PubMed Health⌕ Search

Biomedical subjects

John Browse

Publications and source records attributed to John Browse.

At least 19 recordsLinked to original sources
PubMed2006

Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo.

A fundamental question in animal development is how motile cells find their correct target destinations. During mating in the nematode Caenorhabditis elegans, males inject sperm through the hermaphrodite vulva into the uterus. Amoeboid sperm crawl around fertilized eggs to the spermatheca--a convoluted tube where fertilization occurs. Here, we show that polyunsaturated fatty acids (PUFAs), the precursors of eicosanoid signalling molecules, function in oocytes to control directional sperm motility within the uterus. PUFAs are transported from the intestine, the site of fat metabolism, to the oocytes yolk, which is a lipoprotein complex. Loss of the RME-2 low-density lipoprotein (LDL) receptor, which mediates yolk endocytosis and fatty acid transport into oocytes, causes severe defects in sperm targeting. We used an RNAi screen to identify lipid regulators required for directional sperm motility. Our results support the hypothesis that PUFAs function in oocytes as precursors of signals that control sperm recruitment to the spermatheca. A common property of PUFAs in mammals and C. elegans is that these fats control local recruitment of motile cells to their target tissues.

Animals↗
PubMed2006

Altered rates of protein transport in Arabidopsis mutants deficient in chloroplast membrane unsaturation.

Protein transfer across membranes is mediated by protein machinery embedded in the membrane. The complement of different lipid classes within a membrane is known to influence the efficiency of some protein translocation processes, but very little is known about whether the fatty acid composition of the membrane bilayer also affects protein transport. We investigated this issue using three mutants of Arabidopsis, fad6, fad5, and fad3 fad7 fad8, that have reduced levels of fatty acid unsaturation in their thylakoid membranes. Interestingly, the effect of reduced unsaturation was different for three distinct pathways of protein transport. In thylakoids from all three mutants, transport of the OE17 protein on the DeltapH/Tat pathway was reduced by up to 50% relative to wild-type controls, when assays were run at 10, 20 or 30 degrees C. By contrast, transport of the OE33 protein on the Sec pathway was substantially increased in all the mutants at the three temperatures. Transport of the CF(O)II protein (ATPg) on the 'spontaneous' pathway was largely unaffected by reduced unsaturation of the thylakoid membranes. Experiments with intact chloroplasts from wild-type Arabidopsis and the three mutants confirmed these changes in thylakoid transport reactions and also demonstrated an increased rate of protein import across the chloroplast envelope in each of the mutants. This increased capacity of chloroplast protein import may partially compensate for the reduced capacity of thylakoid transport by the DeltapH/Tat pathway. The fad5, fad6 and fad3 fad7 fad8 mutants used in this study grow normally at 15-20 degrees C, but exhibit reduced photosynthesis and growth, relative to wild-type controls, at low temperatures (4 degrees C). The results reported here indicate that protein transport and chloroplast biogenesis may well contribute to these low-temperature phenotypes.

Arabidopsis↗
PubMed2006

Genetic regulation of unsaturated fatty acid composition in C. elegans.

Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor, NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex regulation of the delta-9 desaturases to achieve optimal fatty acid composition.

Animals↗
PubMed2006

A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function.

