secretariat (4202) 220 390 455 fax: (4202) 220 390 456 e-mail: [email protected] DEPUTY DIRECTOR: Rozvojová 135 Lysolaje 165 00 Prague 6 RNDr. Eva Zažímalová, CSc.  (4202) 220 390 429 fax: (4202) 220 390 446 e-mail: [email protected] SCIENTIFIC SECRETARY: Rozvojová 135 Lysolaje 165 00 Prague 6 RNDr. Martin Vágner, CSc.  (4202) 220 390 414 fax: (4202) 220 390 419 e-mail: [email protected] CHAIRMAN OF THE SCIENTIFIC BOARD: Rozvojová 135 Lysolaje 165 00 Prague 6 RNDr. Jan Martinec, CSc.  (4202) 220 390 416 fax: (4202) 220 390 419 e-mail: [email protected] MEMBERS OF SCIENTIFIC BOARD: RNDr. Lenka Burketová, CSc. RNDr. Noemi Čeřovská,CSc. RNDr. Věra Cenklová, PhD. RNDr. Věra Čapková, CSc. Mgr. Lucie Perry, PhD. RNDr. Radomíra Vaňková, CSc. ADMINISTRATION: Rozvojová 135 Lysolaje 165 00 Prague 6 Naděžda Pulcová  (4202) 220 390 475 fax: (4202) 220 390 474 e-mail: [email protected] 5 IRP identification code AV0Z50380511 B B1. Information on research and development activities of the applicant/institution Specification of the principal research and development (R&D) activity of the applicant/institution The aim of setting up this working site is to perform the scientific research in the field of experimental botany and searching for possibilities of exploitation of its results. The subject of business of IEB AS CR is the scientific research in the field of plant physiology, genetics, plant biotechnologies, biochemistry and molecular biology with the focus on the regulation of growth and development, physiology of photosynthesis, adaptive mechanisms against stress induced by the surroundings and pathogenic agents, molecular genetics of pollinic and somatic cells and mechanisms of mutagenesis and synthesis of marked bioactive substances. Further activities include the participation in programs of development of plant physiology, genetics and biotechnologies directed towards generation of new genetic sources, improvement of new genotypes resistant against biotic and abiotic factors, reproducing of plant material in vitro and its application in cultivation, as well as development of new silvicultural technologies in agriculture and horticulture. Within the scope of its subject of business, the Institute contributes to increasing the level of knowledge and understanding, exploiting the results of research activities and its promotion. The Institute receives, processes and spreads the scientific information by publishing magazines, textbooks, monographies and regular reviews of its research activities. The Institute provides scientific references, standpoints, recommendations and searches. In co-operation with universities, the institute organizes post-graduate studies and raises scientists. The Institute develops international co-operation projects and, in the scope of the subjected activities, organizes scientific conferences, symposia and seminars in the Czech Republic, with international participation. The Institute obtains national and international grants as well as other forms of support of its scientific activities. In order to exploit the results of scientific research together with having the right of their registration and maintaining of patent protection locally and abroad, the Institute administers the property rights to the patents originated in its working site and enters into license agreements with local and foreign subjects. The Institute executes its projects in co-operation with other scientific and professional institutions. A comparison of this Foundation document with the description of the results gained in the Institute of Experimental Botany (IEB) in years 1999-2003 and the research proposal for years 2005-2010 clearly shows that IEB fulfills its research and other tasks. IEB is active in research in the fields of plant physiology, genetics, biochemistry and molecular biology, namely regulation of plant growth and development (metabolism, transport and mechanisms of action of phytohormones and other regulatory substances), regulation of cell cycle, signal transduction, stress physiology, physiology of photosynthesis and interaction with pathogens, DNA damage and repair and mutagenesis, gene mapping and functional genomics, chromosome sorting and use of chromosomes in modern genomics. IEB participates in the programmes preparing new materials for cereal breeding gained by in vitro manipulations or transgenosis, and introduces new apple tree varieties resistant to scab. Researchers in IEB have also started research in the field of potential production of pharmaceutically important proteins in transgenic plants and they have developed new substances on the basis of the structure of phytohormones cytokinins, which inhibit cell division and are tested as medicaments against proliferative diseases. The Izotope Laboratory of IEB synthetises many substances for research, in which colleagues abroad are also interested. 6 IRP identification code AV0Z50380511 The researchers in IEB publish their research results in scientific journals (see the list, pp. 71-102 ) and also help the popularisation of these results and science as such (part 4.3). IEB issues two international journals: Biologia Plantarum and Photosynthetica. Researchers of IEB are also authors or coauthors of a number of textbooks and scripta (part 4.3) and many of them actively participate in lecturing at several universities and supervise the diploma and doctoral theses (part 4.1). IEB has actively developed and supported international collaboration as may be seen from the list of workers from abroad who have worked in the IEB for long periods (see part B4) and from the list of publications (some of them come from collaboration) and grant projects. IEB has organised several symposia and taken part in the organisation of some others. IEB is the representative of Czech plant biologists in the EPSO - European Plant Science Organisation. IEB has several patents and many licences, the list of which may be found in D4, pp. 101102. B2. Contribution of the applicant/institution to the development of knowledge in the disciplines referred to in B1, in national and international context The Institute of Experimental Botany (IEB) is the only institution in the Czech Republic (CR) whose research covers a wide area of plant physiology and genetics and interconnects and integrates these two areas. Similar in orientation is the Institute of Plant Molecular Biology (IPMB) in České Budějovice, but its research in plant physiology is limited to photosynthesis and phytopathology. Earlier the research in the individual laboratories of the IEB was relatively independent, but in the last 10 years we have interconnected and integrated the research of individual teams and different teams participate in the work on common projects. Collaboration with the Universities has also been successfully developed (with Departments of Botany and Genetics at the Palacky University in Olomouc - common "Laboratory of Plant Growth Regulators", the Department of Plant Physiology at Charles University in Prague common laboratory is being prepared, the Institute of Biochemistry and Microbiology at the Institute of Chemical Technology in Prague, the Institute of Botany and Plant Physiology at the Mendel University of Agriculture and Forestry in Brno and the Department of Botany at the Czech University of Agriculture in Prague) as well as collaboration with colleagues abroad. The workers of the IEB take an active part in teaching at the Department of Plant Physiology in Prague and the Departments of Botany and Genetics, respectively, in Olomouc. The researchers of the IEB supervised and supervise a number of diploma and doctoral theses (in the frame of official acreditations or agreements with universities). In the last 5 years we have integrated our research in the field of studies of signal transduction pathways in plants, in which the IEB is the coordinator of a prestige Research Centre, which integrates the best laboratories in the Czech Republic in this field of research. During this period the number of foreign researchers working in the IEB increased - they were colleagues from the Belorussia, Bulgaria, France, Great Britain, Guatemala, Japan, India, Italy, Russia, Serbia, Ukraine and USA, Poland. The Institute has a good tradition in organising international symposia on auxins and cytokinins - the last one was in Prague in 1999. We also participated in the organisation of the 17th Conference of the International Plant Growth Substances Association (IPGSA) in 2001 in Brno. The Institute also produces two major scientific journals: Biologia Plantarum and Photosynthetica. 7 IRP identification code AV0Z50380511 The level of research performed in the IEB is high and the IEB has a leading position within the CR in research on phytohormones and other regulatory substances, signal transduction pathways, pollen biology, cytogenetics, cell cycle and DNA repair. The IEB together with the IPMB were the first in the Czech Republic to study and evaluate the possibility of producing pharmaceutically important proteins in transgenic plants (edible vaccines etc.). The Laboratory of Genetic Manipulations in vitro contributed significantly, with its haploid and other plant materials, to the breeding of new varieties of cereals and grasses. At the Research Station in Střížovice new apple tree varieties resistant to scab were bred - licenses for these varieties are now requested all over the world. The number of publications continues to increase as does their quality and impact (see figure bellow). Several research areas in the IEB also have a high level in an international context fully comparable with that of other European Institutions: The Laboratory of Growth Regulators (Palacky University and IEB) together with the Isotope Laboratory of the IEB have developed groups of substances based on the structure of phytohormones of the cytokinin type, which inhibit the cell cycle and in collaboration with hospitals in CR and abroad were tested as potential medicaments against cancer and other proliferative diseases. These substances are patented and the Institute has a number of license agreements for testing and potential use. This research has received several international awards. Phytohormone research has a long tradition and a high level, particularly research on auxins and cytokinins. The Institute is known as a centre for analytical methods for the determination of phytohormones and some other regulatory substances and for studies on hormone metabolism and transport. In recent years the Laboratory of Molecular Cytogenetics and Cytometry was very successful - they managed to localise some sequences on sorted chromosomes and for the first time described isolation of pure high-molecular weight DNA from sorted chromosomes. Changes in the structure of the cytoskeleton during the cell cyle and division of plant cells and function of some regulatory proteins were also described. In collaboration with the University of Leicester (UK) gene expression at the level of transcriptome in one developing cell (pollen tube) was described for the first time. In collaboration with the Department of Plant Physiology (Charles University, Prague) first indications of the occurrence of an Exocyst complex in plants were found and its possible role in cell morphogenesis proposed. In research on the phosphoinositide signalling system a method of in situ determination of activities of different types of phospholipases was elaborated and a new type of phospholipase cleaving phosphatidylcholine previously unknown in plants was described. The regulatory role of excretion of chloride ions was shown in pollen tube growth and a new model for this regulation proposed. The occurrence of the hormone melatonin was shown in higher plants and a previously unknown rhythm in its level, which depends on the photoperiod was described. The Comet assay was elaborated for studies of DNA damage and is being used in the study of DNA repair and the effect of mutagens. Our research on the role of polyamines in cell division, studies of the role of various signals and PR (pathogenesis related) proteins in the interaction of plants with pathogens as well as the molecular characterisation of plant viruses also has a good European standard. A significant contribution to the research level is made by the Isotope Laboratory of IEB, where they synthetise compounds which inhibit the cell cycle mentioned above as well as substances, both radioactive and non-radioactive, necessary for the research of other teams, which either cannot be bought or are very expensive. A number of foreign colleagues are interested in these substances. The priority results described obtained in the last 5 years document a high quality of research performed in IEB, both at local and international levels. Specificity of the research in IEB resides mainly in the areas of phytohormones and signalling pathways (and their integration), studies of the genome at the level of chromosomes, pollen biology and DNA repair. In these 8 IRP identification code AV0Z50380511 areas the research in IEB is the leading one in the CR. When compared with other European institutions, the quality of research in IEB is good and it contributes to and complements European research. The equipment at IEB for research in molecular biology, biochemistry, cytology and physiology of plants is at a standard European level. Many institutions concentrate in narrow research fields; the specifity of IEB is integration of several research areas of plant biology in one moderate-sized institution and make them complementary. Scientific publications of IEB * impacted papers average impact factor 60 2.2 2.0 50 1.8 40 30 1.6 20 average impact factor number of papers in impacted journals 70 1.4 10 1.2 0 1995 * 1996 1997 1998 1999 2000 2001 2002 2003 year only papers published until November 2003 (currently at least 14 other papers are "in press", some of them will be printed in 2003) 9 IRP identification code AV0Z50380511 B3. Major R&D results achieved and implemented by the applicant/institution in the disciplines referred to in B1 within the last five years (overall characteristics)4 Main results of IEB AS CR 1999 - 2003 The Research Programme of the Institute of Experimental Botany (IEB) for the period 1999 2003 was Physiological and Genetic Basis of Plant Development, Cell Cycle, Morphogenesis, Reactions to Stress Conditions and Biotechnologies. Organisation and Function of the Genome (No.: AV0Z5038910). The programme was aimed at elucidating the basic molecular and cellular mechanisms in the following topics: • The integrated action of phytohormones and growth regulators in plant development control; • The regulation of cell morphogenesis and the role of cytoskeleton and relevant signalling pathways; • The regulation of the cell cycle and the use of synthetic regulators for its inhibition and the inhibition of tumour growth; • The regulation of ontogenesis of the male gametophyte: gene expression, proteosynthesis and signalling; somatic and pollen embryogenesis, anther cultures, haploid production; • Genome organisation and function at the chromosome level; • The detection of regulatory genes, dynamics of damage and repair of genomic DNA under the influence of stress and mutagens; • The factors affecting photosynthesis and water relations in stress conditions; • The reaction of plants to infection and molecular characteristics of viruses. This research has resulted in an improved understanding of the above topics and their biotechnological applications. The tasks of the programme imply that the IEB covers a relatively broad field of research in the physiology and genetics of plants. The main aim was to integrate the individual parts of the programme and the grant proposals were composed and the projects run accordingly. One of the significant steps in the integration of this research was the award to the Research Centre of the project „Signalling Pathways in Plants“ by the Ministry of Education, Youth and Sports of the Czech Republic, No.: LN00A081), co-ordinated by IEB. Some of the results reported in the area of signalling were performed within the framework of this project and are included marked with sign ◘ at the end of the paragraph within this report. The research running in the IEB during the period 1999-2003 in the frame of the Research Programme, the Research Centre and grant projects, can be divided into two main parts: • The regulation of plant growth and development • The structure and function of the genome. In the area of „regulation of plant growth and development“ research was focussed on growth regulators (namely phytohormones: auxins and cytokinins and, to a lesser extent, ethylene and abscisic acid). Some aspects of metabolism, transport and mechanism of action of phytohormones and polyamines were described and characterised. In this area an important achievement was reached in the investigation of auxin carriers and their regulation, and in the characterisation of the role of cytokinin oxidase in the regulation of the level of active cytokinins in cells and tissues. The derivatives of N6-substituted adenine were prepared and 4 Implemented results are those R&D results which have been published, applied in practice and/or protected as an intellectual property according to a specific law (e.g. publications, patents, trademarks, and newly applied technologies). 