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。     

Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.     

Plant Cell Wall Remodelling in the Rhizobium–Legume Symbiosis

Colonization of host cells by rhizobium bacteria involves the progressive remodelling of the plant–microbial interface. Following induction of nodulation genes by legume-derived flavonoid signals, rhizobium secretes Nod-factors (lipochitin oligosaccharides) that cause root hair deformations by perturbing the growth of the plant cell wall. The infection thread arises as a tubular ingrowth bounded by plant cell wall. This serves as a conduit for colonizing bacterial cells that grow and divide in its lumen. The transcellular orientation of thread growth is controlled by the cytoskeleton and is coupled to cell cycle reactivation and cell division processes. In response to rhizobium infection, host cells synthesize several new components (early nodulins) that modify the properties of the cell wall and extracellular matrix. Root nodule extensins are a legume-specific family of hydroxyproline-rich glycoproteins targeted into the lumen of the infection thread. They have alternating extensin and arabinogalactan (AGP) glycosylation motifs. The structural characteristics of these glycoproteins suggest that they may serve to regulate fluid-to-solid transitions in the extracellular matrix. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking and perhaps also by modification of the pectic matrix. Endocytosis of rhizobia from unwalled infection droplets into the host cell cytoplasm depends on physical contact between glycocalyx components of the plant and bacterial membrane surfaces. As endosymbionts, bacteroids remain enclosed within a plant-derived membrane that is topologically equivalent to the plasma membrane. This membrane acquires specialist functions that regulate metabolite exchanges between bacterial cells and the host cytoplasm. Ultimately, however, the fate of the symbiosome is to become a lysosome, causing the eventual senescence of the symbiotic interaction.     

Ultrastructure of soybean nodules. I: release of rhizobia from the infection thread

Root nodules on soybeans (var. Clark 63) were examined by transmission electron microscopy 10-12 days after seed inoculation and planting. The cell infection process appeared identical in both effective nodules, induced by Rhizobium japonicum strain 138 (USDA) and in ineffective nodules, induced by strain 8-0 (Iowa). Electron micrographs are presented which suggest that rhizobia are freed from the infection thread by disintegration of the thread wall and compartmentalization of the distintegrated wall material in membrane-bound vesicles derived from the membrane surrounding the thread. As the thread wall is removed in this manner, the bacteria are released into the host cytoplasm by a process which encloses each in an envelope also dervide from the thread membrane. Any thread wall material remaining around a bacterium after it has dissociated from the thread is removed from the envelope space by vesiculation of the membrane envelope. thus, it appears that endocytosis of both the bacteria and the material composing the infection thread wall occurs during release of rhizobia into the host cell.     

Relationship Between the Membrane Envelope of Rhizobial Bacteroids and the Plasma Membrane of the Host Cell as Demonstrated by Histochemical Localization of Adenyl Cyclase

By using adenyl cyclase as a marker enzyme, the relationship between the membrane envelope of the bacteroids of rhizobia and the plasma membrane of the host cell was demonstrated histochemically. Electron-dense deposits were found on the outer surface of the plasma membrane of the host cell and on the inner surface of the membrane envelopes of the bacteroids, but not in vacuole membranes, endoplasmic reticula, Golgi apparatus, and mitochondrial membranes. The results suggest that the membrane envelopes of the bacteroids are closely related to the host plasma membrane, and that entry of the bacteroids into the cytoplasm is in a manner similar to endocytosis.     

拟菌体包囊膜的动态变化和宿主细胞与细菌的相互关系

本文初次报道紫云英根瘤的超微结构。用根瘤中段的中心组织作实验材料,以显示受根瘤菌侵染的宿主细胞的一般结构。细菌借助于侵入线进入宿主细胞,发育成拟菌体,为包囊膜所裹。一个包囊膜内一般只有一个拟菌体。包囊膜可以与细胞质内的囊泡和小液泡融合而扩增,导致膜对拟菌体的包裹由紧密到疏松的变化。包囊膜和拟菌体表面都有突起,两者的突起相对接触和融合。对拟菌体包囊膜的动态变化与衰老的关系以及宿主细胞和拟菌体之间物质交换的关系进行了讨论。作者指出包囊膜的扩增和电子透明区域的存在,是拟菌体发育成熟的一个阶段,包囊膜和拟菌体通过互相突起、融合沟通的结构,可能是宿主细胞和细菌之间物质交换功能的一种表现。     

Structure of nitrogen-fixing nodules formed by Rhizobium on roots of Parasponia andersonii Planch.

The structure of nitrogen-fixing nodules produced by Rhizobium infection of the non-legume Parasponia andersonii was examined by light and electron (both SEM and TEM) microscopy. Comparisons were made with the nodules previously described on P. rugosa. Like the nodules on different non-legumes formed by other types of endophytes, the Rhizobium nodules on Parasponia resembled modified roots by having a central vascular bundle surrounded by an endophyte-infected zone. The intimate association between the Rhizobium and the host nodule cell was compared with the Rhizobium association found in legumes. The rhizobia were not released from the infection thread as happens in the legume. The infection thread, which propagates the Rhizobium infection to new cells, was transformed within a nodule cell from a darkly stained (light microscopy) or very electron-dense (TEM) structure to a number of thread types. The walls of the threads varied greatly in thickness and often the thread structures were without rigid walls and were only enclosed by a plasma membrane. If the rhizobia are transformed into bacteroids, as in the legumes, it would have to occur when the threads had reached their mature size, when bacterial division had ceased. Nitrogen fixation was considered to occur in all thread types.     

