Metabolism of Glycerolipids in Plastidial and Extraplastidial Membranes of Etiolated Wheat Leaves: In Vivo and In Vitro Studies

Etiolated wheat leaves were fed with [l-¹⁴C]-acetate and chaseexperiments were performed in the dark or under light. In bothconditions, in extraplastidial membranes, phosphatidylglycerol(PG) and phosphatidylcholine (PC) were fatty acid donors, PGand PC providing palmitate and oleate respectively. The labelof linoleate increased only in phosphatidylethanolamine (PE).In etioplasts, PG was also a palmitate donor but PC, ratherpoor in labelled oleate, was an oleate acceptor, contrary towhat was observed in chloroplasts. The galactolipids and sulfoquinovosyldiacylglycerol(SL) remained poorly labelled. When isolated etioplasts were labelled in vitro, during thefirst two hours they incorporated the same amount of [l-¹⁴C]-acetatein their phospholipids, whether they were in the presence orin the absence of extraplastidial membranes. Afterwards, theaddition of a mitochondrial fraction enhanced the label of PG,mainly in palmitate, then in oleate, and to some extent, andonly under light in palmitoleate and linoleate. The mitochondrialfraction might be regarded here as a supplier of labelled precursorto he etioplasts which rapidly accumulated radioactivity inpalmitoyl-PG. In PC of isolated etioplasts, only palmitate wasfairly labelled. The deficiency in labelled oleoyl-PC in plastidsof dark-grown plants and of linoleoyl-PC in extraplastidialmembranes might be the reason for the delay in the labellingof unsaturated galactolipids. ¹ (Received March 9, 1987; Accepted August 21, 1987)     

The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope

Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and [14C]18:0-CoA to form [14C]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, [14C] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of [14C]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, [14C]PC-containing lipid mixtures were incubated with cytosol. Degradation of [14C]PC to [14C]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDP-galactose, there was a time-dependent transfer of [14C]PC from this fraction to chloroplasts, where [14C]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation.     

Desaturation of oleate-[14C] in leaves of barley

Etiolated barley leaves when exposed to light desaturate oleate-[¹⁴C] to linoleate. The production of substantial amounts of radioactive linolenate was found only in very young, tightly rolled leaves. In oleate-[¹⁴C] pulse experiments, radioactive linolenate first appeared in phosphatidylcholine (PC) and only after a lag period did it begin to accumulate in monogalactosyldiacylglycerol (MGDG). The results indicate that in young, immature barley leaves linolenate is synthesized from oleate on the parent lipid, PC, and is then transferred to MGDG.     

Lipids of Ectocarpus fasciculatus (Phaeophyceae). Incorporation of [l-14C]Oleate and the Role of TAG and MGDG in Lipid Metabolism

Lipids and fatty acids of Ectocarpus fasciculatus (Ectocarpales,Phaeophyceae) were analyzed. Major polar lipids are monogalactosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol(SQDG), diacylglycerylhydroxymethyl-N,N,N-trimethyl-rß-alanine(DGTA), phosphatidylcholine (PC), phospha-tidylethanolamine(PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI).Diphosphatidylglycerol (DPG), phosphatidic acid (PA) and phosphatidyl-O-[N-(2-hydroxy-ethyl)glycine](PHEG) were also present in small amounts. Nonpolar lipids mainlyconsist of triacylglycerol (TAG) and diacylglycerol (DAG). Majorfatty acids are 16:0,18:1, 18:3, 18:4, 20:4 and 20:5. The positionaldistribution of fatty acids showed that molecular species ofeukaryotic structure account for 99% in MGDG, 98% in DGDG, 62%in PG and 23% in SQDG. On incubation with [1-¹⁴C]18:1 for 30min, 33% of the total label was detected in TAG, 16% in PG,14% in PE, 10% in PC and 8% in MGDG. During 7 days of chase,the label in TAG, PG, PE and PC decreased and simultaneouslyincreased in MGDG up to 41% of the total. In SQDG, labelledfatty acids were found in prokaryotic as well as in eukaryoticmolecular species. During the experiment, the label shiftedfrom 18:1 to 18:2, 18:3, 18:4 and, to a minor extent, to 20:4and 20:5 acids indicating 18:1 to be processed by elongationand/or desaturation. These results suggest TAG to act as a majorprimary acceptor of exogenous oleate and to be involved in thetransfer of fatty acids to MGDG and other polar lipids. (Received March 24, 1997; Accepted June 11, 1997)     

Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids

The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after ¹⁴CO₂ feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as “carriers” for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after ¹⁴CO₂ and ¹⁴C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids.     