10 IRP identification code AV0Z50380511 tested as inhibitors of key reactions of the cell cycle and as a medicine against serious diseases. One of the compounds designed, prepared and characterised in the IEB is now in the second phase of clinical tests as a potential drug against cancer in several European countries. Attention has also been aimed towards the participation of plant growth regulators in the stress reactions of plants to high levels of radiation, high and low temperatures, water deficits and some viruses. Attention was also focussed on the investigation of signalling pathways and their coupling with cell structures (cytoskeleton) and of cell cycle control. A new type of plant phosphatidylcholine-hydrolysing phospholipase C was characterised and the complex Exocyst (previously unknown in plant cells) was identified. The distribution of gamma-tubulin and F-actin was described during mitosis, when centrioles were not formed and cyclin B2 expression was monitored in the course of cell division. In the area of genomics, the investigation was focussed on: • Characterisation of the genome structure and its variability in some plant species, • Localisation of some sequences and molecular markers using sorted chromosomes, • The preparation of high-molecular weight DNA from sorted chromosomes and chromosome-specific DNA-libraries. In the male gametophyte the expression of genes and regulation of transcription and translation were investigated. In the male gametophyte, the expression of genes and regulation of transcription and translation were investigated; it was the first time when the expression has been analysed on the level of transcriptom in the range of the whole genome of a single developing cell. The expression of genes encoding proteins involved in the regulation of cell cycle was described, and the mechanisms of DNA repair and plant viruses were characterised. Transgenosis has been characterised in relation to the plant protection and for the food industry (potato) and for crop improvement (cereals). Preparation of transgenic plants with controlled expression of genes related to metabolism of cytokinins was started with the aim of increasing longevity and productivity. Research work was also concentrated on androgenesis in vitro and haploid and dihaploid plants were prepared for breeding. Varieties of apple tree resistant to fungal diseases were prepared and an investigation of the possibilities of production of bioactive proteins in plants started. In the following text, a more detailed description of results achieved is divided into two main research areas and these are sub-divided into smaller thematic parts for better understanding. Numbers in parentheses relate to the papers published, as listed on page 71102. List of contents: 1 REGULATION OF PLANT GROWTH AND DEVELOPMENT 1.1 Plant growth regulators 1.1.1 Metabolism and mechanism of action of phytohormones 1.1.2 Transport of auxins and cytokinins 1.1.3 Hormonal regulation of some life processes in plants 1.1.4 Other plant growth regulators 1.1.5 The role of plant growth regulators in plant stress reactions 1.1.6 New methods in the investigation of phytohormones 1.1.7 Potential practical applications of research on plant growth regulators 1.2 Signalling and signal transduction 1.2.1 Signalling mechanisms 1.2.2 The relationship between signalling and the cytoskeleton 1.2.3 Signalling in plant defence mechanisms 11 IRP identification code AV0Z50380511 2 STRUCTURE AND FUNCTION OF THE GENOME 2.1 Functional genomics 2.2 Studies on genome structure using sorted chromosomes 2.3 Genotoxicity and DNA repair 2.4 Molecular aspects of plant virology 2.5 Potential practical applications of genetic research 1 1.1 REGULATION OF PLANT GROWTH AND DEVELOPMENT Plant growth regulators 1.1.1 Metabolism and mechanism of action of phytohormones Using a newly developed method of large-scale synchronisation of tobacco cell suspension (line BY-2), which allows synchronisation up to 30 g FW of cells, the dynamics of cytokinins and activity of cytokinin-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), was determined during the cell cycle progression. Results have confirmed a significant and transient accumulation of cytokinins at the beginning of the S-phase and mitosis and brought new knowledge about fast metabolic regulation of levels of physiologically active cytokinins. It is based on their conversion to storage cytokinins of cis-zeatin and zeatin-O-glucoside type in the premitotic phase of the cell cycle and on the degradation of isoprenoid cytokinins by CKX at the beginning of the S-phase (229). Novel N6-substituted derivatives of adenine were isolated and identified: In the photoautotrophic cell culture of Chenopodium rubrum 6-[2-(β-D-glucopyranosyloxy)benzylamino]purine (oTOG), 6-[2-(β-D-glucopyranosyloxy)benzylamino]-2-methylthiopurine (2MeS-oTOG) a 6-benzylamino-9-β-D-glucopyranosylpurine (BAP9G) were found. Their cytokinin activity was confirmed in the Amaranthus bioassay and their endogenous origin was proven using a novel method based on the incorporation of deuterium in situ from the cultivation medium enriched by D2O into the compounds studied. The appearance of a range of other new cytokinins, e.g. zeatin-9-glucoside-O-glucoside, and aromatic cytokinins was detected in other species (224, 230, 353, 375). For the first time the new group of naturally occurring plant hormones structurally derived from 6-(2- a 3methoxybenzylamino)purine was isolated. The identification was carried out in A. thaliana, poplar leaves, and selected strains of A. tumefaciens using LC-MS. The high biological activity of the compounds isolated was confirmed in three cytokinin biotests (364). In co-operation with laboratory of Prof. Schmülling (Berlin, Germany) the effects of expression of four different genes encoding the cytokinin oxidase/dehydrogenase (CKX) from Arabidopsis thaliana on the development of transgenic tobacco plants were determined. A high increase in CKX activity resulted in a significant lowering of the cytokinin content and, consecutively, to pronounced changes of the phenotype of transformed plants. Cytokinindeficient plants were characterised by dwarf shoots with smaller apical meristems and prolonged plastochron. In contrast, root meristems of transgenic plants were bigger; roots were more branched and grew more intensively. This finding represents the first direct experimental evidence of the physiological relevance of endogenous cytokinins in the control of morphogenesis and meristematic activity in plants based on down-regulation of cytokinin levels (195). The dependence of CKX activity on the level of endogenous cytokinins was found in a cytokinin-autonomous line of A. thaliana, which thus represents a promising model for studies of CKX induction by its substrates (89). The regulatory effects of cytokinins on CKX activity were proved in tobacco cell suspension where increases in cytokinin levels following either the application of exogenous cytokinin or the expression of IPT gene, encoding enzyme for cytokinin biosynthesis, resulted in a significant increase in the activity of CKX, namely of its glycosylated form, and its preferential secretion outside the plasma membrane (336). 12 IRP identification code AV0Z50380511 By a combination of affinity chromatography with immobilised zeatin and liquid chromatography with mass spectrometry detection, the adenosine kinase was isolated from suspension culture of the tobacco cell line BY-2 and characterised (327). Adenosine kinase is one of the enzymes responsible for the formation of cyclic nucleotides in cells. This is the first report of this enzyme in plants and it shows the significance of this enzyme in the metabolism of cytokinins. The existence of the isopentenyl adenosine-5´-monophosphate- (iPMP)-independent biosynthetic pathway for zeatin-type cytokinins was proven (82). This pathway was active in both IPT-transgenic A. thaliana and wild type plants. The investigation of cytokinin biosynthesis de novo in the IPT-transgenic A. thaliana using labelling with deuterium in vivo and mass spectrometry showed that the rate of biosynthesis of zeatin riboside-5’monophosphate was ca. 66-times higher than that for iPMP, which was supposed to be the primary product of isopentenyl transferase from A. tumefaciens. By the double-labelling method, using [2H6]-isopentenyl adenosine and deuterium oxide, the existence of an alternative, iPMP-independent biosynthetic pathway for zeatin and its derivatives was discovered. This pathway operates in both IPT-gene-expressing and wild type plants. A decrease in the activity of the alternative biosynthetic pathway after the application of mevastatin, inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, indicates the terpenoid origin of the side-chain precursor for this iPMP-independent pathway. A second gene encoding cis-zeatin specific O-glucosyltransferase (cisZOG2) was isolated from maize in co-operation with the laboratory of Profs Moks (Corvallis, Oregon, U.S.A.). The gene is significantly expressed in roots and, in contrast to cisZOG1, its expression in kernels is very low. Interestingly, high levels of zeatin riboside O-glucoside were found in kernels using HPLC/MS. Results confirm the existence of specific metabolic pathways regulating levels of cis-zeatin type cytokinins in plants (373). Chloroplasts from tobacco plants (SR1) carrying the gene for β-glucosidase Zm-p60.1 under the CaMV35S promoter (kindly provided by Dr. B. Brzobohatý, Institute of Biophysics, Brno) contained almost unmeasurable levels of cytokinin O-glucosides (5). This implies that the active product of the gene Zm-p60.1 is present in chloroplasts. Chloroplasts from tobacco plants carrying the gene for isopentenyltransferase (IPT) under the promoter of the small subunit of RUBISCO from pea (Pssu) (kindly provided by Dr. Valcke, Diepenbeck, Belgium) had significantly higher cytokinin content than those from control plants. As the Pssu promoter is a nuclear one, it is probable that cytokinins are transported to chloroplasts from the cytoplasm. Chloroplasts from tobacco plants carrying the gene for cytokinin oxidase from Arabidopsis (AtCKX3) under the CaMV35S promoter (received from Prof. T. Schmülling, Berlin, Germany) had lower cytokinin content than those from control plants. For the investigation of cytokinin biosynthesis in chloroplasts, these were immobilised into alginate gel to prolong their longevity at the temperature necessary for metabolic studies (25oC). Immobilisation prolonged the longevity of chloroplasts but, on the other hand, decreased the rate of metabolism, due to diffusion through the gel. Therefore, the usage of immobilised chloroplasts had not proved to be advantageous for metabolic studies. ◘ Cyclin-dependent kinase inhibitors developed in IEB (roscovitine, olomoucine and bohemine) were proved to also inhibit N-glucosylation of cytokinins. They affected especially exogenously applied cytokinins in a species-specific manner, depending on the prevailing activity of individual cytokinin-down-regulating pathways (cytokinin oxidase/dehydrogenase or N-glucosylation) (379). Their short-term effect on the endogenous physiologically active cytokinins was balanced by the mechanisms involved in the maintenance of cytokinin homeostasis, especially by the stimulation of the formation of physiologically inactive ciszeatin derivatives (300, 379). The long-term effect resulted in cytokinin accumulation, however, only of the non-active forms. The growth of seedlings was significantly impaired. Cytokinin-binding proteins which exhibit high binding activity to aromatic cytokinins were isolated and characterised in oat and wheat grains. Although they have very similar molecular weight to native proteins and their subunits, they exhibit very different 13 IRP identification code AV0Z50380511 immunological properties. Their accumulation in maturating wheat grains correlates with an accumulation of aromatic cytokinins, including N6-(3-hydroxybenzyl)adenosine, which was identified in wheat grains using mass spectrometry. On the basis of this correlation and because of the release of proteins bound during germination the physiological function of these proteins was proposed: These binding proteins temporarily immobilise aromatic cytokinins during grain maturation and thus prevent their stimulatory effect on cell division and, consequently, the premature germination. In contrast, cell division during germination of mature grains is stimulated by the release of the bound aromatic cytokinins during degradation of the binding protein (322). 1. 1. 2 Transport of auxins and cytokinins Using model auxin-dependent and cytokinin-autotrophic tobacco cell lines BY-2 and VBI-0, the action of auxins and cytokinins was characterized from the point of view of regulation of their internal and external levels and their transport across the plasma membrane. It was proved that auxin in the cultivation medium functions as an “external mitogen”, which regulates the internal auxin level. The significance of the “fine-tuning” of the internal auxin level for standard progress of cell division was confirmed in studies of auxin carriers. Changes in the activity of the auxin efflux carrier in relation to the commencement of cell division were monitored. The studies of the mechanism of action of a specific inhibitor of the auxin efflux carrier (1-N-naphthylphthalamic acid, NPA) (Fig. 1) resulted in the proposition of a mechanism controlling orientation (polarity) of cell division, based on regulatory NPAbinding-protein controlled targetting of the auxin efflux carrier to a specific region of the plasma membrane (253). Kinetic studies of NPA effects on auxin transport out of the cells revealed much higher effectiveness of NPA than the inhibitors of vesicle-mediated protein traffic in inhibiting auxin efflux. Experimental data were also provided about the behaviour of cytoskeletal structures and endoplasmatic reticulum, arguing against the idea proposed earlier, that the action of NPA and other inhibitors of auxin efflux is more general and based on the impairing of vesicle-mediated protein traffic (344). Some data on the regulation of auxin action on plant development were summarised in the review (377), and data related to auxin transport in papers (394, 395, 401). On the same experimental material, the excretion of cytokinins from the cells into the cultivation medium within the growth cycle was also characterised. Cytokinins were excreted from cells to the cultivation medium in relation to their internal concentration during the whole subcultivation period. It was suggested that, besides metabolic reactions, transport of cytokinins across the plasma membrane may represent another mechanism involved in the regulation of internal cytokinin levels (252). Fig. 1: The effect of 1-N-naphthylphtalamic acid (NPA) on the phenotype of suspensioncultured VBI-0cells. a – cells grown in control medium, day 9; b – cells grown in control medium supplemented with NPA (final concentration 10 µM), day 9. Note abnormal cell division planes. Scale bars = 50 µm. Modified from Petrášek et al. (253). 14 IRP identification code AV0Z50380511 The hypothesis has been tested that more erect leaves in the modern maize hybrid 3394, than in the older variety 307, might be a cause of tolerance of the modern maize to neighbours and, ultimately, the high yield in dense planting. It was found that light controlled leaf angle development in maize via regulation of polar auxin transport and auxin sensitivity in leaf tissues (314). The tissue of a new hybrid differed more in sensitivity (number of receptors) than in affinity (of receptors) towards auxin and it was less sensitive to treatment with antiauxin p-chlorophenoxyisobutyric acid (PCIB). In the maize leaf, at the junction of the blade and the sheath, a specialised structure is formed, called the auricle. The growth of the auricle is controlled by light, probably via polar auxin transport. The mutants with auxinbinding proteins affected differed significantly in the leaf angle. The results suggest the important role of both auxin-binding proteins and polar auxin transport in determining the leaf angle in maize (314). 1. 1. 3 Hormonal regulation of some life processes in plants The optimisation of cultivation protocols and image analysis was a prerequisite for the investigation of morphological characteristics and measurement of internal levels of endogenous phytohormones (IAA, cytokinins, ABA) during the process of somatic and zygotic embryogenesis of Picea abies (36, 76, 399). From the point of view of hormonal changes, the courses of both somatic and zygotic embryogenesis were very much the same and characteristic alterations of hormonal levels occurred in the same phases of embryo ontogenesis. The lower level of auxins and higher level of cytokinins was typical for the early phase of embryo development. During cotyledon and primary root establishment the level of IAA increased temporarily but remarkably. During embryo maturation and desiccation the level of endogenous cytokinins temporarily increased. Somatic and zygotic embryos differed in the relative proportion of individual cytokinins and in the total endogenous cytokinin content, which is one order of magnitude higher in zygotic embryos. These differences disappeared after germination. The dynamics of alterations in hormonal levels implies an important regulatory role of hormones in the process of embryogenesis. Expression of the homologue of the gene ABI3 (abscisic acid insensitive from A. thaliana) was followed in both embryogenic and non-embryogenic lines of Norway spruce differing in their embryogenic capacity. In the embryogenic lines the ABI3 homologue was expressed early in the proliferative phase and its expression increased during maturation, while in the non-embryogenic lines its expression was almost not detectable during all phases of development. Thus, the expression of ABI3 homologue is proportional to the embryogenic capacity of the line. The application of cytokinin N6-benzyladenine (BA) on bean plants increased the water use efficiency because of a higher stimulation of the photosynthetic rate than the transpiration rate (116, 183). The positive effect of BA was also observed during the rehydration of waterstressed bean plants. In sugar beet plants, N6-(m-hydroxy-benzyl)adenosine (HBA) was tested in addition to BA, however, HBA only rarely stimulated the gas exchange (179, 374). BA applied simultaneously with ABA was able to reverse stomatal closing induced by ABA. However, BA applied after ABA was not able to induce re-opening of stomata previously closed by ABA (347). During development of water stress, pre-treatment with ABA markedly decreased gas exchange parameters at the beginning of the experiment but, in its later phase, the effect was compensated by delay in development of water stress. Pretreatment with BA delayed development of water stress and increased photosynthetic rate in waterstressed leaves. At mild water stress BA also reversed the effect of ABA (347). The rate of photosynthesis decreased in cytokinin-overproducing transgenic tobacco plants (Pssu-IPT grafts of shoots on control roots and poorly rooted plants of F1 generation) by 20 - 50 % probably as a result of closed stomata. Activity of photosystem 2 was hardly affected in both transgenic types, nevertheless, in grafts, the activity of photosystem 1 was inhibited up to 70 % (37). Although the number of chloroplasts in cells did not change in transgenic 15 IRP identification code AV0Z50380511 plants, their ultrastructure was markedly changed. Beside distinctive peripheral reticulum and membrane-bound protein bodies of various densities, large crystalloids of lamellar structure were localised within chloroplasts (359). 3D-reconstruction of chloroplasts showed that up to 20 % of total plastid volume could be occupied by crystalloids. Transgenic tobacco plants were more resistant towards mild water stress, probably due to the increased activity of antioxidant enzyme systems (186). A spontaneous mutant of tomato 7B-1 was selected and physiologically characterised. It is a recessive mutant producing higher amounts of ABA, where germination and hypocotyl growth were resistant to mannitol and ABA. The mutant was also resistant to osmotic, salt and low-temperature stresses (160, 161) and showed blue-light-specific resistance to osmotic stress and abscisic acid (232). Therefore, the mutant 7B-1 is an excellent object for study of the role of light in plant responses to osmotic stress. 1. 1. 