肯氏相思根瘤亚显微结构观察

肯氏相思根瘤亚显微结构观察结果表明,刚受侵袭的寄主细胞的细胞核和质体膨大,线粒体内嵴消失成为圆球状体,内质网膜松散;幼年类菌体细胞外形较小,呈圆形和椭圆形,细胞质浓密,染色深而均匀;成熟类菌体外型较大,形态多样,细胞内聚－β－羟基丁酸积累增多,随着根瘤细胞逐渐发育成熟,在类菌体被内可见１至数个类菌体,在根瘤衰老细胞中,类菌体包被周膜解离破裂,流出电子透明物质,本文还对周膜扩增的问题进行了讨论。     

银合欢根瘤细胞的光学显微镜和电子显微镜研究

银合欢接种根瘤菌形成根瘤后,应用光镜和电镜技术观察。银合欢根瘤由分生组织细胞、皮层组织细胞、维管束系统和侵染细胞区域四个不同部分组成。根瘤菌借助于侵染线侵染细胞,释放进入宿主细胞质中,转变成固氮类菌体。最初每个包被膜内只含单独的类菌体,随后较老的侵染细胞中,每个包被膜内含有一个以上的类菌体。因此,成熟根瘤的侵染细胞可见有2~5个类菌体群集包被膜里,并且明显地累积PHB物质,显示电子染色透明颗粒。本文还讨论了上述变化的意义与银合欢根瘤细胞结构和功能的关系。     

灰金合欢根瘤类菌体繁殖及其宿主细胞的超微结构

根瘤细胞早期发育阶段,以宿主细胞器和根瘤菌转化类菌体的数量增多为特征。随后类菌体增殖到填满宿主细胞内的大部分区域。各个类菌体周膜内含有1至几个类菌体。晚期共生发育阶段,类菌体细胞结构和宿主细胞器数量发生了变化。文中还讨论了根瘤的共生固氮作用。     

与玉米混作改善花生铁营养对其根瘤形态结构及豆血红蛋白含量的影响

通过土培方法研究了与玉米混作对花生根瘤形态结构及固氮功能的影响。结果表明,玉米与花生混作能够明显地改善花生铁营养、提高根瘤豆血红蛋白的含量。同时,单作花生根瘤细胞液泡化程度较高,正在发育的根瘤细胞内类菌体数量明显地比混作的花生低。成熟根瘤细胞类菌体周膜外空间(细胞壁以内、周膜外的空间)体积变大。说明单作花生固氮酶活性较低的原因是缺铁抑制了豆血红蛋白的合成和改变了根瘤形态结构以及类菌体的超微结构。     

豆科根瘤类菌体周膜的功能

豆科根瘤类菌体周膜的功能韩善华（四川师范大学细胞生物学研究室成都６１００６６）在豆科根瘤中,侵染细胞里面的细菌（根瘤菌）不仅大小不同,形态各异,而且内部结构也不完全一样,即使在同一品种中,这种现象也依然存在。尽管如此,这些细菌也有共同之处,其中最为引...     

超结瘤大豆根瘤的亚显微结构

电镜观察表明,超结瘤大豆未受侵染的宿主细胞中有一明显增大的细胞核。幼年类菌体为椭圆形,里面有个拟核区,正常类菌体有完善的周膜和ＰＨＢ颗粒,受侵染的寄主细胞中出现类似无效根瘤的异常现象;少数类菌体退化或溶解,还有空周膜及裸露的类菌体,这可能是超结瘤大豆固氮活性较低的原因。     

哈茨木霉发酵液中肽类物质对豇豆根瘤结构和功能的影响

通过树脂吸附、离子交换、薄层层析、高效液相色谱系统等分离、纯化方法,从哈茨木霉(Trichoderm aharzianum)T2-16菌株发酵液中分离得到一种对豆科作物生长具促进作用的物质,通过质谱等方法鉴定为肽类物质。为探明该活性物质对豆科作物的促生机制,用该活性物质对豇豆种子浸种处理后,进行盆栽实验,通过对盆栽实验中根瘤结构与固氮活性变化的研究,结果显示,该活性物质可增加根瘤侵染组织的面积和根瘤细胞中类菌体的数量,降低根瘤细胞液泡化程度,促进根瘤中公共细胞周膜较多形成,加快类菌体的发育成熟,提高根瘤豆血红蛋白的含量,从而提高根瘤的固氮活性。     

