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.     

A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide.A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein.The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots.The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their permeability to oxygen diffusion

The effects of short-term NaCl-salinity on nodules of soybean ( Glycine max L. cv. Kingsoy) were studied on hydroponically-grown plants. Both acetylene reducing activity (ARA) and nodule respiration (O₂ uptake and CO₂ evolution) were immediately inhibited, and the stimulation of them by rising the external partial pressure of O₂ (pO₂) was diminished by the application of 0.1 M NaCl in the nutrient solution. The permeability of the nodule to O₂ diffusion, estimated by O₂ consumption or CO₂ evolution, was significantly lower in the stressed nodules than in the cootrol ones. The respiratory quotient of intact nodules and the ethanol production of excised nodules were increased by low pO₂ and by salt stress. These data confirm that in salt-stressed soybean nodules, O₂ availability is reduced and fermentative pathways are stimulated.     

Metabolic reprogramming in nodules,roots, and leaves of symbiotic soybean in response to iron deficiency

To elucidate the mechanism of adaptation of leguminous plants to iron (Fe)‐deficient environment, comprehensive analyses of soybean (Glycine max) plants (sampled at anthesis) were conducted under Fe‐sufficient control and Fe‐deficient treatment using metabolomic and physiological approach. Our results show that soybeans grown under Fe‐deficient conditions showed lower nitrogen (N) fixation efficiency; however, ureides increased in different tissues, indicating potential N‐feedback inhibition. N assimilation was inhibited as observed in the repressed amino acids biosynthesis and reduced proteins in roots and nodules. In Fe‐deficient leaves, many amino acids increased, accompanied by the reduction of malate, fumarate, succinate, and α‐ketoglutarate, which implies the N reprogramming was stimulated by the anaplerotic pathway. Accordingly, many organic acids increased in roots and nodules; however, enzymes involved in the related metabolic pathway (e.g., Krebs cycle) showed opposite activity between roots and nodules, indicative of different mechanisms. Sugars increased or maintained at constant level in different tissues under Fe deficiency, which probably relates to oxidative stress, cell wall damage, and feedback regulation. Increased ascorbate, nicotinate, raffinose, galactinol, and proline in different tissues possibly helped resist the oxidative stress induced by Fe deficiency. Overall, Fe deficiency induced the coordinated metabolic reprogramming in different tissues of symbiotic soybean plants.     

Formation of nodules on non-nodulating soybean T201 after treatment with 2,4-dichlorophenoxyacetate

The formation of effective root nodules on a non-nodulating line (T201) of soybean (Glycine max (L.) Merr.,) was induced by a treatment with 2,4-dichlorophenoxyacetate (2,4-D). The induced nodules, inoculated with mixed Bradyrhizobium japonicum strains A1017 and IRj2101, had a normal internal structure, red in colour and the cells being filled with bacteroids. Externally, the induced nodules were of unusual shape, being paired or gourd-like in form and were attached to thickened roots. The nodules were capable of acetylene reduction (3.1–3.5 moles g^-1 fresh weight nodules h^-1), allowing the growth of plants with dark green leaves.     

Magnetic force microscopy of iron oxide nanoparticles and their cellular uptake

Magnetic force microscopy has the capability to detect magnetic domains from a close distance, which can provide the magnetic force gradient image of the scanned samples and also simultaneously obtain atomic force microscope (AFM) topography image as well as AFM phase image. In this work, we demonstrate the use of magnetic force microscopy together with AFM topography and phase imaging for the characterization of magnetic iron oxide nanoparticles and their cellular uptake behavior with the MCF7 carcinoma breast epithelial cells. This method can provide useful information such as the magnetic responses of nanoparticles, nanoparticle spatial localization, cell morphology, and cell surface domains at the same time for better understanding magnetic nanoparticle‐cell interaction. It would help to design magnetic‐related new imaging, diagnostic and therapeutic methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

微生物嗜铁素介导的铁摄取

嗜铁素是好氧菌和兼性厌氧菌的一种产物,它是微生物在低铁条件下产生的小分子的、特异性的Fe^3＋螯合因子。大多数的好氧和兼性厌氧微生物至少合成一种嗜铁素,由嗜铁素介导的铁摄取可能是细菌最普遍的一种获取铁元素的方式。     

