Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

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; )     

Reversal of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition of carbon dioxide fixation in spinach chloroplasts and protoplasts by dicarboxylic acids

3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA.     

The Photoinhibition Site of Photosystem I in Isolated Chloroplasts under Extremely Reducing Conditions

Under anaerobic atmosphere where the gas phase was simply replacedby N₂, photo-inhibition of PS I of isolated spinach chloroplastswas insignificant. However, when dithionite was included inthe irradiation mixture, severe photoinhibition of the NADP⁺and the MV photo-reduction occurred. Neither P700 determinedby continuous illumination-induced difference spectroscopy,Fe-S centers determined by EPR under cryogenic temperatures,nor vitamin K-l determined by HPLC analysis were significantlydecreased under these photoinhibition conditions. Although photobleachingof antenna chlorophylls occurred to more or less extent, NADP⁺photoreduction activities were markedly depressed even undersaturating actinic light. The maximal amplitude of the flashinduced absorbance changes of P700 in ms range decreased almostin parallel with the loss of NADP⁺ photoreduction activity.These results indicate that although the Fe-S centers of thephotoinhibited chloroplasts were reducible by continuous illumination,to almost the same extents as that of the control chloroplasts,the efficiency of reduction by each flash was much lower thanthat of the control chloroplasts. The site of photoinhibitionin PS I under extremely reducing conditions is between A₀ andFe-S X. (Received July 28, 1988; Accepted October 31, 1988)     

Malate and Dihydroxyacetone Phosphate-dependent Nitrate Reduction in Spinach Leaf Protoplasts

Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO₃⁻. A similar stimulation of nitrate reduction in the absence of CO₂ fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light-independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO₂ fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship.     

The Metabolism of Proline in Higher Plants: II. L-PROLINE DEHYDROGENASE FROM COTYLEDONS OF GERMINATING PEANUT (ARACHIS HYPOGAEA L.) SEEDLINGS

The L-proline-dependent reduction of NAD⁺ has been obtainedwith a soluble enzyme extracted from acetone powders of thecotyledons of 3- to 5-day-old germinating peanut seedlings.The enzyme has been purified approximately 20-fold. NAD⁺ ismuch more effective as an electron acceptor than NADP⁺, thereaction rate with the latter being only 15 per cent that withthe former. The K_m for L-proline at pH 10.3, with NAD⁺ saturating,is 0.30 mM, and that for NAD⁺, with L-proline saturating, is0.25 mM. NADP⁺ is an excellent competitive inhibitor for NAD⁺with a K₁ of 6.2 µM. L-proline, L-proline methyl ester, and 3,4-dehydro-DL-prolineare equally effective as substrates. Thiazolidine-4-carboxylatecatalyses the reduction of NAD⁺ at 63 per cent the rate withL-proline. D-proline is not a substrate nor an inhibitor. L-prolineamide has 11 per cent the activity of L-proline and N-methyl-L-prolinehas a very slight activity. Other proline derivatives or thelower and higher homologues are completely inactive. Incubation with L-proline-¹⁴C in the presence of NAD⁺ yieldsone product which has a higher Rf than proline using butanol-aceticacid-water as the solvent in paper chromatography. Elution ofthis product and treatment with hydrogen peroxide gives severalproducts of high Rf with the same solvent mixture. None of theproducts is -aminobutyrate or glutamic acid. This eliminateseither P2C or P5C as the reaction product.     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids

Thylakoid-bound and stromal ascorbate peroxidases scavenge thehydrogen peroxide that is photoproduced in PSI of chloroplastthylakoids. The primary oxidation product of ascorbate in thereaction catalyzed by ascorbate peroxidase, the monodehydroascorbate(MDA) radical, is photoreduced by thylakoids [Miyake and Asada(1992) Plant Cell Physiol. 33: 541]. We have now shown thatthe photoreduction of MDA radical in spinach thylakoids is largelydependent on ferredoxin (Fd), as determined by the monitoringthe MDA radical by electron paramagnetic resonance. Further,the reduced Fd generated by NADPH and Fd-NADP reductase couldreduce the MDA radical at a rate of over 10⁶ M^–1 s^–1,indicating that the photoreduced Fd in PSI directly reducesthe MDA radical to ascorbate. Photoreduction of NADP⁺ by spinach thylakoids was suppressedby the MDA radical and conversely that of MDA radical was suppressedby NADP⁺, indicating a competition between the MDA radical andNADP⁺ for the photoreduced Fd in PSI. The ratio of the rateconstant for the photoreduction of MDA radical to that for thephotoreduction of NADP⁺ was estimated to be more than 30 to1. Thus, MDA radical is preferentially photoreduced as comparedto NADP⁺. From these results, we propose that the thylakoid-boundascorbate peroxidase and the Fd-dependent photoreduction ofMDA radical in PSI are the primary system for the scavengingof the hydrogen peroxide that is photoproduced in the thylakoids. (Received December 9, 1993; Accepted February 16, 1994)     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Autocatalysis and light activation of enzymes in relation to photosynthetic induction in wheat chloroplasts

