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Physiological studies on acid metabolism. 5. Effects of carbon dioxide concentration on phosphoenolpyruvic carboxylase activity
... This is converted to P-enolpyruvic acid by the enzyme pyruvate, phosphate dikinase (14), thus completing the cycle. Enzymes catalyzing each of the steps of the cycle except the carboxyl transfer have been described (14,15,36,39). Extracts of plants which have the 0-carboxylation pathway contain these enzymes in synthesis found in vivo (36). ...
... Phosphoenolpyruvate carboxylase (4.1.1.3 1) was assayed at 340 nm (29,39) with NAD-malate dehydrogenase, which was also present in the enzyme extracts. Assays were performed in 100 mM HEPES buffer (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5. ...
... The mechanism postulated to explain the carboxyl transfer in the bundle sheath for Zea and Gomphrena is similar to that postulated for the deacidification process which occurs in plants with crassulacean acid metabolism (39,40). In plants with the P-carboxylation pathway, C 0 2 fixation (acidification) and the carboxyl transfer (deacidification) occur at the same time, while these occur at different times of the day in crassulacean plants. ...
Zea mays and Gomphrena globosa form labeled aspartate and malate (C4-acids) via β-carboxylation of P-enolpyruvate during photosynthesis. Studies of the redistribution of 14C in pulse- and chase-type feedings of 14CO2 indicate that most labeled phosphorylated compounds are formed from the C4-acids. A mechanism involving CO2 as a transitory intermediate is advanced to explain the carboxyl transfer from the C4-acids to 3-phosphoglyceric acid (3-PGA). In this model, CO2 is generated through the oxidative decarboxylation of malic acid by "malic" enzyme, and is refixed by RuDP carboxylase to form 3-PGA. The pattern of labeling of photosynthetic products, the extractable enzyme activities, and the gas exchange properties of these plants appear to be consistent with this proposed sequence of reactions. The location of 14C-labeled compounds was determined by radioautography, and by nonaqueous density gradient separation. Differential grinding was used to study the location of some photosynthetic enzymes. These indicate that CO2 fixation by β-carboxylation occurs in the leaf mesophyll. The carboxyl transfer and the reactions leading to the photosynthesis of starch appear to be confined predominantly to the bundle sheath cells. Rapid transport of C4-acids from the site of CO2 fixation in the mesophyll to the bundle sheath may occur by plasmodesmata.
... This could be derived by decarboxylation of ,c carboxyl labelled oxalacetate. Labelling of oxalacetate by pepcarboxylase has been shown to be almost entirely into the / carboxyl (3,34), however our degradative studies of labelled malate showed equal labelling of the ec and /8 carboxyl groups. A similar distribution of carbon-14 activity in malate has been found in other investigations with root material (2,12). ...
... Inhibition of pepcarboxylase activity by p-chloromercuribenzoate has also been reported for wheat germ preparationis (27), andl the reduction in CO., fixation under low 02. tensions (28,34) indicated that fixation in both the pyruvate and PEP systems depended on the normal functioning of the Krebs tricarboxylic acid cycle. ...
... The enzyme, PEP carboxylase was shown to require a divalent metal ion for activity. Phosphoenolpyruvate was carbox- Walker and Brown (115) showed that the optimum CO^ concentration of PEP carboxylase was low and that high concentrations of CO^ inhibited the reaction noncompetitively. ...
... Walker (114) investigated the enzymatic nature of the formation of malate in succulent plants, and suggested the phosphoenolpyruvate carboxylase and malic dehydrogenase were responsible for synthesis. Walker and Brown(115) showed that the optimum CO^ concentration for PEP carboxylase was low and that high concentrations of CO^ inhibited the reaction non-competitively.Both PEP carboxylase and PEP carboxykinase were demonstrated in the chemoautotrophic bacterium Thiobacillus thiooxidans by Suzuki and Werkman(97,98). The two enzymes also were shown to be present in cell-free extracts of Nocardia corallina and Mycobacterium phlei (heterotrophic organisms) while only PEP carboxykinase was demonstrated in extracts of Rhodospirillum rubrum, a photosynthetic autotroph(11,13,15,78). ...
