CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Study of Energy Storage Processes in Bundle Sheath Cells of Zea mays

Photochemical energy storage in isolated bundle sheath cells from Zea mays was examined. Photoacoustic spectroscopy was used in this study to monitor energy storage processes. The presence of methyl viologen or addition of substrates which activated carbon fixation, prevented energy storage processes through the electron transport system. The energy storage was inhibited completely by dibromothymoquinone (DBMIB) and DCMU, inhibitors of noncyclic electron flow. However, the reductants such as dithiothreitol and ascorbate increased the energy storage. It was concluded that photosystem (PS) I may be reduced by some electron donor(s) other than water and that PSII only partially participates in PSI reduction. It is postulated that the role of PSII is to regulate PSI electron transport and prevent its overoxidation. In the presence of high level of malate, photoacoustic spectroscopy indicated a low energy storage which may be due to induction of energy utilization in carbon assimilation.     

Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K_m was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl₂, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Light-induced Changes of the Electrical Potential in Chloroplasts Associated with the Activity of Photosystem I and Photosystem II

The electric potential changes induced by flashing and continuouslight were measured with microcapillary electrodes in isolatedwhole chloroplasts of Peperomia inetallica. In continuous lightthe chloroplast electrical potential rose in two phases. Theinitial rapid phase coincided in extent with the flash-inducedpotential and was insensitive to the electron transfer inhibitorDBMIB. The subsequent phase was relatively slow (20–30ms) and was inhibited by DBMIB. Electron acceptors of photosystemII (p-phenylendiamine, p-benzoquinone) added to DBMIB-treatedchloroplasts produced a suppression of the flash-induced responseand a considerable increase in the steady level of the potentialin the light. The electrical potential associated with the activityof photosystem II rose in continuous light much more slowlythan that associated with the activity of photosystem I aloneor the activities of both photosystems. Illumination of chloroplastswith successive flashes at a repetition rate 5 Hz in the presenceof oxaloacetate, a terminal acceptor of photosystem I, was accompaniedwith a gradual decline of the flash-induced potential. The specificrole of two photosystems in the light-induced H⁺ transport andthe electrogenesis across the chloroplast thylakoid membranesis discussed.     

Photooxidation by Photosystem II of Tris-washed Chloroplasts

Irradiation of tris-washed chloroplasts with moderate intensities of red light caused a partial bleaching of chloroplast pigments and an inhibition of the hydroquinone-supported photoreduction of NADP. The presence of an electron donor for photosystem 2 (PS2) during the irradiation prevented the bleaching and inhibition. It is concluded that the strong oxidant produced by PS2 accumulates in tris-washed chloroplasts during irradiation and an electron donor for PS2 protects against the photooxidation reactions.     

Ferricyanide Reduction in Photosystem II of Spinach Chloroplasts

Ferricyanide can be reduced in Photosystem II of spinach chloroplasts at 2 separate sites, both of which are sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but only one of which is sensitive to dibromothymoquinone. Data presented in this paper emphasize ferricyanide site II of Photosystem II, which is sensitive to thiol inhibition and may reflect a cyclic pathway around Photosystem II. Ferricyanide reduction sites 1 and 2 also differ from each other in fractions isolated from discontinuous sucrose gradients, from fragmented chloroplasts, and upon trypsin treatment. Sucrose density gradient centrifugation shows that ferricyanide reduction site 1 activity at pH 6 decreases from 30 to 50% in various isolated fractions, while the dibromothymoquinone-insensitive activity at pH 8 (site 2) is stimulated from 15 to 35%.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C₄ plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg²⁺ chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.     

Characterization of Three Photosystem II Mutants in Zea mays L. Lacking a 32,000 Dalton Lamellar Polypeptide

Two fully blocked and one partially blocked photosystem II nuclear mutants have been selected in Zea mays. The fully blocked mutants lack photosystem II activity, variable fluorescence, the light-inducible C-550 signal, the high potential form of cytochrome b-559, and most or all of the low potential form of the cytochrome. The block in these mutants may primarily affect the reducing side of photosystem II, inasmuch as chloroplasts isolated from both mutants exhibit an elevated F695 fluorescence emission peak. The partially blocked mutant exhibits partial photosystem II activity and a reduction, but not the total loss of the variable fluorescence yield, the C-550 signal, and the high potential form of cytochrome b-559. Lamellae isolated from the fully blocked mutants are greatly deficient for a major lamellar polypeptide with an apparent molecular weight of 32,000 daltons, whereas lamellae from the partially blocked mutant show the partial loss of this same polypeptide, suggesting that the 32,000 dalton polypeptide is necessary for the proper function of photosystem II.     

