Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis.

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp.

The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.

Oil-bodies as substrates for lipolytic enzymes.

Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown. One of the questions that arises is whether the protein/phospholipid coat of oil-bodies prevents the access of lipase to the oil core. We have investigated the susceptibility of almond oil-bodies to in vitro lipolysis by various purified lipases with a broad range of biochemical properties. We have found that all the enzymes assayed were capable of releasing on their own free fatty acids from the TAG of oil-bodies. Depending on the lipase, the specific activity measured on oil-bodies using the pH-stat technique was found to range from 18 to 38% of the specific activity measured on almond oil emulsified by gum arabic. Some of these lipases are known to have a dual lipase/phospholipase activity. However, no correlation was found to exist between the ability of a lipase to readily and efficiently hydrolyse the TAG content of oil-bodies and the presence of a phospholipase activity. Kinetic studies indicate that oil-bodies behave as a substrate as other proteolipid organelles such as milk fat globules. Finally we have shown that a purified water-soluble plant lipase on its own can easily hydrolyse oil-bodies in vitro. Our results suggest that the lipolysis of oil-bodies in seedlings might occur without any pre-hydrolysis of the protein coat.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.

Improving palm oil quality through identification and mapping of the lipase gene causing oil deterioration.

The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards.

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 µmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 µmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 µmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 µmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.

Characterization of typo-, regio-, and stereo-selectivities of babaco latex lipase in aqueous and organic media.

The unripe fruit of babaco (Vasconcellea x heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3-diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn-3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids.

Assaying Arabidopsis lipase activity.

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.

Supercooling capacity of seeds and seedlings in Arabidopsis thaliana.

The influence of the water content of seeds and seedlings of Arabidopsis thaliana (Ecotype Columbia:2) on their supercooling capacity was investigated. Equilibration of the seeds to various air relative humidities resulted in final moisture contents ranging from 8 to 82% (dry weight basis). No supercooling point could be detected when the water content remained below 32.5%, and in seeds at just above this moisture level ice crystals started to form at -26 degrees C. However, cooling partly affected the germination of seeds down to a water content of 26.5%. Upon imbibition, the supercooling point of the seeds remained around -21.6 degrees C and rose sharply to -14.7 degrees C when visible germination started. It remained around -13 degrees C during the following 96 h while the water content of the seedlings increased from 155 to 870%. Hydrated seeds (above 32.5% water content), germinated seeds, and seedlings of Arabidopsis cannot survive being frozen.

Lipid transfer in plants.

Plant cells contain cytosolic proteins, called lipid transfer proteins (LTP), which are able to facilitate in vitro intermembrane transfer of phospholipids. Proteins of this kind from three plants, purified to homogeneity, have several properties in common: molecular mass around 9 kDa, high isoelectric point, lack of specificity for phospholipids, and binding ability for fatty acids. The comparison of their amino acid sequences revealed striking homologies and conserved domains which are probably involved in their function as LTPs. These proteins could play a major role in membrane biogenesis by conveying phospholipids from their site of biosynthesis to membranes unable to form these lipids. Immunochemical methods were used to establish an in vivo correlation between membrane biogenesis and the level of LTP or the amount of LTP synthesized in vitro from mRNAs. The recent isolation of a full-length cDNA allows novel approaches to studying the participation of LTPs in the biogenesis of plant cell membranes.

Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana.

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated omega-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized omega-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased omega-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.

Isolation and functional expression in Escherichia coli of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides.

Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine. In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase. Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine. A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants. Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine. A protein extract derived from the E. coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine. Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R. sphaeroides.

Bifunctional lipid-transfer: fatty acid-binding proteins in plants.

A cytosolic protein, able to facilitate intermembrane movements of phospholipids in vitro, has been purified to homogeneity from sunflower seedlings. This protein, which has the properties of a lipid-transfer protein (LTP), is also able to bind oleoyl-CoA, as shown by FPLC chromatography. This finding, in addition to previous observations suggesting that a lipid-transfer protein from spinach leaves can bind oleic acid and that oat seedlings contain a fatty acid-binding protein with similar features than lipid transfer proteins, provides a clear demonstration that plant cells contain bifunctional fatty acid/lipid transfer proteins. These proteins can play an active role in fatty acid metabolism which involves movements of oleyl-CoA between intracellular membranes.

Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.

A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana.

Mutations at the fad7 locus of Arabidopsis thaliana (previously called fadD) cause decreased desaturation of dienoic fatty acids in chloroplast lipids in plants grown at elevated temperatures. This suggested that the fad7 locus encodes a chloroplast omega-3 desaturase that catalyzes the desaturation of lipid-linked 18:2 and 16:2 fatty acids. In order to clone the fad7 gene, it was first genetically mapped relative to the flanking restriction fragment length polymorphism markers 4547 and 2488A on chromosome 3, and yeast artificial chromosomes covering the locus were identified. A putative desaturase cDNA clone that was isolated by low stringency heterologous probing with a cDNA for an endoplasmic reticulum-localized omega-3 desaturase (fad3) hybridized to the yeast artificial chromosomes and could not be resolved from the locus by restriction fragment length polymorphism mapping. Expression of the cDNA in transgenic fad7 mutant plants resulted in restoration of wild type fatty acid composition and suppression of a previously observed effect of the fad7 mutation on chloroplast number, indicating genetic complementation. The structural gene contained seven introns within a coding sequence of 1338 base pairs, which encodes a 446-amino acid polypeptide of 51,172 daltons. The amino-terminal region of the fad7 gene product contained a consensus chloroplast transit peptide. Except for the amino-terminal domain, the deduced amino acid sequence of the fad7 gene product had high homology to the fad3 gene product, indicating that fad7 encodes an omega-3 desaturase and that the two genes arose from a common ancestral gene. There was no apparent effect of growth temperature on the steady-state levels of fad7 mRNA in wild type plants.

Synthesis of phospholipid transfer proteins from maize seedlings.

The synthesis of phospholipid transfer proteins has been studied in vitro after isolation of poly(A)+RNAs from maize seedlings and by in vivo labelling of coleoptiles. After immunoprecipitation of translation products in wheat germ or in reticulocyte lysate systems, the analysis by electrophoresis revealed two bands of molecular mass 9 kDa and 12 kDa. The in vitro synthesized 12 kDa protein is a precursor of the 9 kDa purified protein from maize seedlings as suggested by competition experiments with the pure protein. After immunoprecipitation of in vivo labelled proteins, two bands were detected. One of them, having a molecular mass of 7 kDa, could be related to the in vitro synthesized 9 kDa protein, the other corresponding to the purified protein. Furthermore, biosynthesis of both precursors occurs on membrane-bound polysomes. Presumably a post translational process occurs, yielding to the mature forms.

Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast.

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

Use of the tape stripping technique for directly quantifying esterase activities in human stratum corneum.

The enzymes secreted in the intercellular spaces of stratum corneum (SC), the outermost layer of the epidermis, are thought to be involved in normal desquamation and skin barrier function. Their activity can barely be measured due to the difficulty in isolating enough biological material. Human SC layers were obtained from the forearm of healthy volunteers by the tape stripping technique. Assays for esterase activities were carried out in specially designed plates which contained the SC blotted on tape strips, using various fluorescent methylumbelliferone acyl esters as substrates. Triacylglycerol hydrolase activities were also studied by this method. By using radiolabeled triolein and fluorescent 4-methylumbelliferyl 7-oleate as substrates, true lipase activities could be detected and quantitated in SC at pH 5.5 and 7.5. These activities were shown to be strongly inhibited by tetrahydrolipstatin while this was not the case with 4-methylumbelliferyl 7-heptanoate. The method described here combines the painless tape stripping technique with a sensitive plate assay analysis. Since the whole process needs little manipulation, this method can permit rapid quantitation of multiple enzyme activities from a single strip. Therefore, it will permit the study of the involvement of enzyme activities in epidermis aging and skin pathologies.