ACKNOWLEDGMENTS I am deeply,debted,to Professor,Clarence,L. Baugh for his,direction,of this,thesis,and,to the,other,members,of my committee, Professors Lyle C. Kuhnley and John A. Anderson, for their helpful criticism. The research,for,this,thesis,was,supported,by state organized,research,funds.,The Institute,for,Environmental Chemistry and Office of the Dean, Graduate School, Texas Tech,University. 11 TABLE OF CONTENTS ACKNOWLEDGMENTS,11 LIST OF TABLES ,iv LIST OF FIGURES ,V CHAPTER I.INTRODUCTION,1 II. MATERIALS AND METHODS ,28 III. RESULTS ,34
... Chlorophyll synthesis has been found to be reduced by a 10% concentration of the gas in illuminated etiolated leaves (11). High levels of carbon dioxide are also known to inhibit several reactions associated with the Krebs cycle (8,13). Perhaps, similar reactions are associated with the reduced oxygen production in Chlorella. ...
The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 320 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimize the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units.
... High CO.. concentration in the external atmosplhere was shown to retard the climacteric an'd the ripening of several fruits (12,14,27). At the enzymnatic level it was reported that 10 % CO., inhibite(d markedly succinate oxidation (1,17) and phosphoenolpyruvate carboxylase activity (23). Ranson et al. (17) relatedl inhibition of the succinooxidase enzyme to previous observations on succinate accumulation when inhibitory levels of CO., were encountered in fruits. ...
and the composition of the internal atmosphere has been studied in several fruits. In the case of the banana (Musa sapientum) and the papaya (Carica papaya), Wardlaw and Leonard (24, 25) observed the coincidence of the onset of the climacteric rise with the peak in oxygen concentration inside these fruits. With the respiratory rise the oxygen level dropped, reaching very low values of 1 % at late stages of senescence. Impressed with the marked changes in the gaseous composition, they suggested that the internal oxygen concentration is the controlling mechanism of the climacteric pattern. Trout et al. (22) reported that in mature apples oxygen deficiency depressed respiration and retarded ripening. Observations on the epidermal layers as the tissue that offers major resistance to gaseous diffusion were made by Trout et al. (22) for the apple and by Clendenning (7) for the tomato. Biale (4) included in his review a brief account of the studies dealing with the internal atmospheres of fruits. The avocado fruit is distinguished from other fruits by its low fermentative capacity, shown by depression of CO2 evolution and ripening under anaerobic conditions (3). This study was undertaken, therefore, with the purpose of finding an explanation for the physiological behavior of the avocado in terms of changes in the composition of the internal atmosphere.
ber den mengenmigen Umfang der an sich bekannten Kohlensureaufnahme durch Pflanzenwurzeln existieren nur ungenaue und recht widersprechende Vorstellungen. Diese erste Arbeit behandelt zunchst die experimentellen Schwierigkeiten, die einer Lsung des Problems entgegenstehen, und beschreibt dann eine spezielle Versuchsanordnung, mit deren Hilfe man einer Beantwortung der Frage nher kommen kann. Diese Diskussion scheint fr eine sachliche Beurteilung der in zwei weiteren Arbeiten folgenden Versuchsergebnisse erforderlich.Although it is known that plants do absorb carbon dioxide by their roots, there are little and rather contradictory data on the quantitative importance of this process. This paper deals with the experimental difficulties which are responsible for this lack of information, and demonstrates a special experimental device which might help to solve the problem. It is felt that this discussion is prerequisite for the experimental data to be presented in two subsequent papers.
Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H14CO3− incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent Km (PEP) and Vmax values compared more favourably with those obtained from a C3-species (spinach) than from a C4-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C4 photosynthetic enzyme.
