Biotransformation of the Pentahydroxy Flavone Quercetin by Rhizobium loti and Bradyrhizobium Strains (Lotus)

Lotus rhizobia catabolized quercetin in an arabinose-based medium via a novel form of C-ring cleavage, yielding phloroglucinol and protocatechuic acid. Conservation of the A and B rings of the flavone suggests that a chalcone could be formed as a transient intermediate.     

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Characterization of NADP+-isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules

NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500-fold from the host-plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn²⁺), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol⁻¹ (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The K_m for isocitrate was 20 μ M and for NADP⁺ 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α-ketoglutarate was a non-competitive inhibitor with respect to isocitrate and NADP⁺. Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non-saturating isocitrate concentrations. The potential significance of glutamine regulation of α-ketoglutarate production in a dinitrogen-fixing tissue is discussed.     

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.     

Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas

Three strains of rhizobia isolated from effective root nodules of pea (Pisum sativum L.) collected from the Indian trans-Himalayas were characterized using 16S rRNA, atpD and recA genes. Phylogeny of the 16S rRNA genes revealed that the newly isolated strains were members of the genus Rhizobium with ≥99.9% sequence similarity to the members within the “Rhizobium leguminosarum” group. Phylogenetic analyses based on the concatenated sequences of atpD and recA gene, and 92 core genes extracted from the genome sequences indicated that strains JKLM 12A2^T and JKLM 13E are grouped as a separate clade closely related to R. laguerreae FB206^T. In contrast, the strain JKLM 19E was placed with “R. hidalgonense” FH14^T. Whole-genome average nucleotide identity (ANI) values were 97.6% within strains JKLM 12A2^T and JKLM 13E, and less than 94% with closely related species. The digital DNA-DNA hybridization (dDDH) values were 81.45 within the two strains and less than 54.8% to closely related species. The major cellular fatty acids were C_18:1w7c in summed feature 8, C_{14:0 3OH}/C_{16:1 iso I} in summed feature 2, and C_18:0. The DNA G + C content of JKLM 12A2^T and JKLM 13E was 60.8 mol%. The data on genomic, chemotaxonomic, and phenotypic characteristics indicates that the strains JKLM 12A2^T and JKLM 13E represent a novel species, Rhizobium indicum sp. nov. The type strain is JKLM 12A2^T (= MCC 3961^T = KACC 21380^T = JCM 33658^T). However, the strain JKLM 19E represents a member of “R. hidalgonense” and the symbiovar viciae.     

Danish Rhizobium leguminosarum strains nodulating 'Afghanistan' pea (Pisum sativum)

A wild pea ( Pisum sativum L.) native to Afghanistan normally known to be resistant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection under aseptic conditions. Five types differing in isoenzyme composition pattern were found among 15 isolates from 'Afghanistan' nodules. None were identical with the 'Tom' strain from Turkey, which also forms effective nodules with 'Afghanistan'. The five types were also different with respect to isoenzyme pattern from Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Alleviation of water stress effects in cowpea by Bradyrhizobium spp. inoculation

Experiments were carried out to investigate the effects of different degrees of water stress on cowpea in the presence and absence of Bradyrhizobium spp. inoculation and to evaluate physiological responses to stress. The soil used was Yellow Latosol, pH 6.3 and the crop used was cowpea (Vigna unguiculata (L.) Walp.) cv. ‘IPA 204’. Stress was applied continuously by the control of matric potential (ψ _m ) through a porous cup. The lowered soil ψ _m had a direct effect on the N2 fixation, but the strains Bradyrhizobium introduced by inoculation in the cowpea plants were superior to the indigenous strain demonstrating the importance of inoculation in the stressed plants. At the more negative ψ _m plants inoculated with the strains EI 6 formed associations of greater symbiotic efficiency which helped the cowpea plants to withstand drought stress better than the strain BR 2001 and the uninoculated control. The leghaemoglobin concentration was not inhibited in the drought-stressed plants at ψ _m -70 kPa when inoculated with the strain EI 6, which confered a differential degree of drought resistance in plants. The ψ _w declined in the stressed plants reaching values of -1.0 MPa which was sufficient to cause disturbance in nodulation and biomass production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inoculation with an enhanced N2‐fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max Merr.) response to elevated [CO2]

