Tissue distribution of acetyl-coenzyme a carboxylase in leaves

Acetyl-CoA carboxylase [acetyl-CoA—carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C₃- and C₄-plants. The mesophyll tissues of the C₃-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using ¹²⁵I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.

In the C₄-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.

The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

     

Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO₃ to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO₂-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO₂ and Mg²⁺. In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO₂ levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO₂ demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO₂ fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO₂ levels.     

Primary structure of the biotin-binding site of chicken liver acetyl-CoA carboxylase

Limited proteolysis of chicken liver acetyl-CoA carboxylase by staphylococcal serine proteinase yielded a fragment of 31 kDa which contained the biotinyl active site. This polypeptide was purified by preparative polyacrylamide gel electrophoresis and characterized. The complete amino acid sequence of this polypeptide has been deduced from the nucleotide sequence of cloned DNA complementary to the chicken liver acetyl-CoA carboxylase mRNA. A highly conserved sequence of Met-Lys-Met was found in the biotin-binding site. Appreciable homology was observed among the sequences in close vicinity of the biotin sites of chicken liver acetyl-CoA carboxylase and other biotin-dependent carboxylases including biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase.     

Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings.

A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.     

Use of streptavidin to detect biotin-containing proteins in plants

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.     

Biotin carboxyl carrier protein in barley chloroplast membranes.

Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21 000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained by solubilization of the lamellae in phenol/acetic acid/8 M urea. Feeding barley seedlings with [14C]-biotin revealed that the vitamin is not degraded into respiratory substrates by the plant, but is specifically incorporated into biotin carboxyl carrier protein.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Fat metabolism in higher plants. LVI. Distribution and nature of biotin in chloroplasts of different plant species

A number of bacteria, algae, and higher plant chloroplasts were examined to determine the nature of their biotin-protein complexes. In all tissues studied, the major fraction of the total biotin was bound to protein(s) through a lysine bridge and these proteins accepted ¹⁴CO₂ to form carboxybiotinyl protein(s). The biotinyl protein was present in the soluble protein fraction in the procaryotic organisms, Escherichia coli and Rhodospirillum rubrum. In eucaryotic organisms, such as Chlamydomonas reinhardi and chloroplasts from higher plants, biotinyl protein was associated with chloroplast membranes. The blue-green alga, Anacystis nidulans, showed an intermediate condition, while the filamentous blue-green alga, Anabaena flos-aquae, resembled the higher plant chloroplasts. Although on a chlorophyll basis, stroma lamellae fractions enriched in Photosystem I had a higher biotin protein content than did the grana lamellae fractions, on a protein basis, the biotinyl protein content was rather evenly distributed between the different membrane systems. In dormant embryos of barley and wheat acetyl CoA carboxylase was a soluble protein localized in the proplastids. During germination the biotin protein(s) became associated with the lamellar membrane fraction.     

Effect of path or sink anoxia on sugar translocation in roots of maize seedlings

The subcellular and developmental distribution of β-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of β-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.

β-Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total β-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves.

     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Detection of ligand-induced perturbations affecting the biotinyl group of mammalian acetyl-coenzyme A carboxylase by using biotin-binding antibodies

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

Intact mesophyll protoplasts from Zea mays as a source of phosphoenolpyruvate carboxylase unaffected by extraction: Advantages and limitations

Isolated intact mesophyll protoplasts from Zea mays L. were used as an enzyme source for studying properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1 1 31) just after release from cells into the reaction medium. After the injection of protoplasts into the assay mixture, an initial lag of activity was observed, mainly due to the time necessary for complete disruption of protoplasts by the osmotic shock. The final specific activity obtained was ca 18 μmol mg^-1 of liberated protein min^-1, a value comparable to that usually achieved after arduous purification. Under the assay conditions employed, the chloroplasts were not disrupted and the retention of their proteins, together with the use of purified mesophyll protoplasts, were obviously the reasons for the high specific activity obtained. The activity and properties of phosphoenolpyruvate carboxylase stored in isolated protoplasts were stable for at least 24 h at 5°C. The main difference between the protoplast-derived and the routinely extracted enzyme was the sensitivity to malate inhibition, which was partially lost in the extracted phosphoenolpyruvate carboxylase; no difference was found in the K_m(PEP). The stress imposed by the protoplast isolation procedure diminished the sensitivity of the enzyme to malate inhibition, so that it can be inferred that the real malate sensitivity of pbosphocnolpyruvale carboxylase is even greater and that it is grossly underestimated with routinely extracted enzyme.     

Tissue Distribution and Subcellular Localization of Prephenate Aminotransferase in Leaves of Sorghum bicolor

The tissue and subcellular distribution of prephenate aminotransferase, an enzyme of the shikimate pathway, was investigated in protoplasts from leaves of Sorghum bicolor. Activity was detected in purified epidermal and mesophyll protoplasts, and in bundle sheath strands. After fractionation of mesophyll and epidermal protoplasts by differential centrifugation, 92% of the total prephenate aminotransferase activity was detected in the plastid fraction.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

Transient expression for functional gene analysis using Populus protoplasts

Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.