The respiratory rate of fruits and vegetables can be used as an indicator for designing storage conditions to maximize the longevity of these commodities. One postharvest technique that has been used to prolong the storage life of some of these commodities is the use of a controlled atmosphere. The modulation of respiratory metabolism of such commodities held in controlled atmospheres containing reduced oxygen and/or elevated carbon dioxide levels has been thought of as the primary reason for the beneficial effects on the commodities. However, the mechanism by which elevated carbon dioxide influences the regulation of respiratory metabolism is still obscure and several hypotheses have been proposed for its mode(s) of action. The regulation may be directed towards the glycolytic pathway, the fermentative metabolism, the tricarboxylic acid cycle or the electron transport system, presumably through its influence on the synthesis, degradation, inactivation and/or activation of the respective enzymes. It may also be through the antagonistic effects of carbon dioxide on ethylene action as well as its influence on secondary metabolism through an alteration in cell pH. This article discusses the recent developments on the biochemical and physiological fronts as well as the possible mode(s) of action of elevated carbon dioxide in the regulation of respiratory metabolism in fruits and vegetables.
Three P-enolpyruvate carboxylase isoenzymes (Pc-I, II, and III) were isolated from cotton leaf tissue by DEAE-cellulose column chromatography. One of the isoenzymes (Pc-III) was associated with particulate fractions isolated and purified by both aqueous and nonaqueous methods. Michaelis constants (Km) for P-enolpyruvate were similar among the isoenzymes (~1.0–1.2 × 10−5m and 3.7–5.7 × 10−5m, respectively, at 0.005–0.05 mm and 0.05–2.5 mm PEP). Michaelis constants for Mg2+ and Mn2+ appeared to differ. The Km values for Mg2+ were approximately one order of magnitude higher than for Mn2+. In both cases, the Km for Pc-III (Mn2+, 6.3 × 10−5m; Mg2+, 1.3 × 10−4m) was greater than for Pc-II (Mn2+, 2.8 × 10−5m; Mg2+, 0.7 × 10−4m), and the Km for Pc-II was greater than Pc-I (Mn2+, 0.2 × 10−5m; Mg2+, 0.1 × 10−4m). In general, the order of cation activation at 0.34 mm was Mg++ > Mn2+ > Co2+ > Zn2+, Al2+, Fe2+. Some differences among the isoenzymes for cation requirement were noted. The Km values for bicarbonate were similar for Pc-III (5.6 × 10−5m) and Pc-II (5.3 × 10−5m) and smaller than for Pc-I (14.4 × 10−5m). Bicarbonate concentrations greater than 2.5 mm tended to inhibit all isoenzymes. Pc-I was more thermal stable than Pc-II or Pc-III. All were inactivated by increasing ionic strength (NaCl). Pc-I could be distinguished from Pc-II and Pc-III by polyacrylamide gel electrophoresis. Kinetic and physical properties tended to suggest that Pc-II and Pc-III were similar, but different from Pc-I. None of the isoenzymes is activated or inhibited by acetyl-CoA. At high concentrations (5–30 mm) compounds such as malate, pyruvate, citrate, isocitrate, aconitate, and ADP tended to partially inhibit activity. Pc-II was much more sensitive to these compounds than were Pc-I and Pc-III. Kinetic studies with organic inhibitors suggested that Pc-III may be an allosteric protein. With respect to inhibition by organic acids and nucleotides, it was concluded that the leaf isoenzymes are unlike the P-enolpyruvate carboxylase of the Enterobacteriaceae and that regulation of metabolism mediated by these isoenzymes is to a large extent by spatial compartmentation.
The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 320 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimize the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units.
Phosphoenolpyruvate (PEP) carboxylase was purified over 200-fold from potatoes (Solanum tuberosum). An apparent molecular weight of 265,000 was obtained by sucrose gradient centrifugation. It does not appear to dissociate or aggregate in the presence of substrates, within the pH range of 6–8, or in an ionic strength (0.5) that produces approximately 60% inhibition of enzymic activity. Free sulfhydryl groups on the enzyme are important for activity and apparently also for structural integrity. It is inhibited by p-mercuribenzoate competitively with Mg2+, noncompetitively with PEP; and uncompetitively with HCO3−. Mg2+, Mn2+, and Co2+ are activators and Zn2+ and Ca2+ are inhibitors. Kinetic analysis indicates that Zn2+, Mg2+, and Mn2+ compete for the same site on the enzyme.
