Phosphoenolpyruvate Carboxylase Enzyme Research Articles

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.)

The response of carbohydrate metabolism in 3-year-old Norway spruce plants to an increased amount of nitrogen supply to a N-poor forest soil was investigated in a pot experiment. After 7 months of treatment we found a decreased amount of starch in both needles and roots, together with decreased amounts of sucrose in needles of those plants grown under an enhanced inorganic N supply. In addition, the activity and the protein amount of the anaplerotic enzyme phosphoenolpyruvate carboxylase (PEPC) and the activity of NADP-dependent isocitrate dehydrogenase (IDH) were clearly increased. The activity of sucrose phosphate synthase (SPS) and the pool size of fructose 2,6-bisphosphate (F26BP) were not affected by high supply of inorganic N. These data indicate a shift of carbon flow from starch formation towards an enhanced provision of carbon skeletons for N assimilation and shoot growth. In parallel, we found decreased contents of fungus-specific compounds (ergosterol, mannitol, trehalose) in roots, which are indicators of a decreased colonization by ectomycorrhizal fungi, probably as a result of a changed allocation and partitioning of photoassimilates due to an increased N supply.

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.)

The response of carbohydrate metabolism in 3-year-old Norway spruce plants to an increased amount of nitrogen supply to a N-poor forest soil was investigated in a pot experiment. After 7 months of treatment we found a decreased amount of starch in both needles and roots, together with decreased amounts of sucrose in needles of those plants grown under an enhanced inorganic N supply. In addition, the activity and the protein amount of the anaplerotic enzyme phosphoenolpyruvate carboxylase (PEPC) and the activity of NADP-dependent isocitrate dehydrogenase (IDH) were clearly increased. The activity of sucrose phosphate synthase (SPS) and the pool size of fructose 2,6-bisphosphate (F26BP) were not affected by high supply of inorganic N. These data indicate a shift of carbon flow from starch formation towards an enhanced provision of carbon skeletons for N assimilation and shoot growth. In parallel, we found decreased contents of fungus-specific compounds (ergosterol, mannitol, trehalose) in roots, which are indicators of a decreased colonization by ectomycorrhizal fungi, probably as a result of a changed allocation and partitioning of photoassimilates due to an increased N supply.

Quantitative structure-activity relationships of competitive inhibitors of phosphoenolpyruvate carboxylase

The quantitative structure-activity relationships (QSAR) of all known competitive inhibitors of the enzyme phosphoenolpyruvate carboxylase from C 4 plants were investigated by means of molecular mechanics, the semiempirical quantum chemical methods MNDO and AM1, and the Hansch approach. In the case of phosphoenolpyruvate analogues, the hydrophobicity and steric impediment of the combined cis and trans substituents, the bond distance to the cis substituent along with its volume, dipole moment, the distance between the phosphorus and the carbonyl carbon, and the net electric charges on the phosphate and substituent groups are the main factors that govern their binding to the active site. For the phosphoglycolate analogues, the difference in the HOMO-LUMO energies, the magnitudes of their dipole moments and their non-polar surfaces, and the distance between the phosphorus and the carbonyl carbon are the variables that control their binding to the active site. These results, in conjunction with a discriminant analysis, also suggest that these inhibitors can actually be divided into two groups, according to the way they presumably interact with the active site.

Phosphorus Stress-Induced Proteoid Roots Show Altered Metabolism in Lupinus albus.

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.