4 Other plant growth regulators The control of cell growth and differentiation is one of the possible functions of phenolic compounds in plants. Content and degree of methylation of phenolic substances were found to be the important factors for initiation of sessile oak somatic embryos and for their further development. A higher content of some cell-wall-bound phenolic acids could restrict cell expansion and, consequently, a normal development of somatic embryos (49, 305). Phenolic compounds could influence also the content of some phytohormones and polyamines. The application of the inhibitor of phenylpropanoid biosynthesis to alfalfa suspension cultures resulted not only in the marked decline in the content of phenolic compounds, but also in the decrease in the content of free IAA and IAA-oxidase activity (97). The formation of conjugates of phenolic acids with polyamines had a significant impact on the regulation of the levels of free polyamines in plant cells (304). An experimental system has been developed which enabled comparison of the endogenous levels of polyamines with cytological changes of alfalfa explants cultured on media inducing either (i) the intensive cell division and formation of proembryogenic structures or (ii) enlarged, highly vacuolated cells with limited cell division. Certain polyamines were shown to participate in certain developmental processes. High spermidine and spermine levels might be essential for the development of proembryogenic structures in dividing alfalfa explants, while higher content of putrescine was characteristic for elongating cells (11). Further experiments have also shown the involvement of polyamines in the initiation of sessile oak somatic embryos. The inhibition of phenylpropanoid biosynthesis in sessile oak embryogenic culture resulted in a decrease in the level of conjugated polyamines and in the increase of the content of their free forms. The results indicated an important role for spermidine in the initiation of somatic embryo formation (305). ◘ The dynamics of polyamine content was described in the course of the cell cycle in synchronous meristematic tissue of Vicia faba roots with the aim of confirming the direct participation of spermidine in the control of cell division. Polyamine content was determined in the individual phases of the cell cycle, characterised by flow cytometric analysis. Cells were released from hydroxyurea block in the G1-phase and progression to S-phase coincided with a decrease in the level of free putrescine and spermidine, while the G2-phase was characterised by an increase in free polyamines. The transient prolongation of the Sphase, i.e. the delay in DNA replication, was accompanied by an accumulation of free spermidine and soluble spermidine conjugates (304). The investigation of the dynamics of polyamine content during the growth cycle of tobacco BY-2 cell suspension culture resulted in the finding that the maximum in free spermidine content coincided with the onset of cell mitotic activity. The results obtained suggest the relationship between DNA replication and metabolism of spermidine and extend the knowledge about the role of polyamines in the regulation of plant growth. The accumulation of free polyamines (namely putrescine) induced by aphid feeding on winter wheat plants has also demonstrated the important role of polyamines in plant responses to 16 IRP identification code AV0Z50380511 biotic and abiotic stresses (94). Melatonin has been studied as another plant regulatory compound. This substance is known from animal cells as the regulator of rhythmical and photoperiodical processes. In plants it has been found that the rhythms in melatonin level in the above-ground parts of Chenopodium rubrum plants was dependent on photoperiod. The longer the dark period, the later the maximum of the rhythm occurred. Interestingly, in the case of C. rubrum it always appeared 6 h before the start of the photoperiod (196). The application of melatonin, its analogs and agonists, inhibited flower induction in C. rubrum when applied in concentrations higher than 10-5M 1 h before the start of the inductive darkness. Melatonin has had an inhibitory effect only when applied after the 6th h of the dark period and it did not change either the phase or the period of the rhythm of flowering (323). 1. 1. 5 The role of plant growth regulators in plant stress reactions High irradiation (700 µM m-2 s-1) brought about a decrease in the maximal efficiency of photosystem 2, in quantum yield and in photochemical quenching and stronger photoinhibition in tobacco plants cultivated in vitro in closed vessels in comparison with plants cultivated with a higher supply of CO2 in ventilated “Magenta” vessels. The positive effect of higher CO2 concentrations was obvious not only during the in vitro cultivation (22), but also after the transfer of plants into ex vitro conditions (31, 115). Increased CO2 concentration stimulated plant growth to a higher extent by increasing the velocity of photosynthesis more than by slightly increasing pigment content. At the same time there were increased activities of peroxidase, glucose-6-phosphate dehydrogenase and malic enzyme. Application of abscisic acid (ABA) immediately after the transfer ex vitro weakened the “transplantation shock” and probably decreased formation of active oxygen species. This resulted in a decrease of activities of glutathione reductase, Mn-superoxide dismutase, peroxidase, glucose-6-phosphate dehydrogenase and malic enzyme (263). However, wheat plants growing under the increased CO2 concentration had lower photosynthetic capacity and on the opposite side, plants growing under lower CO2 concentration had an increased photosynthetic capacity. These changes persisted even after return the of plants to natural CO2 concentration (128). The role of antioxidative enzymes was studied in the sensitivity to chilling of some inbred hybrid maize lines. Low temperature induced activities of glutathione reductase and ascorbate peroxidase and, at the same time, increased concentration of carotenoids. Chilling without preceding acclimatisation caused higher changes in enzyme activities than a gradual decrease of temperature. Photosynthetic capacity decreased during senescence (74). The nature of products of radical reactions in lipids did not change during ageing of bean cotyledons, while their amount rose significantly with age. Mass spectrometry showed that the C12 compounds were the most abundant. Young and old cotyledons differed in their composition. Oxidative damage of soluble proteins was studied using antibodies against carbonyl groups and it was found that this damage increased during ageing. Oxidatively damaged proteins were rapidly destroyed. Beside reactive oxygen species, reactive nitrogen species are active as well. The content of nitrotyrosine, determined by specific monoclonal antibodies, increased in old cotyledons. Both enzymatic and non-enzymatic antioxidative system was studied. Ratios of reduced and oxidised ascorbate and glutathione diminished during ageing. Similarly, activities of glutathion reductase, ascorbate peroxidase, SOD and catalase decreased as well as the capacity of the enzymatic and non-enzymatic systems (349). These events resulted in oxidative damage in the course of ageing. Phenolic acids play an important role in the reaction of plants to abiotic stress as was demonstrated in acclimation of soybean roots to low temperature. Low temperature in soybean plants brought about a decrease in the concentration of phenolic acids bound in the cell walls and an increase of free phenolic acids (99, 241). In spruce needles damaged by 17 IRP identification code AV0Z50380511 emissions increased lignification was found together with an increased concentration of conjugated phenolic acids and a lower concentration of phenolic acids bound to cell walls (119). 1. 1. 6 New methods in phytohormone research A new rapid method was developed for parallel extraction, separation and purification of phytohormones of the auxin type, abscisic acid and cytokinins, based on the use of new twoparameter sorbents with the functions of cation exchanger and reverse phase. The method substantially shortens the time needed for phytohormone analysis while at the same time increasing their recovery and enabling subsequent mass spectrometric determination (228). A new method was developed for immunolocalisation of cytokinins by means of antibodies against zeatin riboside and isopentenyladenosine (prepared in the laboratory of Prof. Strnad, IEB) and different fixation procedures for free bases, ribosides and ribotides, respectively. Specific detection is possible only in the case of free bases fixed by formaldehyde. ◘ A new sensitive ESI-MS (electrospray ionisation - mass spectrometry) method was elaborated which enables the use of a cheap and simple (in comparison with MS/MS techniques) mass detector with one quadrupole analyser for quantitative cytokinin analysis (337). Tritium-labelled cytokinins were prepared with very high molar radioactivity (higher than 1TBq/mmol), namely cis- and trans-zeatin, N6-isopentenyladenine, N6-benzyladenine, dihydrozeatin and their ribosides. For trans-zeatin a new synthesis was worked out. Two original syntheses of non-radioactive cis-zeatin were elaborated. The following standards, trans-zeatin-9-glucoside-O-glucoside, glucose esters of 3-indoleacetic and 2,4dichlorophenoxyacetic acids, as well as putrescine and spermidine amide conjugates of tyramic, p-coumaric and ferulic acids were prepared for HPLC/MS and metabolic studies. 1. 1. 7 Potential practical applications of research on plant growth regulators A new series of inhibitors of cyclin-dependent kinases (CDK) was developed as well as a system of modelling enabling targeting of the molecules of CDK inhibitors into the binding site for ATP in the molecules of CDK2 and CDK1. New compounds with high inhibitory activity to CDK1 were designed derived from purines, pyrazolo[4,3-d]pyrimidines and 8azapurines. The most active of these substances with IC5O 0.01µM for CDK2 was given the name olomoucine II (6-(2-hydroxybenzylamino)-2-{[1-(hydroxymethyl)propyl]amino}-9isopropyl purine) and it has the same inhibitory activity towards CDK as purvalanol, until now the most efficient inhibitor. However, olomoucin II is more easily synthesised, it is, as well, soluble under physiological conditions and more effective in the tests of cytotoxicity in tumour cell lines. The inhibitor of CDK of the first generation, roscovitine (6-benzylamino-2-[1-R(hydroxymethyl)propyl]amino-9-isopropyl purine) entered the second phase of clinical tests in several European countries. Among the synthesised compounds there are also substances that do not exhibit pronounced inhibitory activity towards CDK, but they show high cytotoxicity in tumour cell lines. Metabolism of one selected CDK inhibitor, bohemine (6benzylamino-2-[3-hydroxypropylamino]-9-isopropyl purine) was described and it was shown that the main metabolite is its O-glucoside. In addition, a stimulatory effect of CDK inhibitors on production of monoclonal antibodies was described (103, 114, 164, 170, 236, 242, 243, 245, 246, 272, 273, 333, 334). It was demonstrated that both roscovitine and bohemine induce the expression of nonmutated form of protein p53 in tumour cell lines. The protein actively binds to DNA and is capable of activation of some genes, e.g. the one coding the protein p21WAF1. This finding should start development of a new generation of anti-tumour agents the molecular target of which should be the gene/protein p53, which is most often mutated in human tumours (173). 18 IRP identification code AV0Z50380511 A number of complexed substances - derivatives of benzylaminopurine (BAP) (aromatic cytokinins) and selected transition metals (Ni, Cu, Fe, Pd, Pt) - were prepared. Testing biological activity of these compounds showed that complex formation can result in a substantial increase of cytotoxicity against selected tumour lines (33, 175, 192, 248, 269, 367). 1. 2 Signalling and signal transduction 1. 2. 1 Signalling mechanisms The regulatory role of plant proteins dependent on GTP (so called small GTPases) was shown in establishing the cell polarity and in regulation of morphogenesis (79, 199). Microinjection of non-metabolised analogues of GTP/GDP into living polarised plant cells led to the loss of their polarity while microinjection of ATP/ADP was without any immediate effect (158). We suggest that the most important small GTPase in the processes described is the Rop GTPase. Currently the Rab geranylgeranyl transferase complex (GGTasell) is being characterised, which is responsible for posttranslatory prenylations of Rab GTPases. Its subunit was described called "Rab escort protein" (REP), which is very close to the homologue of Rab GDP dissociation inhibitor as described by us earlier. In position 195 of this protein asparagine was detected as a specific amino acid for plant REP. ◘ In Arabidopsis plants three subunits were characterised (Sec6, Exo70G1 and Sec10) of complex Exocyst, which is an effector of small GTPases in polarised/localised exocytosis not yet described in plants. Using antibodies against the subunit Sec6 its localisation was shown in the membrane-bound complex in the tips of growing tobacco pollen tubes (313) (Fig. 2). ◘ Fig. 2: Indirect immunolocalisation of Exocyst Sec6 subunit in proliferating pollen tubes of tobacco (Nicotiana tabacum, cv. Samsun). Plant phospholipases D (PLD) were phyllogenetically analysed in detail (283) and their possible role in regulation of cell expansion was studied. It was shown that the product of PLD activity - phosphatidic acid (PA) - acts as an important signal in the regulation of polar morphogenesis of plant cells and of their growth. The specific inhibitor of PLD - 1-butanol stopped the growth of the pollen tube and application of phosphatidic acid restored it. Growth was partially restored also after taxol application, which suggests binding of PLD to microtubules (348). ◘ Basic components of plant phospholipid signalling pathways were studied. The receptor for inositol-1,4,5-trisphosphate was localised inside the cells at the endoplasmic reticulum. This 19 IRP identification code AV0Z50380511 finding supported the hypothesis on the existence of an alternative source of calcium ions (besides the vacuole) in plant cells (108). For the first time, the regulatory function of phosphorylation of membrane-bound phosphatidylinositol bisphosphate-dependent phospholipase D, isolated from 5-day-old hypocotyls of Brassica oleracea was shown (340). ◘ Three types of signalling enzymes degrading phospholipids - phosphatidylinositol-dependent phospholipase C (PI-PLC) and two types of phospholipase D, PIP2- dependent and independent (PIP2, PLD and PLDα, respectively) - were studied during ripening, germination and early growth of oilseed rape. Activities of membrane-bound forms of all three types of phospholipases changed during ripening in a different way to the activities of soluble forms. PIP2-PLD activity changed in an opposite way to the activities of PLDα and PI-PLC, both during ripening of rape seeds and during germination and growth of seedlings. In fractions isolated from hypocotyls of seedlings most phospholipases were detected in the form bound to the plasma membrane (341). ◘ To monitor phospholipase activity in situ a new method was developed using fluorescencelabelled substrates. These substrates are incorporated into membranes (mainly into the plasmalemma) of living cells (Fig.3). Fluorescently labelled products of phospholipase activity are then quantified using TLC or HPLC. ◘ Fig. 3: Plasmolysed BY-2 tobacco cell with incorporated fluorescent BODIPY FLC5 – phosphatidylinositol -4,5-bisphosphate (PIP2) to the plasma membrane (green colour). Fluorescent shuttle PIP2 carrier stay in cell wall (red colour). Cells with incorporated BODIPY-PIP2 is used for in situ determination of phosphatidylinositol specific phospholipase C activity. Treatment of plant cells with elicitors induced changes in phospholipase activity. Phospholipase A activity was increased after treatment of tobacco cells with cryptogein (elicitor from the pathogen Phytophthora cryptogea) and after treatment of parsley cells with a glycoprotein elicitor isolated from Phytophthora sojae. Under these conditions, the formation of diacylglycerol was decreased. Phosphatidic acid was detected only in minor amounts, which suggests that diacylglycerol was formed from the added phosphatidylcholine by the activity of phospholipase C hydrolysing phosphatidylcholine. These results are the first description of a new plant phospholipase C hydrolysing phosphatidylcholine and suggest its role in signal transduction (260). Oscillations in the excretion (flux) of chloride ions at the tip of pollen tubes were shown to be the main regulator of the growth rate of the pollen tube (197, 276). This excretion of chloride ions is regulated by a harmonic oscillator, which is switched on before growth oscillations can be observed (378). The chloride oscillator is tightly coupled to changes in the pressure potential in the cell and correlates with osmoregulation and cell volume changes and reacts 20 IRP identification code AV0Z50380511 to the signal inositol-3,4,5,6-tetrakisphosphate (Ins3,4,5,6P4), which determines the frequency of the oscillator (197, 276) (Fig. 4). It seems that this oscillator is able to convert the pollen tube to a sort of "pneumatic hammer" which enables the cells to penetrate stigma tissue. ◘ Fig. 4: Proposed minimal model for dynamic Cl− efflux oscillations from the apex of pollen tubes. The Cl− pool at the apex is supplied from Cl− influx along distal regions of the tube and an endogenous source. Spatial coupling of efflux and influx sites predicts a closed loop of flux vectorially traverses the apical region. Cell volume sensing circuits (grey nodes and flowlines) are hypothesized to be concentrated in the apical dome. The Ins(3,4,5,6)P4 cycle and putative Ca2+ cycle are hypothesized to be phaseshifted by 180°. Negative flowlines are denoted as l⎯I , positive flowlines are denoted as ´. The thick outline around the cell denotes the cell wall, the thin interior line denotes the plasma membrane. 1. 2. 2 Coupling of signal transduction to the cytoskeleton Structural and functional analysis of the cytoskeleton in the course of microspore development showed that in critical developmental phases specific cytoskeletal structures are formed, which determine further direction of development. It was shown that microtubules are important in nucleus migration and polarisation of microspores, that actin and tubulin structures take part in the course of microspore asymmetric mitosis and that actin has a significant role in postmitotic differentiation of vegetative and generative cells of the pollen (46, 152). During the induction of pollen embryogenesis the actin cytoskeleton shows significant reorganisation, namely around and inside the vegetative nucleus. Phases of gametophytic pollen development are coupled with the occurrence of developmentally specific fucosylated and especially mannose or hybrid N-glycoproteins (23). Glycoprotein 92 kDa, specific and, at the same time, dominant in the phase of asymmetric microspore mitosis, corresponds in its amino acid sequence to β-galactosidase. Glycoproteins 51 and 59 kDa, which accumulate during pollen maturation are thermostable and glycoprotein 59 kDa belongs to the group of dehydrins. In microspore cultures of potato, certain concentrations and forms of organic nitrogen induce a change of asymmetric mitosis into the symmetric type of division and reorientation of gametophytic development into an early embryogenic one (34). Nucleation and cytoskeleton organisation was studied in plant cells with the absence of centers for nucleation of microtubules, like are e.g. centrosomes in animals. In acentrosomal plant cells the distribution of γ-tubulin was characterised; this protein is known to play a key role in nucleation and organisation of microtubules in animals (85, 299). Beside the occurrence of γ-tubulin in the nucleus, it also was found to be associated with membranes in the form of protein complexes (312) (Fig. 5). Using immunoprecipitation association of γtubulin with the dimers of α- and β-tubulin was proved. Large γ-tubulin complexes, resistant to cleavage by salts, were detected in the microsomal fraction; molecular mass of these complexes was higher than 1 MDa. These complexes were active in microtubule nucleation. Association of γ-tubulin complexes with dynamic membranes obviously enables flexibility of acentrosomal nucleation of microtubules. 