A “quasi-diffusion resistance”, rq, is defined to accommodate the role which the thermochemical and photochemical phenomena of photosynthesis play in the control of CO2 fixation to the terminology and approach of the diffusion resistance analogue for the CO2 exchange of leaves. The relationship of rq to Rabinowitch's classical rectangular hyperbolic model of photosynthesis rate as a function of CO2 concen-tration at the carboxylating surface is discussed. Examination of Kmapp for phos-phopyruvate carboxylase (the predominant carboxylase in maize) suggests, as a reasonable hypothesis for light-saturated maize leaves, that rq may be essentially independent of ambient CO2-concentration up to at least 300 μ1/l. A corollary of the hypothesis is that an increase of diffusion resistance, rather than of rq, may account for the observed curvature of the response curves of light-saturated maize leaf photosynthesis to ambient CO2-concentration. An experiment carried out on fieldgrown maize plants, using a well controlled leaf chamber as a nitrous oxide diffusion porometer, gave evidence which strongly supported the hypothesis.
Phosphoenolpyruvate carboxylase has been partially (50-fold) from extracts of germinating peanut cotyledons. The enzyme catalyzes the irreversible Mg2+-dependent carboxylation of phosphoenolpyruvate to form oxaloacetate. Purified preparations of the carboxylase are completely inactive in the absence of added sulfhydryl compounds, but are readily reactivated by glutathione addition. Carboxylase, active in the presence of small amounts of glutathione, is reversibly inhibited by p-hydroxymercuribenzoate. Co2+ and Mn2+ are 22% and 10% as effective, respectively, as equimolar amounts of Mg2+ for the carboxylation reaction.In the absence of phosphoenolpyruvate, high levels of purified carboxylase failed to catalyze significant incorporation of [14C]bicarbonate into oxaloacetate in the presence of oxaloacetate, orthophosphate, Mg2+ and glutathione. When the enzymic carboxylation of phosphoenolpyruvate was conducted in the presence of [14C]pyruvate, incorporation of 14C-activity into oxaloacetate did not occur. The catalytic action of the carboxylase is not inhibited by avidin.
High concentrations of carbon dioxide inhibit the greening of etiolated plants. In the presence of 20% oxygen, concentrations of carbon dioxide of 10% and above inhibited the production of chlorophyll in etiolated leaves of barley, wheat, and dwarf French bean. On return to air, recovery from this inhibition took place rapidly. High concentrations of carbon dioxide were also inhibitory when illumination was discontinuous (2-msecond flash separated by 3-minute dark period) during which photosynthetic activity was adjudged to be negligible. The inhibition was alleviated by feeding with delta-amino levulinic aid, implying that the site of inhibition was early in the sequence of chlorophyll synthesis. 15 references, 3 figures, 1 table.
The C4 dicarboxylic acid pathway of photosynthetic carbon assimilation (the C4 pathway) is a complex biochemical and physiological elaboration of the common photosynthetic carbon reduction cycle (PCR cycle, C3 pathway; Bassham and Calvin, 1962). We define the C4 pathway as the complete reaction sequence in which CO2 is transferred via the C-4 carboxyl of C4 acids to the reactions of the PCR cycle and there reduced to the level of carbohydrate (Hatch and Slack, 1970; Hatch et al., 1971; Black, 1973; Hatch, 1976a). The distinctive biochemical features of this process are the carboxylation and associated reactions leading to the synthesis of C4 acids, and those concerned with the subsequent decarboxylation of these C4 acids to supply CO2 for the PCR cycle. Unlike the PCR cycle, in which carboxylation and carbon reduction is restricted to the chloroplast, the C4 pathway involves the operation of reactions in cytoplasm, mitochondria, and chloroplasts, and the transport of intermediates between intracellular compartments. In this sense it may be compared with another well-established elaboration of the PCR cycle, the glycolate pathway of photorespiration (see Chap. II,5). However, an additional and distinctive feature of the C4 pathway is the mandatory exchange of photosynthetic intermediates between adjacent cells. These exchanges constitute one of the most rapid and complex forms of symplastic transport known.
