Plant Hormones for Crops Improvement.
Synthesis and Comparative Study of Brassinosteroid plant Hormones and the Development of Measuring Equipment to Monitor Their Effects on Crop Yield and Quality
Tech Area / Field
- AGR-PPR/Plant Protection/Agriculture
3 Approved without Funding
Scientific-Production State Enterprise "MBI-LOTIOS", Belarus, Minsk
- Institute of Bioorganic Chemistry, Belarus, Minsk
- Max-Planck-Institute for Chemical Ecology, Germany, Jena\nRamon Llull University, Spain, Barcelona\nWageningen University, The Netherlands, Wageningen
Project summaryThe main project’s purpose is carrying out the synthesis and comparative study of major natural brassinosteroids as potential ecologically friendly plant growth promoters, and development of method and equipment for monitoring of their action in plants allowing to predict their efficiency in crop yield and quality promotion. Brassinosteroids (BS) are a new class of plant hormones. Their first representative, brassinolide (Bl), has been found in rape pollen about 20 years ago. BS are wide-spread in plants but their content is extremely low (less than10-5 %). Till now about 40 structurally and functionally related steroids which are characterized as BS have been isolated. BS study goes very intensively. Nowadays the world literature on BS includes more than one thousand publications. These are summarised in a recent monograph  written with a participation of members of the project team. This book covers all the aspects of BS study and application and shows the actuality of the research directed to the aims such as ones formulated as project’s purpose. BS demonstrate various kinds of regulatory activities in plants. It has been shown that BS are capable to stimulate plant growth and development, enhance early crop maturation, increase crop yields and improve its quality. This is accomplished by mobilization of the life resources of plants and becomes evident in the enhanced capability of the plant to overcome all kinds of stresses induced by unfavourable growth conditions, and diseases. These effects can be achieved by applying BS at concentrations, which are very close to natural content of BS in plants (10-50 mg/ha).Natural BS are not well studied because of their low accessibility. The biggest part of published results is obtained for unnatural BS analogs and only one natural phytohormone, 24-epibrassinolide (EBl). Bl, one of the most active BS, and 28-homobrassinolide (HBl) are studied much less, and there are no data in the literature on comparative studies of these natural BS with respect to their physiological properties which are important for practical use . Our project is intended to fill this gap that means its actuality for the scientific area. Plant contain very limited amount of BS, and the only way to get sufficient quantities to study their biological properties, is by chemical synthesis. All known synthesis of BS are multi-step (usually more than 15-20 steps) and give low total yields of the desired product (less than 10%). This causes very high cost of synthetic BS, particulary Bl, which nowadays is available for scientific research for ten thousand dollars per one gram. There are only few groups in the world that are able to provide all the necessary BS for wide-scale investigations. One of them is the steroid group from the Institute of Bioorganic Chemistry (IBOC) of the National Academy of Sciences of Belarus, that is involved in this project. Until now, synthesis of the most important natural BS and their analogs have been realised by this group. In collaboration with biological and agricultural scientific institutions, studies of physiological, biochemical and applied aspects of BS have been performed also. As a result of this work a new chemical using EBl as active ingredient has been permitted for application in agriculture since 1992. Other phytohormones like Bl and HBl are in earlier stages of investigation, and there are no data which allow their direct comparison with regard to application in agriculture because of their limited availability. The first opportunity to get these data is presented by this project. An important prerequisite to obtain reliable plant-growth-promoting characteristics of these BS is the availability of well-standardized test-system, which can monitor the growth process at the levels having close connection with total plant productivity. All the known testing procedures that are used for estimation of BS activity, have low prognostic value in this respect because only short-term specific plant responses in specific experimental conditions are measured. A new approach to test the activity of BS will be developed. It is based on the invention of project team participants , that use some electrical parameters of plants for permanent control of changes in growth dynamic, and their “know-how”. Ingeneering of the apparatus and providing it with an automatic system for data treatment is also the task of the project. It will be carried out by group of project participants from the Scientific-Production State Enterprise “MBI-LOTIOS” (SPSE “MBI-LOTIOS”). Further application of the developed test-system to study of agricultural plants treated with BS, followed by analysis of total plant productivity and biochemical parameters that characterize the food value of the production, will bring a solution of the problem formulated in the project. Finally its fulfilment will influence the area significantly because of giving new basic knowledge on intime differences in BS properties and new version of their practical application.
