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Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3–CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase.

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.

In this study, the function of the NAD(P)H dehydrogenase (NDH)-dependent pathway in suppressing the accumulation of reactive oxygen species in chloroplasts was investigated. Hydrogen peroxide accumulated in the leaves of tobacco (Nicotiana tabacum) defective in ndhC-ndhK-ndhJ (ΔndhCKJ) at 42°C and 4°C, and in that of wild-type leaves at 4°C. The maximum quantum efficiency of PSII decreased to a similar extent in both strains at 42°C, while it decreased more evidently in ΔndhCKJ at 4°C. The parameters linked to CO2 assimilation, such as the photochemical efficiency of PSII, the decrease of nonphotochemical quenching following the initial rise, and the photosynthetic O2 evolution, were inhibited more significantly in ΔndhCKJ than in wild type at 42°C and were seriously inhibited in both strains at 4°C. While cyclic electron flow around PSI mediated by NDH was remarkably enhanced at 42°C and suppressed at 4°C. The proton gradient across the thylakoid membranes and light-dependent ATP synthesis were higher in wild type than in ΔndhCKJ at either 25°C or 42°C, but were barely formed at 4°C. Based on these results, we suggest that cyclic photophosphorylation via the NDH pathway might play an important role in regulation of CO2 assimilation under heat-stressed condition but is less important under chilling-stressed condition, thus optimizing the photosynthetic electron transport and reducing the generation of reactive oxygen species.

Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.

Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport.

Tobacco (Nicotiana tabacum L.) cells growing heterotrophically in the light on supplied sucrose (S0) have previously been adapted to grow in 428 mM NaCl (S25). Among the changes occurring in salinity-adapted cell cultures are (a) elevated levels of chlorophyll compared to unadapted cells; (b) decreased levels of starch; (c) alterations in chloroplast ultrastructure, including loss of starch grains, increased thylakoid membrane structure, and the presence of plastoglobules; and (d) increased rates of O2 evolution, CO2 fixation, and photophosphorylation relative to S0 cells. These latter changes apparently derive from the fact that thylakoid membranes in S25 cells contain higher levels of photosystem I- and II-associated proteins as well as thylakoid ATPase components. S25 chloroplasts contain immunologically detectable levels of ribulose-1,5-bisphosphate carboxylase/oxygenase, whereas S0 completely lack the enzyme. These changes taken together suggest that even in the presence of sucrose, S25 cells have acquired a significant degree of salt-tolerant photosynthetic competence. This salt-tolerant photoysynthetic capability manifests itself in plants backcrossed with normal plants for three generations. These plants contain chloroplasts that demonstrate in vitro more salt-tolerant CO2 fixation, O2 evolution, and photophosphorylation than do backcross progeny of plants regenerated from S0 cultures.

The effect of O2 on photosynthesis was determined in maize (Zea mays) leaves at different developmental stages. The optimum level of O2 for maximum photosynthetic rates was lower in young and senescing tissues (2-5 kPa) than in mature tissue (9 kPa). Inhibition of photosynthesis by suboptimal levels of O2 may be due to a requirement for functional mitochondria or to cyclic/pseudocyclic photophosphorylation in chloroplasts; inhibition by supraoptimal levels of O2 is considered to be due to photorespiration. Analysis of a range of developmental stages (along the leaf blade and at different leaf ages and positions) showed that the degree of inhibition of photosynthesis by supraoptimal levels of O2 increased rapidly once the ribulose-1,5-bisphosphate carboxylase/oxygenase and chlorophyll contents were below a critical level and was similar to that of C3 plants. Tissue having a high sensitivity of photosynthesis to O2 may be less effective in concentrating CO2 in the bundle sheath cells due either to limited function of the C4 cycle or to higher bundle sheath conductance to CO2. An analysis based on the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase was used to predict the maximum CO2 level concentrated in bundle sheath cells at a given degree of inhibition of photosynthesis by supraoptimal levels of O2.

A complex between chloroplast-coupling factor 1 (CF1) and subunit III of the membrane-spanning portion of the chloroplast ATP synthase (CF0), isolated as described in the accompanying paper (C.M. Wetzel and R.E. McCarty [1993] Plant Physiol 102: 241-249), has been further characterized. A comparison of the ATPase activities of CF1, CF1-subunit III, and the chloroplast ATP synthase (CF1-CF0) holoenzyme revealed that the properties of CF1-subunit III more closely resemble those of CF1-CF0 than those of CF1. In particular, the Ca2+-ATPase activity after reduction of the enzyme with dithiothreitol was much lower in CF1-subunit III and CF1-CF0 than in CF1, suggesting that the association of the inhibitory [epsilon] subunit is tightened by the presence of either CF0 or subunit III. Cold stability is a property of CF1-CF0 in thylakoid membranes. The ATPase activity of CF1 incubated in the cold in the presence of asolectin liposomes was lost more rapidly than that of either CF1-subunit III or CF1-CF0 incorporated into liposomes. Removal of the [epsilon] subunit from all three preparations resulted in marked stimulation of their ATPase activity. Although subunit III was also removed during depletion of the [epsilon] subunit, it is not known whether the two subunits interact directly. CF1 deficient in the [epsilon] subunit binds to liposomes containing either subunit III or CF0. Taken together, these results provide evidence that the association of CF1 and subunit III of CFo is specific and may play a role in enzyme regulation.

