1,2,4-三氯苯胁迫对萌发大豆种子中活性氧的影响

以盆栽法研究了不同浓度 1,2 ,4 三氯苯 (TCB)胁迫对萌发大豆种子中活性氧代谢的影响 .结果表明 ,10 0～ 30 0 μg·g-1TCB胁迫初期 (1～ 3天 )促使萌发大豆种子呼吸强度升高及其峰值提前出现 ,超氧阴离子自由基 (O2 - )及过氧化氢 (H2 O2 )的积累显著增加 ,同时伴随丙二醛 (MDA)含量升高 ,显示发生膜脂质过氧化作用 .TCB胁迫 1～ 6天使活性氧清除酶功能紊乱 ,其中过氧化物酶 (POD)活性升高 ,超氧化物岐化酶 (SOD)活性开始上升后转为下降 .在萌发大豆种子受TCB胁迫伤害过程中 ,活性氧代谢失衡造成的膜脂质过氧化将起着重要作用 .     

Trypsin-like protease from soybean seeds. Purification and some properties

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.     

CO2激光处理对大豆种子萌发及生理的影响

以大豆(Glycine max Merr.)为实验材料,研究了CO2激光不同功率密度和不同时间处理对大豆种子发芽率、淀粉酶活性、可溶性蛋白质、可溶性糖含量和氨基酸含量的影响。结果表明：(1)16mw／mm^2、18mw／mm^2、20mw／mm^2CO2激光处理均能提高种子发芽率、淀粉酶活力、可溶性蛋白质、可溶性糖含量和游离氨基酸含量,其中18mw／mm^2CO2激光处理效果最好;(2)相同功率(18mw／mm^2)不同时间处理均能提高种子发芽率、淀粉酶活力、可溶性蛋白质和游离氨基酸含量,处理时间以75s效果最好。     

The anaerobic response of soybean

The effect of anoxia on roots of soybean (Glycine max [L.] Merr., variety `Williams') was studied at various levels and the results compared to those from previously studied species. While alcohol dehydrogenase (ADH) activity is induced in a manner similar to other plant species, other aspects of the anaerobic response are unique to soybean. A variety of molecular clones was used to analyze changes in soybean and maize RNA levels. Increased RNA accumulation was observed in both species with a maize ADH clone, while a maize aldolase and one of the two different maize glyceraldehyde-3-phosphate dehydrogenase cDNA clones showed induction only in maize. A maize sucrose synthase 1 clone showed induction in maize but no hybridization to soybean RNA samples. The reduction in the number of anaerobically inducible soybean genes relative to maize is consistent with in vivo and in vitro protein synthesis results. Only four major proteins are labeled during anoxia in soybean, one corresponding to ADH, while maize has been reported to have about 20. In either species, in vitro translation yields similar products with RNA from anaerobic and pre-stress plants, indicative of translational control during anoxia. These results are discussed in relation to the differential tolerance of maize and soybean to anaerobic stress.     

On the mechanism of aging in soybean seeds

Changes in seeds of soybeans (Glycine max [L.] Merr. var. Wayne) which occur during accelerated aging (41 C, 100% relative humidity) showed subsequent loss of vigor, a decline in early respiratory activity, increased leakage of electrolytes, losses of as much as 10% dry weight from imbibing cotyledons, and a decrease in the swelling response of the imbibing system (seed plus H₂O). Each of these changes with aging is interpreted as resulting from deteriorative changes in membranes.     

The performance and phase separated characteristics of an anaerobic baffled reactor treating soybean protein processing wastewater

A laboratory-scale anaerobic baffled reactor (ABR) with four compartments using soybean protein processing wastewater as organic loading rates (OLRs) was investigated for the performance and phase separated characteristics. It was found that the chemical oxygen demand (COD) removal efficiencies were 92-97% at 1.2-6.0kgCOD/m(3)d feeding. The dominated species, propionate and butyrate, were found in the 1st compartment. Acetate was dominated in the 2nd compartment and then decreased in the 3rd and 4th. Meanwhile, 93% volatile fatty acids (VFAs) were removed in the 3rd and 4th compartments. In the 1st compartment, biogas revealed carbon dioxide (CO(2)) and hydrogen (H(2)). The highest H(2) yield was found in the 2nd compartment, thereafter decreased from the 2nd to 4th which corresponded to the increased of the methane (CH(4)) yield. It indicated that the proper anaerobic consortium in each separate compartment was developed along with substrate availability and specific environmental conditions.     

Reversal of the effects of aging in soybean seeds

Accelerated aging predisposed seeds to imbibition injury. Slowing the rate of hydration prevented the loss of germinability due to imbibition injury. Germinability of accelerated aged seeds (50 hours) was increased from 10 to 90% by controlling the rate of imbibition. Slow hydration also prevented seed electrolyte leakage. This may indicate that cell membrane permeability or rupture was a major factor contributing to the loss of germinability after aging.

Reversal of the effects of aging (repair) was accomplished by slowly inbibing and then redrying seeds (priming). This treatment lowered steep water conductivity by a factor of 2 to 5. Priming also increased the per cent germination of low vigor seeds. The mechanism of this reversal was probably metabolic because it depended on temperature, seed moisture, and treatment duration.

Priming doubled the survival of seeds in the accelerated aging vigor test. The `rejuvenation' was accepted as evidence for metabolic repair. Since the `vigor' of seeds was increased by priming, metabolic repair probably included other subcellular components as well as the plasma membrane.

     

大豆豆乳和豆腐产量、品质及有关加工性状的遗传变异

采用全国各地的261个大豆品种为材料,研究豆乳和豆腐产量、品质及有关加工性状的遗传变异.结果表明豆乳和豆腐加工过程中每100g干籽粒平均生产干豆乳71.92g,生产干豆腐51.80g,损失豆渣干物质25.76g,损失乳清干物质20.12g.干豆乳蛋白质、脂肪和总糖的含量分别为51.85%、22.55%和18.42%,干豆腐蛋白质、脂肪和总糖的含量分别为46.22%、24.58%和19.36%.豆乳和豆腐产量、品质及有关加工性状在品种间的差异均达到极显著水平,变异系数都较大,从中筛选出一批优异种质.     

Research on soybean protein wastewater treatment by the integrated two-phase anaerobic reactor

The start-up tests of treating soybean protein wastewater by the integrated two-phase anaerobic reactor were studied. The results showed that the soybean protein wastewater could be successfully processed around 30 days when running under the situation of dosing seed sludge with the influent of approximately 2000 mg/L and an HRT of 40 h. When the start-up was finished, the removal rate of COD by the reactor was about 80%. In the zone I, biogas mainly revealed carbon dioxide (CO₂) and hydrogen (H₂). Methane was the main component in the zone 2 which ranged from 53% to 59% with an average of 55%. The methane content in biogas increased from the zone I to II. It indicated that the methane-producing capacity of the anaerobic sludge increased. It was found that the uniquely designed two-phase integrated anaerobic reactor played a key role in treating soybean protein wastewater. The acidogenic fermentation bacteria dominated in the zone I, while methanogen became dominant in the zone II. It realized the relatively effective separation of hydrolysis acidification and methanogenesis process in the reactor, which was benefit to promote a more reasonable space distribution of the microbial communities in the reactor. There were some differences between the activities of the sludge in the two reaction zones of the integrated two-phase anaerobic reactor. The activity of protease was higher in the reaction zone I. And the coenzyme F₄₂₀ in the reaction zone II was twice than that in the reaction zone I, which indicated that the activity of the methanogens was stronger in the reaction zone II.     

Localizations of aluminum in soybean bacteroids and seeds

Aluminum, long known to be detrimental to soybean productivity, was localized in the polyphosphate granules (PPG) of bacteroids in root nodules of soybean plants. By using energy-dispersive X-ray analysis, bacteroids in early infections were shown to have typical PPG constituents. However, in PPG in older infections and after the bacteroids were digested intracellularly, aluminum was also detected. These results indicate that aluminum accumulates in PPG after a period when organisms have been resident in host cells and that high levels of aluminum were present in the bacteroids at the time of their demise. At least some of the aluminum in these laboratory-grown plants could have come from the seeds used.     

Cessation of assimilate uptake in maturing soybean seeds

In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [¹⁴C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [³H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [³H]glutamine to ethanolinsoluble components, but converted little exogenous [¹⁴C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

Lipid changes during natural aging of soybean seeds

When soybean seeds [ Glycine max (L.) Merr. cv. Wayne] were stored at approximately 4°C and low humidity for 44 months ("natural aging") there was a marked decline in vigor and viability which was associated with a decrease in the proportion of the polyunsaturated fatty acids. Other lipid parameters showed little change. Seeds subjected to high humidity at 40°C for several days ("accelerated aging") experience a comparable decline in vigor and viability, but without the change in fatty acid polyunsaturation. These results indicate that accelerated aging may cause loss of vigor in a manner quite different from natural aging. The accelerated aging treatment is therefore of limited usefulness in investigations of the mechanism of natural seed aging.     

A novel C-terminal proteolytic processing of cytosolic pyruvate kinase,its phosphorylation and degradation by the proteasome in developing soybean seeds

Cytosolic pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGGS407TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS-PAGE, the phosphorylated polypeptides were found to be smaller (20-51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro, the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level. One involves partial C-terminal truncation to generate a 51 kDa pyruvate kinase subunit which might have altered regulatory properties and the other involves phosphorylation and ubiquitin conjugation that targets the protein to the 26S proteasome for complete degradation.     

Changes in soybean phytate content as a result of field growing conditions and influence on tofu texture

It is known that tofu quality tends to vary among soybeans even of the same variety. Cultivation environments can affect the contents of the soybeans. Twenty-seven soybean varieties were grown in a drained paddy field and an upland field, and then their protein and phytate contents were determined using the Fourier transfer infrared spectroscopy (FT-IR) method. The phytate contents of 12 varieties were higher in the drained paddy field than in the upland field. On the other hand, the environmental factor had little effect on the protein contents. In order to determine whether the difference in phytate content affected tofu texture, the hardness of the tofu made from phytate-added soymilk was measured. The tofu texture having more phytate became softer in the range of the common coagulant concentration. We concluded that the difference in the phytate content of the soybeans among the environmental conditions is a factor that causes fluctuation in tofu quality.     

Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes

Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate.     

Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.     

Adenylate metabolism of embryonic axes from deteriorated soybean seeds

RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA.