Evolution of C4 Photosynthesis in Flaveria Species : Isoforms of NADP-Malic Enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C₄ photosynthesis, provides CO₂ to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C₃, C₃-C₄ intermediate, C₄-like, C₄) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C₄ isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C₃ Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C₃ photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C₄ cycle activity. The 62-kD isoform, which is the predominant highly active form in the C₄ species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C₄ metabolism, and a 62-kD form that is necessary for the complete functioning of C₄ photosynthesis.     

Measurement of the Leakage of CO2 from Bundle-Sheath Cells of Leaves during C4 Photosynthesis

During C4 photosynthesis, CO2 is released in bundle-sheath cells by decarboxylation of C4 acids and then refixed via ribulose-1,5-bisphosphate carboxylase. In this study we examined the efficiency of this process by determining the proportion of the released CO2 that diffuses back to mesophyll cells instead of being refixed. This leak of CO2 was assessed by determining the amount of 14CO2 released from leaves during a chase in high [12CO2] following a 70-s pulse in 14CO2. A computer-based analysis of the time-course curve for 14CO2 release indicated a first-order process and provided an estimate of the initial velocity of 14CO2 release from leaves. From this value and the net rate of photosynthesis determined from the 14CO2 fixed in the pulse, the CO2 leak rate from bundle-sheath cells (expressed as a percentage of the rate of CO2 production from C4 acids) could be deduced. For nine species of Gramineae representing the different subgroups of C4 plants and two NAD-malic enzyme-type dicotyledonous species, the CO2 leak ranged between 8 and 14%. However, very high CO2 leak rates (averaging about 27%) were recorded for two NADP-malic enzyme-type dicotyledonous species of Flaveria. The results are discussed in terms of the efficiency of C4 photosynthesis and observed quantum yields.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

