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.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Anatomy, physiology and biochemistry of root nodules of Sprint-2 Fix-, a symbiotically defective mutant of pea (Pisum sativum L.)

A plant-determined pea mutant Sprint-2 Fix^– and the parentalline Sprint-2 were compared for selected physiological and biochemicalparameters. The Fix^– mutation prevented differentiationof Rhizobium leguminosarum bacteria into bacteroids and producedlarge, white, non-fixing nodules. These lacked nitrogenase-linkedrespiration, but had a background rate of CO₂ evolution similarto the normal Fix⁺ nodules. The cortical structure of the ineffectivenodules suggests the existence of an oxygen diffusion barrierand this was supported by a low oxygen concentration in thecentral region (0.5–3.0%), measured using an O₂ sensitivemicro-electrode. Sucrose and starch contents were similar innormal and ineffective nodules while ononitol content was about15 times lower in the Fix^– nodules. The distribution ofstarch was also different in the two nodule types. The activitiesof glutamine synthetase (GS), sucrose synthase (SS), phosphoenolpyruvatecarboxylase (PEPC) and alanine pyruvate aminotransferase (APAT)were markedly higher in Fix⁺ nodules while the activities ofpyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) andglutamate dehydrogenase (GDH) were higher in Fix^– nodules.The data from immunodetection of host nodule proteins confirmedthe reduced levels of sucrose synthase and the almost completeabsence of glutamine synthetase and leghaemoglobin in mutantnodules. There was no significant difference in the amount ofnitrogenase component 1 extracted from the microsymbiont ofnormal and ineffective nodules, but component 2 was hardly detectablein the Fix^– mutant. Key words: Pisum sativum, Fix^– mutant, nodules     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea ( Pisum sativum L. cv. Lincoln) plants were subjected to 50 m M sodium chloride stress for 3 weeks. C₂H₂ reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O₂ uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N₂ fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O₂ availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.     

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.Abbreviation PEP phosphoenolpyruvate     

Root system growth and nodule establishment on pea (Pisum sativum L.)

Development of the root system, appearance of nodules, and relationshipsbetween these two processes were studied on pea (Pisum sativumL., cv. Solara). Plants were grown in growth cabinets for 4weeks on a nitrogen—free nutrient solution inoculatedwith Rhizobium leguminosarum. Plant stages, primary root length,distance from the primary root base to the most distal first-orderlateral root, and distance from the root base to the most distalnodule, were recorded daily. Distribution of nodules along theprimary root and distribution of laterals were recorded by samplingroot systems at two plant stages. Primary root elongation ratewas variable, and declined roughly in conjunction with the exhaustionof seed reserves. First-order laterals appeared acropetallyon the primary root. A linear relationship was found betweenthe length of the apical unbranched zone and root elongationrate, supporting the hypothesis of a constant time lag betweenthe differentiation of first-order lateral's primordia and theiremergence. Decline of the primary root elongation rate was precededby a reduction in density and length of first-order laterals.Nodules appeared not strictly but roughly acropetally on theprimary root. A linear relationship was found between the lengthof the apical zone without nodule and root elongation rate,supporting the hypothesis of a constant time lag between infectionand appearance of a visible nodule. A relationship was foundbetween the presence/absence of nodules on a root segment andthe root elongation rate between infection and appearance ofnodules on the considered root segment. Regulation of both processesby carbohydrate availability, as a causal mechanism, is proposed. Key words: Pisum sativum L, root system, nodules     

Comparison of Enzymes Involved in Carbon and Nitrogen Metabolism in Normal Nodules and Ineffective Nodules Induced by a Pea Mutant E135 (sym 13)

Activities of enzymes involved in carbon and nitrogen metabolismwere examined in nodules of normal pea (Pisum sativum L. cv.Sparkle) and an ineffective plant mutant E135 (sym 13). Specificactivities of some enzymes were lower in ineffective nodulesthan in effective nodules. However, there were no major differencesbetween respective bacteroid fractions. ¹Present address: Department of Life Science, Aichi Universityof Education, Kariya, Aichi, 448 Japan     

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf w was allowed to reach -1.0 MPa over a period of 14 d whilst in the second experiment -1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehdyrogenase, uridine pyro-phosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (-1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (-1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress).Key words: Pisum sativum, water stress, nitrogen metabolism, nodule metabolism, pea, sucrose synthase.      