In order to study the relative contributions of the autocatalytic increase in the level of substrates and the light activation of enzymes to the control of the induction phase or “lag” in wheat chloroplasts, we measured the light-induced reductive activation of fructose 1,6-bisphosphatase, phosphoglycerate kinase, NADP⁺-dependent glyceraldehyde-phosphate dehydrogenase, ribulose 1,5-bisphosphate carboxylase, and phosphoribulokinase in isolated chloroplasts. Each was rapidly activated to levels more than adequate to support the maximum rate of photosynthesis. Induction in wheat chloroplasts is characterized by a period of about 1 min during which no O₂ is evolved. If small quantities of intermediates such as dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerate (PGA) are added, maximum rates of photosynthesis are achieved within the first minute of illumination. The presence of PGA did not affect the activation of any of the above-mentioned enzymes. Each of the enzymes was therefore capable of sustaining maximum rates of photosynthesis in the presence of PGA, even though there was no O₂ evolution from those chloroplasts incubated with CO₂ alone as substrate. The inclusion of PGA did not give rise to abnormally high levels of DHAP, FBP, or fructose 6-phosphate in the stroma. We conclude that the levels of substrates or cofactors are the principal, if not the sole, determinants of the rate of photosynthetic carbon assimilation during induction in wheat chloroplasts.     

Maximum H+/h{nu}PSI Stoichiometry of Proton Transport during Cyclic Electron Flow in Intact Chloroplasts Is at Least Two, but Probably Higher than Two

Effects of antimycin A on 9-aminoacridine (9AA) fluorescencequenching by intact chloroplasts during light-dependent electronflow to different electron acceptors indicated that considerablecyclic electron flow occurs concurrently with linear electrontransport already at low PFDs, when oxygen supported electronflow, but not, when nitrite or methylviologen (MV) were present.Quantum efficiencies of the use of 696 and 675 nm light werecalculated for oxygen-, nitrite- and MV-dependent linear electronflows. Since H⁺/e=3 during linear electron transport [Ivanov(1993) Photosynthesis, p. 111; Kobayashi et al. (1995) PlantCell Physiol. 36: 1613] and comparable 9AA fluorescence quenchingindicates comparable transthylakoid proton gradients, totalproton transport could be calculated and part of it could beassigned to linear and the remainder to cyclic electron transportwhen oxygen was electron acceptor. Quanta of 696 nm light notused to support linear electron flow to oxygen at h/e=2 wereassumed to be available for coupled proton transport duringcyclic electron flow. H⁺/h ratios for cyclic electron transportobtained on this basis were consistently higher than 1 and occasionallyapproached 3. No allowance was made in these calculations foroxidized P700 in the reaction center of PSI, which could notdonate electrons to the cyclic pathway, and for reduced Q_A inthe reaction center of PSII. It therefore appears likely thatmaximum H⁺/h ratios in cyclic electron transport are higherthan values calculated in this work. Our observations with intactchloroplasts agree in principle with those of [Heath (1972)Biochim. Biophys. Acta 256: 645] with thylakoids, who also reportedhigh H⁺/ e ratios in cyclic electron transport. These ratiosare briefly discussed in relation to the H⁺/ATP stoichiometryof ATP production during carbon assimilation of leaves and toprotection of chloroplasts against photoinactivation. ²Present address: Timiriasev Institute of Plant Physiology,Russian Academy of Sciences, Botanicheskaya, 35, Moscow, Russia ³Present address: Department of Forestry, Faculty of Agriculture,Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812 Japan     

Effect of linolenate on photosynthesis by intact spinach chloroplasts I. Inhibition of orthophosphate uptake and of 3-P-glyceraldehyde efflux across the chloroplast envelope

Linolenic acid (C_18:3) inhibited photosynthesis by intact spinachchloroplasts. This inhibition was due neither to a lack of NADPHin chloroplasts nor to a direct inhibition of the enzyme activitiesin the Calvin-Benson cycle. Linolenate inhibited CO₂ fixationand oxygen evolution more effectively than NADP⁺ photoreductionbut did not inhibit the activity of several key enzymes of theCalvin cycle. Linolenate inhibited phosphate influx and 3-phosphoglyceraldehydeefflux across the chloroplast envelope. A hypothesis explainingthe inhibition of photosynthesis by linolenate is presented. ¹ This work is part of a doctoral program which is carried outby L. Mv? Akamba in this laboratory. (Received October 14, 1978; )     