The following results are expected: Task 1: 1) elaboration of new preparative synthetic approach to Bl and HBl; 2) preparation of the most perspective BS for laboratory and field trials with several agricultural plants; Task 2: making measuring equipment for plant-growth monitoring (EPGM) based on measuring of some electric parameters of plants; Task 3: 1) results on structure-productivity relationships for mentioned BS with respect to crop yield promotion and food value; 2) finding the principles to predict efficiency of BS application to certain agricultural plants; Task 4: results on EPGM -based comparative study of plant-growth promotion induced by Bl, HBl and EBl in agricultural plants; Task 5: 1) formulation of recommendations on the application of new natural BS as environmentally friendly (biorational) agents to increase yield and quality of agriculturally produced crops. These results will be used in scientific research for futher clearifying the mechanisms of specific action of BS in different plants, and in agriculture via application of new BS for larger and higher quality crop production. The expected results are considered as a contribution to base and applied science in the field (area) of the biotechnology and ecology. Being directed to application in environmental-protecting biotechnologies for higher-yield and higher-quality agricultural production, they have significant commercial importance. It is supposed that some of the results obtained in the cours of the project elaboration, which involve new technical solutions and/or new methods will be patented.
Members of the project team are experience enough to be able to carry out the project. The team includes 11 main investigators and can be devided in two parts: chemical-biological part (7 researchers, two DSc. and three PhD), which is involved in chemical synthesis of BS and their biological testing, and technical part (4 researchers, two PhD), which is directed to elaboration, engeneering and application of special equipment and new BS-testing technology. Both groups are higly experienced in the corresponding fields, which is confirmed by their publications (more than 300 including 3 monographs), inventions and patents (more than 20), and by the results reached in the specified area until now. Professional skills of the former weapon scientists (Missile Technology and Chemical Weapons) are confirmed by their long working (about 15-29 years) in these areas.We plan the participation of the post-graduate students, laboratory assistant and supporting personnel in the project.
The project fully corresponds to ISTC objectives because the “technical” part of the project team, which combines former weapons scientists, will concentrate their efforts on elaboration and testing of special equipment to fulfil one of the key tasks of the project. They will take part also in the investigations on monitoring of the phytohormone activity. In this way former weapons scientists redirect their talents to peaceful activities and follow all the strategic ideas of ISTC. In particular, the project will contribute to solving national and international problems concerning the environmentally healthy agricultural production; it will support basic and applied research; it will stimulate further integration of CIS weapons scientists into the international scientific comunity
The duration of the project makes up 30 months, the total number of person-months is 260, and the number of person-months of effort of weapons scientist is 135. Two CIS institutions - SPES “MBI-LOTIOS” and IBOC- will participate in the project. Team activities will include: Task 1. Preparative synthesis of brassinosteroids; Task 2. Design and engineering of the equipment for plant growth monitoring (EPGM); Task 3. Comparative study of BS local effects in plants and their correlation with crop yield and quality; Task 4. Testing of the EPGM in long-term studies of BS-influenced plants; Task 5. Study of correlation between BS local effects, EPGM-data, and crop yield and quality measurements. Formulation of the conclusions on the application of the EPGM for BS-effect studies and agricultural application of new BS for mentioned plants
Foreign Collaborators of the project - Prof. A.deGroot (The Netherlands ), Dr. B.Schneider (Germany) and Dr. C.Brossa (Spain) are highly qualified specialists in the area. They will participate in the development of project proposal and work plan, exchanging of information during project implementation, sharing materials and samples, joint seminars, workshops, meetings, consultations, verification of results using independent methods and/or equipment, joint or parallel investigations.
Their participation in this project could garantie: Improvement in the design and execution of projects;
Assurance that projects do not duplicate efforts in the same fields; Guidance on how best to internationally disseminate the results of research and development; Long-term cooperation in science and technology from experience gained during project implementation.