It is of theoretical as well as practical interest to identify the components of the photosynthetic machinery that govern variability in photosynthesis rate (A) and water-use efficiency (WUE), and to define the extent by which the component processes limit A and WUE during developing water-deficit stress. For that purpose, leaf exchange of CO2 and H2O was determined in two growth-chamber-grown wheat cultivars (Triticum aestivum L. cv TAM W-101 and cv Sturdy), and the capacity of A was determined and broken down into carboxylation efficiency (c.e.), light- and CO2-saturated A, and stomatal conductance (gs) components. The limitations on A measured at ambient CO2 concentration (A350) were estimated. No cultivar difference was observed when A350 was plotted versus leaf water potential (Ψw). Light- and CO2-saturated A, c.e., and gs decreased with decreasing leaf Ψw, but of the corresponding photosynthesis limitations only those caused by insufficient c.e. and gs increased. Thus, reduced stomatal aperture and Calvin cycle activity, but not electron transport/photophosphorylation, appeared to be major reasons for drought stress-induced inhibition of A350. WUE measured as A350/gs first increased with stomatal closure down to a gs of about 0.25 mol H2O m−2 s−1 (Ψw = −1.6 MPa). However, it was predicted that A350/gs would decrease with more severe stress due to inhibition of c.e.

Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.

High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20 degrees C) and stress (35 degrees C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b(6)-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity.

The changes in the light-harvesting antenna size of photosystem I were investigated in the green alga Chlamydobotrys stellata during transition from autotrophic to photoheterotrophic nutrition by measuring the light-saturation behavior of hydrogen evolution following single turnover flashes. It was found that during autotrophic-to-photoheterotrophic transition the antenna size of photosystem I increased from 180 to 250 chlorophyll. The chlorophyll (a + b)/P700 ratio decreased from 800 to 550. The electron transport of photosystem I measured from reduced 2,6-dichloro-phenolindophenol to methylviologen was accelerated 1.4 times. In the 77K fluorescence spectra, the photosystem II fluorescence yield was considerably lowered relative to the photosystem I fluorescence yield. It is suggested that the increased light-harvesting capacity and redistribution of absorbed excitation energy in favor of photosystem I is a response of photoheterotrophic algae to meet the ATP demand for acetate metabolism by efficient photosystem I cyclic electron transport when the noncyclic photophosphorylation is inhibited by CO(2) deficiency.

The in situ response of photophosphorylation and coupling factor activity to low leaf water potential (psi(L)) was investigated using kinetic spectroscopy to measure the flash-induced electrochromic absorption change in attached sunflower (Helianthus annuus L. cv IS894) leaves. The electrochromic change is caused by the formation of an electric potential across the thylakoid membrane associated with proton uptake. Since depolarization of the thylakoid membrane following flash excitation is normally dominated by proton efflux through the coupling factor during ATP formation, this measurement can provide direct information about the catalytic activity of the coupling factor. Under low psi(L) conditions in which a clear nonstomatal limitation of net photosynthesis could be demonstrated, we found a strong inhibition of coupling factor activity in dark-adapted leaves which was probably caused by an increase in the energetic threshold for the activation of the enzyme at low psi(L). While this result supported earlier in vitro findings, we further discovered that the light-dependent reduction of coupling factor reversed any observable effect of low psi(L) on the energetics of activation or on photophosphorylation competence. Furthermore, coupling factor was reduced, even in severely droughted sunflower, almost immediately upon illumination. Based on these measurements, we conclude that the nonstomatal limitation of photosynthesis observed by us and others in droughted plants cannot be explained by impaired coupling factor activity.

Photosynthetic oxygen evolution by isolated spinach ( Spinacia oleracea L.) chloroplasts approached complete inhibition in the presence of a 5 mm concentration of sulfur dioxide. A similar inhibition was observed in the presence of equimolar concentrations of bisulfite ions, suggesting a parallel mode of action. In contrast, an equimolar concentration of sulfite ions was markedly less inhibitory and sulfate ions caused negligible inhibition of apparent photosynthesis. The mode of action of sulfur dioxide and related sulfur anions in inhibiting photosynthesis was found to be essentially independent of direct hydrogen-ion effects. Supplements of inorganic pyrophosphate lessened the inhibition of oxygen evolution caused by sulfur dioxide and the sulfur anions. Sulfur dioxide and the sulfur anions were almost equally effective in inhibiting cyclic and noncyclic photophosphorylation in chloroplast suspensions. However, the extent of the inhibition of these photosynthetic reactions does not appear sufficient to account for the inhibition of photosynthetic oxygen evolution by sulfur dioxide.

Oligomycin inhibited the membrane-bound, Ca(2+)-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg(2+)-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.Sensitivity of isolated pea thylakoid Mg(2+)-dependent ATPase to oligomycin was found during summer months but not during the winter. This was traced to a seasonal variation in relative humidity with values of 20% or less during the winter, leading to lack of inhibition in the isolated chloroplasts. The effect of low relative humidity during growth could be reversed by placing plants for 17 to 20 hours in a chamber at 50% relative humidity. The optimum humidity range for sensitivity to oligomycin was between 40 and 60%.With chloroplasts potentially sensitive to oligomycin due to "permissive" growth conditions, the inhibition was modulated by a number of parameters during chloroplast activation or ATPase assay. Preincubation in high concentrations of KCl diminished oligomycin sensitivity, and this effect was reversed by valinomycin. The greatest sensitivity, as well as the highest ATPase rates, occurred only if thylakoids were activated by light and DTT at a relatively high temperature (35 to 40 degrees C). Controlling the levels of uncouplers and running the reaction in darkness rather than in the light, both of which diminished the degree of membrane energization, increased the sensitivity to oligomycin. It is possible that accessibility of the oligomycin binding site is diminished by membrane energization. These results are contrasted with those from studies of variability in sensitivity of yeast mitochondria to oligomycin.

Illuminated pea (Pisum sativum) chloroplasts can convert glutamate to glutamine using ATP generated by photophosphorylation to drive the glutamine-synthetase reaction. Light-dependent glutamine synthesis is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), but only at concentrations higher than are necessary to suppress photoreduction of ferricyanide or phosphoglycerate. Conversely, glutamine synthesis is far more sensitive to antimycin A than is photoconversion of phosphoglycerate to triosephosphate. When 3.8 mm phosphoglycerate is supplied, glutamine synthesis is stimulated in both the presence and absence of antimycin A.These data seem to be consistent with the operation of an endogenous, DCMU-sensitive, phosphorylation process-possibly cyclic-which can support glutamine synthesis in white light under aerobic conditions. The stimulatory effect of phosphoglycerate suggests that noncyclic phosphorylation is initiated or accelerated when this substrate is supplied. This noncyclic process evidently provides ATP over and above the amount required for phosphoglycerate photoreduction, i.e. the ATP/e(2) ratio exceeds 1.0. The additional ATP produced under these conditions is available for glutamine synthesis and lessens its dependence on cyclically (or pseudocyclically) generated ATP.

Stomatal conductance is coupled to leaf photosynthetic rate over a broad range of environmental conditions. We have investigated the extent to which chloroplasts in guard cells may contribute to this coupling through their photosynthetic activity. Guard cells were isolated by sonication of abaxial epidermal peels of Vicia faba. The electrochromic band shift of isolated guard cells was probed in vivo as a means of studying the electric field that is generated across the thylakoid membranes by photosynthetic electron transport and dissipated by photophosphorylation. Both guard cells and mesophyll cells exhibited fast and slow components in the formation of the flash-induced electrochromic change. The spectrum of electrochromic absorbance changes in guard cells was the same as in the leaf mesophyll and was typical of that observed in isolated chloroplasts. This observation indicates that electron transport and photophosphorylation occur in guard cell chloroplasts. Neither the fast nor the slow component of the absorbance change was observed in the presence of the uncoupler carbonylcyanide p-trifluoromethoxy-phenylhydrazone which confirms that the absorbance change was caused by the electric field across the thylakoid membranes. The magnitude of the fast rise was reduced by half in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Therefore, photosystem II is functional and roughly equal in concentration to photosystem I in guard cell chloroplasts. The slow rise was abolished by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone indicating the involvement of the cytochrome b6/f complex in electron transport between the two photosystems. Relaxation of the absorbance change was irreversibly retarded in cells treated with the energy transfer inhibitor, N,N′-dicyclohexylcarbodiimide. The slowing of the rapid decay kinetics by N,N′-dicyclohexylcarbodiimide confirms that the electrical potential across the thyalkoid membrane is dissipated by photophosphorylation. These results show that guard cell chloroplasts conduct photosynthetic electron transport in a manner similar to that in mesophyll cells and provide the first evidence that photophosphorylation occurs in guard cells in vivo.

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO2 was markedly stimulated (647 ± 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K½, = 18 micromolar). Studies with KCl and Na2SO4 showed that the stimulation was due to Na+. Photosynthesis at pH 6.1 was only slightly stimulated by Na+. The response of photosynthesis at pH 8.0 to [Na+] was strongly sigmoidal for dissolved inorganic carbon ([DIC] ≤ 500 micromolar). Cells grown with high total [DIC], but air-levels of CO2, at pH 9.6 showed the same response to low [Na+]. The absence of Na+ could be partially, but not completely overcome, by higher [DIC]. Various methods for examining CO2 or HCO3− use (K½CO2 determination; isotopic disequilibrium; and consideration of HCO3− dehydration rate) were consistent with CO2 use by the cells, but HCO3− use could not be ruled out. Isotopic disequilibrium studies showed that CO2 use was stimulated by Na+. Cells grown on 5% CO2 accumulated DIC against a concentration gradient by a process (or processes) dependent on Na+. No evidence for uptake of Na+ concomitant with DIC uptake could be found. The lack of O2 evolution during the initial and most rapid period of DIC accumulation suggested that the required energy was obtained from cyclic photophosphorylation.

