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J Browse

Publications and source records attributed to J Browse.

At least 37 records · Page 2Linked to original sources
PubMed1998

Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis.

High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75-80% polyunsaturated fatty acids. For the extra-chloroplast membranes in leaf cells and the membranes of non-photosynthetic tissues, values of 60-65% are typical. We report here the production of Arabidopsis double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.

Arabidopsis↗
PubMed1998

A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed.

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Delta9) plus cis-vaccenic acid (18:1Delta11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Delta9-18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Delta9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Delta9-18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Delta9-18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Delta9-18:0-ACP desaturase.

Amino Acid Sequence↗
PubMed1998

A new class of Arabidopsis mutants with reduced hexadecatrienoic acid fatty acid levels.

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Arabidopsis↗
PubMed1997

Jasmonate is essential for insect defense in Arabidopsis.

The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in Arabidopsis and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (fad3-2 fad7-2 fad8) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality ( approximately 80%) from attack by larvae of a common saprophagous fungal gnat, Bradysia impatiens (Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to approximately 12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant-insect interaction. The transcripts of three wound-responsive genes were shown not to be induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione S-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.

Journal Article↗
PubMed1997

Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis.

In animals, fatty acid desaturases catalyze key reactions in the synthesis of arachidonic acid and other polyunsaturated fatty acids. A search of the Caenorhabditis elegans DNA databases, using the sequences of Arabidopsis genes, identified several putative desaturases. Here we describe the characterization of the first of these genes, fat-1. The predicted protein encoded by a fat-1 cDNA showed 32-35% identity with both FAD2 and FAD3 of Arabidopsis. When expressed in transgenic plants, fat-1 resulted in a 90% increase in the proportion of alpha-linolenic acid in root lipids. Wild-type Arabidopsis incorporated omega-6 fatty acids (delta8,11,14-20:3 and delta5,8,11,14-20:4) into membrane lipids but did not desaturate them. By contrast, fat-1 transgenic plants efficiently desaturated both of these fatty acids to the corresponding omega-3 products. These findings indicate that the C. elegans fat-1 gene encodes the first animal representative of a class of glycerolipid desaturases that have previously been characterized in plants and cyanobacteria. The FAT-1 protein is an omega-3 fatty acyl desaturase that recognizes a range of 18- and 20-carbon omega-6 substrates.

Amino Acid Sequence↗
PubMed1997

Low-temperature damage and subsequent recovery of fab1 mutant Arabidopsis exposed to 2 degrees C.

The fab1 mutant of Arabidopsis thaliana, which contains increased levels of saturated fatty acids, was indistinguishable from the wild type when it was grown at 22 or 12 degrees C. During the first 7 to 10 d after transfer to 2 degrees C, the growth and photosynthetic characteristics of the fab1 plants remained indistinguishable from the wild type, with values for the potential quantum efficiency of photosystem II decreasing from 0.8 to 0.7 in plants of both lines. Whereas wild-type plants maintained quantum efficiency of photosystem II at approximately 0.7 for at least 35 d at 2 degrees C, this parameter declined rapidly in the mutant after 7 d and reached a value of less than 0.1 after 28 d at 2 degrees C. This decline in photosynthetic capacity was accompanied by reductions in chlorophyll content and the amount of chloroplast glycerolipids per gram of leaf. Electron microscopic examination of leaf samples revealed a rapid and extensive disruption of the thylakoid and chloroplast structure in the mutant, which is interpreted here as a form of selective autophagy. Despite the almost complete loss of photosynthetic function and the destruction of photosynthetic machinery, fab1 plants retained a substantial capacity for recovery following transfer to 22 degrees C. These results provide a further demonstration of the importance of chloroplast membrane unsaturation to the proper growth and development of plants at low temperature.

Adaptation, Biological↗
PubMed1997

Overexpression of the FAD3 desaturase gene in a mutant of Arabidopsis.

