Some Observations on a Circadian Rhythm in Carbon Dioxide Compensation in Bryophyllum fedtschenkoi

Detached shoots of Bryophyllum fedtschenkoi maintained in continuouslight and at a constant temperature exhibit a circadian rhythmin CO₂ compensation. The rhythm has a period of 21.6±0.1h at 23 °C and its phase can be set and shifted by suitabletreatments. These observations suggest that the ryhthm is trulyendogenous in nature. The phase is set by a light-on stimulus, or a drop in lightintensity. Phase shift can be induced by short periods (3 h)of reduced light intensity as well as similar periods of darknessgiven during the day phase of the rhythm. A change from whitelight to the same number of incident quanta of monochromaticlight of 450 nm (blue), 550 nm (green), or 652 nm (red) alwaysshifted the phase, but the degree of shift was less in red lightthan in blue or green. The plant reacted to achange to blueor green light as though it had been placed in the dark. Althbughred light was apparently ‘recognized’, the changefrom white to red was still sufficient to alter the phase ofthe rhythm. The possible role of phosphoenolpyruvate carboxylase activityin controlling the rhythm is discussed.     

The Damping and Reinitiation of the Circadian Rhythm of CO2 Output in Bryophyllum Leaves in Relation to their Malate Content

The circadian rhythm of CO₂ output in leaves of Bryophyllumfedtschenkoi damps out after 3–4 d in continuous darknessand a CO₂-free air stream at 15°C. The rhythm is reinitiatedafter a single exposure to white light of 2, 4, 6 or 8 h duration,damps out again after a further 3–4 d and can be reinitiatedfor a second time by a further exposure to light. During the exposure to light there is a burst of CO₂ outputconsistent with the decarboxylation of malate, and the rhythmbegins afterwards with an initial high rate of CO₂ fixation.Malate gradually accumulates in the leaves in continuous darknessto attain a maximum value (35 mol m^–3) at the time whenthe circadian rhythm disappears, and decreases to a low value(19 mol m^–3) after a 4 h exposure to light which reinitiatesrhythmicity. These results support the hypothesis that damping of the rhythmof CO₂ output in continuous darkness is due to the accumulationof malate in the leaf cells, eventually reaching such a levelthat its removal from the cytoplasm into the vacuole cannottake place, with the result that PEPc activity, upon which therhythm of CO₂ output depends, remains allosterically inhibited. Key words: CAM, circadian rhythm, Bryophyllum, CO₂-fixation, malate metabolism     

The circadian rhythm of carbon-dioxide metabolism in Bryophyllum: the mechanism of phase-shift induction by thermal stimuli

The detailed characteristics have been established for the phase shifts induced by high-temperature (35° C) stimuli in the circadian rhythm of phosphoenolpyruvate-carboxylase activity in leaves of Bryophyllum fedtschenkoi otherwise kept under constant environmental conditions. The magnitude and direction of the shifts depend upon the duration of the stimulus and its position in the cycle, and are closely similar to those induced by light. An hypothesis is advanced which accounts for all the characteristics of the phase shifts induced by both high-temperature and light stimuli in terms of the leakage of malate from the vacuole to the cytoplasm though “gates” in the tonoplast which are open only during exposure to these stimuli.     

An Endogenous Rhythm in the Rate of Carbon Dioxide Output of Bryophyllum: I. SOME PRELIMINARY EXPERIMENTS

A techinique is described for recording automatically, withthe aid of an infrared gas analyzer, the rate CO₂ output orabsorption by plant material under controlled conditions. An examination of the rate of CO₂ output by excised leaves of16 species of succulent plants in darkness and in a CO_-2-freeatmosphere revealed clearly defined rhythms in only Bryophyllumfedtschenkoi, B. daigremontianum and B. calycinum (pinnatum). Further investigation of the rhythm in leaves of B. fedtschenkoirevealed that: (1) daylength has no effect upon the period ofthe rhythm in subsequent darkness, the phase being set at thetime the lights are extinguished; (2) normal air suppressesthe rhythm; (3) removel of the epidermis and cutting the mesophyllinto pieces 1 cm² does not effect either the phase or periodof the rhythm; (4) continuous illumination at an intensity of3,000 lux inhibits the rhythm which restarts when the lightsare extinguished. The phase of the rhythm can be set at anytime of day according to the time at which the lights are extinguished.The time which elapses between the onset of darkness and thefirst peak decreases as the length of the light treatment isincreased. The endogenuous nature of the rhythm is fully established. Theresults are compared with of other researches.     

