Change And Characteristic Of Root Endopeptidase Isozymes And Degradation Of Large Subunit From Rubisco Under Salt Stress

Endopeptidase, which degrades protein by hydrolyze endopeptide bond, is one kind of most important proteases in cell. In plants, it is widely distributed and plays an important role in different development stages, such as germination and organic senescence, even during the response to stress. An increasing number of references are correlated with the change and function of endopeptidase isozymes during the normal development of plants, but less with that of plants under stress, especially less with the change and biochemical characteristics of root endopeptidase isozymes(REs) in plants under salt stress.To further know the change of endopeptidase and protein degradation in wheat roots under salt stress, root growth and its oxidative damage (such as, the root length, the TBARS content and the rate change of O2-production), the change and biochemical characteristics of endopeptidase isozymes in roots, the degradation of Rubisco and large subunit (LSU) in leaves of wheat(Triticum aestivum L., H6756) under salt stress have been studied. The results of these researches are as follows.1、The changes of root growth and its oxidative damage of wheat under salt stressWheat H6756seedlings were treated by different salt concentration, i.e.O (control),25,75,150,300mmol·L-1NaCl for8days. There was no significant difference on root length between that treated by25mmol·L-1NaCl and the control. But the difference between that treated by300mmol·L-1NaCl and the control was noticeable, the root length of wheat after300mmol·L-1NaCl treatment on the8th day was only66.2%of the homeochronous control. And the differences on root length between that treated by75or150mmol·L-1NaCl and the control were more marked along with the postponement of experimental period or the raise of NaCl concentration. The rate of O2-production in wheat roots under25mmol·L-1NaCl stress on the8th day reached the maximum value which increased by39.2%than that of control, while the rate of O2-production in roots under75mmol·L-1NaCl stress on the6th day reached the maximum value which increased by39.65%than that of control. Under150mmol·L-1NaCl stress, the rates of O2-production of roots reached the maximum values on the2nd day and declined from the4th day to the8th day, but it was still keeping slightly higher than that of control. However, the rate of O2-production in roots under300mmol·L-1NaCl stress was approximately double of the control during8days, maintained on the higher level than that of other treatment. Similarly, the TBARS contents in wheat roots treated with25and75mmol·L-1NaCl have no greatly changed compared with that of control. The TBARS contents in roots of wheat under150mmol·L-1NaCl or300mmol·L-1NaCl stress were raised from0to the4th day and reached the maximum value which increased respectively by22.08%or44.42%than that of the control on the4th day. The TBARS content in wheat roots under150mmol·L-1NaCl stress was increased at first and decreased nearly to the level of the control after the6th day, while the TBARS content of wheat roots treated with300mmol·L-1NaCl was always higher than the others.2、The endopeptidase changes of wheat roots under salt stressWheat H6756seedling were treated by different salt concentration, i.e. O(control),25,75,150,300,400,500mmol·L-1NaCl for8days. Results showed that the specific activities of endopeptidase in roots increased and the soluble protein contents of roots decreased in various degrees during8days. Compared with the control,25,75and150mmol·L-1NaCl treatments raised the specific activity of endopeptidase slightly. However,300mmol·L-1NaCl treatment dramatically increased the specific activity of endopeptidase from0to the4th day, and the endopeptidase specific activity on the8th day reached to1.86times of the control. The endopeptidase specific activities in roots under400and500mmol·L-1NaCl stress were both greatly raised in8days and were higher than that of300mmol·L-1NaCl treatment in same period. The changing trends of soluble protein content in wheat roots under25and75mmol·L-1NaCl stress are similar to the control. The soluble protein content in roots under150mmol·L-1NaCl stress was the lowest on the6th day and increased nearly to the control on the8th day. The content of soluble protein in roots treated with300mmol·L-1NaCl was declined gradually and was only34.2%of the control on the8th day. The content of soluble protein in root under400and500mmol·L-1NaCl stresswere decreased gradually in8days and both were lower than that of300mmol·L-1NaCl treatment in same experimental period.The changes of endopeptidase isozymes in wheat root under salt stress were studied by using natural gradient-polyacrylamide gel electrophoresis (GPAGE) co-polymerized with gelatin in the gel. Five endopeptidase isozymes named as RE (root endopeptidase isozymes)1-5were detected. Of the REs, RE1was detected only in NaCl treated roots, and the activity of RE1was gradually increased, with increasing of salt concentration. RE2and RE5activity were raised at first and then decreased, along with the experimental period or the increasing of salt concentration. Both RE3and RE4activity were raised greatly when NaCl concentration was over300mmol·L-1. The activity of RE5was the weakest one of the five REs. Accordingly, we presumed that REl and RE2were the REs more related to saline stress. In addition, ABA or GA3treatment at appropriate concentration