Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Formation of the fe-s cluster of ferredoxin in lysed spinach chloroplasts

In vitro formation of the ³⁵S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [³⁵S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the ³⁵S-labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, ³⁵S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) ³⁵S-labeled holo-Fd showed an ability to bind Fd-NADP⁺ reductase; (c) the ³⁵S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to ³⁵S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the ³⁵S-labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH₄⁺. NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP. These results confirm the requirement of light for the Fe-S cluster formation observed previously in intact chloroplasts.     

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts : I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THE COUPLING FACTOR COMPLEX

The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e⁻ = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg²⁺ ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C₃ and C₄ mesophyll chloroplasts.     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.     

Thiophosphate analogs of ADP and ATP as substrates in partial reactions of energy conversion in chloroplasts

Thiophosphate analogs of ADP and ATP have been employed in partial reactions of photosynthetic energy conversion in chloroplasts. Substitution of oxygen by sulfur at the α-phosphate yields a pair of diastereomers (ADPαS, ATPαS, A and B forms). Two diastereomeric compounds are also obtained by replacement of oxygen by sulfur in the β-phosphate group of ATP (ATPβS, A and B form) (Eckstein, F. and Goody, R.S. (1976) Biochemistry 15, 1685–1691).The A form of ADPαS is phosphorylated by chloroplasts with a K_m comparable to that of ADP but with a lower V. The B form of ADPαS as well as ADPβS is not a substrate in photophosphorylation and only weakly competes with ADP.The A forms of ADPαS and ATPαS strongly compete with ADP for the tight nucleotide binding site of CF₁ in the light-induced exchange reaction, whereas the B forms display a much smaller competitive effect. The efficiencies of ADPβS and the A isomer of ATPβS are intermediate, and the B form of ATPβS is a weaker competitor.The A forms of ATPαS and ATPβS are hydrolyzed by light-triggered ATPase, whereas the B forms are not. The efficiency of the A isomer of ATPαS is comparable to that of normal ATP, and the A form of ATPβS is cleaved at a lower rate. In trypsin-activated Ca²⁺-dependent ATPase the A form of ATPαS is the only thiophosphate analog to be hydrolyzed.The results indicate a stereospecific interaction of ADP and ATP at the catalytic sites as well as the tight nucleotide binding site of coupling ATPase of chloroplasts.     

PROTON GRADIENT REGULATION 5 contributes to ferredoxin-dependent cyclic phosphorylation in ruptured chloroplasts

Antimycin A-sensitive cyclic electron flow (CEF) was discovered as cyclic phosphorylation by Arnon et al. (1954). Because of its sensitivity to antimycin A, PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like Photosynthetic Phenotype 1 (PGRL1)-dependent CEF has been considered identical to the CEF of Arnon et al. However, this conclusion still needs additional supportive evidence, mainly because of the absence of definitive methods of evaluating CEF activity. In this study, we revisited the classical method of monitoring cyclic phosphorylation in ruptured chloroplasts to characterize two Arabidopsis mutants: pgr5, which is defective in antimycin A-sensitive CEF, and chlororespiratory reduction 2-1 (crr2-1), which is defective in chloroplast NDH-dependent CEF. We observed a significant reduction in CEF-dependent pmf formation and consequently ATP synthesis in the pgr5 mutant, although LEF-dependent pmf formation and ATP synthesis were not impaired at photosynthetic photon flux densities below 130?μmol?m^?2?s^?1. In contrast, the contribution of chloroplast NDH complex to pmf formation and ATP synthesis was not significant. Antimycin A partially inhibited CEF-dependent pmf formation, although there may be further inhibition sites. Unlike in the observation in leaves, the proton conductivity of ATP synthase, monitored as g_H⁺, was not enhanced in ruptured chloroplasts of the pgr5 mutant.     

The relation of the 515 nanometers absorbance change to adenosine triphosphate formation in chloroplasts and digitonin subchloroplast particles

The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K⁺ accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K⁺, addition of Na⁺, or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg²⁺ and inorganic phosphate. NH₄Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change.     

Wheat leaf RNA polymerases. II. Kinetic characterization and template specificities of nuclear, chloroplast, and soluble enzymes

Further comparisons were made of DNA-dependent RNA polymerase (nucleotide triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) activities, partially purified from purified nuclear fragments and chloroplasts and from the soluble phase of young wheat leaves. All three preparations had the same cation specificities for maximal RNA polymerase activity (Mg²⁺ > Mn²⁺ > Ca²⁺) and showed an absolute dependence on an added divalent cation. All three preparations showed the same thermal stabilities and pH optima, very similar pH-activity profiles, and the same type of kinetics with ATP as substrate. Enzyme activities showed negative cooperativity with respect to ATP concentration; the high and low K_m values for ATP were not significantly different for the three preparations.     

Permeability of chloroplast envelopes to mg: effects on protein synthesis

When suspended in media lacking free Mg²⁺, chloroplasts from young pea plants (Pisum sativum CV Progress No. 9) lose 25 to 75% of their stromal Mg²⁺ content to the medium, without breakage. This effect amounts for the inhibition of protein synthesis in the dark by ATP in excess of the Mg²⁺ provided, since free ATP chelates Mg²⁺. The rate of loss is from 1 to 4.5 microgram-atoms Mg²⁺/milligram Chl/hour; and depleted chloroplasts take up Mg²⁺ from the medium at even faster rates, to a total amount not much more than that present originally (0.8 to 1.8 microgram-atoms/milligram Chl with an average of 1.33 ± 0.32 μg-atoms/mg Chl). Leakage is completely prevented by 0.25 to 0.40 millimolar external Mg²⁺. Addition of Mg²⁺ at a level sufficient to prevent leakage from intact chloroplasts results in approximately 20% stimulation in light-driven protein synthesis.     

