The thesis is devoted to the investigation of molecular chaperones / heat shock proteins HSP70 and HSP90 at molecular, cellular and organism levels in supporting stability and plasticity of forming a plant phenotype, both under optimal and unfavorable environmental conditions. For the first time, various roles of these chaperones have been analyzed in a single complex research: their function as a system of non-specific resistance at critical levels of environmental factors; the ability of cytosolic HSP90s to regulate the HSP expression and, thus, control the cellular pool of chaperones, as well as the
participation of HSP90 and HSP70 in supporting the stability and plasticity of growth, development and morphogenesis.
Particularly, it was shown on the wide experimental material that supporting cellular proteostasis under changing environmental conditions occurs through qualitative and quantitative alterations in the cellular pool of chaperones with its increase when approaching the tolerance limits of the organism. The level of constitutive expression of 12 HSP70s and 9 HSP90s in Arabidopsis thaliana and dynamics of their induction in response to high temperature and water deficit revealed the specific contribution of the members with different subcellular localization. Tissue-specificity was determined for AtHSP70-10 and AtHSP90-2 expression. It was proved that the inducible members of these families are an important tool for tolerance - with the leading role of HSP70. Obtained data on induced and cross-tolerance showed that enhanced expression of the inducible HSPs resulted in an expansion of the tolerance range of the organism.
It was determined for the first time that immune-similar cytosolic HSP70s in plants of different taxa with different adaptive potential - A. thaliana (Brassicaceae), Malva silvestris і M. рulchella (Malvacea), Sium sisaroideum (Apiaceae) and Trapa natans (Trapaceae) – had species-specific kinetics of synthesis under critical changes of ecological factors (high temperature, flooding, water deficit), which were closely related to the processes of metabolic, physiological and structural adaptation. The tolerant species were characterized by higher HSP70 base level and more intensive and continous synthesis of both constitutive and inducible proteins. It was demonstrated in the experiment with flooding that the increase in the cellular HSP70 pool is needed to protect protein homeostasis during the change in the developmental trajectory of plant organisms with different genetically programmed pathways of anaerobic adaptation - the metabolic and structural adaptation in S. sisaroideum, and only the metabolic adaptation in
A. thaliana. A method for estimating the state of plants in natural populations and introduced plants using HSP70 as a biomarker was developed.
Participation of HSP70 and HSP90 in stress-reaction and adaptation of plants to unusual for terrestrial organisms factors, such as hypergravity and simulated microgravity (clinorotation), was revealed. The effects of rotation and position were differentiated in the effect of clinorotation, indicating that simulated microgravity has a small, but statistically significant effect on the level of these proteins. It was shown for the first time that
clinorotation caused cross-tolerance to high temperature due to a faster onset and enhancement of cytosolic HSP70 and HSP90 induction.
The evidence for negative feedback autoregulation of HSP synthesis and regulation of plant cell resistance by HSP90 proteins was obtained using HSP90 inhibition by geldanamycin (GDA). GDA treatment of A. thaliana seedlings induced synthesis of HSP70 and HSP90 in the absence of stress. For the first time was determined that GDA treatment of seeds resulted in an increase in the HSP70 and HSP90 basic level in seedlings, as well as an intensification of their stress induction and cell resistance under the influence of proteotoxic factors (high temperature and ionizing radiation).
Three HSP90-dependent molecular mechanisms, which are important for phenotypic variability of plant populations, were demonstrated on A. thaliana: 1) buffering stochastic development; 2) supporting plastic responses to external stimulus; 3) buffering genetic variations, counteracting their phenotypic display. At that, significance of the total activity of the cellular HSP90 pool, as well as of individual cytosolic family members -
constitutive AtHSP90-4 and inducible AtHSP90-1 was shown. It was first established that HSP70 may also affect the stability of growth processes and morphogenesis. Differences in the influence of individual HSPs on growth processes are
determined. It was shown that the effect of these chaperones on the morphogenetic program may occur discretely at the modular level.
