Transport of Metabolites across Isolated Envelope Membranes of Spinach Chloroplasts

Isolated envelope membranes of spinach chloroplasts (Spinacia oleracea L. var. Viroflay) exhibited selective permeability. Metabolites such as 3-phosphoglycerate, bicarbonate, glyoxylate, and acetate were transported rapidly; 6-phosphogluconate, glycolate, glycine, l-malate, and succinate were intermediate; whereas glucose 6-phosphate, fructose 1,6-diphosphate, and sucrose were hardly transported. Transport rates, metabolite accumulations within the membrane vesicles, and the internal water volume of isolated and in situ envelope membranes were compared and found to show similar trends.     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.     

Photochemical Activity of Chloroplasts Isolated from Etiolated Plants

The rates of phosphorylation, ferricyanide, and dye reductionwere determined with chloroplasts isolated from Linum usitatissimumgrown in darkness and subjected to periods of light of differentduration. With an increased period of illumination, the chlorophyllcontent increased as did also the rate of the three processesmeasured, but no correlation between these two factors was observed.Neither was there any correlation between the rate of any photochemicalreaction and the plastoquinone content. It was concluded thatsome unspecified factor, possibly structural, which developsduring illumination must control the rate of the photochemicalreactions.     

Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

The photosystem II core complex (TSF-IIa) is composed of polypeptides of molecular weight 54-, 47-, 42-, and 30 kilodaltons (kD) and cytochrome b-559. After treatment with trypsin or α-chymotrypsin for 20 hours, the TSF-IIa particles still retained their photochemical activity and the light-induced cytochrome b-559 signal, although all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex from 250,000 to 100,000 daltons as determined by gel filtration, and also decreased the protein to chlorophyll ratio by 40%. Chlorophyll a appeared to be associated with the 47- and 42 kD polypeptides. Proteolysis of the complex produced a single chlorophyll a band with a slightly higher electrophoretic mobility. This band was not equivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), but did not completely abolish it.     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

Identification of the Main Species of Tetrapyrrolic Pigments in Envelope Membranes from Spinach Chloroplasts

The chlorophyll precursors protochlorophyllide and chlorophyllide were identified in purified envelope membranes from spinach (Spinacia oleracea) chloroplasts. This was shown after pigment separation by high performance liquid chromatography (HPLC) using specific fluorescence detection for these compounds. Protochlorophyllide and chlorophyllide concentrations in envelope membranes were in the range of 0.1 to 1.5 nmol/mg protein. Chlorophyll content of the envelope membranes was extremely low (0.3 nmol chlorophyll a/mg protein), but the molar ratios of protochlorophyllide and chlorophyllide to chlorophyll were 100 to 1000 times higher in envelope membranes than in thylakoid membranes. Therefore, envelope tetrapyrrolic pigments consist in large part (approximately one-half) of nonphytylated molecules, whereas only 0.1% of the pigments in thylakoids are nonphytylated molecules. Clear-cut separation of protochlorophyllide and chlorophyllide by HPLC allowed us to confirm the presence of a slight protochlorophyllide reductase activity in isolated envelope membranes from fully developed spinach chloroplasts. The enzyme was active only when envelope membranes were illuminated in the presence of NADPH.     

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Temperature-induced Changes in Hill Activity of Chloroplasts Isolated from Chilling-sensitive and Chilling-resistant Plants

The effect of temperature on Hill activity has been compared in chilling-sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photoreduction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea (Pisum sativum L. cv. Massay Gem), a chilling-resistant plant, likewise increased at temperatures below 14 C. A second change in Ea occurred at higher temperatures. The Ea decreased above about 28 C for mung bean, 30 C for maize, and 25 C for pea. At temperatures approaching 40 C, thermal inactivation of Hill activity occurred. These results, when taken together with previous results obtained with the chilling-resistant plant barley, indicate that chloroplasts from both chilling-sensitive and chilling-resistant plants can undergo a change in chloroplast membrane activity at low temperatures above freezing and that the presence of such a change in chloroplast membranes is not necessarily correlated with chilling sensitivity.     

