Photochemical properties of mesophyll and bundle sheath chloroplasts of maize

Several photochemical and spectral properties of maize (Zea mays) bundle sheath and mesophyll chloroplasts are reported that provide a better understanding of the photosynthetic apparatus of C₄ plants. The difference absorption spectrum at 298 K and the fluorescence excitation and emission spectra of chlorophyll at 298 K and 77 K provide new information on the different forms of chlorophyll a in bundle sheath and mesophyll chloroplasts: the former contain, relative to short wavelength chlorophyll a forms, more long wavelength chlorophyll a form (e.g. chlorophyll a 693 and chlorophyll a 705) and less chlorophyll b than the latter. The degree of polarization of chlorophyll a fluorescence is 6% in bundle sheath and 4% in mesophyll chloroplasts. This result is consistent with the presence of relatively high amounts of oriented long wavelength forms of chlorophyll a in bundle sheath compared to mesophyll chloroplasts. The relative yield of variable, with respect to constant, chorophyll a fluorescence in mesophyll chloroplasts is more than twice that in bundle sheath chloroplast. Furthermore, the relative yield of total chlorophyll a fluorescence is 40% lower in bundle sheath compared to that in mesophyll chloroplasts. This is in agreement with the presence of the higher ratio of the weakly fluorescent pigment system I to pigment system II in bundle sheath than in mesophyll chloroplast. The efficiency of energy transfer from chlorophyll b and carotenoids to chlorophyll a are calculated to be 100 and 50%, respectively, in both types of chloroplasts. Fluorescence quenching of atebrin, reflecting high energy state of chloroplasts, is 10 times higher in mesophyll chloroplasts than in bundle sheath chloroplasts during noncyclic electron flow but is equal during cyclic flow. The entire electron transport chain is shown to be present in both types of chloroplasts, as inferred from the antagonistic effect of red (650 nm) and far red (710 nm) lights on the absorbance changes at 559 nm and 553 nm, and the photoreduction of methyl viologen from H₂O. (The rate of methyl viologen photoreduction in bundle sheath chloroplasts was 40% of that of mesophyll chloroplasts.)     

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.     

Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis

The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C₄ plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C₄ dicarboxylic acid pathway—phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase—were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase—enzymes of the reductive pentose phosphate pathway—were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.     

Chlorophyll a forms in mesophyll and bundle sheath chloroplasts of Zea mays leaves grown at low light intensity

Mesophyll and bundle sheath chloroplasts were prepared fromleaves of Zea mays grown at light intensities of 1.1 and 240µW/cm², respectively. The mesophyll chloroplasts thatdeveloped at the low intensity and bundle sheath chloroplatsthat developed at both low and high intensities showed higherratios of chlorophyll a/b and P700/chlorophylls compared withthe normal ratios found for the mesophyll chloroplasts thathad developed at the high intensity. Derivative absorption spectrophotometryat 77?K revealed that the low intensity mesophyll chloroplastscontained more of chlorophyll a forms with longer wavelengthred bands than high intensity mesophyll chloroplasts. More ofthe longer wavelength forms of chlorophyll a were also presentin the bundle sheath chloroplasts that had developed at lowand high intensities. All these four types of chloroplasts showedtwo peaks of fluorescence, one at 687 hra and the other at 733or 738 nm. In addition to these peaks, the high intensity mesophyllchloroplasts showed a shoulder at 697 nm, and the two typesof bundle sheath chloroplasts showed a shoulder at 680 nm. (Received June 17, 1974; )     

Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C₄ groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.     

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.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

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.     

Comparison of photosynthetic activities of spinach chloroplasts with those of corn mesophyll and corn bundle sheath tissue

Bundle sheath and mesophyll chloroplasts from Zea mays showed comparable rates of O₂ evolution, which amounted to about half of the rate observed in spinach (Spinacia oleracea) chloroplasts.

Ratios of 4.5, 4.6, and 6.2 Mn²⁺ atoms per 400 chlorophylls were observed in mesophyll, bundle sheath, and spinach chloroplasts, respectively. These ratios roughly correspond to the observed O₂ evolution rates.