WHEREAS considerable information is available regarding the enzymatic mechanism of carbon dioxide fixation during acidification in succulents1–3, very little seems to be known about the role of enzymes in the diurnal variation in acids and carbohydrate. If the 3-carbon atom acceptor of carbon dioxide is derived from starch or sugar by the Embden–Meyerhof–Parnas series of reactions, aldolase may be expected to play an important part in the processes of acidification and de-acidification. We have studied the activity of aldolase in cactus tissue at different hours of the day and night, and have found a marked diurnal variation. Phosphatase activity was also found to undergo rhythmic alteration.
It is demonstrated that the use of L. plantarum for 14C-malate degradation leads to incorrect data on the isotope distribution in malate, due to the fumarase content of this organism. The significance of this observation for the understanding of the mechanism of dark fixation of CO2 by CAM plants is discussed.
This section will deal with methods for determining enzymes which decarboxylate oxalacetate (OA) or form the latter substance by CO2 fixation. Three types of enzymes will be considered: (a) those enzymes which decarboxylate OA to pyruvate and CO2 (Reaction 1) and which may be considered true decarboxylases, (b) the plant and bacterial enzyme which catalyzes the irreversible formation of OA from phosphoenolpyruvate (PEP) and CO2 (Reaction 2), and which is termed here PEP carboxylase, and (c) the enzyme from animal tissues or microorganisms which catalyzes the reversible formation of OA from PEP, CO2, and a nucleotide diphosphate (Reaction 3) and which is called here PEP carboxykinase.
Plant tissues vary considerably in their content of protein and it may, therefore, be anticipated that some tissues, in, reference to others, may lend themselves more readily to enzyme isolation and purification. The leaves of the tapioca plant contain 20–36% of crude protein (Rogers, 1959), whereas the phylloclades of cactus have only about 7%, both on a dry weight basis (Sanwal, 1960). The nature of the nonprotein material of the starting material also influences the course of enzyme purification. The presence of mucilaginous material in tissues such as cactus interferes with conventional techniques of enzyme enrichment. Whereas, in general, the investigator is concerned with the enzyme-make up of a given tissue and preliminary screening does not find much scope, it is sometimes possible to choose a special tissue where the given enzyme is present in optimum concentration. In the animal tissue, high alkaline phosphatase activity seems to be associated with intestinal mucosa, acid phosphatase with kidney and spleen and, even more so, with prostate and 5′-nucelotidase activity with testis. Germinating seeds constitute a good sources of acid phosphatases (Newmark and Wenger, 1960). Plant tissue and microorganisms are, in general, poor sources of alkaline phosphatase, but Garen and Levinthal (1960) showed that when Escherichia coli was grown in a medium containing limiting amounts of orthophosphate, as much as 6% of the total protein synthesized was alkaline phosphatase. The availability of such a concentrated source facilitated the isolation of the phosphatase and study of its properties.
Since a number of Krebs cycle enzymes are located in mitochondria, a considerable purification can be achieved by isolating the mitochondria. Recent studies (Marcus and Velasco, 1960) have indicated that the enzymes of the glyoxalate cycle are also located in the mitochondria.
Energy required for vital function whether in the animal, the bacterium or the higher plant is obtained by burning fuel. In most plant tissues the fuel is sugar or one of its storage products (either di- or polysaccharides). In other instances it is fat, and in a few plants, protein storage products may be burned to provide energy. The energy available in these molecules, in the form of bond energy, is derived from the sun either directly or indirectly. For this energy to become available to the cell, a series of transformations must occur which, in the case of the sugars and their related products, and for at least part of the fat molecule, involves the formation of phosphorylated compounds. In short, if substrates are to be used as energy sources they must first undergo phosphorylation. This appears to be true for almost all instances of sugar oxidation. There are, however, a few isolated cases in some animal tissues and especially in bacteria where prior phosphorylation may not be required.