Polypeptide Compositions of Stroma, Internal Membranes, and Inner and Outer Envelope Membranes of Amyloplast from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Intact amyloplasts isolated from liquid-cultured white-wildcells of sycamore (Acer pseudoplatanus L.) were further subfractionatedinto internal membranes (d=1.05g/ml), envelope membranes (d=1.12g/ml)and stromal fraction, which contained each characteristic polypeptidecomposition as revealed by the Na-dodecyl sulfate polyacrylamidegel electrophoresis. Absorption spectra of internal and envelopemembranes were distinctly different. By the immunoblotting analysis,it was shown that the amyloplast envelope membranes contain31 kDa P_i-translocator, although it is not the predominant polypeptidecomponent in contrast to the case of chloroplast envelope. Onehundred kDa -l,4-glucan phosphorylase (plastid type) was detectedin the stromal fraction of amyloplasts using the specific antibodyraised against the major form of -l,4-glucan phosphorylase frompotato tuber. Amyloplast envelopes were further separated into inner and outermembrane fractions by the freezing-thawing method originallydeveloped for the separation of chloroplast envelope membranesby Cline and associates (1981) (Proc. Natl. Acad. Sci. USA 78:3595–3599). Nadodecylsulfate gel electrophoretic analysisrevealed that the inner and outer envelope membranes containthe distinctly different polypeptide compositions. ¹Supported by grants from the Ministry of Education, Scienceand Culture (Mombusho) of Japan. This is paper No. 77 in theSeries "Structure and Function of Chloroplast Proteins". ²Recipient of a predoctoral student fellowship from the Japanesegovernment (Mombusho). Permanent address: Department of Biochemistry,Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ³Permanent address: Department of Biology, Southwest AgriculturalUniversity, BeiBei Chongqing, People's Republic of China. Holderof the Chinese Government Scholarship (1987) (Received May 27, 1988; Accepted August 30, 1988)     

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)     

C3、C4和C3-C4中间型植物的进化

介绍了有关C3、C4和C3-C4中间型植物进化的形态学、生理学、分子生物学、遗传学等方面的证据;推断地球上首先出现C3植物,然后是C3-C4中间类型植物,最后出现C4植物.     

Differential Mobility of Pigment-Protein Complexes in Granal and Agranal Thylakoid Membranes of C3 and C4 Plants

