Signal Transduction in Trans-genic Plants
The Transgenic Plants with Decreased Level of Phospholipase C Genes Expression: the Modification of Signal Transduction Processes in Plant Cells
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
National Academy of Sciences of the Republic of Belarus / Institute of Biophysics and Cell Engineering, Belarus, Minsk
- John Innes Centre, UK, Norwich\nPotsdam University / Institut fuer Biochemie und Biologie, Germany, Potsdam
Project summaryThe Project Aim.
To create transgenic plants with the decreased level of expression of phospholipase C genes. To elaborate the concrete proposals of using the double-stranded RNAs technology for down-regulation of plant genes. To study the role of phospholipase C in plant intracellular signalling using the plants obtained.
The information about the role of phospholipase C will provide a basis for the elaboration of the methods to exert control of the processes of growth and differentiation of plants and their adaptation to the environment.
The coordination of cell functioning in tissues and organs is due to chemical and physical cell signalling. A major portion of signal molecules addressing to a cell never penetrates into cell interior. More often they interact with the receptor molecules (antennae) that are located on the cell surface. Receptors distinguish the signals and trigger the intracellular transduction pathways to transfer the information within the cells. Finally, the regulation of intracellular processes as secretion, gene expression, biochemical reactions, growth etc. occurs. The main barrier to stop the outer information flow is cellular plasma membrane. Currently it becomes more and more evident that plasma membrane phospholipids are the key components of the mechanisms of transformation of outer signals into intercellular events to be associated with secondary messengers, small diffusible molecules capable to provide the rapid propagation of a signal within the cell. The number of secondary messenger was found surprisingly small that means the pathways of transfer of intercellular signals are universal. The known messengers are capable to regulate many physiological and biochemical reactions. It is obvious that the study of concrete secondary messengers is a way for understanding the mechanisms of control of cellular activity.
The central position in the system of intracellular signalling is occupied by three messengers Ca2+, inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). The origin of two last compounds takes special notice. They are formed from the plasma membrane component - phosphatidyl-4,5-bisphosphate (PIP2) under the action of phosphoinositide-specific phospholipase C. The phosphoinositide system was shown to be involved in cellular responses induced by various outer signals in plants and algae. The plant cells contain several immunologically different phospholipases C differing in molecular masses, amino acid sequences and biochemical parameters. For a number of plant phospholipases C the nucleotide sequences of cDNA’s are published.
IP3 induces the Ca2+ release from intracellular Ca2+ stores opening the appropriate membrane channels. Ca2+ participates in many signal reactions in plant cells. An increase in Ca2+ concentration is a trigger event in signal transduction of phytochrome photoregulation, phytohormones action, mechanical action and cold shock. Nevertheless, up to now the involvement of phosphoinositides and phospholipase C in intracellular signalling coupled with an increase in Ca2+ concentration in the cytosol were not proved experimentally. The necessity to study this point indicates the evident actuality of the suggested project. Unfortunately, there are not any non-destroying methods to measure phospholipase C activity within the cell and it is impossible to modify this activity by biochemical approaches (for example, by modulators) without any destroy of cell integrity. Therefore, the creation of transgenic plants with directed change in cellular content of phospholipases C is a promising direction to study this enzyme in vivo. Until recently, the only method to decrease the cellular content of some plant enzyme was the down-regulation of expression of this gene using antisense RNA technique. However, the recent studies have shown that the most effective inhibitor of gene expression were double-stranded RNA’s. To study the prospects of double-stranded RNA technique to inhibit the expression of crucial plant genes is one of the actual directions in plant biotechnology. Actually, this sequence-specific technique is an analog of targeted mutagenesis.