Pflanzen mit einem diurnalen Säurerhythmus sind dadurch ausgezeichnet, daß ihr Gehalt an organischen Säuren sich unter natürlichen Bedingungen im Verlauf des Tages verändert, im Verlauf der Nacht aber wieder dem morgendlichen Ausgangswert sich nähert. Besonders ausgeprägt ist eine Säurerhythmik in grünen Gewebeteilen von Crassulaceen ; man bezeichnet deshalb die hier angetroffene Art von Veränderungen häufig auch als „Crassulaceen-Säurestoffwechsel“. Die Rhythmik besteht bei diesen Pflanzen darin, daß der Säuregehalt im Verlauf des Tages sich vermindert (Absäuerung am Licht) und während der Nacht wieder ansteigt (Ansäuerung im Dunkeln); bei längerdauernder Verdunkelung vermindert sich der Gehalt an organischen Säuren wiederum allmählich (Absäuerung im Dunkeln). Diese Form der diurnalen Rhythmik ist nicht auf Crassulaceen beschränkt, vielmehr wird sie in sehr ausgeprägter Form auch bei Cactaceen (z. B. Richards 1915) und succulenten Compositen angetroffen (z. B. Thoday und Jones 1939, Thoday und Richards 1944) und darüber hinaus mindestens angedeutet bei vielen anderen succulenten und nicht-succulenten Pflanzen der verschiedensten Familien (vgl. zusammenfassende Darstellungen bei Small 1929, 1955 und Bennet-Clark 1933a).
The circadian rhythm of CO2 assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO2. During such exposures substantial fixation of CO2 takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40-50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO2 to normal air, a circadian rhythm of CO2 assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO2, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO2 in continuous illumination, appears to be due to the presence of a high concentration of CO2 within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.
The activities of phosphopyruvate carboxylase (E.C. 4.1.1.31), malic enzyme (malate dehydrogenase (decarboxylating) E.C. 1.1.1.40), selected enzymes of TCA cycle and electron transport chain were determined in mitochondrial and supernatant fractions isolated from phylloclades of the cactus Nopalea dejecta, collected at noon and midnight. There were significant and consistent increases in the activities of mitochondrial and supernatant phosphopyruvate carboxylase, mitochondrial aconitate hydratase (E.C. 4.2.1.3) and soluble malate dehydrogenase (E.C. 1.1.1.37) and decreases in mitochondrial malate dehydrogenase and fumarate hydratase (E.C. 4.2.1.2) and in both mitochondrial and soluble malic enzyme in the midnight samples. Of the respiratory chain enzyme complexes tested, cytochrome c oxidase (E.C. 1.9.3.1) and succinate cytochrome c reductase had higher activities at noon, while reduced NAD-cytochrome c reductase and reduced NAD dehydrogenase (E.C. 1.6.99.3) activities were higher at midnight. The rate of succinate oxidation and coupled phosphorylation also varied, with a maximum at noon. The diurnal pattern of alteration in enzymic activities has a bearing on crassulacean acid fluctuation.
The content of various metabolic intermediates has been determined in material from several species of succulent plants known to show a Crassulacean acid metabolism, grown under natural conditions at Kampala, Uganda. Free titratable acidity, malate, citrate and isocitrate, present in considerable amounts, and various keto-acids present in relatively small amounts, have been measured both at dawn and during the later afternoon. In the case of leaf from two species of Bryophyllum natural changes of keto-acid content were followed in detail.