Mainly, usual approaches and methodology of thin organic synthesis, plant physiology and apparatus design and engineering will be used during the Project implementation. New approaches will be elaborated also.
Task 1. Brassinolide (Bl) is one of the most complicated representatives of BS series from synthetic point of view. In this project two new approaches to Bl synthesis will be developed. One of them is based on the Ireland-Claisen rearrangement for stereoselective construction of the side chain of Bl. Such an approach has never been used before in steroid series and could bring not only new efficient synthesis of brassinolide and its key intermediates but also several new Bl analogs, which are interesting as new biologically active substances and haptens for BS immuno assays. Another method for Bl synthesis, which is planned to be developed, is based on the transformation of campesterol, a natural sterol with the same carbon skeleton as in the molecule of Bl. Synthesis of another highly active and practically promising natural brassinosteroid, 28-homobrassinolide (HBl). In the frames of the Task 1 two new efficient synthetic approaches to HBl will be developed. One of them is based on the application of new chiral catalysts for the stereoselective double bond hydroxylation in the side chain of stigmasterol. It will bring higher yield of the desirable product of natural structure and make synthesis more efficient. Another approach is based on the transformation of the unnatural type stereoisomers, which usually are obtained in the reactions of the side chain hydroxylation, into natural ones via chemical transformations. The methodology for purification and structural elucidation of target compounds will be the same as in the previous case of Bl synthesis. NMR, IR, GCMS and X-ray analysis will be used for structural elucidation of synthesized compounds.
Task 2. For identification of BS and estimation of relative activities of certain representatives of the series, several specific test methods have been developed. All of them are bioassays using different plant systems.. It was found that the obtained results do not always followed the relations estimated from the bioassays that limits possible role of the latter ones in use to predict efficiency of new BS applications. To exclude influence of the limitations and to do the prognostic value of testing approach higher with respect to BS crop yield increasing ability, a new method and special equipment for monitoring of growth and development of BS-influenced plants will be developed in this project. The method is based on the permanent checking, recording and treatment of selected electrical parameters of growing plants during different periods of their development to find the most characteristic ones reflecting total plant productivity. Such an approach and methodology have never been used before in BS investigations.
Task 3. To clarify the life periods most characteristic for predicting the total plant response, for the first time the phytohormones Bl, EBl and HBl will be checked in parallel experiments for their effects on developmental and biochemical parameters in different plant species at early stages of plant growth (before flowering), at flowering, and after flowering (before harvesting). Then taking into account the data on the total plant productivity, a preliminary formulation of hormone-plant-effect relationships could be found.
Task 4. Parallel experiments with the same plant species belonging to different types of agricultural plants (cereals, legumes, vegetables and other) will be performed using EPGM. The data on developmental parameters of BS-treated plants obtained in previously described part of work will be compared with the results of monitoring. This will allow to connect visible morphological and biochemical changes in plants with measured by EPGM characteristics. All these together will create a basis for getting conclusions about hormone-plant-effect relationships, prospects and methods of rational application of BS to different agricultural plants.
Task 5. Data on BS local effects at different phases of plant development obtained as a result of fulfillment of the task 3 and EPGM-data for long-term investigation of growing plants obtained as a result of fulfillment of the task 4 will be compared with the corresponding crop yields and biochemical parameters characterizing crop quality. Parameters for certain life periods, which are the most closely connected with the crop yield and its quality, will be chosen as characteristics for further use in screening of practically valuable BS and their analogs. The data on new agriculturally promising BS, brassinolide and homobrassinolide, will be used for their practical implementation. Finding optimal BS combinations with certain plant species (BS plant-specificity) will be further used for ecologically friendly and predictable regulation of crop yields and their quality in agriculture.
 Khripach V.A., Zhabinskii V.N., de Groot Ae. Brassinosteroids - A New Class of Plant Hormones. San Diego: Academic Press, 1999. 456 p.
 SU 1027526 (1982). Bull. isobr. 1983. N25
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