Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl

The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from δ-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from δ-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen-methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. (© 1989 FMC Corporation. All rights reserved.)     

Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules

Summary A method for the separation and purification of bacteroids and mitochondria from nodules of soybean roots is described. Cross contamination between these two oxidative fractions was easily assessible by using NADH oxidase and -hydroxybutyrate dehydrogenase respectively as specific mitochondrial and bacteroid markers. Bacteroid respiration was characterized by substantial endogenous respiration which could be reduced by keeping plants in the dark prior to isolation, and stimulated by uncoupler or organic acids. Nodule mitochondria readily oxidized external NADH and a range of tricarboxylic acid cycle intermediates, with good respiratory control. A major difference between nodule and root mitochondria was the former's high sensitivity to the inhibitors rotenone and cyanide. This indicates a reduced capacity for non-phosphorylating electron transport in nodule mitochondria, which may be related to the large energy demand during ammonia assimilation in nodule cells.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

O2 consumption and superoxide dismutase content in purified mitochondria from soybean root nodules

Soybean nodule mitochondria have been separated from cotnaminating organella on discontinuous Percoll gradients. The preparations appeared highly purified and at least 80% of the mitochondria were estimated to be derived from infected cells. Percoll-purified mitochondria showed important respiratory activity; in the case of succinate, the rate of O₂ consumption was 185 nmol O₂ min⁻¹ mg⁻¹ and the respiratory control and ADP/O ratio reached 2.72 and 1.16, respectively. These organelles also exhibited an active manganese containing superoxide dismutase (9.7 U mg⁻¹), whose purification is reported. These results are consistent with a significant O₂ consumption by host cell mitochondria in vivo and the possibility of a competition for O₂ supply with the bacteroids is discussed.     

Protoporphyrinogen oxidation, a step in heme synthesis in soybean root nodules and free-living rhizobia.

Extracts of the crude bacteroid fraction of symbiotically grown Bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen. This correlates with the higher heme content of the microaerophilic nodules. Furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized membrane fraction, probably of plant origin, that was separable from the bacteroids by Percoll gradient centrifugation.     

Effects of ferulic acid on L-malate oxidation in isolated soybean mitochondria

The effects of ferulic acid on L-malate oxidation in mitochondria isolated from soybean (Glycine max L.) seedlings were investigated. Oxygen uptake and the products of L-malate oxidation were measured under two conditions: pH 6.8 and 7.8. At acidic pH, the activity of the NAD+-linked malic enzyme (L-malate:NAD+oxidoreductase [decarboxylating] EC 1.1.1.39) was favoured, whereas at alkaline pH a predominance of the L-malate dehydrogenase activity (L-malate:NAD+oxidoreductase EC 1.1.1.37) was apparent. Ferulic acid inhibited basal and coupled respiration during L-malate oxidation either at acidic or alkaline pH, reducing also the amounts of pyruvate or oxaloacetate produced. The results suggest that the site of ferulic acid action is situated at some step that precedes the respiratory chain. An interference with the L-malate entry into the mitochondria could be an explanation for the effects of ferulic acid, but the possibility of a direct inhibition of both enzymes involved in L-malate oxidation cannot be ruled out. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of bacteria, transforming bacteria, and bacteroids from soybean nodules

Postnuclei supernatant of soybean (Glycine max cv. Chippewa 64) nodule homogenate was fractionated by stepwise sucrose density gradient centrifugation into supernatant, endoplasmic reticulum and mitochondria, and three distinct bands with 1.22, 1.25, and 1.27 g/cm³ of peak density. Based on their enzymic activities, composition of electron transport components, and ultrastructural characteristics, the lightest band appears to be the mature bacteroids; the intermediate band the transforming bacteria; and the heaviest, the bacteria. The isolation procedure separates nodule symbionts into different functional and developmental fractions, and it may be a valuable tool for studies involving development, regulation, and senescence of bacteroids in the nodule.     

Effect of unsaturated fatty acids on protoporphyrinogen oxidation, a step in heme and chlorophyll synthesis in plant organelles

Oleic acid stimulates enzymatic protoporphyrinogen oxidation by extracts of barley mitochondria and etioplasts. Greater stimulation occurred with Triton X-100 extracts which had been passed over a Sephacryl S-200 column than with crude Triton extracts, suggesting that purification may have removed a lipid factor required for optimal enzymatic activity. Palmitic acid, various phospholipids and detergents, or esters and alcohols of oleic acid did not substitute for free oleic acid. Linoleic acid caused a greater stimulation of protoporphyrinogen oxidation in both crude and purified barley organelle extracts and also caused a slow chemical oxidation of protoporphyrinogen. The stimulating effect of unsaturated fatty acids on enzymatic protoporphyrinogen oxidation may indicate a lipid requirement for this membrane bound enzyme or may also indicate involvement of unsaturated lipid oxidation in plant protoporphyrinogen oxidation.     