Effect of Thiolactomycin on Lipid Synthesis in Higher Plants

The effect of thiolactomycin (TLM), an inhibitor of type IIfatty acid synthase, on lipid synthesis in greening tissueswas examined. Pulse-chase experiments with Na[1-¹⁴C]acetatefor greening Avena leaves showed that continuous administrationof TLM (100µg/ml) decisively reduced phosphatidylcholine(PC) synthesis from acetate and blocked the subsequent conversionof PC to monogalactocyldiacylglycerol (MGDG), whereas temporaladministration of TLM (100 µ/ml) reduced PC synthesisfrom acetate by only 50% and did not block the conversion ofPC to MGDG. In the reduced PC synthesis, the ratio of oleicto palmitic acid decreased at earlier stages of greening, reflectingmore suppression of oleic acid synthesis. In later greeningstages the modulated fatty acid composition recovered to thenormal composition. In further steps, the fatty acid compositionwas not affected by TLM throughout the greening stages. Greeningof either etiolated Avena leaves or etiolated Brassica cotyledonsin the presence of TLM led to a marked decrease in the contentsof MGDG, digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol(PG), but only a small change in the fatty acid compositionof their lipids. The only inhibition characteristic of TLM wasthe desaturation of palmitic to 3-trans-hexadecenoic acid inAvena leaf PG. These results suggest the presence of a mechanismby which the modulated fatty acid composition of lipids is normalizedin the flow of the synthesis. Electron microscopic observationsshowed that Avena chloroplasts developed into round forms ratherthan normal ellipse forms and the thylakoid membranes of Brassicachloroplasts were abnormally swollen everywhere in the presenceof TLM. Photosynthetic oxygen evolution in both tissues wasnot inhibited. (Received December 26, 1986; Accepted April 24, 1987)     

Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.).

To study membrane lipid synthesis during the life-span of a dicotyledon leaf, the second oldest leaf of 10-40-d-old plants of garden pea (Pisum sativum L.) was labelled with [1-(14)C]acetate and the distribution of radioactivity between the major membrane lipids was followed for 3 d. In the expanding second oldest leaf of 10-d-old plants, acetate was primarily allocated into phosphatidylcholine (PC) during the first 4 h of labelling. During the following 3 d, labelling of PC decreased and monogalactosyldiacylglycerol (MGDG) became the most radioactive lipid. In the fully expanded second oldest leaf of older plants, acetate was predominantly allocated into phosphatidylglycerol (PG), which remained the major radiolabelled lipid during the 3 d studied. The proportion of radioactivity recovered in MGDG decreased with increasing plant age up to 20 d, suggesting that, in expanded leaves, MGDG is more stable and requires renewal to a lower extent than PG. When the second oldest leaf approached senescence, labelling of MGDG again increased, indicating an increased need for thylakoid repair. The proportion of acetate allocated into phosphatidylethanolamine and free sterols was largest in leaves of 18-26-d-old plants and in the youngest leaves, respectively. Thus, these results demonstrate that the distribution of newly synthesized fatty acids between acyl lipid synthesis in the chloroplast and extraplastidial membranes strongly varies with leaf age, as do the proportion utilized for sterol synthesis. The findings emphasize the importance of defining the developmental stage of the leaf material used when performing studies on leaf lipid metabolism.     

Cooperative Pathway for Lipid Biosynthesis in Young Pea Leaves: Oleate Exportation from Chloroplasts and Subsequent Integration into Complex Lipids of Added Microsomes

In vitro [1-¹⁴C]-acetate incorporation into pea chloroplastlipids resulted in the synthesis of palmitic and oleic acids.Separation of chloroplasts after the incorporation proved thespecific exportation of [¹⁴C]-oleate towards the external medium.The addition of microsomes resulted in a stimulated exportationand integration of the exported oleate into the phosphatidylcholine(PC). In such experiments, the galactolipids were labelled byboth palmitate and oleate and confirmed the intraplastidialorigin of the fatty acids they incorporated. Isolated chloroplastsalone performed an acylation of PC by labelled oleate. The chloroplasticlocalization of this activity is discussed. (Received August 13, 1981; Accepted November 16, 1981)     

Glycerolipid Synthesis in Avena Leaves during Greening of Etiolated Seedlings IV. Effect of Light on Fatty Acid Desaturation

Etiolated Avena sativa L. leaves were fed with [l-¹⁴C]acetatefor 20 h in the dark and labeled fatty acids in glycerolipidswere chased during 24 h in the light (greening condition) orin the dark, to determine the light effect on the fatty aciddesaturation. Oleate decrease in phosphatidylcholine was thesame in the light and in the dark, showing that oleate desaturationis independent of light (or greening). Linoleate desaturationin galactolipids, especially in monogalactosyl diacylglycerol,was enhanced by light and palmitate desaturation to hexadecenoatein phosphatidylglycerol was strictly light-dependent. (Received May 11, 1983; Accepted August 16, 1983)     

Role of Phospholipid Transfer Protein in the Exchange of Phospholipids between Microsomes and Chloroplasts

When microsomes containing phosphatidylcholine labelled with[1-¹⁴C]-linoleate were incubated with pea or spinach chloroplasts,active transfer of this phospholipid took place in the presenceof phospholipid transfer protein. This transfer also was demonstratedby incubating unlabelled microsomes, chloroplasts and the phospholipidtransfer protein in the presence of [1-¹⁴C]-acetate. The reconstitutedsystems could synthesize fatty acids which were acylated inmicrosomal phosphatidylcholine. The transfer of this phospholipidto chloroplasts is mediated by the transfer protein. Our resultssuggest a role for phospholipid transfer protein in the synthesisof chloroplast lipids. (Received October 25, 1983; Accepted July 18, 1984)     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

Lipids in Cryptomonas CR-1. II. Biosynthesis of Betaine Lipids and Galactolipids

The biosynthesis of lipids in Cryptomonas strain CR-1 was studiedusing radioactive tracers. For studies of general aspects ofthe biosynthesis of lipids, the cells were labelled with [¹⁴C]NaHCO₃or with [l,3-¹⁴]glycerol. In both cases, monogalactosyl diacylglycerol(MGDG) was the most heavily labelled lipid. Phosphatidylcholineand the alanine lipid DGTA were not labelled to specific activitiescomparable to those of MGDG and DGDG. It is improbable thatthe so-called "eukaryotic pathway", which has been suggestedas the pathway for the synthesis of " eukaryotic" molecularspecies of MGDG from PC in higher plants, is operative in Cryptomonascells which contain typical "eukaryotic" MGDG. The homoserinelipid DGTS was labelled to a significant level only in its polargroup. The C-3 and C-4 atoms of methionine, as well as the methylcarbon of methionine, were incorporated into both DGTS and DGTA,whereas the C-l carbon of methionine was incorporated uniquelyinto DGTS. Results of pulse-chase experiments with [3,4-¹⁴C]methionineand [methyl.-^l4C]methionine suggest the conversion of DGTS toDGTA. (Received April 22, 1991; Accepted June 12, 1991)     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

The Effect of Light Intensity, Day Length, and Temperature on Fatty Acid Synthesis and Desaturation in Vicia faba L.

Some effects of light intensity, day length, and temperatureon the fatty acid composition of the major glycerolipids ofleaves of Vicia faba L. (cv. Giant Windsor) were observed. Increasinglight intensity caused an increase in the relative concentrationsof 16 : 1 in PG and 18 : 3 in MGDG and DGDG. Increasing daylength during growth (and continuous illumination of leaf tissue)had no effect on 16 : 1 in PG but caused a decrease in the 18: 3 content of PG, PC, MGDG, and DGDG. Since the quantitiesof these lipids increased under these conditions, the decreasewas not due to photodestruction but to the differences in therelative rates of biosynthesis and desaturation of fatty acids.Incubation of leaf tissue in the dark for 4 d had little effecton the fatty acid composition of MGDG, DGDG, and PG. Temperaturealso controls fatty acid synthesis and desaturation. Above theoptimum growth temperature (20 °C), the 18 : 3 content ofMGDG, DGDG, PG, and PC decreased. In mature leaf tissue, thedegree of unsaturation of MGDG may be modified upward in responseto temperature changes. When plants were grown at 30 °Cand transferred to 20 °C the level of 18 : 3 in MGDG ofthe leaf tissue increased to levels found in plants grown onlyat 20 °C. The level of 18 : 3 in MGDG does not decreaseas rapidly when plants grown at 20 °C were transferred to30 °C. This suggests that the lower temperature induceddesaturation of 18 : 2 to 18 : 3.     

Light-Dependent CO2 Retrieval in Immature Barley Caryopses

Immature detached caryopses from barley (Hordeum vulgare L.var. distichum cv. Midas) were shown to be capable of light-dependentretrieval of internally-produced CO₂. In the first set of experiments,caryopses were radioactively labelled by supplying (U-14C)-sucroseto detached ears in liquid culture. Caryopses were then removedfrom the ear and given a 12 h chase of non-radioactive sucrosein either the light or dark. More ¹⁴C was recovered in the caryopsesafter the chase in the light than in the dark but the differenceswere not significant. In the second set of experiments, ¹⁴C-labelledcaryopses obtained by a 15 min light incubation in ¹⁴CO₂ weremaintained in either the light or dark for 3 h and any redistributionof label between the tissues recorded. The results show thatunder these conditions, photosynthesis in the Chl-containinggreen layer of the pericarp can prevent losses of internally-producedCO₂, since 3 times as much radiocarbon remained in the caryopsesincubated in the light as in the dark. These differences weresignificant at P=0.001. Experiments with the mutant barley Albinolemma, which has no Chi in the pericarp, showed that there waslittle difference between light and dark treatments. This confirmsthe suggestion that photosynthesis in the pericarp of the normalcultivar Midas may be concerned in the refixation of CO₂. Key words: Barley, pericarp, photosynthesis, carbon dioxide     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )