Galactolipid Metabolism Research Articles

Lipid remodeling of contrasting maize (Zea mays L.) hybrids under repeated drought.

The role of recovery after drought has been proposed to play a more prominent role during the whole drought-adaption process than previously thought. Two maize hybrids with comparable growth but contrasting physiological responses were investigated using physiological, metabolic, and lipidomic tools to understand the plants' strategies of lipid remodeling in response to repeated drought stimuli. Profound differences in adaptation between hybrids were discovered during the recovery phase, which likely gave rise to different degrees of lipid adaptability to the subsequent drought event. These differences in adaptability are visible in galactolipid metabolism and fatty acid saturation patterns during recovery and may lead to a membrane dysregulation in the sensitive maize hybrid. Moreover, the more drought-tolerant hybrid displays more changes of metabolite and lipid abundance with a higher number of differences within individual lipids, despite a lower physiological response, while the responses in the sensitive hybrid are higher in magnitude but lower in significance on the level of individual lipids and metabolites. This study suggests that lipid remodeling during recovery plays a key role in the drought response of plants.

Dissection of novel candidate genes for grain texture in Russian wheat varieties.

Wheat grain texture is a complex trait. The crucial role of puroindoline genes in grain texture determination was confirmed. New loci, highly associated with the trait, were identified and dissected. Grain texture is an important milling characteristic of common wheat. Previously, it was shown that the Ha locus on 5DS, containing the puroindoline-a, puroindoline-b and GSP-1 genes, has a crucial role in endosperm texture. However, some other loci were detected on different chromosomes, but no specific genes were proposed to be involved in grain hardness organization. In this study, we used a panel of Russian spring wheat varieties. The grain texture of varieties ranged from soft to hard. Analysis of the flour particle size and flour particle specific surface of wheat varieties cultivated in different environments demonstrated high heritability (0.79–0.8) and strong repeatability of the traits. Genotyping of wheat varieties with allele-specific markers for the puroindoline-a and puroindoline-b genes demonstrated that 25% of the studied varieties carried rare allele Pin-D1k, which lacked both Pina and Pinb (double null allele). Our results confirmed the strong impact of this allele on grain texture. Association analysis with the use of SNP genotyping (Illumina 15 K Wheat) and 2-year phenotyping confirmed the key role played by puroindoline genes (5D) in determining the grain texture of Russian wheat varieties. The analysis also detected significant SNPs on the 1B, 3A, 5B, 6A, 6D, 7B, 7D chromosomes. The best candidate loci for the grain texture were located on chromosomes 5B and 7B (FDR ≤ 0.05). We dissected a number of candidate genes and proposed a possible mechanism for their contribution to endosperm texture determination. These genes are involved in the metabolism of galactolipids (DGDG) and carbohydrates (1,3-β-glucan).

The leaf lipid composition of ectomycorrhizal oak plants shows a drought-tolerance signature

Ectomycorrhizas have been reported to increase plant tolerance to drought. However, the mechanisms involved are not yet fully understood. Membranes are the first targets of degradation during drought, and growing evidences support a role for membrane lipids in plant tolerance and adaptation to drought. We have previously shown that improved tolerance of ectomycorrhizal oak plants to drought could be related to leaf membrane lipid metabolism, namely through an increased ability to sustain fatty acid content and composition, indicative of a higher membrane stability under stress. Here, we analysed in deeper detail the modulation of leaf lipid metabolism in oak plants mycorrhized with Pisolithus tinctorius and subjected to drought stress. Results show that mycorrhizal plants show patterns associated with water deficit tolerance, like a higher content of chloroplast lipids, whose levels are maintained upon drought stress. Likewise, mycorrhizal plants show increased levels of unsaturated fatty acids in the chloroplast phosphatidylglycerol lipid fraction. As a common response to drought, the digalactosyldiacyloglycerol/monogalactosyldiacyloglycerol ratio increased in the non-mycorrhizal plants, but not in the mycorrhizal plants, associated to smaller alterations in the expression of galactolipid metabolism genes, indicative of a higher drought tolerance. Under drought, inoculated plants showed increased expression of genes involved in neutral lipids biosynthesis, which could be related to an increased ability to tolerate drought stress. Overall, results from this study provide evidences of the involvement of lipid metabolism in the response of ectomycorrhizal plants to water deficit and point to an increased ability to maintain a stable chloroplast membrane functional integrity under stress.

