Judith B. St. John

Differential responses of a range of photosynthetic tissues to a substituted pyridazinone, Sandoz 9785. Specific effects on fatty acid desaturation

Abstract The effect of a substituted pyridazinone (4-chloro-5(dimethylamino)-2-phenyl-3(2H)pyridazinone; Sandoz 9785; BASF 13-338) on the formation of fatty acids from radiolabelled precursors has been studied in a number of angiosperms, bryophytes and algae. The labelling of [ 14 C]linolenic acid was decreased by the herbicide in leaves of barley and rye grass and in cucumber cotyledons regardless of whether [ 14 C]acetate,[ 14 C]oleate or [ 14 C]linoleate was used as precursor. A commensurate increase in the labelling of [ 14 C]linoleic acid was also observed in these species. In contrast, the pattern of fatty acid labelling in maize, pea and spinach leaves was unaffected by 0.1 mM Sandoz 9785. More generalized inhibition of the incorporation of radioactivity from [ 14 C]acetate into the fatty acids of bryophytes and algae was seen. Sandoz 9785 did not alter the distribution of radioactivity in different lipid classes of higher plant leaves, nor did it change the proportions of radioactive fatty ac ids in phosphatidylcholine. In contrast to phosphatidylcholine, which never contained more than trace amounts of [ 14 C]linolenate, diacylgalactosylglycerol contained high levels of the radioactive acid. The relative labelling of linolenate was severely reduced in diacylgalactosylglycerol by Sandoz 9785 in sensitive angiosperms. Uptake studies, in which [ 3 H]Sandoz 9785 was employed demonstrated that the uptake of Sandoz 9785 was reflection of water uptake. Following its uptake, Sandoz 9785 was rapidly converted into other compounds in pea but only gradually metabolized in cucumber and ryegrass. The results are interpreted as showing, firstly, that the different sensitivity of higher plants to Sandoz 9785 is due to variations both in uptake and in metabolism. Secondly, Sandoz 9785 specifically inhibits the desaturation of linoleate to linolenate and, thirdly, diacylgalactosylglycerol plays a role in this conversion.

In vitro desaturation of monogalactosyldiacylglycerol and phosphatidylcholine molecular species by chloroplast homogenates

Abstract An in vitro assay was developed for linoleic acid desaturase activities in chloroplast homogenates using exogenous radiolabelled lipid substrates. Activities were stabilized by addition of catalase to an incubation medium also supplemented with NADPH and BSA. The direct in vitro desaturation of molecular species of MGDG by chloroplast homogenates from soybean and Arabidopsis thaliana was consistent with lipid-linked desaturation proceeding without participation of acyl-CoA intermediates. Linoleic acid was also desaturated in PC substrates. 4-Chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazinone, a herbicide known to reduce levels of linolenic acid in certain plant membrane lipids, directly inhibited in vitro desaturation of fatty acids in both MGDG and PC substrates.

Characterization of the adaptation response ofAnacystis nidulans to growth in the presence of sublethal doses of herbicide

Cells of the cyanobacterium,Anacystis nidulans, were cultured in the presence of sublethal doses of the herbicides DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] and terbutryn (a triazine). The responses observed were characteristic of photosynthetic organisms grown under low light conditions. The contents of the accessory pigment phycocyanin increased in relation to chlorophyll. Moreover, each dose of herbicide was correlated with defined changes in the pigment profile. Data obtained from chlorophyll fluorescence measurements indicated that the additional phycocyanin was functionally integrated into phycobilisomes, probably into newly formed phycobilisomes. The concentration of fatty acids in the total polar lipid fraction (per milligram chlorophyll) was greater in adapted than in control cells; nevertheless, the ratio of unsaturated to saturated fatty acids remained unchanged. Measurable rates of photosynthetic electron transport were similar among herbicide-adapted cultures and controls. These data are consistent with the hypothesis that herbicide treatment impaired electron transport, but that function was restored by the adaptation response. Furthermore, this response is conserved among cyanobacteria and higher plants, indicating that this flexibility is extremely significant to photosynthetic function.