A mutant of Arabidopsis contained increased levels of 18:3 fatty acids and correspondingly decreased levels of 18:2. The fatty acid phenotype was strongly expressed in root and seed tissues and this observation, together with other data, suggested that the mutation leads to increased activity of the endoplasmic reticulum 18:2 desaturase encoded by the FAD3 gene. Gel-blot analysis of RNA from wild-type and mutant plants established that FAD3 transcript levels were increased 80% in the mutant relative to the wild type. Genetic analysis demonstrated a linkage between the new mutation and the fad3 locus. Linkage of the mutation to fad3 raises the possibility that the lesion is an alteration to the promoter or another regulatory region of the FAD3 gene, which results in increased transcription.

Arabidopsis↗
PubMed1996

Dissecting desaturation: plants prove advantageous.

Fatty acid desaturases play important roles in controlling the physical properties o f membranes and in the synthesis of signal molecules such as prostaglandins and pheromones. Most desaturases are membrane proteins that have been recalcitrant to characterization by conventional biochemical methods. Only one enzyme o f this class has been characterized from animals or fungi. In this context, plants have proved to be useful sources of experimental materials. Substantial progress has been made in characterizing and manipulating nine classes of desaturases that control the fatty acid composition o f both plant membranes and plant storage lipids, which account for approximately -30% of the calories in the human diet.

Journal Article↗
PubMed1996

An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack.

The activation of defense genes in tomato plants has been shown to be mediated by an octadecanoic acid-based signaling pathway in response to herbivore attack or other mechanical wounding. We report here that a tomato mutant (JL5) deficient in the activation of would-inducible defense genes is also compromised in resistance toward the lepidopteran predator Manduca sexta (tobacco hornworm). Thus we propose the name defenseless1 (def1) for the mutation in the JL5 line that mediates this altered defense response. In experiments designed to define the normal function of DEF1, we found that def1 plants are defective in defense gene signaling initiated by prosystemin overexpression in transgenic plants as well as by oligosaccharide (chitosan and polygalacturonide) and polypeptide (systemin) elicitors. Supplementation of plants through their cut stems with intermediates of the octadecanoid pathway indicates that def1 plants are affected in octadecanoid metabolism between the synthesis of hydroperoxylinolenic acid and 12-oxo-phytodienoic acid. Consistent with this defect, def1 plants are also compromised in their ability to accumulate jasmonic acid, the end product of the pathway, in response to wounding and the aforementioned elicitors. Taken together, these results show that octadecanoid metabolism plays an essential role in the transduction of upstream would signals to the activation of antiherbivore plant defenses.

Animals↗
PubMed1995

Preparation of radioactively labeled synthetic sn-1,2-diacylglycerols for studies of lipid metabolism.

A method was developed for the synthesis of stereo-specific sn-1,2-diacylglycerols radioactively labeled on the glycerol backbone. This method includes the acylation of L-[U-14C]glycerol 3-phosphate with fatty acid anhydrides in the presence of dimethylaminopyridine and the tetraethylammonium salt of the fatty acid used for the acylation. The phosphatidic acid obtained from this synthesis was converted to sn-1,2-diacyl[U-14C]glycerol by the action of phospholipase C. When di10:0-, di-18:1-, di-18:2-, and di-22:1-diacyl[U-14C]glycerols made by this method were provided as substrates to microsome preparations from developing cotyledons of safflower (Carthamus tinctorius), they were all incorporated into phosphatidylcholine by the action of cholinephosphotransferase. The time course of the incorporation into phosphatidylcholine showed no differences for these four substrates and the initial rates were comparable.

Carbon Radioisotopes↗
PubMed1995

Lipid biosynthesis.

Explore the source record for details and available documents.

Acetyl-CoA Carboxylase↗
PubMed1994

Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.

The polyunsaturated fatty acids linoleate and alpha-linolenate are important membrane components and are the essential fatty acids of human nutrition. The major enzyme responsible for the synthesis of these compounds is the plant oleate desaturase of the endoplasmic reticulum, and its activity is controlled in Arabidopsis by the fatty acid desaturation 2 (fad2) locus. A fad2 allele was identified in a population of Arabidopsis in which mutations had been created by T-DNA insertions. Genomic DNA flanking the T-DNA was cloned by plasmid rescue and used to isolate cDNA and genomic clones of FAD2. A cDNA containing the entire FAD2 coding sequence was expressed in fad2 mutant plants and shown to complement the mutant fatty acid phenotype. The deduced amino acid sequence from the cDNA showed homology to other plant desaturases, and this confirmed that FAD2 is the structural gene for the desaturase. Gel blot analyses of FAD2 mRNA levels showed that the gene is expressed throughout the plant and suggest that transcript levels are in excess of the amount needed to account for oleate desaturation. Sequence analysis identified histidine-rich motifs that could contribute to an iron binding site in the cytoplasmic domain of the protein. Such a position would facilitate interaction between the desaturase and cytochrome b5, which is the direct source of electrons for the desaturation reaction, but would limit interaction of the active site with the fatty acyl substrate.

