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.     

Sensitive fluorometric assay for leghemoglobin

A sensitive spectrofluorometric assay for leghemoglobin is based upon the action of hot saturated oxalic acid on heme proteins. The assay will detect 200 ng of leghemoglobin per milliliter and is specific enough to permit estimation in single nodules or extracts of whole roots. The leghemoglobin concentration measured fluorometrically shows a correlation with nitrogenase [C₂H₂] activity, even during nodule senescence, when standard colorimetric assays may overestimate leghemoglobin.     

Nodule Structure and Functioning in Chickpea (Cicer Arietinum) as Affected by Salt Stress

Cicer arietinum L. plants raised in sand culture under natural light were subjected to salinity stress induced by mixture of NaCl, CaCl₂, MgCl₂ and MgSO₄ (40, 60 or 80 meq dm^-3). Acetylene reduction activity (ARA) of nodules, leghemoglobin content and nodule structure were followed 55, 75 and 85 d after sowing. ARA declined significantly under salt treatments and the lowest ARA was observed at day 85 after sowing. Decrease in ARA was consistent with decreased nodule leghemoglobin content. The leghemoglobin content of control plants decreased by 50 % at day 85 indicating senescence of nodules. This senescence was further accelerated by salt treatment after which the leghemoglobin content fell to negligible levels. The structural changes associated with salt stress were mainly reduction in size of the nodules, decreased meristematic zone, reduced number and degradation of symbiosomes, reduced intercellalar spaces and deposition of electron dense material in the intercellular spaces in the cortex of nodules.     

Effetto della rimozione dei fiori sulla crescita delle piante e sul contenuto in leghemoglobina dei tubercoli radicali di pisello e fava

Abstract

Observations on vegetative growth and leghemoglobin contents of root nodules of pea and bean plants after flower bud removal. — These studies found their origin in the papers by MATTIROLO (1899) on the effect of the removal of flowers as they formed in bean plants; he observed that deflowering resulted in extraordinary plant growth, stem branching and flower buds formation as well as in a delayed root nodule senescence. In the light of modern knowledge of the leghemoglobin role in symbiotic nitrogen fixation, the aim of the present research was to ascertain any possible relation between flower bud removal and haeme pigment contents in the root nodules. The experiments were carried out during two growing seasons (1966 and 1970) using Vicia faba L. cv. Regina and Pisum sativum L. cv. Senatore during 1966 and cv. Vittoria in 1970. In both control and test plants the seasonal trends of average plant height, fresh and dry weight of vegetative portions, fresh and dry weight of root system, fresh weight of nodules, root nodule leghemoglobin concentration and total leghemoglobin content per plant, were determined. The data obtained are quoted in Table 2 and reported in Figures 1, 2, and 3. The removal of flower buds caused in both species: an increased plant growth, a marked stem branching, a longer blooming period, an increased flower number, an increased root nodule number and a certain delay in root nodule reabsorption. Deflowering did not significantly extend — at least in the species studied — life span (senescence was delayed only of one week). On the basis of these and of other Authors' results, we conclude that deflowering may actually delay senescence; the size of this delay, however, depends on the plant species considered and is fairly negligible both in pea and bean. The different effects of deflowering and of preventing floral induction on life span extension, are discussed, and these facts lead to consider floral induction as the onset of a chain of processes leading annual plants toward senescence in a more or less delayable, but definitive way. After having stressed the generally accepted importance of leghemoglobin concentration as an index of nodule nitrogen fixing ability, a correlation between biomass increase of test plants and number and total weight increase of root nodules, is put in evidence. No correlation between test plant biomass and the leghemoglobin concentration in root nodules, was however observed. Leghemoglobin concentration in root nodules is known to change in connection with various factors depending either on host plants and on Rhizobium strains and also in connection with several environmental conditions. Any prevented flower onset (ROPONEN and VIRTANEN, 1968) and deflowering (our data) however exerted no significant influence. The effects of flower bud removal were therefore the following: increased stem, leaf and root weights and increased root nodule number; no difference between control and test plants was however observed as regards size and leghemoglobin concentration of root nodules and hence probably no difference as regards their nitrogen fixing ability.     

Natural and dark-induced nodule senescence in chickpea: nodule functioning and H2O2 scavenging enzymes

An investigation was carried out on chickpea (Cicer arietinum L.) cv. C-235 inoculated withRhizobium sp.Cicer strain cv 4 Az^r. Nodule functioning was monitored at 15 d intervals starting from 45 days after sowing (DAS) and inoculation in order to study nodule development and senescence under natural and stress conditions (dark treatments of 18 and 66 h). Maximum rate of N₂-fixation was observed between 50 - 60 DAS. After this acetylene reducing activity (ARA) fell and it was negligible 75 DAS. This decline in ARA with ageing of plants and nodules was accompanied by a decline in leghemoglobin content and greening of the nodules. When 60 % of the nodule tissue had turned green 75 DAS, a sharp increase in nodule peroxidase activity (3.7 fold) was observed whereas the catalase activity was reduced by 50 % in comparison with the control. The glutathione-reductase and ascorbate-peroxidase activity followed a trend parallel to that in N₂-fixation, but the variation was much smaller. The changes in the total soluble carbohydrates, cytosolic proteins and nitrogen content per se were not expressive. Dark treatments induced premature senescence of the nodules as was evident from the marked decrease in ARA. However, the decline in leghemoglobin content was relatively small as compared to ARA. The changes in cytosolic proteins, total soluble carbohydrates, peroxidase activity, catalase activity, glutathione reductase activity and ascorbate peroxidase activity of nodules under dark-induced nodule senescence were almost parallel to those observed under natural senescence.     

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.     

