Frank R. Minchin

The Response of Carbon Metabolism and Antioxidant Defenses of Alfalfa Nodules to Drought Stress and to the Subsequent Recovery of Plants

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity. Exposure of plants to a moderate drought (leaf water potential of −1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N2ase activity (−43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (−2.1 MPa) decreased N2ase (−82%) and SS (−30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N2ase, a complete recovery of Suc, and a decrease of malate (−48%) relative to control. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and N2ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N2ase activity in alfalfa nodules.

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.

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.

Latent and Active Polyphenol Oxidase (PPO) in Red Clover (Trifolium pratense) and Use of a Low PPO Mutant To Study the Role of PPO in Proteolysis Reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzymes own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus

Abstract Hairy root cultures of three species of legume, Lotus corniculatus, Trifolium repens and T. pratense were established using a wild-type strain (C58C1 with pRi 15834) of Agrobacterium rhizogenes . Southern hybridization analysis confirmed that the lines were genetically transformed. Copy numbers of TL-DNA in different lines varied from one to eight. Examination of the transformed root cultures revealed changes in anatomy, morphology and cytology. Plants that had regenerated from hairy roots of L. corniculatus showed changes in morphology, physiology and cytology but no change in several parameters of nitrogen fixation activity.

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 NO3− metabolism in symbioses ofPhaseolus vulgaris L. andVigna radiata (L.) Wilczek during a 3-d exposure to 10 mM NO3−. 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 NO3− 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 NO3−. 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 NO3−, 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 NO3− exposure. Bacteroids ofVigna and the cytosol of both species contained NR prior to NO3− exposure, and activities increased 1.5- to 2-fold during the treatment period. Despite this, NO2− 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 NO3−. In the first stage NO3− is restricted to the nodule cortex and causes a reversible increase in R. In the second stage NO3− may enter the infected region and toxic amounts of NO2− can be generated in nodules having high bacteroid andor cytosol NR activities. This NO2− can irreversibly damage the nodules and accelerate their senescence.

Source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) is a major factor involved in pea response to moderate water stress.

Summary The effect of the source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) on the response of pea plants to a gradual and moderate water stress was studied. Growth declined under water deficit, but nodulated plants were less sensitive to drought than nitrate-fed plants. Stomatal conductance and internal CO 2 concentration also decreased, but both were higher in nitrogen-fixing plants throughout the drought period, leading to better maintenance of carbon assimilation rates under water deficit. Glycolate oxidase, a key enzyme in the photorespiratory cycle, declined by 50% in nitrogen-fixing plants under water deficit, although it was not affected in nitrate-fed plants. Nitrogen assimilation declined during the drought period and was independent of nitrogen source. Free amino acid content declined in leaves of plants grown under both nutrition regimes, reflecting the decrease in nitrogen assimilation. Water stress led to carbohydrate accumulation in pea plants grown with either nitrogen source, but it was higher in nitrogen-fixing plants. Roots showed the greatest carbohydrate and amino acid accumulation in both nutritions regimes, with significantly greater increases in free amino acids in nitrate-fed plants. It is concluded that the nitrogen source is a major factor affecting pea responses to water stress, although the difference in sensitivity seems to be related not to the nitrogen assimilation process but to complex interactions with photorespiratory flux and stomatal conductance.

Respiratory/Carbon Costs of Symbiotic Nitrogen Fixation in Legumes

This chapter presents an overview of the respiratory/carbon costs of symbiotic nitrogen fixation. The various theoretical costings for nitrogen fixation suggest that respiration directly associated with nitrogenase activity will require between 1.77 and 3.01 g C g−1-N (4.35–7.00 mol CO2 mol−1 N2), while respiration of the entire nitrogen-fixing nodules will require between 2.78 and 4.81 g C g−1-N (6.51–11.19 mol CO2 mol−1 N2). Early attempts to measure these costs were beset by methodological problems, but some reliable approaches were developed. Measured values based on root respiration during the period of active nitrogen fixation are in the range of 5–10 g C g−1-N (11.6–23.4 mol CO2 mol−1 N2), with an average value of 6.5 g C g−1-N (15.1 mol CO2 mol−1 N2). On a nodule basis, values in the range of 3–5 g C g−1-N (7–12 mol CO2 mol−1 N2) appear to represent the ‘normal’ for legume nodules, while values below about 2.5 g C g−1-N are likely to be erroneous. The implications of these costings are considered in terms of the need for legumes to carefully regulate nitrogen fixation and the requirement for such regulation systems to be operational in any novel nitrogen-fixing plants.