Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes

González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; Wettstein-Knowles, Penny von; Martı́nez-Force, Enrique

doi:10.1007/s00425-014-2162-7

Cited by 18 publications

(26 citation statements)

References 48 publications

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“…Sunflower (Helianthus annuus L.) wild-type line CAS-6 (Sunflower Collection of Instituto de la Grasa, CSIC, Seville, Spain) was grown and materials collected as described by González-Thuillier et al ( 2015 ).…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

“…The filter was probed with [α-P]dCTP-labelled HaHAD1 and HaHAD2 gene-specific DNA probes, 684 bp and 666 bp, respectively, obtained by PCR amplification with the following pairs of primers: F-atg_HAD1/R-stop_HAD1 for HaHAD1 and F-atg_HAD2/R-stop_HAD2 for HaHAD2 (Table 1 ). Hybridization was performed as detailed previously (González-Thuillier et al 2015 ). Images of radioactive filters were obtained as described by González-Mellado et al ( 2010 ).…”

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

“…The cDNAs obtained from developing seeds and different vegetative tissues as described in González-Thuillier et al 2015were subjected to RT-qPCR with the following gene-specific pair of primers: F-HAD1qpcr/R-HAD1qpcr for HaHAD1 and F-HAD2qpcr/R-HAD2qpcr for HaHAD2 (Table 1 ) using SYBR Green (QuantiteTect™ SYBR Green PCR Kit, Qiagen, Hilden, DE) in an MiniOpticon system (Bio-Rad, Hercules, CA, USA). The reaction was run, a calibration curve constructed and the sunflower actin gene HaACT1 (GenBank Accession number FJ487620) used as the internal reference as detailed previously (González-Thuillier et al 2015 ). Three biological and two technical replicates were carried out on each sample.…”

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

“…Recombinant proteins were submitted to SDS-polyacrylamide gel electrophoresis (PAGE) and the gels stained with Coomassie blue as detailed previously (González-Thuillier et al 2015 ).…”

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

“…For each enzymatic assay three independent experiments were done with each of the three purified proteins. Kinetic parameter analysis was carried out as described in González-Thuillier et al ( 2015 ).…”

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confidence: 99%

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Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes

González-Thuillier

Venegas-Calerón

Sánchez

et al. 2015

Planta

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Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

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Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

“…Recombinant proteins were submitted to SDS-polyacrylamide gel electrophoresis (PAGE) and the gels stained with Coomassie blue as detailed previously (González-Thuillier et al 2015 ).…”

Section: Modelling Of the Three-dimensional Structures Of Hahad1 And mentioning

confidence: 99%

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confidence: 99%

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Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes

González-Thuillier

Venegas-Calerón

Sánchez

et al. 2015

Planta

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Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities

Aznar-Moreno

Venegas-Calerón

Martı́nez-Force

et al. 2016

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The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

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Identification and integrated analysis of mRNAs, lncRNAs, and microRNAs of developing seeds in high oleic acid sunflower (Helianthus annuus L.)

et al. 2021

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Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes

Abstract: 18A major factor contributing to the amount of fatty acids in plant oils is the first

Cited by 18 publications

References 48 publications

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities

Identification and integrated analysis of mRNAs, lncRNAs, and microRNAs of developing seeds in high oleic acid sunflower (Helianthus annuus L.)

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