The photosynthetic performance of plants is crucially dependent on the mobility of the molecular complexes that catalyze the conversion of sunlight to metabolic energy equivalents in the thylakoid membrane network inside chloroplasts. The role of the extensive folding of thylakoid membranes leading to structural differentiation into stacked grana regions and unstacked stroma lamellae for diffusion-based processes of the photosynthetic machinery is poorly understood. This study examines, to our knowledge for the first time, the mobility of photosynthetic pigment-protein complexes in unstacked thylakoid regions in the C₃ plant Arabidopsis (Arabidopsis thaliana) and agranal bundle sheath chloroplasts of the C₄ plants sorghum (Sorghum bicolor) and maize (Zea mays) by the fluorescence recovery after photobleaching technique. In unstacked thylakoid membranes, more than 50% of the protein complexes are mobile, whereas this number drops to about 20% in stacked grana regions. The higher molecular mobility in unstacked thylakoid regions is explained by a lower protein-packing density compared with stacked grana regions. It is postulated that thylakoid membrane stacking to form grana leads to protein crowding that impedes lateral diffusion processes but is required for efficient light harvesting of the modularly organized photosystem II and its light-harvesting antenna system. In contrast, the arrangement of the photosystem I light-harvesting complex I in separate units in unstacked thylakoid membranes does not require dense protein packing, which is advantageous for protein diffusion.In higher plants, the photosynthetic apparatus is compartmentalized in the specialized chloroplast organelle. The molecular machinery for the primary photosynthetic processes, the sunlight-driven generation of metabolic energy equivalents, is harbored in an intricate thylakoid membrane system within the chloroplasts. Recent improvements in electron tomography have led to three-dimensional models of the complex architecture of thylakoid membranes (Mustárdy and Garab, 2003; Nevo et al., 2009; Austin and Staehelin, 2011; Daum et al., 2010; Kouřil et al., 2011). Although important details about the thylakoid structure are still highly controversial, consensus exists about the overall design of this membrane system. The thylakoid membrane consists of two morphologically distinct domains: strictly stacked cylindrical grana regions with a diameter of 300 to 600 nm are interconnected by unstacked stroma lamellae, thus forming a continuous membrane system. The molecular complexes that catalyze energy transformation are distributed heterogeneously between the stacked and unstacked membrane regions. The majority of the PSII complex and light-harvesting complex II (LHCII) are localized in stacked thylakoid regions, whereas PSI and the ATP-synthase complex are lacking from stacked grana (Staehelin and van der Staay, 1996; Albertsson, 2001; Dekker and Boekema, 2005). It is assumed that the fifth photosynthetic protein complex (cytochrome b₆f complex) is homogenously distributed.An essential feature of the thylakoid membrane system is its high flexibility, which is required for adaptability and maintenance of the photosynthetic machinery in plants. Highly responsive to environmental conditions, both the overall thylakoid architecture (e.g. number of grana discs) and the molecular membrane composition can change remarkably to optimize, protect, and maintain the photosynthetic apparatus (Walters, 2005; Anderson et al., 2008; Chuartzman et al., 2008; Dietzel et al., 2008; Betterle et al., 2009; Johnson et al., 2011). The underlying molecular processes require brisk protein traffic between stacked and unstacked thylakoid domains (Kirchhoff, 2008). The role of grana in these transport-based processes is poorly understood.Although photosynthetic energy conversion is possible without grana (Anderson et al., 2008), the fact that stacked thylakoids are ubiquitous in almost all land plants (with the exception of chloroplasts in bundle sheath [BS] cells in some C₄ plants; see below) highlights the evolutionary pressure to preserve this complex structural feature. Recently, the importance of grana formation was highlighted in Arabidopsis (Arabidopsis thaliana) mutants that lack the GRANA-DEFICIENT CHLOROPLAST1 gene; they grow much slower than the wild type and exhibit seed lethargy due to missing grana formation (Cui et al., 2011). The functional advantages of grana formation have been discussed extensively (Trissl and Wilhelm, 1993; Mullineaux, 2005; Anderson et al., 2008). It was hypothesized that grana could (1) increase the thylakoid membrane area, and the pigment concentration, in chloroplasts, (2) avoid energy spillover from PSII to PSI, (3) regulate the balance of energy distribution between PSII and PSI by state transition, and (4) enable transversal exciton energy transfer between adjacent grana discs. Although there are good arguments that these possibilities are important for photosynthetic energy conversion, the basis for the evolutionary development of grana has not been determined (Mullineaux, 2005; Anderson et al., 2008).A less considered aspect of grana formation is that it leads to a concentration of protein complexes (Murphy, 1986; Kirchhoff, 2008). The membrane area fraction that belongs to integral photosynthetic protein complexes is about 70%, making grana discs one of the most crowded biomembranes (Kirchhoff, 2008). Light harvesting by PSII benefits from a high protein-packing density for two reasons. First, a concentration of PSII and LHCII in grana ensures a high concentration of light-absorbing pigments that increase the probability of capturing sunlight, which is a “dilute” energy source on the molecular scale (Blankenship, 2002). Second, it has been demonstrated that a high protein-packing density in grana thylakoids is required for efficient intermolecular exciton energy transfer between LHCII and PSII (Haferkamp et al., 2010). Macromolecular crowding ensures that weakly interacting LHCII and PSII complexes come in close contact, allowing efficient Förster-type energy transfer.Besides these advantages, lateral protein traffic is challenged by macromolecular crowding (Mullineaux, 2005; Kirchhoff, 2008). The molecular mobility of proteins in grana thylakoids is reduced by numerous collisions of the diffusing object in the two-dimensional reaction space of the membrane with obstacles, integral membrane proteins, that increase the apparent diffusion path and, consequently, the diffusion time. The strong impairment of a high protein density in grana thylakoids on protein mobility was demonstrated by computer simulations (Tremmel et al., 2003; Kirchhoff et al., 2004) and by diffusion measurement on isolated grana membranes (Kirchhoff et al., 2008) and intact chloroplasts (Goral et al., 2010) using the fluorescence recovery after photobleaching (FRAP) technique. Processes that are expected to be affected by restricted protein mobility are a regulation of energy distributed between PSII and PSI by state transitions (Lemeille and Rochaix, 2010), the repair of photodamaged PSII (Mulo et al., 2008), membrane remodeling triggered by long-term environmental changes (Walters, 2005; Anderson et al., 2008), and the biogenesis of the thylakoid membrane network (Adam et al., 2011). Recently, evidence has accumulated that photoprotective high-energy quenching also requires large-scale diffusion-based structural reorganization within grana thylakoids (Betterle et al., 2009; Johnson et al., 2011).In contrast to our current understanding of diffusion-based processes in thylakoid membranes, knowledge about the factors that determine the mobility of photosynthetic protein complexes in different thylakoid domains is still fragmentary (Mullineaux, 2008). The protein-packing density is very likely a main element that determines protein mobility (Kirchhoff et al., 2008). However, other factors, like electrostatic interactions between proteins by membrane surface charges (Tremmel et al., 2005) or the size and molecular shape of protein complexes (Tremmel et al., 2003), can contribute significantly. However, data only exist about protein mobility for isolated grana thylakoids (Kirchhoff et al., 2008) and for chloroplasts from the grana-containing C₃ plant Arabidopsis (Goral et al., 2010). The diffusion characteristics of the latter are almost completely determined by granal proteins. Limiting information on protein diffusion exists for stroma lamellae of C₃ plants (Consoli et al., 2005; Vladimirou et al., 2009), and no data are available for agranal thylakoids, which occur in BS cells of some C₄ species.This study fills this gap in the knowledge base by studying lateral protein diffusion in unstacked thylakoid membranes in BS chloroplasts of two NADP-malate enzyme (ME)-type C₄ species, maize (Zea mays) and sorghum (Sorghum bicolor), in comparison with the grana-containing mesophyll (M) chloroplasts. The analysis was also complemented by studies on isolated thylakoid subfragments (grana core, grana, and stroma lamellae) from Arabidopsis. The protein mobility was measured by FRAP (Mullineaux and Kirchhoff, 2007), which has been shown to be a straightforward method to analyze protein diffusion in photosynthetic membranes by utilizing natural chlorophyll fluorescence (Kirchhoff et al., 2008; Goral et al., 2010). The comparison with diffusion characteristics in unstacked versus stacked membrane areas highlights the significance of grana formation on the lateral mobility of photosynthetic pigment-protein complexes.     

