Breding common bean for improved biological nitrogen fixation

Common bean (Phaseolus vulgaris L.), which is an important food crop in the Americas, Africa and Asia, usually is thought to fix only small amounts of atmospheric nitrogen. However, field data indicate considerable genetic variability for total N₂ fixation and traits associated with fixation. Studies have shown that selection to increase N₂ fixation will be successful if: (1) discriminating traits (selection criteria) are measured precisely, (2) variability in germplasm is heritable, (3) selected parents are also agronomically suitable, (4) units of selection facilitate quantification of selection criteria, and (5) a breeding procedure that allows maximum genetic gain for N₂ fixation and recombination with essential agronomic traits is chosen. Breeding lines capable of fixing enough atmospheric N₂ to support seed yields of 1000–2000 kg ha^–1 have been identified and new cultivars with high N₂ fixation potential are being released.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Electron Microscope Study of DNA-Containing Plasms : II. Vegetative and Mature Phage DNA as Compared with Normal Bacterial Nucleoids in Different Physiological States

The nucleoids of Escherichia coli, independently of the physiological state of the bacteria, are shown to be preserved as a fine-stranded fibrillar nucleoplasm by an OsO₄ fixation under defined conditions: acetate-veronal buffer pH 6, presence of Ca⁺⁺ and amino acids, stabilization with uranyl-acetate before dehydration. The same fixation procedure applied to the DNA of vegetative phage reveals a pool of homogeneous fibrillar structure very similar to the nucleoplasm. The "versene test," which produces a coarse coagulation of these plasms, emphasizes the similar behaviour of the pool and the nucleoids. The heads of mature phage are preserved in their true polyhedral shape by the standard fixation procedure, although they may be badly distorted when fixed under different conditions. Lanthanum nitrate and uranyl-acetate are shown to increase markedly the contrast of both phage and cytoplasm. The consequences of the fibrillar structure of the genetic material are discussed in relation to the probable division process.     

Permanganate Fixation of Plant Cells

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO₄ at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO₄ at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO₄, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO₄ at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO₄. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO₄ or OsO₄.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

Histochemical staining of cadmium thiolate clusters in livers of rats treated chronically with cadmium

Summary In livers of rats exposed to varying doses of CdCl₂ 80–90% of the cadmium content present in the fresh tissue is retained if these livers are fixed with a neutral or acid formalin fixative.Cadmium assays during different stages of the staining procedure for protein bound disulphides show the ability of this staining to demonstrate cadmium thiolate clusters next to disulphides. The methods described may also be useful in gaining more insight in the mechanism involved in fixation and staining procedure of some other metals.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.

     

Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

A simple procedure to overcome polyethelene glycol toxicity on whole plants

A procedure is described that can be used to minimize toxic effects of polyethylene glycol (PEG) to plants. The procedure is based on recycling nutrient solutions containing PEG-6000 through two plant cultures. Tomato plants grown in −0.3 megapascals PEG solutions used after two growth cycles exhibited minimal toxic effects. Long-term responses like dry matter production and chlorophyll content as well as short-term responses like CO₂ fixation rates and leaf conductance were severely inhibited by fresh PEG-6000 and only slightly reduced by recycled PEG-6000. Complete osmotic adjustment was obtained with tomatoes grown in recycled but not in fresh PEG solutions.     

Inadequacy of Rubisco initial and total activities to account for observed rates of photosynthetic carbon dioxide assimilation by Scenedesmus ecornis

Both initial and total activity of ribulose-1,5, bisphosphate carboxylase/oxygenase (Rubisco) measured for the green alga Scenedesmus ecornis are affected by the experimental procedure and they are not sufficiently high to account for the rates of ¹⁴C fixation by photosynthesis. The very low β-carboxylase activities detected (less than 3% of the Rubisco total activity) cannot explain the difference in CO₂ fixation. Attempts to obtain possible optimal conditions (pH, duration of activation with Mg²⁺ and HCO^- ₃, absence of proteases, linearity of ¹⁴C fixation with time) did not lead to increased activity yields. The substrate ribulose-1,5-bisphosphate was found to decrease the initial activity at concentrations higher than 25 μM for algae harvested by centrifugation and having thus experienced several minutes of darkness. Deactivation seems to be primarily responsible for this loss of activity. Furthermore, initial and total activities decrease when the delay before freezing increases, suggesting accumulation of an inhibitor from the light-dark transition metabolism during the first minutes of harvesting.     

Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii

A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO₂ fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

Photosynthetic CO2 fixation in spinach chloroplasts inhibited by pine chloroplasts or extracts of pine chloroplasts