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

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)     

Developmental and Varietal Comparisons of Pod Carboxylase Levels in Pisum sativum L.

The activities of phosphoenolpyruvate (PEP) carboxylase andribulose-1, 5-bisphosphate (RuDP) carboxylase have been determinedin the developing pod walls of six genotypes of Pisum sativum.Genotypes were chosen which varied in pod characters such aschlorophyll content and tissue morphology, which it was hopedwould be associated with variation in carboxylase levels. Whilst both enzymes were detected in all genotypes, the levelsof activity varied considerably with pod type and with age.In general RuDP carboxylase activity correlated with chlorophyllconcentration, and yellow podded types had considerably lessactivity than green types. The yellow podded genotypes, however,contained significantly higher levels of PEP carboxylase which,in terms of total carboxylase activity, compensated for thelower RuDP carboxylase levels. The activities of both enzymes were determined within the endocarpand within the mesocarp plus exocarp, using 16-day-old pods.All genotypes showed an enrichment for PEP carboxylase in theendocarp and all but one genotype showed a similar enrichmentfor RuDP carboxylase activity in this layer. The role of the carboxylase enzymes within the pod wall is discussedand it is suggested that their main function may be to maintainan appropriate level of CO₂ within the pod cavity as well asrecycling carbon to the developing seeds. Pisum savitum L., pea, pods, carboxylase levels, genetic variation     

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm Ag⁺, an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.     

Proteolysis during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Plant-controlled ineffective root nodules, conditioned by the in1 gene in Medicago sativa L. cv Saranac, undergo premature senescence and have reduced levels of many late nodulins. To ascertain which factors contribute to premature senescence, we have evaluated proteolysis as it occurs throughout the development of ineffective Saranac (in1Sa) and effective Saranac nodules. Cysteine protease activities with acidic pH optimum and enzyme proteins were present in both genotypes. We found that acidic protease activity was low in effective Saranac nodules throughout their development. In contrast, by 2 weeks after inoculation, acid protease activity of in1Sa nodules was severalfold higher than that of Saranac nodules and remained high until the experiment was terminated 8 weeks later. This increase in protease enzyme activity correlated with an increase in protease protein amounts. Increased protease activity and amount in in1Sa nodules was correlated with a decrease in nodule soluble protein. The time at which in1Sa nodules initially showed increased protease activity corresponded to when symbiosis deteriorated. High levels of phosphoenolpyruvate carboxylase (PEPC) protein were expressed in effective nodules by 12 d after inoculation and expression was associated with low proteolytic enzyme activity. In contrast, although PEPC was expressed in in1Sa nodules, PEPC protein was not found 12 d after inoculation and thereafter. Acidic protease from in1Sa nodules could also degrade purified leghemoglobin. These data indicate that premature senescence and low levels of late nodulins in in1Sa nodules can be correlated in part with increased proteolysis.     

Evidence for carbon flux shortage and strong carbon/nitrogen interactions in pea nodules at early stages of water stress

Symbiotic N₂ fixation in legume nodules declines under a widerange of environmental stresses. A high correlation betweenN₂ fixation decline and sucrose synthase (SS; EC 2.4.1.13) activitydown-regulation has been reported, although it has still tobe elucidated whether a causal relationship between SS activitydown-regulation and N₂ fixation decline can be established.In order to study the likely C/N interactions within nodulesand the effects on N₂ fixation, pea plants (Pisum sativum L.cv. Sugar snap) were subjected to progressive water stress bywithholding irrigation. Under these conditions, nodule SS activitydeclined concomitantly with apparent nitrogenase activity. Thelevels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate,and fructose-6-phosphate decreased in water-stressed nodulescompared with unstressed nodules. Drought also had a markedeffect on nodule concentrations of malate, succinate, and     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.