Primary assimilation of nitrogen in alfalfa nodules: molecular features of the enzymes involved

The primary assimilation of symbiotically fixed nitrogen (N) in alfalfa root nodules involves complex intermingling with carbon (C) metabolism. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understanding how N and C metabolism are controlled in root nodules requires fundamental knowledge of how the plant genes involved are regulated. While significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes controlling other enzymatic steps in this process. To that end we have isolated, purified and characterized the root nodules enzymes aspartate aminotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glutamate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent forms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The deduced amino acid sequence suggested and immunogold labeling showed that nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the deduced amino acid sequence of nodule-enhanced NADH-GOGAT to the N-terminal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organelle ADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels comparable to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RNA blots showed that GS, AAT, PEPC, and NADH-GOGAT mRNAs were enhanced about 15-fold during the development of effective alfalfa nodules. By comparison, the expression of GS, AAT and PEPC mRNAs was reduced by 65% in ineffective nodules. NADH-GOGAT was different from GS, AAT, and PEPC in that expression had an absolute requirement for a factor(s) related to effective nodules. The data suggest that NADH-GOGAT plays a key role in regulating N assimilation. Moreover, plastids in nodules play a major role not only in C metabolism but also in N metabolism.

Structure and expression of a sugarcane gene encoding a housekeeping phosphoenolpyruvate carboxylase.

A gene (SCPEPCD1) encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from the C-4 monocot sugarcane (Saccharum hybrid var. H32-8560). SCPEPCD1 is ca. 6800 bp long, with 10 exons. The entire gene sequence from -1561 to 262 bp downstream of the putative poly(A) addition signal is reported. A low-level, essentially constitutive pattern of expression, amino acid sequence similarities to other 'housekeeping' PEPC enzymes, and the absence of DNA sequence elements conserved in the upstream region of maize and sorghum C-4-specific PEPC genes indicate that SCPEPCD1 encodes a housekeeping PEPC. Despite this, a motif proposed to act as a phosphorylation site in light-mediated activation of photosynthetic PEPC enzymes [10] is present in the SCPEPCD1 protein; evidence is presented for the presence of this site in other housekeeping PEPC proteins.

The evolution of crassulacean acid metabolism inAeonium inferred from carbon isotope composition and enzyme activities.

Variation in crassulacean acid metabolism (CAM) was investigated in thirtyAeonium species under controlled, well-watered conditions. Carbon isotope ratios and the specific activities of the CAM enzymes phosphoenolpyruvate carboxylase (PEPC) and malic enzyme ME_ are highly correlated and indicate a large variation in CAM. The δ13C values range from -13.6 to -27.1‰; PEPC and ME activities vary seven- and five-fold, respectively. Since these observations were made under controlled conditions, the differences are genetically determined and represent adaptations to different habitats. The CAM activity of the species is correlated with the characteristics of their natural habitats. CAM activity is also highly correlated with the growth- and life-forms of the species and accordingly agrees well with the infrageneric classifications which are partly based on these characters. Our results support the hypothesis that the Sempervivoideae have evolved from an EurasiaticSedum, which used C3 photosynthesis or weak CAM, and that evolution withinAeonium was from undifferentiatedSedum-like dwarf shrubs with C3 photosynthesis to woody candelabrum-shaped shrubs exhibiting strong CAM. The monocarpic, rosulate species with intermediate CAM may have evolved via an independent secondary route.

Convergent Induction of Osmotic Stress-Responses

In Mesembryanthemum crystallinum, salt stress induces the accumulation of proline and a specific isoform of the enzyme phosphoenolpyruvate carboxylase (PEPCase) prior to the switch from C(3) to Crassulacean acid metabolism (CAM). To determine whether plant growth regulators initiate or imitate these responses, we have compared the effects elicited by NaCl, abscisic acid (ABA), and cytokinins using PEPCase and proline levels as diagnostic tools. Exogenously applied ABA is a poor substitute for NaCl in inducing proline and CAM-specific PEPCase accumulation. Even though ABA levels increase 8- to 10-fold in leaves during salt stress, inhibition of ABA accumulation does not affect these salt-induced responses. In contrast, the addition of cytokinins (6-benzylaminopurine, zeatin, 2-isopentyladenine) mimic salt by greatly increasing proline and PEPCase amounts. Endogenous zeatin levels remain unchanged during salt stress. We conclude: (a) The salt-induced accumulation of proline and PEPCase is coincident with, but is not attributable to, the rise in ABA or zeatin concentration. (b) For the first time, cytokinins and NaCl are implicated as independent initiators of a sensing pathway that signals leaves to alter PEPCase gene expression. (c) During stress, the sensing of osmotic imbalances leading to ABA, proline, and CAM-specific PEPCase accumulation may be mediated directly by NaCl.