Patatin-like lipolytic acyl hydrolases and galactolipid metabolism in marine diatoms of the genus Pseudo-nitzschia

Diatoms are eukaryotic microalgae that play a pivotal role in biological and geochemical marine cycles. These microorganisms are at the basis of the trophic chain and their lipids are essential components (e.g. eicosapentaenoic acid, EPA) of aquatic food webs. Galactolipids are the primary lipid components of plastid membranes and form the largest lipid family of diatoms. As source of polyunsaturated fatty acids (PUFAs), these compounds are also involved in the synthesis of lipoxygenase (LOX) products such as non-volatile oxylipins and polyunsaturated aldehydes. Here, we report the first identification of two genes, namely PmLAH1 and PaLAH1, coding for lipolytic enzymes in two diatoms of the genus Pseudo-nitzschia. Functional and modeling studies evidence a patatin-like domain endowed with galactolipase and phospholipase activity at the C-terminus of both proteins. Homologues of Pseudo-nitzschia LAH1 genes were retrieved in other diatom species so far sequenced in agreement with conservation of the functional role of these proteins within the lineage.

Unraveling lipid metabolism in maize with time-resolved multi-omics data.

Maize is the cereal crop with the highest production worldwide, and its oil is a key energy resource. Improving the quantity and quality of maize oil requires a better understanding of lipid metabolism. To predict the function of maize genes involved in lipid biosynthesis, we assembled transcriptomic and lipidomic data sets from leaves of B73 and the high-oil line By804 in two distinct time-series experiments. The integrative analysis based on high-dimensional regularized regression yielded lipid-transcript associations indirectly validated by Gene Ontology and promoter motif enrichment analyses. The co-localization of lipid-transcript associations using the genetic mapping of lipid traits in leaves and seedlings of a B73×By804 recombinant inbred line population uncovered 323 genes involved in the metabolism of phospholipids, galactolipids, sulfolipids and glycerolipids. The resulting association network further supported the involvement of 50 gene candidates in modulating levels of representatives from multiple acyl-lipid classes. Therefore, the proposed approach provides high-confidence candidates for experimental testing in maize and model plant species.

Mutation in barley ERA1 (Enhanced Response to ABA1) gene confers better photosynthesis efficiency in response to drought as revealed by transcriptomic and physiological analysis

Farnesylation is a post-translational modification that promotes the interaction between the modified signaling protein and membrane lipids and/or other proteins. Farnesyltransferase is the crucial enzyme involved in this process. Strikingly, plant mutants in the ERA1 (Enhanced response to ABA 1) gene, encoding β-subunit of farnesyltransferase, exhibited ABA-hypersensitivity during seed germination and drought tolerance in several species including Arabidopsis, wheat and soybean. However, the mechanism of ERA1 action has not been resolved yet. Here, we present the potential regulatory role of ERA1 in the drought signaling network in barley. With the aim of decoding the role of the ERA1 gene, we developed a unique barley mutant using TILLING analysis. Mutation in HvERA1 confers semi-dwarf phenotype, ABA-sensitivity during seed germination and drought tolerance. Our transcriptomic analysis suggested a role of HvERA1 in regulation of the crosstalk between ABA and ethylene at the onset of drought. Furthermore, analysis of hvera1.b response to prolonged drought stress linked HvERA1 to the metabolism of galactolipids, that build the chloroplast membranes. It might results in the protection of hvera1.b photosystem and thus, in its better photosynthesis performance under water stress. Together, these results indicate the possible mechanism of the primary cause of the observed alterations in the hvera1.b mutant.

