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J Ohlrogge

Publications and source records attributed to J Ohlrogge.

18 recordsLinked to original sources
PubMed2001

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2000

Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue.

The principal supply of carbon precursors for fatty acid synthesis in leaf tissue has been a much debated topic, with some experiments suggesting a direct supply from the C3 products of photosynthetic carbon fixation and colleagues suggesting the utilization of free acetate (for which concentrations in leaves in the range of 0.05-1.4 mM have been reported). To address this issue we first reassessed the in vivo rate of fatty acid synthesis using a new method, that of [13C]carbon dioxide labeling of intact Arabidopsis plants with the subsequent analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS). This method gave an average value of 2.3 mmoles carbon atoms h-1 mg chlorophyll-1 for photosynthetic tissues. The method was extended by isotopic dilution analysis to measure the rate of fatty acid synthesis in the dark. There was negligible fatty acid synthesis (< 5% of the rate in the light) in the dark. In addition, the method allowed an estimate of the absolute rate of fatty acid degradation of about 4% of the total fatty acid content per day. With the in vivo rate of fatty acid synthesis in the light defined, if the bulk tissue acetate concentration available for fatty acid synthesis is 1 mM, this acetate pool can sustain fatty acid synthesis for approximately 60 min. When the leaves of Arabidopsis, barley and pea were given a 5 min pulse of [14C]carbon dioxide, the label rapidly appeared in fatty acids with a lag phase of less than 2-3 min. Continuous labeling with [14C]carbon dioxide, for up to 1 h, showed a similar result. Furthermore, 14C-label in free acetate was less than 5% of that in fatty acids. In conclusion, these data suggest that either the bulk pool of acetate is not involved in fatty acid synthesis or the concentration of acetate must be less than 0.05 mM under strong illumination.

Acetic Acid↗
PubMed2000

Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis.

Public databases now include vast amounts of recently acquired DNA sequences that are only partially annotated and, furthermore, are often annotated by automated methods that are subject to errors. Maximum information value of these databases can be derived only by further detailed analyses that frequently require careful examination of records in the context of biological functions. In this study we present an example of such an analysis focused on plant glycerolipid synthesis. Public databases were searched for sequences corresponding to 65 plant polypeptides involved in lipid metabolism. Comprehensive search results and analysis of genes, cDNAs and expressed sequence tags (ESTs) are available online (http://www.canr.msu.edu/lgc). Multiple alignments provided a method to estimate the number of genes in gene families. Further analysis of sequences allowed us to tentatively identify several previously undescribed genes in Arabidopsis. For example, two genomic sequences were identified as candidates for the palmitate-specific monogalactosyldiacylglycerol desaturase (FAD5). A candidate genomic sequence for 3-ketoacyl-acyl-carrier protein (ACP) synthase involved in mitochondrial fatty acid biosynthesis was also identified. Biotin carboxyl carrier protein (BCCP) in Arabidopsis is encoded by at least two genes, but the most abundant BCCP transcript so far has not been characterized. The large number (>165,000) of plant ESTs also provides an opportunity to perform "digital northern" comparisons of gene expression levels across many genes. EST abundance in general correlated with biochemical and flux characteristics of the enzymes in Arabidopsis leaf tissue. In a few cases, statistically significant differences in EST abundance levels were observed for enzymes that catalyze similar reactions in fatty acid metabolism. For example, ESTs for the FatB acyl-ACP thioesterase occur 21 times compared with 7 times for FatA acyl-ACP thioesterase, although flux through the FatA reaction is several times higher than through FatB. Such comparisons may provide initial clues toward previously undescribed regulatory phenomena. The abundance of ESTs for ACP compared with that of stearoyl-ACP desaturase and FatB acyl-ACP thioesterase suggests that concentrations of some enzymes of fatty acid synthesis may be higher than their acyl-ACP substrates.

Databases, Factual↗
PubMed2000

Microarray analysis of developing Arabidopsis seeds.

To provide a broad analysis of gene expression in developing Arabidopsis seeds, microarrays have been produced that display approximately 2,600 seed-expressed genes. DNA for genes spotted on the arrays were selected from >10,000 clones partially sequenced from a cDNA library of developing seeds. Based on a series of controls, sensitivity of the arrays was estimated at one to two copies of mRNA per cell and cross hybridization was estimated to occur if closely related genes have >70% to 80% sequence identity. These arrays have been hybridized in a series of experiments with probes derived from seeds, leaves, and roots of Arabidopsis. Analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approximately 25% of the 2, 600 genes were expressed at ratios > or =2-fold higher in seeds than leaves or roots and 10% at ratios > or =10. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases, and transcription factors. The Arabidopsis arrays were also found to be useful for transcriptional profiling of mRNA isolated from developing oilseed rape (Brassica napus) seeds and expression patterns correlated well between the two species.

Arabidopsis↗
PubMed2000

Unraveling plant metabolism by EST analysis.

Large-scale single-pass sequencing of cDNAs prepared from specific plant species or tissues has evolved as an inexpensive and efficient gene-discovery tool that can be used to identify novel cDNAs encoding enzymes of specific plant metabolic pathways. Collections of expressed sequence tags from metabolically active tissues can provide quantitative estimates of gene expression levels and thus are being exploited to unravel plant metabolic and regulatory networks.

Expressed Sequence Tags↗
PubMed2000

Fatty acid synthesis: from CO2 to functional genomics.

For over 25 years there has been uncertainty over the pathway from CO(2) to acetyl-CoA in chloroplasts. On the one hand, free acetate is the most effective substrate for fatty acid synthesis by isolated chloroplasts, and free acetate concentrations reported in leaf tissue (0.1-1 mM) appear adequate to saturate fatty acid synthase. On the other hand, a clear mechanism to generate sufficient free acetate for fatty acid synthesis is not established and direct production of acetyl-CoA from pyruvate by a plastid pyruvate dehydrogenase seems a more simple and direct path. We have re-examined this question and attempted to distinguish between the alternatives. The kinetics of (13)CO(2) and (14)CO(2) movement into fatty acids and the absolute rate of fatty acid synthesis in leaves was determined in light and dark. Because administered (14)C appears in fatty acids within < 2-3 min our results are inconsistent with a large pool of free acetate as an intermediate in leaf fatty acid synthesis. In addition, these studies provide an estimate of the turnover rate of fatty acid in leaves. Studies similar to the above are more complex in seeds, and some questions about the regulation of plant lipid metabolism seem difficult to solve using conventional biochemical or molecular approaches. For example, we have little understanding of why or how some seeds produce >50% oil whereas other seeds store largely carbohydrate or protein. Major control over complex plant biochemical pathways may only become possible by understanding regulatory networks which provide 'global' control over these pathways. To begin to discover such networks and provide a broad analysis of gene expression in developing oilseeds, we have produced microarrays that display approx. 5000 seed-expressed Arabidopsis genes. Sensitivity of the arrays was 1-2 copies of mRNA/cell. The arrays have been hybridized with probes derived from seeds, leaves and roots, and analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approx. 10% of the genes were expressed at ratios > or = 10-fold higher in seeds than in leaves or roots. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases and transcription factors. The arrays were also found to be useful for analysis of Brassica seeds.

Acetyl Coenzyme A↗
PubMed1999

Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos

The metabolic factors that determine oil yield in seeds are still not well understood. To begin to examine the limits on triacylglycerol (TAG) production, developing Cuphea lanceolata, Ulmus carpinifolia, and Ulmus parvifolia embryos were incubated with factors whose availability might limit oil accumulation. The addition of glycerol or sucrose did not significantly influence the rate of TAG synthesis. However, the rate of (14)C-TAG synthesis upon addition of 2.1 mM (14)C-decanoic acid (10:0) was approximately four times higher than the in vivo rate of TAG accumulation in C. lanceolata and two times higher than the in vivo rate in U. carpinifolia and U. parvifolia. In C. lanceolata embryos, the highest rate of (14)C-TAG synthesis (14.3 nmol h(-1) embryo(-1)) was achieved with the addition of 3.6 mM decanoic acid. (14)C-Decanoic acid was incorporated equally well in all three acyl positions of TAG. The results suggest that C. lanceolata, U. carpinifolia, and U. parvifolia embryos have sufficient acyltransferase activities and glycerol-3-phosphate levels to support rates of TAG synthesis in excess of those found in vivo. Consequently, the amount of TAG synthesized in these oilseeds may be in part determined by the amount of fatty acid produced in plastids.

Journal Article↗
PubMed1999

Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling.

Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [(13)C(2)(18)O(2)]Acetate was incubated with spinach (Spinacia oleracea) leaves and the (18)O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an (18)O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the (18)O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of (18)O or, less likely, complete loss of (18)O, but not a 50% loss of (18)O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of "prokaryotic" and "eukaryotic" lipids have both been confirmed.

Acetates↗
PubMed1998

The biosynthesis of erucic acid in developing embryos of brassica rapa

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.

Journal Article↗
PubMed1997

Why do mitochondria synthesize fatty acids? Evidence for involvement in lipoic acid production.

The function of acyl carrier protein (ACP) in mitochondria isolated from pea leaves has been investigated. When pea leaf mitochondria were labeled with [2-14C] malonic acid in vitro, radioactivity was incorporated into fatty acids, and, simultaneously, ACP was acylated. [1-14C]Acetate was much less effective as a precursor for fatty acid synthesis, suggesting that mitochondria do not possess acetyl-CoA carboxylase. The incorporation of radioactivity from [2-14C]malonate into fatty acids and the labeling of ACP were inhibited by cerulenin and required ATP and Mg2+. These findings indicate that plant mitochondria contain not only ACP, but all enzymes required for de novo fatty acid synthesis. Over 30% of the radioactive products from pea mitochondria labeled with [2-14C]malonate were recovered in H protein, which is a subunit of glycine decarboxylase and contains lipoic acid as an essential constituent. In similar experiments, the H protein of Neurospora mitochondria was also labeled by [2-14C]malonate. The labeling of pea H protein was inhibited by addition of cerulenin into the assay medium. Together, these findings indicate that ACP is involved in the de novo synthesis of fatty acids in plant mitochondria and that a major function of this pathway is production of lipoic acid precursors.

Acyl Carrier Protein↗
PubMed1997

Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds.

Acetyl-coenzyme A carboxylase (ACCase) occurs in at least two forms in rapeseed (Brassica napus): a homomeric (HO) and presumably cytosolic isozyme and a heteromeric, plastidial isozyme. We investigated whether the HO-ACCase of Arabidopsis can be targeted to plastids of B. napus seeds. A chloroplast transit peptide and the napin promoter were fused to the Arabidopsis ACC1 gene and transformed into B. napus, with the following results. (a) The small subunit transit peptide was sufficient to provide import of this very large protein into developing seed plastids. (b) HO-ACCase in isolated plastids was found to be biotinylated at a level comparable to extraplastidial HO-ACCase. (c) In vitro assays of HO-ACCase in isolated plastids from developing seeds indicate that it occurs as an enzymatically active form in the plastidial compartment. (d) ACCase activity in mature B. napus seeds is normally very low; however, plants expressing the SSU/ACC1 gene had 10- to 20-fold higher ACCase activity in mature seeds, suggesting that plastid localization prevents the turnover of HO-ACCase. (e) ACCase over-expression altered seed fatty acid composition, with the largest effect being an increase approximately 5% by the expression of HO-ACCase in plastids.

Acetyl-CoA Carboxylase↗
PubMed1997

Antisense expression and overexpression of biotin carboxylase in tobacco leaves.

The plastid acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis and in most plants is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase (BC), the biotin carboxyl carrier protein, and the alpha and beta subunits of the carboxyltransferase. To gain insight into the subunit organization of this heteromeric enzyme complex and to further evaluate the role of ACCase in regulating fatty acid synthesis, BC expression was altered in transgenic plants. Tobacco (Nicotiana tabacum) was transformed with antisense-expression and overexpression tobacco BC constructs, which resulted in the generation of plants with BC levels ranging from 20 to 500% of wild-type levels. Tobacco plants containing elevated or moderate decreases in leaf BC were phenotypically indistinguishable from wild-type plants. However, plants with less than 25% of wild-type BC levels showed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid content than wild-type plants. A comparison of leaf BC and biotin carboxyl carrier protein levels in plants with elevated and decreased BC expression revealed that these two subunits of the plastid ACCase are not maintained in a strict stoichiometric ratio.

Acetyl-CoA Carboxylase↗
PubMed1995

Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase.

Expression of a plant lauroyl-acyl carrier protein (ACP) thioesterase in an Escherichia coli strain deficient in beta oxidation results in the accumulation of free fatty acids in the culture. Overall fatty acid production by the cultures is increased severalfold, particularly in the late log and stationary stages of growth. In control E. coli cells, malonyl-ACP levels and rates of fatty acid synthesis are highest during rapid logarithmic growth and decline to undetectable levels in stationary stage. In contrast, in cells expressing plant acyl-ACP thioesterase, malonyl-ACP levels remain high in late log and stationary stage in association with the continued fatty acid production. In addition, the biotin carboxyl carrier protein component of acetyl-CoA carboxylase is expressed at higher levels in cultures expressing the acyl-ACP thioesterase. The data presented indicate that removal of the acyl-ACP products of fatty acid synthesis results in increased production of both malonyl-ACP and fatty acids, which may in turn result from higher activity and/or expression of acetyl-CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1995

Lipid biosynthesis.

Explore the source record for details and available documents.

Acetyl-CoA Carboxylase↗
PubMed1994

Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.

High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequences similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing project is provided.

Amino Acid Sequence↗