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; )     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts.

1. Commercially available [2-14C]pyruvate and [2-14C]malonate were found to contain 3-6% (w/w) of [14C]acetate. 2. The contaminating [14C]acetate was efficiently utilized for fatty acid synthesis by isolated chloroplasts, whereas the parent materials were poorer substrates. 3. Maximum incorporation rates of the different substrates examined were (ng-atoms of C/h per mg of chlorophyll): [1-14C]acetate, 2676; [2-14C]pyruvate, 810; H14CO3-, 355; [2-14C]malonate, 19. 4. Products of CO2 fixation were probably not a significant carbon source for fatty acid synthesis in the presence of exogenous acetate.     

On the light dependence of Fatty Acid synthesis in spinach chloroplasts

The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg²⁺ concentration with the ionophore A 23187. In contrast to CO₂ fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT-activated CO₂ fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO₂ fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation.     

Phytol synthesis from geranylgeraniol in spinach chloroplasts

The reduction of /2-¹⁴C/-geranylgeranylpyrophosphate to phytylpyrophosphosphate is shown for the first time in chloroplasts. The esterification of exogenous /2-¹⁴C/-geranylgeranylpyrophosphate with endogenous chlorophyllide and the stepwise reduction of the pigment bound geranylgeraniol to phytol was also proved for spinach chloroplasts for the first time.     

Long chain fatty acid synthesis in developing castor bean seeds II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system

Particles spun down at 10,000 ? g from developing castor beanseeds were capable of synthesizing LFAs from sucrose, a physiologicalprecursor transferred from leaves as a photosynthetic product.Tracer experiments, in combination with inhibitor effects, intermediatedilutions and cofactor requirements, indicated the operationof the following path: sucroseUDPGG-1-PG-6-PGAPpyruvateacetyl-CoAmalonyl-CoALFA.The whole path appears to be associated with 10,000 ? g particles,because repeated washings were unsuccessful in dissociatinga partial path from the particles, depsite of disorganizingthe structure of the particles. Based on the occurrence of freehexose(s) and the utilization of UDPG similar to that of sucroseor G-1-P in this reaction, it is probable that hexose phosphateis formed from sucrose via UDPG and fructose, although the conversionof sucrose to hexose phosphates via glucose and furctose isnot ruled out. Inhibitor experiments showed that ATP is self-supportingover the whole path, the ATP formed in the glycolytic path beingconsumed in a acetyl-CoA carboxylation step. Since oxidizedpyridine nucleotides are as available as reduced ones for LFAsynthesis, they seem to shuttle between the reduction in theconversion of sucrose to acetyl-CoA and the oxidation in LFAsynthesis from acetyl-CoA. From the pattern of the LFAs synthesized,NAD⁺ is available for the synthesis of saturated LFAs (18: 0,16: 0). whereas NADP⁺ is available for that of unsaturated LFAs(18: 1, 16: 1). (Received July 23, 1973; )     

Reconstitution of amino acid synthesis by combining spinach chloroplasts with other leaf organelles

By adding leaf peroxisomes to purified intact chloroplasts, glycine synthesis was reconstituted. On adding leaf mitochondria, serine synthesis was also reconstituted. However, aromatic amino acid synthesis which was effected by purified chloroplasts was not enhanced on adding peroxisomes or mitochondria although the rate in whole leaves was considerably higher.     

Biosynthesis of -linolenic acid by disrupted spinach chloroplasts

A disrupted spinach chloroplast preparation readily synthesized $\text{[}{}^{14}\text{C]α-linolenate}$ from [2-¹⁴C]acetate under anaerobic conditions. It can be shown by degradation data that [¹⁴C]oleate is not a precursor of [¹⁴C]linolenate and that $\text{cis}$ 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [¹⁴C]linolenate.     

Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis

During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

The incorporation of [14C]acetate into the constituent fatty acids of monogalactosyldiglyceride by isolated spinach chloroplasts

The incorporation into diglycerides of the acyl products synthesized from acetate by spinach chloroplasts was greatly stimulated by the addition of glycerol 3-phosphate. When UDP-galactose was added also, monogalactosyldiglycerides became the major products. Palmitate biosynthesis was stimulated about twofold by these additions, while oleate biosynthesis decreased slightly, so that oleate:palmitate ratios were in the range 0.6 to 0.8 rather than about 1.6 when glycerol 3-phosphate and UDP-galactose were not added. On the other hand, Triton X-100 greatly stimulated both oleate and palmitate biosynthesis to give oleate:palmitate ratios of about 2.0. The proportions of oleate and palmitate in the newly synthesized diglycerides, or in monogalactosyldiglycerides when exogenous UDP-galactose was added, did not always reflect the proportions of these two fatty acids synthesized from acetate. When oleate:palmitate ratios were ?1, equal amounts were incorporated into diglycerides or into monogalactosyldiglycerides. When oleate:palmitate ratios were <1, incorporation of palmitate into diglycerides and monogalactosyldiglycerides exceeded that of oleate. 1-Oleoyl, 2-palmitoyl glycerol compounds were the principal products under all conditions but 1,2-dipalmitoyl compounds were also quantitatively important when glycerol 3-phosphate alone, or glycerol 3-phosphate together with UDP-galactose, was added. The distribution of label in the constituent glycerol and fatty acid moieties when monogalactosyldiglycerides were synthesized from diglycerides is consistent with galactosylation occurring without modification or exchange of fatty acids. The distribution of 16- and 18-carbon acyl residues between the 1 and 2 stereospecific positions of newly synthesized monogalactosyldiglyceride was typical of the endogenous polyene monogalactosyldiglycerides. However when palmitate synthesis was in excess of oleate synthesis some palmitate was esterified in position 1, whereas in the endogenous monogalactosyldiglycerides hexadecatrienoate is confined to position 2.     

Reductive carboxylation and amination of keto acids by spinach chloroplasts

1. In spinach chloroplasts, the occurrence of malic enzyme,isocitrate dehydrogenase, glutamate dehydrogenase and alaninedehydrogenase was confirmed. 2. In the presence of ammonia, pyruvate and -ketoglutarate andpyruvate were photoreductively aminated to glutamate and alanine,respectively. 3. In the absence of ammonia, pyruvate and -ketoglutarate werephotoreductively carboxylated to malate and isocitrate, respectively. 4. Photoreductive carboxylation of pyruvate and -ketoglutaratewas suppressed by molecular oxygen. Inhibition was partly dueto oxidation of photoreduced NADP or NAD. (Received August 4, 1969; )