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The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

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Abstract

The β-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

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Abbreviations

ACP:

Acyl carrier protein

DAF:

Days after flowering

DH:

β-Hydroxyacyl-acyl carrier protein dehydratase

ENR:

Enoyl-acyl carrier protein reductase

FAS:

Fatty acid synthase

KAS:

β-Ketoacyl-acyl carrier protein synthase

KR:

β-Ketoacyl-acyl carrier protein reductase

LB:

Luria–Bertani growth media

IPTG:

Isopropyl-1-thio-β-d-galactopyranoside

MCAT:

Malonyl-coenzyme A:acyl carrier protein transacylase

Ni–NTA:

Nickel–nitrilotriacetic acid

Q-PCR:

Quantitative PCR

U:

Units

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Acknowledgments

Our thanks are due to Marianne Mortensen for her skillful technical assistance. This work was supported by the Ministerio de Ciencia e Innovación and FEDER, project AGL2008-01086/ALI and the Danish Natural Sciences Research Council Grant Number 272-05-0401.

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Authors and Affiliations

  1. Instituto de la Grasa, CSIC, Avenida Padre Garcia Tejero 4, 41012, Seville, Spain

    Damián González-Mellado, 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. Damián González-Mellado
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  2. Penny von Wettstein-Knowles
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  3. Rafael Garcés
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  4. Enrique Martínez-Force
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Correspondence to Enrique Martínez-Force.

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González-Mellado, D., von Wettstein-Knowles, P., Garcés, R. et al. The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower. Planta 231, 1277–1289 (2010). https://doi.org/10.1007/s00425-010-1131-z

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