Comparative protein accumulation patterns in soybean somatic and zygotic embryos

Summary The relative maturity and competence of somatic embryos is often estimated on the basis of their morphologic similarity to various stages of immature zygotic embryo development. Morphologic abnormalities noted in soybean [Glycine max (L.) Merr.] somatic embryos are similar to those observed in zygotic embryos maturing in vitro and may reflect common interruptions of normal developmental processes. We provide here a more objective means of assessing the point(s) at which cultured embryos deviate from the normal embryogenical pathway by comparing the accumulation of the embryo-specific marker proteins (11S and 7S storage globulins, soybean agglutinin, and seed lipoxygenase) between somatic and immature zygotic embryos maturing in culture to zygotic embryos maturingin planta. Immature (heart-stage) soybean (cv. ‘McCall’) zygotic embryos were removed from the testa and cultured for 5, 15, or 45 days in nien modified Linsmaer-Skoog salts, 5% sucrose liquid medium. Somatic embryos were induced from immature cotyledon explants on a medium containing either naphthalene acetic acid or 2,4 dichlorophenoxyacetic acid (10 mg·liter⁻¹). The measured level of the marker proteins present in cultured embryos never exceeded those observed in mature soybean seeds. During the culture period, immature zygotic embryos accumulated significant levels of all marker proteins except a 29 kDa soybean agglutinin associated with the final stages of seed maturationin planta. Somatic embryos of all morphologic classes exhibited similar levels of the marker proteins suggesting that morphology may not accurately represent the developmental state of the culture-derived embryos. Somatic embryos induced on naphthalene acetic acid-containing medium accumulated detectable levels of all maturation-specific marker proteins except the 7S β and 29-kD soybean agglutinin antigen and seemed similar in most respects to the cultured zygotic embryos. Embryos induced on 2,4-dichlorophenoxyacetic acid accumulated none of the mature 7S or 11S storage globulin subunits nor any soybean agglutinin antigen, and yet the synthesis of 7S and 11S precursor polypeptides was similar in both naphthalene acetic acid-and 2,4-dichlorophenoxyacetic acid-induced somatic embryos. These observations are consistent with the view that embryos induced on high 2,4-dichlorophenoxyacetic are arrested at a relatively earlier developmental stage than naphthalene acetic acid-induced embryos of similar morphology and may indicate that some external signal (e.g., abscisic acid or desiccation or both) is necessary for the transition to the late maturation stage of seed ontogeny.     

High-frequency direct somatic embryogenesis from leaf tissues of <Emphasis Type="Italic">Drimiopsis kirkii</Emphasis> Baker (giant squill)

Whole plants were regenerated from excised leaves of Drimiopsis kirkii Baker (Lily of the Valley) through direct somatic embryogenesis. An initial exposure to a low level of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 μM) in the medium was essential in inducing the direct formation of somatic embryos. A high concentration of 2,4-D (4.52 μM) in the proliferation medium reduced embryogenesis and enhanced callus formation. The presence of kinetin in the medium enhanced the somatic-embryogenesis-inducing effect of 2,4-D (0.45 μM). The maximum embryogenesis rate (4,026 somatic embryos per gram of leaf) was obtained in explants cultured for 30 d in medium supplemented with 2.33 μM kinetin and 0.45 μM 2,4-D (embryo induction medium). Kinetin (4.65 μM) also enhanced embryo germination (97.6%), but the presence of α-naphthalene acetic acid in the medium drastically reduced embryo germination. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.     

Rapid and sensitive detection by PCR of mpcI gene in the rhizosphere

Abstract: A simple and sensitive detection system, using polymerase chain reaction (PCR) and a soil microcosm, was developed to detect a bacterial catabolic gene in the rhizosphere. The inoculated population of Alcaligenes eutrophus JMP134, a phenol and 2,4-dichlorophenoxy acetic acid utilizer, was readily detected by this technique, which permitted taking of samples from specific locations of root (including rhizosphere) and soil. The number of JMP134 viable cells (10²–10³ cells), typically picked up by the nitrocellulose filter strip method, yielded sufficient amount of the target DNA to be detected by PCR. Primers encoding metapyrocatechase I (MPC I; catechol 2,3-dioxygenase) enabled the discrimination of at least five viable cells of JMP134 among the indigenous microorganisms inhabiting bush bean roots. This simplified PCR detection procedure facilitated monitoring of the specific degradative gene in the rhizosphere in only 5 h.     

Collagenase in Stomach of Crown-of-thorns Starfish Acanthaster planci

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.     

Division and growth of mesophyll cells isolated from Psoralea bituminosa leaves

Mesophyll cells have been isolated from Psoralea bituminosa plant by gentle homogenization in a liquid nutrient medium. Between 60 and 70% of the cells can be isolated from leaves using this method, of which 50 to 60% can be recovered morphologically intact. Under light the separated cells have rates of oxygen evolution under light of 3500 μl O₂ mg^?1 chlorophyll h^?1 (measured with a Clark-type electrode). During growth, this rate decreases rapidly, as does cell pigments (chlorophylls and carotenoids). As a first step in obtaining a photoautotrophic cell suspension, growth factors affecting cell division in free sugar medium were investigated. The starting culture contained between 1 and 2 × 10⁶ cells ml^?1. The best increase in cell number was obtained on a medium composed of Joshi and Ball's elements and vitamins and containing 1 mg l^?1 of naphthalene acetic acid, 0.1 mg l^?1 of benzylaminopurine and the grinding juice. The optimum culture pH was between 5 and 5.3.     

