Studies On Mechanism Of γ-Aminobutyric Acid Accumulation And Antioxidant Activity In Germinated Foxtail Millet Under Hypoxia Stress And Salt Stress

Foxtail millet (Setaria italica L.) is one of main foodstudff corp and widel cultivated in the word, it includes nutritional components, such as starch, protein, lipid, vitamins and minerals, besides it includes functional components which possesses series of functions. Germination is a method that can modify the presence of nutrients and antinutrients of cereals. There are significant changes during seeds germination, including interconversion and production of new compounds. Especially, Germination can increase the content of functional components of cereals and enhance their healthful functions using biochemical technique, such asγ-aminobutyric acid (GABA) and antioxidant matter. GABA, a non-protein amino acid, acts as an inhibitory neurotransmitter in the brain and spinal cord of mammals and possesses series of functions, such as regulation of blood pressure and heart rate et al. It has been reported that GABA synthesis in plants is promoted by various environmental stresses, e.g. hypoxia and salt stress, etc. In this paper, mobilization of stored reserves and the dynamic changes of GABA in foxtail millet cultivars during germination were investigated. The mechanism of GABA accumulation in germinated foxtail millet was carried out under hypoxia stress and salt stress, the changes of reactive oxygen species (ROS), antioxidative enzymes and antioxidant matter was studied under hypoxia and salt stress, and the relationship among GABA content and antioxidative enzymes, antioxidant matter were analyzed. The main results were as follows:1. The physiological activity and chemistry components significantly changes during 60 h germination in foxtail millet cultivars at 25℃in darkness. The sprout length increased 8-14 mm, respiratory rate was improved 3.51-5.02 fold, the dried matter lost 4.19-5.52%, starch content decreased 43-60%, reducing sugar and free amino acid increased 0.90-1.19 and 1.28-2.22 fold, respectively, GABA content increased 92.32-161.42% compared to before-germination. The difference of GABA content in millet cultivars was significant during germination; GABA content in JG-34 among millet cultivars was maximal, tthe maximum of GABA content was 13.54 mg/100g, which makes this millet a potentially promoting source for GABA-enriched foods.2. Under hypoxia stress, GABA increment of germinated foxtail millet (JG-34) culturing in citrate solution was higher than in acetate buffer solution, moreover, citrate buffer at 0.01 mol-L'1 was most effective as culture medium. Box-Behnken experimental design showed that the optimal conditions for GABA accumulation during millet germination were at a temperature of 33℃; an air flow rate of 1.9 L/min; and a pH value of 5.8. Under these conditions, the maximal observed production of GABA (26.96 mg-l00g-1FW) was obtained, which was almost 5 fold of material. Analysis of variance indicated that culture temperature and aeration ratio affect significantly GABA content of germinated millet, there was a significant interaction between temperature and pH. Box-Behnken design indicated that the optimal culture components for GABA accumulation were:Glu at a concentration of 1.2 mg/ml, PLP at a concentration of 50μmol/L and CaCl2 at a concentration of 2.5 mmol/L. Under the optimal conditions, the maximal production of GABA (42.87 mg/100g FW) was obtained. The effect of Glu on GABA content of germinated millet was higher than PLP and Ca2+, however, the interaction of PLP and Ca2+ affect significantly GABA content.3. The GAD activity and GABA content increased significantly with the time extent and NaCl concentration rising under salt stress. The maximal GAD activity and GABA content occurred at the moment of 48 h under 100 mmol/L NaCl, at the same time, GAD activity and GABA content increased 71% and 1.08 fold, respectively. Ca2+ stimulated GAD activity and promoted GABA accumulation under salt stress. GAD activity raised 79.91% and GABA content increased 40.99% when Ca2+ concentration was 5 mmol/L under NaCl stress. GAD activity and GABA accumulation decreased when added exogenous LaCl3 (Ca2+ inhibitor) and EGTA (Ca2+ chelator) under NaCl stress. Through compared the influence of hypoxia and salt stress on GAD activity and GABA accumulation of millet, it indicted that the promoted effect of hypoxia to GAD activity and GABA accumulation of millet was greater than salt stress, however, Ca2+ promoting effect to GAD activity and GABA accumulation under hypoxia were higher than under salt stress.4. ABA induced GAD activity and GABA accumulation. When it was 36 h under 75μM ABA treatment, GAD activity of germinated millet increased 92.89%, when the treatment time was 60 h, GAD accumulation reached to 28.77 mg/100g, which increased 1.30 fold compared to pre-treatment. Moreover, endogenous ABA content in germinated millet sharply enhanced under