An antisense construct targeting the C₄ isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO₂ to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO₂ assimilation rates at high intercellular CO₂ were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO₂ (the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C₄ cycle rate, bundle sheath leak rate, and bundle sheath CO₂ concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C₃ and C₄ cycles that exist during environmental perturbations by light and CO₂ can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C₄ pathway could potentially be improved through a reduction in C₄ cycle activity or increased C₃ cycle activity.In the leaves of a range of plants including maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and millet (Pennisetum americanum), a biochemical pathway known as C₄ photosynthesis has evolved to concentrate CO₂ at the site of Rubisco such that Rubisco can operate at close to its maximal activity and photorespiration is reduced, enhancing the rate of photosynthesis in air (Hatch, 1987; Sage, 2004). In most C₄ plants, CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells into four-carbon acids, which diffuse to an inner ring of bundle sheath cells, where they are decarboxylated and the CO₂ is refixed by Rubisco. Plants using the C₄ photosynthetic mechanism have been subdivided into three primary subtypes, the NADP-malic enzyme (ME), NAD-ME, and phosphoenolpyruvate carboxykinase types, according to the decarboxylating enzyme used to generate CO₂ from C₄ acids in the bundle sheath cells (Hatch, 1987). Flaveria bidentis is a typical NADP-ME dicot in which malate and Asp contribute equally in the transfer of CO₂ to bundle sheath cells (Meister et al., 1996). Presumably, in most C₄ plants, the reactions that facilitate the appropriation, transformation, transport, and eventual concentration of CO₂ in the bundle sheath cell chloroplasts (C₄ cycle) are balanced with the reactions that incorporate CO₂ into usable carbon compounds for energy (C₃/Calvin cycle) such that energy is not lost or wasted as environmental conditions fluctuate. This process is important in maintaining the efficiency of the CO₂-concentrating mechanism and of C₄ photosynthesis overall. The nature of the controlling mechanisms for balance and coordination between the C₃ and C₄ cycles is still unclear, however, and concrete evidence for the coordinated regulation of primary carboxylation in the mesophyll and decarboxylation of C₄ acids in the bundle sheath has not been forthcoming. A key approach to revealing these mechanisms has been the use of antisense RNA in the C₄ dicot F. bidentis to reduce levels of key photosynthetic enzymes, including Rubisco (Furbank et al., 1996), NADP-malate dehydrogenase and pyruvate phosphate dikinase (Furbank et al., 1997), Rubisco activase (von Caemmerer et al., 2005), carbonic anhydrase (Cousins et al., 2006), and PEPC protein kinase (Furumoto et al., 2007). This has proven to be a valuable method to help gain insight into enzyme function and regulation during C₄ photosynthesis and to potentially alter the balance between the C₃ and C₄ cycles.In this study, we targeted the gene encoding the chloroplastic C₄ isozyme of NADP-ME in F. bidentis (Marshall et al., 1996) with an antisense construct designed to reduce its activity in vivo. This isoform is thought to catalyze the decarboxylation of l-malate to pyruvate and CO₂ and of NADP to NADPH in bundle sheath chloroplasts during C₄ photosynthesis (Ashton, 1997; Drincovich et al., 2001), allowing the CO₂ to be fixed into the C₃ cycle by Rubisco and pyruvate to return back to mesophyll cells to be recycled into PEP. These antisense lines were generated for two purposes. First, these plants could be used to confirm the identity of the gene encoding the NADP-ME isozyme involved in C₄ photosynthesis. Several other functioning isoforms of NADP-ME have also been identified within Flaveria spp.: a chloroplastic but potentially nonphotosynthetic NADP-ME form and a cytosolic NADP-ME (Marshall et al., 1996; Drincovich et al., 1998; Lai et al., 2002). The specific role and regulation of a C₄ NADP-ME isozyme in F. bidentis is of interest in relation to the “transfer” or “generation” of a functioning C₄ cycle to C₃ plants (Sheehy et al., 2007; Furbank et al., 2009). A greater understanding of the balance and interactions between this enzyme and others in the C₄ and C₃ cycles will aid in deciding the expression locations and levels needed for C₃ plants to gain a functional CO₂-concentrating mechanism.The second use of these antisense plants was to investigate the degree of coordination between the C₄/C₃ cycles in F. bidentis and the possibility of manipulation to improve photosynthetic efficiency. As mentioned above, the mechanisms of regulation (if any) of the C₃ pathway enzymes such as Rubisco in response to the activity and CO₂ supply rate of the C₄ cycle are unknown. It is similarly unclear how much the reactions of the C₃ cycle affect the rates of the initial CO₂-fixing reactions (carbonic anhydrase and PEPC). Leakiness (ϕ), defined as the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply, reflects the coordination of the C₄ and C₃ cycles by describing the amount of overcycling of the C₄ cycle that has to occur to support a given rate of net CO₂ assimilation (Furbank et al., 1990; von Caemmerer and Furbank, 1999). As a major C₄ enzyme functioning within the bundle sheath, a reduction in NADP-ME should affect both the C₄ cycle rate and the bundle sheath CO₂ concentration (C_s), possibly disrupting the enzymatic balance and coordination in F. bidentis. Here, we have designed experiments to simultaneously look at in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ), facilitating estimates of ϕ, C₄ cycle rate, and the possible range of C_s within transgenic α-NADP-ME and antisense Rubisco small subunit (α-SSu) F. bidentis plants (Furbank et al., 1996). These measurements aim to show the impact of our perturbations of the C₃/C₄ balance, highlighting possible communication pathways between the cycles and also other possible targets for future genetic manipulation to improve the rate and/or efficiency of photosynthesis in C₄ plants.     

Photoinhibition of Photosynthesis in Broken Chloroplasts as a Function of Electron Transfer Rates during Light Treatment

Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O₂ evolution in the presence of NH₄Cl) and photosystem II activity (measured as O₂ 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 O₂). 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 O₂ 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.     