Leaf membrane lipids of the Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis 1 (fab1) mutant contain a 35% to 40% increase in the predominant saturated fatty acid 16:0, relative to wild type. This increase in membrane saturation is associated with loss of photosynthetic function and death of mutant plants at low temperatures. We have initiated a suppressor screen for mutations that allow survival of fab1 plants at 2 degrees C. Five suppressor mutants identified in this screen all rescued the collapse of photosynthetic function observed in fab1 plants. While fab1 plants died after 5 to 7 weeks at 2 degrees C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22 degrees C and to subsequently produce viable seed. Three of the suppressors had changes in leaf fatty acid composition when compared to fab1, indicating that one mechanism of suppression may involve compensating changes in thylakoid lipid composition. Surprisingly, the suppressor phenotype in one line, S31, was associated with a further substantial increase in lipid saturation. The overall leaf fatty acid composition of S31 plants contained 31% 16:0 compared with 23% in fab1 and 17% in wild type. Biochemical and genetic analysis showed that S31 plants contain a new allele of fatty acid desaturation 5 (fad5), fad5-2, and are therefore partially deficient in activity of the chloroplast 16:0 Delta7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2 degrees C, indicating that the fad5-2 mutation is the suppressor in the S31 (fab1 fad5-2) line. Based on the biophysical characteristics of saturated and unsaturated fatty acids, the increased 16:0 in fab1 fad5-2 plants would be expected to exacerbate, rather than ameliorate, low-temperature damage. We propose instead that a change in shape of the major thylakoid lipid, monogalactosyldiacylglycerol, mediated by the fad5-2 mutation, may compensate for changes in lipid structure resulting from the original fab1 mutation. Our identification of mutants that suppress the low-temperature phenotype of fab1 provides new tools to understand the relationship between thylakoid lipid structure and photosynthetic function.

Alleles↗
PubMed2006

Dietary manipulation implicates lipid signaling in the regulation of germ cell maintenance in C. elegans.

Reproduction in C. elegans relies on continuously proliferating germ cells which, during germline development, exit mitosis, undergo meiosis and differentiate into gametes. Supplementing the diet of C. elegans with dihommogamma-linolenic acid (20:3n-6, DGLA), a long chain omega-6 polyunsaturated fatty acid, results in sterile worms that lack germ cells. The effect is remarkably specific for DGLA, as eicosapentaenoic acid (20:5n-3, EPA) and other long-chain polyunsaturated fatty acids with similar physical properties have little or no effect on fertility. Germ cells undergoing mitosis during larval stages are especially sensitive to DGLA, but exposure to DGLA during adulthood also reduces germ cells and brood size, in part by inducing inappropriate apoptosis of meiotic germ cells. Mutant strains with defects in fatty acid desaturation and elongation display altered susceptibility to DGLA, indicating that the sterility effect of the dietary lipid depends on the amount of DGLA present in membranes as well as on the capacity to convert DGLA to other fatty acids. We propose that DGLA produces a signal that interacts with one or more pathways regulating germ cell survival. Our DGLA findings are the first report of a role for a specific fatty acid affecting the development and maintenance of germ cells in C. elegans.

8,11,14-Eicosatrienoic Acid↗
PubMed2006

A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis.

It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial fatty acids usually results in low levels of desired fatty acids in transgenic oilseeds. The abundance of derivatized fatty acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor fatty acid hydroxylase (FAH12) to identify genes that can boost hydroxy fatty acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of hydroxy fatty acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.

Arabidopsis↗
PubMed2006

Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling.

In Arabidopsis, jasmonate is required for stamen and pollen maturation. Mutants deficient in jasmonate synthesis, such as opr3, are male-sterile but become fertile when jasmonate is applied to developing flower buds. We have used ATH1 oligonucleotide arrays to follow gene expression in opr3 stamens for 22 h following jasmonate treatment. In these experiments, a total of 821 genes were specifically induced by jasmonate and 480 genes were repressed. Comparisons with data from previous studies indicate that these genes constitute a stamen-specific jasmonate transcriptome, with a large proportion (70%) of the genes expressed in the sporophytic tissue but not in the pollen. Bioinformatics tools allowed us to associate many of the induced genes with metabolic pathways that are probably upregulated during jasmonate-induced maturation. Our pathway analysis led to the identification of specific genes within larger families of homologues that apparently encode stamen-specific isozymes. Extensive additional analysis of our dataset identified 13 transcription factors that may be key regulators of the stamen maturation processes triggered by jasmonate. Two of these transcription factors, MYB21 and MYB24, are the only members of subgroup 19 of the R2R3 family of MYB proteins. A myb21 mutant obtained by reverse genetics exhibited shorter anther filaments, delayed anther dehiscence and greatly reduced male fertility. A myb24 mutant was phenotypically wild-type, but production of a myb21myb24 double mutant indicated that introduction of the myb24 mutation exacerbated all three aspects of the myb21 phenotype. Exogenous jasmonate could not restore fertility to myb21 or myb21myb24 mutant plants. Together with the data from transcriptional profiling, these results indicate that MYB21 and MYB24 are induced by jasmonate and mediate important aspects of the jasmonate response during stamen development.

