Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport

 Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C₄ plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed. Received: 25 January 2000 / Accepted: 30 March 2000     

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C₄ phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro ³²P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH₄Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca²⁺ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca²⁺ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C₄ MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C₄-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C₄ mesophyll cytosol.Abbreviations BS bundle-sheath - CAM Crassulacean acid metabolism - DHAP dihydroxyacetone phosphate - FPLC fast-protein liquid chromatography - Glc6P glucose 6-phosphate - I_0.5 50% inhibition constant - MC mesophyll cell(s) - MP me-sophyll protoplast(s) - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC protein-Ser/Thr kinase - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - Pyr pyruvate - Ser serine The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH¹³CO₃ was followed by ¹³C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ~5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH¹³CO₃ treatment, ¹³C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ~6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.     

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 mm malate depending on the Mg²⁺ 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 mm Mg²⁺ than at 5–10 mm Mg²⁺ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg²⁺ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg²⁺ 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 $\text{NADPH}\text{NADP}$ 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 HCO₃^?, 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 CO₂ fixation in maize bundle sheath chloroplasts during C₄ photosynthesis.     

Photodestruction of Chlorophyll inZea mays L. Leaves Under Different CO2 Concentration

Photodestruction of chlorophyll (Chl) inZea mays leaves, after their irradiation with high photon fluence rate (5000 μmol m⁻² s{−1}), was determined in fragments of whole leaves (WL) and also in fraction of mesophyll cells (MC) and bundle sheath cells (BSC) after their mechanical separation. The lag phase and the phase of photooxidation of Chl in MC chloroplasts were shorter than in BSC. Duration of both phases was reduced when the leaves were placed in 0 % CO₂ concentration in the atmosphere, while the increase of CO₂ concentration up to 0.3 % totally protected Chl against photodestruction in BSC within the 9 h experiment. During that period of time Chl was destructed by about 30 % in MC.     

Distribution and Properties of Glycolate Oxidase from Bundle Sheath and Mesophyll Cells of Green Amaranth Leaves (<Emphasis Type="Italic">Amaranthus retroflecsus</Emphasis>)

Separation of mesophyll and bundle sheath cells (MC and BSC) from the leaves of green amaranth (Amaranthus retroflexus L.) showed that glycolate oxidase (GO, EC 1.1.3.35) is located predominately in BSC (on the average, 84.5% of the total activity). Three peaks of GO activity were detected following the elution from a DEAE-fractogel column. The first peak corresponded to the isoform located in BSC, the second peak had dual location, and the third one was associated with MC fraction. Elaborated flow sheet of GO purification from the amaranth leaves produced highly purified (by 63.5 times) isoforms from MC and BSC with specific activity of 0.54 EU/mg protein. It was also shown that GO from MC has greater affinity for glycolate, with the K _M values for GO from BSC and MC being 58 and 20 µM, respectively. Intermediates of the Krebs cycle were shown to affect the GO activity from MC and BSC: succinate suppressed and isocitrate activated GO.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 622–627.Original Russian Text Copyright © 2005 by Eprintsev, Ivent’ev, Popov.     

Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1 M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn²⁺ and Cu²⁺ in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn²⁺ and Cu²⁺ in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.     

Maize F<Subscript>1</Subscript> hybrid differs from its maternal parent in the development of chloroplasts in bundle sheath,but not in mesophyll cells: Quantitative analysis of chloroplast ultrastructure and dimensions in different parts of leaf blade at the beginning of its senescence

The quantitative changes of chloroplast ultrastructure and dimensions in mesophyll (MC) and bundle sheath (BSC) cells, associated with the onset of leaf senescence, were followed along the developmental leaf blade gradient of the third leaf of maize (Zea mays L.). To ascertain whether the rapidity of structural changes associated with the transition of chloroplasts from mature to senescent state is a heritable trait, the parental and the first filial generations of plants were used. The heterogeneity of leaf blade, associated with the development of maize leaf (with the oldest regions at the apex and the youngest ones at the base) was clearly discernible in the ultrastructure and dimensions of chloroplasts; however, there were differences in the actual pattern of chloroplast development between both genotypes as well as between both cell types examined. While the course of MC chloroplasts’ development at the onset of leaf senescence in maize hybrid followed that of its parent rather well, this did not apply for the BSC chloroplasts. In this case, each genotype was characterized by its own distinguishable developmental pattern, particularly as regards the accumulation of starch inclusions and the associated changes of the size and shape of BSC chloroplasts.     

Expression and properties of the mitochondrial and cytosolic forms of fumarase in germinating maize seeds

Fumarase (EC 4.2.1.2) catalyzes reversible interconversion of malate and fumarate. It is usually associated with the tricarboxylic acid cycle in mitochondria, although the cytosolic form has also been detected. We investigated the expression of two fumarase genes and activities of the mitochondrial and cytosolic isoforms of fumarase in maize (Zea mays) scutellum during germination. Both isoforms were purified to electrophoretic homogeneity. The cytosolic form had low optimum pH (6.5) and high affinity to malate (K_m 5 μM) when compared with the mitochondrial form (optimum pH 7.0, K_m 50 μM). The cytosolic form was strongly activated by Mg²⁺ and even more by Mn²⁺, whereas the mitochondrial form was moderately activated by Mg²⁺ and Mn²⁺ was less effective. The highest fumarase activity in scutellum and a high expression of the gene encoding the cytosolic form were observed during the maximal activity of the glyoxylate cycle. In leaves, the localization of fumarase is only mitochondrial and only one fumarase gene is expressed. It is concluded that the function of cytosolic fumarase in maize scutellum can be related to metabolism of succinate formed in the glyoxylate cycle.     