The cyanelle from the photosynthetic biflagellate protist Cyanophora paradoxa has been studied in terms of its photosynthetic properties. Structurally, the cyanelle resembles unicellular cyanobacteria. The cyanelle is readily released from the host cell by means of the French press. The isolated cyanelle shows typical photosystem I and photosystem II activities as well as phenazine methosulfate-mediated photophosphorylation. The kinetic parameters Km and Vmax were determined for CO2 fixation in the cyanelle and cells of C. paradoxa and compared to a cyanobacterium. The determined values were not much different, although the cyanobacterium had a significantly greater rate of CO2 fixation, and the cyanelle was least active in this regard. Photosystem I chlorophyll-protein complex is readily isolated from the thylakoid membrane. In all these respects, the photosynthetic apparatus of the cyanelle resembles that of cyanobacteria. No nitrogen fixation activity was observed. Attempts to regenerate the isolated cyanelle were not successful, but in some cases, an unidentified cyanobacterium grew up in standing cultures of C. paradoxa cyanelles. Buoyant density data indicate that the strain of C. paradoxa we have investigated differs from that employed by others, since our strain shows a value of 1.716 grams per cubic centimeter and others report values of 1.695 and 1.691.

Isolated maize bundle sheath chloroplasts showed substantial rates of noncyclic photophosphorylation. A typical rate of phosphorylation coupled to whole-chain electron transport (methylviologen or ferricyanide as acceptor) was 60 mumol per hour per milligram chlorophyll) with a coupling efficiency (P/e(2)) of 0.6. Partial electron transport reactions driven by photosystem I or II supported phosphorylation with P/e(2) values of 0.2 to 0.3. Thus, two sites of phosphorylation seem to be associated with the photosynthetic chain in much the same way as in spinach chloroplasts.Isolated bundle sheath cells were capable of photosynthetic electron transport with membrane permeant electron carriers (but not with ferricyanide) at rates which were similar to those found in isolated chloroplasts. ATP formation also occurred during electron transport when ADP and phosphate were present in the cell suspension. The rates of photophosphorylation reactions in cells were about 30 to 40% of those found in isolated chloroplasts (maximum rate in cells 80 mumoles ATP per hour per milligram chlorophyll with diaminodurene as electron carrier), with the exception of endogenous photophosphorylation (photophosphorylation without added electron carriers) whose rate was three to four times higher in cells than in chloroplasts. The endogenous photophosphorylation in cells appeared to be coupled to pseudo-cyclic, rather than cyclic, electron transport. It was accompanied by O(2) uptake (when the catalase inhibitor KCN was present), was sensitive to dichlorophenyldimethylurea and methylamine, but was totally insensitive to 20 micromolar antimycin which completely inhibited succinate-supported oxidative phosphorylation in the cells. The implications of these and other phenomena associated with photophosphorylation in bundle sheath cells are discussed.

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N',N'-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH(2)OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach ( Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O 2 evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl- p -benzoquinone, and 2,6-dichlorophenolindophenol or as O 2 uptake with methyl viologen as acceptor. Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl- p -benzoquinone (photosystem II) from H 2 O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations. The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O 2 evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O 2 evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

Both transmittance changes in a weak beam of green light (light scattering) and the slow decay of chlorophyll a fluorescence were used as indicators of the energy state of leaves of a Crassulacean acid metabolism plant, Kalanchoe pinnata, at frequent intervals during 12-hour light/12-hour dark cycles. To induce light scattering and fluorescence changes, leaves were exposed to red light for 6 minutes. When measurements were made during the light period, the leaves were kept in darkness for 6 minutes before illumination. In the middle of the light period, when malic acid decarboxylation was very active and stomatal conductance was low, light scattering changes were small and indicated that the energy state of leaves was low. This result was supported by determination of adenylate levels. Light scattering and ATP/ADP ratios increased during the late light period when the tissue was deacidified. Illumination produced maximum light scattering changes between the 2nd and 5th hour of the dark period, when rates of dark CO2 fixation were highest. Light scattering and fluorescence measurements taken from leaves, which were illuminated with red or far-red light in the presence or absence of O2 showed that, in addition to linear electron transport, K. pinnata has the potential for both cyclic and pseudocyclic electron transport. The results are relevant with regard to the high ATP demand during Crassulacean acid metabolism.