超结瘤大豆根瘤的亚显微结构

电镜观察表明,超结瘤大豆未受侵染的宿主细胞中有一明显增大的细胞核。幼年美菌体为椭圆形,里面有个拟核区,正常类菌体有完整的周膜和PHB颗粒。受侵染的寄主细胞中出现类似无效根瘤的异常现象：少数类菌体退化或溶解,还有空周膜及裸露的类菌体,这可能是超结瘤大豆固氮活性较低的原因。     

豆科根瘤拟菌体周膜来源和扩增方式的多样性

豆科植物根瘤内的共生拟菌体需有周膜包围。田菁［Ｓｅｓｂａｎｉａ ｃａｎｎａｂｉｎａ （Ｒｅｔｚ．） Ｐｅｒｓ．］、大豆［Ｇｌｙｃｉｎｅ ｍ ａｘ （Ｌ．） Ｍｅｒｒ．］和豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）的根瘤拟菌体周膜内有多个拟菌体。在周膜形成和周膜内的拟菌体由１ 个到多个的根瘤发育过程中,要有大量的膜来源和周膜的扩增才能适应这种生理变化。同一种根瘤内周膜扩增有周膜间的镶嵌融合,周膜与内质网及其小泡、小液泡膜融合等多种方式。细胞化学表明：周膜、质膜和内质网膜具有相同型的ＡＴＰ酶,周膜是一种嵌合膜,与质膜、内质网膜和液泡膜有共性。最后对周膜来源和扩增方式多样性的生理意义进行了讨论     

用ESR方法研究大豆根瘤细胞和类菌体对环境的敏感性

用ＥＳＲ波谱方法研究了完整大豆根瘤里的根瘤细胞及根瘤细胞中的类菌体对环境的敏感性,分别比较了三种不同类型（快生,慢生,超慢生）的根瘤菌侵染所结的根瘤,测量了不同的发育时期和不同氧化时间ＥＳＲ波谱的变化,观察到根瘤细胞及根瘤内类菌体的豆血红蛋白和钼铁蛋白的ＥＳＲ波谱由氧化而产生的改变,表明了大豆根瘤细胞和根瘤内类苗体所处的状态以及对环境敏感性的不同。     

Relationship between bacteroid poly-beta-hydroxybutyrate accumulation and nodule functioning in the Galega orientalis-Rhizobium galegae symbiosis under diamine treatment

Exposure of Galega orientalis plants to diamines putrescine (Put) and cadaverine (Cad) at concentrations from 0.01 to 2.0 m M significantly altered carbon and nitrogen metabolism in their root nodules. Correlative studies of bacteroid poly- β -hydroxybutyrate (PHB) content and acetylene-reduction capacity of the nodules revealed a negative relationship between these parameters. Utilisation of PHB deposits by bacteroids and high acetylene reduction activity was observed when applying low diamine concentrations. The increase in PHB accumulation in response to high diamine levels was accompanied by a considerable decline in nodule nitrogenase activity. Supplying isolated Galega bacteroids with various diamine concentrations significantly modified bacteroid oxygen consumption, which might be associated with alterations in carbon flux to the bacteroids. Finally, modulation of the bacteroid content upon Put and Cad treatment was examined. The results are discussed in terms of possible causes of the diamine-induced changes in nodule metabolism.     

Metabolism of Rhizobium Bacteroids

Nitrogen fixation within legume nodules results from a complex metabolic exchange between bacteria of the family Rhizobiaciae and the plant host. Carbon is supplied to the differentiated bacterial cells, termed bacteroids, in the form of dicarboxylic acids to fuel nitrogen fixation. In exchange, fixed nitrogen is transferred to the plant. Both the bacteroid and the plant-derived peribacteroid membrane tightly regulate the exchange of metabolites. In the bacteroid oxidation of dicarboxylic acids via the TCA cycle occurs in an oxygenlimited environment. This restricts the TCA cycle at key points, such as the 2-oxoglutarate dehydrogenase complex, and requires that inputs of carbon and reductant are balanced with outputs from the TCA cycle. This may be achieved by metabolism through accessory pathways that can remove intermediates, reductant, or ATP from the cycle. These include synthesis of the carbon polymers PHB and glycogen and bypass pathways such as the recently identified 2-oxoglutarate decarboxylase reaction in soybean bacteroids. Recent labeling data have shown that bacteroids synthesize and secrete amino acids, which has led to controversy over the role of amino acids in nodule metabolism. Here we review bacteroid carbon metabolism in detail, evaluate the labeling studies that relate to amino acid metabolism by bacteroids, and place the work in context with the genome sequences of Mesorhizobium loti and Sinorhizobium meliloti. We also consider a wider range of metabolic pathways that are probably of great importance to rhizobia in the rhizosphere, during nodule initiation, infection thread development, and bacteroid development. Referee: Dr. Robert Ludwig, Department of Molecular, Celluar, and Developmental Biology, Sinheimer Laboratories, University of California, Santa Cruz, CA 95064     

Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules.

Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.     

DNA synthesis and fragmentation in bacteroids during Astragalus sinicus root nodule development

Histo- and cytochemical techniques were used to study the DNA replication and fragmentation patterns in bacteroids formed by Mesorhizobium huakuii subsp. rengei in nodules of Astragalus sinicus. DNA replication was detected by the incorporation of 5-bromo deoxy-uridine. Signals denoting DNA synthesis were observed in plant nuclei within the nodule meristem and in bacteroids near the meristem. The TUNEL (TdT-mediated dUTP nick-end labeling) assay was used to measure DNA fragmentation. In nutrient-depleted 1-mpi (month(s) post inoculation) nodule sections, some bacteroids were in vacuoles, and DNA fragmentation signals were observed only in such bacteroids. In contrast, 1-mpi nodule sections without nutrient depletion showed neither bacteroid localization in vacuoles nor DNA fragmentation signals. The bacteroid translocation into vacuoles upon nutrient starvation might results from autophagy of the plant. In 2-mpi nodule sections, bacteroids with DNA fragmentation signals appeared within the cytoplasm of some nodule cells in the senescence zone.     

Acetoacetyl Coenzyme A Reductase and Polyhydroxybutyrate Synthesis in Rhizobium (Cicer) sp. Strain CC 1192

Biochemical controls that regulate the biosynthesis of poly-3-hydroxybutyrate (PHB) were investigated in Rhizobium (Cicer) sp. strain CC 1192. This species is of interest for studying PHB synthesis because the polymer accumulates to a large extent in free-living cells but not in bacteroids during nitrogen-fixing symbiosis with chickpea (Cicer arietinum L.) plants. Evidence is presented that indicates that CC 1192 cells retain the enzymic capacity to synthesize PHB when they differentiate from the free-living state to the bacteroid state. This evidence includes the incorporation by CC 1192 bacteroids of radiolabel from [¹⁴C]malate into 3-hydroxybutyrate which was derived by chemically degrading insoluble material from bacteroid pellets. Furthermore, the presence of an NADPH-dependent acetoacetyl coenzyme A (CoA) reductase, which was specific for R-(−)-3-hydroxybutyryl-CoA and NADP⁺ in the oxidative direction, was demonstrated in extracts from free-living and bacteroid cells of CC 1192. Activity of this enzyme in the reductive direction appeared to be regulated at the biochemical level mainly by the availability of substrates. The CC 1192 cells also contained an NADH-specific acetoacetyl-CoA reductase which oxidized S-(+)-3-hydroxybutyryl-CoA. A membrane preparation from CC 1192 bacteroids readily oxidized NADH but not NADPH, which is suggested to be a major source of reductant for nitrogenase. Thus, a high ratio of NADPH to NADP⁺, which could enhance delivery of reductant to nitrogenase, could also favor the reduction of acetoacetyl-CoA for PHB synthesis. This would mean that fine controls that regulate the partitioning of acetyl-CoA between citrate synthase and 3-ketothiolase are important in determining whether PHB accumulates.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

PHB颗粒在红豆草根瘤细菌发育中的动态变化

红豆草根瘤胞间隙和侵入线中另有个别细菌含有PHB颗粒,而且数量很少,一个细菌通常仅有一个。随着细菌被从侵入线中释放到寄主细胞中,这些PHB颗粒立即消失。幼龄细菌不含PHB颗粒,成熟细菌一般也不含这种内含物。当细菌衰老时,它们又再度出现,并大量增加,而后很快减少,直至完全消失。从未发现这种颗粒存在于解体细菌中,尽管它们处于各种不同的解体状态。PHB颗粒在细菌发育中的变化表明,它的多少不仅与根瘤细菌发育密切有关,而且也受制于根瘤品种。