Differential lipidome remodeling during postharvest of peach varieties with different susceptibility to chilling injury.

Peaches ripen and deteriorate rapidly at room temperature. Therefore, refrigeration is used to slow these processes and to extend fruit market life; however, many fruits develop chilling injury (CI) during storage at low temperature. Given that cell membranes are likely sites of the primary effects of chilling, the lipidome of six peach varieties with different susceptibility to CI was analyzed under different postharvest conditions. By using liquid chromatography coupled to mass spectrometry (LC-MS), 59 lipid species were detected, including diacyl- and triacylglycerides. The decreases in fruit firmness during postharvest ripening were accompanied by changes in the relative amount of several plastidic glycerolipid and triacylglyceride species, which may indicate their use as fuels prior to fruit senescence. In addition, levels of galactolipids were also modified in fruits stored at 0°C for short and long periods, reflecting the stabilization of plastidic membranes at low temperature. When comparing susceptible and resistant varieties, the relative abundance of certain species of the lipid classes phosphatidylethanolamine, phosphatidylcholine and digalactosyldiacylglycerol correlated with the tolerance to CI, reflecting the importance of the plasma membrane in the development of CI symptoms and allowing the identification of possible lipid markers for chilling resistance. Finally, transcriptional analysis of genes involved in galactolipid metabolism revealed candidate genes responsible for the observed changes after cold exposure. When taken together, our results highlight the importance of plastids in the postharvest physiology of fruits and provide evidence that lipid composition and metabolism have a profound influence on the cold response.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses.

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.

Design and synthesis of fluorescent galactolipid probes

Fluorescent galactolipid analogues (1, 1a, and 2) have been synthesized with a pyrene group attached to the amino-terminal of a hexanoyl chain bound to an otherwise normal galactolipid structure. The synthetic lipids are obtained from peracetyl galactose by a general and versatile procedure based on the trichloroacetimidate methodology. The intense violet fluorescence (376 and 395nm) and good photostability of pyrene make these compounds highly suitable as probes to study galactolipid metabolism. As proof of concept, we report that compound 2 is a valid tool to detect galactolipase activity in enzymatic preparations of potato tubers.

A family 1 galactosyl hydrolase performs galactosyltransferase activity requiring Mg2+ and conferring freezing tolerance (617.8)

The two most abundant photosynthetic thylakoid membrane lipids are monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Understanding galactolipid metabolism is therefore critical to understanding thylakoid formation and subsequent photosynthetic activity. The first enzymatic activity described to synthesize DGDG was named galactolipid: galactolipid galactosyl transferase (GGGT), as it removed a galactosyl head group from MGDG and transferred it to another MGDG making DGDG. Though it is now understood that a separate enzyme, DGD1, synthesizes most of the DGDG in vivo, GGGT was re‐identified in Arabidopsis as the gene product of SENSITIVE TO FREEZING 2 (SFR2). SFR2 was shown to protect Arabidopsis from freezing damage, to be located on the chloroplast outer envelope and to be similar to family‐1 β‐glycosidases. Here, we present a model of the 3‐dimensional structure of SFR2 based on its homology to family‐1 β‐glycosidases and show the functional significance of several unique regions. Using the model, we identify residues required for SFR2 activity that are not conserved among family‐1 β‐glycosidases. We experimentally define pH and temperature optimums for enzymatic activity in vitro. Unexpectedly, we show that SFR2 is a metalloenzyme, unlike its family members. Our data suggest that while SFR2 retains the core structure and active site residues of a family‐1 β‐glycosidase, it has unique residues, regions, and co‐factors required to alter its function to that of a transferase.Grant Funding Source: Supported by the Department of Energy

Biochemical and Molecular-Genetic Characterization of SFD1’s Involvement in Lipid Metabolism and Defense Signaling

