Detection and Differentiation of Tomato Cell Death Essay

The lesions, yellowing, abnormal growth, and drying of tomato leaves at the early stage of the plant affect its fruit bearing. This phenomenon is ascribed to cell death which caused primarily of either â€Å"programmed cell death† or as consequences of the plant’s spontaneous response with pathological agents. Although cell death is an integral part of the plant’s development, extraneous loss of the cell results to the aforementioned consequences. Thus, proper regulation of cell death must be done. Since apoptosis and necrosis can possibly occur in plants, the determination of the type of cell death is crucial in the identification of the appropriate technique for its regulation. In this study, sterilized Solanum lycopersicoides seeds will be germinated at 25  °C culture laboratory. Prior to experimentation, the generated cells will be washed and a two-millimolar pyruvate will be added for ATP production. Then, the cells will be exposed to 2. 5 micromolar of oligomycin for ATP depletion. Also, to limit energy generation to cytosolic ATP production cell will be incubated in the 5 millimolar glucose with 2. 5 micromolar oligomycin. After this, cells will be incubated with staurosporine. Meanwhile, the cell death will be analyzed with respect to morphological criteria, intracellular proteolysis, and DNA fragmentation through conventional agarose gel electrophorosis or field inverted gel electrophoresis. While death detection of the cell will be done by means of Enzyme-Linked Immuno Sorbent Assay, ATP measurement will be done through luminometry. Moreover, phosphatidyl serine traslocation analysis will be done by means of Annexin-V-FLUOS technique to be followed by confocal microscopy and fluorescent-activated cell sorting. Detection and Differentiation of Tomato Cell Death Introduction The term â€Å"apoptosis† was derived from a Greek word which literally corresponds to â€Å"falling off† or â€Å"dropping off†, as analogous to abscission to signify cell death as integral part of every organism’s life cycle (Gewies, 2003). In the mid-nineteenth century, it has been noted that cell death occurs in parallel with physiological functions for every multi-cellular organism (Gewies, 2003). In connection to this, in 1964, expert postulated that cell death occurs not accidentally, but rather in a controlled sequence of steps (Gewies, 2003). Meanwhile, cell death is classified either as apoptosis or necrosis based on morphological and biochemical changes undergone by the cell (Schulze-Osthoff, 2008). As such, plasma membrane of the cell may suffer necrosis due to extreme physiological conditions like hypothermia and hypertonic environment (Schulze-Osthoff, 2008). This plasma membrane damage can also be induced by pathological agents and viruses. On the other hand, the cell can incur apoptosis even at normal physiological conditions, thus, often called as â€Å"programmed cell death† or â€Å"cellular suicide† (Schulze-Osthoff, 2008). The â€Å"programmed cell death† involves intricate biochemical processes; pathogens and environmental stresses attack every cell by means of chemical signals. For example, death signals can be originated from malfunction in DNA repair mechanism, cytotoxic drug treatment, ligation of cell surface receptors, and irradiation (Gewies, 2003). In relation to this, plant responses to inhibit pathogenic growth and disease development by means of protective genes activation which in turn, through chemical reactions, kills the infected cells. The cellular death process then is directed by specific signals and independent biochemical processes in every cell (Dickman, Park, Oltersdorf, Li, Clemente, and French, 2001). Hence, understanding the intricacy of cell death requires an intensive knowledge on chemical principles behind apoptotic or necrotic process. Literature Review Apoptosis, on the basis of pathological and physiological conditions, serves a crucial role in the development of multicellular organisms and regulates cell populations in different tissues (Gewies, 2003). Apoptotic processes direct biological processes such as elimination of harmful cells, differentiation, immune system regulation, and homoeostasis (Gewies, 2003). Hence, apoptotic program dysfunction may lead to pathological conditions like viral infections, cancer, and even AIDS (Gewies, 2003). On the other hand, necrosis occurs when the cell’s inability to regulate homeostasis led to the passage of extraneous water and cellular ions into the cell which results to swelling and lysis (Schulze-Osthoff, 2008). As a consequence, the organelles are then exposed to the extracellular fluid. In contrast, apoptosis may arise even at normal cell condition or tissue homeostasis (Schulze-Osthoff, 2008). This involves chromatin accumulation, cytoplasmic and nuclear condensations, cytoplasm and nucleus transformation into apoptotic