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。     

大豆根瘤细菌周膜的冷冻复型研究

用冷冻复型电镜技术研究了中国丰收１１号大豆根瘤中的细菌周膜。细菌周膜的断裂面上有颗粒状物质,但Ｐ面和Ｅ面有所不同,前者颗粒密度较大。即使都在Ｐ面或Ｅ面上,不同的细菌或同一细菌不同部位的颗粒密度也不一样。在细菌周膜与细菌细胞壁之间有一个环形腔隙,腔的大小随细菌和细菌部位不同而异。腔中不仅有泡状和管状结构,有时也有类寄主细胞质物质。细菌周膜表面有近似半球形或嵴形隆起,它们可能是腔中管泡状结构压迫细菌周     

Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognize the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteroid membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.     

Role of Endoplasmic Reticulum in the Formation of Peribacteroid Membranes

The relation between the endoplasmic reticulum and peribacteroid membranes during the development of infected cells of Chinese soybean (Glycine max L. cv. Harvest 11) root nodules by transmission electron microscopy was observed. After the host cells are infected by bacteria, the ultrastructures of the infected cells appear to have many changes, such as that their cytoplasm becomes thicker, the vacuoles decrease in size and organelles rapidly increase in number, among these organelle changes are more obvious than the others. However, changes of endoplasmic reticulum is mostly striking. It is not only increases greatly in number but often swells and forms wider inter-spaces. The swelling of endoplasmic reticulum is especially conspicuous at its ends and often form various vesicles. Sometimes, the front part of the endoplasmic reticulum also forms a gourd-shaped structure, which together with the vesicles usually contain fibrillar material. After they are released from the endoplasmic reticulum to the host cytoplasm, they continuously move towards neighbouring bacteria and close to the peribacteroid membranes. The gourd-shaped structures always locate near but never fuse with the peribacteroid membranes. However, the vesicles can do that and form a kind of papillae, often containing fibrillar material, on the peri bacteroid membranes. These papillae and their fibrillar material gradually disappear whilst the membrane of the vesicle derived from endoplasmic reticulum becomes one part of the peribacteroid membrane by way of fusing with the latter to form a papilla on it.     

Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea

Abundance and distribution of plant cell surface proteins of the hydroxyproline-rich glycoprotein (HRGP) class were studied in the pea- Rhizobium symbiosis using immunoblot analysis. The MAC 265-epitope was especially abundant in pea root nodules containing nitrogen-fixing Rhizobium bacteria. A 180-kDa MAC 265-HRGP dominated in pea shoot plasma membranes, while almost no MAC 265-HRGP was detected in root plasma membranes. We show here that a major difference between the plant-derived peribacteroid membrane of the symbiosomes and the root plasma membrane was the presence of a 100-kDa MAC 265-HRGP in the former. Arabinogalactan proteins (AGPs), as recognized by the monoclonal antibodies MAC 207 and JIM 8, were not detected in the peribacteroid membrane, while two isoforms (100 and 220 kDa) were detected in shoot and root plasma membranes. Specific MAC 265-HRGP isoforms were found in the peribacteroid space fraction of the symbiosomes and thus as soluble proteins in the interface between the symbionts. The abundance of the MAC 265-epitope was much reduced in non-nitrogen-fixing nodules when this phenotype resulted from a dicarboxylate transport mutation in Rhizobium . There was no reduction in the abundance of the MAC 265-epitope in non-fixing phenotypes resulting from a mutation in the plant. The results suggest that bacterial signals related to the bacterial ability to fix nitrogen, might be responsible for the regulation of HRGP expression in root nodules.     

Isolation and purification of proteins from the symbiosome membrane of yellow lupine root nodules

A unique feature of the symbiotic association between legume plants and rhizobia is the plant-derived membrane which separates the symbionts within root nodule; this membrane is termed the peribacteroid membrane (PBM). Although this membrane plays a vital role in facilitating transport and other processes in nodules, little is known about the proteins that are associated with and are an integral part of it. The objective of this work was to apply modern methods of protein purification to the characterisation of proteins of peribacteroid membrane from nodules of yellow lupine (Lupines luteus). The 17-kDa protein was isolated from purified peribacteroid membrane using size exclusion and ion exchange chromatography (FPLC). The N-terminal amino acid sequence of this protein was determined; the sequence does not match any of the previously reported lupine and other legume sequences. Following detergent solubilisation of purified peribacteroid membrane, integral proteins of 15 to 20 kDa were purified by size exclusion chromatography.     

