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。     

Soybean root nodule ultrastructure during dark-induced stress and recovery

Summary Root-nodules of soybean plants dark-stressed for 8 days and then allowed to recover for up to 17 days were examined by transmission electron microscopy. Control nodules possessed all the ultrastructural features characteristic of infected and uninfected nodule cells. Minimal changes in the appearance of host cells and bacteroids occurred during the first four days of dark stress. After 8 days of dark stress, damage was observed in the cellular and organelle membranes; however, very few changes were observed in the bacteroids. Nodule structure continued to degrade during the first two days of recovery after which time nodules either recovered or completely degraded. In the former case, structural integrity returned to all nodule cells. In the latter case all structural integrity of the host cell disappeared; however, bacteroids appeared intact suggesting that they remained viable.Published as Paper no. 7974, Journal Series, Nebraska Agriculture Research Division.     

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.     

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.     

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。     

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。     

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.     

The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum

A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.     

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv `Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10¹⁰ cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10¹⁰ bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10¹⁰ bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.     

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.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Localization of ATPese and Dynamic Change in its Activity within Root Nodules of Legumes

The study on localization of ATP-ase in root nodules of Astragalus sinicus and Sesbania cannabina shows that the particles of lead phosphate precipitates resulting from the reaction of ATP hydrolytic enzyme are distributed in cell wall, plasmolemma, cytoplasm and peribacteroid membranes etc. of the host cells. This ATP-ase plays an important role in the transportation and absorption of substances. Owing to the need of photosynthates provided for the nitrogen fixation of root nodules, the active reaction of ATP-ase in bacteroids varies with their developments. The numbers of bacteroids having ATP hydrolytic enzyme in young root nodules are significantly less than those in senescent nodules. Possibly, this active reaction is related with the physiological function of nitrogen fixation of bacteroids at different development stages.     

Viability of Bradyrhizobium japonicum bacteriods

Homogenates from soybean nodules, formed by 12 strains of Bradyrhizobium japonicum, were plated into yeast-extract mannitol agar containing 3 or 37 g mannitol 1^-1. Viable counts ranged from 8.298 to 11.265 log₁₀ cells-gram nodule^-1. When monitored over the life cycle of the symbiosis, the viability of strains USDA 110 and USDA 123 increased with days after planting (DAP), and at 70 DAP was 95% and 81%, respectively. By contrast, the viability of USDA 38 bacteroids decreased with time, and at 70 DAP was only 1.9%. At 49 DAP, nodules induced by USDA 38 had significantly fewer bacteroids per peribacteroid membrane than those formed by USDA 110 or USDA 123, and at 70 DAP, 27% of the USDA 38 bacteroids showed some degree of degeneration. Viable counts of USDA 123 and USDA 110 bacteroids, isolated from the nodules of 12 different cultivars, ranged from 10.963 to 11.463 and from 10.683 to 11.117 log₁₀ viable cells-gram nodule^-1, respectively. Varying the osmolarity of the medium had no predictable effect on bacteroid viability. When surface-sterilized nodules of IPAGO 587 (high bacteroid viability) and USDA 38 (low bacteroid viability) were inoculated into a nonsterile silt loam soil, at rates equivalent to 5.0×10⁸ and 5.0×10⁶ viable bacteroids g^-1 soil, respectively, and then incubated at 28° C for 60 days, 4.3×10⁴ and 1.5×10⁴ surviving cells g^-1 soil, respectively, were recovered. Thus, despite differences due to host and strain variation, bacteroid viability appears to be unrelated to persistence of individual strains following an annual legume crop cycle.Journal paper No. 14930, Agricultural Experiment Station University of Minnesota, St. Paul, MN 55108, USA     

Cell wall (outer membrane) of bacteroids in nitrogen-fixing peanut nodules

Rhizobia in peanut nodules are transformed from rod-shaped cells to extremely large spherical bacteroids. The invading rhizobia shed their outer membranes soon after their release into the host cells. The outer membranes are seen to be peeled off and replaced by new outer membranes before rhizobia are differentiated into nitrogen-fixing bacteroids. Evidence is presented that peanut bacteroids, in spite of their spheroplast-or protoplast-like appearance, do possess both the inner and the outer membrane.     

Fate of Nodule-Specific Polysaccharide Produced by Bradyrhizobium japonicum Bacteroids

A polysaccharide produced by Bradyrhizobium japonicum bacteroids in nodules (NPS) on soybean (Glycine max [L.] Merr.) roots is different in composition and structure from the extracellular polysaccharide produced in culture by this organism. Isogenic strains either capable or incapable of NPS synthesis supported similar rates of plant growth and nitrogenase activity, indicating that polysaccharide deposition was not detrimental. The possibility that NPS may have some protective or nutritional role for bacteroids was considered. Analysis of disintegrating nodules over periods of 1 to 3 months indicated greater recovery of viable bacteria from NPS+ nodules prior to the breakdown of NPS. During and after the breakdown of NPS, the decline in viable bacteria was similar for NPS+ and NPS- strains. Bacteroid destruction in senescing nodules may be accelerated by exposure to proteolytic enzymes in host cytoplasm; however, highly purified NPS had no significant effect on the in vitro activity of partially purified proteases, so protection of bacteroids via this mechanism is unlikely. B. japonicum USDA 438 did not utilize NPS as a carbon source for growth in liquid culture. In vitro assays of NPS depolymerase activity in cultured bacteria and bacteroids were negative using a variety of strains, all of which contained extracellular polysaccharide depolymerase. It seems highly unlikely that B. japonicum can utilize the polysaccharide it synthesizes in nodules, and NPS breakdown in senescing nodules is probably caused by saprophytic fungi.     

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.     

箭舌豌豆根瘤液泡中细菌周膜来源的研究

电镜观察结果表明,幼龄箭舌豌豆根瘤侵染细胞的细胞质较少,中央是一些体积较大的液泡。细胞质中侵入线经常可见,由侵入线释放出来的细菌均有细菌周膜。这些细菌只位于细胞质中,不出现在液泡里面。成熟根瘤中的侵染细胞与此不同,它们中有大量的成熟侵染细胞,细胞质丰富,里面充满大量细菌,中央常有一个大液泡。当中央液泡发育到一定程度时,位于其附近的细菌可通过液泡膜内吞、液泡膜与细菌周膜融合及液泡膜破裂3种途径进入液泡,后一种途径常伴有寄主细胞质。液泡中的细菌绝大部分裸露在外,只有个别细菌具有细菌周膜且多位于液泡膜的破损处附近,因此细菌周膜可能是原来就有的。     

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.     

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.