Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme’s apparent V_max.     

The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen-fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O₂ regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H₂ evolution in N₂:O₂) and nodule respiration (CO₂ evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H₂ evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O₂ concentration (O_i), measured by a spectro-photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, O_i levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O₂ at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady-state rates of gas exchange, linear increases in pO₂ from 20 to 100% at 4% min^?1 resulted in recoveries of H² and CO² evolution, indicating that O_i limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O₂ available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O₂, O_i values measured at supra-ambient pO₂ were greater than those at 20% O₂ and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO₂ (O_c) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady-state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above-ambient pO₂.     

Tissue-specific expression of the alternative oxidase in soybean and siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.     

Vascular transport and soybean nodule function: II. A role for phloem supply in product export*

Abstract The ureide content of soybean (Glycine max (L.) Merr.) nodules was unaffected by variations in the transpirational rate, while whole plant manipulations designed to decrease phloem supply to nodules resulted in lower rates of nitrogenase activity and an increase in the ureide content of the nodules. The rate of ureide export from the nodule was estimated from the exponential rate of decrease in the pool size of ureides in nodules, following exposure to an N₂-free atmosphere (Ar:O₂). Export was greatly reduced under treatments which reduced phloem supply to the nodule. A water budget for nodules suggested that the delivery of water to the nodule via mass flow in the phloem was comparable to that required for export of ureides from the nodule in the xylem from the nodule. Therefore, we suggest that xylem export from nodules is related to the phloem supply to the nodule rather than to the transpirational flux in the parent root. This suggestion is related to the reported decreases in nodule permeability to gases under conditions of phloem deprivation.     

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active “oxidized” form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the “reduced” active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.     

Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Effects of incubation temperature and oxygen tension on nitrogenase activity of legume root nodules

Summary Acetylene reduction and H₂ evolution by legume root nodules from several plant species depended on incubation temperature; some nodules were active from 2 to 40°C. Acetylene reduction rates differed between plant species, with maximum activity at temperatures between 20 and 30°C forVicia faba, V. sativa, Trifolium pratense, T. subterraneum, Medicago truncatula and soybean, at 35°C forM. sativa and at 40°C for cowpea. OnlyM. sativa and cowpea reduced substantial amounts at 37.5°C. Temperatures from 2 to 10°C only slightly lessened activity ofT. subterraneum andV. sativa nodules. Nitrogenase functioned at temperatures which prevent establishment of other aspects of the symbiosis. The rate of acetylene reduction was constant for several hours at temperatures below 15°C, and activity continued for several days at 2°C for some species, but declined with time at warmer temperatures. Some nitrogenase was denatured at warmer temperatures, but the O₂ tension in the assay vial also affected activity. In closed assay vessels nodule respiration decreased the pO₂ and reduced nitrogenase activity. Activity was restored by adding O₂ or regassing assay vials with air or Ar/O₂. When the pO₂ was maintained, acetylene reduction and H₂ evolution by detached soybean nodules continued unchanged for 6 h.     

Carbon Metabolism in Relation to Cellular Organization of Soybean Root Nodules and Respiration of Mitochondria Aided by Leghemoglobin

Effects of leghemoglobin on the respiration of bacteroids andmitochondria were examined by following oxygen uptake. Oxyleghemoglobinprepared from soybean nodules promoted the uptake of oxygenby bacteroids isolated aerobically from soybean nodules. Uptakeof oxygen by both the nodule mitochondria and the hypocotylmitochondria was also stimulated by the oxyleghemoglobin. Thestimulatory effect of leghemoglobin on the respiration of thenodule mitochondria became highly significant as the amountof mitochondria injected into assay vial increased. The infected cells, the uninfected cells and the cortex tissuewere isolated by enzymatic maceration from soybean nodules,and some enzyme activities were measured in these three fractions.Activities of alcohol dehydrogenase, pyruvate decarboxylaseand phosphoenolpyruvate carboxylase in the infected cells werelower than in the uninfected cells and in the cortex tissue.Uricase activity was low in the cortex tissue and was highestin the uninfected cells. These results suggested that ethanol production is more activein the uninfected cells and the cortical cells than in the infectedcells, and that respiration of the mitochondria in the infectedcells of soybean nodules is more active, aided by the leghemoglobin. (Received August 23, 1986; Accepted November 14, 1986)     

Functional properties of the Ustilago maydis alternative oxidase under oxidative stress conditions

The mitochondrial respiratory chain of Ustilago maydis contains two terminal oxidases, the cytochrome c oxidase (COX) and the alternative oxidase (AOX). To understand the biochemical events that control AOX activity, we studied the regulation and function of AOX under oxidative stress. The activity of this enzyme was increased by both pyruvate (K₀₅ = 2.6 mM) and purine nucleotides (AMP, K₀₅ = 600 μM) in mitochondria using succinate as respiratory substrate. When U. maydis cells were grown in the presence of antimycin A, the amount of AOX in mitochondria was markedly increased and its selectivity towards AMP and pyruvate changed, suggesting that post-translational events may play a role in the regulation of AOX activity under stress conditions. Addition of antimycin A to isolated mitochondria induced the inactivation of AOX, the formation of lipid peroxides and the loss of glutathione from mitochondria. The two last processes are probably related with the time dependent inactivation of AOX, in agreement with the inhibition of the enzyme by tert-butyl hydroperoxide. Our results suggest that the in vivo operation of AOX in U. maydis depends on the mitochondrial antioxidant machinery, including the glutathione linked systems.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

