Short-term metabolic responses of soybean root nodules to nitrate.

Soybean (Glycine max L. Merr.) plants exposed to 10 mM KNO(3) for a 4 d period were used to test the correlation between nitrogenase activity, gene expression and sucrose metabolism. Nitrate caused the down-regulation of sucrose synthase (SS) transcripts within 1 d, although a decline in nodule SS activity and an increase in nodule sucrose content only occurred after 3-4 d. In a second experiment, plants were exposed to (15)N-labelled nitrate for 48 h to determine the time period during which nitrate was taken up, and to relate this to the decline in apparent nitrogenase activity (H(2) production in air) and the reduction in SS gene transcript levels. The peak of nitrate uptake appeared to be between 8 h and 14 h whilst apparent nitrogenase activity began to decline at about 17.5 h. The SS mRNA signal declined markedly between 14 h and 24 h. The correlative association of these factors is clear. However, SS activity per se does not appear to be related to the initial decline in apparent nitrogenase activity as a result of nitrate uptake. These findings, therefore, do not support the hypothesis that the regulation of nodule function is mediated by the regulation of SS activity.

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Stress-induced legume root nodule senescence. Physiological, biochemical, and structural alterations.

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.

Stress-Induced Declines in Soybean N2 Fixation Are Related to Nodule Sucrose Synthase Activity.

Soybean (Glycine max L.) plants were subjected to a number of treatments (drought, 10 mM nitrate, 150 mM NaCl, shoot meristem removal, and removal of approximately 50% of the nodules) to test the hypothesis that metabolic responses contribute to the regulation of N2 fixation. Nitrogenase activity was correlated with the activity of nodule sucrose synthase (SS), but not with that of glutamine oxoglutarate amino transferase. Leghemoglobin levels and other enzyme activities were not significantly or consistently affected by the treatments. SS mRNA was greatly reduced in nodules of drought-, salt-, and nitrate-treated plants; however, this was not correlated with changes in soluble carbohydrate, starch, amino acids, or ureides. Leghemoglobin mRNA was only slightly affected by the treatments. The time course of drought stress showed a decline in the SS transcript level by 1 d, but levels of leghemoglobin, glutamine synthetase, and ascorbate peroxidase mRNA were not markedly affected by 4 d. SS activity at 4 d was reduced by 46%. We propose that N2 fixation in soybean nodules is mediated by both the oxygen-diffusion barrier and the potential to metabolize sucrose via SS. The response to environmental perturbation may involve down-regulation of the nodule SS gene.

N2 Fixation, Carbon Metabolism, and Oxidative Damage in Nodules of Dark-Stressed Common Bean Plants.

Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.

Involvement of Activated Oxygen in Nitrate-Induced Senescence of Pea Root Nodules.

The effect of short-term nitrate application (10 mM, 0-4 d) on nitrogenase (N2ase) activity, antioxidant defenses, and related parameters was investigated in pea (Pisum sativum L. cv Frilene) nodules. The response of nodules to nitrate comprised two stages. In the first stage (0-2 d), there were major decreases in N2ase activity and N2ase-linked respiration and concomitant increases in carbon cost of N2ase and oxygen diffusion resistance of nodules. There was no apparent oxidative damage, and the decline in N2ase activity was, to a certain extent, reversible. The second stage (>2 d) was typical of a senescent, essentially irreversible process. It was characterized by moderate increases in oxidized proteins and catalytic Fe and by major decreases in antioxidant enzymes and metabolites. The restriction in oxygen supply to bacteroids may explain the initial decline in N2ase activity. The decrease in antioxidant protection is not involved in this process and is not specifically caused by nitrate, since it also occurs with drought stress. However, comparison of nitrate- and drought-induced senescence shows an important difference: there is no lipid degradation or lipid peroxide accumulation with nitrate, indicating that lipid peroxidation is not necessarily involved in nodule senescence.

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules.

Various forms of stress result in decreased O(2) permeability or decreased capacity to consume O(2) in legume root nodules. These changes alter the nodule interior O(2) concentration (O(i)). To determine the relationship between O(i) and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O(i) by varying external pO(2) while monitoring internal H(2) concentration (H(i)) with microelectrodes. O(i) was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O(i), H(i) approached a new steady state, with a time constant averaging 23 s. The rate of H(2) production by nitrogenase was calculated as the product of H(i), nodule surface area, and nodule H(2) permeability. H(2) permeability was estimated from O(2) permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O(i) was nearly optimal for nitrogenase activity (H(2) production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO(2) (40-100 kPa) reduced H(i) to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO(2). The mechanism of nitrogenase inhibition may differ between brief and prolonged O(2) exposures.

Short-term inhibition of legume N2 fixation by nitrate : I. Nitrate effects on nitrate-reductase activities of bacteroids and nodule cytosol.

The hypothesis of NO2 (-) toxicity as the causative factor of NO3 (-) inhibition of nitrogenase (N2ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO3 (-) induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO2 (-) did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO2 (-) assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO2 (-) in vivo. This is probably because NO3 (-) access is restricted to the nodule cortex. Accumulation of NO2 (-) at levels that are damaging for N2ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO2 (-) is not responsible for the initial NO3 (-)-induced decline of N2ase activity, and that toxic amounts of NO2 (-) only build up in nodules following longer exposures to NO3 (-), when this anion is actively reduced by bacteroid and cytosol enzymes.