Two hours after the addition of l-methionine-dl-sulfoximine to the cell suspension, glutamine synthetase activity was inhibited by more than 90% in air-grown Chlamydomonas reinhardii. Cells continued to take up NH(3) from the medium provided that the concentration of dissolved CO(2) was high (equilibrated with 4% CO(2) in air). This NH(3) uptake, about 30% of the control, is discussed in terms of glutamate dehydrogenase activity. Without CO(2), or with a low CO(2) level, a NH(3) excretion was observed, the rate of which depended on the actual concentration of the dissolved CO(2). Experiments with (15)NH(3) demonstrated that no NH(3) uptake was masked by this excretion and inversely that no excretion occurred during the uptake in the conditions where it took place. Furthermore, the NH(3) excretion observed in the absence of CO(2) increased when O(2) concentration rose to 15% and was inhibited when 10 millimolar isonicotinic acid hydrazide was supplied to the algal suspension. Thus, NH(3) excretion in the presence of l-methionine-dl-sulfoximine seems to be related to a photorespiratory process inasmuch as it presents the same properties with regard to the O(2) and the isonicotinic acid hydrazide effects. These results favor the hypothesis that NH(3) produced in the medium originates from the glycine to serine reaction. On the other hand, partial inhibition (50%) of photosynthesis by l-methionine-dl-sulfoximine was attributed to uncoupling between electron transfer and photophosphorylation due to NH(3) accumulation into the cell.

The isolation of the chloroplast ATP synthase complex (CF(0)-CF(1)) and of CF(1) from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil alpha subunit migrates ahead of the beta subunit and epsilon-migrates ahead of subunit II of CF(0) when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF(1) isolated from D. bardawil resembles the CF(1) isolated from Chladmydomonas reinhardi in that a reversible, Mg(2+)-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg(2+)-ATPase of the D. bardawil CF(1) is more resistant to glycerol inhibition than the octylglucoside-activated, Mg(2+)-ATPase of spinach CF(1) or the ethanol-activated, Mg(2+)-dependent ATPase of the C. reinhardi CF(1). Both cyclic photophosphorylation and ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) are more sensitive to high concentrations of NaCl than is the spinach complex.

Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O(2) evolution in the presence of NH(4)Cl) and photosystem II activity (measured as O(2) evolution in the presence of either silicomolybdate plus 3-(3,4-diphenyl)-1,1 dimethylurea or parabenzoquinone) showed similar decreases in activity in response to a photoinhibitory treatment (8 minutes of high light given in the absence of an electron acceptor other than O(2)). Photosystem I activity was less affected. Photoinhibition of silicomolybdate reduction was largely reversible by an 8 minute dark incubation following the light treatment. Decreasing the O(2) concentration during photoinhibition below 2% increased photoinhibition of whole chain electron transport. Addition of superoxide dismutase to the reaction medium did not affect photoinhibition. Photoinhibition of both photosystem I and photosystem II activity increased as the rate of electron transfer during the treatment increased, and was largely prevented when 3-(3,4-diphenyl)-1,1-dimethylurea was present during the photoinhibition period. Noncyclic photophosphorylation was decreased as a consequence of whole chain electron transfer photoinhibition. Since diphenyl carbazide added after light treatment did not relieve photoinhibition of dichlorophenol indophenol reduction, we conclude that the site of inhibition is located within or near the photosystem II reaction center.

Relatively high concentrations of monovalent salts (150 millimolar) stimulated light-saturated uncoupled rates of O 2 evolution linked to oxaloacetic acid (OAA) reduction by intact chloroplasts 2-to 3-fold. In contrast, monovalent salts partially inhibited light-saturated rates of O 2 evolution coupled to CO 2 fixation and uncoupled rates of nitrite reduction. In the presence of high salt concentration, light-saturated rates of electron transport were about equivalent for all three terminal electron acceptors. It is inferred that exogenous monovalent salts have at least two effects on photosynthetic electron transport, independent of photophosphorylation and CO 2 metabolism: a partial inhibitory effect common to OAA, NO 2 − and CO 2 reduction and a marked stimulatory effect unique to the photoreduction of OAA. The stimulation of electron transport to OAA was effected by certain exogenous monovalent salts (KCl or NaCl, but not LiCl). Divalent salts (MgCl 2 or CaCl 2 ) and high osmotic strength were ineffective. The salt-induced stimulation was eliminated by low concentrations of phosphate or sulfate (≥ millimolar) and by higher concentrations of magnesium (≥30 millimolar). These results suggest that the ion content of the medium (or cytosol) is potentially important in modulating photosynthetic electron transport events in intact chloroplasts.

The ATP content of 7-day-old Avena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. l-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, i.e. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.

Glycidate (2,3-epoxypropionate) stimulated CO 2 fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate. Glycolate formation was not inhibited by glycidate, which is in agreement with the observation that preactivated ribulose-1,5-bisphosphate oxygenase is not inhibited by this compound. Furthermore, glycidate had no effect on electron transport (NADP reduction) and photophosphorylation, nor was a change in the coupling ratio observed.