21 IRP identification code AV0Z50380511 Fig. 5: Immunofluorescence localisation of microtubules (green), Golgi membranes (red) and DNA (blue) in mitotic cell of Vicia faba. Golgi membranes are accumulated in close vicinity of spindle poles. Specific changes of cytoskeleton during somatic embryogenesis in Norway spruce were described. F-actin was detected in dividing cells of the embryo head in the whole course of mitosis. Transitory co-localisation of actin microfilaments with the preprophase microtubule bundle was observed. F- actin was not detected in kinetochore microtubule bundles in the course of metaphase and anaphase. A high intensity actin signal was seen in the spindle in the late anaphase in the equatorial plane between separating chromatids (303). ◘ Regulatory aspects important for cytoskeleton organisation were also studied. Activation of the cell cycle in zygotic embryos in the course of imbibition and its relation to tubulin expression and microtubule orientation were analysed in Vicia faba and Medicago falcata (19). Gradual polymerisation of microtubules was seen coupled with reactivation of the cell cycle in those cells, which entered the cycle from the G1 phase, in which the embryo cells were present in dormant seeds. Cell cycle reactivation was similarly coupled with significant reorientation of the microtubular cytoskeleton in the induction phase of somatic embryogenesis of alfalfa (11). ◘ 1. 2. 3 Signalling in plant defence mechanisms The ability of a plant to withstand unfavourable external conditions can be demonstrated in the synthesis of the so-called small heat-shock proteins (sHSP). In collaboration with the Department of Plant Physiology (Faculty of Natural Sciences, Charles University, Prague) a new type of their regulation by means of the level of ATP in the cells was described (118), in which ATP inhibits interaction of the sHSP complex with the substrate. In the phase of acute stress, when the ATP level was decreasing, the affinity of the protecting sHSP towards the cell components endangered by stress was increased. After the end of the acute phase of the stress the increased ATP level loosened these components from the complex and renaturation of HSP70, occurred. Defence mechanisms of plants against pathogens (especially viruses) were studied with special attention to the induction of systemic acquired resistance (SAR) and induced systemic resistance (ISR) by synthetic and natural inducers. Synthesis of pathogenesisrelated proteins (PR-proteins) was also studied (9, 189, 267, 301, 302). The composition 22 IRP identification code AV0Z50380511 and amount of induced PR-proteins (9) as well as their localisation in tissues (301, 302) depended on the applied inducer. In sugar beet (9, 302) and wheat, the most effective inducer was benzothiazol (BTH) and also glycinebetaine and salicylic acid were active inducers. Isozymes of β-1,3-glucanases and chitinases were found both in leaves and in roots after treatment with chemical inducers, infection with Polymyxa betae and beet mosaic yellow vein virus (BNYVV) (Fig. 6). The results show the possible role of these enzymes in resistance to rhizomania. BTH application induced resistance to tobacco mosaic virus (TMV) and potato virus Y (PVY) (266). Nine different inducers including BTH were efficient in wheat and strongly decreased growth of the fungus Blumeria graminis. In contrast to the inducers, herbicides with auxin-like activity increased the content of TMV in tobacco leaf discs but did not induce PR-proteins (121). Enzymes of the biosynthesis of viral RNA precursors, their regulation, subcellular localisation and relation to plant resistance were also studied (9, 40, 41, 122, 265, 266, 267, 361). Correlation between the activities of the key enzymes of metabolic pathways and the virus reproduction was shown for glucose-6-phosphate dehydrogenase (G6PDH) (9, 40, 41, 265, 266, 267, 361) and ribonucleases (9, 122, 267, 361). In the synthesis of the virus chloroplastic isozyme of G6PDH was predominantly active (265). Activities of these pathways also increased considerably after BTH application, which suggests possible competition between virus synthesis and defence reactions (267). In contrast antivirus factor (AVF) decreased infectivity of TMV by lowering activities of the above mentioned enzymes (363). Fig. 6: Immunohistological localisation of basic chitinase Ch4 in healthy (left) and rhizomania-diseased (right) root tissues. In sections of infected roots (right) (P.betae cystosori- Pb), Ch4 was found mainly in endodermis cells (arrowheads) and xylem vessels, but in some rhizodermal cells the accumulation of Ch4 was also detected (arrowheads). In healthy control plants (left), slight staining indicates the constitutive presence of Ch4. Patterns were determined in at least 10 plants and were remarkably consistent. Infection of control and transgenic tobacco plants carrying the Pssu-IPT construct with potato potyviruses A (PVA) or Y (PVY) resulted in different responses. Infection with PVY caused in control and F1 transgenic plants a decrease in photosynthesis and in parameters of chlorophyll fluorescence kinetics and an increase in activities of phosphoenolpyruvate carboxylase, NADP-malic enzyme and pyruvate dikinase. The effect of PVA was negligible (348). Electron microscopy showed aggregates of virus particles in the vicinity of chloroplasts. Pssu-ipt grafts were highly resistant to virus infection and infection with PVA even stimulated their photosynthesis (348). 23 IRP identification code AV0Z50380511 2 STRUCTURE AND FUNCTION OF THE GENOME 2. 1 Functional genomics Using methods of in vitro transcription, translation and N-glycosylation we positively demonstrated that the model tobacco pollen-specific N-glycoprotein p69 is encoded by the ntp303 gene (130). We proved the phylogenetic conservation of this gene at the levels of 1-D and 2-D SDS-PAGE protein spectra of pollen tube-walls in 16 plant species of 15 genera (162). The ntp303 transcript is synthesised during pollen maturation after the first haploid mitosis, accumulated in the non-translatable form and was utilised after germination during the progametic phase of male gametophyte development. We observed the kinetics of ntp303 transcript synthesis and, for the first time in plants, we described a new form of mRNA storage RNP complexes. ntp303 transcripts are stored in the form of translationally silent EPP particles („EDTA-puromycin resistant particles“), high molecular weight RNP complexes resistant to polysome-destabilising agents EDTA and puromycin (Fig. 7) (95, 96, 168). Fig. 7: Potential model of the developmental regulation of ntp303 mRNA subcellular distribution. Newly synthesized ntp303 mRNA is released from the nucleus in the transport form of mRNPs. Between stages 3 and 5 ntp303 mRNA is distributed evenly between polysomes and EPPs. All polysomes associated with ntp303 mRNA are formed at this stage and are translationally silent. EPPs are proposed to be the long term storage compartment formed by aggregation of individual ntp303 mRNPs probably with other proteins. Between stages 5 and 6, polysomes associated with ntp303 mRNA are still present in the vegetative cell but their amount does not increase. ntp303 mRNA synthesized at this time remains in the transient form of “free” mRNPs with only small portion of them combining into EPPs. In the final period of maturation between stages 6 and dry pollen the synthesis of ntp303 mRNA is complete, but a massive redistribution of ntp303 mRNA from “free” mRNPs and polysomes to EPPs occurs. For the first time in the plant kingdom, we performed a genome-wide analysis of the gene expression of a single developing cell at the transcriptome level. Mainly, we focussed on changes in the gene expression during ontogenesis. As a model system, we used the vegetative cell of Arabidopsis pollen. The gametophytic gene expression was quantified and actively transcribed genes were assorted to functional categories. Gametophytic and sporophytic transcriptomes were compared and the extent of their overlap determined (321, 326). We have also described putative gametophytic transcription factors and clusters of coexpressed genes containing candidates for putative regulons, expression of which is controlled by particular gametophytic transcription factors (388). The dynamics of expression of another plant translationally regulated and constitutively transcribed D1 protein was studied. The translation intensity of D1 protein, one of the key proteins of photosystem II, during the first two days following stress treatment remained unchanged and high. Substantial decrease of its synthesis was observed the third day after stress application, when other physiological and biochemical parameters were stabilised at 24 IRP identification code AV0Z50380511 the control level (166, 171, 240). For proteomic studies, new original methods were developed for mitochondrial isolation and fractionation and protein extraction from small amounts of starting material (grams instead of kilograms). We demonstrated that heat stress (40o C) immediately induced intensive expression of 9 low molecular weight proteins: 8 sHSPs and 1 transcription factor. Qualitative and quantitative representation of all these proteins, which form part of the membrane fraction, remained stable during the first 12 hours of stress treatment 2. 2 Studies on genome structure using sorted chromosomes Although many authors believe in intraspecific variation in nuclear genome size, the results obtained in the genus Musa (26), Sesleria (107) and Agave (342) do not support this assumption. The genus Musa was used as a model to study the evolution of polyploid species. The size of the nuclear genome has been established in wild diploid species and in diploid and triploid parthenocarpic cultivars for the first time (26). Systematic genome analysis resulted in the isolation and characterisation of a number of repetitive DNA sequences which constitute a significant part of the banana genome (271). These include a new type of „monkey“ retrotransposon (83). Fig. 8: Analysis of genomic distribution of five repetitive DNA sequences (pSc119.2, GAA microsatellite, Afa family repeat, and 5S rDNA) on chromosomes of tetraploid wheat (Triticum durum cv. Langdon; 2n = 4x = 28) using fluorescence in situ hybridization (FISH). Labelled probes were detected either with fluorescein (yellow-green signals) or Cy3 (red signals); chromosomes were counterstained with DAPI (red pseudolocour for single colour FISH; blue pseudocolour for double colour FISH). For each chromosome type, two representative examples are given. In collaboration with the laboratory of Prof. B. Vyskot (Institute of Biophysics, Brno), the evolution of sex chromosomes was studied in plants. Nuclear genome size and genomic distribution of genes for ribosomal RNA have been established in species of the genus Silene (190). Development of a method for flow cytometric sorting of sex chromosomes facilitated physical mapping of MROS genes, expressed in male plants of S. latifolia, to X chromosome and autosomes (172). Also, the Y chromosome has been found to harbour a 25 IRP identification code AV0Z50380511 MADS box gene, which has been duplicated from an autosome (331). Analyses of crop plant genomes are hampered by the genome size and complexity. In order to simplify the genome analysis, methods for chromosome sorting have been developed in barley (27), wheat (129, 222, 244) and rye (325). The genomic distribution of some repetitive DNA sequences was first established by the method of sorted chromosomes (104, 207, 325) and, using the same method, the chromosomal localisation of molecular markers was determined (27, 325) (Fig. 8). The development of a method for preparation of high molecular weight DNA from sorted chromosomes and the construction of chromosome-specific DNA libraries cloned in BAC vectors (360) have been a significant success. Flow-sorted chromosomes have also been used to map nuclear genomes in legumes. In field bean, new repetitive DNA sequences were isolated and characterised (28) and new microsatellite molecular markers for chromosome 1 have been isolated (255). Flow sorted chromosomes were used for physical mapping and integration of genetic and physical maps in garden pea (251) and chickpea (275). 2. 3. Genotoxicity and DNA repair Fig. 9: Images illustrating the induction of DNA damage in tobacco nuclei as expressed in the Comet assay. Control nucleus (A) and nuclei with different levels of DNA damage (B, C, D). For DNA repair studies, the Comet assay was modified (Fig. 9) to enable the detection of various types of DNA damage and their localisation by the FISH method (fluorescence in situ hybridization) in specific sequences, for example telomeric regions or repetitive sequences (110). The development of the modified method of the Comet assay was necessary for assessment of efficiency and kinetics of DNA repair and, when the Arabidopsis thaliana 26 IRP identification code AV0Z50380511 genome sequencing is completed and knock-out mutants are available, has aided studies on particular repair pathways. The sensitivity to chemical mutagens causing DNA damage was tested. The repair of these DNA damages can be achieved by various mechanisms involving various repair enzymes. The effects of chemical mutagens (alkylation mutagens N‘-methyl-nitrosourea a methyl methanesulfonate; radiomimetic bleomycin causing doublestrand breaks in DNA; mitomycin C inducing cross-link bonds in DNA; plant morphoregulator maleinhydrazide) aid the verification of the mutant phenotype (177). These results led to studies of mutants defective in various repair pathways, mostly the ones defective in genes involved in homologous recombination: AtSpo3-11, AtT3B, AtARF1, AtTop6B, AtRad9, AtRad17, AtBRCA1, AtFANDC2, or non-homologous end-to-end joining of double-strand breaks (NHEJ): AtKu70, AtKu80. So far, the characterisation of AtTop6B topoisomerase mutant has been published (239). Beside the analyses of Arabidopsis repair mutants, the adaptation to genotoxic stress has been proved in Vicia faba and Arabidopsis after application of various alkylation mutagens. The effect of alkylation mutagens is not solely connected to O6-guanine alkylation as had been expected, based on analogy with other organisms, until now (80). Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations and was highly correlated (r = 0.99) with the increased DNA damage in the nuclei of the leaves. 24 h after irradiation a complete repair of DNA damage induced by gammairradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose (181). By contrast, DNA damage induced by the monofunctional alkylating agent ethyl methanesulphonate persisted over a 72 h period (91). The plant growth regulator and herbicide, maleinhydrazide (MH), induced, in sprouting tobacco plants, a high frequency of somatic mutations and recombination events, but no significant increase in DNA damage measured by the Comet assay. MH represents the first chemical agent which has proved to be highly mutagenic but does not cause DNA damage as measured by the Comet assay in the same experimental system (90, 318). 2. 4. Molecular aspects of plant virology The influence of viral infection caused by two different Potyviruses, Potato virus Y (PVY) and Potato virus A (PVA) on plant metabolism and photosynthetic apparatus of Nicotiana tabacum L. cv. Samsun and cv. Petit Havana SR1 was studied. The main emphasis was focussed on the activities of enzymes that catalyse anaplerotic metabolic pathways phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), and pyruvate phosphate dikinase (PPDK, EC 2.7.9.1). PVY infected tobacco plants responded with enhanced activities of all three enzymes. NADP-ME was the most sensitive to viral infection; the enzyme activity was 5 times higher in Samsun and 6-fold higher in SR1, compared with control plants. The activities of PEPC and PPDK were enhanced 2-3 times in both cultivars. In contrast to PVY, PVA infection affected enzyme activities insignificantly, although a moderate increase of activities was observed. No significant difference between both tobacco cultivars was found (352). The entire nucleotide sequence of coding regions of Potato mop-top virus (PMTV) isolate named 54-15 was determined. The genome was found to be highly conserved when the nucleotide sequences and their derived respective amino acid sequences were compared with other known isolates of PMTV, even though the PMTV isolates exhibited different symptoms in host plants and originated from different parts of Europe. However, several changes in nucleotide and amino acid sequences in parts of RNA coding for “triple-geneblock” (TGB) proteins and in the “read-through” part of the coat protein (CP) RNA were detected. The phenotypic differentiation of these isolates could be attributed to these mutations. In order to confirm this hypothesis, the TGB and read-through CP regions from several other isolates were sequenced, namely 54-19 and 54-10. The comparisons of sequences obtained with sequences of other isolates available in databases imply high 27 IRP identification code AV0Z50380511 genetic stability and slow evolution within the PMTV population. The most remarkable evolutionary event observed would be the disruption of the fourth open-reading frame (ORF) on TGB-coding RNA, identified as a gene for cysteine-rich protein in other PMTV isolates (308). 2. 5. Potential practical applications of genetic research To test the concept of plant derived edible vaccine, an agroinfection system has been set up in tobacco and tomato in order to characterise molecular and immunological features of viral proteins of human papilloma virus HPV-16. In this system, the production of 0.5 – 1 % of total protein was achieved in the case of oncoprotein E7, which can potentially be used as a therapeutic vaccine after the onset of cervical cancer and in the case of coat protein L1, that could serve as prophylactic vaccine. L1 protein is produced as structural capsomeres. The conditions influencing formation of the pseudovirions are under study, as well as the immunogenicity of produced proteins. Gene coding for the coat protein of Potato virus A (PVACP) was cloned. This gene was also modified, so that the epitopes of structural and nonstructural proteins (L2 and E7, respectively) from human papilloma virus (HPV), those attached to the N-terminus and/or Cterminus of PVACP protein (modified genes: PVACP+E7, L2+PVACP and L2+PVACP+E7), could be expressed. New apple varieties bearing the Vf resistance against scab, the most widespread and harmful disease caused by the fungus Venturia inaequalis, have been bred. Eleven selections were applied for Plant Breeder’s Rights in the Czech Republic under the numbers MAL 7121, 7123, 7125, 7983, 8137, 8138, 8139, 8140, 8641, 8642, 8643 and 4 varieties were applied for Community Plant Variety Rights in the European Union under file numbers 1999/0804, 2001/0082, 2003/1163, 2003/1164. Plant patents / Plant Breeder’s Rights granted and licence agreements for commercialisation of the varieties concluded are listed in the section 13. In addition testing agreements with option were concluded in Australia (5 var.), South Africa (5 var.), New Zealand (1 var.) and the USA (10 var.). Within the breeding of resistant varieties with compact, columnar growth habit derived from Wijcik McIntosh mutant, one selection was applied for Plant Breeder’s Rights in the Czech Republic (MAL 07120) and option agreements were concluded for 3 selections with a German society for the European Union and for 8 selections in the USA. The resistant varieties bred at the Střížovice Station were commercialised mainly for organic fruit growing in Europe and regular royalties from the licences became an important financial source for the Institute. In the follow up breeding of varieties with multiple durable resistance, breeding material was selected for testing Vf homozygosity and combination of Vf with polygenically encoded resistance using molecular markers. Using the method of microprojectile bombardment, the genes DAP A and PHYT (gene dapA codes for dihydropicolinate synthase form insensitive to feedback inhibition and increasing lysine content and gene PHYT codes for phytase, which affects phosphorus content) were transferred to the cell genomes of immature zygotic embryos of barley, from which transgenic plants were