Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ACP:

Acyl carrier protein

DAF:

Days after flowering

DAG:

Diacylglycerol

FAS:

Fatty acid synthases

PBS:

Phosphate-buffered saline

TAG:

Triacylglycerol

References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Battey JF, Ohlrogge JB (1990) Evolutionary and tissue-specific control of expression of multiple acyl-carrier protein isoforms in plants and bacteria. Planta 180:352–360

    Article  CAS  PubMed  Google Scholar 

  • Beisson F, Koo AJK, Ruuska S, Schwender J, Pollard MR, Thelen JJ, Paddock T, Salas JJ, Savage L, Milcamps A, Mhaske VB, Cho Y, Ohlrogge JB (2003) Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol 132:681–697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucl Acids Res 28:235–242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB (2003) Disruption of the FATB gene in arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell 15:1020–1033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB (2006) The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase. Plant Cell 18:3576–3593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dörmann P, Voelker VA, Ohlrogge JB (1995) Cloning and expression in Escherichia coli of a novel thioesterase from arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins. Arch Biochem Bioph 316:612–618

    Article  Google Scholar 

  • Dörmann P, Voelker TA, Ohlrogge JB (2000) Accumulation of palmitate in arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1. Plant Physiol 123:637–644

    Article  PubMed  PubMed Central  Google Scholar 

  • Ghosh SK, Bhattacharjee A, Jha JK, Mondal AK, Maiti MK, Basu A, Ghosh D, Ghosh S, Sen SK (2007) Characterization and cloning of a stearoyl/oleoyl specific fatty acyl–acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia). Plant Physiol Biochem 45:887–897

    Article  CAS  PubMed  Google Scholar 

  • González-Thuillier I, Venegas-Calerón M, Garcés R, Wettstein-Knowles P, Martínez-Force E (2015) Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-(acyl carrier protein)-reductase genes. Planta 241:43–56

    Article  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modelling. Electrophoresis 18:2714–2723

    Article  CAS  PubMed  Google Scholar 

  • Hawkins DJ, Kridl JC (1998) Characterization of acyl-ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola. Plant J 13:743–752

    Article  CAS  PubMed  Google Scholar 

  • Hiltunen JK, Schonauer MS, Autio KJ, Mittelmeier TM, Kastaniotis AJ, Dieckmann CL (2009) Mitochondrial fatty acid synthesis type II: more than just fatty acids. J Biol Chem 284:9011–9015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Holak TA, Kearsley SK, Kim Y, Prestegard JH (1988) Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations. Biochemistry 27:6135–6142

    Article  CAS  PubMed  Google Scholar 

  • Jones A, Davies HM, Voelker TA (1995) Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases. Plant Cell 7:359–371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim Y, Kovrigin EL, Eletr Z (2006) NMR studies of Escherichia coli acyl carrier protein: dynamic and structural differences of the apo- and holo-forms. Biochem Bioph Res Co 341:776–783

    Article  CAS  Google Scholar 

  • Kuo TM, Ohlrogge JB (1984) Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli. Arch Biochem Bioph 230:110–116

    Article  CAS  Google Scholar 

  • Lambalot RH, Walsh CT (1995) Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem 270:24658–24661

    Article  CAS  PubMed  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Li Q, Khosla C, Puglisi JD, Liu CW (2003) Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein. Biochemistry 42:4648–4657

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Moreno-Pérez AJ, Sánchez-García A, Salas JJ, Garcés R, Martínez-Force E (2011) Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition. Plant Physiol Biochem 49:82–87

    Article  PubMed  Google Scholar 

  • Moreno-Pérez AJ, Venegas-Calerón M, Vaistij FE, Salas JJ, Larson TR, Garcés R, Graham IA, Martínez-Force E (2012) Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds. Planta 235:629–639

    Article  PubMed  Google Scholar 

  • Ohlrogge JB, Jaworski JG (1997) Regulation of fatty acid synthesis. Annu Rev Plant Biol 48:109–136

