Expression of Lauroyl–Acyl Carrier Protein Thioesterase in <i>Brassica napus</i> Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation

Eccleston, Victoria S.; Ohlrogge, John B.

doi:10.1105/tpc.10.4.613

Cited by 119 publications

(64 citation statements)

References 26 publications

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“…The obligatory induction of b-oxidation and the glyoxylate cycle enzyme activities in leaves of these plants was a reasonable suggestion since levels in non-fatty tissues are much lower than in germinating seeds (Gerhardt 1983) and may not be capable of eciently degrading a large amount of medium-chain fatty acid. The recent report that MCOX activity was speci®cally induced in leaves of MCTE-expressing plants appeared to substantiate this conclusion (Eccleston and Ohlrogge 1998). The failure to accumulate an unusual fatty acid in leaves has also been observed for Arabidopsis plants engineered to produce ricinoleic acid (Broun et al 1998).…”

Section: Discussionmentioning

confidence: 87%

“…The third explanation is that a post-transcriptional mechanism could result in higher levels of speci®c enzymes from pre-existing transcripts. This has been suggested to explain the increase in protein levels of fatty acid synthesis enzymes without a parallel increase in transcript levels in developing seeds of the transgenic B. napus producing lauric acid (Eccleston and Ohlrogge 1998). Unfortunately, in that study it was not possible to make the same conclusions for the fatty-acid-cataboliz ing enzymes.…”

Section: Discussionmentioning

confidence: 91%

“…Since ICL activity is not normally detectable in green leaves, its presence in leaves expressing the MCTE was taken as evidence that fatty acid catabolic processes were induced in response to the production of laurate. Recently, it has been reported that acyl-CoA oxidase (ACOX) activity, which is speci®c for medium-chain fatty acyl-CoAs (Hooks et al 1996) is induced in leaves, whereas ACOX activity with longchain fatty acyl-CoAs, such as palmitoyl-CoA, is not (Eccleston and Ohlrogge 1998).…”

Section: Introductionmentioning

confidence: 99%

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No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

Hooks

Fleming

Larson

et al. 1999

Planta

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Leaves from transgenic Brassica napus L. plants engineered to produce lauric acid show increased levels of enzyme activities of the pathways associated with fatty acid catabolism (V.A. Eccleston and J.B. Ohlrogge, 1998, Plant Cell 10: 613-621). In order to determine if the increases in enzyme activity are mirrored by increases in the expression of genes encoding enzymes of beta-oxidation, which is the major pathway of fatty acid catabolism in plants, the medium-chain acyl-acyl carrier protein (ACP) thioesterase MCTE from California bay (Umbellularia california) was over-expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana (L.) Heynh. Arabidopsis was the most suitable choice for these studies since gene expression could be analyzed in a large number of independent MCTE-expressing lines using already well-characterized beta-oxidation genes. Levels of MCTE transcripts in leaves varied widely over the population of plants analyzed. Furthermore, active MCTE was produced as determined by enzymatic analysis of leaf extracts of MCTE-expressing plants. These plants incorporated laurate into triacylglycerol of seeds, but not into lipids of leaves as shown by gaschromatographic analysis of total fatty acid extracts. The expression levels of the beta-oxidation and other genes that are highly expressed during developmental stages involving rapid fatty acid degradation were measured. No significant difference in gene expression was observed among MCTE-expressing plants and transgenic and non-transgenic controls. To eliminate the possibility that post-translational mechanisms are responsible for the observed increases in enzyme activity acyl-CoA oxidase activity was also measured in leaves of MCTE-expressing plants using medium and long chain acyl-CoA substrates. No significant increases in either medium- or long-chain acyl-CoA oxidase activities were detected. We conclude that endogenous beta-oxidation is sufficient to account for the complete degradation of laurate produced in rosette leaves of Arabidopsis expressing MCTE.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

Hooks

Fleming

Larson

et al. 1999

Planta

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“…Total root tissue was collected from plants grown for 6 weeks in sealed tissue culture boxes containing 50 mL of growth media (1ϫ Murashige and Skoog salts, 1ϫ B vitamins, and 0.5% [w/v] agarose). Oilseed rape (Brassica napus cv 212/ 86, line 18) was grown in a green house (Eccleston and Ohlrogge, 1998). Seeds were collected from oilseed rape siliques 25 to 30 d after flowering and leaves were collected from the same plants of the same age.…”

Section: Plant Material Rna Extraction and Probe Synthesismentioning

confidence: 99%

Microarray Analysis of Developing Arabidopsis Seeds

Girke¹,

Todd²,

Ruuska³

et al. 2000

Plant Physiology

317

202

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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 Ͼ10,000 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 Ͼ70% 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 Ն 2-fold higher in seeds than leaves or roots and 10% at ratios Ն 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.

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“…We previously observed that high lauric acid production in transgenic canola induces b-oxidation pathways for lauric acid breakdown (Eccleston and Ohlrogge 1998). Thus, low levels of unusual monoene accumulation could be due to the breakdown of products from the introduced desaturases.…”

Section: Discussionmentioning

confidence: 98%

What limits production of unusual monoenoic fatty acids in transgenic plants?

Suh

Schultz

Ohlrogge

2002

Planta

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Unusual monounsaturated fatty acids are major constituents (greater than 80%) in seeds of Coriandrum sativum L. (coriander) and Thunbergia alata Bojer, as well as in glandular trichomes (greater than 80% derived products) of Pelargonium x hortorum (geranium). These diverged fatty acid structures are produced via distinct plastidial acyl-acyl carrier protein (ACP) desaturases. When expressed in Arabidopsis thaliana (L.) Heynh. under strong seed-specific promoters the unusual acyl-ACP desaturases resulted in accumulation of unusual monoene fatty acids at 1-15% of seed fatty acid mass. In this study, we have examined several factors that potentially limit higher production of unusual monoenes in transgenic oilseeds. (i) Immunoblots indicated that the introduced desaturases were expressed at levels equivalent to or higher than the endogenous delta9 18:0-ACP desaturase. However, the level of unusual fatty acid produced in transgenic plants was not correlated with the level of desaturase expression. (ii) The unusual desaturases were expressed in several backgrounds, including antisense 18:0-ACP desaturase plants, in fab1 mutants, and co-expressed with specialized ACP or ferredoxin isoforms. None of these experiments led to high production of expected products. (iii) No evidence was found for degradation of the unusual fatty acids during seed development. (iv) Petroselinic acid added to developing seeds was incorporated into triacylglycerol as readily as oleic acid, suggesting no major barriers to its metabolism by enzymes of glycerolipid assembly. (v) In vitro and in situ assay of acyl-ACP desaturases revealed a large discrepancy of activity when comparing unusual acyl-ACP desaturases with the endogenous delta9 18:0-ACP desaturase. The combined results, coupled with the sensitivity of acyl-ACP desaturase activity to centrifugation and low salt or detergent suggests low production of unusual monoenes in transgenic plants may be due to the lack of, or incorrect assemble of, a necessary multi-component enzyme association.

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Expression of Lauroyl–Acyl Carrier Protein Thioesterase in Brassica napus Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation

Cited by 119 publications

References 26 publications

No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

Microarray Analysis of Developing Arabidopsis Seeds

What limits production of unusual monoenoic fatty acids in transgenic plants?

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