derived. The transformation frequency in anther cultures of these primarily transformed plants was low (215). The presence of DAP A gene was confirmed by molecular analysis in four androgenous regenerants, one of which was capable of producing seeds. The gene PHYT was detected in more than 70% of androgenous regenerants. A tetrahaploid transgenic line of barley, carrying a phytase gene, has been derived, which we consider to be a significant success. The analysis of transgenic potato plants transformed with the introduced bacterial gene LbPFK (phosphofructokinase of Lactobacillus) was performed in field tests (yield characteristics). Among the transgenic plants, phenotypically standard plants prevailed; sole 27.5% of transformed KIamýk cultivar and 21,4% of transformed Korela cultivar, respectively, showed a deviation from normal phenotype in either growth or flowering. The transformed 28 IRP identification code AV0Z50380511 lines generally displayed a yield equal or higher (by 3 to 32%) compared with control lines. The levels of reducing sugars in harvested tubers of transgenic plants was higher than that observed in tubers of control plants. During the cold-storage of tubers (160 days), however, the amount of reducing sugars in transgenic tubers decreased, while in control tubers it was increasing. By the end of a storage period, the amount of reducing sugars was lower in transgenic tubers than in control non-transgenic tubers. One of the transgenic lines, which is considered the most interesting, contained only 0,17% of reducing sugars after a period of 160 days cold-storage. LIST OF ABBREVIATIONS ABA ABI3 ABPs BAP (BA) BNYVV BY-2 BTH CaMV35S CDK (cdc2) CHN/O analysis CKX (AtCKX3) CP 2D-HPLC DsRed EDTA ELISA ESI-MS EPP EST FPLC FRET GDP GTP GTPase GFP G6PDH GC/MS GUS HPLC HPLC/MS (LC/MS) (s)HSP IAA IEB IPT IPMP IR spektrometry LC MALDI-TOF MH MS NMR NPA ORF PAGE abscisic acid ABA- insensitive (3) A. thaliana mutant auxin binding proteins N6-benzylamino purine beet necrosis yellow vein virus tobacco cell culture benzothiadiazole promoter from cauliflower mosaic virus cyclin-dependant kinase (2) elementary analysis cytokininoxidase/reductase (from A. thaliana) coat protein two-dimensional HPLC Discosoma species Red (fluorescent red dye from species Discosoma) ethylenediamine tetraacetic acid enzyme-linked immunosorbent assay mass spectrometry with „electrospray“ ionisation EDTA, puromycin-resistant particles expressed sequence tag fast protein liquid chromatography fluorescence resonance energy transfer guanosine bisphosphate guanosine trisphosphate enzyme cleaving guanosine trisphosphate green fluorescent protein glucose-6-phosphate dehydrogenase gas chromatography with mass spectrometric detection β-glucuronidase high performance liquid chromatography high performance liquid chromatography with mass spectrometric detection (small) heat-shock proteins indolyl-3-acetic acid Institute of Experimental Botany isopentenyl transferase isopentenyladenosine-5´-monophosphate infrared spectrometry liquid chromatography „matrix-aided laser desorption ionisation-time of flight“- type of mass spectrometry maleinhydrazide mass spectrometry nuclear magnetic resonance 1-N-naphthylphthalamic acid open reading frame polyacrylamide gel electrophoresis 29 IRP identification code AV0Z50380511 PEPC PCIB PIP2 PLC PI-PLC PC-PLC PCR PLD PMTV PPDK PR proteins Pssu-IPT PVA PVY R/FR RNAi RT-PCR SAR SDS-PAGE SOD TGB TLC TMV VC YFP phosphoenolpyruvate carboxylase p-chloroisobutyric acid phosphatidylinositol-4,5-bisphosphate phospholipase C phospholipase C hydrolysing phosphatidyl inositol phospholipase C hydrolysing phosphatidyl choline polymerase chain reaction phospholipase D potato mop-top virus phosphopyruvate dikinase pathogenesis related proteins gene IPTcoding for isopentenyl transferase under the control of promoter of small subunit of Rubisco from pea potato virus A potato virus Y red light 660 nm/ far-red light 730 nm small interfering RNA real time polymerase chain reaction acquired systemic resistance polyacrylamide electrophoresis with addition of sodium dodecylsulfate superoxid dismutase triple gene block thin layer chromatography tobacco mosaic virus research center yellow fluorescent protein 30 IRP identification code AV0Z50380511 C5. Objectives of IRP Plans for the period 2005-2010 The traditional areas of research in the IEB have been focussed previously on two main topics: (i) plant growth and development; (ii) plant genomics. Both these research areas have been intensively studied on the Institute’s grounds and acknowledged in previous years. For plant growth and development, future research will be aimed mainly towards: • studies on metabolism, transport and function of various regulatory metabolites (mainly phytohormones) • signal transduction (phosphoinositide signalling system, small GTPases, Exocyst complex and formin-associated proteins). In plant genomics, priority will be given to the exploration of plant genome structure and, namely, the determination of the function of genes of the male gametophyte, as well as of some genes during the cell cycle, and interaction of plants and viruses. Both these areas of basic research in the IEB will be conducted so as to contribute significantly to the understanding the mechanisms of regulation of plant growth and development, and, consequently, to practical utilisation of the results obtained as well. The main goal of research in the IEB is to integrate the two above mentioned areas of research and thus achieve a complex view on the mechanisms of the basic life processes within a plant cell. The plant cell will be studied at various levels: • those comprising the structure of the genome, • the regulation of gene expression, • the functional organisation of signalling pathways, • the mechanisms of plant growth regulation, • the physiological behaviour of the plant cells studied (studies on the cell cycle, endogenous rhythmicity, cell polarity etc.). Such complex research forms the basis for understanding the principles of the plant cell and subsequent plant organ development and, finally, the whole plant organism itself. Promising areas of research will be preferred, and projects likely to lead to future breakthroughs in given topics will be supported with high priority. These topics include: • molecular cytogenetics (structure and evolution of the genome, construction of cytogenetic maps using sorted chromosomes) • characterisation of transcriptom of the male gametophyte • phosphoinositide signalling system in plant cells • mechanism of function and regulation of the activity of auxin and cytokinin carriers • various aspects of metabolism of cytokinins • molecular mechanisms of nucleation and reorganisation of microtubules during the cell cycle • production of pharmacologically-active proteins by transgenic plants. The coordination of the projects gives a basis for fruitful cooperation between the teams in the IEB, where the genome studies are an information source for further studies on the signal and regulatory molecules and, eventually, for a deeper understanding of the development of plant cells. The process of signal transduction is closely tied to cell 31 IRP identification code AV0Z50380511 structures, which further affect translation and protein transport to respective effectors. The experimental work will be focussed on a limited number of experimental materials (above all Arabidopsis thaliana, model tobacco cell lines, and – in special cases – the others), so that a complex view on the course and control of key developmental processes is achieved. Close connection among the teams in the IEB, together with methodological knowledge and the support of highly trained personnel guarantees a modern approach to basic research into the life processes in a plant cell. The coordination of the two main research topics is documented below. For the purposes of easier understanding, the sections do not strictly correspond to the running projects of the respective teams but the names of key researchers responsible for individual areas are given in parentheses. List of contents: 1 REGULATION OF GROWTH AND DEVELOPMENT 1.1 Plant growth regulators 1.1.1 Metabolism of phytohormones and other plant growth regulators 1.1.2 Transport of phytohormones and other plant growth regulators 1.1.3 Mechanism of action of phytohormones and other plant growth regulators 1.1.4 Hormonal regulation of some processes of growth and development and stress reactions 1.1.5 Potential practical applications of the research of plant growth regulators 1.2 Signalling and signal transduction 2 STRUCTURE AND FUNCTION OF THE GENOME 2.1 Basics of structural and functional genomics 2.2 Molecular aspects of plant virology and phytopathology 2.3 Contribution to the organisation of international scientific activity at the A. thaliana research 2.4 Potential practical applications 1 REGULATION OF GROWTH AND DEVELOPMENT 1.1 Plant growth regulators 1.1.1 Metabolism of phytohormones and other plant growth regulators The detailed understanding of the mechanisms of regulation of levels of plant growth regulators is a necessary prerequisite for the understanding and for targetted control of plant growth and development. This knowledge is indispensable for design of novel synthetic growth regulators, for preparation of transgenic plants with controlled biosynthesis and metabolism of plant hormones, and for their use in plant biotechnologies. Investigation of control of levels and action of cytokinins will include studies of (h) the role of hormonal interactions in the regulation of cytokinin levels, (i) mechanisms of hormonal homeostasis, (j) the targetting of enzymes involved in the biosynthesis and metabolism of cytokinins and its effects on intracellular cytokinin levels and cytokinin secretion to the apoplast, (k) selected cytokinin metabolic pathways involved in regulation of cytokinin levels in plants (spatial and temporal localisation, physiological significance, reaction to stresses), (l) identification and role of specific cytokinin-binding proteins in the formation of a 32 IRP identification code AV0Z50380511 storage pool of immobilised cytokinins. For separation of plant hormones (auxins, cytokinins, abscisic acid), HPLC/MS, GC/MS and 2D-HPLC with on line UV- and fluorescence detectors will be used. Activities of enzymes involved in cytokinin metabolism will be determined by conversion of radio-labelled substrates and HPLC with flow-through radioactivity determination. LC and FPLC will be used for purification of cytokinin-binding proteins; their binding activity will be determined by equilibrium dialysis and ultrafiltration using radio-labelled ligands. (Motyka, Vaňková, Dobrev, Kamínek) The development and stability of chloroplasts is under the strong influence of cytokinins. Changes in cytokinin levels in plastids can regulate functioning of plastids and delay their senescence. Consequently, the effectiveness of photosynthetic apparatus is enhanced. Therefore, the cytokinin content will be measured in chloroplasts from tobacco plants carrying the gene for zeatin-O-glucosyl transferase (obtained from Prof. D. W. Mok, Corvallis, Oregon, USA). Biosynthesis of cytokinins in chloroplasts isolated from tobacco leaves will be studied using incubation with radioactive precursors (pyruvate, mevalonate, D-xylose) and after treatment with inhibitors of both isoprenoid biosynthetic pathways, lovastatin and fosfidomycin. Using immunocytochemical method changes will be followed in the levels and localisation of cytokinins in leaves and isolated chloroplasts from control plants and from plants transformed by genes, products of which affect cytokinin levels. (Vaňková, Macháčková) Recent findings about the appearance of cytokinins in animal cells imply that cytokinins may have a function, which is not known yet, also in animal cells. To investigate this phenomenon, the modern analytical methods developed in the IEB will be used. The appearance of cytokinins in animal tissues will be studied using LC/MS, with special attention to the production and differentiation of blood cells (preliminary results showed kinetin as a native cytokinin in some types of blood cells). The biosynthetic pathway of cytokinins in these cells will be elucidated using incorporation of deuterium in vitro. (Strnad, Doležal) In last years, new native cytokinins, some of them displaying high biological activity and metabolic stability, have been isolated and characterised in the IEB. This very successful research topic will be further elaborated so that new N6-substituted derivatives of adenine will be isolated from plants (and animals) and identified, their internal concentrations will be measured by HPLC/MS and their biological activity will be determined by cytokinin biotests. The metabolism of these compounds will be studied in relation to their potential utilisation in plant biotechnologies; synthetic analogues of these compounds will also be prepared. Other groups of organic compounds, structurally derived from N6-substituted adenine, will be synthesised, as well as their complexes with selected transition metals; their structure will be determined by NMR, X-ray diffraction, CHN/O analysis, magnetic susceptibility, MS-, IRand UV-spectrometry. Final structures will be derived from quantal-chemical computations. (Strnad, Doležal) Besides phytohormones, plant growth and development and the resistance of plants against stresses is influenced also by polyamines. The metabolism of polyamines will be investigated at the level of activities of biosynthetic enzymes (ornithine and arginine decarboxylases), of contents of putrescine, spermidine and spermine, and of determination of diamine and polyamine oxidases (the degradation enzymes), during the growth cycle of tobacco BY-2 cell suspension culture. The metabolism of polyamines will be mapped during the cell cycle of synchronous tobacco BY-2 cell suspension culture using inhibitors of various biosynthetic pathways. (Cvikrová, Gemperlová) Even more than in most of the other research areas, the progress in the research of phytohormones depends on the improvements of analytical methods for their determination. Immunodiagnostics of the new generation will be prepared for determination of cytokinins, as well as other phytohormones (namely abscisic, indole-3-acetic, and jasmonic acids, and brassinosteroids). The aim is to obtain both high-specificity antibodies for individual 33 IRP identification code AV0Z50380511 phytohormones for ELISA assays and for immunochemical detection of these compounds at the cell level, and generic broad-specificity antibodies for detection of metabolites of certain groups of phytohormones (applicable especially with immunoaffinity chromatography). (Strnad) 1.1.2 Transport of phytohormones and other plant growth regulators Mechanism(s) of transport of phytohormones (auxins and cytokinins) and other growth regulators (polyamines) will be studied at the cell level (translocation of these compounds across cell membranes) in relation to (i) modulation of intracellular levels of these compounds, and (ii) regulation of basic processes of cell development (cell and growth cycles, establishment and maintenance of cell polarity). The investigation of auxin carriers will be concentrated on: (h) mechanism of regulation of their activity, (i) localisation of carriers and their dynamics, (j) connection(s) with cytoskeletal structures and the endomembrane system, (k) mechanism of action of phytotropins (i.e. inhibitors of auxin efflux carriers), (l) characterisation of specific inhibitors of auxin influx carrier(s). Experimental approaches will involve transformation of plant cells using vectors bearing genes for auxin carriers under constitutive and controllable promoters, and in translation fusion with reporter genes for fluorescence proteins (GFP, YFP, DsRed); fluorescence and confocal microscopy in vivo; immunofluorescence techniques; determination of kinetic parameters of transport of auxins across cell membranes. All experimental data will be related to regulation of growth (or cell) cycle of the cells of model tobacco lines cultivated in vitro. A. thaliana wild type and mutant plants will be used as a reference material for transformation and transport assays. On the same experimental material the possible cytokinin carriers will be searched for with the emphasis on mechanism(s) of control of their activity (kinetic parameters, effects of anticytokinins, screening of possible inhibitors, etc.). (Zažímalová, Petrášek, Perry) The accumulation of polyamines and their transport across the plasma membrane will be characterised in relation to the cell cycle. Next progression will be analogous to that for auxin carriers. (Cvikrová, Zažímalová) 1.1.3 Mechanism of action of phytohormones and other plant growth regulators Hybrids of maize differ in toleration to the density of growth. The degree of this toleration is probably connected with the leaf angle and thus with their ability to utilise light energy. The role of auxin-binding proteins (ABPs) in growth and development of leaf angle will be characterised. The mechanism of how light regulates ABPs and polar auxin transport will be elucidated. Experimentation will include growth and molecular analysis, with the aim to determine whether differential development of leaf angle in modern and older maize hybrids and maize abp mutants is caused by differential expression of the ABP genes and/or amount of ABPs in leaf tissues. The questions of whether and how light affects levels of ABPs, and how possible changes in ABPs influence auxin- and light-induced growth and leaf angle development in maize seedlings will be addressed. A homologue of the ATHB2 gene (coding for a HD-ZIP protein) will be isolated from maize. The gene is specifically expressed by changes in R:FR (red:far-red light) and by auxin, and it is believed that ATHB2 is a bridge between light and auxin signalling. Consequently, the expression of the gene as a function of light and auxin will be studied in the above mentioned maize hybrids. Physiological characterisation of mutants abp15 and abm19 (T-DNA-mutants of Arabidopsis) will be finished and the primary changes due to the mutation in the genes ABP15 and ABM19 will be described. Two hypotheses will be tested: (a) mutants abp15 and abm19 are affected in transduction of the light signal, which results in changes in hormone levels and signalling, and thus in different growth under light and dark conditions, (b) the above mentioned mutants are primarily affected in growth in the darkness, and 34 IRP identification code AV0Z50380511 consequently also the growth in light and sensitivity to hormones are changed. The sequences of genes ABP15 and ABM19 in the Arabidopsis genome will be identified and mapped. Possible products of these genes will be searched for and their function in regulation of growth and development of Arabidopsis will be investigated. (Fellner) A range of plant hormone-like compounds, both naturally occurring and synthetic, affect animal cells as well. To be able to use these compounds e.g. in pharmacy, their effects and mechanism(s) of action must be well understood. Therefore, molecular mechanisms of action of plant and synthetic growth regulators in animal tissue cultures will be characterised, especially in relation to their potential anti-tumour capacity. Pharmacological properties of these compounds as well as their effects on the cell cycle and its regulatory proteins will be analysed. Highly effective compounds will be selected, co-crystallised with selected enzymes or receptors, and X-ray crystallography will be used to determine the binding mode. Both cellular and molecular effects of these compounds will be analysed and proteins with