Developmental induction, purification, and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbellularia californica

Hm, Davies; Anderson, L; Chen, Fan; Dj, Hawkins

doi:10.1016/0003-9861(91)90588-a

Cited by 69 publications

(41 citation statements)

References 12 publications

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“…There are several published studies of acyl-ACP thioesterases from a variety of plant species (7,8,12,17,18,24), and recently the purification and cloning of a 12:0-ACP thioesterase from California bay has been reported (6,30). We used as a starting point for our purification the protocol described by McKeon and Stumpf (12), but found that in our laboratory it did not yield the degree of purity reported by those authors.…”

Section: Discussionmentioning

confidence: 96%

“…Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) nia bay (Umbellularia californica), which accumulates high levels of lauric acid (12:0) in its seed triacylglycerols. Recently, a 12:0-ACP-specific thioesterase has been isolated from this species and shown to redirect fatty acid synthesis to the production of laurate in transgenic plants (6,18,30). In plants that accumulate the longer-chain fatty acids, the specificity of the acyl-ACP thioesterase may also play a role in determining the final fatty acid composition of the storage oil.…”

Section: Introductionmentioning

confidence: 99%

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Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Knutzon¹,

Bleibaum²,

Nelsen³

et al. 1992

Plant Physiol.

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Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34-and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50-to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16-and 18-carbon acyl-ACP substrates.Fatty acids are important components of membrane phospholipids in all plant tissues and are also stored in the form of triacylglycerols in the seeds of many species for use as an energy source upon germination. Fatty acid biosynthesis in plants is catalyzed by a series of soluble, discrete enzymes (23) that are localized in the chloroplasts of leaves (16) nia bay (Umbellularia californica), which accumulates high levels of lauric acid (12:0) in its seed triacylglycerols. Recently, a 12:0-ACP-specific thioesterase has been isolated from this species and shown to redirect fatty acid synthesis to the production of laurate in transgenic plants (6,18,30). In plants that accumulate the longer-chain fatty acids, the specificity of the acyl-ACP thioesterase may also play a role in determining the final fatty acid composition of the storage oil.We are interested in studying acyl-ACP thioesterases with specificities for 16-and 18-carbon fatty acids. Acyl-ACP thioesterase activities have been characterized at various stages of purification from a number of plant species that accumulate 16-and 18-carbon fatty acids in their triacylglycerols, including avocado (17, 24), safflower (Carthamus tinctorius) (12), squash (8), and rapeseed (7). The enzymes show distinct preferences for 18:1-ACP over 18:0-ACP and 16:0-ACP substrates and are relatively inactive on acyl-CoAs.To investigate further the role of acyl-ACP thioesterase in the determination of seed oil composition, we have purified and obtained partial amino acid sequence of an 18:1-ACP thioesterase from developing safflower seeds. Two different classes of cDNA clones encoding 18:1-ACP thioesterase have been isolated and characterized. The substrate specificity of the protein encoded by each clone has been examined by ...

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Knutzon¹,

Bleibaum²,

Nelsen³

et al. 1992

Plant Physiol.

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“…We previously isolated a 12:O-ACP thioesterase and obtained a cDNA from 12:O-producing seed of California bay trees (Umbe//u/aria californica, Lauraceae; Davies et al, 1991;Voelker et al, 1992). When expressed in developing oil seed of Arabidopsis, the bay enzyme redirects the resident long-chain fatty acid synthase to 12:O production.…”

Section: Introductionmentioning

confidence: 99%

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

Jones¹,

Davies²,

Voelker³

1995

Plant Cell

284

258

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“…These authors were able to separate the activity of a long-chain TE from a mediumchain TE, suggesting that there is medium-chain TE in-volved in oil synthesis. Subsequent purification of the 12:O-ACP TE from U. californica by Davies et al (1991) led to the cloning of a TE cDNA ( U c FatBZ), which was expressed in seeds of Arabidopsis and Brassica (Voelker et al, 1992).…”

mentioning

confidence: 99%

Two Novel Thioesterases Are Key Determinants of the Bimodal Distribution of Acyl Chain Length of Cuphea palustris Seed Oil

Dehesh¹,

Edwards²,

Hayes³

et al. 1996

Plant Physiol.

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l h e seed oil of Cuphea palusfris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatBl and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0-and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:O in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:O and 14:o. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.The end products of plant fatty acid synthetase activities are usually 16-and 18-carbon fatty acids (Harwood, 1988). There are, however, severa1 plant families that store large amounts of 8-to 14-carbon (medium-chain) fatty acids in their oilseed. Several mechanisms have been proposed for the synthesis of medium-chain fatty acids in plants. A specific acyl-ACP TE might terminate fatty acid synthesis by hydrolyzing the thioester bond of a particular acyl-ACP, resulting in the release of both ACP and free fatty acid (Stumpf, 1987). Alternatively, a specific 3-ketoacyl-ACP synthase (condensing enzyme) or an acyl-ACP acyltransferase might be involved (Harwood, 1988). At the time these mechanisms were proposed, however, there were limited experimental data to support these hypotheses. More recently, studies with Umbellularia californica (California bay), a plant that produces seed oil rich in lauric acid, have demonstrated the existence of a medium-chain-specific isozyme of acyl-ACP TE in the seed plastids (Pollard et al., 1991). These authors were able to separate the activity of a long-chain TE from a mediumchain TE, suggesting that there is medium-chain TE in-

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Developmental induction, purification, and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbellularia californica

Cited by 69 publications

References 12 publications

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

Two Novel Thioesterases Are Key Determinants of the Bimodal Distribution of Acyl Chain Length of Cuphea palustris Seed Oil

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