Effects Of Ascorbic Acid And Calcium Signal On Cell Death In Tobacco Suspension Cells Induced With Elicitin

Plants develop a complex variety of defense responses to survive pathogen attack, one of these is the hypersensitive cell death (HCD). In the HCD process, a series of events, such as oxidative burst, ion flow was not understood.Elicitins are a group of proteinous elicitors 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. Here, we report the effects of ASA and calcium channel in hypersensitive cell death in tobacco suspension cell cultures induced with Elicitin.1Ascorbic acid (ASA) can reduce the death of cells and the accumulation of H₂O₂ in tobacco suspension cells induced with Elicitin.The ability of elicitin to cause tobacco suspension cell death was investigated using trypan blue after 24h incubation. After 24h, 100 nM elicitin induced the death of tobacco cells to 69.6%. ASA were added for 10min prior to 100 nM parA1 treatment, The proportion of dead cells after 24h of culture was 46.4% in tobacco suspension cells treated by ASA+Elicitin.It decline 23.2% with alone to deal with Elicitin.Elicitin could induce H₂O₂ changes in tobacco cell suspension, and two phases were obvious after Elicitin treatment within 5h. Elicitin triggered a significant H₂O₂ production after 2h of treatment. Then, Elicitin -induced H₂O₂ accumulation decreased after adding ascorbic acid (ASA). The results show that the ASA could reduce Elicitin-induced cell death through abatement of H₂O₂ accumulation.2 Effects of ascorbic acid on CAT ,APX activity and expression of gene in tobacco suspension cells induced with Elicitin.Ascorbate peroxidase (APX) and peroxidase (CAT) are important tioxidant enzyme. They play a fundamental role in limiting the damage caused by this H₂O₂. In order to analysis the impact of CAT and APX activity in regulating the accumulation of H₂O₂.we determined two kinds of enzyme activity and expression of gene.The results showed that APX and CAT enzyme activity of tobacco cells without any treatment has been in a higher state. After Elicitin-induced cell death 2 h, APX and CAT activity decreased rapidly in all of the time period.Their enzyme activity has been very low. APX and CAT activity by the ASA pre-treatment of Elicitin-induced tobacco cells although lower than that of control, but higher than that induced by Elicitin in tobacco suspension cells. We found a similar phenomenon of genes encoding two enzymes of the inducible expression by RT-PCR. The results show that H₂O₂ accumulation may be relate with two kinds of enzyme activity.And the ASA may be reduce the Elicitin-induced cell death through improving two types of enzyme activity.3 Effect of ascorbic acid on expression of hsr203J and hin1 gene in tobacco suspension cells induced with Elicitinhsr203J and hin1 were expressed in tobacco suspension cells at extents coincident with the HR levels. The expression of hsr203J and hin1 gene was analyzed in time-course experiments using semi-quantitative RT-PCR. The results show that, ASA can impaired transcripts accumulation of hin1 and hsr203J in tobacco suspension cells induced with Elicitin.4 Effect of calcium channel on cell death in tobacco suspension cells induced with Elicitin.To assess the involvement of calcium channel in elicitin -induced signal transduction, Lanthanum chloride and EGTA, calcium channel inhibitor or chelator were used. Treatment with elicitin in the presence of La3+ and EGTA resulted in far fewer dead cells. This result indicate that cell death-treated with Elicitin is regulated by calcium channel.5 Effect of calcium inhibitor on H₂O₂ accumulation and hsr203J hin1 gene in tobacco suspension cells induced with Elicitin. We added to calcium inhibitors in tobacco suspension cells, determining the situation of H₂O₂ with fluorescence photometric method. The results show that, EGTA completely inhibited H₂O₂ accumulation induced with Elicitin, LaCl3's inhibition was only partial.hin1and hsr203J were expressed in tobacco suspension cells at extents coincident with the HR levels. The expression of hin1and hsr203J gene was analyzed in time-course experiments using semi-quantitative RT-PCR. The results show that, Lanthanum chloride and EGTA can impaired transcripts accumulation of hin1 and hsr203J in tobacco suspension cells induced with Elicitin.The above results show that Ca²⁺ signals involved in Elicitin -induced tobacco cell death.