Arabidopsis↗
PubMed2006

A role for caleosin in degradation of oil-body storage lipid during seed germination.

Caleosin is a Ca(2+)-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing alpha- and delta-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.

Arabidopsis↗
PubMed2006

A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation.

As a model for analyzing the production of novel fatty acids in oilseeds, we used the genetic and molecular techniques available for Arabidopsis to characterize modifying mutations affecting the accumulation of hydroxy fatty acids in the seeds of Arabidopsis plants that express a transgene for the castor bean fatty acid hydroxylase, FAH12. We developed a high-throughput analytical system and used it to identify three complementation classes of mutations with reduced hydroxy fatty acid accumulation from among Arabidopsis M3 seed samples derived from chemical mutagenesis. We identified one of the mutations by positional cloning as a single base pair change in a gene encoding NADH:cytochrome b5 reductase (CBR1, At5g17770). When expressed in yeast, the mutant form of the enzyme was less active and less stable than the wild-type enzyme. Characterization of homozygous mutant lines with and without the FAH12 transgene (FAH12 cbr1-1 and cbr1-1, respectively) indicated that the only detectable consequence of the cbr1-1 mutation was on desaturase and hydroxylase reactions in the developing seed. The leaf and root fatty compositions, as well as the growth, development and seed production of mutant plants were indistinguishable from wild type. Interestingly, while the cbr1-1 mutation reduced the accumulation of hydroxy fatty acids in seeds by 85%, the effects on 18:1 and 18:2 desaturation reactions were much less (<25% and <60%, respectively). These results suggest that there is competition in developing seeds among the several reactions that utilize reduced cytochrome b5.

Amino Acid Sequence↗
PubMed2005

Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: syringyl lignin and sinapate/sinapyl alcohol derivative formation.

A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with kenz approximately 10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at approximately 100 microg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this "knockout" had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry.

Alcohols↗
PubMed2005

Jasmonate: an oxylipin signal with many roles in plants.

Jasmonic acid is an oxylipin signaling molecule derived from linolenic acid. So far, jasmonate (JA) (including the free acid and a number of conjugates) has been shown to regulate or co-regulate a wide range of processes in plants, from responses to biotic and abiotic stresses to the developmental maturation of stamens and pollen in Arabidopsis. This review focuses on discoveries in several of these areas. Most work described is from studies in Arabidopsis. While the results are expected to be broadly applicable to other higher plants, there are cases where related but distinct phenotypes have been observed in other species (e.g., tomato). Investigation of JA action in wound- and insect-defense responses has established that this compound is an essential component of the systemic signal that activates defense genes throughout the plant. It is possible that JA acts indirectly through the production of reactive oxygen species including hydrogen peroxide (H2O2). The availability of Arabidopsis mutants deficient in JA synthesis has been central to the identification of additional roles for JA in defense against microbial pathogens and in reproductive development. Currently, the key issues in JA action are to understand the role of the skip/cullin/F-box ubiquitination complex, SCF(COI1), and to identify additional protein components that act in the early steps of JA signaling.

Cyclopentanes↗
PubMed2005

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids.

