| Endopeptidases are one kind of the most important proteases in cell.In plants,they play an important role in the different development stages,such as germination and organic senescence,even during the response to stress.They also contribute to the turnover and reuse of proteins,the maintaining of cell homeostasis and the regulated proteolysis in cell signal transduction.An increasing number of references are correlated with the changes and function of endopeptidase(EP) isozymes during the normal development of plants,but less is known about that of EP isozymes associated with plant under stress,especially with vegetable under salt stress.Meanwhile,the previous reports about SNP(sodium nitroprusside,an exogenous nitric oxide donor) focused primarily on describing the alleviation of NO to plant damage,the positon and function of NO in signal transduction, however,the effect of SNP on EP isozymes in plant under salt stress remain unknown. Non-heading Chinese cabbage(Brassica campestris L.ssp.chinensis Makino) is one of the important popularized vegetable,it plays a considerable role in the vegetable production and supply in people daily life of China especially the south of China.Shulii is a new kind of non-heading Chinese cabbage cultivated recently.The changes and biochemical characters of EP isozymes in leaves of Shul(u|¨) under salt stress and with SNP treatment were studied in this paper.1,The changes and characters of EP isozymes in seedling leaves of Shulii under salt stressShul(u|¨) seedling were treated by different salt concentration,i.e.50,100,150,200,250, 300,500 mmol·L-1NaC1 for one day,Compared with the leaves of CK,the leaves treated with 200 mmol·L-1 NaCl began to wilt,and the symptom of wilting were obviously increasing as the raising of salt concentration;the changes of EP isozymes in leaves under 100 mmol·L-1NaCl stress showed great difference with that under 200 mmol·L-1NaCl stress. The changes of soluble protein content,EPs activity and EP isozymes in Shul(u|¨) leaves during salt stress were studied mainly by gradient gel electrophoresis,and the characteristics of EPs were further investigated.The results showed that the activity of EPs gradually increased accompanied by the decreasing of soluble protein content in leaves under 100 mmol·L-1 salt stress during 4 days;The EPs activity in leaves under 200 mmol·L-1 salt stress increased significantly compared with that of CK or 100 mmol·L-1 salt treatment on fourth day,most of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was obviously degraded and the leaves began to senescing.Furthermore,five EPs(EP1～EP5) in leaves were detected by gradient-PAGE co-polymerized gelatin in the gel,among five EPs,EP3 and EP5 are cysteine proteases,EP1 and EP2 were serine proteases,but EP4 was not inhibited almost by all the EP inhibitors in this study.The optimum pH and temperature of EPs in crude extracts were pH6.0 and 40～50℃.EP3, EP4 and EP5 still had high activities after them incubated at 60℃for lh.The activity of EP4 increased under 100 mmol·L-1 salt stress,but the other EPs were almost invariable; However,under the 200 mmol·L-1 salt stress,the activities of EP2,EP4 and EP5 increased and that of EP1 and EP3 declined obviously in the leaves.It suggested that EP2,EP4 and EP5 might play an important role in salt stress-induced leaf senescence.2,The effect of SNP on the EP isozymes in Shulii seedling leaves under salt stressThe growth of Shuli(u|¨) leaves was inhibited obviously under 200mmol·L-1NaCl treatment. However,the morphology of leaves was closed to that of CK when the Shul(u|¨) seedling was treated additionally with low concentration of SNP(25 and 50μmol·L-1);on the contrary, high concentration of SNP(above 100μmol·L-1) treatment aggravated the damage to leaves. After salt treatment for 5 days,the soluble protein content in Shul(u|¨) leaves decreased to 76% of that of CK the activities of EPs increased;When low concentration(10,25 and 50μmol·L-1) of SNP employed,the activities of EPs decreased until the concentration of SNP exceeded 100μmol·L-1.To further study the effect of SNP on the EP in Shulii seedling leaves under salt stress, the followed experiments,the treatments with 300 mmol·L-1 NaCl combined 25,50,100,200,400μmol·L-1 SNP were employed.The results showed that leaves turned yellow after 3 days of salt treatment only.Meanwhile,the total activities of EPs increased and Rubisco began to degrade,only three EPs(EP2,EP4 and EP5) were detected,the activity of EP4 increased,but that of EP2 and EP5 declined obviously;the symptom of yellowing in leaves was alleviated,but there was no phenomenon reversed by 25μmol·L-1 SNP treatment for 3days accompanied by the increasing activities of EP2,EP4 and EP5;when the concentration of SNP exceeded 50μmol·L-1,the leaves began to death,the soluble protein content declined obviously associated with the decreasing of activities of three EP isozymes (EP2,EP4 and EP5),but EP4 showed stability.It showed that SNP treatment at certain low concentration had an alleviation to the leaf senescing promoted by salt stress,however,this function could be affected as the increasing of salt concentration.Moreover,it showed that EP2,EP4 and EP5 might play an important role in the protein degradation during leaf senescing promoted by salt stress.3,The changes of EP isozymes of Shul(u|¨) leaf during natural senescenceTo further know the changes of EP isozymes in Shul(u|¨) leaves during senescence,the changes of both EP isozymes and soluble protein content in leaves during natural senescence were studied.The results showed that the protein content decreased until two weeks of leaf unfolding. Meanwhile,the degradation of protein was associated with the increasing activities of EPs. The results using the native PAGE showed that the bands of EP1 and EP2 were linked with each other,so do that between EP4 and EP3 due to the high activity of EP4.Rubisco was obviously degradated,it seemed that Rubisco was degradated into two bands which were detected clearly in the gel.In Shul(u|¨) plant on four-leaf period,the difference of EP isozymes in different position leaves was investigated.Five EP isozymes were detected in all leaves at different position, but in the 4th leaf,both EPs activities and content of Rubisco were low,and the degradation of Rubisco were not detected;Five EP isozymes had high activities accompanied by the degradation of Rubisco in the leaves at other positions,especially in the 1st leaf,It suggested that the leaves at 1st,2nd and third position were in different process of senescence.In a word,the activities of EP1,EP2 and EP4 increased during Shul(u|¨) leaf senescence, but the activities of EP3 and EP5 decreased during the late stage of senescence. Consequently,EP1,EP2 and EP4 might play an important role on protein degradation in Shul(u|¨) leaf senescence.
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