The Arabidopsis thaliana SFD1 (suppressor of fatty acid desaturase deficiency1) gene (also known as GLY1) is required for accumulation of 34:6 (i.e., 18:3–16:3) monogalactosyldiacylglycerol (MGDG) and for the activation of systemic acquired resistance (SAR), an inducible defense mechanism that confers resistance against a broad spectrum of pathogens. SFD1, which has been suggested to be involved in lipid-based signaling in SAR, contains a putative chloroplast transit peptide and has glycerol-3-phosphate synthesizing dihydroxyacetone phosphate (DHAP) reductase (also referred as glycerol-3-phosphate dehydrogenase) activity. The goals of this study were to determine if the DHAP reductase activity and chloroplast localization are required for SFD1’s involvement in galactolipid metabolism and SAR signaling. The crystal structure of a Leishmania mexicana glycerol-3-phosphate dehydrogenase was used to model SFD1 structure and identify Lys194, Lys279, and Asp332 as potential catalytic site residues in SFD1. Mutational analysis of SFD1 confirmed that Lys194, Lys279, and Asp332 are critical for SFD1’s DHAP reductase activity, and its involvement in SAR. SFD1 proteins with these residues individually substituted by Ala lacked DHAP reductase activity and were unable to complement the SAR defect of the sfd1 mutant. The SFD1–Ala279 protein was also unable to restore 34:6-MGDG content when expressed in the sfd1 mutant. In vivo imaging of a green fluorescent protein-tagged SFD1 protein demonstrated that SFD1 is targeted to the chloroplast. The N-terminal 43 amino acids, which are required for proper targeting of SFD1 to the chloroplast, are also required for SFD1’s function in lipid metabolism and SAR. Taken together, these results demonstrate that SFD1’s DHAP reductase activity is required in the chloroplast for lipid metabolism and defense signaling.

Lead Alters the Developmental Profile of the Galactolipid Metabolic Enzymes in Cultured Oligodendrocyte Lineage Cells

Lead is a neurotoxicant that can cause myelin deficits. Galactolipids are expressed during differentiation of oligodendrocyte lineage cells and accumulate in myelin. To examine the impact of lead on oligodendroglial differentiation, galactolipid metabolism in cultured oligodendrocyte lineage cells exposed to the metal was studied. Oligodendrocyte progenitor cells obtained from newborn rat pups were exposed to 1 μM lead acetate for 24 h prior to maintenance of the cells in medium containing the metal salt for 0, 2, or 6 days of differentiation. Lead caused approximately 50% reduction in levels of the galactolipid biosynthetic transferases, UDP-galactose:ceramide galactosyltransferase and 3′-phosphoadenosine-5′-phosphosulfate:galactocerebroside sulfotransferase, as compared to sodium-treated controls, in cultures of oligodendrocyte lineage cells following 2 days of differentiaition. The activities of the galactolipid catabolic hydrolases, galactocerebroside-β-galactosidase and arylsulfatase A, were reduced by 20%. Following 6 days of differentiation, lead-exposed cells exhibited levels of all the enzymes, except for arylsulfatase A, similar to those of the control cells. These results are consistent with the lead-induced delay of oligodendrocyte differentiation, as evidenced by the emergence of stage-specific immunochemical markers and the observed change in the developmental activity profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase. The activity of arylsulfatase A in lead-treated 6-day oligodendrocytes was significantly less than that found in control cultures. This effect is consistent with the lead-induced reduction of arylsulfatase A in human fibroblasts caused by mis-sorting the newly-synthesized enzyme. The perturbation of galactolipid metabolism by lead during developmental maturation of oligodendrocytes may represent a contributing mechanism for lead-induced neurotoxicity.