Lipid Composition of Plasma Membranes Isolated from Lactating Bovine Mammary Gland

The light and heavy plasma membranes (PM) isolated from lactating bovine mammary glands contained 38~43% lipid of which 41~44% was phospholipid and 47~52% neutral lipid. The contents of phospholipid and neutral lipid were somewhat higher in the light PM than in the heavy PM. Cholesterol was contained 55 ~60% of neutral lipid and the ratio of cholesterol to phospholipid was 0.64 to 0.69. Phospholipid was composed of sphingomyelin (Sph) 29~38%, phosphatidylcholine (PC) 27~35%, phosphatidylethanolamine (PE) 16~20%, phosphatidylserine 10%, and phosphatidylinositol 6~7%. The content of Sph was higher in the heavy PM than in the light PM, while the values of PC and PE were opposite. The major fatty acids of lipid components were palmitic acid, stearic acid, and oleic acid and those of Sph were palmitic acid, stearic acid, C23:0 and 24:0. The fatty acid composition of individual lipid classes differed significantly from each other but were similar between the light and heavy PMs. Tetracosapentaenoic acid (C24:5) was the major fatty acid of the diacylglycerol fraction. The results indicated that the lipid composition, especially phospholipid components, of bovine mammary gland PMs was different from those of milk fat globule membranes which is derived from the PM of mammary secretory cells.     