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.     

Transient Energy Coupling Between Rhizobia and Legume Cells Mediated by the Peribacteroid Membrane ATPase Proton Pump

We present a model for the metabolic coupling between rhizobia and plant cell in the nitrogen-fixing legume root nodules. The symbiosome, an organelle-like structure formed by the modified rhizobia (the bacteroids) enclosed by a plant cell derived peribacteroid membrane, is an unique structure in which two energized membranes are closely packed: the inner bacteroid membrane and the peribacteroid membrane that possesses an ATPase proton pump. The model is based on the following points: (i) The permeability for hydrogen ions of the outer membrane of the rhizobia. (ii) The reversibility of the ATPase proton pump of the peribacteroid membrane [Szafran, M. M. and Haaker, H. (1995) Plant Physiol. 108, 1227–1232]. (iii) The relative affinites for oxygen of the bacteroid and plant mitochondria terminal oxidases, and the prevailing oxygen concentration inside the nodule, which results in aerobic metabolism for the bacteroid, but in quite fermentative catabolism for the host plant cell. We propose that the bacteroid can transiently supply free energy to the plant cell in the form of protonmotive force by the movement of hydrogen ions from the bacteroid periplasmic space to the plant cytoplasm through the peribacteroid membrane ATPase. The proposed hydrogen ion flux could be dependent on the phosphorylation potential in both the plant cell cytoplasm and the bacteroid, and the simultaneous ion movements to avoid the development of opposite . It could be important in situations of transient ATP depletion inside plant cell, which involves the block of ammonia assimilation and, subsequently, the inhibition of bacteroid nitrogenase.     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Protein composition of the peribacteriod space in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum biovar viciae

Proteins in the peribacteroid space (PBS) between the bacteroid outer membrane and the peribacteroid membrane in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum PRE were analysed by two-dimensional (2-D) gel electrophoresis. Most of the detectable proteins were found to migrate to identical positions; however the level of accumulation of some of these appear to be determined by the host plant. When a different R. leguminosarum strain (RB1) was used to inoculate P. sativum , the majority of the isolated PBS proteins were found to migrate in the 2-D gel to identical positions as those of the other two combinations ( R. leguminosarum PRE x P. sativum and R. leguminosarum PRE x V. faba ).     

An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF).

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.     

The peribacteroid membrane

The objective of this review is to summarise current knowledge about the structure and function of the peribacteroid membrane from the root nodules of leguminous plants. The information is presented in terms of development of this symbiotic membrane from its origin, through proliferation and in the mature state. There are clear indications that the peribacteroid membrane has a distinct structure and function at each developmental stage. The mature peribacteroid membrane has been the most intensively studied. The lipid and protein content of the mature peribacteroid membrane is discussed with particular emphasis on genetic and functional studies of the proteins. The mechanism and control of peribacteroid membrane biogenesis is also discussed. There is evidence for a specific biogenetic pathway for this membrane which requires both symbiotic partners for its correct functioning.     

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix⁻ (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.     

A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor.

Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. Immunocytochemical study showed that the protease inhibitor is exclusively localized in infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.     

细菌周膜与液泡膜和液泡内含物的关系

箭舌豌豆根瘤中有丰富的侵入线,从侵入线释放出来的细菌都有细菌周膜。有时液泡中也有细菌,但它们中的绝大多数没有细胞周膜,只有个别例外,而且结构较清晰。细菌结构越好,它的细菌周膜就越完整。因此,液泡中细菌所具有的细菌周膜并非由液泡膜和液泡内含物形成。     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes

The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules. Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two-dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry. The proteins of 46 spots could be identified by database search. The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known from COPI-coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis.