Electron Microscopic Observation on the Infected Cells During Pea (Pisum sativum L.) Nodule Senescence

Ultrastructural changes of the infected cells have been observed by transmission electron microscopy during pea root nodule senescence. The infected cells and bacteroids of pea nodules ultimately senesce, their senescence has certain laws and features. Firstly, peribacteroid membrane were loosened, leaving a large electron-empty space with fibrillar and vesicular material. Then bacter0id cytoplasm lost features and aggregated into some clustered electron- dense material. At next stage bacteroids were structurally emtpy and appeared like “ghost” cells. Companying bacteroid senescence, host cytoplasm changed from dark to light in electron density and cell organelles gradually decreased. After the host cell tonoplasts and plasmalemma broke down, the infected cells showed a chaotic state of bacteroids and host cell debrises. Finally, infected cells disintegrated completely. Sometimes some young bacteria were seen in the intercellular spaces surrounded by degenerating cells, in the degenerating cytoplasm. A few infection threads were also found among the disintegrated bacteroids, even some of them were releasing the bacteria into the degenerating host cytoplasm.     

Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.     

Bacteroid distributions in alfalfa nodules upon dark-induced senescence and subsequent partial rejuvenation

Photosynthate availability directly controls the maturation, senescence and distribution of bacteroids (inoculum Rhizobium meliloti 102 F28) in alfalfa ( Medicago saliva L. cv. Buffalo) nodules. Mature, dinitrogen-fixing bacteroids were located principally in the middle section (region) of 6- to 8-week-old nodules in light-grown alfalfa plants. Upon dark treatment of the plants, bacteroids in the middle region of a nodule were induced to senescence while those in the tip region began to mature faster. Senescence and deterioration of bacteroids in the basal region of a nodule also were more advanced in the dark-treated plants. Sugar supplied exogenously during the dark period retarded the senescence process. Exposure of the dark-treated plants to light partially restored nitrogenase activity. The distribution of bacteroids in the rejuvenated nodules was similar to that of the light-grown plants.     

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。     

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。     

Poly-beta-hydroxybutyrate Utilization by Soybean (Glycine max Merr.) Nodules and Assessment of Its Role in Maintenance of Nitrogenase Activity

Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-β-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer. Changes in poly-β-hydroxybutyrate content and in activities of nitrogenase, β-hydroxybutyrate dehydrogenase, and isocitrate lyase in nodule bacteroids were investigated under conditions in which the supply of carbohydrate from the soybean plants was interrupted. The poly-β-hydroxybutyrate content of bacteroids did not decrease appreciably until the carbohydrate supply from the host plants was limited by incubation of excised nodules, incubation of plants in the dark, or by senescence of the host plant. Isocitrate lyase activity in bacteroids was not detected until poly-β-hydroxybutyrate utilization appeared to begin. The presence of a supply of poly-β-hydroxybutyrate in nodule bacteroids was not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant. An unusually high activity of β-hydroxybutyrate dehydrogenase was observed in bacteroid extracts but no significant change in the activity of this enzyme was observed as a result of apparent utilization of poly-β-hydroxybutyrate by nodule bacteroids.     

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate–glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.     

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。     

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.     

Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.     

A novel ankyrin-repeat membrane protein, IGN1, is required for persistence of nitrogen-fixing symbiosis in root nodules of Lotus japonicus

Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix(-) mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8-9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.     

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：刚受侵染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;幼年类菌体细胞外型较小,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（PHB）累积增多;随着根瘤细胞逐渐发育成熟,在类菌体包被内可允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。     

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Effect of water stress on the reduction of nitrate and nitrite by soybean nodules

The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from −0.6 to −1.4 megapascals, and then decreased when −1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.     

Forms and Fates of Rhizobial Bacteria Present in the Infected Host Cells of Nodules

There were two forms of rhizobial bacteria present in infected host cells of nodules. One was bacteroids which were enclosed in peribacteroid membrane originated from the infected host cells. The other was rhizobia as vegetative cells. The infected host cells were occupied by most of the bacteroids and a certain number of the vegetative cells respectively. With the nodule senescence, there were two kinds of fate of the bacteria: The bacteroids degenerated togather with the infected host cells at the same time and further disintegrated completely, so it is not possible that the disintegrated bacteroids could be returned into soil to revive: the vegetative cells did not disintegrate and die when the infected host cells senesced, eventually could be turned back into soil. The vegetative cells may play an important role, on the one hand, in cycle between legume and soil, on the other hand, maintain rhizobia in natural balance of population ecosystem.     

根瘤菌在宿主细胞内的存在形式及其命运

根瘤菌在根瘤宿主细胞内有两种形式：一种为拟菌体、被宿主细胞来源的财膜包裹;另一种为自由生活的营养细胞。前者色大多数,后者只有少数。随着根瘤的衰老,其命运是：拟菌体及其宿主细胞同时衰老以致最终解体,拟菌体不能再入土壤复生;以自由生活的营养细胞形式存在的细菌,不随其宿主细胞的解体而亡,可回复到土壤,一方面在豆科植物和土壤之间循环,一方面维持根瘤菌在土壤中天然的群体生态平衡。