MANIPULATION OF GENETIC INFORMATION IN STUDYING PLANT PERFORMANCE

V. SUMMARY AND CONCLUSIONIn this investigation, methods of manipulating genetic information affecting plant performance under stress condition were studied. The above mentioned goal was achieved by observing the cellular modifications occurred in the flow of information, known as signaling cascades; in response to heat stress on plants. This investigation started with studying the accumulation of heat responsive genes/proteins in tobacco cells, followed by studying the upstream changes and regulation in cell membrane fluidization and cytoskeleton remodeling and ended with comparative bioinformatics analysis between tobacco and other biological systems. The activation of HAMK, heat activated MAP kinase, and HSP70 were used as end point markers. By understanding heat signaling response inside the cell, our knowledge of heat thermo-tolerance will be enriched and will be of great help in trials for producing heat tolerant plants. In this study tobacco BY-2 cells were used as a plant model system. The obtained results were summarized as follow:A) Results of studying the activation of heat responsive genes:1- By analyzing the growth rate and cell viability of tobacco BY-2 cells, 7 days old cultures were found to be at the optimum age for conducting our research. At this age, the accumulation and domination of HSP 101, HSP90, HSP70 and sHSPs were observed after 3 hours exposure to heat shock on tobacco BY-2 cell. However, the absence of many regular proteins which normally found in room temperature was noticed.2- By studying the response of HSF1 and HSF2 to heat, it was found that HSF1 is the predominant heat inducible HSF, while HSF2 responds to the developmental signals and constitutively expressed.3- HSP70 transcripts and HSP70 protein accumulation were observed during heat-shock in tobacco cells. Heat-induced increase in HSP70 transcripts was determined by Rt-PCR; while accumulation of HSP70 protein during heat shock was determined by both immuno-blotting with anti-HSP70 rabbit polyclonal antibody and by ELISA.4- The existence of a plant homologue of mammalians heat shock protein HSP27 was discovered in plants. This protein was rapidly increased and phosphorylated during heat shock in tobacco cells.B) Results of studying up-stream regulation of heat signaling:1- The results showed that heat activated MAP kinase (HAMK) was transiently activated within 2 min. in tobacco BY-2 cells exposed to 37C.2- It was proved that heat-shock-activation of HAMK was mediated through a mitogen activated protein kinase/kinase (MAPKK) related to MEK protein family which indicated that HAMK activation is a part of heat-triggered MAPK cascade.3- Moreover, results suggested that HAMK was an essential component of HSF1 and HSF2 phophorylation and Heat shock-induced HSP70 accumulation.4- Results suggested that Heat-induced phosphorylation and activation of both mitogen-activated protein kinase-activated protein kinase2 (MAPKAPK2) and HSP27 required the activity of HAMK.5- It was indicated that heat-induced accumulation of HSF, HSP70 and HSP27 proteins required membrane fluidization and reorganization of cytoskeleton.6- The obtained results suggested that protein kinase C (PKC) mediated the upstream regulation of HAMK during heat shock.C) Bioinformatics analysis and predictions :1- The bioinformatics analysis of HSFs revealed conserved domain of tobacco HSFs in other organisms which indicated that HSFs is amongst the most conserved proteins used by the cell to protect it-self from the improper conditions.2- The possible phophorylation sites of HSF1 were predicted by its structure using computer based phosphorylation prediction software, Netphosphos 2.0.3- By searching the HSP27 and other sHSPs conserved domains using CDART, it was revealed that the conserved (Alpha Crystalli) domain can be found in most of them indicating similar conserved function. Other similar domains architectures of HSP27 were predicted in different organisms.4- By comparing the DNA binding domain (DBD) of HSP70 and its counterparts in different species, similar conserved primary structure was revealed. In addition, different sequenced plants showed a conservative nature of the DBD of HSP70 indicating similar function in all of them.