Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules

Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv `Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark-stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence.     

Carbon Metabolism Enzymes of Rhizobium meliloti Cultures and Bacteroids and Their Distribution within Alfalfa Nodules

Several carbon metabolism enzymes were measured in cultured cells and bacteroids of Rhizobium meliloti 102F51 and in alfalfa root nodule cytosol. The enzyme activity levels of the pentose phosphate pathway were much higher than those of the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways in extracts of cultured cells. The pattern of enzyme activities in the bacteroids was different from that of cultured cells.     

Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.     

Involvement of Activated Oxygen in Nitrate-Induced Senescence of Pea Root Nodules

The effect of short-term nitrate application (10 mM, 0-4 d) on nitrogenase (N2ase) activity, antioxidant defenses, and related parameters was investigated in pea (Pisum sativum L. cv Frilene) nodules. The response of nodules to nitrate comprised two stages. In the first stage (0-2 d), there were major decreases in N2ase activity and N2ase-linked respiration and concomitant increases in carbon cost of N2ase and oxygen diffusion resistance of nodules. There was no apparent oxidative damage, and the decline in N2ase activity was, to a certain extent, reversible. The second stage (>2 d) was typical of a senescent, essentially irreversible process. It was characterized by moderate increases in oxidized proteins and catalytic Fe and by major decreases in antioxidant enzymes and metabolites. The restriction in oxygen supply to bacteroids may explain the initial decline in N2ase activity. The decrease in antioxidant protection is not involved in this process and is not specifically caused by nitrate, since it also occurs with drought stress. However, comparison of nitrate- and drought-induced senescence shows an important difference: there is no lipid degradation or lipid peroxide accumulation with nitrate, indicating that lipid peroxidation is not necessarily involved in nodule senescence.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

Presence of Host-Plasma Membrane Type H-ATPase in the Membrane Envelope Enclosing the Bacteroids in Soybean Root Nodules

An improved method is described for the isolation of membrane envelope enclosing the bacteroids (peribacteroid membrane) from soybean (Glycine max L.) root nodules. The ATPase activity of the peribacteroid membrane from infected roots is compared with that of the plasma membrane from uninfected roots. The two ATPases are similar in terms of their vanadate sensitivities, pH optima, and mineral cation requirements, and show antigenic cross-reactivity. However, the ATPase of peribacteroid membrane is more sensitive to stimulation by NH₄⁺. ATP-dependent proton translocation across the peribacteroid membrane was demonstrated in broken protoplasts of infected cells, by the use of fluorescence microscopy with acridine orange. It is suggested that acidification of the peribacteroid space by the peribacteroid membrane ATPase results in the conversion of NH₃ to NH₄⁺ in this space and thereby facilitates the removal of fixed-nitrogen from the bacteroid.     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Modulation of Sucrose and Starch Metabolism by Salicylic Acid Induces Thermotolerance in Spring Maize

Russian Journal of Plant Physiology - Two inbred lines of spring maize (Zea mays L.), CML 32 (stress tolerant) and LM 11 (stress susceptible) were taken to study the effect of salicylic acid (SA)...     

Observations on the Composition and Metabolism of the Nitrogen-Fixing Root Nodules of Alnus

Nodules of Alnus glutinosa (Alder) were exposed to excess ¹⁵Neither before or after detachment from the plant. The solublenitrogen compounds were extracted from the nodules and the extractsfractionated by chromatography on an ion exchange resin. Theamino-acid composition of the extracts was thus determined,and it was confirmed that citrulline is the predominant amino-acidpresent, being accompanied by smaller amounts of aspartic, glutaniic,and -aminobutyric acids, arginine, and other constituents. Thehighest atom per cent, excess ¹⁵N always found in glutarnicacid and the next highest in citrulline or aspartic acid. Ammoniacontained a smaller proportion of ¹⁵N than these compounds andarginine showed only very small enrichment. When the citrullinewas degraded to ammonia and ornithine it was found that theammonia liberated was richer in ¹⁵N than even glutamic acid.The significance of these findings in relation to the fixationand further metabolism of nitrogen by the alder nodule is discussed.     

烟草愈伤组织生长和衰老期间呼吸代谢的改变

前人(Ross和Thorpe 1973,Thorpe和Laishley 1973,Brown和Thorpe 1982)曾报道了烟草愈伤组织芽形成期间呼吸速率、线粒体活性、底物代谢途径和有关呼吸酶活性的变化。我们对烟草愈伤组织呼吸代谢的研究证明:组织分化和芽原基的形成与HMP途径运行升高相联系(毕玉蓉和梁厚果1987);愈伤组织的呼吸链存     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Carbon Metabolism in Relation to Cellular Organization of Soybean Root Nodules and Respiration of Mitochondria Aided by Leghemoglobin

Effects of leghemoglobin on the respiration of bacteroids andmitochondria were examined by following oxygen uptake. Oxyleghemoglobinprepared from soybean nodules promoted the uptake of oxygenby bacteroids isolated aerobically from soybean nodules. Uptakeof oxygen by both the nodule mitochondria and the hypocotylmitochondria was also stimulated by the oxyleghemoglobin. Thestimulatory effect of leghemoglobin on the respiration of thenodule mitochondria became highly significant as the amountof mitochondria injected into assay vial increased. The infected cells, the uninfected cells and the cortex tissuewere isolated by enzymatic maceration from soybean nodules,and some enzyme activities were measured in these three fractions.Activities of alcohol dehydrogenase, pyruvate decarboxylaseand phosphoenolpyruvate carboxylase in the infected cells werelower than in the uninfected cells and in the cortex tissue.Uricase activity was low in the cortex tissue and was highestin the uninfected cells. These results suggested that ethanol production is more activein the uninfected cells and the cortical cells than in the infectedcells, and that respiration of the mitochondria in the infectedcells of soybean nodules is more active, aided by the leghemoglobin. (Received August 23, 1986; Accepted November 14, 1986)     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Acetylene-induced Changes in the Oxygen Diffusion Resistance and Nitrogenase Activity of Legume Root Nodules

Legume symbioses such as pea, lucerne and clover exhibit a substantialdecline in nodular respiration and nitrogenase activity whensubjected to gas streams containing C₂H₂. Assuming a constantrate of O₂ diffusion into the nodule this decrease in respiratoryO₂ consumption would lead to an increase in internal O₂ concentrationwhich would inactivate nitrogenase. No such inactivation hasbeen observed indicating that a change in diffusion resistanceis involved in the C₂H₂ response. Root nodules of C₂H₂ responsivesymbioses are distinguished by their tolerance to high (80 percent) O₂ levels. The nitrogenase of soya-bean and sainfoin,which do not respond to added C₂H₂, is denatured at O₂ levelsabove 40 per cent. Even in O₂-tolerant systems (e.g. pea) theenzyme is damaged if the increase in O₂ levels is rapid (ca.45 s) indicating that an induced change rather than a ‘permanent’feature is involved. The O₂-tolerant, C₂H₂-responsive behaviourpattern of white clover nodules was reversed by pulse feedingwith nitrate-N indicating that the overall C: N ratio in thenodules is involved in determining the response.     

Aspartate Aminotransferase in Alfalfa Root Nodules : II. Immunological Distinction between Two Forms of the Enzyme

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.