Phase resetting of the circadian rhythm of carbon dioxide assimilation inBryophyllum leaves in relation to their malate content following brief exposure to high and low temperatures,darkness and 5% carbon dioxide

Leaves ofBryophyllum fedtschenkoi show a persistent circadian rhythm in CO₂ assimilation when kept in continuous illumination and normal air at 15°C. The induction of phase shifts in this rhythm by exposing the leaves for four hours at different times in the circadian cycle to 40° C, 2° C, darkness and 5% CO₂ have been investigated. Exposure to high temperature has no effect on the phase at the apex of the peak but is effective at all other times in the cycle, whereas exposure to low temperature, darkness or 5% CO₂ is without effect between the peaks and induces a phase shift at all other times. The next peak of the rhythm occurs 17 h after a 40° C treatment and 7–10 h after a 2° C, dark or 5% CO₂ treatment regardless of their position in the cycle. When these treatments are given at times in the cycle when they induce maximum phase shifts, they cause no change in the gross malate status of the leaf. The gross malate content of the leaf in continuous light and normal air at 15% shows a heavily damped circadian oscillation which virtually disappears by the time of the third cycle, but the CO₂ assimilation rhythm persists for many days. The generation of the rhythm, and the control of its phase by environmental factors are discussed in terms of mechanisms that involve the synthesis and metabolism of malate in specific localised pools in the cytoplasm of the leaf cells.     

An Endogenous Rhythm in the Rate of CO2 Output of Bryophyllum: II. THE EFFECTS OF LIGHT AND DARKNESS ON THE PHASE AND PERIOD OF THE RHYTHM

The effects of light, darkness, and changes in light intensityon the phase and period of the endogenous rhythm in the rateof CO₂ output of excised leaves of Bryophyllum fedtschenkoihave been examined. The duration, intensity, and wavelength of a short light treatment,and the point in the cycle at which it is administered, determinethe degree of phase shift induced in a rhythm persisting indarkness. When light treatments of 3 and 6 hours' duration,at an intensity of 3,000 lux, are applied between the peaksthe phase is completely reset, the first post-treatment peakoccurring 18–19 hours after the end of the treatment.The degree of phase shift is therefore determined not by theduration of the treatment but by the time at which the treatmentterminates. One hour's illumination has little or no effect.The phase is unaffected when light treatments of up to 5 hours'duration at an intensity of 3,000 lux are applied at the crestof a peak. Over the range 8-3,000 lux the intensity of lightduring a 6-hour treatment applied between the peaks does notaffect the efficiency with which that treatment completely resetsthe phase. At an intensity of 2 lux, however, the phase delayis equal to the duration of the treatment. A 6-hour red-light treatment (850 ergs/cm.²/sec.) applied betweenthe peaks completely resets the phase whereas blue light (10,860ergs/cm.²/sec.) has no effect on the phase but induces a slightprotraction of the period. Moreover, continuous red light inhibitsthe rhythm, which recommences in blue light. A rhythm is induced in illuminated leaves when the light intensityis either gradually or suddenly reduced by at least 80 per cent.Whether a given intensity of illumination inhibits or permitsthe persistence of a rhythm depends upon the light intensityby which it is immediately preceded. A rhythm will persist in illuminated leaves for approximatelyas long as in leaves in darkness and the phase shows no correlationwith time of day. The period is unaffected by the intensityof white light (from 0-500 lux) to which the leaves are subjected.The duration of a short dark treatment, and the point in thecycle at which it is applied, determine the degree of phaseshift induced in a rhythm in illuminated leaves. The phase isreset when 3-, 6-, and 9-hour dark treatments are applied atthe crest of a peak, the amount of phase shift increasing toa maximum with 9 hours' darkness. The phase shift is not equalto the duration of the treatment. The phase is unaffected when3- and 6-hour dark treatments are applied between the peaks. The variation in the sensitivity of the phase of a rhythm persistingin darkness to short light treatments is in the opposite senseto that of a rhythm persisting in light to short dark treatments.The phase of a rhythm in illuminated leaves is completely resetwhen the leaves are transferred to continuous darkness commencingeither at the crest of, or between, the peaks. The results are discussed and compared with those of other authors.     