bodies that encapsulate nuclear material, ribosomes, and mitochondria (Schulze-Osthoff, 2008). While in vivo necrosis results to damaged tissues causing inflammation, the apoptotic bodies formed by in vivo apoptosis are engulped by adjacent ephitelial cells or macrophages (Schulze-Osthoff, 2008). Conversely, the apoptotic bodies formed by in vitro apoptosis undergo â€Å"secondary necrosis† or final swelling and bursting (Schulze-Osthoff, 2008). Every human body has an estimated 1014 cells that are in continuous progress (Schulze-Osthoff, 2008). In fact, hundreds of thousands cells are generated through mitosis in every second but almost equal number suffers apoptosis due to specific tasks and homeostasis regulation (Gewies, 2003). For instance, the elimination of the tail, and the separation of fingers and toes of a tadpole during its metamorphosis are all attributed to cell death (Schulze-Osthoff, 2008). In addition, newly formed or perilous lymphocytes are destroyed through cell death (Schulze-Osthoff, 2008). Furthermore, programmed cell death or PCD has been observed in variety of species such as in mammals, metazoans, nematodes, insects, cnidaria, plants, and even in unicellular organisms (Gewies, 2003). Thus, cell death is scientifically viewed as essential in the functionality maintenance of an organism. Even though plants have the capability to protect themselves from pathogenic invaders through cell death, viral pestilence and antibiotic stressors, most often, are the cause of loss in tomato harvest (Xu, Rogers, and Roossink, 2004). As defensive response, cell death occurs only in the infected sites or termed as hypersensitive response (Morel and Dangl, 1997). Other means of plants’ defense are through cell wall reinforcement, phytoalexin synthesis, and defense-related genes activation (Kazan, Murray, Goulter, Llewellyn, and Manners, 1998). In hypersensitive response, the pathogen is restricted to a specific part of the plant through localized necrotic reactions (Taliansky, Ryabov, Robinson, and Palukaitis, 1998). Significance Researches showed that at some points PCD of plants and animals is similar. As such, jus like animal cells, plant cells generate apoptotic bodies during apoptosis (Greenberg, 1996). Also, DNA fragmentation is both observed in plants and animals apoptosis (Greenberg, 1996). Moreover, antiapoptotic gene, homologous to dad 1, in animal cells was also detected in plant cells (Greenberg, 1996). However, despite these similarities, differences were also noted. For instance, unlike animal cells, plant cells do not exhibit phagocytotic characteristics. In fact, dead cells of the plants may still perform important functions for the whole architectural organization of the plant (Greenberg, 1996). Hence, further exploration on the nature of PCD in plants should be done to gain an intensive understanding on the underpinning principles behind plant cell death. Similarly, yellowing, abnormal growth, and drying of tomato leaves at the early stage of the plant directly affect its photosynthetic activities. These observations are ascribed to cell death which caused primarily of either â€Å"programmed cell death† or as consequences of the plant’s spontaneous response with pathological agents (Greenberg, 1996). Although cell death is an integral part of the plant’s development, extraneous loss of the cell results to the aforementioned consequences. Thus, proper regulation of cell death must be done. Since apoptosis and necrosis can possibly occur in plants, the determination of the type of cell death is crucial in the identification of the appropriate technique for its regulation. Therefore, it is an imperative to determine the possible type of death, under specific physiological conditions, experienced by tomato cells in order to employ the appropriate intervention in regulating cell death. Experimental Design Sterilized Solanum lycopersicoides seeds will be germinated at 25  °C culture laboratory (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). Prior to experimentation, the generated cells will be washed and in the absence of glucose, a two-millimolar pyruvate will be added for ATP production (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). Then, the cells will be exposed to 2. 5 micromolar of oligomycin for ATP depletion. Also, to limit energy generation to cytosolic ATP production cell will be incubated in the 5 millimolar glucose and 2. 5 micromolar oligomycin concoctions (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). After this, cells will be incubated with staurosporine or STS, a cell death inducer. Meanwhile, the cell death will be analyzed with respect to morphological criteria, intracellular proteolysis, and DNA fragmentation through conventional agarose gel electrophoresis or field inverted gel electrophoresis (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). While death detection of the cell will be done by means of Enzyme-Linked Immuno Sorbent Assay or ELISA of Roche Technology, ATP measurement will be done through luminometrical technology of Boehringer Mannheim Biochemicals (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). Moreover, phosphatidyl serine or PS traslocation analysis will be done by means of Annexin-V-FLUOS technique to be followed by confocal microscopy and fluorescent-activated cell sorting or FACS analysis (Leist, Single, Castoldi, Kuhnle, and Nicotera, 1997). Cell Death Detection and Differentiation The following instrumental techniques will be utilized in this study for the detection of tomato cell death, and for the apoptotic and necrotic death differentiation. Agarose Gel Electrophoresis of Nucleic Acids Nucleic acids are nucleotide polymers joined by diester bonds of the sugar units (Devor, 2005). These linkages between nucleotides give a negative overall charge to the nucleic acid polymer. Molecules with net electrical charges move predictably under electrical field. Hence, when nucleic acids are subjected to semi-solid gel matrix, they move toward the positive pole (Devor, 2005). In an agarose matrix, the mobility of nucleic acids can be formulated by treating its viscosity as gel density with respect to its entire length (Devor, 2005). This migration is then expressed as a negative exponential function of the radius of nucleic acid (Devor, 2005). ELISAPLUS Cell Death Detection ELISAPLUS is a one-step colorimetric technique of detecting cell death. It can differentiate necrosis from apoptosis with relative quantification (Roche Applied Science, 2007). This can be done without cell staining. ELISAPLUS can be utilized for culture supernatants, plasma, lysates, and serum (Roche Applied Science, 2007). About three hours after induced apoptosis, histone-complexed DNA fragments can be detected through immunochemical method (Roche Applied Science, 2007). On the other hand, the histone-complexed DNA fragments are determined directly in the culture supernatant (Roche Applied Science, 2007). Annexin-V-FLUOS Annexin-V-FLUOS, employed for microscopic and cytometric analysis, is done by means of direct fluorescence staining (Roche Applied Science, 2007). This technique can differentiate necrotic from apoptotic cells and typically used for apoptotic detection of membrane-altered cells especialy in PS-translocation (Roche Applied Science, 2007). In line with this, freshly isolated cells and suspension or adherent cell lines are the appropriate samples for this test (Roche Applied Science, 2007). As such, the PS of the cell surface and necrotic cells are stained by FLUOS or green dye and Annexin-V-Alexa or red dye respectively (Roche Applied Science, 2007). Lastly, about 15 minutes after induced apoptosis, determination test is already done (Roche Applied Science, 2007). References Devor, E. J. (2005). IDTutorial: Gel Electrophoresis. Integrated DNA Technologies. Retrieved March 6, 2009, from http://www. idtdna. com/Support/Technical/TechnicalBulletinPDF/Gel_Electrophoresis. pdf Dickman, M. B. , Park, Y. K. , Oltersdorf, T. , Li, W. , Clemente, T. and French, R. (2001). Abrogation of Disease Development in Plants Expressing Animal Antiapoptotic Genes. Proceedings of the National Academy of Sciences, 19, 12, 6957-6962. Gewies, A. (2003). Introduction to Apoptosis. Apo Review. Retrieved March 6, 2009, from http://www. celldeath. de/encyclo/aporev/apointro. pdf Greenberg, J. T. (1996). Programmed Cell Death: A Way of Life for Plants. Proceedings of the National Academy of Sciences, 93, 12094-12097. Kazan, K. , Murray, F. R. , Goulter, K. C. , Llewellyn, D. J. and Manners, J. M. (1998). Induction of Cell Death in Transgenic Plants Expressing a Fungal Glucose Oxidase. Molecular Plant-Microbe Interactions, 11, 6, 555-562. Leist, M. , Single, B. , Castoldi, A. F. , Kuhnle, S. , and Nicotera P. (1997) Intracellular ATP Concentration: A Switch Deciding Between Apoptosis and Necrosis. Journal of Experimental Medicine, 185, 1481–1486. Morel, J. B. and Dangl, J. L. (1997). The Hypersensitive Response and the Induction of Cell Death in Plants. Cell Death and Differentiation, 4, 671-683. Roche Applied Science. (2007). Apoptosis, Cell Death and Cell Proliferation, 3rd ed. Mannheim, Germany: Roche Diagnostics GmbH. Schulze-Osthoff, K. (2008). Apoptosis, Cell Death and Cell Proliferation, 4th ed. Roche Applied Science. Mannheim, Germany: Roche Diagnostics GmbH. Taliansky, M. E. , Ryabov, E. V. , Robinson, D. J. and Palukaitis, P. (1998). Tomato Cell Death Mediated by Complementary Plant viral Satellite RNA Sequences. Molecular Plant-Microbe Interactions, 11, 12, 1214-1222. Xu, P. , Rogers, S. J. and Roossink, M. (2004). Expression of Antiapoptotic Genes bcl-xl and ced-9 in Tomato Enhances Tolerance to Viral-Induced Necrosis and Antibiotic Stress. Proceedings of the National Academy of Sciences, 101, 4, 15805-15810.