Chloroplasts isolated from pine needles were found to be inactive with respect to CO₂ fixation. Since it was suspected that pine needles may contain substances inhibitory to photosynthesis, studies were carried out using photosynthetically active isolated spinach chloroplasts and chloroplasts isolated from pine needles. When isolated pine chloroplasts were suspended in buffer and were added to isolated spinach chloroplasts they inhibited photosynthetic CO₂ fixation. When the pine chloroplasts were separated from the medium by centrifugation, the separated pine chloroplasts severely inhibited CO₂ fixation by isolated spinach chloroplasts, but the supernatant solution from the pine chloroplasts was not inhibitory. As little as 5% pine chloroplasts (based on chlorophyll content) produced 50% inhibition of CO₂ fixation by the spinach chloroplasts. Studies of fixation of ¹⁴C-labelled CO₂ by spinach chloroplasts were carried out in which after 5 min photosynthesis the pine chloroplasts were added. It was found that the subsequent inhibition of spinach CO₂ fixation was neither due to any effect on the rate of export of photosynthetic metabolites from the chloroplasts to the medium, nor to a direct effect on the RUBP carboxylase reaction. The principal effect was found to be an inhibition of the conversion of fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate to the respective monophosphates and inorganic phosphate. From this finding it was concluded that a principal effect of the inhibition by pine chloroplasts is probably an inhibition either directly or indirectly of the bisphosphatase enzymes in the spinach chloroplasts. Based on its distribution between organic and aqueous acidic or neutral solutions, the inhibitory factor of the pine chloroplasts must be lipophilic. Most of the factor could be transferred to an aqueous phase in a strongly alkaline solution. Following subsequent acidification of the aqueous phase the activity could be completely transferred back into the organic phase. This procedure allowed for separation of the inhibitory factor from most of the pigments and other lipophilic substances present in the pine chloroplasts and yielded a preparation which could be subsequently fractionated by thin layer chromatography. UV absorption was found in two fast moving spots and at the origin. The fastest running spot from the thin layer chromatography plate was found to be the one containing most of the inhibitory activity.     

Notes or Technic

A progressive silver staining method is described, which permits microscopic examination of the sections during the staining process. After formaldehyde fixation, dehydration and embedding in paraffin or celloidin, fine fibers and synaptic endings may be demonstrated. After formaldehyde fixation and mordanting in 3% K₂Cr₂O₇, myelinated fibers and mitochondria are specifically stained.

The unique feature of this method is, that the silver solution (0.5% protargol) is mixed with the reducing solution: 1.6% Rochelle salts, containing traces of Ag NO₃, MgSO₄, and K₂S (U.S.P.). The sections are placed directly into this mixture, which is then warmed to 45-55° C. Sections are removed when progressive staining is completed, washed in water, dehydrated and mounted.

In the fiber stain, nerve fibers and synaptic endings are dark brown or black, and nuclear chromatin is deep brown, against a pale yellow background. When the myelin sheath procedure is followed, the fiber bundles are deep brown, and the intensity of the staining remains the same for specific tracts, aiding in their identification.     

异养微生物固定CO2的合成生物学研究进展

人类活动造成大气二氧化碳（CO₂）浓度不断升高,使当今世界面临着气候变化的重大危机。微生物CO₂固定为实现地球“碳中和”提供了一条有前景的绿色发展路线。与自养微生物相比,异养微生物具有更快的生长速度和更先进的遗传工具,但是其固定CO₂的能力还很有限。近年来,基于合成生物学技术强化异养微生物CO₂固定受到诸多关注,主要包括优化能量供给、改造羧化途径以及基于异养微生物间接固定CO₂。本综述将围绕上述3个方面重点讨论异养微生物CO₂固定的研究进展,为将来更好地利用微生物CO₂固定技术实现“碳达峰、碳中和”提供参考。     

Diurnal variation in n(2) fixation and photosynthesis by aquatic blue-green algae

Rates of ¹⁴CO₂ fixation, O₂ evolution, and N₂ fixation (acetylene reduction) by natural populations of blue-green algae recovered from Lake Mendota were measured at frequent intervals between sunrise and sunset. Photosynthesis and N₂ fixation were depressed during midday when light intensity was greatest. As the light intensity rose, most of the algal population migrated to deeper, light-limited waters where radiation damage would be diminished. As the relative rate of N₂ fixation compared to CO₂ fixation increases with depth, it is suggested that the algae maintain balanced growth by migrating vertically via buoyancy regulation. High concentrations of dissolved O₂ in lake water may inhibit N₂ fixation by enhancing photorespiration. Several factors such as photosynthetic rate, light intensity, dissolved O₂, species composition, and vertical and horizontal migration all affect observed rates of in situ N₂ fixation.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Dark microbial CO2 fixation in temperate forest soils increases with CO2 concentration

Dark, that is, nonphototrophic, microbial CO₂ fixation occurs in a large range of soils. However, it is still not known whether dark microbial CO₂ fixation substantially contributes to the C balance of soils and what factors control this process. Therefore, the objective of this study was to quantitate dark microbial CO₂ fixation in temperate forest soils, to determine the relationship between the soil CO₂ concentration and dark microbial CO₂ fixation, and to estimate the relative contribution of different microbial groups to dark CO₂ fixation. For this purpose, we conducted a ¹³C‐CO₂ labeling experiment. We found that the rates of dark microbial CO₂ fixation were positively correlated with the CO₂ concentration in all soils. Dark microbial CO₂ fixation amounted to up to 320 µg C kg^?1 soil day^?1 in the Ah horizon. The fixation rates were 2.8–8.9 times higher in the Ah horizon than in the Bw1 horizon. Although the rates of dark microbial fixation were small compared to the respiration rate (1.2%–3.9% of the respiration rate), our findings suggest that organic matter formed by microorganisms from CO₂ contributes to the soil organic matter pool, especially given that microbial detritus is more stable in soil than plant detritus. Phospholipid fatty acid analyses indicated that CO₂ was mostly fixed by gram‐positive bacteria, and not by fungi. In conclusion, our study shows that the dark microbial CO₂ fixation rate in temperate forest soils increases in periods of high CO₂ concentrations, that dark microbial CO₂ fixation is mostly accomplished by gram‐positive bacteria, and that dark microbial CO₂ fixation contributes to the formation of soil organic matter.