The Identification of Photosynthetic Subpathways of Some C4 and CAM Plants

The photosynthetic subpathways of five C4 plants and one CAM plant were distinguished according to their chemical, physiological and cytological characteristics. Based on C4 acid decarboxylation enzymes, four C4 plants of Setaria glauca, Sporobolus indicus, Zoysia tenuifolia and Leptochloa chinensis all exhibited the functional high activities of PEP carboxykinase and aspartate aminotransferase as seen in the known PEP-CK subtype. The δ13C value of –12.43% in leaves of L. chinensis was also consistent with that range among PEP-CK subtype. So, these species were classified into PEP-CK subtype. However, their chloroplasts in bundle sheath cells were evenly distributed, not as that displayed centrifugally or centripetally in three typical subtypes. The even arrangement of chloroplasts in bundle sheath cells was likely to be an evolutional intermediate from centripetal (NAD ME type) to centrifugal types (NADP-ME and most PEP-CK types). The high activities of NAD-malic enzyme and aspartate aminotransferase, accompanied with the centripetally located chloroplasts, 0.057 of quantum yield and tile δ13C value of –15.3% in leaves of C4 dicot Euphobia hirta indicated characteristics of NAD-ME subtype. Moreover, CAM plant Aloe vera clearly fell into PEP-CK sybtype because of its high activity of PEP-CK both in whole leaf and green tissue.     

Carbon Isotope Ratios of Epidermal and Mesophyll Tissues from Leaves of C3 and CAM Plants

The ¹³C values for epidermal and mesophyll tissues of two C₃plants, Commelina communis and Tulipa gesneriana, and a CAMplant, Kalancho daigremontiana, were measured. The values forthe tissues of both C3 plants were similar. In young leavesof Kalancho, the epidermis and the mesophyll showed S13C valueswhich were nearly identical, and similar to those found in C3plants. However, markedly more negative values for epidermalcompared to mesophyll tissue, were obtained in the mature Kalancholeaf. This is consistent with the facts that the epidermis ina CAM leaf is formed when leaves engage in C₃ photosynthesisand that subsequent dark CO₂ fixation in guard cells or mesophyllcells makes only a small contribution to total epidermal carbon. (Received January 27, 1981; Accepted May 14, 1981)     

Reconstitution of the Functional Membranes from Chloroplasts with the Deficient Crista Membranes from Mitochondria

The basic mechanisms of phosphorylation of chloroplasts and mitochondria are identical. The identity may be proved by membrane combination. There are two ways to get the combination as shown in figure 1. One way is, as previously reported, to combine deficient membranes from chloroplasts with ctista membranes from mitochondria and the reconstituted membranes thus obtained greatly enhance photophosphorylated activities. The other way, i.e., to combine deficient crista membranes with thylakoid membranes, has also been successful, as shown in this paper. The reconstituted membranes obtained in this way can carry out oxidative phosphorylation in the dark as well as shown in Table 2. There are some relationship between the ATP formation from oxidative phosphorylation of reconstituted membranes and the protein of deficient crista membranes added, as shown in Table 3. When the quantity of combined chloroplast membranes is kept constant, the amount of ATP formation varies, within certain limits, with the amount of deficient crista membranes as shown in Table 3. But the reconstituted oxidative phosphorylation activity of membranes formed by combinating thylakoid with deficient crista membranes is lower than reconstituted photophosphorylation activity of combination in the opposite direction, i.e. by combinating deficient thylakoid membranes and crista membranes of mitochondria (compare Table 4 and 3).