high affinity to them will be characterised in detail (proteomic MS analysis) with the aim of describing the molecular mechanisms of action of these compounds in cells. (Strnad, Doležal) 1.1.4 Hormonal regulation of some processes of growth and development and stress reactions High concentration of cytokinins (resulting from the expression of the ipt gene under Pssu promoter) gives rise to the formation of a range of structural and functional anomalies. The elucidation of these anomalies may contribute to the understanding of cytokinin action. Anomalies in ultrastructure of cell organelles (crystalloids and spherical protein formations in chloroplasts, peripheral reticulum and interactions between chloroplasts, mitochondria and peroxisomes) of transgenic Pssu-ipt tobacco will be described using electron microscopic analysis of ultra-thin sections and 3D computer reconstruction of organelles. Isolation and analysis of crystalloids will be done by spectrofluorometry, SDS-PAGE, and immunodetection using specific antibodies. A complex study will be carried out involving virological, physiological, ultrastructural, and biochemical aspects of viral infection of transgenic tobacco with elevated content of endogenous cytokinins. Attention will focus on the role of enzymes of antioxidant and anaplerotic pathways, expression of pathogenesis related (PR)-proteins and endogenous cytokinins during viral infection. (Synková) Hormones very often interact in the regulation of physiological processes. The action of cytokinins is often antagonistic to the action of ABA. In the research of possible antagonistic actions of ABA and cytokinins in the regulation of stomatal opening and, in consequence, leaf gas exchange, the interaction between cytokinins and ABA on the activity of stomatal guard cells and metabolic interaction during water stress-induced ABA synthesis will be investigated. The research will continue on plants with elevated ABA levels as a consequence of water stress and on transgenic plants with modified cytokinin content. The role of pigments of the xanthophyll cycle will be determined in plant protection against photoinhibition and as precursors in ABA synthesis induced by water stress under low and high irradiance, as affected by cytokinins and in transgenic plants with an elevated cytokinin content. During development of water stress changes in activities of antioxidative enzymes and the effect on these activities of ABA and cytokinins will be followed. (Wilhelmová, Pospíšilová) The use of mutants with changed content of and/or sensitivity to hormones is advantageous for elucidation of the function of individual hormones in growth or developmental processes. Genetic analysis will be performed on the 7B-1 mutant of tomato, characterised by changed sensitivity and reaction to ABA, increased resistance to osmotic stress, and decreased ability to react to blue light. The locus will be mapped by classical methods or using molecular markers. The 7B-1 gene will be cloned by the most suitable molecular methods followed by functional analysis. (Fellner) Cytokinins are well-known for their strong effect on the delay of senescence. The 35 IRP identification code AV0Z50380511 hypothesis that the induction of plant senescence is related to free radical reactions and their products will be tested. The accumulation and characteristics of free radical products and activities of the antioxidative system (enzymatic and low-molecular) will be investigated. Further, regulation of leaf senescence induction will be studied using genetically manipulated plants with changed concentrations of cytokinins. The changes in the formation of the inductor of senescence – ethylene, will be tested in relation to the production of nitric oxide and nitrated proteins. (Wilhelmová) There are more, mutually interacting hormones taking part in the regulation of complex developmental processes in plants. To elucidate the mechanism(s) of hormonal regulation it is necessary to monitor changes in levels of all hormones involved in the duration of such a process. New possibilities of modern analytical methods for endogenous phytohormones (LC-MS, GC-MS) enabling the detection of a higher number of different compounds (cytokinins) with higher sensitivity (IAA, ABA, cytokinins) will be utilised in studies of somatic embryogenesis. Cellular and organ distribution of endogenous phytohormones will be elucidated by immunolocalisation (auxins, cytokinins). The role of the homologue of the ABI3 (abscisic acid insensitive 3) gene will be determined in embryogenic cultures of spruce, and the possible control of its expression and its potential use as a marker of embryogenesis will be followed. (Vágner, Fischerová) 1.1.5 Potential practical applications of the research of plant growth regulators Cytokinins prolong the leaf vitality and longevity and increase the flow of assimilates into set grains. This knowledge will be utilised for the preparation of transgenic plants with increased level of cytokinins and enhanced productivity. Transgenic plants will be prepared with controlled expression of genes involved in biosynthesis and metabolism of cytokinins, suitable for increased crop productivity. Genes encoding the cytokinin biosynthetic enzyme (isopentenyl transferase, ipt) and cytokinin degradation (cytokinin oxidase, Atckx3, in sense and antisense orientation) under control of chemically and developmentally inducible promoters (induction by alcohol [PalcA]; induction of leaf senescence [PSAG12]) will be used for transformation of the model plant (A. thaliana) and wheat. The idea is to enhance plant productivity and longevity by delaying leaf senescence, prolongation of leaf photosynthetic activity and assimilation of nitrogen. Plants will be transformed using the method of floral dipping in a suspension of transformed clones of Agrobacterium tumefaciens bearing the above specified genes and promoters obtained from collaborating laboratories. (Hoyerová, Kamínek) For cultivation of animal cell cultures, bovine serum albumin (BSA) is frequently used. This may be dangerous with respect to Creutzfeld-Jacob disease. The serum can be replaced with hydrolysates of some plant proteins. Therefore, innovative “safe“ protein-less (BSAfree) cultivation media, containing hydrolysates of wheat proteins (mixtures of peptides), will be prepared and optimised. Their use for stationary and mixed cultures of animal cell lines in biopharmacy will be tested. (Franěk) 1.2 Signalling and signal transduction The regulation of morphogenesis is often connected with cell endomembrane system, and with the transport of material from cytoplasm to membranes or between individual types of membranes. With cell membrane system a range of signalling systems is coupled. The analysis will be performed of factors regulating plant cell morphogenesis with the special focus on small regulatory GTPases (Rab, Rho, Arf) and interacting regulators and protein complexes. The plant geranylgeranyl transferase complex responsible for post-translational Rab GTPase prenylation will be characterised. The characterisation will be continued of subunits of the plant Exocyst complex, especially the Exo70 one which, in contrast to other eukaryotes, is present in multiple isoforms in plants. The complex of formin-associated proteins will be studied in relation to the dynamics of the plant cytoskeleton and the mobility and localisation of endomembrane compartments. (Žárský, Hála) 36 IRP identification code AV0Z50380511 The participation of PLD and phosphatidic acid (PA) will be analysed with special emphasis on the control of metabolism of components of endomembrane metabolism in the plant cell. Special attention will be concentrated on PLD as a potential microtubule-associated protein (MAP) regulating an interaction of microtubules with the plasmalemma and at the same time intervening in the actin dynamics. A new topic will be an analysis of the influence of PLD on membrane compartments – especially the Golgi apparatus. Mutants of Arabidopsis will serve as a basic experimental material, recombinant plant proteins will be expressed in bacteria and their interactions will be observed in vitro or in the yeast two-hybrid system. Relevant antibodies will be used also for immunofluorescence localisation in cells. In parallel, the expression of relevant genes for these proteins in fusion with GFP and functional analysis will be performed on transgenic plants as well as using detailed phenotype analysis of mutants (predominantly Arabidopsis). (Žárský) Molecular and biochemical characterisation of a novel plant enzyme – phosphatidylcholinehydrolysing phospholipase C (PC-PLC) - will be performed in relation to its potential role in plant signalling systems. Functional analysis will continue. Classical approaches to molecular genetics (cloning of homologous sequences, expression and biochemical characterisation of recombinant protein) will be used to describe proteins highly homologous with bacterial PC-PLC. Intracellular localisation of the protein will be determined using PCPLC-GFP construct, promoter will be studied using GUS constructs and functional analysis of protein(s) will be performed using RNAi. (Martinec) The revealing of mechanisms of signal transduction is of key importance for the understanding of the interactions between plants and environmental factors (physical, chemical, or pathogens). The molecular basis of aluminium toxicity in plants will be elucidated from the point of view of the role of phospholipid-cytoskeleton signalling. The role of phospholipases will be studied in situ using fluorescence-labelled substrates and specific inhibitors for individual phospholipases. Tobacco cell culture will be used for these experiments. Interaction between phospholipases and the cytoskeleton will be observed using purified plant actin and tubulin. Proteins showing differential binding with actin before and after treatment with aluminium ions will be further tested for phospholipase activity and identified using specific antibodies and MALDI-TOF mass spectrometry. Constructs with GFP of genes for phospholipases detected as above will be prepared and fusion proteins visualised in A. thaliana during stress reaction to aluminium ions. Direct protein-protein interactions will be proven by fluorescence resonance energy transfer (FRET) technology using GFP-fusion proteins. A similar experimental approach (namely measurement of phospholipase activities in situ) will be used to understand the role of phospholipid signalling systems in biotic stress, especially in the defence mechanisms of Brassica napus against the fungal pathogen Leptosphaeria maculans. (Martinec, Burketová) Plant defence mechanisms against plant pathogenic agents will be studied, with the emphasis on their induction by synthetic and natural environment-friendly compounds, with perspective of the application to crop plants with minimised impact on the environment. Both the early response of plants to infection and/or inductor (the role of phospholipases) and the involvement of signalling pathways (pathway of jasmonic and salicylic acids) and the production of defence compounds (PR-proteins) in the process will be characterised. (Burketová, Šindelářová) 2. 2.1 GENOME STRUCTURE AND FUNCTION Basics of structural and functional genomics Isolated chromosomes are an ideal material for investigation of structure and function of genome, the knowledge of which is the prerequisite for understanding of regulation of growth and development. Three groups of plant species will be used for the analysis of plant genome structure and its evolution. 37 IRP identification code AV0Z50380511 In Banana, construction of a fine physical cytogenetic map will be a main goal. The map will help to unravel chromosome changes accompanying evolution and speciation within the genus Musa. In the same genus, dynamics of repetitive DNA sequences accompanying evolution of cultivated forms will be followed. In Legumes, procedures based on the use of sorted chromosomes will be used for targetted isolation of molecular markers and physical mapping with the aim of aiding the cloning of agronomically important genes. In cereals, the main focus will be on the use of flow-sorted chromosomes for high-resolution physical cytogenetic mapping and construction of unique chromosome-specific BAC libraries. Clones selected from these libraries will be used to develop physical maps from regions of interest and, in collaboration with other laboratories, important genes will be isolated. New avenues for using sorted chromosomes in highthroughput physical mapping of ESTs and in vitro (HAPPY) mapping will be explored. (Doležel, Šimková, Lysák, Valárik) Pollen tube, as a single intensively growing cell, is one of the most suitable materials for the investigation of transcription. The male gametophyte of tobacco will be used for studies on regulation of transcription and translation. A detailed analysis of previously described EPP particles containing stored ntp303 transcripts will be performed. The role of the cytoskeleton on the developmentally induced translational regulation of gene expression in tobacco male gametophyte will be characterised. Currently available bioinformatic tools and reverse genetic resources will be exploited to reveal, on a genome-wide scale, the network of gametophytic transcription factors that regulate the start and flawless progress of the male gametophyte developmental programme during Arabidopsis pollen maturation. (Honys, Čapková) Studies on the mechanisms underlying regulation of pollen development will be oriented on analysis of the properties and functions of glycoproteins specific for the critical developmental stages. Emphasis will be placed on sequencing, regulation of expression and on the role of thermostable and dehydrin-like pollen proteins as potential markers and genetic sources of tolerance to abiotic stresses. (Hrubá, Tupý) New genes that encode proteins associated with microtubules and microtubule organising proteins will be cloned in order to describe molecular mechanisms of plant microtubule nucleation and acentrosomal organisation, one of the, so far, not well explained phenomena in cell biology. (Cenklová, Binarová) The majority of processes in organisms proceed in a rhythmical way. Therefore, the investigation of mechanisms of rhythmicity is nowadays in the centre of interest. Using real time RT-PCR, we will study the expression of genes connected with rhythmicity and flowering (especially the gene CONSTANS) in Chenopodium rubrum. The influence of light regime, various phytohormones and melatonin on the expression of these genes will be studied in connection with flowering. Some CONSTANS-like genes expressed in flower buds will be studied as well. (Štorchová, Kolář, Macháčková) Both mutagens in the environment and UV-irradiation often cause DNA damage. Majority of organisms, including plants, possess mechanisms to eliminate this damage, so-called DNA repair. The interactions among DNA repair pathways will be studied in A. thaliana by characterisation of T-DNA knock-out mutants and various types of DNA damage repair in mutants will be analysed by the method of Comet Assay. The kinetics of the DNA repair and its connection to the cell cycle will be observed. The main goal of our research will be to induce the adaptation to genotoxic stress and to induce programmed cell death by DNA damage. (Angelis) Together with the DNA repair in Arabidopsis, the DNA repair of DNA double-strand breaks will be monitored in Physcomitrella patens, the moss, which has an extremely high level of homologous recombination, compared with other plant species. The aim of this project is to develop an expression system for desired proteins by using targetted integration into the coding sequences („gene targetting“) into the plant genome. (Angelis) 38 IRP identification code AV0Z50380511 2.2 Molecular aspects of plant virology and phytopathology There is a possibility that in the future plants could produce a range of pharmacologically important proteins. For this purpose, it is necessary to elaborate reliable systems of transgenosis and to optimise the expression of transgenes. Using the effective system of agroinfection, the possibility of producing pharmaceutically useful proteins, such as scFv antibodies, viral proteins for vaccination, and others will be tested. In cooperation with specialised teams, the immunological features of these substances will be confirmed. The existing experience with the production of the coat proteins of human papillomavirus will be used in confirming the possibility of using pseudovirions as the means of targetting DNA sequences to the plant genome (and the development of DNA vaccines). In „edible vaccine“ development, the expression of defined sequences in edible tissues of transgenic plants will be tested in each step. Emphasis will be put on defining the yield and stability, as well as storage possibilities and dispersal of the protein in a given tissue. In addition to production tomato plants, other plants will be tested for their feasibility of production and edibility. (Angelis) Plant viruses harm a range of crops. The knowledge of mechanisms of interaction between viruses and plants can help to defend against viruses effectively. Molecular and biological features of potato viruses will be characterised on the basis of: • the known and complete nucleotide and amino acid sequences of selected isolates, • the prediction of a 3-D structure, • the phylogenetic analysis and the studies of protein-protein, protein-RNA interactions, • the preparation and use of antibodies to nonstructural viral proteins. The results obtained will lead to a detailed description of the viral life cycle and introduction of simple and reliable methods of detection and classification. (Čeřovská, Moravec) Other types of research will concentrate on protein and genome analysis of the fungus Venturia inaequalis and apple varieties resistant to this pathogen in order to gain knowledge of the molecular mechanisms underlying plant-pathogen interactions, and to identify molecular markers of qualitative and quantitative resistance to the plant pathogen. These markers will be used for further breeding. (Juříček, Juříčková, Tupý) 2.3 Contribution to organisation on international scientific activity concerning research on Arabidopsis thaliana Following the 2000 completion of the Arabidopsis genome sequence by the Arabidopsis Genome Initiative, the international community of scientists has developed a long-range plan for the „Multinational Coordinated Arabidopsis thaliana Functional Genomics Project“. The mission of this project is to achieve complete understanding of the biology of the flowering plant A. thaliana, which is used as a model plant. Arabidopsis researchers representing 14 countries from all around the world involved in the project have already established the Multinational Arabidopsis Steering Committee (MASC). Eastern European Arabidopsis research groups and laboratories are not yet involved in the Project and are not represented on MASC. After correspondence with Dr. Rebecca Joy, coordinator of the MASC, Dr. Fellner has initiated a project called Eastern Europe Arabidopsis Community (EEAC). The goal of this project is to involve Eastern European countries in international Arabidopsis research and in the Arabidopsis Functional Genomics Project. (Fellner) 2.4 Potential practical applications Some of the approaches leading to practical use of our research have already been mentioned above. They include: • production of pharmaceutically useful proteins (scFv antibodies, viral proteins for vaccination, other edible vaccines), • expression of verified sequences in edible tissues of transgenic plants (besides 39 IRP identification code AV0Z50380511 • • production tomato plants, other feasible plant species will be tested), development of pseudovirion system of human papillomavirus for oral administration of DNA/vaccines, establishment and further development of the methods of production of pharmaceutically important proteins in liquid culture of the moss Physcomitrella patens (this will be achieved by extending the possibilities of targetted modification of the genome of the moss due to homologous recombination, which will be studied in connection with DNA repair in plants). The possible use of modified plant vectors for the production of therapeutically effective substances will be looked into (use of suppressors of post-transcriptional gene silencing for optimal expression of recombinant proteins in plants). (Angelis) The appearance of heavy metals in the environment and their harmful effects on organisms are one of the serious ecological problems. The genotoxic effect of heavy metals, contaminating the soil in the area of North Bohemia, will be explored, as well as the genotoxic effect of by-products of water disinfection by chlorine and chloramin. The Comet Assay, hellow DNA test for the estimate of DNA damage and apoptosis, somatic mutation frequency test and homologous recombination test on tobacco (Nicotiana tabacum var. Xanthi) will be used to study the above-mentioned effects. Potato plants will be used for in situ experiments in contaminated soil. (Stavreva, Gichner) Transgenosis is potentially very useful tool how to introduce required properties (as e.g. resistance to insects or herbicides, some qualitative or quantitative features) into plants. Transgenic potato plants with improved agricultural characteristics will be produced: • selected potato cultivars carrying a modified gene for phosphofructokinase from Lactobacillus bifidus (gene LbPFK), adapted for translational apparatus of a plant; these plants will be transformed with a vector carrying no selection marker of antibiotic resistance, • potato plants carrying silk proteinase inhibitor gene from Galleria mellonella (protection against pathogenic microorganisms). (Navrátil) The transgenosis technique of microprojectile bombardment of transgenes will be further optimised. The expression and penetration of introduced genes will be monitored in the progeny of transgenic plants. Both molecular and phenotypical analyses will be performed in haploid, dihaploid, polyhaploid and diploid populations. (Ohnoutková) The process of breeding needs quality and homogeneous starting material. For this purpose the material obtained by androgenesis in vitro is very suitable. The key factors inducing and affecting the sporophyte development of male gametes – by androgenesis in vitro – will be explored in seedy plants. The ability of dioecious plants with heterogametous male sex to androgenously develop microspores with different sex chromosomes will also be studied. The in vitro method for obtaining haploid, dihaploid and polyhaploid Solanum plants will be introduced. This method will enable hybridisation between wild-type diploid species carrying the resistance genes and tetraploid cultivars of potato. Isogenisation of some Triticale with desired gene (chromosomal) translocations will be achieved by induced androgenesis. Together with other applied research teams and breeding stations, dihaploid lines of promising barley and wheat will be prepared. (Ohnoutková) The long-term project on breeding, advanced testing, legal protection and commercialisation through licensing of apple varieties resistant against fungal diseases and with low requirements for plant protection chemical use and, therefore, suitable for organic farming will continue. The project also includes breeding of varieties with a compact, columnar growth habit derived from McIntosh Wijcik mutant. The aim of the programme is to improve growing characteristics of the trees, marketing quality of fruit and more durable resistance to scab by combining the Vf resistance derived from Malus floribunda with polygenic tolerance of genetic sources we have selected previously. The genetic background of resistance of selected hybrids will be characterised with the use of molecular markers. (Tupý, Juříček) 40 IRP identification code AV0Z50380511 C6. Strategies and methods to be applied to carry out IRP Due to the complexity of the research proposal, only the more general methods and approaches are described in detail. Specific methods and approaches are, when necessary, described directly in the research proposal (part C5). The research will run in parallel in all studied areas and it will be our aim both to integrate all the directions as much as possible and to use all available methods, apparatuses and approaches most effectively. It will be an obvious part of most research directions to improve the existing methods and to develop new ones. 1. Determination of the concentrations of phytohormones and other growth regulators Determination of the levels of phytohormones (namely auxin, cytokinins,ABA and ethylene) and other regulators (polyamines, melatonin) is an integral part of the research on regulation of plant growth and development. The methods for determination of these substances are well established in the IEB and on a very good level. Recently, a new method of extract purification on two-parameter columns was elaborated and we can now determine cytokinins, IAA and ABA in one extract. Liquid chromatography is used for the determination, with mass spectrometric detection for cytokinins and fluorimetric detection for auxin. Gas chromatography is used for ABA and ethylene determinations. At present, a new determination method is being developed for ABA and IAA using GC/MS. A method was developed for immunohistochemical localisation of cytokinins (free bases) in vivo and a similar method will be developed for IAA as well. There will be new antibodies produced against further types of cytokinins, IAA, ABA jasmonic acid and brassinosteroids. These antibodies will be used for preparation of immunoaffinity columns for extract purification, for ELISA tests and for imunohistochemical determination in situ. 2. Studies of transport, metabolism and mechanism of action of growth regulators For the studies of transport and metabolism of hormones, radioactively labelled substances will often be used. These are, in many cases, synthesised in the Izotope laboratory of IEB. (it mostly concerns substances labelled with tritium and with very high specific radioactivity). For detection of these substances, HPLC will be used with a radiometric detector. The Isotope laboratory will synthesise not only labelled substances, but also substances needed for various research directions, especially new groups of CDK inhibitors. The mechanism of action of phytohormones will be studied at the level of binding sites (receptors). For these studies, radioactively labelled ligands will be used and also transgenic tobacco cell cultures with changed expression of the gene for the receptor. A similar strategy will be used in the studies of auxin and cytokinin influx and efflux carriers. In this case, inhibitors of auxin transport (TIBA, NPA, brefeldin) will be also used. Other inhibitors will be used for studies of cytokinin biosynthesis in chloroplasts, inhibitors of both pathways of isoprenoid biosynthesis, lovastatin and fosfidomycin, respectively. Also, in studies of polyamine synthesis inhibitors, difluoromethyl ornithine and cyclohexyl amine - will be used. 3. Use of mutants In several research directions, mutants will be used. In most cases it will be mutants of Arabidopsis thaliana, namely T-DNA mutants abp15 and abm19 with changed reaction to light and changed sensitivity to auxin, mutants with altered auxin transport and mutants defective in reparation genes: AtSpo3-11, AtArf1, Att3B, AtTop6B, AtRad17, AtBrcA1 and AtFandC2 and mutants in non-homologous recombination of double-strand DNA breaks AtKu70 and AtKu80. Spontaneous tomato mutant 7b-1, with altered sensitivity to ABA and increased resistance to osmotic stress, will also be used. 41 IRP identification code AV0Z50380511 4. Transgenic plants Besides mutants, transgenic plants are also an important tool in modern plant biology.. In the phytohormone research, transgenic plants of A. thaliana and tobacco carrying the genes for isopentenyl transferase (IPT), cytokinin oxidase (CKX), and proteins involved in auxin transport will be used under the control of various promoters – constitutive (CaMV35S promoter) and inducible (promoters Palc and PSAG12 as well as dexamethason-inducible one). We will also use transgenic tobacco plants with the genes for zeatin-O-glucosyl transferase or specific glucosidase (Zm-P60.1) and transgenic tobacco and Arabidopsis plants with increased expression of genes for proteins of signalling pathways. Plants carrying constructs of various genes with green or yellow fluorescent protein (GFP, YFP) or rsRed will also be prepared and these will be used for studies of the expression of studied genes and their localisation using fluorescence microscopy. Plants with fusions of promoters of some genes and GUS (β-glucuronidase) will also be constructed for this purpose. In addition, cereals will be prepared carrying genes PHYT (for phytase, increasing the phosphate availability) and DAP A (for dihydropicolinate synthase A, increasing lysine content) and also potato plants carrying the gene for phosphofructokinase, which lowers level of reducing sugars. Last, but not least, transgenic plants will be used for elaborating a system of production of edible (oral) vaccines (namely tomato). Transformation will be carried out using both Agrobacterium tumefaciens and gene gun. 5. Microscopic techniques In the area of phytohormone research, immunohistochemical determination of cytokinin free bases and of auxins (see above) will be performed. In signalling studies the activity of phospholipases in vivo will be measured using fluorescently labelled substrates from the BODIPY Comp. The study of expression of the constructs with fluorescent proteins (GFP, YFP, dsRed) will also be performed at microscopic level. Not just a fluorescent microscope will be used, but also a two-photon confocal microscope and techniques such as FRET (fluorescence resonance energy transfer) and FRAP (fluorescence recovery after photobleaching). Microscopic analysis will be also used in studies of cytoskeleton (staining of actin and tubulins), especially in the course of cell cycle and in signalling. Image analysis techniques are elaborated for qualitative and quantitative evaluation of microscopic images. 6. Methods of plant genomics Besides those approaches already described, the method of DNA chips (microarrays), hybridisation techniques (Western, Northern blots), PCR and RT-PCR will be used for studies of gene expression. Functional analysis of some genes will be performed using small interfering RNA (RNAi). For some genes recombinant proteins will be prepared in yeasts and used for further studies. Antibodies will be prepared against some proteins and these will be used in localisation studies in situ. Functional interaction of proteins will be studied in the two-hybrid system. Flow cytometry will be used to sort chromosomes and sorted chromosomes from various plants will be used for the preparation of pure, chromosome specific, high molecular DNA and for construction of chromosome specific cDNA BAC libraries. Sorted chromosomes will also be used for localisation studies of some genes using FISH (fluorescence in situ hybridisation). 42 IRP identification code AV0Z50380511 D4. List of major implemented R&D results related to the subject of IRP achieved by the members of research team, within the period of 1999-2003 PUBLICATION LIST 1999-2003: 1999: IMPACTED JOURNALS: no. title 1 Angelis K.J., Dušínská M., Collins A.R.: 2 3 4 5 6 7 8 9 10 11 Single cell gel electrophoresis: Detection of DNA damage at different levels of sensitivity. Electrophoresis 20: 2133-2138, 1999. Angelis K.J., McGuffie M., Menke M., Schubert I.: Studies of DNA repair in various plants using the comet assay. Neoplasma 46: 72–73, 1999. Auer C. A., Motyka V., Březinová A., Kamínek M.: Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol. Plant. 105: 141-147, 1999. Bavrina T.V., Lozhnikova V.N., Macháčková I., Gryanko T.I.: Tobacco transformants to study the role of phytohormones in flowering and seed formation. Russ. J. Plant Physiol. 46: 189-193, 1999. Benková E., Witters E., Van Dongen W., Kolář J., Motyka V., Brzobohatý B., Van Onckelen H.A., Macháčková I.: Cytokinins in tobacco and wheat chloroplasts: occurrence and changes due to light/dark treatment. Plant Physiol. 121: 245-251, 1999. Bílková J., Albrechtová J., Opatrná J.: Histochemical detection and image analysis of nonspecific esterase activity and the amount of polyphenols during annual buds development in Norway spruce. J. Exp. Bot. 336: 1129-1138, 1999. Blažková J., Krekule J., Macháčková I., Procházka S.: Auxin and cytokinins in the release of apical dominance in pea – a differential response due to bud position. J. Plant Physiol. 154: 691-696, 1999. Bögre L., Calderini O., Binarová P., Mattauch M., Till S., Kiegerl S., Jonak C., Pollaschek C., Barker P., Huskisson N.S., Hirt H., Heberle-Bors E.: A MAP kinase is activated late in mitosis and becomes localized to the plane of cell division. Plant Cell 11: 101-113, 1999. Burketová L., Šindelářová M., Ryšánek P., Šindelář L.: Changes in ribonuclease and glucose-6-phosphate dehydrogenase activities induced by beet necrotic yellow vein virus in sugar beet. Biol. Plant. 42: 423 – 430, 1999. Burketová L., Šindelářová M., Šindelář L.: Benzothiadiazole as an inducer of β-1,3-glucanase and chitinase isozymes in sugar beet. Biol. Plant. 42: 279-287, 1999. Cvikrová M., Binarová P., Cenklová V., Eder J., Macháčková I.: Reinitiation of cell division and polyamine and aromatic monoamine levels in alfalfa explants during the induction of somatic embryogenesis. Physiol. Plant. 105: 330-337, 1999. 43 IF 1999 3.447 0.448 2.460 0.094 4.434 2.482 1.143 10.463 0.414 0.414 2.460 IRP identification code AV0Z50380511 12 13 14 15 16 17 18 19 20 21 22 23 24 Cvikrová M., Binarová P., Eder J., Vágner M., Hrubcová M., Zoń J., Macháčková I.: Effect of inhibition of phenylalanine ammonia lyase activity on growth of alfalfa cell suspension culture: Alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins, and polyamines. Physiol. Plant. 107: 329-337, 1999. Cvrčková F., Žárský V.: Ntrop1, a tobacco (Nicotianna tabacum) cDNA encoding a Rho subfamily GTPase expressed in pollen (accession No AJ222545) (PGR 99-079). Plant Physiol. 120: 633, 1999. Čeřovská N., Moravec T., Filigarová M., Ryšlavá H., Grosclaude J.: Partial antigen characterization of different potato virus Y-NTN isolates with monoclonal antibodies by means of competitive binding tests and immunoblotanalysis. Acta Virologica 43: 391-393, 1999. Dewitte W., Chiappetta A., Azmi A., Witters E., Strnad M., Rembur J., Noin M., Chriqui D., Van Onckelen H.: Dynamics of cytokinins in apical shoot meristems of a day neutral tobacco during floral transition and flower formation. Plant Physiol. 119: 111-122, 1999. Dršata J., Netopilová M., Tolman V.: Stereoisomers of 4-fluoroglutamic acid: influence on Escherichia coli glutamate decarboxylase. Pharmazie 54: 713-714, 1999. Ehrenbergová L., Vaculová K., Zimolka J., Müllerová E.: Výnosové znaky a jejich vztahy k jakostním ukazatelům zrna bezpluchého ječmene jarního. Rostl. Výr. 45: 53-59, 1999. Ephritikhine G., Fellner M., Vannini C., Lapous D., Barbier-Brygoo H.: The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and its partially rescued by exogenous brassinosteroid. Plant J. 18: 303-314, 1999. Fujikura Y., Doležel J., Číhalíková J., Bögre L., Binarová P.: Vicia faba germination: Synchronized cell growth and localization of nucleolin and alpha-tubulin. Seed Sci. Res. 9: 297-305, 1999. Gichner T., Ptáček O., Stavreva D.A., Plewa M.J.: Comparison of DNA damage in plants as measured by single cell gell electrophoresis and somatic leaf mutation induced by monofunctional alkylating agents. Environ. Mol. Mutagen. 33: 279-286, 1999. Gichner T., Velemínský J.: Monitoring the genotoxicity of soil extracts from two heavily polluted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays. Mut. Res. 426: 163-166, 1999. Haisel D., Pospíšilová J., Synková H., Čatský J., Wilhelmová N., Plzáková Š.: Photosynthetic pigments and gas exchange of in vitro grown tobacco plants as affected by CO2 supply. Biol. Plant. 42: 463-468, 1999. Hrubá P., Tupý J.: N-glycoproteins specific for different stages of microspore and pollen development in tobacco. Plant Sci. 141: 29-40, 1999. Jansa J., Gryndler M., Matucha M.: Comparison of the lipid profiles of arbuscular mycorrhizal (AM) fungi and soil saprophytic fungi. Symbiosis 26: 247-264, 1999. 44 2.460 4.434 0.476 4.434 0.446 0.192 5.098 0.942 1.998 2.107 0.414 1.015 0.766 IRP identification code AV0Z50380511 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Lebeda A., Křístková E., Doležal K.: Peroxidase isozyme polymorphysm in Cucurbita pepo cultivars with various morphotypes and different level of field resistance to powdery mildew. Scientia Horticulturae 81: 103-112, 1999. Lysák M.A., Doleželová M., Horry J.P., Swennen R., Doležel J.: Flow cytometric analysis of nuclear DNA content in Musa. Theor. Appl. Genet. 98: 1344-1350, 1999. Lysák M.A., Číhalíková J., Kubaláková M., Šimková H., Künzel G., Doležel J.: Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chrom. Res. 7: 431-444, 1999. 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Acta Virologica 47(1): 37-40, 2003. 309 Doležel J., Bartoš J., Voglmayer H., Greilhuber J.: Nuclear DNA content and genome size of trout and human. Cytometry 51: 127-128, 2003. 310 Doležel J., Carter N.P., Ferguson-Smith M.: Chromosomes go with the flow. Chrom. Res. (in press). Doležel J., Kubaláková M., Bartoš J., Macas J.: 311 Flow cytogenetics and plant genome mapping. Chrom. Res. (in press) 312 Dryková D., Cenklová V., Sulimenko V., Volc J., Dráber P., Binarová P.: Plant γ-tubulin interacts with α-,β-tubulin dimers and forms membrane associated complexes. Plant Cell 15(2): 465-480, 2003. 313 Eliáš M., Drdová E., Žiak D., Bavlnka B., Hála M., Cvrčková F., Soukupová H., Žárský V.: The exocyst complex in plants. Cell Biol. Int. 27(3): 199-201, 2003. Fellner M., Horton L.A., Cocke A.E., Stephens N.R., Ford E.D., Van 314* Volkenburgh E.: Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings. Planta 216: 366-376, 2003. 315 Filek M., Biesaga-Koscielniak J., Marcinska I., Krekule J., Macháčková I., Dubert F.: The effects of electric current on flowering of grafted of non-vernalized winter rape. Biol. Plant. 46(4): 625-628, 2003. 66 0.583 0.583 1.556 1.582 0.984 0.567 0.660 1.933 1.828 1.828 10.751 1.017 2.960 0.583 IRP identification code AV0Z50380511 316 Franěk F., Eckschlager T., Katinger H.: 1.734 317 0.848 318 319 320 321 322 323 324 325 326 327 328 329 Enhancement of monoclonal antibody production by lysine-containing peptides Biotechnol. Progr. 19(1): 169-174, 2003. Franěk F., Eckschlager T., Kohout L.: 24-Epibrassinolide at subnanomolar concentrations modulates growth and production characteristics of a mouse hybridoma. Collect. Czech Chem. Commun. 68(11): 2190-2200, 2003. Gichner T. : DNA damage induced by indirect and direct acting mutagens in catalasedeficient transgenic tobacco. Cellular and acellular Comet assays. Mutation Res. 535(2): 187-193, 2003. Gichner T., Patková Z., Kim J.K.: DNA damage measured by the Comet assay in eight agronomic plants. Biol. Plant. 47(2): 185-188, 2003/2004. Gichner T.: Differential genotoxicity of ethyl methanesulphonate, N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays. Mutat. Res. – Genet. Toxicol. Env. Mutag. 