Mapping QTLs for tissue culture response in soybean (Glycine max (L.) Merr.)

Hempseed is rich in polyunsaturated fatty acids (PUFAs), which have potential as therapeutic compounds for the treatment of neurodegenerative and cardiovascular disease. However, the effect of hempseed meal (HSM) intake on the animal models of these diseases has yet to be elucidated. In this study, we assessed the effects of the intake of HSM and PUFAs on oxidative stress, cytotoxicity and neurological phenotypes, and cholesterol uptake, using Drosophila models. HSM intake was shown to reduce H₂O₂ toxicity markedly, indicating that HSM exerts a profound antioxidant effect. Meanwhile, intake of HSM, as well as linoleic or linolenic acids (major PUFA components of HSM) was shown to ameliorate Aβ42-induced eye degeneration, thus suggesting that these compounds exert a protective effect against Aβ42 cytotoxicity. On the contrary, locomotion and longevity in the Parkinson’s disease model and eye degeneration in the Huntington’s disease model were unaffected by HSM feeding. Additionally, intake of HSM or linoleic acid was shown to reduce cholesterol uptake significantly. Moreover, linoleic acid intake has been shown to delay pupariation, and cholesterol feeding rescued the linoleic acid-induced larval growth delay, thereby indicating that linoleic acid acts antagonistically with cholesterol during larval growth. In conclusion, our results indicate that HSM and linoleic acid exert inhibitory effects on both Aβ42 cytotoxicity and cholesterol uptake, and are potential candidates for the treatment of Alzheimer’s disease and cardiovascular disease.     

Localization of myo-inositol phosphate synthase (GmMIPS-1) during the early stages of soybean seed development

As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.     

Protein synthesis at low temperatures in two soybean cultivars differing by their cold sensitivity

The effect of low temperatures (14°C/8°C, day/night) on polypeptide synthesis in leaves of two soybean ( Glycine max [L.] Merr.) cvs (Verdon and Maple Arrow) differing in cold sensitivity was investigated. The two cultivars were initially characterized in terms of cold tolerance according to their growth at the young plant stage at 14°C/8°C. Verdon was found to be more tolerant than Maple Arrow. In vivo [³⁵S]-methionine labeled polypeptides were resolved by two-dimensional electrophoresis. Autoradiograms were computer analyzed to evidence and quantify significative changes occurring after 5 days at 14°C/8°C, and to compare the response of the two cultivars. Most of the observed changes were quantitative. The two cultivars essentially exhibited a common modified polypeptide pattern in response to cold temperatures, but the changes were quantitatively more pronounced in the most tolerant cultivar. Computer analysis of two-dimensional electrophoresis gels allowed, for the first time, characterization of cultivar differences in terms of protein pattern under cold conditions.     

Responses to sucrose and glutamine by soybean embryos grown in vitro

Immature soybean (Glycine max [L.] Merr. cv. Ransom) embryos were grown in vitro in the presence of different concentrations of sucrose and glutamine to examine how availability of carbohydrate and nitrogen affects dry matter accumulation and embryo composition. Embryos were transferred to fresh medium every 4 days to maintain sucrose and glutamine concentrations of the culture medium. In all experiments, accumulation of dry matter and protein content increased when the sucrose concentration of the culture medium was increased from 1.5 to 150 mM: however, a relatively greater enhancement of dry matter than of protein accumulation resulted in a lower protein concentration at 150 than at 1.5 mM sucrose. Both content and concentration of protein were increased by the increases in glutamine supply to concentrations exceeding 68% protein at 120 mM glutamine. In combination with 150 mM sucrose, however, oil increased as glutamine supply was increased from 0.6 to 6 mM and then decreased as glutamine supply was increased from 6 lo 120 mM. Varying the concentration of sucrose available during seed development also affected embryo composition. Decreased availability of sucrose during either the early or late portion of the culture period resulted in lower accumulation of dry mailer as well as oil. Protein concentration was actually higher for embryos transferred from 150 to 1.5 nM sucrose than for those remaining in 150 mM throughout the culture period: however, the greater percentage of protein was due lo a decrease in accumulation of dry weight. In addition, embryo composition was affected by altering the availability of glutamine during culture, indicating that variation in the level of nitrogen assimilate delivered during seed development can change embryo composition. Decreasing the glutamine concentration of the medium lowered both protein and oil content. In contrast, increasing the glutamine concentration of the medium from 0.6 to 6 mM 8 days after initiation of culture increased the protein content and concentration of the embryo while oil content was not affected.     

