Characteristics And Mechanisms Of Apoptosis In Tobacco Cells Induced By Parasiticein

Hypersensitive response (HR) and necrosis are very common phenomena in plant -pathogen interactions. The necrotic death, named'necrapoptosis', is commonly distinguished from HR. From a mechanistic point of view, HR is a kind of programmed cell death (PCD) accompanied by some morphological changes that appear to be similar to those described during apoptosis in animals. Elicitins are a group of proteinous eliciors produced by oomycete pathogen. Parasiticein used in this work has been identified from Phytophthora parasitic and expressed in E. coli. Application of parasiticein to tobacco plants can induce cell death and systemic acquired resistance to pathogens. How parasiticein performs those functions have been unclear. Here in this work, we use tobacco suspension cells to observe the morphological and biochemical characteristics of PCD after induced by parasiticein. Also we analyzed the mechanisms of PCD in tobacco cells induced by parasiticein. The main results are as follows.1. We have established a tobacco suspension cell model system to study plant cell death and obtained a very convenient method to conserve tobacco suspension cells.It is very important to establish useful model systems for the study of plant cell death in this work. First of all, we obtained a steady tobacco suspension cell system following callus and suspension culture, starting with Nicotiana tabacum (NC89). We found that the optimal conditions for the growth of tobacco cells is MS+6-BA 0.2 mg/L+NAA 2.0 mg/L, combined with 3% sucrose and 5% original cell content. The optimal temperature is 26℃with rotate speed of 140 rpm , which made the maximal dry weight of cell products to be 46.2 g/L cultured 10 days later.Secondly, we analyzed the method to conserve tobacco cells. When suspension cells cultured in liquid medium are transferred to MS solid medium, they grow very well. Cells conserved on solid medium can grow 60-75 days without altering the culture medium. And cells can grow very well at temperatures through 15℃to 30℃. When conserved cells were transferred to the liquid medium, the transition period was shortened about 20-30d.2. Tobacco cells induced by parasiticein showing morphological and biochemical properties of PCD.PCD comprises a series of genetically controlled events. Activation of PCD leads to distinct morphological features in the cell, such as nuclear condensation, membrane blebbing, and cell shrinkage. Using tobacco suspension cells and parasiticein, we observed serial reactions of the dead cells.2.1 Tobacco suspension cells were subjected to 100nmol/L parasiticein and used to observe morphological aspects of dead cell by phase contrast microscopy. In the early phase, dead cells showed that the cell membrane retracts and moves away from the cell wall leaving a visible gap. In the later phase the cytoplasm shrinkage. The same aspects can be obtained after stained with trypan blue.2.2 Using fluorescence microscopy, chromatin morphology was observed by DAPI staining. Chromatin had a granular appearance with lobated nuclei in some cells treated with parasiticein.2.3 The ultrastructural study was performed on the tobacco suspension cells treated with parasiticein. Changes in nuclear morphology and vacuolar membrane was observed under transmission electron microscopy. Chromatin condensation and vacuolar membrane disruption were observed clearly in treated cells but were not in control.2.4 DNA laddering was found in dead cells after separated on a 2.0% (w/v) agarose gel. Those data suggest that parasiticein-induced cell death shows many morphological and biochemical properties of apoptosis.3. Mechanisms of tobacco suspension cells during PCD induced by parasiticein. The PCD mechanism was elucidated in tobacco suspension cells subjected to parasiticein. Signaling molecules, including the ROS superoxide and hydrogen peroxide have been shown to modulate PCD. In this paper we tested ROS, H2O2 and some enzymes that related with ROS to show their relations with parasiticein-induced PCD.3.1 During the process of parasiticein-induced apoptosis in tobacco suspension cells, we observed ROS changing greatly treated with 100nmol/L parasiticein by DCFH-DA staining. The kinetics of H2O2 burst within 12 h treatment was biphasic: first phase peaked at 4 hour and second peaked at 8 hour post-treatment.3.2 At the same time, activities of some enzymes that are related to ROS were test. Activities of enzymes such as SOD, POD and CAT have changed separately. Those results indicate a correlation of SOD, POD and CAT with PCD induced by parasiticein.3.3 Also we use ascorbic acid salt to test the correlation of oxidation with PCD. The mortality of suspension cell was greatly reduced after the cells were treated by ascorbic acid salt before the parasiticein induced the cells, and the oxidation enzymes activity also decreased in some degree separately. Those results elucidated that oxidation is very important to PCD induced by parasiticein.3.4 Using K252-a, an inhibitor of protein kinase, we found that the death of the cells induced by parasiticein was partially inhibited by K252-a.Those data show us that the parasiticein-induced cell death may associate with the protein kinase signaling.4.ConclusionBase on the results described, we conclude that parasiticein-induced cell death shows many morphological and biochemical properties of PCD. And many signals involved in PCD such as ROS and kinase cascade.