Short-term inhibition of legume N2 fixation by nitrate : II. Nitrate effects on nodule oxygen diffusion.

A comparison was made of changes in nitrogenase (N2ase; EC 1.18.6.1) activity, oxygen diffusion resistance and NO 3 (-) metabolism in symbioses ofPhaseolus vulgaris L. andVigna radiata (L.) Wilczek during a 3-d exposure to 10 mM NO 3 (-) . Bacteroids fromPhaseolus nodules lacked nitrate reductase (NR;EC 1.7.99.4) but those fromVigna nodules had elevated amounts of the enzyme. The nodule cytosol of both species contained assimilatory NR (EC 1.6.6.1). Both symbioses showed a C2H2-induced decline in N2ase activity, the extent of which remained constant with NO 3 (-) exposure forPhaseolus but became greater forVigna. Nitrate application for 3 d reduced maximum (pre-decline) rates of C2H2-reduction activity by 83% and 36% inPhaseolus andVigna, respectively. Nitrogenase-linked respiration (NLR) closely paralleled N2ase activity as the carbon costs of N2ase were not significantly altered by NO 3 (-) . The relationship between NLR and increases in external O2 concentration from 21 to 60% was used to characterize the oxygen diffusion resistance (R) of nodules from both species. In absolute terms the minimum R ofPhaseolus nodules increased with NO 3 (-) , whereas the ability to adjust this R in response to O2 was lost after 2d. ForVigna nodules the increase in minimum R was much smaller and the adjustment ability was retained for the 3-d period of NO 3 (-) exposure. Bacteroids ofVigna and the cytosol of both species contained NR prior to NO 3 (-) exposure, and activities increased 1.5- to 2-fold during the treatment period. Despite this, NO 2 (-) was not detected in nodules ofPhaseolus, and showed only a very small accumulation in the cytosol ofVigna nodules. It is proposed that nodules have a two-stage response to applied NO 3 (-) . In the first stage NO 3 (-) is restricted to the nodule cortex and causes a reversible increase in R. In the second stage NO 3 (-) may enter the infected region and toxic amounts of NO 2 (-) can be generated in nodules having high bacteroid andor cytosol NR activities. This NO 2 (-) can irreversibly damage the nodules and accelerate their senescence.

The influence of calcium on potassium fluxes across the root of Ricinus communis.

The effect of calcium on the flux of potassium to the exudate of detached root systems of Ricinus communis has been investigated. Previous analyses have indicated the presence of a water dependent and a water independent flux of potassium which vary with the concentration of potassium in the bathing medium, in the presence of 0.1 mM CaCl2. In the present study it has been observed that at a higher concentration of calcium in the bathing medium (2.5 mM CaCl2) the water dependent flux of potassium is greatly reduced while the water independent flux is not affected. It is proposed that the differential effect of calcium on these two fluxes is a reflection of the degree of dependence of these fluxes on the permeability of the plasmamembranes within the root.

A mathematical analysis of water and solute transport across the root of Ricinus communis.

A mathematical analysis of the relationship between the flux of water, f H2O, and the flux of potassium f K, to the xylem of exuding root systems of Ricinus communis, is presented. Previous analyses (Baker and Weatherley, 1969; Minchin and Baker, 1969) have indicated the presence of a water dependent and a water independent f K both of which vary with the external concentration of potassium, Cm, supplied as potassium nitrate.The present analysis reveals that whereas at Cm values<1 mM both components of f K contribute ions to the osmotically active solutions within the osmotic barrier, at Cm values>1 mM only the water dependent f K is responsible for the osmotic work. This suggests that the ions are released within different regions of the stele. It is proposed that at cm values<1 mM both components are released from the inner stelar tissues whilst at higher Cm values the water dependent f K is released from the outer stelar tissues. This requires that the solute permeability of the plasmalemma of the outer stelar tissues increases markedly at or about Cm values of 1 mM.It is postulated that the required separation of the two f K components within the stelar symplasm at Cm values>1 mM is due to the water independent f K being in a bound state, possibly being transported along a chain of binding sites whilst the water dependent f K is in a free state within the aqueous phase of the cytoplasm.

Water dependent and water independent fluxes of potassium in exuding root systems of Ricinus communis.

The flux of water, [Formula: see text], to the xylem of exuding root systems of Ricinus communis was controlled using a range of mannitol concentrations permitting the influence of this water flux on the potassium flux, f K, to be studied. The relationship between [Formula: see text] and f K thus obtained was investigated, for a number of external concentrations of potassium, Cm, supplied as potassium nitrate. An analysis of these data indicated the presence of a water dependent and a water independent f K both of which varied with Cm. The water dependent f K shows a parabolic relationship with Cm for Cm values <1 mM followed by a sharp inflection and decline at higher Cm values whereas the water independent f K shows an hyperbolic relationship over the same range of Cm values.Uptake of potassium by exuding root systems was measured and shown to be dependent on the solute potential of the medium. The uptake was also shown to exhibit a dual absorption isotherm the kinetics of which indicate a low Km system (system 1) and a high Km system (system 2). The Km value obtained for system 1 is very similar to that obtained for the water independent f K. It is postulated that the water independent f K is contributed by that portion of f K arriving in the stele via the cortical symplast and is directly dependent on Cm. The water dependent f K is contributed by those ions moved across the root in response to centripetal water movement through the cortical cell walls.