Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Photoreduction of oxygen in mesophyll chloroplasts of c(4) plants: a model system for studying an in vivo mehler reaction

Mesophyll chloroplasts of three C₄ sub types, Panicum miliaceum (NAD-malic enzyme), Panicum maximum (PCK), and Zea mays (NADP-malic enzyme), were prepared from protoplast extracts and used to study the photoreduction of O₂. The processes of O₂ uptake and evolution in these preparations, which lack ribulose 1,5-bisphosphate carboxylase/oxygenase, were studied simultaneously using stable isotopes of O₂ and mass spectrometry. The responses of O₂ uptake to O₂ tension and addition of various substrates (3-phosphoglycerate, pyruvate, and oxaloacetate) were studied in detail. The addition of photosynthetic substrates differing in ATP to NADPH demands indicated that photoreduction of O₂ in these chloroplast preparations is linked to ATP production and strongly regulated by NADP⁺ levels. The results clearly indicate that photoreduction of O₂ could be of physiological relevance in balancing the ATP to NADPH requirements of C₄ mesophyll chloroplasts.     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization

The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-³H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([³H]H₂DIDS) and by using SDS-PAGE. Assay of ³²Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C₃ counterpart and can be regarded as C₄ types.     

Intracellular localization of certain photosynthetic enzymes in bundle sheath cells of plants possessing the C4 pathway of photosynthesis.

Bundle sheath cells were enzymatically isolated from representatives of three groups of C₄ plants: Zea mays (NADP malic enzyme type), Panicum miliaceum (NAD malic enzyme type), and Panicum maximum (phosphoenolpyruvate (PEP) carboxykinase type). Cellular organelles from bundle sheath homogenates were partially resolved by differential centrifugation and on isopycnic sucrose density gradients in order to study compartmentation of photosynthetic enzymes. A 48-h-dark pretreatment of the leaves allowed the isolation of relatively intact chloroplasts. Enzymes that decarboxylate C₄ acids and furnish CO₂ to the Calvin cycle are localized as follows: NADP malic enzyme, chloroplastic in Z. mays; NAD malic enzyme, mitochondrial in all three species; PEP carboxykinase, chloroplastic in P. maximum. The activity of NAD malic enzyme in the three species was in the order of P. miliaceum > P. maximum > Z. mays. There were high levels of aspartate and alanine aminotransferases in bundle sheath extracts of P. miliaceum and P. maximum and substantial activity in Z. mays. In all three species, aspartate aminotransferase was mitochondrial whereas alanine aminotransferase was cytoplasmic. Based on the activity and localization of certain enzymes, the concept for aspartate and malate as transport metabolites from mesophyll to bundle sheath cells in C₄ species of the three C₄ groups is discussed.     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

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.     

Controls on na influx in corn roots

We have investigated the effects of hyperpolarization and depolarization, and the presence of K⁺ and/or Ca²⁺, on ²²Na⁺ influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na⁺ influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K⁺. However, added Ca²⁺ suppresses Na⁺ influx by 60%. In washed tissue which has recovered, Na⁺ influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na⁺ influx is completely eliminated by low concentrations of K⁺, leaving the same level and kind of Na⁺ influx seen in freshly excised roots. The K⁺-sensitive energy linkage appears to be by the carrier for active K⁺ influx. Calcium is equally inhibitory to Na⁺ influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na⁺ uptake was about 25% of ²²Na⁺ influx, but proportionately the response to K⁺ and Ca²⁺ was about the same.

The constancy of K⁺-insensitive Na⁺ influx under conditions known to hyperpolarize and depolarize suggests that if Na⁺ transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of ²²Na⁺ for endogenous Na⁺. The data suggest that an inwardly directed Na⁺ current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange.

     

Effects of O(2) Tension and Temperature during Light Uptake of CO(2) on the Dark Release of CO(2) by Excised C(3) and C(4) Leaves

A study was conducted on a C₄ (Panicum maximum) and a C₃ (Panicum bisulcatum) species to determine the nature of the dark release of ¹⁴CO₂ with respect to its responses to changes in temperature and O₂ tension during light CO₂ uptake of ¹⁴CO₂.     

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.     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO₃⁻ and Mg²⁺ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C₃); P. maximum (C₄ phosphoenolpyruvate carboxykinase); P. milioides (C₃/C₄); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C₃); P. miliaceum (C₄ NAD malic enzyme); Zea mays and Sorghum bicolor (C₄ NADP malic enzyme); Moricandia arvensis (C₃/C₄); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C₃ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO₂ and Mg²⁺ activation, but which can be converted to an activatable state upon exposure of the leaf to light.     

Differences in cold lability of pyruvate, Pi dikinase among C4 species

The cold lability of pyruvate, Pi dikinase in crude leaf extractswas studied in a number of C⁴ plants. The survey included C⁴monocots and dicots and species representingthe three C⁴ subgroups:NADP-malic enzyme, NAD-malic enzyme, and PEP-carboxykinase types. In some species (e.g., Digitaria sanguinalis, Sorghum bicolorand Echinochloa crus-galli), the enzyme was very sensitive tocold treatment (half life of about 8 min at 0?C and 10 to 15min at 10?C). In other species (Panicum miliaceum, Panicum maximumand Panicum texanum), the enzyme was very cold tolerant (retentionof 60 to 85% activity after 60 min at 0?C and 90% activity after60 min at 10?C). Among the plants examined, the most cold sensitivepyruvate,Pi dikinase was found among species of the NADP-malicenzyme subgroup. (Received March 9, 1979; )     

Light-Dependent Uptake of Pyruvate by Mesophyll Chloroplasts of a C4 Plant, Panicum miliaceum L.

Light-dependent active uptake of pyruvate was reported in mesophyllchloroplasts of a C₄ plant, Panicum miliaceum [Ohnishi and Kanai(1987) Plant Cell Physiol. 28: 1]. The present study tried toclarify the energy source of this active uptake. Preilluminationof the mesophyll chloroplasts increased over tenfold their pyruvateuptake in the light and dark. This indicates that light itselfis not essential for the enhancement. The pyruvate uptake capacity(the initial uptake rate) of the mesophyll chloroplasts increasedon illumination and reached a steady-state level after a fewminutes; this rise was faster under higher light intensities.When the chloroplasts were returned to darkness, the uptakecapacity decayed with a half-life of about 1 min; this was independentof the light intensity of preillumination. Illumination of thechloroplasts also increased the stromal pH from about 7 to 8and the stromal ATP level from about 5 to 15–25 nmol.(mg chl)^–1. The change of the former during dark-to-lightand light-to-dark transitions occurred within 2 to 5 min, whilethe change of the latter took place much faster within 1 min.The steady-state levels of the pyruvate uptake capacity andstromal pH were saturated at a light intensity of 3 µE.m^–2.s^–1,while the ATP level increased with a further increase in thelight intensity. The former two parameters also showed similarsensitivity to the inhibition by carbonylcyanide-m-chlorophenylhydrazone,while a higher concentration of the inhibitor was needed toreduce the ATP level. Nitrite at 4 mM inhibited the light-dependentpyruvate uptake and stromal alkalization but had little effecton the stromal ATP level, while 2 mM arsenate decreased thestromal ATP without significant effects on pyruvate uptake andstromal pH. The good correlation of pyruvate uptake and stromalpH suggests that the active pyruvate uptake by the mesophyllchloroplasts is primarily driven by the pH gradient across theenvelope. (Received August 15, 1986; Accepted December 8, 1986)     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

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.     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

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

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