Subcellular Localization of Enzymes of Carbon and Nitrogen Metabolism in Nodules of Medicago sativa

Alfalfa (Medicago sativa L. cv. Vernal) nodules were separatedinto host plant fractions and fractions of rhizobial originby differential centrifugation and sedimentation equilibriumcentrifugation. Both NAD- and NADP-linked isocitrate dehydrogenase(70%, 90%) and glutamate dehydrogenase activities (90%, 83%)were located primarily (percent total nodule activity) in thefractions of plant origin and their specific activities werehighest in the fractions of plant origin. More than 50% of thenodules' total activity of both glutamine synthetase and NAD-glutamatesynthase and greater than 90% of the total glutamate oxaloacetatetransaminase activity was located in plant fractions. However,the fractions of rhizobial origin had the highest specific activitiesof glutamine synthetase and glutamate synthase. (Received September 5, 1981; Accepted December 7, 1981)     

Bacteroids Isolated from Ineffective Nodules of Pisum sativum Mutant E135 (syml3) Lack Nitrogenase Activity but Contain the Two Protein Components of Nitrogenase

Pea mutant E135 (sym15) forms ineffective (Fix^–) nodulesthat lack nitrogen fixing activity. To determine the developmentalstep blocked in E135 nodules we studied the nitrogenase activitiesin isolated bacteroids and in cell-free extracts of bacteroids,and measured the two components of nitrogenase protein in bacteroids.Bacteroids prepared anaerobically from E135 nodules showed noacetylene reduction activity in the presence and absence ofmyoglobin. Furthermore, no acetylene reduction activity by cell-freeextracts of E135 bacteroids was detected in the presence ofATP-generating system and dithionite. However, immuno-blottinganalyses revealed the presence of nitrogenase components I andII in E135 nodule bacteroids. These results suggest that a hostplant gene is involved in the expression of nitrogenase activityin symbiotic bacteria. (Received May 11, 1998; Accepted August 7, 1998)     

Comparison of the Protein Composition and Enzymatic Activities during Development between Effective and Plant-Determined Ineffective Nodules in Pea

The protein composition and enzymatic activities during developmentof ineffective nodules, produced by mutant E135 (sym 13) ofpea (Pisum sativum L.), were compared with those of the nitrogen-fixingnodules of the normal parent, the Sparkle cultivar. The proteincomposition of 3-week-old E135 nodules, as determined by SDS-polyacrylamidegel electrophoresis, was quite similar to that of Sparkle nodules.After 4 weeks, however, the intensities of bands of 15-, 38-,and 87-kDa polypeptides were lower in the case of E135 nodules.Western blot analysis using a "nodule-specific" antiserum revealedthat most nodulins could be detected in 3-week-old E135 nodules,but a 35.5-kDa nodulin disappeared after 5 weeks and severalnovel peptides ranging in molecular weight from 26 to 31 kDaappeared after 6 weeks in E135 nodules. The activities of glutaminesynthetase, glutamate synthase, alanine-pyruvate aminotransferase,sucrose synthase, and phosphoenolpyruvate carboxylase increasedduring development of Sparkle nodules, but such increases werenot found in E135 nodules after 5 weeks. These results showthat the nodules of E135 begin to develop normally but differfrom those of Sparkle within 4 weeks, indicating that, duringearly stages of nodule development, the protein compositionand activities of enzymes involved in carbon and nitrogen metabolismare not regulated by the presence or absence of nitrogenaseactivity. (Received February 26, 1993; Accepted May 19, 1993)     

Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).

For most samples, thin layer chromatography revealed major peaks of radioactivity at the R_F values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [³H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule.

     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Carbon Metabolism Enzymes of Rhizobium meliloti Cultures and Bacteroids and Their Distribution within Alfalfa Nodules

Several carbon metabolism enzymes were measured in cultured cells and bacteroids of Rhizobium meliloti 102F51 and in alfalfa root nodule cytosol. The enzyme activity levels of the pentose phosphate pathway were much higher than those of the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways in extracts of cultured cells. The pattern of enzyme activities in the bacteroids was different from that of cultured cells.     

Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: α-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.     

Symbiotic Tissue Degradation Pattern in the Ineffective Nodules of Three Nodulation Mutants of Pea (Pisum sativum L.)

Three symbiotic mutants of pea (Pisum sativum L.) forming ineffectivenodules were investigated for aberrations in nodule structureusing light and transmission electron microscopy. The mutantswere ordered according to the timing of the nodule developmentblock. In the mutant RisfixO, symbiotic tissue development isarrested before the late symbiotic zone (LSZ) forms, while theinfected cells of the LSZ of RisfixT lose the wild-type structureafter full differentiation. In contrast to the bacteroid degradationvia an electron-dense stage in RisfixO, lysis of symbiosomecontents prevails in RisfixT nodules. Enhancement of the lyticfunction of symbiosomes in RisfixT may be interpreted in termsof the symbiosome—lysosome homology. The weakened controlover symbiotic development in RisfixO may be responsible forthe abundant spread of the infection threads and their enlargement. Cells from the LSZ of RisfixV undergo fast collapse, resemblingdefence necrosis, after differentiation. In contrast to thenodules of RisfixO and RisfixT, degraded nodules of RisfixVdo not function as a sink for photosynthates and a source ofthe nodulation regulatory factor. This is indicated by the absenceof further starch accumulation after collapse, and by hypernodulation.Copyright1995, 1999 Academic Press Garden pea, developmental mutant, Pisum sativum, Rhizobium leguminosarum, root nodule, symbiosis, ultrastructure     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

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.     

