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

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Abstract

Main conclusion

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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Abbreviations

ACP:

Acyl carrier protein

DAF:

Days after flowering

DAG:

Days after germination

ENR:

Enoyl-ACP reductase

FAS:

Fatty acid synthase

HAD:

β-Hydroxyacyl-ACP dehydratase

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Acknowledgments

We thank Dr. Alicia M. Muro-Pastor for help with Southern blot analysis and A. González-Callejas and B. Lopez-Cordero for skilful technical assistance. This work was supported by the “Ministerio de Economia y Competitividad” and FEDER project, AGL2011-23187. IGT was supported by a JAE-CSIC contract, in part financed by the European Social Fund.

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Author notes

  1. Irene González-Thuillier

    Present address: Department of Plant Biology and Crop Science, Rothamsted Research, Harpenden, AL5 2JQ, Herts, UK

Authors and Affiliations

  1. Instituto de la Grasa (CSIC), Edificio 46, Campus Universitario Pablo de Olavide, Carretera de Utrera Km 1., 41013, Seville, Spain

    Irene González-Thuillier, Mónica Venegas-Calerón, Rosario Sánchez, Rafael Garcés & Enrique Martínez-Force

  2. Biology Department, Copenhagen University, Ole Maaloees Vej 5, 2200, Copenhagen N, Denmark

    Penny von Wettstein-Knowles

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  1. Irene González-Thuillier
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Correspondence to Mónica Venegas-Calerón.

Electronic supplementary material

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425_2015_2410_MOESM1_ESM.tif

Fig. S1 Phylogenetic comparison of plant and algae β-hydroxyacyl-[ACP]- dehydratase homologues of the E. coli FabZ protein. H. annuus sequences are shown in bold (TIFF 59094 kb)

425_2015_2410_MOESM2_ESM.tif

Fig. S2 Purification of H. annuus recombinant HAD proteins. Coomassie blue-stained SDS-PAGE of empty pQE80L vector, recombinant HaHAD1, HaHAD2 and HaHAD F166V from XL1-Bl grown at 37 °C. S, soluble fraction; AF, affinity purified using one passage through a Ni2+-charged HisTrap FF column; kDa, size markers (TIFF 781 kb)

425_2015_2410_MOESM3_ESM.tif

Fig. S3 Activity of H. annuus β-hydroxyacyl-[ACP]-dehydratase proteins, HaHAD1, HaHAD2 and HaHAD1. The in vitro reverse reactions were performed in 17.9 mM potassium phosphate buffer, 1.6 mM DTT at pH 6.8, 25 °C with HaHAD1 (5 μg ml−1), HaHAD2 (5 μg ml−1) and HaHAD1 F166V (5 μg ml−1). The reaction was started by adding the substrate (crotonyl-CoA) at different concentrations (20-100 μM) and following the decrease in absorbance at 260 nm as crotonyl-CoA was hydrated. Results correspond to the average of three independent determinations (TIFF 31640 kb)

425_2015_2410_MOESM4_ESM.tif

Fig. S4 Schematic representation of E. coli fatty acid biosynthesis comparing the effect on the ratio of saturated to unsaturated fatty acids expressing H. annuus β-hydroxyacyl-[ACP]-dehydratases, HaHAD1 or HaHAD2 with functional EcFabZ. Proteins involved in fatty acid biosynthesis in E. coli are represented. FabZ, β-hydroxyacyl-[ACP]-dehydratase Z; FabA, β-hydroxydecanoyl-[ACP]-dehydratase A; FabB, β-ketoacyl-[ACP]-synthase I; FabI, enoyl-[ACP]-reductase. Symbols in grey define the bacterial pathway; in black represent the effect of expression of H. annuus β-hydroxyacyl-[ACP]-dehydratases in the E. coli cells (TIFF 1684 kb)

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González-Thuillier, I., Venegas-Calerón, M., Sánchez, R. et al. Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes. Planta 243, 397–410 (2016). https://doi.org/10.1007/s00425-015-2410-5

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