Three-dimensional organization of uninfected tissue in soybean root nodules and its relation to cell specialization in the central region

Summary The three-dimensional structure of uninfected tissue in the central infected region of soybean nodules has been studied using several methods. Transverse and longitudinal sections have been examined. A view of the surface of the central region has been obtained by partially clearing nodules and removing their cortex. A three-dimensional reconstruction of the central region has been assembled using transverse sections. It was found that some of the uninfected cells participate in forming characteristic structural aggregates. A centrally located spherical region with a diameter half that of the entire infected region is filled with interconnected aggregates of uninfected cells. A set of branching interconnected planes of uninfected cells emanate from the sphere and extend out to the surface of the infected region. The planes are oriented generally in a longitudinal direction but are sometimes at other angles. These planes divide the nodule into compartments of various sizes and shapes. The planes are perforated by irregularly shaped groups of infected cells. Uninfected cells also often occur arranged in lines oriented approximately radially in the central region. Irregularly shaped fine aggregates of uninfected cells occur outside the central sphere formation and are interconnected by narrow lines of uninfected cells. All of these types of formations could provide uninfected paths all or part of the way from the center of the central region to the cortex. The planes and lines often contain uninfected cells elongated perpendicular to the surface of the central region, suggesting that the routes may serve in the transport of substances from the inside to the surface of the central region. The distribution of plasmodesmata also appears to favor transport from one uninfected cell to another.Abbreviation TER tubular endoplasmic reticulum     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Phosphoserine aminotransferase in soybean root nodules : demonstration and localization

Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine-pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules.     

Vacuolation and infection thread in root nodules of soybean

Profuse vacuolation takes place in the soybean root nodule cells where infection threads carry rhizobia. After the rhizobia are released the disappearance of the infection thread is attributed to its degradation within large vacuoles which result from fusion of small vacuoles.     

Reduction of ferric leghemoglobin in soybean root nodules

Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions.

     

Reversible dark-induced senescence of soybean root nodules

Nodule senescence was induced in intact soybean [Glycine max. (L.) Merr., cv Woodworth] plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

Immunochemical studies on xanthine dehydrogenase of soybean root nodules

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was purified from root nodules of soybean (Glycine max) and used to prepare a polyclonal rabbit antiserum. Monospecificity of this antiserum was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipate. During root nodule development of soybean, only one form of XDH was detected on an immunological basis. Titration of XDH by immunoelectrophoresis showed that a remarkable increase in the amount of XDH occurred between two and four weeks after inoculation, in parallel with the increase in enzyme activity. Localization of XDH by immunofluorescence indicated that the enzyme was present exclusively in uninfected cells where it appeared to be associated with discrete organellelsAbbreviations IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis - XDH xanthine dehydrogenase     

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg^-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

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     

Asparaginase and asparagine transaminase in soybean leaves and root nodules

Asparaginase activity (≤1 μmol/mg protein · hr) was detected in extracts of soybean (Glycine max [L.] Merr.) leaf blades, but, even after efforts to optimize extraction and assay of the enzyme, specific activity was not sufficient to metabolize the estimated amount of asparagine translocated to leaves. Asparagine transaminase activity with glyoxylate or pyruvate was at least 52 and 62 nmol/mg protein · hr, respectively. This estimate of transaminase activity is based on the analysis of the reaction product α-ketosuccinamate. Formation of glycine and alanine was confirmed by amino acid analysis. α-Ketosuccinamate deamidase had a specific activity of 85 nmol/mg protein · hr in leaf blade extracts.     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999