Stress-induced changes in the free amino acid composition in transgenic soybean plants having increased proline content

Following drought stress at supraoptimal temperature the increase in proline (Pro) content in transgenic (T) soybean [Glycine max (L.) Merr. cv. Ibis] plants overexpressing the gene coding for the last enzyme of Pro biosynthesis, L-Δ¹-pyrroline-5-carboxylate reductase, was much greater than in wild type (W) plants (105-fold versus 19-fold after 7 d). Under control conditions arginine accounted for nearly 60 % of the total free amino acid content. After stress treatment the content of Pro was more than 50 % in both T and W genotypes, and at the end of recovery the γ-aminobutyrate content reached 27 and 53 % in the W and T plants, respectively. Without stress treatment there was only a 2-fold difference between T and W in the tyrosine content. However, during the stress period and the subsequent recovery a similar difference was found for many amino acids. The present results indicate that manipulating of the content of a single amino acid influences the whole free amino acid composition in soybean.     

FREE AMINO ACID CHANGES DURING GERMINATION OF TELIOSPORES OF THE WHEAT BUNT FUNGUS,TILLETIA CARIES

Teliospores were aerated and agitated in a mineral salts medium and their free amino acid contents were analyzed at eight different times, from shortly after imbibition of water until just before germ tube emergence. In addition to the common amino acids, eight unidentified ninhydrin-positive components were detected. About 50 % or more of nearly each of the amino acids diffused out of the spores during the initial phase of germination. These released amino acids were actively taken up by the spores during the latter stages of germination. The free amino acids in largest amounts in the dormant spores of T. caries were arginine 15.0, glutamic acid 6.3, and alanine 3.7 μmoles per g dry spores. Together these three amino acids accounted for about 71 % of the total free amino acids in dormant spores of T. caries and T. controversa. The total amounts of free amino acids in spores of common bunt were much higher than in spores of dwarf bunt.     

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.

Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.

Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.

     

Relationships between cell membrane stability, exudate content and infectivity of Bradyrhizobium japonicum strain 639 to boron starved soybean plants

The influence of boron starvation on the root exudates content in soybean seedlings (Glycine max. L. Merr.) and the effect of exudates pretreatment on the pre-infection processes in symbiotic system Br. japonicum strain 636 and soybean were investigated. Root cell membrane stability of boron starved soybean plants (-B) decreased compared to the control. The concentrations of all analyzed metabolites (reducing sugars, free amino acids, organic acids, soluble phenols and total flavonoids) from root exudates of -B plants were lower than the control concentrations. Analysis of polyphenols after HPLC chromatography of root exudates showed significant difference of peak numbers between chromatograms of exudates obtained from boron starved and from control plants. Bacterial culture treatment with root exudates from -B plants showed decreased growth, chemotaxis and attachment ability toward the host root compared to the control exudate treatments. These changes were accompanied by decreased nodulation and acetylene reduction activity of boron starved soybean plants.     

湖南道地白芷氨基酸含量与组成分析

白芷是我国常用中药材的一种,关于白芷镇痛的有效成分,主要认为是异欧前胡素、欧前胡素等香豆素类具有解痉、镇痛等作用的挥发油类化学成分,但白芷镇痛的成分及其机理尚不十分清楚,是否有其他物质也具有一定的药理作用也不得而知,比如说某些药效性氨基酸。该研究采用自动氨基酸分析仪测定了湖南道地茶陵白芷的蛋白质类氨基酸和游离氨基酸的含量,并分析其氨基酸组成。结果表明:总氨基酸检出除Asn以外的其他16种蛋白质氨基酸,含量最高的Arg占总氨基酸的31.21%,接近1/3;必需氨基酸总量达27.01%,其中含量最高的是Leu,占总必需氨基酸的24.14%;药效氨基酸比例较高,在酸水解产物总氨基酸和游离氨基酸中分别达到73.89%和85.78%,其中Arg的含量最高,达1.383g·100g-1,占比为42.24%,游离氨基酸中γ-氨基丁酸、鸟氨酸含量也较高。此外,还含有少量的高半胱氨酸和鹅肌肽等游离非蛋白质氨基酸和短肽;必需氨基酸的组成接近WHO/FAO的建议摄入值,但Met+Cys的RC值最小,为第一限制性氨基酸。这些药效性氨基酸和游离氨基酸可能是茶陵白芷具有良好药效的一个因素,而且茶陵白芷的氨基酸组成和配比较合理、符合人体需要,在强化含硫氨基酸的基础上具有开发成为新型药食同源保健性食品的潜力。研究结果可为进一步研究白芷的药理作用提供依据。     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