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (<or=16 carbons) in the light. No detectable 14C-acyl-CoA intermediates were formed during 14C-laurate elongation, whereas 14C-acyl-ACP accumulated to 2.3 microm. These data indicate that the elongation of exogenous medium-chain FAs to 16- and 18-carbon FAs occurs primarily in the chloroplasts, most likely via the enzymes of de novo FA synthesis. An Arabidopsis mutant with a T-DNA insertion in At4g14070 (AAE15) was reduced 80% in 14C-laurate elongation into 16- and 18-carbon FAs. AAE15 has sequence similarity to long-chain acyl-CoA synthetases and a predicted N-terminal plastidial targeting sequence. Direct acyl-ACP-forming activity from FA and ACP was observed in extracts of Arabidopsis leaves and isolated chloroplasts but aae15 plants had markedly reduced in vitro acyl-ACP synthesis activity. Together these results demonstrate that plants possess a mechanism for direct activation of FA to ACP in the plastid via an acyl-ACP synthetase encoded by At4g14070.

Arabidopsis↗
PubMed2004

Specific polyunsaturated fatty acids drive TRPV-dependent sensory signaling in vivo.

A variety of lipid and lipid-derived molecules can modulate TRP cation channel activity, but the identity of the lipids that affect TRP channel function in vivo is unknown. Here, we use genetic and behavioral analysis in the nematode C. elegans to implicate a subset of 20-carbon polyunsaturated fatty acids (PUFAs) in TRPV channel-dependent olfactory and nociceptive behaviors. Olfactory and nociceptive TRPV signaling are sustained by overlapping but nonidentical sets of 20-carbon PUFAs including eicosapentaenoic acid (EPA) and arachidonic acid (AA). PUFAs act upstream of TRPV family channels in sensory transduction. Short-term dietary supplementation with PUFAs can rescue PUFA biosynthetic mutants, and exogenous PUFAs elicit rapid TRPV-dependent calcium transients in sensory neurons, bypassing the normal requirement for PUFA synthesis. These results suggest that a subset of PUFAs with omega-3 and omega-6 acyl groups act as endogenous modulators of TRPV signal transduction.

Animals↗
PubMed2004

Identification of the Arabidopsis palmitoyl-monogalactosyldiacylglycerol delta7-desaturase gene FAD5, and effects of plastidial retargeting of Arabidopsis desaturases on the fad5 mutant phenotype.

Hexadeca 7,10,13-trienoic acid (16:3Delta(7,10,13)) is one of the most abundant fatty acids in Arabidopsis (Arabidopsis thaliana) and a functional component of thylakoid membranes, where it is found as an sn-2 ester of monogalactosyldiacylglycerol. The Arabidopsis fad5 mutant lacks activity of the plastidial palmitoyl-monogalactosyldiacylglycerol Delta7-desaturase FAD5, and is characterized biochemically by the absence of 16:3Delta(7,10,13) and physiologically by reduced chlorophyll content and a reduced recovery rate after photoinhibition. While the fad5 mutation has been mapped, the FAD5 gene was not unambiguously identified, and a formal functional characterization by complementation of fad5 mutant phenotypes has not been reported. Two candidate genes (At3g15850 and At3g15870) predicted to encode plastid-targeted desaturases at the fad5 chromosomal locus were cloned from fad5 plants and sequenced. A nonsense mutation changing codon TGG (Trp-98) into TGA (stop) was identified in At3g15850 (ADS3), whereas the fad5 At3g15870 allele was identical to wild type (after correction of a sequencing error in the published wild-type genomic At3g15870 sequence). Expression of a genomic clone or cDNA for wild-type At3g15850 conferred on fad5 plants the ability to synthesize 16:3Delta(7,10,13) and restored leaf chlorophyll content. Arabidopsis carrying a T-DNA insertion in At3g15870 had wild-type levels of both 16:3Delta(7,10,13) and chlorophyll. Together, these data formally prove that At3g15850 is FAD5. Interestingly, the fad5 phenotype was partially complemented when extraplastidial Delta9-desaturases of the Arabidopsis desaturase (ADS) family were expressed as fusions with a plastidial transit peptide. Tight correlation between leaf 16:3Delta(7,10,13) levels and chlorophyll content suggests a role for plastidial fatty acid desaturases in thylakoid formation.

Amino Acid Sequence↗
PubMed2004

The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis.

Long-chain acyl-CoA synthetase (LACS) activities are encoded by a family of at least nine genes in Arabidopsis (Arabidopsis thaliana). These enzymes have roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. Here, we show that the LACS2 gene (At1g49430) is expressed in young, rapidly expanding tissues, and in leaves expression is limited to cells of the adaxial and abaxial epidermal layers, suggesting that the LACS2 enzyme may act in the synthesis of cutin or cuticular waxes. A lacs2 null mutant was isolated by reverse genetics. Leaves of mutant plants supported pollen germination and released chlorophyll faster than wild-type leaves when immersed in 80% ethanol, indicating a defect in the cuticular barrier. The composition of surface waxes extracted from lacs2 leaves was similar to the wild type, and the total wax load was higher than the wild type (111.4 microg/dm(2) versus 76.4 microg/dm(2), respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 +/- 1.7 nm compared with 33.0 +/- 2.0 nm for the wild type. In vitro assays showed that 16-hydroxypalmitate was an excellent substrate for recombinant LACS2 enzyme. We conclude that the LACS2 isozyme catalyzes the synthesis of omega-hydroxy fatty acyl-CoA intermediates in the pathway to cutin synthesis. The lacs2 phenotype, like the phenotypes of some other cutin mutants, is very pleiotropic, causing reduced leaf size and plant growth, reduced seed production, and lower rates of seedling germination and establishment. The LACS2 gene and the corresponding lacs2 mutant will help in future studies of the cutin synthesis pathway and in understanding the consequences of reduced cutin production on many aspects of plant biology.

Arabidopsis↗
PubMed2004

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana.

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2003

Biochemical and molecular characterization of ACH2, an acyl-CoA thioesterase from Arabidopsis thaliana.

By using computer-based homology searches of the Arabidopsis genome, we identified the gene for ACH2, a putative acyl-CoA thioesterase. With the exception of a unique 129-amino acid N-terminal extension, the ACH2 protein is 17-36% identical to members of a family of acyl-CoA thioesterases that are found in both prokaryotes and eukaryotes. The eukaryotic homologs of ACH2 are peroxisomal acyl-CoA thioesterases that are up-regulated during times of increased fatty acid oxidation, suggesting potential roles in peroxisomal beta-oxidation. We investigated ACH2 to determine whether it has a similar role in the plant cell. Like its eukaryotic homologs, ACH2 carries a putative type 1 peroxisomal targeting sequence (-SKL(COOH)), and maintains all the catalytic residues typical of this family of acyl-CoA thioesterases. Analytical ultracentrifugation of recombinant ACH2-6His shows that it associates as a 196-kDa homotetramer in vitro, a result that is significant in light of the cooperative kinetics demonstrated by ACH2-6His in vitro. The cooperative effects are most pronounced with medium chain acyl-CoAs, where the Hill coefficient is 3.8 for lauroyl-CoA, but decrease for long chain acyl-CoAs, where the Hill coefficient is only 1.9 for oleoyl-CoA. ACH2-6His hydrolyzes both medium and long chain fatty acyl-CoAs but has highest activity toward the long chain unsaturated fatty acyl-CoAs. Maximum rates were found with palmitoleoyl-CoA, which is hydrolyzed at 21 micromol/min/mg protein. Additionally, ACH2-6His is insensitive to feedback inhibition by free CoASH levels as high as 100 microm. ACH2 is most highly expressed in mature tissues such as young leaves and flowers rather than in germinating seedlings where beta-oxidation is rapidly proceeding. Taken together, these results suggest that ACH2 activity is not linked to fatty acid oxidation as has been suggested for its eukaryotic homologs, but rather has a unique role in the plant cell.

Amino Acid Sequence↗