could inhibite protein degradation by reducing the high endopeptidase activities of wheat roots under NaCl treatment.3、 Characterization of endopeptidase and two endopeptidase isozymes in wheat roots under salt stressOn the basis of the previous experiments, the total roots of the wheat H6756treated with300mmol·L-1NaCl for6days were used as test materials. The results showed that the total endopeptidase specific activity of wheat roots under salt stress was significantly stimulated at pH4and50℃compared with other conditions, and the specific activity of total endopeptidase incubated at4-40℃for1hour maintained at high level with no greatly changed, but the activity decreased when the incubation temperature was raised above40℃, and the activity incubated at80℃still had68.7%of that at4℃. Additionally, the activity was inhibited markedly by EGTA, EDTA and PMSF, but activated obviously by ATP.The activity of RE2could be detected at wider range of pH values (pH3-10) and temperature levels (20-80℃), the optimal pH was6and the optimal temperature was50℃, and it still presented high activity even by incubation at70℃for1hour. So RE2possessed the well stability of pH and thermal. The activity of RE1could be detected at the range of pH3-9and30-70℃, the optimal pH was5and the optimal temperature was40℃. Compared with RE2, RE1had no well thermal stability, and lost most activity after1hour incubation at temperatures above50℃. Be tested by different class-specific inhibitors, RE1was characterized as serine-protease, and RE2was characterized as serine-protease and metal-protease.According to results by natural GPAGE without gelatin, western blot and protease inhibitor test, RE1was finally identified to be20S proteasome. In addition, it showed that the caseinolytic activity of RE1(20S proteasome) was higher at pH5and200mmol·L-1HAc-NaAc than other buffer conditions.We also found the amounts of20S proteasome gradually decresed, with the concomitant increase of NaCl concentration or the postponement of experimental period, by GPAGE and western blot.4、 The effect of salt on the degradation of Rubisco large subunit(LSU) in wheat leavesH6756seedlings were treated with different salt concentration, i.e. O(control),25,75,150,300mmol·L-1NaCl for8days. The seedling under25or75mmol·L-1NaCl stress hardly showed great difference with the control. But compared with the control, the growth period of seedling treated with150or300mmol·L-1NaCl had been delayed, and the symptom was obviously aggravated along with the postponement of experimental period or the raise of NaCl concentration. On the8th day, the wheat seedling under300mmol·L-1NaCl stress only had two leaves, while the control had three leaves.In contrast to the control, the soluble protein content of first wheat leaf under different concentration NaCl stress was all significantly decreased from0to the4th day and maintained basically on a stabile level from the4th day to the8th day. And the endopeptidase specific activity of first wheat leaf treated with different concentration NaCl was raised gradually, but the effect was lessened from the6th day to the8th day. The endopeptidase specific activity in first leaf under300mmol·L-1NaCl treatment was higher than other treatments, while the soluble protein content of the treatment was the least. It is shown that NaCl treatment induced the enhancement of endopeptidase specific activity and aggravated the degradation of wheat leave protein, resulted in declining the soluble protein content of wheat leaves. According to results above all, it could be concluded that roots and the first leaf of wheat under NaCl stress have similar features in the changes of the soluble protein content and the endopeptidase specific activity, and it further indicated that the wheat H6756have good salt tolerance and could resist or adjust to the environment change via accelerating the rate of protein turnover.In addition, the degradation product of LSU in vivo with molecular weight of51kDa was detected by SDS-PAGE and western-blotting, and the contents of Rubisco and LSU were decreased continuously in natural growth period. The protein contents of51kDa degradation product of LSU under different concentration NaCl stress were increased gradually and reach to the maximum on the6th day, then decreased noticeably from the6th day to the8th day. In contrast to the control and other NaCl treatment, the contents of LSU and51kDa fragment of LSU in wheat leaves under150or300mmol·L-1NaCl stress were significantly declined from the6th day. And, the51kDa degradation fragment of LSU in vivo was hardly be detected in wheat leaves treated with300mmol·L-1NaCl on the8th day. The content of Rubisco and the LSU degradation of wheat leaves treated with25,75mmol·L-1NaCl were similar to those of control. These results suggested that high concentration (150,300mmol·L-1) NaCl accelerate degradation of Rubisco, LSU and the degradation product of LSU in vivo by significant increase of endopeptidase specific activity. Once LSU (53kDa) had been degraded to51kDa product, the degrade speed of51kDa fragment would be accelerated. The result suggests that a polypeptide of2kDa approximately on N-terminal of LSU maybe have an important role in stability of Rubisco structure.All the results are contributing to comprehensive understanding about endopeptidase isozymesa and protein degradation in plant roots and leaves, and are also helpful to further clarify the relationship between plant endopeptidase, protein degradation and salt response.