Effect of Salt Stress on Carbon Metabolism and Bacteroid Respiration in Root Nodules of Common Bean (Phaseolus vulgaris L.)

Abstract: In the present work, we examined the effect of salinity on growth, N fixation and carbon metabolism in the nodule cytosol and bacteroids of Phaseolus vulgaris, and measured the O₂ consumption by bacteroids incubated with or without the addition of exogenous respiratory substrates. The aim was to ascertain whether the compounds that accumulate under salt stress can increase bacteroid respiration and whether this capacity changes in response to salinity in root nodules of Phaseolus vulgaris. The plants were grown in a controlled environment chamber, and 50, 100 mM or no NaCl (control) was added to the nutrient solution. Two harvests were made, at the vegetative growth period and at the beginning of the reproductive period. The enzyme activities in the nodule cytosol were reduced by the salt treatments, while in the bacteroid cytosol the enzyme activities increased at high salt concentrations at the first harvest and for ADH in all treatments. The data presented here confirm that succinate and malate are the preferred substrates for bacteroid respiration in common bean, but these bacteroids may also utilize glucose, either in control or under saline conditions. The addition of proline or lactate to the incubation medium significantly raised oxygen consumption in the bacteroids isolated from plants treated with salt.     

Carbon Partitioning in Split Root Systems of Barley: Relation to Metabolism

We tested four hypotheses for the control of partitioning ofphotoassimilated C-11 between the two halves of split root systemsof young barley plants. Our data supported the hypothesis thatphloem is unloaded without the use of metabolic energy, sinceseveral metabolic inhibitors applied to one half of a splitroot system reduced respiratory oxygen uptake without alteringimport of C-11. The hypothesis that rate of import C-11 is directlyrelated to metabolic activity in the root was rejected, since(a) certain inhibitors reduced respiration but not import and(b) exogenous sucrose reduced import into the root half to whichit was supplied. Our data were consistent with the hypothesisthat import is related to the total ability (metabolism plusstorage) of the sink to use sucrose. Treatments that would haveled to greatly decreased use of sucrose (iodoacetate inhibition)decreased import before those which would have led to a smallerdecrease in sucrose use (FCCP inhibition). These data, and thereduction in import to a root half supplied exogenously withsucrose, supported the hypothesis that the size of soluble sugarpools within the roots is, in the short-term, inversely proportionalto rate of import, the soluble sugar pools thus acting as amediator between rate of sucrose use and supply from the phloem.     

Peroxidase Activity of Leghemoglobin of Bean (Vicia faba L.) Nodules in Relation to Tert-Butyl Hydroperoxide

Applied Biochemistry and Microbiology - Like many other hemoglobins, leghemoglobin (Lb), the hemoglobin of legume nodules, demonstrates peroxidase activity and can oxidize various substances with...     

毛萼田菁茎瘤豆血红蛋白含量与固氮活性的关系

在个体瘤发育的不同阶段和植株生长的不同时期,以及在不同的光照条件下,分别测定茎瘤的固氨活性和豆血红蛋白含量。结果表明,毛萼田菁茎瘤的固氨活性和豆血红蛋白含量呈正相关,豆血红蛋白在茎瘤的共生固氮中起着重要的作用。     

Tricarboxylic Acid Cycle Activity in Mitochondria from Soybean Nodules and Cotyledons

Infected cells of soybean (Glycine max) nodules require NADH,ATP, and 2-oxoglutarate for ammonia assimilation. The role ofmitochondria in nodule metabolism was investigated by determiningtheir respiratory properties and comparing them with cotyledonmitochondria. Nodule mitochondria oxidized malate at a ratetwice that of any other NAD-linked substrate although theirmalic enzyme activity was very low, accounting for only 12%of malate oxidation at pH 6.4 compared to 56% for cotyledonmitochondria. The reduction of NAD⁺ in mitochondria of noduleson adding malate (determined by fluorescence) was rapid andreached a stable level, whereas in cotyledon mitochondria theNADH level declined rapidly as oxaloacetate accumulated. Anoxaloacetate scavenging system in the mitochondrial reactionmedium increased malate oxidation by cotyledon mitochondria4-fold, but increased that of nodule mitochondria by less than50%. This demonstrates that the efflux of oxaloacetate by theoxaloacetate carrier is highly regulated by the extra-mitochondrialoxaloacetate concentration in cotyledon mitochondria comparedto nodule mitochondria. The activity of TCA cycle enzymes, exceptmalate and succinate dehydrogenases, was low in nodule mitochondria.Their oxaloacetate export during malate oxidation was rapid.The aspartate amino transferase activity associated with nodulemitochondria was sufficient to account for significant formationof 2-oxoglutarate from oxaloacetate and glutamate. These resultssuggest that nodule mitochondria operate a truncated form ofthe TCA cycle and primarily oxidize malate to provide oxaloacetateand ATP for NH₃ assimilation. Key words: Glycine max (L.), nitrogen fixation, gluconeogenesis, respiration     