Indirect measures of N2 fixation in common bean (Phaseolus vulgaris L.) under field conditions: The role of lateral root nodules

The role of lateral root nodules in N₂ fixation and the relationships between total shoot N and several traits which influence or control N₂ fixation in common bean (Phaseolus vulgaris L.)i.e., acetylene reduction value, specific nodule activity, leghemoglobin concentration, total leghemoglobin and nodule mass, were investigated in field studies. Significant variation among bean lines was observed for all the traits measured. Lines varied for the proportion of total N accumulated up to the R3 growth state, thus measurements of total shoot N near maturity (e.g., R7) provided a better estimate of total N₂ fixation than measurements taken at an early growth stage. Nodule mass was correlated with acetylene reduction and total leghemoglobin, and total leghemoglobin was correlated with acetylene reduction value. Total shoot N at R7 was correlated with seasonal means of nodule mass and number, acetylene reduction value and total leghemoglobin. For all traits except total leghemoglobin, values for lateral roots were more highly correlated with total shoot N than were values for either crown roots or the whole root system. Seed yield was most highly correlated with nodule mass of the lateral roots. These results will be useful in devising breeding strategies for improved N₂ fixation of the host plant.     

Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.     

Cadmium-induced senescence in nodules of soybean (Glycine max L.) plants

The relationship between cadmium-induced oxidative stress and nodule senescence in soybean was investigated at two different concentrations of cadmium ions (50 and 200 μM), in solution culture. High cadmium concentration (200 μM) resulted in oxidative stress, which was indicated by an increase in thiobarbituric acid reactive substances content and a decrease in leghemoglobin levels. Consequently, nitrogenase activity was decreased, and increases in iron and ferritin levels were obtained. Senescent parameters such as ethylene production, increased levels of ammonium and an increase in protease activity were simultaneously observed. Glutamate dehydrogenase activity was also increased. Peroxidase activity decreased at the higher cadmium concentration while the lower cadmium treatment produced changes in peroxidase isoforms, compared to control nodules. Ultrastructural investigation of the nodules showed alterations with a reduction of both bacteroids number per symbiosome and the effective area for N₂-fixation. These results strongly suggest that, at least at the higher concentration, cadmium induces nodule senescence in soybean plants.     

Contribution of Protein Conformation to Heme Stereochemistry and Reactivity. Low-Temperature Magnetic Circular Dichroism Data

Abstract

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O₂ complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N_epslion(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those inthe protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Uptake hydrogenase in Rhizobium and nodule leghemoglobin in cow pea miscellany hosts

Two effective strains of green gram rhizobia S24 (slow growing and Hup⁺) and M11 (fast growing and Hup^-) were tested for leghemoglobin production in nodules and effectivity on six species of cow pea miscellany hosts. Both strains nodulate green gram [Vigna radiata (L.) (Wilczek)], black gram [Vigna mungo (L.) (Hepper)], cow pea [Vigna unguiqulata (L.)], moth bean [Vigna aconitifolia (Jacq.) (Marechel)], Cluster bean [Cyamopsis tetragonoloba (L.) (Taub.)] and pigeon pea [Cajanus cajan (L.)]. In all these hosts, nodules formed by strain M11 contained 1.5 to 2 times more leghemoglobin than the nodules formed by strain S24. Gel electrophoresis of nodule contents of different host species showed a high concentration of a fast-moving ferricoxy leghemoglobin in the nodules of plants inoculated with strain M11 as compared to that of strain S24. Strain M11, however, was relatively less effective than strain S24 on black gram, cow pea and moth bean and was at par with the later on green gram, cluster bean and pigeon pea. Hydrogen recycling ability of the strain S24 was observed in nodules of all the host species. The effective functioning of strain S24 at low levels of leghemoglobin suggests an involvement of recycling hydrogenase in maintaining an appropriate oxidation-reduction potential in nodules.Abbreviations Lb Leghemoglobin - Cvr cultivar     

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

Circular dichroism of soybean leghemoglobin.

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Enhancement in leghemoglobin content of root nodules by exclusion of solar UV-A and UV-B radiation in soybean

The impact of exclusion of solar UV-B (280–320 nm) and UV-A+B (280–400 nm) radiation on the root nodules was studied in soybean(Glycine max var. MACS 330). Soybean plants were grown in the tropical region of Indore (Latitude-22.4°N), India under field conditions in metal cages covered with polyester exclusion filters that specifically cut off UV-B (<320 nm) and UV-A+B (<400 nm) radiation; control plants were grown under ambient solar radiation. Leghemoglobin content was analyzed in the root nodules on the 50^th day after emergence of seedlings. Exclusion of UV radiations significantly enhanced the leghemoglobin content in the nodules on fresh weight basis; 25% and 45% higher amount of leghemoglobin were present in the nodules after the exclusion of UV-B and UV-A+B radiation respectively. Analysis by native and SDS-PAGE showed high intense bands of leghemoglobin after the exclusion of UV-A+B as compared to control. Exclusion of UV radiation also enhanced the growth of roots as well as aerial parts of the plants. UV Exclusion increased nodulation by increase in the number and size of nodules. The results are discussed in the light of advantage of exclusion for enhancing protein/nitrogen content in the plants.     

Methods of Isolation and Some Characteristics of Leghemoglobins from Alfalfa (Medicago setiva L. Vernal) Nodules

The methods of isolation of leghemoglobin from alfalfa nodules and some characteristics of the proteins were described. It is indicated that the purified leghemoglobin massage contains precursor mRNA through in vitro translation of leghemoglobin mRNA and pulse chase experiment. The immunoprecipitation between leghemoglobin components from root nodules of legumes and IgGs against alfalfa leghemoglobins showed that it is possible to be of closer relationship among several kinds of legumes.