Maize Bundle Sheath Research Articles

NADP-malic enzyme from maize leaf: Regulatory properties

The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 m m malate depending on the Mg 2+ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 m m Mg 2+ than at 5–10 m m Mg 2+ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg 2+ concentration at 3 m m malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg 2+ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of NADPH NADP increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO 3 −, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO 2 fixation in maize bundle sheath chloroplasts during C 4 photosynthesis.

A comparison based on delayed light emission and fluorescence induction of intact chloroplasts isolated from mesophyll protoplasts and bundle-sheath cells of maize.

Chloroplasts or cells from maize (Zea mays) bundle sheath show a very low intensity of delayed light emission compared with mesophyll protoplasts or chloroplasts. The bundle-sheath chloroplasts retain only the fast (less than 1 ms) component of the emission. They also fail to show fluorescence induction in contrast to the mesophyll, which behaved normally. The mesophyll material could be made to resemble the bundle-sheath chloroplasts in respect of both phenomena by adding to it 1-(3',4'-dichlorophenyl)-3,3-dimethyl-urea and hydroxylamine, together. It is concluded that Photosystem II, that gives rise to both effects, is not active in the bundle sheath but may be present in an inhibited form.

Photosynthetic Electron Transport in Isolated Maize Bundle Sheath Cells

Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (mueq/hr . mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 mueq/hr . mg Chl. This was increased to 200 to 350 mueq/hr . mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 mueq/hr . mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 mueq/hr . mg Chl).When the tissue fragments were illuminated in a weakly buffered suspension, a reversible rise in the medium pH was observed which apparently originated from H(+) translocation in the thylakoids. The kinetics of the pH changes was rather slow (t((1/2)) > 15 seconds for pH rise; > 30 seconds for dark decay) but the extent of H(+) uptake was substantial (0.1 to 0.3 mueq/mg Chl). All of the electron transport reactions tested, including partial reactions which involve only photosystem I or photosystem II, invariably supported H(+) uptake. This suggests that two sites of energy conservation are associated with the photosynthetic chain in the bundle sheath chloroplasts (as in spinach chloroplasts) and that both of these sites are functional in vivo. The pH changes observed in the absence of exogenous electron carriers were abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea or by anaerobiosis, indicating that the underlying endogenous electron transport was strictly a pseudocyclic reaction. There was no evidence of endogenous cyclic electron flow which might contribute to the energy metabolism of the bundle sheath cells.

Inhibition of Photorespiration and Increase of Net Photosynthesis in Isolated Maize Bundle Sheath Cells Treated with Glutamate or Aspartate

Net photosynthetic (14)CO(2) fixation by isolated maize (Zea mays) bundle sheath strands was stimulated 20 to 35% by the inclusion of l-glutamate or l-aspartate in the reaction mixture. Maximal stimulation occurred at a 7.5 mm concentration of either amino acid. Since the photosynthetic rate and the glutamate-dependent stimulation in the rate were equally sensitive to a photosynthetic electron transport inhibitor, 3-(p-chlorophenyl)-1,1-dimethylurea, it was concluded that glutamate did not stimulate CO(2) fixation by supplying needed NADPH (NADH) through glutamate dehydrogenase. Treatment of the bundle sheath strands with glutamate inhibited glycolate synthesis by 59%. Photorespiration in this tissue, measured as the O(2) inhibition of CO(2) fixation (the Warburg effect), was inhibited by treatment with glutamate. The stimulation in net photosynthetic CO(2) fixation probably results from the decrease in photorespiratory CO(2) loss. This metabolic regulation of the rate of glycolate synthesis and photorespiration observed with isolated bundle sheath strands could account for the inability to detect rapid photorespiration in the mature intact maize leaf.

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.The unappressed membranes (stroma lamellae and bundle sheath plastid membranes) were enriched in heavy (60-40 kilodaltons) peptides and depleted in light (31-20 kilodaltons) peptides as compared to stacked grana membranes. The heavier peptides were tentatively identified as subunits of chloroplast coupling factor. These peptides in unappressed membranes were much more resistant to removal by washing with ethylenediaminetetraacetate (under conditions of low ionic strength) than they were in granal membranes.Ribulose-1,5-diphosphate carboxylase was identified on the gels and was localized exclusively in the bundle sheath cells. It is suggested that sodium dodecyl sulfate electrophoresis is a simple method to test for the localization of carboxylase in various C(4) plastid fractions.

Photosystem II in Symbiotic Algae

Symbiotic brown algae (zooxanthellae), Gymnodinium microadriaticum (= Symbiodinium microadriaticum), were isolated from the coral polyp Pocillopora damicornis and from the mantle of the clam Hippopus hippopus collected from coral reefs near Lizard Island on the Great Barrier Reef. Passage of the coral and clam zooxanthellae through a Yeda Press at 2000 and 21 000 p.s.i., respectively, yielded preparations of chloroplast lamellae with the ratio of chlorophyll α to chlorophyll c ranging from 0.95 to 1.2. The chloroplast preparations photoreduced 2,6-dichlorophenolindophenol (DCPIP) at rates of 1.66 (coral zooxanthellae chloroplasts) and 1.96 (clam zooxanthellae chloroplasts) micromoles DCPIP reduced per minute per milligram total chlorophyll in white light. Rates were 30-50% lower in red light. The photoreduction was inhibited more than 99% by 2.5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Comparisons made with maize chloroplasts indicated that the clam zooxanthellae chloroplasts required only slightly higher light intensities for maximum rates of photoreduction of DCPIP than the grana-containing maize mesophyll chloroplasts. The coral zooxanthellae chloroplasts required a still higher light intensity for near saturation of the photoreduction of DCPIP, but not as high as that required by maize bundle sheath chloroplasts. Chloroplasts prepared from green algae isolated from the tissues of a tunicate of unknown species also photoreduced DCPIP but at low rates. Light saturation for the reaction was attained at around the same intensity as for the clam zooxanthellae chloroplasts. Based on the photosynthetic rate of zooxanthellae isolated from the clam Tridacna maxima and the number of cells contained in the mantle, it was concluded that the photosynthetic potential of the mantle of T. maxima, on either a chlorophyll or area basis, was about the same as that of a leaf of a C3 plant.