Diurnal changes in malate-decarboxylation activity in the leaves of a CAM plant, Bryophyllum daigremontianum

Bryophyllum diagremontianum plants grown under light-dark regimeswere exposed to one more cycle of the regime or to continuousdarkness for 24 hr. Photosynthetic O₂ evolution by leaf segmentsfrom these plants was investigated in the presence of 15 mMNaHCO₃ (CO₂-dependent O₂ evolution) or in the absence of CO₂(malate-dependent O₂ evolution). The malate-dependent O₂ evolutionserved as an index of the activity of malate decarboxylation.Malate content was respectively 67, 64 and 85 µmoles/g.fwin leaves measured at 7 hr 30 min in light and 6 hr 26 min inthe dark from plants under the light-dark regime (light 12 hr/dark12 hr) and those measured at 6 hr 26 min in the dark from plantsunder the continuous dark regime. The malate- and CO₂-dependentphotosynthetic O₂ evolutions in the same leaves were 9.7 and22, 0.2 and 17, and 16 and 26 µmoles/g.fw.hr, respectively.Thus, the diurnal change in capacity for malate-dependent O₂evolution was relieved by continuous dark treatment. These results suggest that the diurnal change in malate decarboxylationin this crassulacean acid metabolism plant does not occur byan endogenous rhythm. This further indicates lack of an endogenousrhythm for the influx-efflux of malate across the vacuole andin malate decarboxylation enzyme activity. (Received August 1, 1979; )     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

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.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Antagonism Between Malate and Ethylene in the Regulation of Avena sativa Coleoptile Elongation

Etiolated Avena sativa L. coleoptile sections were used to determinethe influence of C₂H₄ on in vivo and in vitro rates of CO₂ fixation,and to measure the influence of various permutations of C₂H₄,CO₂, and malate on growth. Whereas 1 mM malate or 320 µI^-1 CO₂ stimulated growth by approximately 100 per cent, inhibitionof growth by 10-8 µ I_-1 C₂H₄ was substantial only in thepresence of malate or CO₂ The increase in growth rate in responseto these two agents was eliminated by the simultaneous applicationof C₂H₄. The in vivo rate of dark [¹⁴C]bicarbonate fixationand in vitro enzymic assays of fixation were not measurablyinhibited by C₂H₄. These results are discussed in the lightof evidence which indicates that CO₂-stimulated growth is mediatedby dark fixation. The data do not support the view that C₂H₄inhibition of growth results from an inhibition of fixation,but suggests that C₂H₄ may inhibit some step in the processby which malate stimulates growth.     

The Anisotropy of the Mesophyll and CO2 Capture Sites in Vicia faba L. Leaves at Low Light Intensities

Experiments are reported on the spatial distributions of isotopiccarbon within the mesophyll of detached leaves of the C₃ plantVicia faba L. fed ¹⁴CO₂ at different light intensities. Eachleaf was isolated in a cuvette and ten artificial stomata providedspatial continuity between the ambient atmosphere (0.03–0.05%v/v CO₂) and the mesophyll from the abaxial leaf side. Paradermalleaf layers exhibited spatial profiles of radioactivity whichvaried with the intensity of incident light in 2 min exposures.At low light, when biochemical kinetics should limit CO₂ uptake,sections through palisade cells contained most radioactivity.As the light intensity was increased to approximately 20% offull sunlight, peak radioactivity was observed in the spongycells near the geometric mid-plane of the mesophyll. The resultsindicate that diffusion of carbon dioxide within the mesophyllregulated the relative photosynthetic activity of the palisadeand spongy cells at incident photosynthetically active lightintensities as little as 110 µE m^–2 s^–1 whenCO₂ entered only through the lower leaf surface. Key words: CO₂ capture sites, Vicia faba L., Artificial stomata     