Development of Enzymes Involved in Photosynthetic Carbon Assimilation in Greening Seedlings of Maize (Zea mays)

Upon illumination of dark-grown maize seedlings (5 days old) with incandescent light, there occurred a nearly simultaneous increase, after a certain lag period, in the activities of enzymes engaged in the C₄ pathway and the Calvin-Benson cycle. The light-induced biosynthesis of chlorophyll (a and b) precedes the increase in enzyme activities and proceeds without lag phase. A diphasic feature in the elevation of enzyme activities as a function of the intensities of light provided was observed; the increase in enzyme activities was enhanced by light intensities greater than 10³ ergs per square centimeter per second in comparison with light of lower intensities. Under light intensities greater than 10³ ergs per square centimeter per second, the simultaneous addition of levulinic acid, which inhibited chlorophyll formation, markedly reduced the increase of enzyme activities. However, neither the diphasic light effect nor the inhibitory effect of levulinic acid was observed with ribulose-1,5-bisphosphate carboxylase. The enzyme activities in the dark-grown maize seedlings were enhanced by a brief irradiation with the red light and the red light effect was reversed by the following far red light treatment. The red light-induced increase in the enzyme activities did not accompany chlorophyll synthesis, and was completely inhibited by cycloheximide, indicating that enzyme synthesis rather than activation might be involved. Light may play a dual role in enzyme induction; one is as an energy source through the photosystems at high intensities and the other is presumably as a signal mediated by phytochrome at low intensities.     

Effects of Irradiance-Sulphur Interactions on Enzymes of Carbon,Nitrogen, and Sulphur Metabolism in Maize Plants

The effect of sulphur deprivation and irradiance (180 and 750 µmol m^–2 s^–1) on plant growth and enzyme activities of carbon, nitrogen, and sulphur metabolism were studied in maize (Zea mays L. Pioneer cv. Latina) plants over a 15-d-period of growth. Increase in irradiance resulted in an enhancement of several enzyme activities and generally accelerated the development of S deficiency. ATP sulphurylase (ATPs; EC 2.7.7.4) and o-acetylserine sulphydrylase (OASs; EC 4.2.99.8) showed a particular and different pattern as both enzymes exhibited maximum activity after 10 d from the beginning of deprivation period. Hence in maize leaves the enzymes of C, N, and S metabolism were differently regulated during the leaf development by irradiance and sulphur starvation.     

Influence of s-Triazines on Some Enzymes of Carbohydrates and Nitrogen Metabolism in Leaves of Pea (Pisum sativum L.) and Sweet Corn (Zea mays L.)

Foliar applications of 2 milligrams per liter of 2-chloro-4,6-bis (ethylamino)-s-triazine, 2-methylmercapto-4-ethylamino-6-isobutylamino-s-triazine, and 2-methoxy-4-isopropylamino-6-butylamino-s-triazine caused increases in the activities of starch phosphorylase, pyruvate kinase, cytochrome oxidase, and glutamate dehydrogenase 5, 10, and 15 days after treatment in the leaves of 3-week-old seedlings of pea (Pisum sativum L.) and sweet corn (Zea mays L.). The results indicate that sublethal concentrations of s-triazine compounds affect the physiological and biochemical events in plants which favor more utilization of carbohydrates for nitrate reduction and synthesis of amino acids and proteins.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Light Microscopy Study of Nodule Initiation in Pisum sativum L. cv Sparkle and in Its Low-Nodulating Mutant E2 (sym 5)

We compared nodule initiation in lateral roots of Pisum sativum (L.) cv Sparkle and in a low-nodulating mutant E2 (sym 5). In Sparkle, about 25% of the infections terminated in the epidermis, a similar number stopped in the cortex, and 50% resulted in the formation of a nodule meristem or an emerged nodule. The mutant E2 (sym 5) was infected as often as was the parent, and it formed a normal infection thread. In the mutant, cell divisions rarely occurred in advance of the infection thread, and few nodule primordia were produced. Growing the mutant at a low root temperature or adding Ag⁺ to the substrate increased the number of cell divisions and nodule primordia. We conclude that, in the E2 line, the infection process is arrested in the cortex, at the stage of initial cell divisions before the establishment of a nodule primordium.     

Interaction Between a Non-Nodulating and an Ineffective Mutant of Rhizobium trifolli Resulting in Effective (Nitrogen-Fixing) Nodulation

A noninvasive (non-nodulating) mutant of Rhizobium trifolii when mixed with an ineffective (non-nitrogen-fixing) mutant gives rise to effective (nitrogen-fixing) nodules.     

Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots

Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P₃] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-³²P]Ins(1,4,5)P₃ and [2-³H]Ins(1,4,5)P₃ as tracers. Ins(1,4,5)P₃ was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P₂] whereas inositol(1,4)bisphosphate [Ins(1,4)P₂] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P₄. Dephosphorylation of Ins(1,4,5)P₃ to Ins(4,5)P₂ was dependent on Ins(1,4,5)P₃ concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P₃ to Ins(4,5)P₂ and Ins(1,4,5,X)P₄ was inhibited by 55 micromolar Ca²⁺. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P₃ and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.