Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.     

Permeability and Ultrastructure of Bundle Sheath Cells Isolated from C4 Plants: Structure-Function Studies and the Role of Plasmodesmata

Bundle sheath cells from leaves of C₄ plants can be isolated as strands surrounding vascular tissue. In this form these cells are highly permeable to metabolites and, as a consequence, they have a variety of experimental uses. The present paper reports on anatomical and ultrastructural features of isolated bundle sheath cell strands in relation to their integrity and permeability. This analysis shows that the cells retain a high degree of structural integrity during isolation. The plasmodesmata that originally connected the bundle sheath cytosol with mesophyll cells are apparently also retained in their entirety. However, at the external surface (mesophyll side) a membranous sac was commonly observed protruding from the end of plasmodesmata. The functional integrity of cells and the molecular weight exclusion limit for entry of compounds was assessed by following plasmolysis and cytorrhysis induced by polyethylene glycol solutions of varying molecular weights. Other evidence for the retention of cell compartment semipermeability is also provided.     

Photosynthetic and Carbohydrate Metabolism in Isolated Leaf Cells of Digitaria pentzii

Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C₄ species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO₂ fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO₂ per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The K_m(HCO₃⁻) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO₃⁻ concentrations. After 2 hours of photosynthesis by mesophyll cells in ¹⁴CO₂ and phosphoenolpyruvate, 88% of the incorporated ¹⁴C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated ¹⁴C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO₂ fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5-phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C₄ photosynthesis which places the Calvin cycle in the bundle sheath and C₄ acid formation in mesophyll cells.     

Distribution of carboxylation and decarboxylation enzymes in isolated mesophyll cells and bundle sheath strands of C 4 plants

Mature leaves of Cyperus rotundus, Cyperus polystachyos, Digitaria decumbens, and Digitaria sanguinalis were separated, using pectinase and cellulase, into pure preparations of mesophyll cells and bundle sheath strands. Assays on these distinct leaf cell types show a clear compartmentation of phosphoenolpyruvate carboxylase, >98%, into mesophyll cells and of ribulose-1, 5-diphosphate carboxylase and malic enzyme, >98%, into the bundle sheath strands. The results clearly establish that the major CO₂ uptake in mesophyll cells is via a β-carboxylation and that both a decarboxylation and a carboxylation reaction occurs in the bundle sheath strands of plants using C₄-dicarboxylic acid photosynthesis.     

Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Generation and maintenance of concentration gradients between the mesophyll and bundle sheath in maize leaves

(1) Experiments have been carried out to test the proposal that intercellular transport of carbon occurs by diffusion during photosynthesis in C-4 plants. (2) The intercellular distribution of metabolites has been compared in different conditions. A partial separation of the mesophyll and bundle sheath was obtained by homogenisation in liquid N₂, followed by filtration through nylon nets with differing aperture. (3) Concentration gradients between the bundle sheath and mesophyll were found for 3-phosphoglycerate, triose phosphates, malate and pyruvate during photosynthesis. These gradients are shown to be large enough to allow rapid intercellular transport by diffusion. They disappear when photosynthesis is prevented by removal of light or CO₂. (4) The concentration gradients for triose phosphates and 3-phosphoglycerate are due to the differing capacity of the bundle sheath and mesophyll to reduce 3-phosphoglycerate. (5) The distribution of carbon between the malate/pyruvate and 3-phosphoglycerate/triose phosphate shuttles is flexible, and may be controlled by phosphoenolpyruvate carboxylase. (6) The maintenance of these large concentration gradients has consequences for the regulation of sucrose synthesis and the Calvin cycle.     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

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.     

Pyruvate, pi dikinase in bundle sheath strands as well as in mesophyll cells in maize leaves

Mesophyll protoplasts and bundle sheath strands were isolated from maize leaves. Light microscopic observation showed the preparations were pure and without cross contamination. Protein blot analysis of mesophyll and bundle sheath cell soluble protein showed that the concentration of pyruvate orthophosphate dikinase (EC 2.7.9.1) is about one-tenth as much in the bundle sheath cells as in mesophyll cells, but about eight times greater than that found in wheat leaves, on the basis of soluble protein. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was barely detectable in the bundle sheath cells, while ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and NADP-dependent malic enzyme (EC 1.3.1.37) were exclusively present in the bundle sheath cells and were absent in the mesophyll cells. Whereas pyruvate, Pi dikinase was previously considered localized only in mesophyll cells of C₄ plants, these results clearly demonstrate the presence of appreciable quantities of the enzyme in the bundle sheath cells of the C₄ species maize.     

Fluorescence and Delayed Light Emission from Mesophyll and Bundle Sheath Cells in Leaves of Normal and Salt-Treated Panicum miliaceum

A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem II unit sizes than do bundle sheath chloroplasts. The larger photosystem II units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCl levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to DCMU, salinity altered photosystem II in bundle sheath cells but not in mesophyll cells. This result may indicate different ionic distributions in the two cell types or, alternatively, different responses of the two chloroplast types to environmental change.     

Spectral, Physical, and Electron Transport Activities in the Photosynthetic Apparatus of Mesophyll Cells and Bundle Sheath Cells of Digitaria sanguinalis (L.) Scop

Isolated mesophyll cells and bundle sheath cells of Digitaria sanguinalis were used to study the light-absorbing pigments and electron transport reactions of a plant which possesses the C₄-dicarboxylic acid cycle of photosynthesis. Absorption spectra and chlorophyll determinations are presented showing that mesophyll cells have a chlorophyll a-b ratio of about 3.0 and bundle sheath cells have a chlorophyll a-b ratio of about 4.5. The absorption spectrum of bundle sheath cells has a greater absorption in the 700 nm region at liquid nitrogen temperature, and there is a relatively greater amount of a pigment absorbing at 670 nm in the bundle sheath cells compared to the mesophyll cells. Fluorescence emission spectra, at liquid nitrogen temperature, of mesophyll cells have a fluorescence 730 nm-685 nm ratio of about 0.82 and bundle sheath cells have a ratio of about 2.84. The reversible light-induced absorption change in the region of P₇₀₀ absorption is similar in both cell types but bundle sheath cells exhibit about twice as much total P₇₀₀ change as mesophyll cells on a total chlorophyll basis. The delayed light emission of bundle sheath cells is about one-half that of mesophyll cells. Both mesophyll cells and bundle sheath cells evolve oxygen in the presence of Hill oxidants with the mesophyll cells exhibiting about twice the activity of bundle sheath cells, and both activities are inhibited by 1 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. Ferredoxin nicotinamide adenine dinucleotide phosphate reductase is present in both cells although it is about 3- or 4-fold higher in mesophyll cells than in bundle sheath cells. Glyceraldehyde 3-P dehydrogenases, both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, are equally distributed in the two cell types on a chlorophyll basis. Malic enzyme is localized in the bundle sheath cells.     

Photosynthetic Metabolism of Aspartate in Mesophyll and Bundle Sheath Cells Isolated from Digitaria sanguinalis (L.) Scop., a NADP-Malic Enzyme C(4) Plant

Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4)-dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate.     

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.     

玉米叶肉细胞和维管束鞘细胞中光合产物的分析

玉米苗照光后,叶肉细胞和维管束鞘细胞大量积累淀粉和可溶性糖(包括蔗糖),其中淀粉95％以上在维管束鞘细胞中。阻断光合产物输出时,两类细胞中蔗糖和淀粉积累都显著增加。离体维管束鞘细胞也能合成蔗糖。离体玉米叶内原生质体饲喂NaH~(14)CO_3并照光后,通常90％以上的~(14)C参入到有机酸和氨基酸中,3～10％参入糖和淀粉中。玉米叶肉原生质体具有直接利用CO_2合成碳水化合物的能力。     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

Nitrogen Assimilation Pathways in Leaf Mesophyll and Bundle Sheath Cells of C(4) Photosynthesis Plants Formulated from Comparative Studies with Digitaria sanguinalis (L.) Scop

Nitrogen assimilation in crabgrass Digitaria sanguinalis (L.) Scop., was studied by comparing leaf extracts with isolated mesophyll cell and bundle sheath strand extracts. The results show that both nitrate and nitrate reductase are localized in mesophyll cells; glutamine synthetase is nearly equally distributed in the mesophyll and bundle sheath; approximately 67% of the glutamate synthase activity is in the bundle sheath and 33% is in the mesophyll; and 80% of the glutamate dehydrogenase activity is in the bundle sheath, with the NADH-dependent form exhibiting a 2.5-fold higher activity than the NADPH-dependent form.     

Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.