Wavelength effects on photosynthetic carbon metabolism in Chlorella

To study the wavelength-effect on photosynthetic carbon metabolism,¹⁴C-bicarbon-ate was added to Chlorella vulgaris 1 lh suspensionunder monochromatic blue (456 nm) and red (660 nm) light. Thelight intensities were so adjusted that the rates of ¹⁴CO₂ fixationunder blue and red light were practically equal. Analysis of¹⁴C-fixation products revealed that the rates of ¹⁴CO₂ incorporationinto sucrose and starch were greater under red light than underblue light, while blue light specifically enhanced ¹⁴CO₂ incorporationinto alanine, aspartate, glutamate, glutamine, malate, citrate,lipid fraction and alcohol-water insoluble non-carbohydratefraction. Pretreatment of the algal cells in phosphate mediumin the dark, which was essential for the blue light enhancementof PEP carboxylase activity, was not necessary to induce theabove wavelength effects. Superimposition of monochromatic bluelight at low intensity (450 erg.cm^–2.sec^–1) on thered light at saturating intensity caused a significant decreasein the rate of ¹⁴CO₂ incorporation into sucrose and increasein incorporation into alanine, lipid-fraction, aspartate andother related compounds, indicating that the path of carbonin photosynthesis is regulated by short wavelengdi light ofvery low intensity. Possible effects of wavelength regulationof photosynthetic carbon metabolism in algal cells are discussed. ¹ Part of this investigation was reported at the XII InternationalBotanical Congress, Leningrad, 1975 and the Japan-US CooperativeScience Seminar "Biological Solar Energy Conversion", Miami,1976. Requests for reprints should be addressed to S. Miyachi,Radioisotope Centre, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. ⁴ Present address: Department of Chemistry, Faculty of PharmaceuticalSciences, Teikyo Univ., Sagamiko, Kanagawa, Japan. (Received August 6, 1977; )     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma     

Measurement of the Carbon Dioxide Compensation Point and the Rate of Loss of 14CO2 in the Light and Dark in Some Bryophytes

The CO₂ compensation point at 25 °C and 250 µEinsteinsm^–2 s^–1 wasmeasured for 27 bryo-phyte species, andwas found to be in the range of 45–160 µl CO₂ I^–1air. Under the same conditions Zea mays gave a value of 11 µlI^–1 and Horde um vulgare 76 µI^–1. The rate of loss of photosyntheticallyfixed ¹⁴CO₂ in the light and dark in six bryophytes (three mosses,two leafy liverworts, one thalloid liverwort) was determinedin CO₂-free air and 100% O₂. The rate of ¹⁴CO₂ evolution inthe light was less than that in the dark in CL₂-free air, butin 100% O₂ the rate in the light increased, so that in all butthe leafy liverworts it was greater than that in the dark. Raisingthe temperature tended to increase the rate of 14CO₂ evolutioninto CO₂-free air both in the light and dark, so that the light/dark(L/D) ratio did not greatly vary. The lower rate of loss of¹⁴CO₂ in the light compared tothe dark could be due to partialinhibition of ‘dark respiration’ reactions in thelight, a low rate of glycolate synthesis and oxidation, or partialreassimilation of the ¹⁴CO₂ produced, or a combination of someor all of these factors.     

Studies on the Role of Photosynthesis in the Photoperiodic Induction of Flowering in the Short-Day Plants Kalanchoe blossfeldiana Poellniz and Xanthium pensylvanicum Wallr: I. THE REQUIREMENT FOR CO2 DURING PHOTOPERIODIC INDUCTION

It has been established that Kalanchoe blossfeldiana and Xanthiumpensylvanicum require CO₂ during the light period of short daysfor successful photoperiodic induction of flowering, even ifall but the induced leaf are held in normal air. In X. pensylvanicumfloral induction in normal air was independent of the starchstatus of the leaves but when reserves were reduced, lack ofCO₂ in the light suppressed floral induction to an even greaterextent. Injection into the induced leaf (Kalanchoe) or leaftip feeding (Xanthium) of carbohydrates, organic and amino acidsor several other metabolites failed to substitute for the CO₂requirement for induction. A small response was produced by10 mg ml^–1 sucrose in X. pensylvanicum while in normalair 25 parts 10^–6 ATP reduced the time to flowering inK. blossfeldiana and 10^–4 M proline was inhibitory. Anexperiment on the light requirement established a need for redlight ( max 660 nm) during photoperiods but red light alonedid not facilitate maximal induction. It is concluded that someearly, possibly labile, product of photosynthetic CO₂ fixationis essential to floral induction in these species.     

14CO2 Pulse-Chase Labelling in Clusia minor L.

Conditions and maintenance of growth were chosen so that plantsof Clusia minor L. were obtained which showed the C₃- and CAM-modes of CO₂-exchange, respectively. C. minor is known to accumulateconsiderable amounts of citric acid in addition to malic acidduring the dark-phase of CAM. ¹⁴CO₂-pulse-chase experiments were performed with these plants.Patterns of labelling during the pulse and redistribution oflabel during the chase in the C₃-mode were as expected for C₃-photosynthesis.Pulse-labelling in the CAM-mode during the last hour of thelight period, during the first part of the dark period and duringthe last hour of the dark period always led to an almost exclusiveincorporation of label into malate. Redistribution of labelfrom malate after the pulse at the end of the dark period duringthe chase in the subsequent light period followed the patternexpected for light-dependent reassimilation of CO₂ remobilizedfrom malate in CAM during the light period. During the chasesin the dark period, label was transferred from ^l4C-malate tocitrate. This suggests that during accumulation of citric acidin the dark period of CAM in C. minor, citrate is synthesizedin the mitochondria from malate or oxaloacetate after formationof malate via phosphoenolpyruvate carboxylase. The experiment also showed that no labelled compounds are exportedfrom leaves in the CAM-mode during the dark period. In plantsof the C₃-mode the roots proved to be strong sinks. Key words: Clusia minor, labelling, pulse-chase, ¹⁴CO₂     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

Photosynthesis with Single-Rooted Amaranthus Leaves. I. Changes in the Activities of RuBP-1,5-bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and the Amounts of Intermediates in Photosynthetic Metabolism in Response to Changes in the Source-Sink Balance

C₄ model plants composed of single-rooted Amarantus cruentusL. leaves were developed to study source-sink relationships.The photosynthetic activity of CO₄ fixation in the rooted leavescultured under the control condition (10-h light/14-h darkness,730 µmol photons m^–2s^–1) remained constantand high. When the leaves kept for 8 or 9 d under the controlcondition were exposed to continuous light (CL) for 4 d, theactivity decreased steeply. The starch content increased significantlyduring the first 2 d in CL whereas the sucrose content increasedalmost linearly during the 4 d in CL. The close relationshipbetween the decrease in photosynthetic activity and the increasein sucrose indicates that the activity was subjected to feedbackinhibition when the end product was forced to accumulate inthe leaves. In the treated leaves, the initial activity of ribulose-1,5-bisphoshatecarboxylase decreased significantly. A large increase in ribulose-1,5-bisphosphateand decrease in 3-phosphoglycerate occurred following the deactivationin RuBPcase, which was fully restored by the preincubation withCO² and Mg²⁺ and/or P₁ and Mg²⁺. The activity of phosphoenolpyruvatecarboxylase also decreased but not as rapidly as RuBPcase. Thealanine and pyruvate levels decreased markedly but the oxaloacetate,glutamine and asparagine levels significantly increased whilephosphoenolpyruvate and glutamate were kept at almost constantlevels. The malate level first increased slightly and then decreasedsignificantly. The mechanism of coordinated decrease in carboxylationreactions between phtosynthetic carbon reduction cycle and C₄metabolism in response to change in the source-sink balanceis discussed. (Received May 24, 1999; Accepted September 1, 1999)     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )