Manipulation of galactolipid Fatty Acid composition with substituted pyridazinones

The fatty acid composition of the major lipids of the chloroplast membranes, the mono- and digalactosyl diglycerides, can be definably altered with various substituted pyridazinones. Galactolipid fatty acid composition of wheat (Triticum aestivum L.) can be altered so that there is a decrease in linolenic acid accompanied by an increase in linoleic acid without a shift in the relative proportion of saturated to unsaturated fatty acids; the fatty acid composition can be shifted toward a higher proportion of saturated fatty acids; or the fatty acid composition of the monogalactosyl diglycerides can be altered in preference to the digalactosyl diglycerides. Also, the light-mediated parallel accumulation of chlorophyll and linolenic acid can be separated with a substituted pyridazinone. The substituted pyridazinones may be useful tools in clarifying the role the galactolipids and their component fatty acids play in the structure and function of chloroplast membranes in higher plants.     

Nitrogen deficiency in Arabidopsis affects galactolipid composition and gene expression and results in accumulation of fatty acid phytyl esters

Nitrogen is an essential nutrient for plants because it represents a major constituent of numerous cellular compounds, including proteins, amino acids, nucleic acids and lipids. While N deprivation is known to have severe consequences for primary carbon metabolism, the effect on chloroplast lipid metabolism has not been analysed in higher plants. Nitrogen limitation in Arabidopsis led to a decrease in the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) and a concomitant increase in digalactosyldiacylglycerol (DGDG), which correlated with an elevated expression of the DGDG synthase genes DGD1 and DGD2. The amounts of triacylglycerol and free fatty acids increased during N deprivation. Furthermore, phytyl esters accumulated containing medium-chain fatty acids (12:0, 14:0) and a large amount of hexadecatrienoic acid (16:3). Fatty acid phytyl esters were localized to chloroplasts, in particular to thylakoids and plastoglobules. Different polyunsaturated acyl groups were found in phytyl esters accumulating in Arabidopsis lipid mutants and in other plants, including 16:3 and 18:3 species. Therefore N deficiency in higher plants results in a co-ordinated breakdown of galactolipids and chlorophyll with deposition of specific fatty acid phytyl esters in thylakoids and plastoglobules of chloroplasts.     

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

Light-induced changes in the lipid composition and ultrastructure of plastids from potato tubers

Sandelius, A. S. and Liljenberg, C. 1982. Light-induced changes in the lipid composition and ultrastructure of plastids from potato tubers. – Physiol. Plant. 56: 266–272.
Amyloplasts and starch containing plastids from green tissue – amylochloroplasts – from potato tubers ( Solanum tuberosum L., var. King Edward) were separated from other cell organelles by sedimentation in a discontinuous sucrose gradient. Their lipid composition was analysed with emphasis on galactolipids and phospholipids and the fatty acid compositions of these lipids. Irradiation of the tubers caused increased ratios of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and of total galactolipids to total phospholipids in the plastid membranes. Furthermore, the degree of unsaturation of the fatty acids increased in all lipid classes analysed, this effect being most prominent in the galactolipids. The ultrastructural studies made on tuber tissue revealed that irradiation caused a change in starch grain size distribution concomitant with formation of membrane structures resembling grana within the envelope. In many cases prolamellar bodies and plastoglobuli were present.     

Lipid biosynthesis by intact mesophyll and bundle sheath chloroplasts from maize

The two dimorphic forms of chloroplast isolated from maize leaves utilized acetate for fatty acid biosynthesis and had similar requirements for cofactors. The oleate:palmitate ratio of the fatty acid products was lower for bundle sheath chloroplasts as was acetate incorporation into total fatty acids. Galactose from UDP-galactose was incorporated into galactolipids by both morphological forms to give monogalactosyl diacylglycerol and digalactosyl diacylglycerol in the ratio of 4:1.     

Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris)

Primary leaves of Phaseolus vulgaris show concomitant changes in phospholipid, galactolipid, chlorophyll and fresh weight during leaf development from 3 to 32 days after planting. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol show only small changes on a mole per cent lipid phosphate basis during leaf development. The chloroplast lipids, phosphatidyl glycerol, monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) all show marked increases and decreases which are coincident with chloroplast development. The decline in the leaf content of chloroplast polar lipids and chlorophyll become evident upon reaching maximal leaf size. The molar ratio of galactolipids (MGDG/DGDG), reaches a maximum value of 2.3 in expanding leaves, but steadily declines during senescence to a minimum value of 1.5 at abscission. The declining ratio is caused by a preferential loss of MGDG in the senescing leaves.     