Hydroxy- and non-hydroxy-galactolipids in developing rat CNS

Rat spinal cord (1–24 weeks postnatal) was analysed by HPLC for various species of galactolipids that accumulate in mammalian myelin during development. Cerebral tissue of the same animals was taken as reference. The levels of the major galactolipids, galactosylceramide (GalCer) and its sulfated analog (SGalCer), increased linearly during the first 2 months after birth. At 3 months, constant levels were reached that were approx. 4-fold (GalCer) and 2.5-fold (SGalCer) higher than in cerebral tissue of corresponding age. The accumulation of galactoglycerolipids slightly preceded that of galactosphingolipids. Levels of galacto-glycerolipids were much lower (4% of galactospingolipids in 3- and 2.5% in 6-month-old spinal cord on weight basis) and decreased upon CNS maturation. During the first postnatal month, the ratio of non-hydroxy- over hydroxy-species (NFA/HFA) of cerebral GalCer declined from 2.2 to 0.5 whereas the NFA/HFA ratio for cerebral SGalCer increased from 1.0 to 1.8 in the same period. Through development the hydroxy-species contributed 56–60% to GalCer and 28–41% to SGalCer in spinal cord, whereas in cerebrum of 24-week-old rats 73% of GalCer and 48% of SGalCer was α-hydroxylated in the ceramide moiety. These data point to different developmental programs with respect to galactolipid metabolism of oligodendrocytes in high- (spinal cord) as compared to low-myelinated (cerebral) areas of rat CNS.

Metabolism of radiolabelled galactolipids in senescent barley leaves

The metabolism of galactolipids in senescent primary leaves of barley was followed upon the incorporation of [1- 14C]oleic acid during greening of etiolated shoots. In presenscent leaves the bulk of the label was present in apolar compounds of which 36% were galactolipids and 57% phospholipids. In segments induced to senesce in continouos darkness the loss of the predominant 18:3/18:3 molecular species of galactolipids was accompanied by the temporary accumulation of 18:3/18:3 phosphatidylcholine, a species which was almost absent in the presenescent leaves. A large proportion of radiocactivity lost in the galactolipids was eventually released as CO 2. Label was also transferred to sugars and other polar compounds. A pathway of galactolipid breakdown featuring the conversion of the diacylglycerol cores to phosphatidylcholine followed by the release of free fatty acids in glyoxisome-like microbodies and gluconeogenesis via β-oxidation and the glyoxylic acid cycle is proposed.

Biosynthèse des acides gras polyinsaturés des glycérolipides de la feuille d'olivier

The patterns of incorporation of [l‐14C]‐acetate into the glycerolipid fatty acids of leaves of olive plants (Olea europea L. cv. Chétoui) suggested a specific pathway for a‐linolenic acid biosynthesis. The results confirmed the involvement of phosphati‐dylcholine in galactolipid metabolism, and seemed to exclude the role of that mole‐cule as a substrate for desaturation of oleate to linoleate. The two oleate desaturation steps seemed to occur rapidly on the diacylgalactosylglycerol molecule for biosynthesis of galactolipid linolenate. In addition, the results indicated a slow sequen‐tial desaturation of oleate to linolenate via linoleate in the phospholipid molecules (phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol).

Differential Effects of the Substituted Pyridazinone Herbicide Sandoz 9785 on Lipid Composition and Biosynthesis in Photosynthetic and Non-Photosynthetic Marine Microalgae

A comparison was made of the effects of the substituted pyridazinone herbicide, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H) pyridazinone (Sandoz 9785; BASF 13–338) on the growth rate and lipid class synthesis of two species of photosynthetic (Chroomanas salina and Nannochloropsis oculata), and one species of non-photosynthetic (Crypthecodinium cohnil), marine microalgae. The growth rate, but not the total lipid content, of all three species was reduced by the herbicide. The percentage distribution of radioactivity incorporated by C. cohnii into lipid classes generally resembled the relative abundance of the classes and the presence of Sandoz 9785 resulted in only small changes in lipid class proportions or in the distribution of radioactivity. In C. salina, the proportion of radioactivity located in monogalactosyldiacylglycerol (MGDG) increased from 9.2% to 20.3%, and that in digalactosyldiacylglycerol (DGDG) from 6.3% to 12.7%, in the presence of the herbicide. In N. oculata the proportion of radioactivity recovered in DGDG, but not MGDG, was increased significantly by the presence of Sandoz 9785. The results suggest that Sandoz 9785 alters galactolipid metabolism in photosynthetic microalgae but has no great effect on lipid synthesis in non-photosynthetic species.

Regulated galactolipid synthesis and cell surface expression in Schwann cell line D6P2T.

Clonal cell line D6P2T, subcloned from an ethylnitrosourea-induced tumor line D6 of the rat peripheral nervous system, has been characterized with particular attention to galactolipid metabolism. Galactosylcerebroside and sulfatide synthesis and expression on the cell surface are highly regulated in D6P2T cells by mechanisms involving serum- and cyclic AMP-mediated pathways. These cells also express 2',3'-cyclic nucleotide 3'-phosphohydrolase (Wolfgram protein W1a) and laminin. In contrast, myelin basic protein and antigen HNK-1 were not detected. Line D6P2T appears to be a semi-differentiated Schwann cell model, which offers interesting possibilities for studies of galactolipid synthesis, transport, and sorting.

Characterization of galactosyltransferases in spinach chloroplast envelopes

Two galactosyltransferases involved in the galactolipid metabolism of spinach chloroplast envelopes were studied by specific assays: UDPgalactose: 1,2-diacylglycerol galactosyltransferase, which synthetizes monogalactosyldiacylglycerol from UDPgalactose plus diacylglycerol; and galactolipid: galactolipid galactosyltransferase (GGGT), which forms di- , tri- and tetragalactosyldiacylglycerol by dismutation of galactosyl groups between two galactosyldiacylglycerols. In the assay developed for UDPgalactose: 1,2-diacylglycerol galactosyltransferase, chloroplast envelope membranes were mixed with liposomes of phosphatidylcholine, and the mixture was treated with phospholipase C (from Bacillus cereus). The diacylglycerol produced from phosphatidylcholine is used by UDPgalactose: 1,2-diacylglycerol galactosyltransferase for synthesis of monogalactosyldiacylglycerol in the presence of UDPgalactose. Several characteristics of this enzyme were studied, including the effect of substrate concentrations, pH and temperature. UDPgalactose: 1,2-di-acylglycerol galactosyltransferase did not require cations for activity, but was stimulated by Mg 2+ and Mn 2+. Of the mono- and divalent cations tested only Zn 2+, Cd 2+ and Fe 2+ were inhibitory. Specific inhibitors for UDPgalactose: 1,2-diacylglycerol galactosyltransferase were UDP and N-ethylmaleimide. In contrast to UDPgalactose: 1,2-diacylglycerol galactosyltransferase, galactolipid: galactolipid galactosyltransferase was strongly stimulated by a series of mono- and divalent cations, most stimulatory being Mn 2+ Ba 2+, Ca 2+ and Mg 2+. Galactolipid: galactolipid galactosyltransferase was specifically inhibited by low concentrations of Zn 2+ (1 mM) and by chelating anions. UDPgalactose: 1,2-diacylglycerol galactosyltransferase synthetized monogalactosyldiacylglycerol from various molecular species of diacylglycerol; the highest activity was measured with distearoylglycerol and dioleoylglycerol. On the other hand, digalactosyldiacylglycerol synthesis by galactolipid: galactolipid galactosyltransferase proceeded most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol.

Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress

Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from -0.6 megapascal to -1.7 megapascals after 4 days of stress; then to -3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-(14)C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids.

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/ n-hexane (80:55, v v ) and 2-propanol/ n-hexane/water/methanol (80:55:15:10, v v ). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH 2 column (7-μm particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.