Alleles↗
PubMed1993

Mutants of Arabidopsis deficient in the synthesis of alpha-linolenate. Biochemical and genetic characterization of the endoplasmic reticulum linoleoyl desaturase.

The overall fatty compositions of leaf and root lipids from a mutant of Arabidopsis thaliana were characterized by reduced levels of linolenate (18:3) and correspondingly elevated levels of linoleate (18:2) as a consequence of a single nuclear mutation at the fad3 locus. Comparison of the fatty acid compositions of individual lipids from wild type and mutant plants showed that chloroplast lipids were largely unaffected by the mutation, whereas each of the phospholipids synthesized on the endoplasmic reticulum in the mutant exhibited a marked reduction in the proportion of 18:3 relative to wild type. These and other results indicate that the fad3 mutants are deficient in the activity of an endoplasmic reticulum 18:2 desaturase. In root tissues, this enzyme appears to account for over 80% of the 18:3 present. However, in young leaves of the mutant, phosphatidylcholine (the major phospholipid) contains 16% 18:3 compared with 29% in the wild type, and the proportion of 18:3 in this lipid increases with plant age in both wild type and mutant plants. These results reflect an exchange of lipid between the chloroplast and endoplasmic reticulum that allows the chloroplast desaturases to provide highly unsaturated lipids to the extrachloroplast membranes of leaf cells.

Arabidopsis↗
PubMed1993

Arabidopsis requires polyunsaturated lipids for low-temperature survival.

Mutants of Arabidopsis that contain reduced levels of polyunsaturated fatty acids showed growth characteristics at 22 degrees C that were very similar to wild type. By contrast, at 12 degrees C, the mutants failed to undergo stem elongation during reproductive growth although they produced normal flowers and fertile seeds. After transfer to 6 degrees C, rosette leaves of the mutants gradually died, and the plants were inviable. These different responses of the mutant plants at 12 degrees C and 6 degrees C suggest that distinct functions may be affected at these two temperatures. The gradual development of symptoms at 6 degrees C and other lines of evidence argue against a general collapse of membrane integrity as the cause of the lethal phenotype. Rather, they indicate that the decrease in polyunsaturated membrane lipids may initially have relatively limited effects in disrupting cellular function.

Journal Article↗
PubMed1993

Isolation of signaling mutants of tomato (Lycopersicon esculentum).

As a first step towards developing a genetic system for investigating signaling processes in plants, we have developed a screen for signaling mutants deficient in a wound response. We have isolated two mutants of tomato that lack detectable production of proteinase inhibitors induced systemically in leaves by wounding. The mutants are deficient in the induction of both proteinase Inhibitor I and proteinase Inhibitor II but can be induced to respond at near wild-type levels by methyl jasmonate, a known elicitor of inhibitor production in tomato. While completely deficient in systemic production of proteinase inhibitors, both mutants produce some proteinase inhibitor in wounded leaves. This evidence suggests the existence of two signaling pathways, one local and one systemic, that regulate the induction of proteinase inhibitor synthesis in response to wounding.

Acetates↗
PubMed1992

A preparation and purification of [1-14C]acetylcarnitine.

[1-14C]Acetylcarnitine was prepared from [1-14C]acetate and L-carnitine using acetyl-CoA synthetase and carnitine acetyltransferase. The product was purified by ion-exchange and thin-layer chromatography. Conversion of [1-14C]acetate to [1-14C]acetylcarnitine was better than 90% and overall recovery of the pure product was greater that 80%.

Acetates↗
PubMed1992

Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.

Chloroplasts↗