This study tested the hypothesis that inoculation of soybean (Glycine max Merr.) with a Bradyrhizobium japonicum strain (USDA110) with greater N₂ fixation rates would enhance soybean response to elevated [CO₂]. In field experiments at the Soybean Free Air CO₂ Enrichment facility, inoculation of soybean with USDA110 increased nodule occupancy from 5% in native soil to 54% in elevated [CO₂] and 34% at ambient [CO₂]. Despite this success, inoculation with USDA110 did not result in greater photosynthesis, growth or seed yield at ambient or elevated [CO₂] in the field, presumably due to competition from native rhizobia. In a growth chamber experiment designed to study the effects of inoculation in the absence of competition, inoculation with USDA110 in sterilized soil resulted in nodule occupation of >90%, significantly greater ¹⁵N₂ fixation, photosynthetic capacity, leaf N and total plant biomass compared with plants grown with native soil bacteria. However, there was no interaction of rhizobium fertilization with elevated [CO₂]; inoculation with USDA110 was equally beneficial at ambient and elevated [CO₂]. These results suggest that selected rhizobia could potentially stimulate soybean yield in soils with little or no history of prior soybean production, but that better quality rhizobia do not enhance soybean responses to elevated [CO₂].     

Metabolism of [13C]-labelled photosynthate in plant cytosol and bacteroids of root nodules of Glycine max

Well-nodulated soybean ( Glycine max L. Merr. cv. Akisengoku) plants were allowed to assimilate ¹³CO₂. Plant cytosol and bacteroid fractions were isolated from nodules, and the kinetics of [¹³C]-labelling of soluble carbohydrates, organic acids and amino acids were investigated.
The concentrations of all metabolites, with the exception of trehalose and 3-hydroxy-butyrate, were 10- to 1000-fold higher in plant cell cytosol than in bacteroids. The major portion of trehalose was found in bacteroids and 3-hydroxybutyrate only in bacteroids. Sucrose was most highly labelled with ¹³C in nodules, and the levels and time-course of labelling of sucrose were in good agreement with those of respired CO₂ from the nodules. The levels and time-courses of labelling of sucrose were closely similar in cytosol and bacteroids. Glucose was less labelled than sucrose and the level of labelling was consistently higher in cytosol than in bacteroids. The levels of [¹³C]-labelling of organic acids and amino acids in nodules were lower than those of sucrose and of respired CO₂. Tricarboxylic acid cycle intermediates, particularly succinate, were considerably less labelled in bacteroids than in the cytosol. All amino acids detected were also much more rapidly labelled in the cytosol. The results are discussed in relation to the utilization and possible compartmentation of carbon substrates in nodule tissues.     

Differentiating Indigenous Soybean Bradyrhizobium and Rhizobium spp. of Indian Soils

Soybean is extensively cultivated worldwide and is the largest source of biologically fixed nitrogen among legumes. It is nodulated by both slow and fast growing rhizobia. Indigenous soybean rhizobia in Vertisols of central India were assessed for utilization of 35 carbon sources and intrinsic resistance to 19 antibiotics. There was greater utilization of trehalose and raffinose by fast growers (87 and 73 % by fast vs. 35 and 30 % by slow growers); but slow growers had higher ability to utilize glucosamine (75 % by slow vs. 33 % by fast growers). A larger proportion of slow growers were resistant to vancomycin, polymyxin-B and rifampicin (70, 65 and 55 %) compared to fast growers (13, 7 and 7 % each). Among the two 16S rRNA sequence types in the slow growers, those belonging to Bradyrhizobium spp. utilized glucosamine while those belonging to Rhizobium radiobacter did not. All the fast growers had 16S rRNA homology to R. radiobacter and majority could not utilize glucosamine. It is suggested that during initial isolations and screening of rhizobia in strain selection programmes, using carbon sources like glucosamine and antibiotics like vancomycin, polymyxin-B and rifampicin in the media may provide a simple way of distinguishing Bradyrhizobium strains from R. radiobacter among the slow growers.     

牛角花齿蓟马为害对苜蓿无性系根茎叶及同化产物分配的影响

为探索同化产物分配利用与苜蓿耐蓟马的关系,本试验以扦插的抗蓟马苜蓿无性系R-1和感蓟马苜蓿无性系 I-1为材料,研究不同虫口牛角花齿蓟马为害对苜蓿的抗性、根、茎和叶生长特性及可溶性糖含量的影响.结果表明: 随着虫口压力的增大,R-1和I-1苜蓿的受害指数升高;在相同虫口压力下,R-1苜蓿的受害指数显著低于I-1.受蓟马为害后,R-1和I-1苜蓿株高降低、叶面积减少、茎秆变细、节间长变短、节间数增加,根颈和主根直径加粗、侧根增多.在低虫口密度下,随虫口压力增大,R-1和I-1苜蓿地上部生物量增加,根冠比下降,分配到茎的生物量比例升高;在高虫口密度下,地上部生物量随虫口压力增大而减少,根冠比增加,分配到根系的生物量比例升高;R-1根冠比和茎生物量比例随虫口压力变化曲线的拐点均为每枝条5头,I-1根冠比和茎生物量比例随虫口压力变化曲线的拐点均为每枝条3头.在低虫口压力下,R-1苜蓿茎和叶中的可溶性糖含量随虫口压力增加而升高;在高虫口压力下,茎和叶中的可溶性糖含量随虫口压力增加而下降;根中可溶性糖含量随虫口压力增加持续下降.I-1根、茎和叶中的可溶性糖含量均随虫口压力增加持续下降.牛角花齿蓟马为害后,R-1根、茎和叶的农艺性状及抗性比I-1好,对同化产物的分配利用率高.     

Comparison of acetylene-reduction and nitrogen-15 techniques for the determination of nitrogen fixation by field bean (Vicia faba) nodules

Field bean (Vicia faba L.) cv. Maris Bead seeds were inoculated with Rhizobium Catalogue No. 1001, supplied by Rothamsted Experimental Station, and grown in sand culture supplied with a complete nutrient solution which included nitrate at either 1.5 or 6.0 mM. Nodules were detached from the roots at intervals during plant development and their rates of nitrogen fixation estimated by both acetylene reduction and ¹⁵N gas technique. There was a constant relationship, independent of nitrate supply, between the results obtained by these two methods at all samplings. The amounts of acetylene reduced divided by a factor of 5.75 gave the amount of true nitrogen fixation; this factor is about twice the theoretical value. It is suggested that this discrepancy arose because, with acetylene, all the electrons available to the nitrogenase were used to form ethylene, whereas during normal fixation only about half the electron supply was used to fix nitrogen, the remainder having been consumed in the production of hydrogen gas.     

Symbiotic effectiveness of spontaneous antibiotic-resistant mutants of Rhizobium sp. Cicer nodulating chickpea (Cicer arietinum)

Spontaneous streptomycin-resistant mutants were isolated from two fast growing gum-producing strains Ca85 and Ca401 and from two moderately growing strains Ca181 and Ca534 of Rhizobium sp. Cicer. The nodulation ability and symbiotic effectiveness of the mutants relative to parent strains were evaluated on chickpea (Cicer arietinum) grown in sterilized chillum jars. Some mutants of strains Ca85 and Ca401 showed Nod phenotype whereas some mutants of strains Ca181 and Ca534 showed Nod(+) fix(-) phenotype. Other mutants also showed decreased nodule number and reduction in nitrogenase activity as well as in shoot dry weight as compared to inoculation with parental strains. The results showed that acquisition of streptomycin resistance in Rhizobium sp. Cicer strains is associated with decreased symbiotic effectiveness in chickpea, suggesting that antibiotic-resistant mutants first should be analyzed for symbiotic effectiveness before using these mutants for ecological studies or nodulation competitiveness.     

Effects of atmospheric CO2 enrichment and root restriction on leaf gas exchange and growth of banana (Musa)

The effects of atmospheric CO₂ enrichment and root restriction on photosynthetic characteristics and growth of banana (Musa sp. AAA cv. Gros Michel) plants were investigated. Plants were grown aeroponically in root chambers in controlled environment glasshouse rooms at CO₂ concentrations of 350 or 1 000 μmol CO₂ mol^-1. At each CO₂ concentration, plants were grown in large (2001) root chambers that did not restrict root growth or in small (20 1) root chambers that restricted root growth. Plants grown at 350 μmol CO₂ mol^-1 generally had a higher carboxylation efficiency than plants grown at 1 000 μmol CO₂ mol^-1 although actual net CO₂ assimilation (A) was higher at the higher ambient CO₂ concentration due to increased intercellular CO₂ concentrations (C_i resulting from CO₂ enrichment. Thus, plants grown at 1 000 μmol CO₂ mol^-1 accumulated more leaf area and dry weight than plants grown at 350 μmol CO₂ mol^-1. Plants grown in the large root chambers were more photosynthetically efficient than plants grown in the small root chambers. At 350 μmol CO₂ mol^-1, leaf area and dry weights of plant organs were generally greater for plants in the large root chambers compared to those in the small root chambers. Atmospheric CO₂ enrichment may have compensated for the effects of root restriction on plant growth since at 1 000 μmol CO₂ mol^-1 there was generally no effect of root chamber size on plant dry weight.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.