Calcium modifies Cd effect on runner bean plants

The effect of different Ca concentrations in the growth medium on the toxicity of 25 μM CdSO₄ was studied in runner bean plants (var. Pi kny Ja ) at two different growth stages of primary leaves. In young plants growing in a medium with low level of Ca a treatment with Cd for 12 days resulted in Ca accumulation in roots, a strong reduction of the leaf area, a decreased monogalactosyl diacylglycerol/digalactosyl diacylglycerol ratio and efficiency of the photosynthetic apparatus. In leaves of older plants growing under the same conditions, and surviving Cd treatment, a high accumulation of Ca but a low one of Cd, chlorosis of leaves, a decrease of the ratio monogalactosyl diacylglycerol/digalactosyl diacylglycerol and photosynthetic activity were shown. At a high level of Ca in the nutrient medium plant roots showed a remarkably high specificity to accumulate Cd but the toxic effect of the metal on plant growth parameters and content of pigments was decreased. No changes were observed in the level of galactolipids, but changes in fluorescence quenching were recorded. Calcium deficit enhanced the effect of Cd toxicity, including primary photochemistry, whereas excess Ca reduced toxic effects, while it is increasing the nonphotochemical quenching of excitation energy.     

Requirement of galactolipids for photosystem I activity in lyophilized spinach chloroplasts.

1. The effect of monogalactosyl diacylglycerol and digalactosyl diacylglycerol on reconstitution of Photosystem I activity in heptane-extracted and galactolipase-treated spinach chloroplasts was investigated. 2. Both galactolipids, in a molar ratio with chlorophyll of 2.5, partially restored Photosystem I activity in heptane-extracted chloroplasts. An addition of saturating amounts of plastocyanin caused complete reactivation of Photosystem I. 3. Similarly, with galactolipase-treated chloroplasts, both galactolipids partially restored Phostosystem I activity and additional amounts of plastocyanin were required for complete reactivation. 4. The action of galactolipids on partial reconstitution of Photosystem I supports the suggestion of their structural role in the restoration of thylakoid membranes.     

Requirement of galactolipids for Photosystem I activity in lyophilized spinach chloroplasts

1. The effect of monogalactosyl diacylglycerol and digalactosyl diacylglycerol on reconstitution of Photosystem I activity in heptane-extracted and galactolipase-treated spinach chloroplasts was investigated.2. Both galactolipids, in a molar ratio with chlorophyll of 2.5, partially restored Photosystem I activity in heptane-extracted chloroplasts. An addition o saturating amounts of plastocyanin caused complete reactivation of Photosystem I.3. Similarly, with galactolipase-treated chloroplasts, both galactolipids partially restored Photosystem I activity and additional amounts of plastocyanin were required for complete reactivation.4. The action of galactolipids on partial reconstitution of Photosystem I supports the suggestion of their structural role in the restoration of thylakoid membranes.     

A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition

The aim of the present investigation was to find factors critical for the co-existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 ± 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins.     

Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis

Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.     

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono‐ and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl‐MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl‐MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12‐oxo‐phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA‐containing galactolipids in the plant kingdom. While acyl‐MGDG was found to be ubiquitous in green tissue of plants ranging from non‐vascular plants to angiosperms, OPDA‐containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non‐oxidized and OPDA‐containing acyl‐MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl‐MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.     

Sulfoquinovosyldiacylglycerol and phosphatidylglycerol bilayers share biophysical properties and are good mutual substitutes in photosynthetic membranes

From cyanobacteria to higher plants, photosynthetic membranes are composed of two galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), and two negatively charged lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). In many environments, plants and algae grow in a shortage of nutrients, leading to the development of nutrient-saving mechanisms. For example, at the cellular level, in phosphate starvation, these mechanisms include conversion of phospholipids into phosphorus-free lipids. In photosynthetic membranes, PG is supposed to be replaced by SQDG in phosphate starvation whereas the opposite occurs in sulfur deprivation. All biological data confirm a complementary relationship between SQDG and PG and suggest the importance of maintaining the total amount of anionic lipids in photosynthetic membranes. Using neutron diffraction on reconstituted SQDG or PG lipid membranes, we demonstrate that, despite chemically different headgroups, PG and SQDG have similar physicochemical properties. With an equivalent diacylglycerol backbone, PG and SQDG membranes have a similar bilayer thickness and bending rigidity. They also have essentially the same response to hydration in terms of repulsion and interaction forces. The results presented here establish that SQDG and PG are good substitutes to each other in nutrient starvation conditions to maintain the chloroplast functional organization and its photosynthesis activity.     

Chemical compositions and physical states of chloroplast lipids related to atrazine resistance in Conyza canadensis L.

A comparison of the chemical composition and physical states of chloroplast lipids, of atrazine-resistant (R) and sensitive (S) biotypes of Conyza canadensis L. (horseweed), in the rosetta stage showed: (1) the R biotype contains lower amounts of polar lipids in its thylakoids, as expressed on a chlorophyll basis, than the S biotype. (2) The chloroplasts of the R biotype have higher contents of monogalactosyl diacylglycerol (MGDG) and lower contents of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG), than those of the S biotype. (3) The chloroplast total lipids exhibit a higher degree of unsaturation in the R biotype. This is due to a higher level of linolenic acid, and a lower level of palmitic acid in the glycolipids. The fatty acid compositions of the phospholipids, except that of PG, do not differ significantly. (4) The lipid matrix of the thylakoid membranes of the R biotype is more fluid than that of the S biotype, as measured by the fluorescence polarization technique. The results are discussed in terms of whether these differences are responsible for the herbicide resistance.     

Lipid composition of chloroplast membranes from weed biotypes differentially sensitive to triazine herbicides

Chloroplasts were isolated from triazine-sensitive and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.), common lambsquarter (Chenopodium album L.), and redroot pigweed (Amaranthus retroflexus L.). Chloroplast lipids were extracted and analyzed for differences among sensitive and resistant biotypes. The distribution of lipid between major lipid classes differed in chloroplasts from resistant and susceptible biotypes. Chloroplasts from resistant biotypes contained higher proportions of monogalactosyl diglyceride and phosphatidyl ethanolamine and lower proportions of digalactosyl diglyceride and phosphatidyl choline than did chloroplasts from susceptible biotypes. Monogalactosyl diglyceride and phosphatidyl ethanolamine were also quantitatively higher in membranes of resistant versus susceptible biotypes. The major lipid classes of resistant chloroplast membranes contained lipids comparatively richer in unsaturated fatty acids with the exceptions of digalactosyl diglyceride from all three biotypes and phosphatidyl ethanolamine from common groundsel. Results correlated changes in triazine sensitivity with qualitative and quantitative differences in the lipid composition of chloroplast membranes.     

Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28°C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Influence of S-ethyl Dipropylthiocarbamate (EPTC) on the Fatty Acid Composition of Wheat Leaf Galactolipids

Winter wheat (Triticum sativum L. cv. Nisu) was grown in sand which contained 0, 0.25, 0.5 and 1.0 mg S-ethyl dipropylthiocarbamate (EPTC) per kg air dry sand. The galactolipids of leaves of 21-day-old seedlings were isolated by preparative thin layer chromatography. The fatty acids of the mono- and digalactosyl diglycerides were analysed by gas liquid capillary chromatography. The major fatty acids of the wheat leaf galactolipids were palmitic, palmitoleic, stearic, oleic, vaccenic, linoleic and linolenic acids (in the monogalactosyl diglyceride fraction of untreated plants 22.5, 2.4, 3.1, 5.2, 2.5, 51.1 and 1526.6 μg and in the digalactosyl diglyceride fraction 108.8, 2.3, 10.4, 9.9, 8.2, 42.3 and 1120.7 μg/g leaf fresh weight, respectively). Total fatty acid content of the mono- and digalactosyl diglyceride fractions was decreased by 85 and 87%, respectively, at 1 mg EPTC/kg sand, while decrease in the fresh weight of the leaves was 79%. The content of linoleic and linolenic acids/g fresh weight of the leaves was decreased in the monogalactosyl diglyceride fraction by 27 and 43%, respectively, while the content of all other fatty acids was increased. In the digalactosyl diglyceride fraction the content of both linoleic and linolenic acids/g leaf fresh weight was decreased by 55%. The content of palmitic and vaccenic acids was also decreased, whereas the content of other fatty acids remained at the level of the untreated samples. The general quality of the fatty acids in the mono- and digalactosyl diglyceride fractions was altered slightly by EPTC.