The Investigation Of Endopeptidases And 20S Proteasome In Wheat Germination Seed Endosperms And Salt Stress Root Tips

Wheat cultivar Yangmai 158 (Triticum aestivum L., Yangmai 158) is the experimental material of this study, the changes of cysteine endopeptidases and their biochemical characteristics were investigated in wheat endosperm during seed germination and subsequential seedling growth, and enzymatic properties of wheat endosperm 20S proteasome and the response of which to this germination process were also investigated; simultaneity, the effect of salt-induced oxidative damage to the 20S proteasome was also studied in wheat root tips under salt stress. The main contents of this dissertation as followings:1. The investigation of cysteine endopeptidases and 20S proteasome in wheat endosperm after seed imbibitionThe endopeptidases (EPs) in wheat endosperm after seed imbibition were characterized by 4-20% gradient PAGE with gelatin copolymerized into the gel. Four cysteine EPs (EP1, EP2, EP3 and EP4) were detected in wheat endosperm during the 7 d growth after seed imbibition. The results showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0℃. All EPs were completely inhibited by 25μM E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.The 20S proteasome from wheat endosperm was purified by three sequential centrifugations and gradient PAGE (GPAGE) to apparent homogeneity, as judged by GPAGE and western blotting. The results showed that the purified 20S proteasome could clearly cleave the peptidyl-arylamide bonds in model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, used for reflecting its chymotrypsin-like and trypsin-like activity respectively. Both of optimum pH were 8.0, but the optimum temperatures of chymotrypsin-like and trypsin-like activity were 55℃and 37℃respectively, the activities above were clearly inhibited by MG115 or PMSF. The results also showed that polyubiquitinated proteins remained constant from 0 to 7 d after seed imbibition. But the caseinolytic activity and amount of 20S proteasome associated with aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached the highest level on 4 DAI and kept the level till 7 DAI. With the mRNA level increase ofβ5-subunit for 20S proteasome from 2 DAI, the caseinolytic activity and amount of 20S proteasome increased accordingly from 3 DAI. In addition, the main storage proteins of wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidences above suggested that the main role of 20S proteasome may not be to degrade massive proteins of wheat endosperm after seed imbibition.2. Effect of salt-induced oxidative damage to the 20S proteasome in wheat root tips under salt stressIn order to study the effect of salt-induced oxidative damage to the 20S proteasome in wheat root tips under salt stress, the root tips from wheat seedlings treated with 200 mM NaCl for 12,24,48 and 72 h were used for studying the content of H2O2, production rate of O2-, the degree of lipid peroxidation and plasma membrane integrity; and the carbonyl level, caseinolytic activity and protein amount of 20S proteasome and other biochemical characteristics. The contents of carbonylated and ubiquitinated proteins (Ub-P) in root tips were also investigated. The results showed that the contents of H2O2 and TBARS, production rate of O2-, and the quantity of cell death in root tips all gradually increased along with the stress time prolonged. Histochemical detection of lipid peroxidation and loss of plasma membrane integrity found that the degree and distribution of the histochemical staining patterns of these two events were aggravated and extended along with the prolonged treatment time. Caseinolytic activities of the total and the 20S proteasome both gradually increased, with a concomitant increase of the carbonyl level of total proteins and the 20S proteasome during the salt treatment. But the amounts of 20S proteasome and Ub-P decreased oppositely during this process. Metal-catalyzed oxidation of soluble proteins from control root tips validated that oxidative treatment could increase the caseinolytic activity of 20S proteasome, but oppositely decrease its protein amount. These results indicated that the amount and activity of 20S proteasome could be contrarily altered by mild oxidative modification in wheat root tips under salt stress.