大豆突变新品系部分品质性状的分析

为丰富大豆种质资源,改良大豆品质,对现有的大豆品种进行改良,以获得新的大豆品系。大豆品种高蛋白东农42和高脂肪东农163经NaN3诱变处理后,分别在其M6代品系中取32份和54份材料进行实验,对它们的农艺性状(株高、百粒重等)和部分品质(脂肪、水溶蛋白和碱溶蛋白)进行了分析。结果表明:(1)所有品系的品质性状的变异系数差别较大,说明在后代中进行品质性状筛选是有效的;(2)突变品系内脂肪含量与水溶性蛋白含量呈显著负相关。     

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H-pyrazolo-[3,5- d ]pyrimidine-4-o1) to intact nodulated roots of ureide-forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H₂ and CO₂ evolution, short term effects of AP application were examined in a Hup ⁻ soybean symbiosis [ Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H₂ evolution following AP application was not due to altered electron allocation between N₂ and H⁺ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Whole-genome sequence diversity and association analysis of 198 soybean accessions in mini-core collections

We performed whole-genome Illumina resequencing of 198 accessions to examine the genetic diversity and facilitate the use of soybean genetic resources and identified 10 million single nucleotide polymorphisms and 2.8 million small indels. Furthermore, PacBio resequencing of 10 accessions was performed, and a total of 2,033 structure variants were identified. Genetic diversity and structure analysis congregated the 198 accessions into three subgroups (Primitive, World, and Japan) and showed the possibility of a long and relatively isolated history of cultivated soybean in Japan. Additionally, the skewed regional distribution of variants in the genome, such as higher structural variations on the R gene clusters in the Japan group, suggested the possibility of selective sweeps during domestication or breeding. A genome-wide association study identified both known and novel causal variants on the genes controlling the flowering period. Novel candidate causal variants were also found on genes related to the seed coat colour by aligning together with Illumina and PacBio reads. The genomic sequences and variants obtained in this study have immense potential to provide information for soybean breeding and genetic studies that may uncover novel alleles or genes involved in agronomically important traits.     

Influence of accelerated and natural aging on free radical levels in soybean seeds

Embryonic axes and cotyledons of three soybean ( Glycine max L. cv. Gudzon) seed lots designated as "normal", "naturally aged" and "acceleratedly aged" were analyzed for their organic free radical contents. No signals of free radicals were found in ESR spectra from cotyledonary material of the three samples investigated. High levels of organic free radicals were observed in the embryonic axes. There were significant differences in the free radical contents of the embryonic axes: the relative values of free radicals were 100, 190 and 170% for samples aged normally, naturally and in an accelerated manner, respectively. These results point to the physiological role of the embryonic axes during natural or accelerated aging of seeds, notwithstanding that the axis is a small part as compared to the cotyledons. It is suggested that lipid peroxidation in embryonic axes may play an important role in the seed deterioration during aging.     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

Evidence for the involvement of jasmonic acid in the control of the stem-growth habit of soybean plants

Soybean plants show diversity in stem-growth habit which ranges from the determinate type to the indeterminate type. Stem growth of determinate plants abruptly terminate near the beginning of flowering. The possible involvement of jasmonic acid (JA) in the control of the stem growth-habit was examined in indeterminate and determinate isolines of soybean [ Glycine max (L.) Merril cv. Harosoy]. JA-like activities in leaves of both isolines were very low 20 days before the commencement of flowering. The activity increased rapidly thereafter and reached a maximum near the time of flowering. Although the activities in leaves of both isolines fluctuated in a similar manner, the activity in the determinate isoline was much higher than that in the indeterminate isoline after flowering. The presence of JA in the leaves of the determinate isoline was confirmed by purification by high-performance liquid chromatography and by mass spectrometry. Exogenous application of JA to cultured shoot apices of the indeterminate isoline strongly inhibited growth. These results suggest that jasmonic acid is a major endogenous factor that controls the growth habit of soybean plants.     

Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants

A number of independently derived transgenic soybean plants expressing a chimeric β-glucuronidase (GUS) gene under the control of the 355 CaMV promoter and a nopaline synthase polyadenylation signal were recovered using direct DNA transfer via electric discharge particle acceleration. Expression of GUS in R, plants was localized using thin tissue sections. Many tissue types expressed GUS at various levels. Pericycle cells in root, parenchyma cells in xylem, and phloem tissues of stem and leaf had high levels of enzyme activity. Procambium, phloem, and cortex cells in root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib, expressed low or no GUS activity. Intermediate levels of GUS activity were detected in leaf mesophyll cells, certain ground tissue cells in stem and leaf midrib, and in trichome and epidermal guard cells. Thus, we conclude that the 35S CaMV promoter is cell-type specific and is developmentally regulated in soybean.     

Cytological analysis of early microspore divisions and embryo formation in cultured soybean anthers

Young inflorescences of two Brazilian soybean cultivars (IAS 5 and RS 7) were subjected to 4 °C pretreatment for 0, 5, and 10 days. Cytological examinations of the t in vitro anthers were done during the first four weeks of culture. The cold pretreatment had no clear effect on the frequencies of symmetrical-binucleate microspores or multinucleate grains. The multinucleate grains might originate either by symmetrical or assymmetrical division. The best medium for callus and embryo induction was B5 long containing 2.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ benzyladenine. The frequency of anthers/calli with embryos was about 2% in all cultivars. Histological analysis of such embryos showed that they were similar to zygotic embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

增强UV-B辐射对大豆胚轴DNA损伤、修复和蛋白质含量的影响

大气平流层臭氧层减薄引起到达地表的 UV- B辐射增强。为探讨在增强 UV- B辐射下植物细胞 DNA的损伤修复和蛋白质含量的关系 ,利用 3H- Td R掺入法 ,研究了在 8.2 2 k J/(m2 d)和 12 .4 2 k J/(m2 d) U V- B辐射 (相当于兰州地区大气平流层臭氧减薄约 12 %和 2 0 % )胁迫下 ,大豆胚轴细胞 DNA合成和非按期合成 (UDS)变化 ,并测定了胚轴蛋白质含量变化 ,结果显示 ,UV- B辐射导致 DNA损伤 ,并诱导了 DNA损伤的修复 ,胚轴细胞 UDS效应增强 ,U DS指数增大。低 UV- B辐射强度下 ,胚轴蛋白质含量增加 ,可能是 U V- B诱导了一些与抗性有关的基因表达 ,导致一些新的与抗性有关的蛋白质合成 ;在高强度 UV- B辐射下 ,U DS指数与低强度辐射下无显著差异 (P=0 .0 5 ) ,但蛋白含量较低强度辐射下显著下降 (P=0 .0 5 ) ,说明高强度 UV- B辐射加重了 DNA损伤 ,而修复并未加强 ,并且高强度辐射抑制基因的正常表达和蛋白质合成。这些蛋白质的合成可能与大豆对 UV- B辐射的抗性有关。     

Analysis of apoplastic solutes in the cortex of soybean nodules

Various techniques were used to extract solutes from the free space of intact soybean [ Glycine max (L.) Merr.] nodules. A variety of solutes (carbohydrates, amino acids, organic acids, ions) was found, but the major solute obtained with all methods was allantoic acid. Most work was done with a technique involving vacuum infiltration of intact detached nodules with water. This approach provided rapid sampling of the apoplastic solutes, and the results indicated that solutes were not derived from the xylem and phloem of ruptured vascular bundles. Infiltration of intact nodules with Fast Green showed dye penetration only to the barrier in the inner cortex, indicating that infected tissues did not contribute to solute composition. Although allantoic acid was the only ureide which could be detected in solute samples, no evidence was obtained for the presence of allantoinase in the cortical apoplast. The results suggest the transport of allantoic acid by an apoplastic route in nodules or the release of allantoic acid to the cortical apoplast in response to treatments which disrupt ureide export. Calculated values for solute concentrations in the cortical apoplast were in the hundred millimolar range, suggesting that apoplastic solutes may represent a significant osmotic component in the nodule cortex.