The Inhibition of the Activity of Apple Mitochondria by Oxaloacetate

The inhibitory effect of oxaloacetate (OAA) on the activityof mitochondria isolated from the peel of Cox's Orange Pippinapples has been investigated. A given concentration of OAA causesa longer inhibition of succinate than of malate oxidation andthe rate of disappearance of OAA is faster in the presence ofmalate than in that of succinate. Mg⁺⁺⁺, Al⁺⁺⁺, ATP, and glutamateaccelerate the disappearance of inhibition by OAA; Ca⁺⁺ reinforcesthe inhibition. It is established, by estimation of the oxoacids in the reaction mixtures, that the relief from inhibitionis directly due to removal of OAA. The fall in rate of O₂ uptakewith time, using succinate or malate as substrate, is accompaniedby an accumulation of OAA, and the inhibition of succinate oxidationby malate is due to the increased OAA production when malateis oxidized. Some OAA is broken down non-enzymically to formpyruvate and the rate of breakdown is enhanced by Mg⁺⁺; someis metabolized via the Krebs cycle; some disappear in a coupledreaction between pyruvic and malic dehydrogenases to form citrateand malate, and some can be removed by transamination. It issuggested that all these may be factors in a regulatory actionof OAA on the operation of the Krebs cycle. It is relevant inthis connexion that very small amounts of OAA inhibit the activityof the Krebs cycle when they are produced at the active siteswithin the mitochondrion.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

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

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

Effects of univalent cations on the formation of nitrate reductase and nitrite reductase in rice seedlings

An investigation was made to determine the effects of univalentcations as activators on the formation of nitrate reductaseand nitrite reductase in rice seedlings. K⁺ functioned moreeffectively as a univalent cation activator than did other univalentcations examined. Substitution of Rb⁺ for K⁺ resulted in stimulationof nitrate reductase formation at about half the rate obtainedwith K⁺. There was no effect on nitrite reductase formation.Na⁺ could be partially substituted for K⁺ in the formation ofboth enzymes. NH₄⁺ slightly inhibited formation of the enzymes.In the absence of univalent cations, enzyme formation proceededat a slower rate during the initial 15-hr period, but thereafterproceeded at a higher rate. This delayed formation was not observedin the presence of K⁺. Results from inhibitor experiments suggestthat K⁺ stimulates the formation of nitrate reductase and nitritereductase. In conclusion, when nitrate nitrogen is supplied to rice plantsutilization of the nitrogen may be accelerated by increasedformation of enzymes involved in nitrate assimilation in thepresence of K⁺. (Received February 21, 1969; )     

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)     

The Role of Phosphoenolpyruvate in Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of Maize

Phosphoenolpyruvate (PEP) was transported together with H⁺ inC₄ mesophyll chloroplasts. Medium alkalization and stromal acidificationdue to pyruvate uptake into maize mesophyll chloroplasts inthe light were partially inhibited by adding PEP. Thus, theH⁺ taken up by H⁺/pyruvate cotransport into mesophyll chloroplastsis released together with PEP in vivo. (Received August 5, 1994; Accepted October 3, 1994)     

Vieillissement de l'appareil photosynth?tique II. Effet synergique de la lumi?re et du vieillissement in vitro sur les activit?es photochimiques de chloroplastes isol?s d'?pinard

The consequences of chloroplast ageing in vitro were furtherinvestigated, especially on the photochemical activities ofthese organelles. Ageing of chloroplasts in dark was accompanied by decreasesin activities for photohydrolysis and cyclic and non-cyclicsyntheses of ATP, photoreduction of NADP⁺ and O₂ evolution;but there was no decrease in ferricyanide photoreduction. Therates of decrease in these activities were comparable to therate of increase in chloroplast volume. Complete inhibitionswere reached when maximum chloroplast swelling had occurred,i.e. after 5 to 6 hr of incubation at 20?C in a Tris-NaCl (pH8) medium. Ageing in the light resulted in much accelerateddecreases in activities tested; the loss of capacity for light-inducedshrinkage was also accelerated by the light during ageing. Thus,light acts synergetically towards the ageing process. Moreover,light and, to a less extent, dark ageing, resulted in an uncouplingof chloroplast photophosphorylation and associated electronflow measured by ferricyanide photoreduction. The part of theelectron flow which is induced by coupling (+ ADP, Pi, MgCl₂,pH 8) or by uncoupling (+ NH₄C1, pH 7) was found to be verysensitive to light ageing. The NADP⁺ photoreduction loss wasrestored by addition of the ascorbate-DCPIP electron donor system,suggesting that ageing interferes with the integrity of photosystemII. In many respects, these effects of ageing are comparable withthe action of detergents and fatty acids on the structure andphotochemical activities of chloroplasts, especially in thatthey attack the energy transducing mechanism in chloroplasts. (Received May 24, 1969; )