High Stearic Acid Soybean Mutant Induced by X-ray Irradiation

Seeds of soybean [Glycine max (L.) Merr. var. Bay] were subjected to X-ray irradiation (21.4 kR), and the M2 generation was evaluated for the stearic acid content in the seed oil. Treatment with X-ray irradiation significantly increased genetic variability in the stearic acid content of the oil from Bay variety in comparison with the control plants. Among the 2513 M₂ plants tested, one mutant named M25 was selected for its stearic acid content of 20.8%, about seven-fold higher than that of the original variety. An inverse relationship of stearic acid with oleic and linoleic acids was observed. Mutant M25 always had higher stearic acid content under different environmental conditions in the M₃ generation.     

Genomic Regions Associated with Amino Acid Composition in Soybean

Soybean [Glycine max (L.) Merr.] is the single largest source of protein in animal feed. However, few studies have been conducted to evaluate genomic regions controlling amino acid composition in soybean. It is important to study the genetics of amino acid composition to achieve improvements through breeding. The objectives of this study were to determine the ratios between essential to non-essential (E:NE) and essential to total (E:T) amino acids, and to identify genomic regions controlling essential and non-essential amino acid composition in soybean seed. To achieve these objectives, 101 F₆-derived recombinant inbred lines (RIL) developed from a cross of N87-984-16 × TN93-99 were used. Ground soybean seed samples were analyzed for amino acids using a near infrared spectroscopy (NIRS) instrument. A significant (p < 0.01) difference among the RIL was found for amino acid composition. Heritability estimates on an entry mean basis ranged from 0.13 for His to 0.67 for Tyr. A total of 94 polymorphic simple sequence repeat (SSR) molecular genetic markers were screened in DNA from progenies. Single factor ANOVA was used to identify candidate quantitative trait loci (QTL), which were then confirmed by QTL Cartographer. At least one QTL for each amino acid was detected in this population. QTL linked to molecular markers Satt143, Satt168, Satt203, Satt274 and Satt495 were associated with most of the amino acids. Phenotypic variation explained by an individual QTL ranged from 9.4 to 45.3%. QTL detected for amino acids in soybean in this experiment are expected to be useful for future breeding programs targeting development of improved soybean amino acid composition for human and animal nutrition.     

Possible phytotoxic metabolites in culture filtrates of Diaporthe phaseolorum var. sojae

Filter-sterile culture filtrates of an isolate of Diaporthe phaseolorum var. sojae (Phomopsis sojae), causal agent of pod and stem blight and seed decay of soybeans (Glycine max), grown on Czapeks-dox broth significantly (P = 0.05) inhibited germination of cabbage (Brassica oleracea), cantaloupe (Cucumis melo), onion (Allium cepa), soybean, and wheat (Triticum vulgare) seeds and wilted soybean seedling cuttings within 24 hr. Inhibition of seed germination and severity of soybean seedling wilt increased with increased concentrations of culture filtrates and increased incubation time of the cultures.     

Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter

Thermoanaerobacter brockii, T. ethanolicus, T. thermohydrosulfuricus, T. finnii, and Thermoanaerobacter strain SEBR 5268 (an isolate from an oil-producing well) were studied for their ability to oxidize proteinaceous compounds that included gelatin, peptides, and casamino acids. All bacteria tested used peptides and amino acids, but only slightly. However, in the presence of thiosulfate all the Thermoanaerobacter species showed a substantial improvement in growth and/or the production of acetate, isovalerate, isobutyrate, and sulfide. Propionate was a minor product of peptide or amino acid oxidation. The reduction of thiosulfate during growth on peptides by members of the Thermoanaerobacter species is a trait that closely resembles that of archaeal hyperthermophiles during growth on peptides and amino acids with elemental sulfur as electron acceptor.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Differential expression of β-conglycinin genes in nodulated and non-nodulated isolines of soybean

Nodulated (T202) and non-nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7-day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS-PAGE. The β-subunit of β-conglycinin could scarcely be detected in the non-nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′- and α-subunits of β-conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β-subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β-subunit in the non-nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)-subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β-subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations. Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse-grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO₃–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO₃–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N₂ fixation by the non nodulated T201 plants. However, when adequate NO₃ was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO₃ was supplied, the subunit proportion of the seed storage protein was similar in non-nodulating and nodulating isolines. On the other hand, when plants received a low level of NO₃ (2 mM), the β-conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO₃–the proportion of β-conglycinin increased markedly. The results indicate that non-nodulated T201 has a normal, non-defective, β-subunit gene and that limited N availability decreases accumulation of β-conglycinin, whereas high N availability increases the proportion of β-conglycinin in soybean seeds, irrespective of whether N was derived from N₂ fixation or from NO₃ absorption.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

A Peroxisomal Long-Chain Acyl-CoA Synthetase from Glycine max Involved in Lipid Degradation

Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination.     

外源水杨酸对NaCl胁迫下大豆种子萌发和幼苗生长生理的影响

以大豆种子、幼苗为试验材料,采用砂培的方法,研究了0.2mmol·L-1外源水杨酸(SA)对100mmol·L-1 NaCl胁迫下大豆种子萌发、幼苗形态及生物量、膜脂过氧化和抗氧化酶活性的影响。结果显示:NaCl胁迫下,大豆种子萌发和幼苗生长受到显著抑制,且随着胁迫时间的延长(0~3d),大豆幼苗相对电解质渗漏率、硫代巴比妥酸活性产物(TBARS)含量显著升高,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性均明显降低。外源SA促进NaCl胁迫下大豆种子萌发和根茎生长,增加幼苗生物量积累,降低幼苗叶片相对电解质渗漏率和TBARS含量,增强其叶片SOD、CAT、APX活性。研究表明,NaCl胁迫能显著抑制大豆种子萌发和幼苗生长,而一定浓度的外源SA能有效提高NaCl胁迫下大豆种子活力及幼苗抗氧化酶活性,减轻膜脂过氧化程度,缓解NaCl胁迫所造成的伤害,提高大豆幼苗抗盐胁迫的能力。     

野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…     

Cloning and Structure Analysis of rbcS Gene from Wild Soybean

About 1 kb fragment of rbcS (ribulose 1, 5-bisphosphate carboxylase small subunit) gene in wild soybean (Glycine soja, Ji 50017) was amplified from total DNA by PCR assay. Sequence analysis of the fragment indicated that 1089 bp sequenced included the whole coding region for Rubisco small snbunit. The rbcS gene in wild soybean encoded a precursor composed of a transit peptide of 55 amino acids and a mature protein of 123 amino acids. There were two introns found in the rbcS gene as other dicotyledonous species previously sequenced. Comparison of DNA sequences showed high degree homology of rbcS genes between wild soybean and cultivated soybean (Glycine max var. wayne). Some changes of amino acids emerged from the diverse nucleotides did not affect the function of the small subunit. These results may contribute some basic data in molecular biology to study the origin and evolution of soybean.     

河蚬汤中氨基酸组分的分析

利用全自动氨基酸分析仪测定了传统保肝食品--河蚬汤中的总氨基酸和游离氨基酸含量.结果表明,河蚬汤中含有17种常见(色氨酸在酸解时遭破坏)氨基酸,还有鸟氨酸和牛磺酸两种非蛋白质组成氨基酸,总含量为297.4 mg·g-1.其中鸟氨酸含量最高,占总氨基酸的12.4%,且87%的鸟氨酸以结合态形式存在.河蚬汤中游离氨基酸含量...     

Biochemical changes during the fermentation of Prosopis africana seeds for ogiri-okpei production

Biochemical changes during fermentation of seeds of Prosopis africana for production of ogiri-okpei, a food condiment popular among people of West Africa were studied. Fermentation resulted in a net increase in concentrations of total soluble sugars and free amino acids, both reaching a peak after 72 h of fermentation but declining thereafter. Corresponding increases were observed in amylase and protease activities, respectively. Lipase activity was observed to be very strong, increasing throughout the duration of fermentation. Analyses of amino and fatty acid composition using an amino acid analyzer and gas liquid chromatography, respectively, revealed a wide variety of amino acids including glutamine > cystine > arginine and the fatty acids stearic > Arachidic > linolenic > linoleic in the unfermented seed in the highest concentrations. Fluctuations in the concentrations of these compounds were observed during the fermentation. At the end of 96 h fermentation, glutamine > cystine > lysine and an unidentified fatty acid > arachidic > linolenic acids were found in the highest concentrations. Marked increases in composition with increasing period of fermentation were observed for Ca, P, K, Mn, and Z. Transformations of amino acids, fatty acids, and minerals during the fermentation of this seed revealed during this study will contribute towards the development of an industrial process for ogiri-okpei as well as an understanding of its contribution to the nutrition of its consumers.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.