Influence of the project proposed on the progress in the field of the development:
– the understanding of the phospholipase C role will promote the progress in biotechnology of plants since it gives a key to elaborate the methods of control of growth and development of plants, for example of change in time of mature and keeping the plant production and also plant reaction on the action of stresses (cold, drought etc.), ecologically unfavorable pollution (high salt concentrations, heavy metal ions) and etc. (high illumination for example);
– the transgenic Nicotiana plumbaginifolia plants with decreased content of the products phospholipase C genes are the model systems, which will promote the progress in the field of phospholipid transduction in plants that is still poorly understood;
– the fragments of phospholipase C genes received can be used later on to obtain the full-sized gene sequences of phospholipase C isoforms in Nicotiana plumbaginifolia, which are still not cloned;
– the concrete proposals of using the double-stranded RNAs technology for down-regulation of phospholipase C genes will be the input in development of this technology for plants.
The laboratory of photoregulation of cellular processes of IPB NASB fulfills all experimental stages of the project. Scientific team consists of 1 Doctor of Biological Science, 4 Candidates of Science (Ph.D.), 3 scientific workers of high qualification, 1 technician, 1 aspirant and auxiliary personnel. “Weapon” specialists (5 persons) have the knowledge of constructing the biological weapon and of its action on the organisms, among them 3 persons are the research workers, who were involved in military project concerning the action of high-intensive laser illumination on biological objects. Two groups are the research team. The group of plant genetic engineering (5 research worker including 2 Ph.D.) will create the Nicotiana plumbaginifolia transgenic plants with decreased content of phospholipase C isoforms. The plant physiology group (4 research workers including 2 Ph.D.) will study the transgenic plants created by first group to evaluate the role phospholipase C isoforms in signal transduction in the cytosol of plant cells. Both groups are of high qualification in appropriate fields that is approved by their publications.
During the study of 7 tasks of the project the following results will be obtained:
1. The fragments of various phospholipase C genes of Nicotiana plumbaginifolia using PCR technique will be produced.
2. The genetic constructions for synthesis of double-stranded and antisense RNA’s for the fragments of various phospholipase C genes of Nicotiana plumbaginifolia in plant cells will be created.
3. The Nicotiana plumbaginifolia transgenic plant lines with the decreased level of expression of phospholipase C genes will be obtained and the concrete proposals of using the double-stranded RNAs technology for down-regulation of plant genes will be elaborated.
4. The differential expression of phospholipase C genes in cells of initial and transformed plants will be studied.
5. The expression of phytochrome A gene in the course of photomorphogenesis of plants with decreased level of the expression of phospholipase C genes will be investigated.
6. The physiological, biophysical and biochemical parameters of plants with the decreased level of expression of phospholipase C genes will be studied.
7. The mechanism of the increase in [Ca2+]cyt induced by the action of physical and chemical factors in cells of initial and transformed plants will be studied. The general scheme of Ca2+ signal transduction will be derived.
The global sum. The general scheme of Ca2+ signal transduction in plant cells with determination of the role of phospholipases C system in this process will be generated. The input in development of technology of using the double-stranded RNAs for down-regulation of plant genes activity will be done.
The scientific importance. The evaluation of the role of phospholipase C isoforms in intracellular signalling in plant cells will be a substantial contribution to understanding the processes underlying intracellular signalling that is poorly studied at the moment. The scientific importance of the project lies also in obtaining information on the possibilities of double-stranded RNA’s to use for inhibition of the expression of plant genes. The successful completion of the project opens to research team the long-term prospects of international cooperation in the field of genetic engineering.
The practical importance. The transgenic plants with changed content of various isoforms of phospholipase C are the perfect model to study the concrete functions of enzyme isoforms in the course of development of plant response to stress factors of environment such as salinity and flood of soil and plant adaptation to various light condition of growth. The obtained data can be easily extrapolated to other plants including those of agricultural importance. Using biotechnology this point gives at later time the improved varieties of agriculture plants optimally adapted to concrete climate conditions of a region.
Application of project results. The scientific information on the role of phospholipase C in the processes of signal transduction is of great importance to research in the field of physiology and biochemistry of plants. The applicability of the method of double-stranded RNA’s to inhibit plant genes expression is of interest for scientific and applied activity in the field of plant biotechnology. The cloned fragments of phospholipase C genes can be used in research laboratories (collaborator in particular) to get the full-sized sequences of Nicotiana plumbaginifolia phospholipase C genes. Beyond a doubt the elaborated genetic constructions and the obtained transgenic plants will be used in research laboratories to study cellular regulation.
Meeting ISTC Goals and Objectives.
The proposed research corresponds ISTC aims:
1. Gives the participants of the project engaged in weapon the possibility for reorientation of their abilities for peaceful activities aiming at study: a) the possibility of targeted inhibition of plants genes using double-stranded RNA’s technology; b) the process of signal transduction in plants occurring with involvement of lipid mediators, which is poorly understood at the moment.
2. Encourages the integration of the projects participants into international scientific community (participation in international scientific conferences and symposia on actual problems of plant sciences).
3. Supports the fundamental and applied researches in peaceful purposes, especially in the field of the environment protection (the study of transduction processes in plants helps to understand the ways of actions of outer stress factors on plant properties).
Data on works volume. Total labor costs will be 306.5 man/months, 155 from them are labor costs of weapon scientists and specialists. The duration of the project is 36 months. The project consists of the following experimental tasks:
1. Production of the fragments of various phospholipase C genes of Nicotiana plumbaginifolia using PCR technique.
2. Creation of genetic constructions to synthesize the double-stranded and antisense RNA’s for the fragments of various phospholipase C genes of Nicotiana plumbaginifolia in plant cells.
3. Production of Nicotiana plumbaginifolia transgenic plant lines with the decreased level of expression of phospholipase C genes and working-out of the concrete proposals of using the double-stranded RNAs technology for down-regulation of plant genes.
4. The study of differential expression of phospholipase C genes in the cells of initial and transformed plants.
5. The study of the expression of phytochrome A gene in the course of photomorphogenesis of plants with decreased level of the expression of phospholipase C genes;
6. The study of physiological, biochemical and biophysical parameters of plants with the decreased level of expression of phospholipase C genes.
7. The investigation of the mechanism of increase in [Ca2+]cyt induced by the action of physical and chemical factors in cells of initial and transformed plants. The derivation of general scheme of Ca2+ signal transduction in plant cells.
Role of Foreign Collaborators.
In the framework of this project the long-term collaboration with leading scientists from Germany who are currying out the researches in the same field is planned. They provided the consulting during project preparation and producing the working plan. In the course of the project the following volume of cooperation is planned:
– information exchange with foreign collaborator when carrying out the project;
– presentation of comments toquarter, annual and final reports to submit to ISTC;
– joint using of certain installation and samples;
– cross-check-up of results obtained during implementation of the project;
– participation in working inspections organized by ISTC;
– performing the collaborative experiments;
– holding the joint working seminars;
– preparation of collaborative publications.
Technical Approaches and Methodology.
The laboratory of photoregulation of cellular processes has experience in the field of plant genetic engineering. The proposed project is also the continuation of researches of the laboratory, which was involved during a number of last years in the study of signal transduction in animal and plant cells. The accumulated experience in the study of phosphoinositides metabolism and Ca2+ transduction is substantial to solve the tasks of this project. As the objects to study the plants of Nicotiana plumbaginifolia of wild type and those of transgenic type expressing apoaequorin will be used. The well-known methods of molecular cloning, genetic transformation of plants and molecular-genetic analysis of transgenic plants will be taken. A rather substantial set of genes fragments will be obtained using PCR without the isolation of full-sized genes. At a later time this set can be used to get the full-sized gene sequences with help of the possibilities of collaborator laboratory. The determination of phospholipase C activity in plant tissue homogenates will be performed by radioisotope method and also by the direct measurements of IP3 and DAG with help HPLC. Ca2+ translocation will be registered by chemiluminescence method routinely used in this laboratory during last 10 years. To evaluate the reliability and efficiency of researches the scientific experience of collaborator will be used.