Inhibition by dibromothymoquinone of photosynthetic electron transfer in chloroplasts of differing ultrastructure

The effect of the plastoquinone antagonist, dibromothymoquinone, on the photoreduction of ferricyanide and plastocyanin by maize mesophyll, maize bundle-sheath and Euglena gracilis chloroplasts has been investigated. Maximum inhibition of FeCN and plastocyanin reduction by mesophyll chloroplasts was obtained at dibromothymoquinone concentrations of 5 × 10 −7 m. At higher concentrations dibromothymoquinone acted as an electron shuttle, increasing the rate of reduction of both substrates. In contrast, little inhibition of FeCN and plastocyanin reduction by bundle-sheath chloroplasts occurred at 5 × 10−7 m dibromothymoquinone, and above this concentration of inhibitor, the extent of inhibition increased, with no shuttle effect being observed. Euglena chloroplasts showed a response intermediate between that of mesophyll and bundle-sheath chloroplasts. The presence of a shuttle effect caused by dibromothymoquinone appears to be directly related to the presence of a proton pump in the chloroplast preparations. Plastocyanin is reduced by photosystem 2 alone and shows some of the properties of a class III electron acceptor, although the rates of reduction observed were much lower than those observed with lipophilic class III acceptors.

Membrane polypeptides of some higher plant chloroplasts

Sodium dodecylsulfate-polyacrylamide gel electrophoresis of membrane polypeptides of the mesophyll cell chloroplasts of barley, pea, and maize show similar profiles, with the polypeptides falling into two major groups: those associated with a membrane fraction enriched in Photosystem I (called Group I polypeptides) and those associated with a membrane fraction enriched in Photosystem II (called Group II polypeptides a, b, and c). In contrast to these profiles, the polypeptides from the extensively unstacked membranes of chloroplasts from the chlorophyll-deficient mutant strains of barley and pea as well as those obtained from the agranal bundle sheath cell chloroplasts of maize are deficient in the Group II polypeptides b and c. It is proposed that these polypeptides are required for membrane stacking in higher plant chloroplasts. These Group II polypeptides b and c are not required for Photosystem II activity since both the barley and pea mutant chloroplasts and the maize bundle sheath chloroplasts possess Photosystem II activities.

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 C(4) 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 H(2)O. (The rate of methyl viologen photoreduction in bundle sheath chloroplasts was 40% of that of mesophyll chloroplasts.).

Photosynthetic Carbon Metabolism in Isolated Maize Bundle Sheath Strands

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO(2) per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic (14)CO(2) fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath (14)CO(2) fixation. After 5 minutes of photosynthesis in (14)C-bicarbonate, 95% of the incorporated (14)C was found in compounds other than C(4)-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of (14)C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of "malic" enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP(+)-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize.

The Warburg effect in maize bundle sheath photosynthesis

Bundle sheath strands isolated from maize are capable of photosynthesizing glycolate in the presence of an inhibitor of glycolate oxidase. Glycolate synthesis is stimulated by high oxygen concentrations and suppressed by high levels of CO 2. This data, together with the reversible oxygen inhibition of bundle sheath photosynthesis reported earlier [Ref. 6], demonstrates the occurrence of the Warburg effect in maize bundle sheath photosynthesis. These results also suggest a mechanism to account for the inhibition of photo-respiration in intact maize leaves.

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.Chloroplast fragments isolated from bundle sheath cells showed a light-dependent reduction of potassium ferricyanide, 2, 6-dichlorophenolindophenol, mammalian cytochrome c, plastocyanin, and Euglena cytochrome c(552). These activities were inhibited by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea at 1.25 micromolar. However, the photoreduction of NADP from water was extremely low or absent, except in chloroplasts from very young leaves, and the capacity for NADP reduction appeared to be related to the degree of grana formation.Photosystem I activity was present in bundle sheath chloroplast preparations at all stages of leaf growth and senescence examined. However, the activity was lower than in isolated mesophyll chloroplasts. NADPH diaphorase activity was comparable in both types of chloroplast.Chloroplasts isolated from bundle sheath cells of plants grown under a variety of conditions, including continuous and intermittent light, high and low light intensities, and high temperature, exhibited photosystem II activity.

Oxygen inhibits maize bundle sheath photosynthesis

Oxygen inhibits photosynthetic CO 2 fixation in isolated maize bundle sheath strands. This inhibition is rapidly and completely reversible and is relieved by increased levels of CO 2. These observations provide evidence that the relative oxygen-insensitivity of photosynthesis in intact maize leaves results from a CO 2 concentrating mechanism.