538(1-2): 171-179, 2003. Honys D., Twell D.: Comparative analysis of Arabidopsis pollen transcriptome. Plant Physiol. 132(2): 640-652, 2003. Kamínek M., Trčková M., Fox J.E., Gaudinová A.: Comparison of cytokinin-binding proteins from wheat and oat grains. Physiol. Plant. 117(4): 453-458, 2003. Kirschner J., Štěpánek J., Mes T.H.M., den Nijs J.C.M., Oosterveld P., Štorchová H., Kuperus P.: Principal features of the cpDNA evolution in Taraxacum (Asteraceae, Lactuceae): a conflict with taxonomy. Plant Syst. Evol. 239(3-4): 231-255, 2003. Kolář J., Johnson C.H., Macháčková I.: Exogenously applied melatonin (N-acetyl-5-methoxytryptamine) affects flowering of the short-day plant Chenopodium rubrum L.. Physiol. Plant. 118(4): 605-612, 2003. Kubaláková M., Valárik M., Bartoš J., Vrána J., Číhalíková J., MolnárLáng M., Doležel J.: Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46: 893-905, 2003. Lalanne E., Honys D., Johnson A., Borner G., Dupree P., Grossniklaus U., Twell D.: AtPIGC and AtPIGA, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. Plant Cell 2003, (accepted). Laukens K., Lenobel R., Strnad M., Van Onckelen H., Witters E.: Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase. FEBS Lett. 533(1-3): 63-66, 2003. Lexa M., Genkov T., Malbeck J., Macháčková I., Brzobohatý B.: Dynamics of endogenous cytokinin pools in tobacco seedlings: a modeling approach. Ann. Bot. 91: 585-597, 2003. Kuthanová A., Gemperlová L., Zelenková S., Eder J., Macháčková I., Opatrný Z., Cvikrová M.: Cytological changes and alterations in polyamine contents induced by cadmium in tobacco BY-2 cells. Plant Physiol. Biochem. (accepted) 67 1.636 0.583 1.636 5.800 1.565 1.114 1.565 1.815 10.751 3.912 1.476 1.582 IRP identification code AV0Z50380511 330 Mandák B., Pyšek P., Lysák M., Suda J., Krahulcová A., Bímová K.: 1.476 331 5.271 332 333 334 335 336 337 338 339 340 341 342 343 344 Variation in DNA-ploidy levels of Reynoutria taxa in the Czech Republic. Ann. Bot. 92(2): 265-272, 2003. Matsunaga S., Isono E., Kejnovský E., Vyskot B., Doležel J., Kawano S., Charlesworth D.: Duplicative transfer of a MADS box gene to a plant Y chromosome. Mol. Biol. Evol. 20(7): 1062-1069, 2003. Matucha M., Forczek S.T., Gryndler M., Uhlířová H., Fuksová K., Schroder P.: Trichloroacetic acid in Norway spruce/soil-system I. Biodegradation in soil. Chemosphere 50(3): 303-309, 2003. Moravcová D., Kryštof V., Havlíček L., Moravec J., Lenobel R., Otyepka M., Strnad M.: Pyrazolo[4,3-d]pyrimidines as new generation of cyclindependent kinase inhibitors. Bioorg. Med. Chem. Lett. 13(18): 2989-2992, 2003. Moravec J., Kryštof V., Hanuš J., Havlíček L., Moravcová D., Fuksová K., Kuzma M., Lenobel R., Otyepka M., Strnad M.: 2,6,8,9-Tetrasubstituted purines as new CDK1 inhibitors. Bioorg. Med. Chem. Lett. 13(18): 2993-2996, 2003. Moravec T., Čeřovská N., Boonham N.: The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVYNTN) using a three-primer PCR based in the coat protein gene. J. Virol. Meth. 109: 63-68; 2003. Motyka V., Vaňková R., Čapková V., Petrášek J., Kamínek M., Schmulling T.: Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol. Plant. 117(1): 11-21, 2003. Naganowska B., Doležel J., Świecicki W.K.: Development of molecular cytogenetics and physical mapping of ribosomal RNA genes in Lupinus. Biol Plant. 46(2): 211-215, 2003. Novák J., Vlasáková V., Tykva R., Ruml T.: Degradation of juvenile hormone analog by soil microbial isolate. Chemosphere 52: 151-159, 2003. Novák O., Tarkowski P., Tarkowská D., Doležal K., Lenobel R., Strnad M.: Quantitative analysis of cytokinins in plants by liquid chromatography/single qadrupole mass spectrometry. Anal. Chimica Acta 480(2): 207-218, 2003. Novotná Z., Hynek R., Martinec J., Potocký M., Valentová O.: Plant PIP2-dependent phospholipase D activity is regulated by phosphorylation. FEBS Lett. 554(1-2): 50-54, 2003. Novotná Z., Martinec J., Profotová B., Žďárová Š., Kader J.-C., Valentová O.: In vitro distribution and characterization of membrane associated PLD and PIPLC in Brassica napus. J. Exp. Bot. 54: 691-698, 2003. Palomino G., Doležel J., Méndez I., Rubluo A.: Nuclear genome analysis in Agave tequilana Weber through flow cytometry. Caryologia 56(1): 37-46, 2003. Pechová R., Kutík J., Holá D., Kocová M., Haisel D., Vičánková A.: The ultrastructure of chloroplasts, content of photosynthetic pigments, and photochemical activity of maize (Zea mays L) as influenced by different concentrations of the herbicide amitrole. Photosynthetica 41(1): 127-136, 2003. Petrášek J., Černá A., Schwarzerová K., Elčkner M., Morris D.A., Zažímalová E.: Do Phytotropins Inhibit Auxin Efflux by Impairing Vesicle Traffic? Plant Physiol. 131: 254-263, 2003. 68 1.461 2.051 2.051 1.938 1.565 0.583 1.461 2.114 3.912 2.852 0.267 0.773 5.800 IRP identification code AV0Z50380511 345 Pokorná J., Schwarzerová K., Zelenková S., Petrášek J., Janotová I., 3.015 346 0.583 347 348 349 350 351 352 353 354 355 356 402 357 Čapková V., Opatrný Z.: Sites of actin filament initiation and re-organization in cold-treated tobacco cells. Plant Cell Environ., in press 2004. Pospíšilová J.: Participation of phytohormones in the stomatal regulation of gas exchange during water stress. Biol. Plant. 46(4): 491-506, 2003. Pospíšilová J.: Interaction of cytokinins and abscisic acid during regulation of stomatal opening in bean leaves. Photosynthetica 41: 49-56, 2003. Potocký M., Eliáš M., Novotná Z., Profotová B., Valentová O., Žárský V.: Phosphatidic acid produced by PLD is necessary for pollen tube growth. Planta 217(1): 122-130, 2003. Procházková, D., Wilhelmová, N.: Changes in antioxidative protection in bean cotyledons during natural and continuous irradiation-accelerated senescence. Biol. Plant. 48: accepted, 2004 Roman B., Satovic Z., Požárková D., Macas J., Doležel J., Cubero J.I., Torres A.M.: Development of a composite map in Vicia faba, breeding applications and future prospects. Theor. Appl. Genet. (in press). Roux N., Toloza A., Radecki Z., Zapata-Arias F.J., Doležel J.: Rapid detection of aneuploidy in Musa using flow cytometry. Plant Cell Rep. 21: 483-490, 2003. Ryšlavá H., Muller K., Semorádová Š., Synková H., Čeřovská N.: Photosynthesis and activity of phosphoenolpyruvate carboxylase in Nicotiana tabacum L. leaves infected by Potato virus A and Potato virus Y. Photosynthetica (in press) Sáenz L., Jones L.H., Oropeza C., Vláčil D., Strnad M: Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.). Plant Growth Regul. 39(3): 205-211, 2003. San Martin A.P.M., Adamec L., Suda J., Mes T.H.M., Štorchová H.: Genetic variation within the endangered species Aldrovanda vesiculosa (Droseraceae) as revealed by RAPD analysis. Aquatic Bot. 75(2): 159-172, 2003. Schröder P., Matucha M., Forczek S.T., Uhlířová H., Fuksová K., Albrechtová J.: Uptake, translocation and fate of trichloracetic acid in Norway spruce/soil system. Chemosphere 52(2): 437-442, 2003. Schwarzerová K., Pokorná J., Petrášek J., Zelenková S., Čapková V., Janotová I., Opatrný Z.: The structure of cortical cytoplasm in cold-treated tobacco cells: the role of the cytoskeleton and the endomembrane system. Cell Biol. Int. 27(3): 263-265, 2003. Stirk W.A., Novák O., Strnad M., van Staden J.: Cytokinins in makroalgae. Plant Growth Regul. 41(1):13-24, 2003. Strnad M., Kohout L.: Simple brassinolide analogue 2α,3α-dihydroxy-17β-(3methylbutyryloxy)-7oxa-B-homo-5α-androstan-6-one inducing the bean second internode splitting. Plant Growth Regul. 40(1): 39-47, 2003. 69 0.773 2.960 0.583 2.264 1.340 0.773 0.850 1.014 1.461 1.017 0.850 0.850 IRP identification code AV0Z50380511 358 Sun J., Niu Q.W., Tarkowski P., Zheng B., Tarkowská D., Sandberg G., 5.800 359 0.773 360 361 362 363 364 365 366 367 368 369 370 Chua N.-H., Zuo J.: The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyltransferase that is involved in de novo cytokinin biosynthesis. Plant Physiol. 131: 167-176, 2003. Synková H., Pechová R., Valcke R.: Changes in th echloroplast structure in PSSU-ipt tobacco during plant ontogeny. Photosynthetica 41: 117-126, 2003. Šimková H., Čihalíková J., Vrána J., Lysák M., Doležel J.: Preparation of high molecular weight DNA from plant nuclei and chromosomes isolated from root tips. Biol. Plant. 46(3): 369-373, 2003. Šindelář L., Šindelářová M.: Hexokinases of tobacco leaves: changes in the cytosolic and non-cytosolic isozyme complexes induced by tobacco mosaic virus infection. Biol. Plant. 47: 413-419, 2003/2004. Šindelářová M., Šindelář L.: Changes in glucose-6-phosphate dehydrogenase, ribonucleases and esterases and content of viruses in potato virus Y infected tobacco superinfected with tobacco mosaic virus. Biol. Plant. 47: 99-104, 2003/2004. Šindelářová M., Šindelář L.: Influence of antiviral factor on tobacco mosaic virus RNA biosynthesis in tobacco. Biol. Plant. 46(1): 95-100, 2003. Tarkowská D., Doležal K., Tarkowski P., Åstot C., Schmülling T., Holub J., Fuksová K., Sandberg G., Strnad M.: Identification of new aromatic cytokinins in Arabidopsis thaliana, poplar leaves and Agrobacterium tumefaciens strains by LC-(+)ESI-MS and capillary liquid chromatography/frit - fast atom bombardment mass spectrometry. Physiol. Plant. 117(4): 579-590, 2003. Tarkowská D., Kotouček M., Doležal K.: Elecrochemical reduction of 6-benzylaminopurine at mercury electrodes and its analytical application. Coll. Czechoslovak Chem. Comm. 68(6): 1076-1093, 2003. Trávníček B., Lysák M.A., Číhalíková J., Doležel J.: Karyo-taxonomic study of the genus Pseudolysimachion (Scrophulariaceae) in the Czech Republic and Slovakia. Folia Geobot. (in press). Trávníček Z., Maloň M., Zatloukal M., Doležal K., Strnad M., Marek J.: Mixed ligand complexes of platinum(II) and palladium(II) with cytokininderived compounds Bohemine and Olomoucine: X-ray structure of [Pt(BohH(+)-N7)Cl-3]center dot 9/5H(2)O {Boh=6-(benzylamino)-2-[(3(hydroxypropyl)-amino]-9-isopropylpurine, Bohemine} J. Inorg Biochem. 94: (4) 307-316, 2003. Tykva R., Šimek P., Benetová B., Holík J., Hlaváček J., Havlíček L.: Comparison for following the metabolism of oostatic peptides in Neobellieria bullata by mass spectrometry and radiolabelling. Coll. Czechoslovak Chem. Comm. (in press) Tykva R., Vlasáková V., Novák J., Havlíček L.: RadioHPLC for ecotoxicity assessment of insect growth regulators. J. Chromatography A (in press) Überal I., Vrána J., Bartoš J., Šmerda J., Doležel J., Havel L.: Isolation of chromosomes from Picea abies L. and their analysis by flow cytometry. Biol. Plant. (in press). 70 0.583 0.583 0.583 0.583 1.565 0.848 0.564 2.204 0.848 3.098 0.564 IRP identification code AV0Z50380511 371 Vagera J., Novotný J., Ohnoutková L.: 0.632 372 5.850 373 374 375 376 377 378 Induced androgenesis in vitro in mutated populations of barley, Hordeum vulgare L. Plant Cell, Tissue Org. Cult. (in press) 2003 Valárik M., Bartoš J., Kovářová P., Kubaláková M., de Jong J.H., Doležel J.: High-resolution FISH of super-stretched flow sorted plant chromosomes. Plant J. (in press) Veach Y., Martin R.C., Mok D.W.S., Malbeck J., Vaňková R., Mok M.C.: O-glukosylation of cis-zeatin in maize: characterization of genes, enzymes, and endogenous cytokinins. Plant Physiol. 131: 1374-1380, 2003. Vomáčka L., Pospíšilová J.: Rehydration of sugar beet plants after water stress: effect of cytokinins. Biol. Plant. 46(1): 57-62, 2003. Werner T., Hanuš J., Holub J., Schmülling T., van Onckelen H., Strnad M.: New cytokinin metabolites in ipt transgenic Arabidopsis thaliana plants. Physiol. Plant. 118(1): 127-137, 2003. Werner T., Motyka V., Laucou V., Smets R., van Onckelen H., Schmülling T.: Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in regulating shoot and root meristem activity. Plant Cell 15: 2532-2550, 2003. Zažímalová E., Napier R.: Points of regulation for auxin action. Plant Cell Rep. 21(7): 625-634, 2003. Zonia L., Feijó J.A.: State and spectral properties of chloride oscillations in pollen. Biophys. J. 84(2): 1387-1398, 2003. OTHERS: 379 Blagoeva E., Dobrev P. I., Vaňková R.: 380 381 382 383 384 Inhibition of cytokinin-glucose conjugation in derooted radish, tobacco and bean seedlings. Bulg. J. Plant Physiol. 29: (1-2), in press. Coufal D., Matucha P., Uhlířová H., Lomský B., Forczek S.T., Matucha M.: GUHA analysis of coniferous forest damage: Effects of trichloroacetic acid, sulphur, fluorine and chlorine on needle loss of Norway spruce. Neural Network World 13: 89-102, 2003. Dobrev P.I., Motyka V.: Stanovení aktivity cytokininoxidasy/dehydrogenasy v rostlinách pomocí HPLC spojené s průtokovým radioaktivním detektorem. (In Czech) Biol. listy 68: 163-166, 2003. Doležel J., Kubaláková M., Vrána J., Bartoš J.: Flow cytogenetics. In: Goodman R.M. (ed.): Encyclopedia of Plant and Crop Science. Marcel Dekker Inc., New York (in press) Doležel J., Macas J., Lysák M., Neumann P., Kubaláková M., Nouzová M., Šimková H., Koblížková A., Požárková D., Číhalíková J., Lucretti S.: Sorting of Plant Chromosomes and Construction of Chromosome-Specific DNA Libraries. In: Speel, E.J.M. and Hopman, A.H.N. (eds.): Chromosome Analysis Protocols, Humana Press, Totowa, USA (in press). Doležel J., Šafář J., Janda J., Bartoš J., Kubaláková M., Číhalíková J., Šimková H., Sourdille P., Bernard M., Chalhoub B.: Development of flow cytogenetics for wheat genome mapping. In: Proceedings of the Tenth International Wheat Genertics Symposium. Pp. 6568. Istituto Sperimentale per la Cerealicoltura, Rome, 2003. 71 5.800 0.583 1.565 10.751 1.340 4.643 - - - - - IRP identification code AV0Z50380511 385 Galbraith D.W., Bartoš J., Doležel J.: - 386 - 387 388 389 390 391 392 393 394 395 396 397 Flow cytometry and cell sorting in plant biotechnology. In: Sklar L.A. (ed.): Flow Cytometry in Biotechnology. Oxford Univ. Press, Inc., New York (in press) Gaudinová A., Vaňková R., Dobrev P.I., Motuyka V.: Extrakce a stanovení aktivity cytokinin N- a O-glukosyltransferas v rostlinných pletivech. (In Czech) Biol. Listy 68: 176-179, 2003. Genkov T., Vágner M., Dubová J., Malbeck J., Moore I., Brzobohatý B.: Increased ethylene production can account for some of the phenotype alterations accompanying activation of a cytokinin biosynthesuis gene, during germination and early seedluing development in tobacco. In: Biology and Biotechnology of the Plant Hormone Ethylene III, Eds.: Vendrell M., Klee H., Pech J.C., Romojaro F., Proc. of the NATO Advanced Workshop on Biology and Biotechnology of the Plant Hormone Ethylene, 23-27 April 2002, IOS Press, Amsterdam, 2003. Honys D., Twell D.: Male gametophyte. In Goodman R.M. 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Proc. of the NATO - Russia Workshop, May 2002, Moscow, Kluwer, Dordrecht, The Netherlands, pp. 79-87 (in press) Krinke O., Novotná Z., Valentová O., Martinec J.: Fluorometrická metoda pro in vitro měření ligandem otevíraných iontových kanálů pro Ca2+ v rostlinách. Biol. listy 68(3): 195-199, 2003. Lebeda, A., Doležalová, I., Dziechciarková, M., Doležal, K., Frček, J.: Morphological variability and isozyme polymorphisms in maca and yacon. Czech J. Genet. Plant Breed. 39: 1-8, 2003. Matucha M., Gryndler M., Forczek S.T., Uhlířová H., Fuksová K., Schröder P.: Chloroacetic acids in environmental processes. Environ. Chem. Letters 1,127-130,2003. Morris D.A., Friml J., Zažímalová E.: The transport of auxins. In Davies P.J. (ed): Plant Hormones: Biosynthesis, Signal Transduction, Action! New edition. Kluwer Acad. Publ., NY. (in press). Morris D.A., Zažímalová E.: Physiological and molecular genetic aspects of auxin transport: recent developments. In: Macháčková I., Romanov G.A. (eds): Phytohormones in Plant Biotechnology and Agriculture. Kluwer Acad. Publ. In press 2003. Pejchar P., Valentová O., Martinec J.: Stanovení aktivit rostlinných fosolipas in situ s využitím fluorescenčně značených substrátů. Biol. listy 68(3): 227-234, 2003. Pospíšilová J., Dodd I.C.: Role of plant growth regulators in stomatal limitation of photosynthesis during water stress. In: Pessarakli, M. (ed.): Handbook of Photosynthesis, Second Edition, Revised and Expanded. Marcel Dekker, New York , in press. 72 - - - - - - - IRP identification code AV0Z50380511 398 Uhlířová H., Novotný R., Matucha M.: - 399 - 400 401 Projevy poškození lesních dřevin pod vlivem abiotických stresů. Seminář „Vliv abiotických a biotických stresů na vlastnosti rostlin“, Výzk. úst. rostlinné výroby a ČZU Praha, 8.10.2003, Sborník přednášek, str. 76-89, 2003. Vágner M., Fischerová L., Špačková J., Vondráková Z.: Somatic embryogenesis of Norway spruce. In: Protocols of Somatic Embryogenesis – Woody Trees, Eds. Jain S.M., Gupta P.K., Kluwer Acad. Publ. (in press) Vaňková R., Gaudinová A.: Využití dvojrozměrné fluorescenční spektroskopie pro stanovení množství a viability buněk tabáku. Biol. listy 68(3): 250-253, 2003. Zažímalová E., Petrášek J., Morris D.A.: The dynamics of auxin transport in tobacco cells. Bulg. J. Plant Physiol., Spec. Iss., 207-224, 2003. - - * - indicates the papers of M. Fellner published during his study stay abroad without adress of IEB (they are listed above as his research became the part of research plan for years 2005-2010) PATENTS AND LICENCES 1999-2003: patents: FUKSOVÁ, K., HAVLÍČEK, L., KRYŠTOF, V., LENOBEL, R., STRNAD, M.: Azapurine derivatives. PO12166GB NJN. HANUŠ, J., KRYŠTOF, V., HAJDÚCH, M., VESELÝ, J., STRNAD, M.: Substituted nitrogen heterocyclic derivatives and pharmaceutical use thereof. WO 00/43394. DOLEŽAL, K., POPA, I., HOLUB, J., LENOBEL, R., WERBROUCK, S., STRNAD, M.: Heterocycklické sloučeniny na bázi N6-substituovaného adeninu, způsoby jejich přípravy, tyto deriváty pro použití jako léčiva, kosmetické přípravky a růstové regulátory. PV 2001-2818. HAVLÍČEK, L., KRYŠTOF, V., SIGLEROVÁ, V., LENOBEL, R., VAN ONCKELEN, H., RERNEMAN, Z., SLEGERS, H., ESMANS, E., STRNAD, M., WERMEULEN, K.: Purine derivatives, process for their preparation and use thereof. WO 01/49688. MORAVCOVÁ. D., HAVLÍČEK, L., LENOBEL, R., KRYŠTOF, V., STRNAD, M.: Novel pyrazolo-pyrimidine derivatives with antiinflamatory, anticancer, immunosuprresive and neurogenerative properties and their use thereof. EP 24128-099. MORAVCOVÁ. D., HAVLÍČEK, L., LENOBEL, R., KRYŠTOF, V., STRNAD, M.: Disubstituted pyrazolo-pyrimidine derivatives with CDK inhibitory activity and their use thereof. EP 37456099. DOLEŽAL, K., POPA, I., ZATLOUKAL, M., LENOBEL, R., HRADECKÁ, D., VOJTĚŠEK, B., ULDRIJAN, S., MLEJNEK, P., WERBROUCK, S., STRNAD, M.: "Substituční deriváty N6adenosinu, způsob jejich přípravy, jejich použití pro přípravu léčiv, kosmetických přípravků a růstových regulátorů, kosmetické přípravky a růstoví regulátory tytzo přípravky obsahující. PV 2002-4273 DOLEŽAL, K., POPA, I., HOLUB, J., LENOBEL, R., WERBROUCK, S., STRNAD, M.: "Heterocyclic compounds based on N6-substituted adenine. PCT/CZ02/00045 73 IRP identification code AV0Z50380511 Apple varieties - patents and licences: Patents: Maďarsko – 3 plant patents: TOPAZ P 9800450; RAJKA P 9800585; RUBINOLA P 9800586 Plant Breeder’s Rights (analogy of plant patents): EU – 5 certificates ”Community Plant Variety Rights“: RUBINOLA EU 5824; GOLDSTAR EU 7380; OTAVA EU 7381; RAJKA EU 8880; LENA EU 10713 Švýcarsko – 4 Plant Breeder’s Rights: RUBINOLA 00.20.1293; OTAVA 01.20.1393; RAJKA 02.20.1474; GOLDSTAR 01.20.1394 Polsko – 4 Plant Breeders Rights: TOPAZ OS 00001; RUBINOLA OS 00002; RAJKA OS 00003; GOLDSTAR OS 00004 Slovakia – 4 Plant Breeders Rights: 4528; SVATAVA 4755; GOLDSTAR 5161; RAJKA 5169 Czech Republic – 2 Plant Breeders Rights: BIOGOLDEN 647; LENA 54/2002 Licences - foreign: country Belgium Netherlands Germany Poland U.S.A. Great Britain total no of licenses 1 2 3 2 1 1 10 no of varieties 1 7 3 3 1 1 16 year 1999 2002,2003 2001,2003 2000,2001 1999 2000 Licences in CR : country CR no of licenses 41 no of varieties 4 74 year 1999-2003