Micropropagation of Sterculia urens Roxb. — an endangered tree species

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl^–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.     

Embryogenesis and cardenolide formation in tissue cultures of Digitalis lanata

Digitalis lanata strain VII from filament callus grew in small cell colonies in nutrient media with high auxin activity. Upon increasing the cytoki     

Biological monitoring of dinitrotoluene by gas chromatographic–mass spectrometric analysis of 2,4-dinitrobenzoic acid in human urine

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower μg l⁻¹ range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 μg l⁻¹ urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 μg l⁻¹ urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.     

一株番茄表面着生醋酸菌的分离鉴定及产酸条件优化

醋酸菌是食醋酿造过程中的关键菌种,性能优良的菌种对于产品品质的提升意义重大。以分离自番茄表面的产醋酸菌为研究对象,通过生理生化指标鉴定、16S rRNA编码基因比对及系统发育树构建等方法对其种类进行鉴定,并通过单因素实验、正交实验对鉴定为醋酸菌的菌株进行培养条件优化。结果表明,所分离的3株醋酸生产菌中,BQ-1被鉴定为醋酸杆菌属(Acetobacteraceae),在以酵母粉为主要氮源,蔗糖为主要碳源的培养基中,其最高产酸量为1823 g·L-1。由于该菌株在番茄表面具有很强的生长能力,因此有望应用于番茄果醋的酿造。     

Inertness of horseradish peroxidase to 2,4-dichlorophenoxyacetic acid

The possibility of mutual effects of 2,4-D and horseradish (Armoracia lapathifolia L.) peroxidase on each other has been explored by four procedures. (i) Compounds I, II, and III of horseradish peroxidase (HRP) and H₂O₂ were exposed to 2,4-D. (ii) Extracts from batchwise operations of HRP + H₂O₂ and 2,4-D were analyzed for oxidation products by means of thin layer chromatography. (iii) The velocity of the IAA oxidase reaction with HRP as catalyst, and (iv) K_m and V_s of the overall peroxidation of guaiacol by HRP + H₂O₂, were determined in the absence and presence of 2,4-D. The results failed to show any effect of 2,4-D; only at very high concentrations did 2,4-D slightly inhibit the oxidation of IAA by one isoperoxidase. It is concluded that 2,4-D does not promote growth in plants by hampering a peroxidase-catalyzed IAA oxidation. It seems probable that 2,4-D perturbs the isoperoxidase pattern by acting at some step prior to the release of the enzyme from its site of synthesis.     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

Selection for increased vernolate tolerance in tobacco (Nicotiana tabacum L.) cell cultures

Vernolate (S-propyl N, N-dipropylthiocarbamate) has been used to select thiocarbamate-tolerant tobacco cell lines. Cross-tolerance to other thiocarbamate herbicides was observed. Tolerance was initially unstable in the absence of continued selection, but shoot regeneration produced plants in which some tolerance was stably expressed. Morphological abnormalities in the regenerated plants are described.     

Studies on the energy-linked Ca2+ accumulation in pig heart mitochondria - role of Mg2'ons

Comparative intracellular distribution of Ca2+, Mg2+ and adenine nucleotides has been studied in pig heart by differential centrifugation or fractional extraction and has shown that Mg2+ and ATP are associated mainly with soluble fractions whereas Ca2+ and ADP are more tightly bound to subcellular structures. Ca2+ accumulation and Ca2+ stimulated respiration were studied in pig heart mitochondria under different energetic conditions in the absence or presence of phosphate. Ca2+ concentrations of about 1200 nmoles/mg protein inhibit Ca2+ accumulation, site I substrate oxidation and induce an efflux of mitochondrial Mg2+. These deleterious effects of Ca2+ on respiration occur even in the absence of phosphate or oxidizable substrate; they are completely prevented by ruthenium red only, and partially prevented by the addition of M2+ to the medium. The kinetics of Ca2+ uptake become of the sigmoidal type when Mg2+ is present. This cation strongly inhibits the rate of Ca2+ uptake in the presence of added phosphate and decreases the affinity of Ca2+ for its transport system. In the absence of phosphate, Mg2+ has no effect on Ca2+ uptake. The possible physiological implications of these findings are discussed     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Global Analysis of the Genes Involved in the Thermotolerance Mechanism of Thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

醋酸菌耐酸机理及其群体感应研究新进展

醋酸菌(acetic acid bacteria,AAB)是一类严格好氧的革兰氏阴性细菌,因其乙醇氧化生成醋酸能力强、高耐醋酸等特性而成为食醋发酵的主要工业菌种。醋酸菌的耐酸性对于高酸度食醋生产具有重要意义。随着醋酸菌的蛋白组学及基因组学研究的深入,其糖代谢、蛋白质代谢、脂代谢及应激响应等分子机制或过程也得到更多的阐释;葡糖醋杆菌中有关群体感应系统的研究报道则为从信号通路角度探索醋酸菌的耐酸机制提供了新的思路,进而对于高耐酸醋酸菌的选育以及醋酸发酵工艺的优化具重要的参考意义。本文在简介蛋白组、基因组研究的基础上,着重综述醋酸菌群体感应的研究进展。