The Study On Biochemical Characteristics Of An Endopeptidase Responding To Cell Death In Wheat Root Tip Under Salt Stress

Salt is an important environmental factor that effect plant growth and production. Salt stress will reduce the water potential and lead to an imbalance of intracellular ions. Wheat root tip is the first part of sensing salt stress, and do some adaptive responses for this stress. So it has great significance to explore the physiological and biochemical changes, especially in protein levels of wheat root tip cells under salt stress.Endopeptidases are short for EPs, taking part in regulation of protein degradation of plant senescence, and its activity increase significantly in the aging period. On account of that, we used the wheat(Triticum aestivum L. cv.3E-158)as the material, preliminary studied in a certain concentration of salt stress and different concentrations of Hemin (exogenous CO donor), the oxidative damage,aging conditions, and the types of endopeptidase in wheat root tip, researching deeply on the biochemical characteristics and protease isozymes activity under salt stress. The key findings are as follows.When the wheat root grew to6-8cm, we began salt treatment of200mmol·L-1NaCl. The results showed that with the salt-stress time, the growthes of roots were inhibited significantly, and the stress inhibition of the forth day had come to28.8%; The content of TBARS in wheat root tip cells were2times than the corresponding control (C) in the first day of stress, and it remained at a relatively high level with the extension of salt-stress time. The rate of superoxide anions (O2(?)) production also continued to grow with the extension of process. We used the corresponding treatment of wheat root tip cells to do genome-wide analysis by DNA gel electrophoresis, found that it happened DNA ladder on the second salt-stress day, and it also showed obscure DNA ladder on the forth salt-stress day. The above results show that salt stress of200mmol·L-1NaCl can inhibit plant’s growth, also can damage its physiological and biochemical activities.To study the changes of isozymes in wheat root-tip cells under salt stress, we adopted an electrophoresis method using gelatin as substrate gradient associated activity staining. The results showed that on the third day under salt stress of200mmol·L-1NaCl, there was an endopeptidase whose activity enhanced significantly, and the enzyme activity had no clear trend of lowering but enhanced with the extension of salt-stressed time. We analysised biochemical features on this enzyme, found that it had activity not only in wide pH from pH3.0to pH10.0and but also in wide temperature from20℃to80℃, this evidence explained that this enzyme had special biochemical features,. Further studying showed that the enzyme’s optimum pH is pH6.0, the optimum temperature is40℃.We continued to analysis the enzyme’s thermal stability in the condition of pH6.0and40℃, putting crude enzyme solution in4℃--50℃for1hour, found the activity remained unchanged by electrophoresis. Endopeptidase inhibitor analysis revealed that enzyme activity was being inhibited by serine-type endopeptidase inhibitor PMSF, metal-ion type endopeptidase inhibitor EDTA and EGTA, in which PMSF had the strongest inhibition.We treated the wheat with200mmol·L-1NaCl and different concentrations of exogenous CO donor (Hemin) for four days, the results showed that under the influence of1μmol·L-1and5μmol·L-1Hemin, compared with the salt-stress group of no Hemin, the length of wheat root increased5.3%and1.3%, the content of TBARS were reduced10.7%and13.6%respectively in root-tip cells, the rate of superoxide anions production had also decreased by17.1%and28.2%in root-tip cells, intracellular soluble protein content increased by41.3%and22.5%. But under the influence of0.1μmol·L-1Hemin, the above indicators had no significant difference. We also found that under the higher concentration of10μmol·L-1and20μmol·L-1Hemin’s influence, the above indicators were not shown to alleviate the phenomenon of cell death, but there was some trends of increasing injury. Therefore, we infer that the concentration of1μmol·L-1and5μmol·L-1Hemin should be the suitable concentration to alleviate the wheat root-tip cells death phenomenon under salt stress, of which5μmol·L-1Hemin has the most significant effect. We studied the effect on the change of endopeptidase EP activity with the influence of Hemin under salt stress, adopting an electrophoresis method using gelatin as substrate gradient associated activity staining. The results showed that under the influence of0.1umol·L-1μmol·L-1and5μmol·L-1Hemin, EP’s activity decreased, of which0.1μmol·L-1and5μmol·L-1Hemin has the more significant affect.