Light-induced Changes of the Electrical Potential in Chloroplasts Associated with the Activity of Photosystem I and Photosystem II

The electric potential changes induced by flashing and continuouslight were measured with microcapillary electrodes in isolatedwhole chloroplasts of Peperomia inetallica. In continuous lightthe chloroplast electrical potential rose in two phases. Theinitial rapid phase coincided in extent with the flash-inducedpotential and was insensitive to the electron transfer inhibitorDBMIB. The subsequent phase was relatively slow (20–30ms) and was inhibited by DBMIB. Electron acceptors of photosystemII (p-phenylendiamine, p-benzoquinone) added to DBMIB-treatedchloroplasts produced a suppression of the flash-induced responseand a considerable increase in the steady level of the potentialin the light. The electrical potential associated with the activityof photosystem II rose in continuous light much more slowlythan that associated with the activity of photosystem I aloneor the activities of both photosystems. Illumination of chloroplastswith successive flashes at a repetition rate 5 Hz in the presenceof oxaloacetate, a terminal acceptor of photosystem I, was accompaniedwith a gradual decline of the flash-induced potential. The specificrole of two photosystems in the light-induced H⁺ transport andthe electrogenesis across the chloroplast thylakoid membranesis discussed.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme

The degree of C₄ photosynthesis was assessed in four hybrids among C₄, C₄-like, and C₃-C₄ species in the genus Flaveria using ¹⁴C labeling, CO₂ exchange, ¹³C discrimination, and C₄ enzyme activities. The hybrids incorporated from 57 to 88% of the ¹⁴C assimilated in a 10-s exposure into C₄ acids compared with 26% for the C₃-C₄ species Flaveria linearis, 91% for the C₄ species Flaveria trinervia, and 87% for the C₄-like Flaveria brownii. Those plants with high percentages of ¹⁴C initially fixed into C₄ acids also metabolized the C₄ acids quickly, and the percentage of ¹⁴C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to ¹²CO₂. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C₄ and C₃ cycles. Synthesis and metabolism of C₄ acids by the species and their hybrids were highly and linearly correlated with discrimination against ¹³C. The relationship of ¹³C discrimination or ¹⁴C metabolism to O₂ inhibition of photosynthesis was curvilinear, changing more rapidly at C₄-like values of ¹⁴C metabolism and ¹³C discrimination. Incorporation of initial ¹⁴C into C₄ acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C₄ acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C₄ photosynthesis but that the carbon accumulation and reduction portions of the C₄ and C₃ cycles are well coordinated.     

Analyzing the Light Energy Distribution in the Photosynthetic Apparatus of C4 Plants Using Highly Purified Mesophyll and Bundle-Sheath Thylakoids

The chlorophyll fluorescence characteristics of mesophyll and bundle-sheath thylakoids from plant species with the C4 dicarboxylic acid pathway of photosynthesis were investigated using flow cytometry. Ten species with the NADP-malic enzyme (NADP-ME) biochemical type of C4 photosynthesis were tested: Digitaria sanguinalis (L.) Scop., Euphorbia maculata L., Portulaca grandiflora Hooker, Saccharum officinarum L., Setaria viridis (L.) Beauv., Zea mays L., and four species of the genus Flaveria. This study also included three species with NAD-ME biochemistry (Atriplex rosea L., Atriplex spongiosa F. Muell., and Portulaca oleracea L.). Two C4 species of unknown biochemical type were investigated: Cyperus papyrus L. and Atriplex tatarica L. Pure mesophyll and bundle-sheath thylakoids were prepared by flow cytometry and characterized by low-temperature fluorescence spectroscopy. In pure bundle-sheath thylakoids from many species with C4 photosynthesis of the NADP-ME type, significant amounts of photosystem II (PSII) emission can be detected by fluorescence spectroscopy. Simulation of fluorescence excitation spectra of these thylakoids showed that PSII light absorption contributes significantly to the apparent excitation spectrum of photosystem I. Model calculations indicated that the excitation energy of PSII is efficiently transferred to photosystem I in bundle-sheath thylakoids of many NADP-ME species.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.     