Structure, Regulation and Biosynthesis of Phosphoenolpyruvate Carboxylase from C4 Plants

This review attempts to summarize the large body of information on the structure, regulation and biosynthesis of the enzyme phosphoenolpyruvate carboxylase in C4 plants which has accumulated particularly since the appearance of the last review in 1987. Among the major discoveries are the involvement of protein phosphorylation-dephosphorylation cascade in the light activation of the enzyme, extraction and characteristics of PEPC-protein serine kinase, dynamic changes in oligomeric state of the enzyme in response to pH or temperature, isolation of multiple cDNAs encoding different forms of PEPC and cloning and expression of maize/sorghum PEPC in transgenic tobacco or transformed E. coli cells. Further experiments using advanced techniques of biochemistry and molecular biology would help in understanding the molecular mechanism of reaction, regulation of enzyme activity, gene expression and evolutionary pattern of C4 PEPC.

Tansley Review No. 37 Circadian rhythms: their origin and control.

This article reviews the circadian rhythm of carbon dioxide metabolism in leaves of the Crassulacean plant Bryophyllum (Kalanchoë) fedtsckenkoi which persists both in continuous darkness and a CO2 -free atmosphere, and in continuous light and normal air. Under both conditions the rhythm is due to the periodic activity of the enzyme phosphoenolpyruvate carboxylase (PEPc). The physiological characteristics of the rhythm are described in detail and, from these characteristics, hypotheses are advanced to account for both the generation of the rhythm and the regulation of its phase and period by environmental factors. The periodic activity of PEPc is ascribed to the periodic accumulation of an allosteric inhibitor, malate, in the cytoplasm and its subsequent removal either to the vacuole in continuous darkness, or by metabolism in continuous light. Also involved in the generation of the rhythm is a periodic change in the sensitivity of PEPc to malate inhibition due to the periodic phosphorylation and dephosphorylation of PEPc which changes its K1 by a factor of 10 from 30 to 0.3 mM and vice versa. This periodic phosphorylation of PEPc is apparently achieved by the periodic synthesis and breakdown of a PEPc kinase which phosphorylates the enzyme on a serine residue; dephosphorylation is achieved by a type 2A phosphatase which shows no rhythmic variation. The induction of phase shifts in the rhythm in continuous darkness and CO2 -free air has been explained in terms of light and high-temperature activated gates or channels in the tonoplast which, when open, allow malate to diffuse between the vacuole and cytoplasm. For the rhythm in continuous light and normal air phase, control by environmental signals can be attributed to changes in the malate levels in critical cell compartments, or in particular cell populations such as the stomatal guard cells, due to regulation of the malate synthesizing enzyme system involving PEPc, and malic enzyme which is responsible for malate metabolism. The role of the stomata in the generation of the rhythm is also discussed. The biochemical events which appear to give rise to the well-studied circadian rhythms in leaf movement in Samanea and Albizza, in luminescence in Gonyaulax polyedra and in the synthesis of the chlorophyll a/b binding protein are also reviewed in an attempt to identify similarities between these events and those involved in the Bryophyllum rhythm. Finally, the somewhat similar nature of the genes apparently responsible for circadian rhythmicity in Neurospora and Drosophila are discussed, and suggestions made for utilizing anti-sense nucleic acid technology in the further elucidation of the critical biochemical events involved in the basic, temperature-compensated circadian oscillator in living organisms. CONTENTS Summary 347 I. Introduction 348 II. Occurrence of circadian rhythms 348 III. Physiological characteristics of circadian rhythms 349 IV. Biochemical and molecular events involved in the circadian rhythm in Bryophyllum leaves 362 V. Biochemical and molecular events involved in the origin and control of circadian rhythmicity in other organisms 366 VI. Genetic studies 370 VII. Conclusion 371 References 372.