    Article  CAS  Google Scholar 

  • Pérez-Vich B, Garcés R, Fernández-Martínez JM (1999) Genetic control of high stearic acid content in the seed oil of the sunflower mutant CAS-3. Theor Appl Genet 99:663–669

    Article  PubMed  Google Scholar 

  • Pleite R, Martínez-Force E, Garcés R (2006) Increase of the stearic acid content in high-oleic sunflower (Helianthus annuus) Seeds. J Agric Food Chem 54:9383–9388

    Article  CAS  PubMed  Google Scholar 

  • Pollard MR, Anderson L, Fan C, Hawkins DJ, Davies MH (1991) A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica. Arch Biochem Bioph 284:306–312

    Article  CAS  Google Scholar 

  • Rawlings M, Cronan JE (1992) The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes. J Biol Chem 267:5751–5754

    CAS  PubMed  Google Scholar 

  • Rock CO, Garwin JL (1979) Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J Biol Chem 254:7123–7128

    CAS  PubMed  Google Scholar 

  • Salas JJ, Ohlrogge JB (2002) Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Arch Biochem Biophys 403:25–34

    Article  CAS  PubMed  Google Scholar 

  • Sánchez-García A, Moreno-Pérez AJ, Muro-Pastor AM, Salas JJ, Garcés R, Martínez-Force E (2010) Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation. Phytochemistry 71:860–869

    Article  PubMed  Google Scholar 

  • Serrano-Vega MJ, Venegas-Calerón M, Garcés R, Martínez-Force E (2003) Cloning and expression of fatty acids biosynthesis key enzymes from sunflower (Helianthus annuus L.) in Escherichia coli. J Chromatog B 786:221–228

    Article  CAS  Google Scholar 

  • Serrano-Vega MJ, Garcés R, Martínez-Force E (2005) Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.). Planta 221:868–880

    Article  CAS  PubMed  Google Scholar 

  • Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40

    Article  CAS  PubMed  Google Scholar 

  • Suh MC, Schultz DJ, Ohlrogge JB (1999) Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis. Plant J 17:679–688

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thelen JJ, Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng 4:12–21

    Article  CAS  PubMed  Google Scholar 

  • Voelker T (1996) Plant Acyl-ACP Thioesterases: chain-length determining enzymes in plant fatty acid biosynthesis. Genetic Eng 18:111–133

    Article  CAS  Google Scholar 

  • Wakil SJ (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28:4523–4530

    Article  CAS  PubMed  Google Scholar 

  • White SW, Zheng J, Zhang Y-M, Rock CO (2005) The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74:791–831

    Article  CAS  PubMed  Google Scholar 

  • Wu P-Z, Li J, Wei Q, Zeng L, Chen Y-P, Li MR, Jiang HW, Wu G-J (2009) Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas. Tree Physiol 29:1299–1305

    Article  CAS  PubMed  Google Scholar 

  • Zornetzer GA, Fox BG, Markley JL (2006) Solution structures of spinach acyl carrier protein with decanoate and stearate. Biochemistry 45:5217–5227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by the “Ministerio de Economia y Competitividad” and FEDER (Project AGL2014-53537-R and JAE-CSIC to J.A. A-M).

Author information

Authors and Affiliations

  1. Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA

    Jose A. Aznar-Moreno

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

    Mónica Venegas-Calerón, Enrique Martínez-Force, Rafael Garcés & Joaquín J. Salas

Authors
  1. Jose A. Aznar-Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mónica Venegas-Calerón
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Enrique Martínez-Force
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rafael Garcés
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joaquín J. Salas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joaquín J. Salas.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aznar-Moreno, J.A., Venegas-Calerón, M., Martínez-Force, E. et al. Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities. Planta 244, 479–490 (2016). https://doi.org/10.1007/s00425-016-2521-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-016-2521-7

Keywords

Search

Navigation