Signal Mechanisms Of Exogenous Nitric Oxide On The Promotion Of Wheat Seeds Germination

Effects of nitric oxide (NO) donor, sodium nitroprusside (SNP) on the germination and activities of hydrolytic enzymes in wheat (Triticum aestivum L.) seeds were researched. Treatment with SNP dramatically promoted imbibition and germination of wheat seeds, increased the elongation of roots and shoots, accelerated the degradation of reserve starch and the liquefaction of endosperms under osmotic stress. After osmotic stress removed, the wheat seed treated with SNP could also retain higher viability. Further studies showed that SNP could induce amylase activities apparently, enhance protease activities slightly, while exhibit no roles in esterase. The effects of nitric oxide (NO) donor, sodium nitroprusside (SNP) on the antioxidant metabolism in germinating wheat seeds were also investigated. Treatment with SNP dramatically promoted the activities of CAT, APX and the contents of proline simultaneously, inhibited activities of LOX, decreased the contents of H₂O₂ and MDA. All of these functions were beneficial for improving the antioxidant capacity during germination of wheat seed under osmotic stress.Moreover, SNP could strongly increase the content of sugars, such as fructose, glucose and sucrose in wheat seeds during early germination (12 h) under normal conditions. When the seeds treated with SNP with or without PTIO, an NO scavenger, and different exogenous sugars, including fructose, glucose, sucrose and sorbitol, mannitol as controls, it was found that there might be existing a cross-talking between NO and sugars signaling in the activation of amylase in the early stage of germination, while GA acted no such actions in the same period.The effects of nitric oxide (NO) and gibberellic acid (GA₃) on the responses of amylases in wheat seeds were further investigated during the first 12 h of germination. GA3 had no effects on the activities of α-amylase (EC 3.2.1.1) or p-amylase (EC 3.2.1.2), either in intact seeds or embryoless halves within 12 h. In contrast, addition of sodiumnitroprusside (SNP), an NO donor, was able to induce a rapid increase in P-amylase activity without affecting a-amylase. Furthermore, the rapid response of p-amylase to SNP in wheat seeds could be attributed to NO and was approximately dose-dependent. Some other aspects of SNP induction of amylase isozymes were also characterized. Further investigations showed that SNP might play an interesting role in the dissociation of free p-amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced the release of bound P-amylase from glutenin and its crude enzyme preparation. However, the slight increase in protease induced by SNP might not be responsible for this action. Interestingly, based on the fact that the rapid response of p-amylase to NO also existed in seeds of other species, such as barley, soybean, rice and watermelon, it might be a universal event in early seed germination.Activities of P-amylase in seeds of three wheat species and their germination index were investigated at the same time. It showed that there was a positive correlationship between activities of p-amylase and the index of germination in wheat seeds. Experiment of p-amylase inhibitors also showed us P-amylase played a key role in wheat seeds germination.In this paper, characterization of beta-amylase isozymes induced by NO was further investigated. The pI of the SNP-induced beta-amylases, Type II, as determined by IEF, was in the range 6.0-7.0, and that of the intrinsically active ones, Type I, in the range 4.8-6.0. The optimum pH of these amylases was found to be 4.5-6.5, and they were stable in the pH range 3.0-9.0. The optimum temperature of these enzymes was 40°C, but they active even up to 55 °C. Further investigations using protein synthesis inhibitors, such as actidione D and chloromycetin, showed that beta-amylase isozymes induced by NO were not synthesized de novo.