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor δ-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60°C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[¹⁴C]glutamate and 3,4-[³H]glutamate but not from 1-[¹⁴C]glutamate, and thus, operation of the C₅ pathway could not be confirmed.     

Enzymes of alpha,alpha-Trehalose Metabolism in Soybean Nodules

Metabolism of trehalose, α,d-glucopyranosyl-α,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and β-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.     

Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO₂ (1-80%, v/v) in N₂ for up to 28 days after planting. At the extremes of sub- and supra-ambient pO₂, the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO₂. Short-term changes in pO₂ (transferring plants grown with sub- or supra-ambient pO₂ in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO₂ was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO₂ with those for their structural adaptation to an under supply of O₂ indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O₂) compared to those grown in air. Progressive decline in pO₂ resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O₂ level.     

Assimilation of 15NH3 by Root Nodules Detached from Soybean Plants

The assimilation of ¹⁵NH₃ by crude breis prepared from crushedsoybean nodules was examined. The highest enrichment during60 min of reaction time with ¹⁵N was found in alanine and thenext highest in the amide-N of asparagine and glutamate. Thelabelling of allantoic acid was relatively low, although itwas higher than that of other amino compounds. Nodule breiswere separated into a bacteroid fraction and a supernatant plantfraction, and the ¹⁵NH₃ incorporation into the main nitrogencompounds by each fraction was determined. The bacteroid fractionwas much more efficient in converting ¹⁵NH₃ into glutamate,alanine and glycine than the supernatant fraction, while forallantoic acid, the supernatant fraction showed a greater ability.The incorporation of ¹⁵NH₃ into allantoic acid was stronglyinhibited by the addition of azaserine or allopurinol, and enhancedby organic acid compounds, especially fumarate, succinate andmalate. The mode of ureide formation in the course of ammoniaassimilation in the soybean nodule is discussed. ¹Present address: Department of Pharmacology, Nara Medical University,Kashihara, Nara 634, Japan. (Received February 2, 1981; Accepted May 16, 1981)     

Inhibition of Nitrogen Fixation in Non-Legume Root Nodules by Hydrogen and Carbon Monoxide

The effect of the presence of hydrogen and of carbon monoxideon the fixation of nitrogen in detached root nodules of non-legumeshas been studied, fixation being measured by the use of ¹⁵N.Parallel tests on legumes (pea and soya bean) have been included.Fixation in the nodules of Casuarina, Alnus, and Myrica is inhibitedin the presence of substantial proportions of hydrogen, to adegree resembling that shown in legumes. Fixation in Alnus andMyrica is arrested in the presence of small proportions of carbonmonoxide, and here again the sensitiveness is of the same orderas in legumes.     

The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions. The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 10⁸ per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 10⁸ per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod-shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 10⁸ per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis. Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N-fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ-aminobutyric acid and ethanolamine were decreased and the most of the others increased. In the hydrolysate of the non-soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light-grown and dark-grown pea plants were very similar. Hydroxyproline and α, γ-diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.     

Efficiency of Root Respiration in Relation to Growth Rate, Morphology and Soil Composition