Physiological studies on N2-fixing root nodules ofDatisca cannabina L. andAlnus nitida Endl. from Himalaya region in Pakistan

Summary The nodulation and the morphology and physiology of the nodules were studied onDatisca cannabina, a perennial herb from northern Pakistan andAlnus nitida, a nodulated tree in the same locality. Both species bear coralloid clusters of actinorhizal nodules. The main free amino acid inD. cannabina nodules was arginine while the predominant free amino acid inA. nitida nodules was citrulline. The infectivity of crushed nodules of both types of plants on their respective host was about 10⁶ infective particles per gram of nodule fresh wt. In cross-inoculation experiments crushed nodule inoculum fromA. nitida failed to induce nodulation onD. cannabina seedlings but the crushed nodule inoculum fromD. cannabina caused low nodulation on seedlings ofA. nitida (10³ infective particles. g. nodule fresh wt.).The activity of nitrogenase, hydrogenase and respiration (O₂ uptake) were measured in detached nodules, nodule homogenates and the 20 m residue and 20 m filtrate preparations from the nodules of both species. Both species showed similar patterns of activities except that only the nodule homogenate and 20 m residue preparations fromD. cannabina showed pronounced enhancement of the O₂ uptake by succinate which was further stimulated by ADP. This has in part been explained by the presence of mitochondria in close connection with the endophyte.     

An osmotic hypothesis for the regulation of oxygen permeability in soybean nodules

Nodule permeability (P) controls the amount of O₂ entering the nodule, and thereby the rates of both nodule respiration and N₂ fixation. P may be regulated by changes in the effective thickness of a water-filled diffusion barrier in the nodule cortex. Regulation of diffusion barrier thickness was hypothesized to result from changes in the water content of intercellular spaces. Modulation of intercellular water would be a response to osmotic potential gradients in the tissue. To test this hypothesis, preliminary experiments examined three classes of solutes (soluble sugars, free amino acids, and ureides) in nodules of intact plants exposed to 10 or 21 kPa O₂ for 24 h. Neither soluble sugars nor free amino acids in nodules were responsive to O₂ treatments. However, nodule ureides accumulated after exposure to 10kPa O₂ for 24 h. A symplastic increase in nodule ureides under the 10kPa O₂ treatment compared to the 21 kPa O₂ treatment may have removed water from intercellular spaces in the nodule cortex and increased P. In addition, the nodule cortex of intact plants was infiltrated with water, polyethylene glycol (PEG), KC1, or Na-succinate solutions to determine the effect of intercellular water and osmoticants on dinitrogenase activity and P. Results from infiltrating the apoplast of the nodule cortex with osmotic solutions indicated that both increases in intercellular water and decreases in the apoplastic water potential decrease dinitrogenase activity and P. Furthermore, the inability to recover dinitrogenase activity and P following the infiltration of the cortex with PEG compared to either KCl or Na-succinate treatments may indicate that recovery was dependent upon removal of the solute from the apoplast.     

Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria,microsomes and plasma by high-performance liquid chromatography

Reduced and oxidized coenzyme Q₁₀ (Q₁₀H₂ and Q₁₀) in guinea-pig liver mitochondria were rapidly extracted and determined by high-performance liquid chromatography (HPLC). The percentages of Q₁₀H₂ as compared to the total (sum of Q₁₀ and Q₁₀H₂) were increased by the addition of respiratory substrates such as succinate, malate and β-hydroxybutyrate (State 4). The levels of Q₁₀H₂ in State 4 were increased more extensively with electron-transport inhibitors such as KCN, NaN₃ and antimycin A. These results indicate that the method for determining Q₁₀H₂ and Q₁₀ by HPLC is quite useful for investigation of the physiological function of coenzyme Q in mitochondria and other organelles. The reduced and oxidized coenzyme Q levels of rat liver mitochondria, which contain both coenzyme Q₉ and coenzyme Q₁₀, were measured simultaneously. The results suggest that coenzymes Q₉ and Q₁₀ play a similar role as an electron carriers. The liver microsomes of guinea-pig contained approx. 133 nmol total coenzyme Q₁₀ per g protein. The Q₁₀H₂ levels of microsomes were increased from 46.5 to 67.5 and 64.8% with NADH and NADPH, respectively. The plasma levels of total coenzyme Q were 0.92 μg/ml for man, 0.35 μg/ml for guinea-pig and 0.27 μg/ml for rat. The reduced coenzyme Q were also present in those plasma samples. The levels of reduced coenzyme Q were 51.1, 48.9 and 65.3%, respectively.     

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O₂ evolution and O₂ uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m^?2 s^?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO₃‐dependent O₂ evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O₂ uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO₃‐dependent O₂ evolution or p‐benzoquinone (BQ)‐dependent O₂ evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.     

Cytosolic pyruvate kinase: subunit composition,activity, and amount in developing castor and soybean seeds,and biochemical characterization of the purified castor seed enzyme

Antibodies against Brassica napus cytosolic pyruvate kinase (PK_c) (EC 2.7.1.40) were employed to examine PK_c subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PK_c activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g⁻¹ FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PK_c activity and the relative amount of the immunoreactive 56-kDa PK_c polypeptide. PK_c from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg⁻¹ protein. Gel filtration and SDS-PAGE indicated that this PK_c exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PK_c subunit best matched three putative PK_cs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of V _max /K _m for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PK_c exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.