溶磷性大豆根瘤内生菌的筛选、抗性及系统发育和促生

对采自河南省不同地区的大豆根瘤进行内生菌分离纯化、溶磷性筛选试验。根据能否产生溶磷圈及溶磷圈直径(D)、菌落直径(d)和D/d值大小确定菌株溶磷能力,采用钼锑抗比色法测定培养液中有效磷含量;平板筛选法对筛选菌株进行耐盐性、耐酸碱、重金属等抗性测定,并对筛选菌株进行理化特性、16S r DNA、rec A序列和系统发育分析。结果表明,从分离纯化的324株内生菌中筛选出36株具有溶磷特性,其中20株有较强溶磷性。菌株DD291发酵液中可溶性磷含量最高(452 mg/L),发酵液p H与对照相比均有不同程度下降,最大降幅达2.92。大部分溶磷性内生菌具有较强耐盐碱性,对Pb2+、Cr6+和Cu2+有较高耐受性,对Ni2+和Hg2+抗性较弱。结合细胞形态、生理生化、16S r DNA、rec A序列和系统发育分析结果,菌株确定为Bacillus cereus,Enterobacter cancerogenus,E.cloacae和Pseudomonas putida。部分溶磷菌株对大豆的生长有促进作用,显示出潜在的应用前景。     

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O₂. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O₂ in soybean root nodules.     

Proteomic analysis on symbiotic differentiation of mitochondria in soybean nodules

Symbiotic interactions between legume plants and rhizobia induce specific metabolisms and intracellular organelles in nodules. For surveying symbiotic differentiation of a key organelle, mitochondria, protein constituents of soybean nodule and root mitochondria were compared after two-dimensional (2-D) electrophoresis, and the proteins were characterized in combination with matrix-assisted desorption/ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry and N-terminal amino acid sequencing. Of the proteins that were detected only in nodule mitochondria, phosphoserine aminotransferase, flavanone 3-hydroxylase, coproporphyrinogen III oxidase, one ribonucleoprotein and three unknown proteins were identified. Seven up-regulated, eight down-regulated and two strongly suppressed protein spots in nodule mitochondria were also assigned protein identities. The physiological roles of these differential expressions were discussed in relation to nodule-specific metabolisms in soybean nodules.     

Reduction of ferric leghemoglobin in soybean root nodules

Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions.

     

Vacuolation and infection thread in root nodules of soybean

Profuse vacuolation takes place in the soybean root nodule cells where infection threads carry rhizobia. After the rhizobia are released the disappearance of the infection thread is attributed to its degradation within large vacuoles which result from fusion of small vacuoles.     

Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.     

Immunochemical studies on xanthine dehydrogenase of soybean root nodules

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was purified from root nodules of soybean (Glycine max) and used to prepare a polyclonal rabbit antiserum. Monospecificity of this antiserum was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipate. During root nodule development of soybean, only one form of XDH was detected on an immunological basis. Titration of XDH by immunoelectrophoresis showed that a remarkable increase in the amount of XDH occurred between two and four weeks after inoculation, in parallel with the increase in enzyme activity. Localization of XDH by immunofluorescence indicated that the enzyme was present exclusively in uninfected cells where it appeared to be associated with discrete organellelsAbbreviations IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis - XDH xanthine dehydrogenase     

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg^-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

大豆根瘤细菌周膜的冷冻复型研究

用冷冻复型电镜技术研究了中国丰收１１号大豆根瘤中的细菌周膜。细菌周膜的断裂面上有颗粒状物质,但Ｐ面和Ｅ面有所不同,前者颗粒密度较大。即使都在Ｐ面或Ｅ面上,不同的细菌或同一细菌不同部位的颗粒密度也不一样。在细菌周膜与细菌细胞壁之间有一个环形腔隙,腔的大小随细菌和细菌部位不同而异。腔中不仅有泡状和管状结构,有时也有类寄主细胞质物质。细菌周膜表面有近似半球形或嵴形隆起,它们可能是腔中管泡状结构压迫细菌周