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O2 limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O2‐limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO2 fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O2 regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH‐glutamate synthase (NADH‐GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.

Variations in carboxylase activity in marine phytoplankton cultures, ß-carbolxylation in carbon flux studies

Inorganic carbon may be assimilated through the Calvin-Benson cycle via the enzyme ribulose-1.5-bisphosphate carboxylase (Rubisco) andlor by 0-carboxylation [via the enzymes phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) or pyruvate carboxylase]. Here, carboxylase activity measurements for marine phytoplankton are described. Two indices measuring carboxylase activity in marine phytoplankton were used. The first measures Rubisco activity per unit chlorophyll [R/Chl; nmol CO2 (pg chl a+b+c)-' h-'] while the second is the ratio of P-carboxylase activity to Rubisco activity, expressed as % (PC/R), which reflects the proportion of inorganic carbon fixed by these 2 groups of carboxylases. These ratios were studied in (1) different algal species in culture, (2) dunng the different growth phases of a culture, and (3) after a light-dark transition to measure the time response of carboxylase activities. These indices were different from one species to another at the same stage of growth. In autotrophic cells, P-carboxylation remained low (PC/R<40). The PCIR ratio increased significantly when R/Chl began to decrease at the end of the growth phase of a culture of Skeletonerna costaturn. The heterotrophic dinoflagellate Crypthecodinjurn cohnii, grown on an organic medium, incorporated inorganic carbon in the dark through PEPCK activity. The wlde range in PC/R ratio observed among the species confirm that in phytoplankton there may exist a continuum between autotrophy and heterotrophy. From a carbon budget point of view the 2 mechanisms are not equivalent. Rubisco fixation uses light as an energy source and results in gross production; 0-carboxylation also fixes inorganic carbon but as energy source uses metabolites synthesized by other pathways in the cell or from the external medium.

Properties of Phosphoenolpyruvate Carboxylase and Carbohydrate Accumulation in the C3-CAM IntermediateSedum telephiumL. Grown Under Different Light and Watering Regimes

A comparison of the activity and properties of the enzyme phosphoenolpyruvate carboxylase (PEPC) was made for plants of Sedum telephium L. grown under low (70 μmol m−2 s−1) or high (500μmol m−2 s−1) PPFD and subjected to varying degrees of water stress. Under well-watered conditions only plants grown under high PPFD accumulated titratable acidity overnight and the extractable activity of PEPC was almost 2-fold higher in these plants than in plants grown under low PPFD. Increasing drought stress resulted in a substantial increase in the activity of PEPC extracted both during the light and dark periods and a decrease in the sensitivity to inhibition by malic acid. The magnitude of these changes was determined by the severity and duration of drought and by light intensity. A comparison of the kinetic properties of PEPC from severely droughted plants revealed that plants droughted under high PPFD had a lower Km for PEP than plants under low PPFD. Addition of 2·0 mol m−3 malate resulted in an increase in the Km for PEP, with plants draughted under low PPFD having a significantly higher Km in the presence of malic acid compared to those under high PPFD. Response to the activator glc-6-P, which lowered the Km for PEP, also varied between plants grown under the two light regimes. Under well-watered conditions PEPC extracted from plants under high PPFD was more sensitive to activation by glc-6-P than those under low PPFD. After the severe drought treatment, however, the Km for PEP in the presence of glc-6-P was similar for enzyme extracted from plants grown under both light regimes. Soluble sugars and starch were depleted overnight and were both possible sources of substrate for PEPC. With increasing drought, however, the depletion of starch relative to soluble sugars increased under both light regimes. The properties of PEPC and the characteristics of carbohydrate accumulation/depletion are discussed in relation to the regulation of CAM in S. telephium grown under different light and watering regimes.

Malate content of picoliter samples of Raphanus sativus cytoplasm.

Malate, which plays many essential roles in plant metabolism, is a potent in vitro inhibitor of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC). Because PEPC activity leads to malate biosynthesis, malate is assumed to attenuate its own synthesis in situ. To test this hypothesis, we measured directly the malate content of picoliter samples of Raphanus root-hair cytoplasm using quantitative histochemical techniques. We also obtained an estimate for malate accumulation in these cells. These values were compared with the PEPC activity of individual root hairs (less than 2 ng). The results indicate that high cytoplasmic malate concentration does not severely inhibit PEPC in situ. We suggest that the focus for studies on the regulation of organic anion accumulation be on the interactive effects of malate and other PEPC effectors.

Variation in crassulacean acid metabolism within the genus Sedum: Carbon isotope composition and water status dependent phosphoenolpyruvate carboxylase activity

Summary Variation in Crassulacean acid metabolism (CAM) was examined in a large group of Sedum species, using two criteria: 13 C/ 12 C ratio and water status dependent specific activity of the CAM key enzyme phosphoenolpyruvate carboxylase (PEP-C). A large amount of variation in CAM was found within the genus Sedum . In three European species C3-like photosynthesis was found, as indicated by water status independent, low specific PEP-C activity and δ 13 C values of around -30‰. Three Mexican species showed water status independent high specific PEP-C activity and δ 13 C values of -15 to -22‰, indicating constitutive CAM. In the majority of European species the specific PEP-C activity was water status dependent; δ 13 C values ranged from -25 to -31‰. Specific PEP-C activity and carbon isotope ratios are significantly correlated, species with high specific activities showing less negative δ 13 C values. The Michaelis constant for PEP of the PEP-C enzymes of these Sedum species was determined. The observed values differed from 120-800 µmo1. The observed variation in CAM was compared with taxonomic relations of the examined species, leaf thickness, Km(PEP) and life cycle length.

Chromosomal location and copy number in wheat and some of its close relatives of genes for enzymes involved in photosynthesis

Homologous probes for the wheat coding sequences of the enzymes phosphoribulokinase, phosphoglycerate kinase (both chloroplast and cytosolic forms), chloroplast fructose-1,6-bisphosphatase and the small subunit of ribulose-1,5-bisphosphate carboxylase were used to determine the copy number and chromosomal location of the genes encoding these enzymes by restriction fragment length polymorphism analysis. Heterologous probes were similarly used to characterize the genes for the enzymes glyceraldehyde phosphate dehydrogenase (both chloroplast and cytosolic forms), phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase. Several of the genes are present in single copies per haploid genome, and the different enzymes are encoded by loci dispersed on different chromosomes. The significance of these findings is discussed in relation to gene expression and control of copy number.

Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Involvement in Protein and Oil Biosynthesis during Soybean Seed Development

During soybean [Glycine max (L.) Merr.] seed development, sucrose translocated to the seed is metabolized to precursors for protein and oil, the major components of mature soybean seed. while breeders have had success in selecting for increased seed protein or oil concentration, there is little understanding the metabolic events that regulate the partitioning of carbon into these seed components. Enzyme reactions that may determine carbon partitioning to seed protein and oil were examined in six soybean genotypes exhibiting genotypic differences in concentration of seed protein and oil. Developing seed were assayed for the enzymes phosphoenolpyruvate (PEP) carboxylase, pyruvate kinase, malate dehydrogenase, NADP‐malic enzyme, and aspartate and alanine aminotransferases. Correlations between these data and the protein and oil concentrations of each sample were determined. There was no significant difference between the correlation of pyruvate kinase activity with the oil and that with the protein concentration of the seed over development. The correlation of PEP carboxylase activity with protein was generally higher than with oil, suggesting that much of the oxaloacetate (OAA) produced may be used for synthesis of protein precursors. However, the high r values between PEP carboxylase and oil suggested that OAA may also be converted to malate and then to pyruvate (a precursor for oil) via a transhydrogenase system. Therefore, PEP carboxylase and pyruvate kinase activities appeared to contribute to a complex interaction that regulates the metabolic flow of glycolytic carbon into precursors for both protein and oil biosynthesis during soybean seed development.

Effect of varying environments on photosynthetic parameters of C3, C3-C4 and C4 species of Panicum.

CO2 exchange characteristics and the activity of the carboxylating enzymes phosphoenolpyruvate carboxylase (PEP-C, E.C. 4.1.1.31) and ribulose 1,5-bisphosphate carboxylase (RuBP-C, E.C. 4.1.1.39) during one year in the greenhouse and at two levels of light and temperature in growth chambers were determined in the C3-C4 intermediate species P. milioides Nees ex. Trin. These results were compared with those of P. bisulcatum Thumb. (C3) and P. maximum Jacq. (C4). Under all tested conditions, and even when the influence of leaf surface temperature on photosynthetic rates and CO2 compensation points were measured, the biochemical and physiological behaviour of the C3-C4 intermediate was more similar to that of the C3 plant than the C4 species. The C4 plant P. maximum, however, responded positively, mainly in terms of PEP-C activity and photosynthetic rate, to the regime of high light and temperature. The results presented indicate that in the C3-C4 Panicum grown in high light and temperature no direct relationships between a low CO2 compesation point and superior growth are evident. It has still to be clarified why in nature a photosynthetic-photorespiratory pathway leading to an intermediate CO2 compensation value has evolved in P. milioides.

Low Bundle Sheath Carbonic Anhydrase Is Apparently Essential for Effective C4 Pathway Operation

Bundle sheath cells from leaves of a variety of C(4) species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C(4) pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO(2)] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C(4) function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C(4) pathway.

Variation for Activity of Nodule Nitrogen and Carbon Assimilating Enzymes in Alfalfa1

The nodule enzymes glutamate synthase (GOGAT) and phosphoenolpyruvate carboxylase (PEPC) are important in the assimilation of symbiotically fixed N in alfalfa (Medicago sativa L.). The objectives of this study were: to evaluate several alfalfa germplasm sources for variability in nodule GOGAT and PEPC activities, and to determine the relationships between N2 fixation and plant yield traits with nodule GOGAT and PEPC enzyme activities. Approximately 160 plants from each of six alfalfa germplasms were grown in sand culture in the glasshouse and assayed for nodule GOGAT and PEPC enzyme activities. Twenty‐four genotypes (four from each germplasm) were selected to represent a range in GOGAT and PEPC activities. These genotypes were evaluated in glasshouse and field clonal studies for GOGAT and PEPC activities per gram nodule fresh weight, acetylene reduction activity (ARA), nodule mass, nodule color, nodule soluble protein concentration, and plant yield. Variability for nodule GOGAT and PEPC activities within each of the six germplasms was large and normally distributed. The 12 high GOGAT genotypes (2 from each germplasm) were 44% greater GOGAT activity than the 12 low genotypes. The 12 high PEPC genotypes were 27% greater in PEPC activity than the 12 low genotypes. Both groups of high enzyme activity genotypes were significantly greater than the low genotypes in ARA, nodule soluble protein concentration, and field shoot yield. Both GOGAT and PEPC activities were positively correlated (r=0.61**, significant at the 0.01 probability level). Also, GOGAT and PEPC were each correlated with ARA (r=0.43*, significant at the 0.05 probability level, and 0.45*, respectively), nodule color score (r=0.46** and 0.73**) soluble protein concentration (r= 0.77** and 0.90**). Plant yield and N2 fixation measured in the field were not correlated with nodule GOGAT and PEPC activities measured in the glasshouse. The relationships between GOGAT and PEPC with other plant traits indicated that these enzymes are associated with the host‐Rhizobium meliloti Dang. symbiosis and should be explored as possible selection criteria to include in an alfalfa breeding program.