NADP-Malate Dehydrogenase in the C4 Plant Flaveria bidentis (Cosense Suppression of Activity in Mesophyll and Bundle-Sheath Cells and Consequences for Photosynthesis)

Flaveria bidentis, a C4 dicot, was transformed with sorghum (a monocot) cDNA clones encoding NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) driven by the cauliflower mosaic virus 35S promoter. Although these constructs were designed for over-expression, many transformants contained between 5 and 50% of normal NADP-MDH activity, presumably by cosense suppression of the native gene. The activities of a range of other photosynthetic enzymes were unaffected. Rates of photosynthesis in plants with less than about 10% of normal activity were reduced at high light and at high [CO2], but were unaffected at low light or at [CO2] below about 150 [mu]L L-1. The large decrease in maximum activity of NADP-MDH was accompanied by an increase in the activation state of the enzyme. However, the activation state was unaffected in plants with 50% of normal activity. Metabolic flux control analysis of plants with a range of activities demonstrates that this enzyme is not important in regulating the steady-state flux through C4 photosynthesis in F. bidentis. Cosense suppression of gene expression was similarly effective in both the mesophyll and bundle-sheath cells. Photosynthesis of plants with very low activity of NADP-MDH in the bundle-sheath cells was only slightly inhibited, suggesting that the presence of the enzyme in this compartment is not essential for supporting maximum rates of photosynthesis.     

C4 Photosynthesis Promoted Species Diversification during the Miocene Grassland Expansion

Identifying how organismal attributes and environmental change affect lineage diversification is essential to our understanding of biodiversity. With the largest phylogeny yet compiled for grasses, we present an example of a key physiological innovation that promoted high diversification rates. C₄ photosynthesis, a complex suite of traits that improves photosynthetic efficiency under conditions of drought, high temperatures, and low atmospheric CO₂, has evolved repeatedly in one lineage of grasses and was consistently associated with elevated diversification rates. In most cases there was a significant lag time between the origin of the pathway and subsequent radiations, suggesting that the ‘C₄ effect’ is complex and derives from the interplay of the C₄ syndrome with other factors. We also identified comparable radiations occurring during the same time period in C₃ Pooid grasses, a diverse, cold-adapted grassland lineage that has never evolved C₄ photosynthesis. The mid to late Miocene was an especially important period of both C₃ and C₄ grass diversification, coincident with the global development of extensive, open biomes in both warm and cool climates. As is likely true for most “key innovations”, the C₄ effect is context dependent and only relevant within a particular organismal background and when particular ecological opportunities became available.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

Chemical Modification of the Membrane-bound ATP Synthetase of Spinach Chloroplasts with Pyridoxal Phosphate in Light

Photosynthetic activities of the thylakoid membranes modifiedwith pyridoxal phosphate (PLP) and sodium borohydride in lightwere studied and compared with those modified in the dark. PLPmodified the membrane-bound chloroplast coupling factor 1 (CF₁)and inhibited photophosphorylation. Only PLP modification inlight stimulated basal electron transport. This stimulationof electron transport was prevented by the presence of ATP orcarbonylcyanide m-chlorophenylhydrazone in the modificationmixture. Magnesium ion was required for PLP modification. Theextent of lightinduced proton uptake was decreased by PLP modificationin light. N,N'-Dicyclohexylcarbodiimide lowered the stimulatedelectron transport to the basal level of unmodified chloroplastsand restored proton uptake. When chloroplasts were modified with 4 mM PLP in light and dark,11.6 and 11.0 mol of PLP were incorporated into mol of CF₁,respectively. ATP could bind with high affinity to CF₁ isolatedafter PLP modification in light. The results indicate that PLP modifies membrane-bound CF₁ whichhas a conformation altered by energization of the thylakoidsin light, and causes an apparent uncoupling of phosphorylation(stimulation of basal electron transport). The results suggestthat this uncoupling is induced by the loss of the regulatoryfunction of CF₁ for proton translocation after PLP modificationin light. ¹ Presented at the ISRACON on Control Mechanisms in Photosynthesis.Aug. 31-Sept. 4, 1980, Acre, Israel (Received June 22, 1981; Accepted August 28, 1981)     

Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)