Root growth respiration and root maintenance respiration rate of the following species were determined: Hypochaeris radicata L. ssp. radicata L., H. radicata ssp. ericetorum Van Soest, Plantago lanceolata L., P. major L. ssp. major, P. major ssp. pleiosperma Pilgcr, P. maritime L., Senecio viscosus L., S. vulgaris L. and Urtica dioica L. A high root growth respiration (i.e. the amount of oxygen consumed for synthesis of a given weight of root material) implied a high maintenance respiration rate (i.e. the amount of oxygen consumed per unit of time and dry weight, but not connected with growth). High values of both components reflect a low efficiency of root respiratory processes. The efficiency of root respiration, as determined by the values for root growth respiration and root maintenance respiration rate could not be demonstrated to be of advantage in adaptation to soil conditions, as e.g. nitrogen content, moisture content and pH. It is concluded that (he degree of ‘wasteful utilization of sugars’ in roots, i.e. such consumption of sugars as cannot be related to structural growth, storage of carbohydrates or maintenance processes, depends on imbalance of transport of sugars from the shoot to the roots with utilization of sugars for synthesis of root material. The results are discussed in relation to Brouwer's explanation for the equilibrium between the growth of shoots and of roots. Root growth rate in the present species appears limited by a factor produced in the shoot under light conditions, and which factor is distinct from carbohydrates. The evidence presented shows that relatively inefficient root respiration does not imply a low growth rate. In regulation of plant growth the growth rate itself and also the shoot to-root ratio may be more important than the regulation of the efficiency of energy metabolism.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Responses of Respiration to Increases in Carbon Dioxide Concentration and Temperature in Three Soybean Cultivars

The purpose of this experiment was to determine how respirationof soybeans may respond to potential increases in atmosphericcarbon dioxide concentration and growth temperature. Three cultivarsof soybeans (Glycine max L. Merr.), from maturity groups 00,IV, and VIII, were grown at 370, 555 and 740cm³m^-3carbon dioxideconcentrations at 20/15, 25/20, and 31/26°C day/night temperatures.Rates of carbon dioxide efflux in the dark were measured forwhole plants several times during exponential growth. Thesemeasurements were made at the night temperature and the carbondioxide concentration at which the plants were grown. For thelowest and highest temperature treatments, the short term responseof respiration rate to measurement at the three growth carbondioxide concentrations was also determined. Elemental analysisof the tissue was used to estimate the growth conversion efficiency.This was combined with the observed relative growth rates toestimate growth respiration. Maintenance respiration was estimatedas the difference between growth respiration and total respiration.Respiration rates were generally sensitive to short term changesin the measurement carbon dioxide concentration for plants grownat the lowest, but not the highest carbon dioxide concentration.At all temperatures, growth at elevated carbon dioxide concentrationsdecreased total respiration measured at the growth concentration,with no significant differences among cultivars. Total respirationincreased very little with increasing growth temperature, despitean increase in relative growth rate. Growth respiration wasnot affected by carbon dioxide treatment at any temperature,but increased with temperature because of the increase in relativegrowth rate. Values calculated for maintenance respiration decreasedwith increasing carbon dioxide concentration and also decreasedwith increasing temperature. Calculated values of maintenancerespiration were sometimes zero or negative at the warmer temperatures.This suggests that respiration rates measured in the dark maynot have reflected average 24-h rates of energy use. The resultsindicate that increasing atmospheric carbon dioxide concentrationmay reduce respiration in soybeans, and respiration may be insensitiveto climate warming. Glycine max L. (Merr.); carbon dioxide; respiration; temperature; climate change     

Aging of Soybean Seeds in Relation to Metabolism at Different Relative Humidities

The present study was carried out to clarify the relationshipsbetween respiration, moisture content and the aging of dry soybean(Glycine max) seeds under different storage conditions. Ethanolicfermentation increased with increasing relative humidity (RH)during storage. However, at 75% RH which corresponded to a watercontent of 15.2% in seeds, the production of ethanol ceasedafter one month of storage. The ethanol that had accumulatedwithin the container was re-absorbed by the seeds themselvesand disappeared entirely within 3 months, unless the seeds wereexposed to HCN gas. Similar changes were also found for methanol.The activity of mitochondria increased only in seeds storedat 75% RH, it reached a maximum after the third month of storageand then rapidly declined. The amounts of both CoA and acetylCoA in seeds that had been stored for 4 months were far lowerat 75% RH than at 53% RH, and the viability of soybean seedsstarted to decrease after the fourth month at 75% RH. Therefore,a major cause of the rapid deterioration of seeds at 75% RHat room temperature seems to be the rapid development of mitochondrialrespiration, which is accompanied by the consumption of CoAand its acetyl derivatives. By contrast, the slow deteriorationof seeds during long-term storage at 33% to 68% RH and/or atsubzero temperatures seems to be due to the toxic action ofacetaldehyde, an intermediate in ethanolic fermentation, whichcan slowly occur. (Received April 10, 1995; Accepted July 10, 1995)