The structure of infection threads,bacteria and bacteroids in pea and clover root nodules

Summary The bacteria and infection threads of the pea root nodule were examined by light and electron microscopy. The bacteria in the infection thread are enclosed in a microcapsule. This capsule disappears when the bacterium is released into the host cytoplasm. The membrane envelope which surrounds the bacteria in the host cell is shown to be derived from the plant cell membrane. The infection of the host cell is by means of a process akin to pinocytosis and the bacteria are confined to vacuoles in the host cytoplasm. As each membrane envelope contains only one bacterium, the envelopes must divide with the bacteria. The bacteria increase 40fold in volume from the infection thread to the stage of the mature bacteroid. The mature infected host cell contains few organelles. The mitochondria become confined to the periphery of the cell. Differences of membrane structure in gram negative bacteria found by other workers have been attributed to fixation artifacts.     

The origin of the membrane envelope surrounding the bacteria and bacteroids and the presence of glycogen in clover root nodules

Summary Thin sections of clover nodules were examined by electron microscopy. The emergence of the bacteria from the infection thread was found to occur by a process of phagocytosis. No evidence was found, using glutar-aldehyde-osmium as a fixative, that there is any connection between the membrane envelope and the endoplasmic reticulum.Electron dense granules, previously observed in clover nodule bacteria, were identified as glycogen granules. These glycogen granules accumulated in bacteria and bacteroids in some ineffective nodules.     

Development of the bacteroid in the root nodule of barrel medic (Medicago tribuloides Desr.) and subterraneum clover (Trifolium subterraneum L.)

Summary The development of the bacteriod is traced from thin sections of slices of nodules fixed in KMnO₄ and OsO₄. While in the infection thread the Rhizobium cell has the ultrastructure characteristic of gram-negative bacteria, with two unit membranes bounding a granular cytoplasm containing dense bodies, a nucleoid area and inclusion granules. A 10–12 fold increase in size, a loss of inclusion granules and the formation of a membrane envelope around each Rhizobium cell follows the dispersal of the rhizobia through the host cytoplasm. As the bacteriods develop there is a loss of fibrillar material from the nucleoid region and changes occur in the distribution of ribosome-like particles in both host and bacterial cells. When fully differentiated and presumably fixing nitrogen the bacteroids from the red zone of subterraneum clover nodules but not barrel medic have a well developed intra-cytoplasmic membrane system.     

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。     

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

Specific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl-esterified pectin and non-esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium-induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.     

红豆草根瘤侵入线的超微结构研究

用透射电镜对红豆草根瘤侵入线的超微结构进行了观察研究.结果表明,(1)红豆草根瘤侵入线由胞间隙和胞间层细胞壁内陷形成,它们的体积较小,多为管状,基质丰富,含菌很少,常有分叉和1个以上的基质区,而且不同基质区的电子密度、细菌数量和侵入线壁厚度都不相同.(2)红豆草根瘤的侵入线十分丰富,它们不仅大量存在于根瘤分生细胞和幼龄侵染细胞中,也经常出现在发育成熟的侵染细胞内.(3)红豆草根瘤中有一种近似圆形的特殊结构,表面由一层膜包围,其内电子密度较低且无固定结构,且只位于侵染细胞的细胞质中,常在侵入线附近,从不出现在侵染细胞的液泡内和非侵染细胞里面.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Fine structure changes in the development of the nodules of Trifolium subterraneum L. and Medicago tribuloides Desr.

Summary Electron microscope observations of thin sections of nodules of subterranean clover and barrel medic, after fixation in KMnO₄ or OsO₄, show that following infection there is a marked increase in the amount of endoplasmic reticulum, in the number of ribosomes, Golgi bodies, mitochondria and proplastids in the host cells.As the infection thread approaches the nucleus, large gaps appear in the nuclear membrane. During the formation of the membrane envelopes around the rhizobia, after their release from the infection thread, the reticulum changes from a predominantly plate-like to a vesicular form. As the bacteroids develop the plastids of the host cells become filled with starch, and become aligned, with the mitochondria, against the cell walls of the host cells. Plastids in noninvaded cells also become starch-filled. Bacteroids and host cells enlarge further and finally the bacterioids occupy most of the cytoplasm of the host cell, except for the nuclear region and vacuole. With OsO₄ fixation the nucleoplasm, predominantly fibrillar before infection, with a dense staining nucleolus, becomes packed with dense ribosome-like (150 A° diameter) granules. No such changes occur in the nuclei of non-infected cells. In the proplastids and plastids many small, electron dense particles (60 A° diameter) (phytoferritin?) are observed.     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

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。     

Specific targeting of membrane nodulins to the bacteroid-enclosing compartment in soybean nodules

Rhizobium bacteroids in nodule cells are surrounded by the peribacteroid membrane (pbm), which is derived from the host plasma membrane during infection. The pbm was purified from R. japonicum 61A76-induced soybean nodules and analyzed by comparing it with the host cell plasma membrane for the presence of nodulins, nodule-specific plant proteins. Nodulins were found in pbm by reacting Western blots with a nodule-specific antiserum raised against the pbm. Peribacteroid fluid (the fluid enclosed in the pbm) was also found to contain several nodulins. The pbm nodulins were confirmed to be of plant origin by in vitro translation of poly(A)⁺ nodule mRNA followed by immunoprecipitation by the nodule-specific antiserum. Antibodies raised against a synthetic peptide corresponding to a repeated domain in nodulin-24, a pbm nodulin, and the nodule-specific pbm antiserum reacted exclusively with the pbm. The absence of pbm-nodulins in the plasma membrane suggests that the infected cells direct the intracellular transport of the pbm nodulins exclusively to this de novo synthesized subcellular compartment essential for symbiotic nitrogen fixation.     

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。