Identification of covalently bound fatty acids on acylated proteins immoblized on nitrocellulose paper

A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.

Anesthetics alter the lipid composition of barley-root membranes

The question of whether membrane expansion, which is caused by anesthetics in animal systems, alters the lipid composition of plant cell membranes was investigated. We have measured the effects of several anesthetics on the relative amounts of the principal fatty acids from the polar lipids of barley (Hordeum vulgare L.) root membranes. Procaine, dibucaine, tetracaine, chloroform and, to a lesser degree, methanol increased the proportions of palmitic, stearic and oleic acids and decreased the proportions of linoleic and linolenic acids. Ethanol had no significant effect. Total amounts of the fatty acids from the polar lipids of roots in procaine solution decreased markedly so that all of the acids decreased in amount. The anesthetic was effective as soon as the roots were introduced to the solution and the changes progressed at constant rates for 6 h. Only the polar membrane lipids were altered; other lipids were not affected. Increased hydrostatic pressure of about 1.0 MPa largely prevented the anesthetic effects, including the decrease in the total amounts of the fatty acids. Hydrostatic pressure as high as 2 MPa had no effect per se on the membrane lipid composition. These results indicate that anesthetics cause expansion of the root membranes which results in the lipid changes. That a compositional change in the membrane lipids involves a conformational change such as expansion is an indication of the nature of the link between changes in the membrane lipids and changes in function of areas where hydrophilic ions permeate.

Action of R-40244 on chloroplast pigments and polar lipids

Abstract The herbicide R-40244 [1-(m-trifluoromethylphenyl)-3-chloro-4-chloromethyl-2-pyrrolidinone] blocked the accumulation of chloroplast pigments in cabbage (Brassica oleracea L.) and annual ryegrass (Lolium multiflorum L.). The total amount of polar (membrane) lipids synthesized and the proportion of unsaturated 18-carbon fatty acids were progressively reduced in shoots of annual ryegrass as the concentration of R-40244 increased from 1 to 100μM. Blocked chloroplast pigment accumulation and perturbations of membrane lipids were related to reduced growth of annual ryegrass. Growth and membrane lipids of cabbage were much less affected by R-40244. Light was not required for the action of R-40244 on membrane lipids. The combined data suggest that the accumulation of chloroplast pigments is blocked by R-40244 in all species sensitive to R-40244, but that species differ in sensitivity to action on membrane lipids. In sensitive species, where both pigments and membrane lipids are affected, the action on membrane lipids may limit growth independently of effects on pigments.

Lipid Molecular Species Composition of Granal and Stromal Lamellae

We are investigating the lipid biosynthetic pathways involved in both the development of chloroplast membranes and the lipid-protein interactions in a mature chloroplast. We report the molecular species compositions of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) from highly purified granal and stromal lamellae of Splrodela oligorrhiza. Characteristic differences in the lipids suggest that constituent lipid molecular species (formed via different pathways) are associated with the specialized structure and function of the chloroplast membrane systems.

Multiple Pathways of Linolenic Acid Synthesis Operate and Interact in Leaf Tissue

Different biosynthetic pathways have been proposed for trienoic fatty acids(FA) in 18: 3/16: 3 and 18: 3/18: 3 monogalactosyldiacylglycerol (MGDG) molecular species1. In the ‘prokaryotic’ pathway, 18: 1/16: 0 MGDG is synthesized within the chloroplast and desaturated in situ to form 18: 3/16: 3 MGDG. A ‘eukaryotic’ pathway involving microsomal phosphatidylcholine (PC) as substrate for desaturation of 18: 1 to 18: 2 provides the diglyceride precursor for 18: 3/18: 3 MGDG. After galactosylation, the MGDG is further desaturated in the chloroplast to form 18: 3/18: 3. We have used a recently identified mutant of Arabidopsis thaliana (L.) (‘JBl’) deficient in 18: 3,2 and secondly 4-chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazinone (BASF 13-338, Sandoz 9785) which inhibits 18: 2 desaturation3 to obtain definitive evidence for the synthesis of 18: 3 via these different pathways.