Structural Associations Between Mitochondria and Chloroplasts in the Bundle Sheath Cells of Portulaca oleracea

Mitochondria and chloroplasts are structurally associated inthe bundle sheath cells of Portulaca oleracea L., an NAD malicenzyme type C₄ plant. These associations occur in some restrictedregions where the mitochondrial cristae extend inwards. Exposureof plants to 1.0 µ11^–1 SO₂ for 3 h induced shrinkageof mitochondria of the bundle sheath cells, which further visualizedthe structural association between the organelles. C₄ plant, chloroplast, mitochondrion, Portulaca oleracea, sulphur dioxide     

Centripetal Disposition of Bundle Sheath Chloroplasts During the Leaf Development of Eleusine coracana

Distribution of chloroplasts in bundle sheath cells was examinedby light and electron microscopy during the leaf developmentof finger millet (Eleusine coracana Gaertn.), an NAD malic enzymetype C₄ plant with centripetal arrangement of bundle sheathchloroplasts. Young chloroplasts are almost evenly distributedalong the cell walls in bundle sheath cells of folded immatureleaves. In elongating leaves and above the elongation zone thebundle sheath chloroplasts tend to lie along the radial wallsand the walls adjacent to the vascular bundle. They furthermigrate near to the vascular bundle and finally establish acentripetal arrangement. Mitochondria, microbodies and nucleusmigrate along with the chloroplasts. Etioplasts and other organellesare centripetally located in the bundle sheath cells of etiolatedseedlings grown in the dark. Bundle sheath chloroplast, C₄ plant, chloroplast, chloroplast orientation, Eleusine coracana, finger millet     

Cytokinins from Zea mays

A number of adenine derivatives with cytokinin activity were isolated from immature sweet corn (Zea mays) kernels. The following structures were assigned: 9-β-d-ribofuranosylzeatin, 9-β-d-ribofuranosylzeatin 5′-monophosphate, 6-(1-carboxy-2-hydroxypropylamino)-9-ribofuranosylpurine, 6-(2,3,4-trihydroxy-3-methylbutylamino)purine, 2-hydroxy-6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine, 6-(3,4-dihydroxy-3-methylbutylamino)purine, a 9-glycoside of zeatin(identity of sugar moiety not established), and 6-(1,2-dicarboxyethylamino)-9-β-d-ribofuranosylpurine.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

Photochemical Characteristics of Mesophyll and Bundle Sheath Chloroplasts from C4 Plants

Mesophyll and bundle sheath chloroplasts were isolated by differential grinding from the leaves of two NADP-ME C₄ plants, Setaria italica Beauv. cv. H-1, Pennisetum typhoides S & H. cv. AKP-2, and a NAD-ME C₄ species Amaranthus paniculatus L. The mesophyll chloroplasts of C₄ plants possessed slightly lower K_m for ADP and Pi than those of bundle sheath chloroplasts. The Hill reaction activities and noncyclic photophosphorylation rates of the bundle sheath chloropiasts from S. italica and P. typhoides were less than one-fifth of those by the mesophyll chloroplasts. But the bundle sheath chloroplasts of A. paniculatus exhibited high rates of Hill reaction, cyclic as well as noncyclic photophosphorylation. The pigment- and eyiochrome composition suggested a relative enrichment of PS 1 in bundle sheath chloroplasts of S. italica and P. typhoides. The chain exists in both mesophyll and bundle sheath chloroplasts. As much as 35–52% of leaf chlorophyll was located in the bundle sheath chloroplasts. The photochemical activities of bundle sheath chloroplasts are significant though a major part of leaf photochemical potential is associated with the mesophyll chloroplasts.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.