Runqiang Yang

GABA shunt and polyamine degradation pathway on γ-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia.

GABA shunt and polyamine degradation pathway on γ-aminobutyric acid (GABA) accumulation in germinating fava bean under hypoxia was investigated. GABA content, GAD and DAO activity were significantly increased under hypoxia treatment. Glu and polyamine contents enhanced largely and thus supplied as sufficient substrates for GABA formation. In contrast, GABA content decreased, mainly in the embryo, after removing the hypoxia stress. DAO activity, Glu and polyamines contents decreased, while an increment of GAD activity was observed. This indicated that GAD activity can be not only regulated by hypoxia, but by the rapid growth of embryo after the recovery from hypoxia stress. When treated with AG, DAO activity was almost inhibited completely, and the GABA content decreased by 32.96% and 32.07% after treated for 3 and 5 days, respectively. Hence, it can be inferred that about 30% of GABA formed in germinating fava bean under hypoxia was supplied by polyamine degradation pathway.

Factors Influencing Diamine Oxidase Activity and γ-Aminobutyric Acid Content of Fava Bean (Vicia faba L.) during Germination

Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation.

Effect of NaCl stress on health-promoting compounds and antioxidant activity in the sprouts of three broccoli cultivars

Abstract Health-promoting compounds, antioxidant and myrosinase activity in the sprouts of three broccoli cultivars under 40 mM, 80 mM and 160 mM NaCl were investigated. LangYan (LY) sprouts had the richest health-promoting compounds among the evaluated cultivars. Treatment of 40 mM and 80 mM NaCl significantly decreased the content of ascorbic acid and total phenolic as well as antioxidant activity, but did not affect glucoraphanin, sulforaphane and myrosinase activity compared to the control. However, 160 mM NaCl treatment significantly enhanced the level of total phenolic, glucoraphanin, sulforaphane, antioxidant and myrosinase activity, while significantly decreased ascorbic acid content. The results suggest that 160 mM NaCl treatment would enhance antioxidant activity and sulforaphane yield in broccoli sprouts. The health-promoting value of broccoli sprouts depends on plant genotype and could be affected by NaCl stress.

Comparative proteomic and physiological analyses reveal the protective effect of exogenous calcium on the germinating soybean response to salt stress.

Calcium enhances salt stress tolerance of soybeans. Nevertheless, the molecular mechanism of calciums involvement in resistance to salt stress is unclear. A comparative proteomic approach was used to investigate protein profiles in germinating soybeans under NaCl-CaCl2 and NaCl-LaCl3 treatments. A total of 80 proteins affected by calcium in 4-day-old germinating soybean cotyledons and 71 in embryos were confidently identified. The clustering analysis showed proteins were subdivided into 5 and 6 clusters in cotyledon and embryo, respectively. Among them, proteins involved in signal transduction and energy pathways, in transportation, and in protein biosynthesis were largely enriched while those involved in proteolysis were decreased. Abundance of nucleoside diphosphate kinase and three antioxidant enzymes were visibly increased by calcium. Accumulation of gamma-aminobutyric acid and polyamines was also detected after application of exogenous calcium. This was consistent with proteomic results, which showed that proteins involved in the glutamate and methionine metabolism were mediated by calcium. Calcium could increase the salt stress tolerance of germinating soybeans via enriching signal transduction, energy pathway and transportation, promoting protein biosynthesis, inhibiting proteolysis, redistributing storage proteins, regulating protein processing in endoplasmic reticulum, enriching antioxidant enzymes and activating their activities, accumulating secondary metabolites and osmolytes, and other adaptive responses. Biological significance Soybean (Glycine max L.), as a traditional edible legume, is being targeted for designing functional foods. During soybean germination under stressful conditions especially salt stress, newly discovered functional components such as gamma-aminobutyric acid (GABA) are rapidly accumulated. However, soybean plants are relatively salt-sensitive and the growth, development and biomass of germinating soybeans are significantly suppressed under salt stress condition. According to previous studies, exogenous calcium counters the harmful effect of salt stress and increases the biomass and GABA content of germinating soybeans. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unknown. This paper is the first study employing comparative proteomic and physiological analyses to reveal the protective effect of exogenous calcium in the germinating soybean response to salt stress. Our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. The functions of those significantly changed proteins are also analyzed. The physiological and comparative proteomic analyses revealed the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. The findings from this paper are beneficial to high GABA-rich germinating soybean biomass. Additionally, these findings also might be applicable to the genetic engineering of soybean plants to improve stress tolerance.

Accumulation and Identification of Angiotensin-Converting Enzyme Inhibitory Peptides from Wheat Germ

The incubation conditions of wheat germ for angiotensin I-converting enzyme inhibitory activity (ACEI) elevation and peptide accumulation were investigated, and five ACE inhibitory peptides were obtained. The effect of individual factors such as incubation time, temperature, initial pH, and liquid to solid ratio on ACEI and peptide concentration of incubation medium was evaluated, respectively. The combinations of four factors were further optimized using a Box-Behnken design. Under the best incubation condition (pH 4.4 with a liquid to solid ratio 8.14 mL/g at temperature 47 °C, for 7 h), maximum ACEI (92.16%) and peptide concentration (88.12 mg/g) were obtained, which were 6.2- and 2.4-fold, respectively, as compared to the unincubated wheat germ. After they were purified, five ACE inhibitory peptides, VEV, W, NPPSV, QV, and AMY, were identified by liquid chromatography/tandem mass spectrometry. The IC(50) were 115.20, 94.87, 40.56, 26.82, and 5.86 μM, respectively.

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

BACKGROUND γ-Aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. RESULTS The molecular mass of the enzyme estimated by Sephadex G-100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg(2+), Cu(2+), Fe(3+), aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA-Na(2)), L-cysteine and β-mercaptoethanol. The K(m) value of DAO was 0.23 mmol L(-1) for putrescine and 0.96 mmol L(-1) for spermidine. However, the enzyme did not degrade spermine. CONCLUSION DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine.

Nitric oxide mediates isoflavone accumulation and the antioxidant system enhancement in soybean sprouts

In this study, we investigated the relationships between endogenous NO signal transduction pathways, the antioxidant system and isoflavone accumulation induced by UV-B radiation in soybean sprouts. Results showed that UV-B-triggered NO generation induced isoflavone accumulation by up-regulating the activity and gene expression of key enzymes (phenylalanine ammonia lyase, PAL; chalcone isomerase, CHI; chalcone synthase, CHS; isoflavone synthase, IFS) that participate in isoflavone biosynthesis and enhanced the antioxidant system by regulating levels of antioxidants (glutathione reductase, GR; glutathione S-transferase, GST; ascorbate peroxidase, APX; glutathione GSH; ascorbic acid, ASC), antioxidant enzyme activities (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT) and their gene expression. These effects were inhibited by the addition of a specific NO-scavenger, carboxy-PTIO (cPTIO). The inhibition was reversed through application of the exogenous NO donor, SNP. Overall, NO is an essential signaling molecule, mediating UV-B-induced isoflavone accumulation and the antioxidant system enhancement in soybean sprouts.

Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.).

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58 kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40°C with a K(m) value for glutamic acid (Glu) of 2.63 mM. The enzyme was inhibited significantly by Cu(2+), Fe(3+), Mg(2+), Ba(2+), aminoxyacetate, EGTA, Na(2)EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca(2+) 0.2mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787 bp long, containing a 1527 bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74 kDa and a pI of 5.41 (GenBank accession number: JX444699).

Organ-Specific Proteomic Analysis of NaCl-Stressed Germinating Soybeans

A comparative proteomic approach was employed to explore proteome expression patterns in germinating soybeans under NaCl stress and NaCl-aminoguanidine treatment. The proteins were extracted from 4-day-old germinating soybean cotyledons and noncotyledons (hypocotyl and radicle) and were separated using two-dimensional polyacrylamide gel electrophoresis. A total of 63 and 72 differentially expressed proteins were confidently identified by MALDI-TOF/TOF in the noncotyledons and cotyledons, respectively. These identified proteins were divided into ten functional groups and most of them were predicted to be cytoplasmic proteins in noncotyledons. Moreover, γ-aminobutyric acid was accumulated while the major allergen (Bd 30K protein) was reduced in the germinating soybeans. The proteins involved in energy metabolism and in protein processing in endoplasmic reticulum were enriched under NaCl stress. Meanwhile, the negative effect of stress was aggravated once polyamine degradation was inhibited. Redistribution of storage proteins under stress indicated that storage proteins might not only function as seed storage reserves but also have additional roles in plant defense.

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS After purification a single protease of molecular weight 61-63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg(2+), Mn(2+), Ba(2+) and iodoacetic acid and stimulated by Li(+), Ca(2+), Cu(2+), β-mercaptoethanol and dithiothreitol, while Zn(2+), L-cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K(m) of 0.562 mg mL(-1) with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION The purified enzyme from wheat germ was identified as a cysteine protease.