植物吸收利用铁的机理

根据植物铁营养的一些研究进展,论述了植物对铁的吸收和运输机理以及HCO3^-,N、P等因素对铁利用效率的影响。     

Mitogen-activated protein kinase is involved in the symbiotic interaction betweenBradyrhizobium japonicum USDA110 and soybean

Mitogen-activated protein kinase (MAPK) plays an important role in mediating the intracellular transmission and amplification of extracellular stimuli. We examined whether MAPK is involved in the signaling process during the early step of nodule formation. A genistein induced culture filtrate (GCF) ofBradyrhizobium japonicum was prepared for inducing an early response by soybean root hairs via Nod factor. Upon treatment, several types of deformations were seen, demonstrating that GCF contains active Nod factor molecules. In-gel kinase assays showed that treating soybean roots with GCF induced the rapid activation of two protein kinases (molecular masses of 47 kD and 44 kD), which phosphorylate myelin basic protein (MBP). To identify the activated kinase, we prepared an antibody againstGMK1 (Glycine max MAP kinase 1), based on information from SIMK (an alfalfa MAP kinase) and a soybean EST database. An immunocomplex kinase assay with the GMK1-specific antibody revealed that the 47-kD kinase in GCF-treated seedlings is indeed GMK1. Consistent with many other MAP kinases, GMK1 is likely to be under post-translational regulation. Considering these results and previous reports from soybean, GMK1 seems to be a signaling mediator with a broad range of stimuli, including a fungal elicitor, wounding, and the symbiotic interaction between soybean and B.japonicum.     

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     

缺硼对大豆根瘤结构和功能的影响

在营养液培养条件下以普通结结瘤大豆Ｂｒａｇｇｃｖ．「Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒ」及其超结瘤突变体ｎｔｓ３８２为实验材料,运用光学显微方法研究了硼对大豆根瘤结构的影响,并测定了根瘤固氮酶活性结果表明,缺硼使根瘤结构受到严重破坏,并使固氮酶活性显著下降,缺硼使根瘤结构受到破坏是导致固氮酶活性下降的可能原因。     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

A high-affinity iron transport system of Rhizobium meliloti may be required for efficient nitrogen fixation in planta

We have analyzed the ability of single site insertion mutants of Rhizobium meliloti 1021 defective in various components of a high-affinity iron transport system to produce nodules, fix nitogen and promote plant growth. Our results indicate that a high-affinity iron transport system may significantly increase the ability of the differentiated form of the bacterium to fix nitrogen and induce an increase in plant growth.Abbreviations EDDA ethylenediamine-N,N-bis(2-hydroxyphenylacetic acid) - CAS chrome azurol S     

Expression analysis of LacZ gene placed in the locus of Cnot7 exhibits its activity in osteoblasts in vivo and in mineralized nodules in vitro

CCR4-NOT complex 7 (Cnot7) was identified as a regulator of gene expression in yeast and evolutionally conserved in mammals. Cnot7 deficient male mice exhibit abnormality in spermatogenesis. As these mice contained construct to express LacZ, we followed the expression patterning in these animals. LacZ was expressed in osteoblasts located in the primary spongiosa in adult mice. Cellular analysis indicated that LacZ is expressed in osteoblasts but not in osteoclasts. In the mineralized nodules formed in the culture of bone marrow cells obtained from Cnot7 +/- mice, LacZ expression was mainly observed in the cells forming mineralized nodules but not in un-mineralized area scattered around the periphery of the nodules. LacZ blue positive cells were gradually depositing minerals along its time course of the in vitro mineralization assay. Cnot7 expression was enhanced by the treatment with BMP. These data suggest that Cnot7 is expressed in osteoblasts and is associated with mineralization.     

The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen-fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O₂ regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H₂ evolution in N₂:O₂) and nodule respiration (CO₂ evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H₂ evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O₂ concentration (O_i), measured by a spectro-photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, O_i levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O₂ at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady-state rates of gas exchange, linear increases in pO₂ from 20 to 100% at 4% min^?1 resulted in recoveries of H² and CO² evolution, indicating that O_i limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O₂ available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O₂, O_i values measured at supra-ambient pO₂ were greater than those at 20% O₂ and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO₂ (O_c) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady-state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above-ambient pO₂.