Rates of electron transport from water to methylviologen (photosystem I and II) in both types of corn chloroplasts were about one-third that in spinach. Compared to spinach, transport rates from reduced diaminodurene to methylviologen (photosystem I) were about one-third and greater than one-half in mesophyll and bundle sheath material, respectively.

In both types of corn chloroplasts, electron flow from photosystem II to P700 was abnormal. This observation, together with the low rates of all activities, suggests that damage occurred during isolation. Such damage may limit the quantitative significance of observations made with these materials (including the following data).

Measurements of flash yields of O₂ evolution or O₂ uptake showed that the size of the photosynthetic unit was the same in photosystems I and II and in all three types of chloroplasts (about 400 chlorophylls per equivalent).

Similarity of the photochemical cross-section of the two photosystems in the three preparations was also found in optical experiments: that is the half-times of the fluorescence rise in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (photosystem II) and of the photooxidation of P700 (photosystem I).

The ratio of P700 to chlorophyll appeared to be about 2-fold higher in bundle sheath chloroplasts than in the other materials (1/200 versus 1/400).

     

Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia

Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C₄ dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4×, 6×, and 8×, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Isolation of Mesophyll Cells and Bundle Sheath Cells from Digitaria sanguinalis (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology

A technique is described for the separation of mesophyll and bundle sheath cells from Digitaria sanguinalis leaves and evidence for separation is given with light and scanning electron micrographs. Gentle grinding of fully differentiated leaves in a mortar releases mesophyll cells which are isolated on nylon nets by filtration. More extensive grinding of the remaining tissue yields bundle sheath strands which are isolated by filtration with stainless steel sieves and nylon nets. Further grinding of bundle sheath strands in a tissue homogenizer releases bundle sheath cells which are collected on nylon nets. Percentage of purity derived from cell counts and yield data on a chlorophyll basis are given.     

Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass)

Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C₄ grass Digitaria sanguinalis (L.) Scop. Two-dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.

Protein patterns were also compared with C₄ plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C₄ species were found to be more like bundle sheath proteins of another C₄ species than like mesophyll proteins of the same species.

     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.     

Differences in Lipid Composition between Undifferentiated and Mature Maize Chloroplasts

Lipid compositions of undifferentiated maize (Zea mays) chloroplasts, capable of fixing CO₂, were compared with the lipid compositions of mature chloroplasts, which do not fix CO₂, located in both the mesophyll and bundle sheath cells. The major lipids found in all three chloroplast types were the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride, followed by decreasing amounts of sulfolipid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol, and diphosphatidyl glycerol. Quantitative differences in lipid components were observed among the chloroplast types. The mesophyll and bundle sheath maize chloroplasts differed in their chlorophyll a/chlorophyll b ratios (2.27 and 4.13 respectively) and their content of glycolipid relative to chlorophyll (51.8% glycolipid to 20.9% chlorophyll and 84.5% glycolipid to 10.1% chlorophyll respectively). A comparison between the lipid compositions of maize mesophyll chloroplasts and mesophyll chloroplasts obtained from spinach, sugar beet, and tobacco showed many similarities.     

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.     

Photoreduction and Oxidation of Cytochrome f in Bundle Sheath Cells of Maize

The photo-oxidation of cytochrome f (cytochrome c₅₅₄) in bundle sheath cells isolated from leaves of maize (Zea mays var. DS 606A) has been compared with that in intact maize leaf and in isolated pea leaf cells (Pisum sativum L.). In all cases, illumination with red light caused a negative absorbance change at 554 nm which was attributed to the oxidation of cytochrome f. The extent of this change was greater using monochromatic red light at wavelengths above 700 nm compared with wavelengths below 700 nm. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea abolished this difference in bundle sheath cells. After illumination for 1 minute or longer in bundle sheath cells, reduction of cytochrome f in the dark was rapid only if the wavelength of the illuminating light was below 700 nm. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea, reduction was slow after illumination at all wavelengths.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.