Identification of promoter elements involved in the cytosolic Ca(2+)-mediated photoregulation of maize cab-m1 expression.

Changes in cytoplasmic Ca2+ levels are involved in the regulation of several plant genes. However, to our knowledge, no regions of genes or specific cis elements have been shown to be involved in the regulation of plant gene expression by cytosolic Ca2+ signaling. The maize (Zea mays) gene cab-m1, which encodes a light-harvesting chlorophyll a/b-binding apoprotein, is positively photoregulated in mesophyll cells (MC) but not in bundle-sheath cells (BSC). This gene is highly preferentially expressed in maize MC versus BSC. In situ transient expression assays have revealed that exposure of tissues to ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, blocks the photostimulation of cab-m1 full promoter (-1026 to + 14) activity in MC of leaf segments of dark-grown maize seedlings. EGTA has no effect on expression in BSC. These results suggest that light-induced elevation of the cytosolic Ca2+ concentration in MC is required for the enhancement of cab-m1 expression in MC. Deletion of the sequence from -1026 to -360 completely abolished Ca2+ responsiveness of cab-m1 expression in MC. On the other hand, a 54-bp fragment in the 5' flanking region (-953 to -899 relative to the translation start site) conferred Ca2+ responsiveness on a -359 core promoter: reporter gene, suggesting that Ca2+ signaling is mediated via specific sequences in this short fragment. Furthermore, possible involvement of Ca(2+)-calmodulin in the signal transduction chain for regulating cab-m1 expression was suggested by the results of inhibitor experiments.     

Ultrastructure and Dimensions of Chloroplasts in Leaves of Three Maize (Zea mays L.) Inbred Lines and their F1 Hybrids Grown Under Moderate Chilling Stress

Influence of moderate chilling stress on vascular bundle sheath cell (BSC) and especially mesophyll cell (MC) chloroplasts of mature maize leaves was studied by electron microscopy and stereology. Plants of two inbred lines of maize, differing in their photosynthetic activity, and their F₁ hybrids were cultivated during autumn in heated or unheated glasshouse. Generally, chilling temperatures resulted mainly in the decrease in stereological volume density (VD) of both granal and intergranal thylakoids of MC chloroplasts, while the ratio of granal to all thylakoids (granality) was less affected. The VD of peripheral reticulum and plastoglobuli usually increased after cold treatment of plants. The volume of MC chloroplasts usually increased under chilling stress, the shape of the chloroplasts changed only slightly. The ultra-structure of chloroplasts differed between individual genotypes; chilling-stressed hybrid plants showed positive heterosis particularly in the granal thylakoids' VD of MC chloroplasts.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase     

Maize and pigweed response to nitrogen supply and form

As nitrogen management practices change to achieve economic and environmental goals, effects on weed-crop competition must be examined. Two greenhouse experiments investigated the influence of N amount and form on growth of maize and redroot pigweed (Amaranthus retroflexus L.). In Experiment 1, maize and pigweed were grown together in a replacement series (maize: pigweed ratios of 0:4, 1:3, 2:2, 3:1, 4:0) under three NH₄NO₃-N supplies (0, 110, and 220 mg N kg^-1 soil). Maize was planted into established pigweed and plants were harvested 24 days after maize germination. Pigweed responded more to supplemental N than maize and accumulated 2.5 times as much N in shoots at the high N supply. Competition effects were not significant. Maize and pigweed were grown separately in Experiment 2 and supplied 220 mg N kg^-1 as either Ca(NO₃)₂ or (NH₄)₂SO₄ plus a nitrification inhibitor (enhanced ammonium supply, EAS). In maize, EAS treatment did not affect shoot growth and reduced root growth 25% relative to the NO₃-N treatment. In pigweed, shoot and root growth were restricted 23 and 86% by EAS treatment, respectively. Total plant N accumulation under EAS treatment was higher in maize, less in pigweed. Under EAS treatment, pigweed leaves were crinkled and chlorotic; leaf disks extracted in 70% ethanol, pH 3, contained less malate and oxalate but more NH₄ ⁺ compared to the NO₃-N treatment. Maize leaf disk malate levels were generally higher compared to pigweed but were less due to EAS treatment. Ammonium level in maize leaf disks was unaffected by N form and EAS treatment increased oxalate levels. Final bulk soil pH was generally lower in pots where pigweed were grown and tended to be lower due to EAS. Leaf disk malate levels and soil pH were positively associated. Results indicate that pigweed is more likely to compete with maize when high levels of NO₃-N are provided. Enhancing the proportion of N supplied as NH₄ ⁺ should restrict the growth of NH₄ ⁺-sensitive pigweed.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC–MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C₃–C₄ photosynthetic plant.