ABA, NaCl, ABA and NaCl mixed treatments respectively. The maximal value of endogenous ABA content was occurred when ABA treated 36 h, the changes curve of GAD activity was consistent with endogenous ABA, at the same time, GABA content increased. Endogenous ABA content reached to its peak value when it was 36 h under NaCl treatment, which was prior to peak of GAD activity which treatment time was 48 h. It was indicted through correlation analysis that GABA content and sprout length, GAD activity and endogenous ABA content were significantly positive correlation. This phenomenon showed that endogenous ABA regulated the accumulation of GABA accumulation under salt stress and exogenous ABA treatment. Treatment of salt stress combined exogenous ABA resulted in the accumulation of GABA by activating GAD activity.5. The membrane lipid peroxidation (MDA content and relative electron leakage) and ROS (H2O2 content) affected by hypoxia stress and salt stress. The MDA content, REL and H2O2 content markedly increased with aeration ratio reducing under hypoxia stress, the activities of antioxidative enzymes corresponding increased significantly. SOD and POD activity presented firstly increasing and then decreasing, CAT activity showed to decline with aeration ratio decreasing. The promoted effect of SOD and POD activity under hypoxia were higher than CAT. The MDA, REL and H2O2 content ascend with the NaCl concentration increase, The change trend of SOD and POD activity were like parabola, however, CAT activity have no changes under lower NaCl concentration, it rapid rose under higher NaCl concentration. After the exogenous Ca2+ treatment, the MDA, REL and H2O2 content decreased and the activities of SOD, POD and CAT further increased in germinated foxtail millet under hypoxia and salt stress. Exogenous LaCl3 and EGTA cause the inhibition of antioxidative enzymes activities and the increase of ROS contents, and the inhibiting effect of EGTA treatment was more dominant than that of LaCl3, which indicated that exogenous Ca2+ treatment alleviated injury occurred by hypoxia and salt stress and increased the adaptability of germinated seedlings to hypoxia and salt stress. Correlation analysis showed that GABA content and SOD, POD and CAT activity in germinated millet presented significant positive correlation, there are significant correlations between MDA, REL and SOD, POD and CAT, H2O2 content was negatively correlated with activities of SOD, POD, CAT and content of GABA, which indicted that hypoxia and salt stress caused the increase antioxidative enzyme (SOD, POD and CAT) activities and GABA content which relieved the harm of hypoxia and salt stress to germinated foxtail millet.6. The content of total phenolic and total flavonoids of germinated millet appeared the trend of firstly increasing later decreasing under hypoxia stress, after 12 h treatment, the total phenolic and total flavonoids content increased 18.95 and 57.86% respectively compared to pre-treat, AsA content slowly increased with the extent of treatment time, which enhanced 61.79% compared with pre-treat at the 60 h treatment. Reducing power of germinated millet increased after 36 h treated under hypoxia stress; scavenging OH, DPPH free radical activity and anti-membrane lipid peroxidation in linoleic acid system increased by different extent under hypoxia stress, but the superoxide radical scavenging activity of germinated millet were very lower under hypoxia.7. Salt stress significantly enhanced the content of total phenolic, total flavonoids and AsA. After 36 h treatment, total phenolic and AsA increased 20.61 and 99.26% than pre-treat, After 48 h treatment, the total flavonoids content was 1.99 fold compared with pre-treat. The reducing power of germinated millet was 2 fold when treated 48 h under salt stress, scavenging-OH activity markedly enhanced under salt stress, the peak value occurred when 24 h treatment, increasing 91.18% compared to pre-treat; NaCl stress promoted anti-O2" activity, which increased 2.88 fold after 60 h; DPPH radical scavenging activity were in increasing trend under salt stress; Anti-membrane lipid peroxidation in linoleic acid system reached to 83% treated 36 h under salt stress, which increasing 1.68 fold compared with pre-treat.8. Correlation analysis showed that the contribution of activities of SOD and POD and content of AsA and GABA to antioxidative activity were higher than total phenolic and total flavonoids under hypoxia stress. The correlation between antioxidative enzymes and antioxidant capacity under NaCl stress was higher than under deionized water treatment, the correlation of total phenolic to antioxidant capacity was higher than AsA, the correlation of between total flavonoids, GABA and antioxidant capacity was highest among all determined items. The results showed that hypoxia and salt stress promoted the antioxidative enzymes and antioxidant of germinated foxtail millet, which increased antioxidant capacity of germinated millet.