Overnight changes in free titratable acidity could, in all cases, be largely attributed to malate accumulation, but citrate also increased considerably in some species. Diurnal fluctuations in isocitrate were not observed. Concentrations of both oxaloacetate and α-oxo-glutarate increased markedly overnight. In the two Bryophyllum species, pyruvate concentrations were maximal during photodeacidification. The phosphopyruvate content of B. crenatum leaf increased slightly during photodeacidification but was low during dark acidification. No isocitrate lactone was detected in B. calycinum leaf.
Observed oxygen uptake rates would account for the complete oxidation during photodeacidification of only a small proportion of the malate decarboxylation product. Probable pathways involved in the formation and utilization of malate are discussed.
Fragments cut from Kalanchoe leaves during their acidifying phase, when infiltrated with labelled glucose in the dark, accumulated label in malic acid and other intermediates of the TCA cycle. The course of the accumulation of label in malate in relation to $^{14}$CO$\_{2}$ release was in accord with the long accepted view that the acid is produced under natural conditions in acidifying leaves at the expense of reserve carbohydrate in reactions involving CO$\_{2}$-fixation. When the acidifying leaves were supplied with pyruvate-1-$^{14}$C, -2-$^{14}$C or -3-$^{14}$C the labelled carbon atoms were incorporated into acids of the tricarboxylic acid cycle and related amino-acids and to a small extent into carbohydrates. The results suggest that in the main the infiltrated pyruvate was utilized in three ways, namely; (a) oxidation by the pyruvic oxidase system to yield CO$\_{2}$ and an acetyl residue which was incorporated into citrate; (b) $\beta $- carboxylation to yield oxaloacetate or malate; and (c) dark fixation of CO$\_{2}$ released from pyruvate in reaction (a) or in subsequent transformations of the products of reactions (a) and (b). When the pyruvates were supplied in the presence of high environmental concentrations of CO$\_{2}$ the total $^{14}$C incorporation was reduced. The reduction was most marked in the case of pyruvate-1-$^{14}$C. This and other observations support the conclusion that the dark fixation of CO$\_{2}$ released in one or other way from the infiltrated pyruvate was responsible for a substantial fraction of the carbon incorporated into the leaves from the pyruvate. In the presence of the high CO$_{2}$ concentration no label was incorporated into carbohydrate. When the labelled pyruvates were infiltrated into fragments from deacidifying leaves the $^{14}$C accumulation in acids in the cells was markedly reduced, but the incorporation into sugars was increased several-fold. The reduction of acid labelling appeared to result mainly from a reduction of the incorporation by reactions (b) and (c). Labelled fumarate was metabolized in the acidifying leaves to yield other acids of the TCA cycle and related amino acids. In addition there was a slow accumulation of $^{14}$C into carbohydrates. In all of these experiments the time course of the accumulation of $^{14}$C in individual acids was in accord with the operation of a tricarboxylic acid cycle in the leaf cells, and with the view that malate, citrate and isocitrate were present, for the greater part, in storage pools that equilibrated relatively slowly with pools at metabolically active sites within the cells. It is suggested that in acidifying leaves the $^{14}$C entering the cells was largely incorporated into the storage pools of acids and released relatively slowly for further metabolism, whereas in the deacidifying leaves the storage mechanism was less active and acid became more readily available for further metabolism including conversion into carbohydrates.
Measurements of gaseous exchange between green callus tissue cultures of Kalanchoe crenata and air showed that photosynthesis occurred in the green callus. Feeding experiments with 14CO2 showed that 14C was incorporated by green callus into sugars and sugar phosphates in the light, and some 14C was incorporated into organic acids. Organic acids were the main products of dark fixation of 14CO2 by green callus but no activity was detected in sugars or sugar phosphates. The products of 14CO2 fixation in colourless callus were primarily organic acids both in the light and dark and no activity was detected in sugars or sugar phosphates.Green callus did not show a diurnal fluctuation of acid content. This, coupled with the observations that green callus did not show a net fixation of CO2 in the dark, suggested that the green callus did not exhibit crassulacean acid metabolism. Green callus, however, contained greater quantities of malate, citrate and isocitrate than did colourless callus. Enzyme assays suggested that the presence of PEP carboxylase in green callus and its absence in colourless callus might in part contribute to the higher acid levels of green callus.
P-enolpyruvate carboxylase prepared from leaves of several plants is activated by mM concentrations of glucose-6-phosphate. At 2 mM glucose-6-phosphate the activation was due to the lowering of K0.5 for P-enolpyruvate, without appreciable effects on the maximum velocity. Glucose-6-phosphate activation of P-enolpyruvate carboxylase purified from leaves of Crassulacean plants reverses the substrate level inhibition of this enzyme by malic acid. The role of activation and inhibition is discussed in relation to the control of malic acid synthesis in Crassulacean leaves.
Photosynthesis often reaches its maximal rate only after an initial induction period of some minutes. Simple induction phenomena in whole plants and intact chloroplasts have common features which imply that they are brought about by the same mechanism. The Osterhout–Haas Hypothesis explains induction in terms of light activation of a catalyst and building up of intermediates. This proposal is examined in the light of contemporary work and it is concluded that the major factor is the accumulation of metabolites brought about by the autocatalytic action of the Benson-Calvin cycle. Light activation of ribulose diphosphate carboxylase is seen as an important factor in autocatalysis but normally too rapid a process, in itself, to account for delays of several minutes.
Carbon dioxide and oxygen gas exchange of illuminated Amaranthus and Phaseolus leaves was measured from 0–600 ppm of CO2 in an open system.
At low oxygen concentration (2% O2) the ratio of CO2 uptake to O2 evolution came close to 1.
At high oxygen partial pressure (42% O2) the O2 compensation point of an Amaranthus leaf was increased and oxygen evolution was depressed. Accordingly the CO2/O2 quotients were variable; the lowest value of 1,9 differed significantly from 1,0.
The oxygen and carbon dioxide compensation points of a Phaseolus leaf were increased at high oxygen concentration (42% O2) and oxygen evolution as well as carbon dioxide uptake were reduced. Therefore the ratios CO2 over O2 varied and differed greatly from 1,0.
It was concluded that the nature of photosynthates is regulated by the gas composition around the leaves.
Enzymes involved in malic acid production via a pathway with 2 carboxylation reactions and in malic acid conversion via total oxidation have been demonstrated in mitochondria of Bryophyllum tubiflorum Harv. Activation of the mitochondria by Tween 40 was necessary to reveal part of the enzyme activities. The temperature behavior of the enzymes has been investigated, revealing optimal activity of acid-producing enzymes at 35 degrees . Even at 53 degrees the optimum for acid-converting enzymes was not yet reached. From the simultaneous action of acid-producing and acid-converting enzyme systems the overall result at different temperatures was established. Up to 15 degrees the net result was a malic acid production. Moderate temperatures brought about a decrease in this accumulation, which was partly accompanied by a shift to isocitrate production, while at higher temperatures total oxidation of the acids exceeded the production.
The plant surfaces which are exposed to the atmosphere, such as leaves, stems, fruits, and petals, are covered with a hydrophobic, water repellent substance called wax, of which the leaf waxes have received most attention. The outer surface of leaf epidermis is covered with a substance called cutin which is usually impregnated with wax; together they comprise the cuticle. The insoluble polymer cutin is composed of cross-linked hydroxy fatty acids (Kolattukudy 1975), which are released upon hydrolysis. The wax is a complex mixture of lipophilic substances such as hydrocarbons, wax esters, alcohols, and ketones (Tulloch 1976a). The cuticular wax plays an important role in preserving the water balance of the plant by reducing evaporation from the leaf surface. The hydrocarbons together with other waxy components serve as a barrier to the passage of water in and out of the cell, thus preventing water inundation or dehydration (Misra et al. 1984a; Weete et al. 1978). Other protective functions may include minimizing mechanical damage to leaf cells and inhibiting fungal and insect attack. The structural and functional roles of leaf epicuticular waxes along with their biosynthesis and some analytical aspects have been discussed in an excellent review by Eglinton and Hamilton (1967).
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