The anaerobic starch breakdown into end-products in the green alga Chlamydomonas reinhardtii F-60 has been investigated in the dark and in the light. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide- p -trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigated. Anaerobic starch breakdown rate (13.1 ± 3.5 micromoles C per milligram chlorophyll per hour) is increased 2-fold by FCCP in the dark. Light (100 watts per square meter) decreases up to 4-fold the dark rate, an inhibition reversed by FCCP. Stimulation of starch breakdown by the proton ionophore FCCP points to a pH-controlled rate-limiting step in the dark, while inhibition by light, and its reversal by FCCP, indicates a control by energy charge in the light. In the dark, formate, acetate, and ethanol are formed in the ratios of 2.07:1.07:0.91, and account for roughly 100% of the C from the starch. H 2 production is 0.43 mole per mole glucose in the starch. Glycerol, d-lactate, and CO 2 have been detected in minor amounts. In the light, with DCMU and FCCP present, acetate is produced in a 1:1 ratio to formate, and H 2 evolution is 2.13 moles per mole glucose. When FCCP only is present, acetate production is lower, and CO 2 and H 2 evolution is 1.60 and 4.73 moles per mole glucose, respectively. When DCMU alone is present, CO 2 and H 2 photoevolution is higher than in the dark. Without DCMU, CO 2 and H 2 evolution is about 100% higher than in its presence. In both conditions, acetate is not formed. In all conditions in the light, ethanol is a minor product. Formate production is least affected by light. The stoichiometry in the dark indicates that starch is degraded via the glycolytic pathway, and pyruvate is broken down into acetyl-CoA and formate. Acetyl-CoA is further dissimilated into acetate and ethanol. In the light, acetate is produced only in the presence of FCCP and, when photophosphorylation is possible, it is used in unidentified reactions. Ethanol formation is inhibited by the light in all conditions.

Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions.

Glandless cotton plants (Gossypium hirsutum L. cv. Coker 100) were subjected to the influence of high CO(2)-bicarbonate. The content of protein decreased with no accompanying increase in its degraded products. The decrease in protein was correlated with the low content of chlorophyll and also with the reduced activity of carbonic anhydrase. The initiation of these correlations coincided with the time when the control leaves contained the highest enzyme activity during leaf growth. The high concentration of bicarbonate directly restricted the rate of photophosphorylation and that of the Hill reaction in isolated chloroplasts. The amount of ATP in leaves treated in vivo also diminished. High CO(2) as bicarbonate, however, did not directly inhibit the activity of carbonic anhydrase in vitro.

Chlorophyll fluorescence transients from mesophyll and guard cell chloroplasts of variegated leaves from Chlorophytum comosum were compared using high resolution fluorescence spectroscopy. Like their mesophyll counterparts, guard cell chloroplasts showed the OPS fluorescence transient indicating the operation of the linear electron transport and the possible generation of NADPH in these organelles. They also showed a slow fluorescence yield decrease, equivalent to the MT transition in mesophyll, suggesting the formation of the high energy state and photophosphorylation. Unlike the mesophyll chloroplasts, the fluorescence from guard cell chloroplasts lacked the increment of the SM transition, indicating that the two types of chloroplasts have some metabolic differences. The presence of CO2 (supplied as bicarbonate, pH 6.7) specifically inhibited the MT-equivalent transition while its absence accelerated it. These observations constitute the first specific evidence of a guard cell chloroplast response to CO2. Control of photosynthetic ATP levels in the guard cell cytoplasm by CO2 may provide a mechanism regulating the availability of high energy equivalents at the guard cell plasmalemma, thus affecting stomatal opening.

Growth and shorter term incorporation measurements with both wild type Chlamydomonas reinhardtii and a mutant (F-60, lacking phosphori-bulokinase activity) indicate that the rate of glycolate utilization is always relatively low. Growth support with external glycolate is restricted to cells with full photosynthetic capacity. A high concentration of glycolate is required for optimal growth support and incorporation of [14C]glycolate. Glycolate incorporation is low at pH 3.8 even with the relatively free permeability. The F-60 mutant can take up only small quantities of glycolate in spite of photosynthetic electron transport and photophosphorylation competencies. This requirement for photosynthetic carbon metabolism indicates a significant difference in the glycolate pathway of this alga. No growth condition significantly increases glycolate incorporation rates. There is no evidence that one of the primary enzymes, glycolate dehydrogenase, is limiting utilization; measurements of glycolate uptake and excretion do not always correlate with its activity. Since the maximal utilization rate of glycolate is low, control of glycolate formation is important in preventing the loss of this fixed carbon from the algal cell.

Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p-phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p-phenylenediamine is completely inactive. When photosystem II is not inhibited, p-phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations.

The synthesis of Mg-protoporphyrin-IX from exogenous protoporphyrin-IX, in a crude plastid pellet extracted from greening cucumber cotyledons was found to require l-glutamate as a cofactor. It has now been shown that glutamate acts in the presence of contaminating mitochondria to provide an ATP regenerating system. With purified plastids, Mg chelatase is not stimulated by glutamate; instead, it requires a high concentration of ATP and is greatly stimulated by added phosphoenolpyruvate and pyruvate kinase. GTP, UTP, CTP, and ITP will not substitute for ATP. ADP in the absence of an ATP generating system is completely ineffective, whereas it is slightly inhibitory in the presence of 10 mm ATP. AMP is strongly inhibitory in the reaction; 50% inhibition is obtained at approximately 3.5 mm AMP in the presence of 10 mm ATP.

Some photosynthetically stimulated acidification of the medium by oat ( Avena sativa L. cv Garry) leaf protoplasts required respiration. The requisite respiration (a) had a low apparent affinity for O 2 , (b) was blocked by cyanide plus salicylhydroxamic acid, (c) characterized protoplasts and mitochondria isolated from protoplasts, (d) could be induced in leaf segments, and (e) appeared to result from an inhibition of mitochondrial respiration that included the cytochrome pathway. Carbon monoxide and cyanide prevented acidification of weakly photosynthesizing suspensions. Salicylhydroxamic acid had no effect on acidification, indicating a specific dependence upon cyanide-sensitive respiration. Photosynthesis stimulated acidification through stable products, and exogenously supplied O 2 stimulated acidification. The acidification response to O 2 was additive to the response to photosynthesis at subsaturating levels of light, indicating a common mode of action. Oligomycin prevented stimulation of acidification by low levels of photosynthetic activity; this stimulation appeared to be due to O 2 -induced increases in mitochondrial energy production. Oligomycin only partially inhibited stimulation of acidification by higher levels of light; this stimulation appeared to be partially dependent upon photophosphorylation. Therefore, oligomycin-sensitive acidification of the medium appeared to reflect changes in mitochondrial energy production in photosynthesizing protoplasts.

Methyl viologen and phenazine methosulfate (photosystem I electron acceptors), 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU, electron-transport inhibitor), and methylamine (photophosphorylation uncoupler) were used to study the dependence of nitrite reduction on electron transport in chloroplasts.DCMU, methyl viologen, and phenazine methosulfate markedly inhibited, whereas methylamine stimulated NO(2) (-) reduction in isolated, intact spinach (Spinacia oleracea L.) chloroplasts. The addition of DCMU to leaf sections of spinach and corn, (Zea mays L. var. XL81), incubated with No(3) (-), caused no inhibition of nitrate reduction but inhibited nitrite reduction leading to the accumulation of NO(2) (-) in the light. The addition of methylamine to comparable leaf sections did not affect either nitrate or nitrite reduction.WE CONCLUDED THAT: (a) nitrite reduction is functionally associated with the electron transport arising from the light reactions of the chloroplast and this provides additional support for the localization of nitrite reductase in the chloroplast; (b) nitrite reduction is associated with photosystem I and ferredoxin is the most likely donor in leaf tissue; and (c) ATP is not involved directly in nitrite reduction. However, ATP synthesis, by regulating electron flow to photosystem I, can affect nitrite reduction in the light.

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.

The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5'-triphosphate synthesis if added in the final, adenosine 5'-triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg(2+)-dependent adenosine 5'-triphosphatase of chloroplasts, and the Ca(2+)-dependent adenosine 5'-triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation.

The ability of intact spinach ( Spinacia oleracea ) chloroplast preparations to catalyze CO 2 fixation and photophosphorylation was examined. Under conditions optimal for CO 2 fixation, only poor photophosphorylation was observed. Conditions optimal for photophosphorylation were found to be highly inhibitory to the CO 2 -fixing capacity of the intact chloroplast preparation. A method for following the activity of phosphoribulokinase in the intact chloroplast preparation was developed, and conditions for optimal activity were defined. The enzyme was found to be activated 2- to 4-fold by preillumination with a half-time of less than 15 seconds. Activation was inhibited by magnesium ions and selectively by inhibitors of photosynthetic electron transport. We concluded that activation was due to the effect of a photoproduced reductant in a site preceding ferredoxin in the electron transport chain. The photoactivated state of the enzyme decayed in the dark with a half-time of about 8 minutes.

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix 14CO2 when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial 14CO2 fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of 14CO2 fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no 14CO2 fixation occurred when 3-phosphoglycerate was used. 14CO2 fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O2 (25-50 micromolar) stimulated 14CO2 fixation, but the activity decreased with increasing O2 concentrations. The fixation of 14CO2 in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.

Leaves of C 3 plants which exhibit a normal O 2 inhibition of CO 2 fixation at less than saturating light intensity were found to exhibit O 2 -insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O 2 -insensitive photosynthesis has been reported in nine other C 3 species and usually occurred when the intercellular CO 2 pressure was about double the normal pressure. A lack of O 2 inhibition of photosynthesis was always accompanied by a failure of increased CO 2 pressure to stimulate photosynthesis to the expected degree. O 2 -insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O 2 and CO 2 insensitive at physiological CO 2 pressures. Postillumination CO 2 exchange kinetics showed that O 2 and CO 2 insensitivity was not the result of elimination of photorespiration. It is proposed that O 2 and CO 2 insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO 2 assimilation.

This study describes a specific Cu2+ and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu2+ and pyocyanine in the absence of ADP, Mg2+, and HPO42−. The inhibition, which is dependent on free Cu2+ as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu2+ causes an O2-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent 3H-N-ethylmaleimide labeling of the γ subunit of coupling factor 1 by the Cu2+-light pretreatment.

Briefly sonicated Chlamydomonas reinhardi cells are capable of both cyclic and noncyclic photophosphorylation and, in each case, the maximum rates approach those reported for higher plant chloroplasts. Photophosphorylation coupled to ferricyanide reduction occurs with a P/2e ratio approaching unity.The conditions for optimum activity are similar to those reported for spinach or swiss chard chloroplasts; the major difference is the extreme sensitivity to salt and relative insensitivity to methylamine. Cell preparation, sonication, and assays were all performed at room temperature under conditions suitable for screening a large number of potential mutants deficient in photophosphorylation activity. This method was easily adapted to Euglena gracilis strain Z but not adaptable for Chlorella vulgaris or Scenedesmus obliquus strain D(3).

Nitrogen assimilation in crabgrass Digitaria sanguinalis (L.) Scop., was studied by comparing leaf extracts with isolated mesophyll cell and bundle sheath strand extracts. The results show that both nitrate and nitrate reductase are localized in mesophyll cells; glutamine synthetase is nearly equally distributed in the mesophyll and bundle sheath; approximately 67% of the glutamate synthase activity is in the bundle sheath and 33% is in the mesophyll; and 80% of the glutamate dehydrogenase activity is in the bundle sheath, with the NADH-dependent form exhibiting a 2.5-fold higher activity than the NADPH-dependent form.Isolated crabgrass mesophyll cells reduce NO(2) (-) coupled to the photochemical production of O(2) but are inactive with NO(3) (-). The NO(2) (-) -dependent O(2) evolution is light-dependent; inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea; stimulated by photophosphorylation uncouplers; and exhibits a stoichiometry of O(2) evolved to NO(2) (-) reduced of 1.45 and 0.67 in coupled and uncoupled experiments, respectively. Isolated bundle sheath strands are inactive in O(2) evolution with NO(3) (-) or NO(2) (-).Based on these results, plus literature data, two schemes for crabgrass leaf nitrogen assimilation are presented, depending on whether the plant is using ammonium or nitrate as its nitrogen source. It is proposed that the increased nitrogen use efficiency in crabgrass and other C(4) plants is due partially to a "division of labor" between mesophyll and bundle sheath cells, where NO(3) (-) and NO(2) (-) reductase in mesophyll cells act as nitrogen reduction traps in an analogous fashion to phosphoenolpyruvate carboxylase acting as a CO(2) trap during C(4) photosynthesis.

The transformation of protochlorophyllide (PChle) into chlorophyllide (Chle) has been studied in isolated etioplasts from Zea mays. ATP (1.5mm) prevented the transformation of photoconvertible PChle 650 to PChle 630 in aged etioplasts. Curve analysis indicated that the ATP effect on photoconvertibility could be entirely accounted for by changes in the proportions of PChle 630 and PChle 650 and examination of the isolated pigments revealed that only unphytylated PChle could be activated for photoconversion by ATP. In etioplasts aged for 5 hours, ATP also stimulated photoconversion of PChle 630 into Chle 670. The process was temperature-sensitive and involved PChle 650 and Chle 680 as intermediates. AMP alone had no effect, but inhibited ATP retardation of PChle loss. ADP had a similar but lesser effect than ATP. The ADP response, but not the ATP response, was considerably enhanced in the presence of an ATP-generating system (phosphoenolpyruvate/pyruvate kinase). UTP, GTP, and CTP gave 40 to 50% of the ATP response with intact etioplasts. In envelope-free etioplasts, ATP gave the greatest response but the other nucleotides were now 80% as effective as ATP. After primary photoconversion, ATP stimulated resynthesis of PChle 650. It is proposed that ATP both gives the holochrome the ability to bind to the PChle molecule and enables additional association of the pigment-protein complex to form PChle 650.

Isolated, intact spinach (Spinacia oleracea L. var. "Long Standing Bloomsdale") chloroplasts were heated in the dark and the effect of this treatment on photosynthetic activities was determined at 25 degrees C. Dark incubation of the chloroplasts for 10 minutes at 35 degrees C and pH 8.1 resulted in a 50% decline in CO(2) photoassimilation. This decline in photosynthetic performance was dependent upon time, temperature, and medium pH with the optimum effect at acidic pH values. Photosynthetic decline was not observed if MgATP, MgADP, or a mixture of fructose 1,6-bisphosphate, aldolase, and oxaloacetate or ribose 5-phosphate and oxaloacetate was added prior to but not after the temperature pretreatment. A chloroplast preparation reconstituted with thylakoids and stroma from pretreated (35 degrees C, 10 minutes, pH 8.1) intact chloroplasts and supplemented with ferredoxin, ADP, and NADP was photosynthetically competent, indicating that ATP-coupled electron flow and the enzymes comprising the Benson-Calvin cycle remained stable during the dark treatment. In contrast, exposure of isolated thylakoids to 35 degrees C for 10 minutes uncoupled photophosphorylation from NADP and ferricyanide reduction. We propose that the decline of intact chloroplast photosynthesis is the result of a decrease in the content of or a change in the ratios of the adenine nucleotides. Maintenance of an adequate supply of adenine nucleotide is the effect of the externally added MgATP or of chloroplastic respiration of a sugar phosphate.