Light Activation of Mg-dependent Adenosine 5'-Triphosphatase in Isolated Euglena Chloroplasts

Enhancement of Mg²⁺-dependent ATPase activity in Euglena gracilis chloroplasts by light in the presence of a sulfhydryl compound has been demonstrated. A number of uncouplers and energy transfer inhibitors were studied for their effects on the light enhancement of ATPase activity simultaneously with their effects on photophosphorylation. Results suggest that the light-enhanced ATPase activity in Euglena chloroplasts is an energy-initiated process and that the energy is supplied through electron flow upon illumination of the chloroplasts. However, by studying the difference in their response toward the various uncouplers and inhibitors, it seems that the two processes (photohydrolysis of ATP and photophosphorylation) share only the latter part of their energy-transferring pathway.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Photoreduction of oxygen in mesophyll chloroplasts of c(4) plants: a model system for studying an in vivo mehler reaction

Mesophyll chloroplasts of three C₄ sub types, Panicum miliaceum (NAD-malic enzyme), Panicum maximum (PCK), and Zea mays (NADP-malic enzyme), were prepared from protoplast extracts and used to study the photoreduction of O₂. The processes of O₂ uptake and evolution in these preparations, which lack ribulose 1,5-bisphosphate carboxylase/oxygenase, were studied simultaneously using stable isotopes of O₂ and mass spectrometry. The responses of O₂ uptake to O₂ tension and addition of various substrates (3-phosphoglycerate, pyruvate, and oxaloacetate) were studied in detail. The addition of photosynthetic substrates differing in ATP to NADPH demands indicated that photoreduction of O₂ in these chloroplast preparations is linked to ATP production and strongly regulated by NADP⁺ levels. The results clearly indicate that photoreduction of O₂ could be of physiological relevance in balancing the ATP to NADPH requirements of C₄ mesophyll chloroplasts.     

A proposed mechanism for the stimulatory effect of bicarbonate ions on ATP synthesis in isolated chloroplasts

The effect of bicarbonate ions on induction of Mg²⁺-ATPase activity, on the N-ethylmaleimide inhibition of phosphorylation and on energy-dependent adenine nucleotide exchange has been examined with pea seedling chloroplasts. Incubation of chloroplasts with N-ethylmaleimide in the presence of 15 millimolar bicarbonate in the light results in enhanced inhibition of ATP synthesis when the preillumination pH is maintained between 7.0 and 7.5. Bicarbonate also enhances Mg²⁺-ATPase activity when it is included in the light-triggering stage at pH 7.0. The conditions (medium pH, bicarbonate concentration, etc.) for demonstrating the bicarbonate-induced enhancement of the N-ethylmaleimide inhibition and ATPase activity are similar to those required for the direct effect of bicarbonate on phosphorylation. Bicarbonate, under the same conditions, does not affect adenine nucleotide exchange (binding or release). It is concluded that the stimulatory effect of bicarbonate on ATP synthesis may be related to its ability to alter directly the conformation of the chloroplast coupling factor under conditions (suboptimal pH) where the enzyme shows minimal activity.     

Activation of fructose 1,6-bisphosphatase in darkened intact chloroplasts by NADPH

When intact chloroplasts are incubated in the dark with dihydroxyacetone phosphate, an increase in fructose 1,6-bisphosphatase activity occurs which resembles the reductive activation observed in illuminated chloroplasts. Under optimum conditions, the activity increases to about 150 μmol · h^?1 · mg^?1 chlorophyll within 60 min. The dark activation of the enzyme is reversed by electron acceptors such as oxaloacetate, nitrite, and 3-phosphoglycerate plus ATP. Activation is most marked under strictly anaerobic conditions, being strongly inhibited by O₂. It is concluded that NADPH, generated from dihydroxyacetone phosphate in situ in the reaction catalyzed by NADP⁺-dependent glyceraldehyde phosphate dehydrogenase, can provide electrons for the reductive activation of fructose 1,6-bisphosphatase in the dark.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[¹⁴C] and geranylgeranyl pyrophosphate-[¹⁴C] were biosynthesized from mevalonic acid-[2-¹⁴C] by cell-free enzyme extracts of pea (Pisum sativum) cotyledons containing MgCl₂, MnCl₂, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[¹⁴C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2-¹⁴C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

Rates and products of long-chain Fatty Acid synthesis from [1-C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants

Chloroplasts highly active in the synthesis of long-chain fatty acids from [1-¹⁴C]acetate were prepared from leaves of Solanum nodiflorum, Chenopodium quinoa, Carthamus tinctorius, and Pisum sativum. These preparations were used to test whether the various additions to incubation media found to stimulate the synthesis of particular lipid classes in vitro by Spinacia oleracea chloroplasts were applicable generally. Chloroplasts from 18:3 plants incorporated a greater proportion of radioactivity into unesterified fatty acids under control conditions than did those from 16:3 plants. Supplying exogenous sn-glycerol 3-phosphate or Triton X-100 to chloroplasts increased the synthesis of glycerolipids in all cases and accentuated the capacity of chloroplasts from 18:3 plants to accumulate phosphatidic acid rather than the diacylglycerol accumulated by chloroplasts from 16:3 plants. The UDP-galactose-dependent synthesis of labeled diacylgalactosylglycerol was much less active in incubations of chloroplasts from 18:3 plants also containing sn-glycerol 3-phosphate and Triton X-100 compared with similar incubations from 16:3 plants. Exogenous CoA stimulated total fatty acid synthesis in all chloroplast preparations and the further addition of ATP diverted radioactivity from the unesterified fatty acid to acyl-CoA. The results have been discussed in terms of the two pathway hypothesis for lipid synthesis in leaves.