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

• Published in: Planta Vol. 241; no. 1; pp. 43 - 56
• Main Authors: González-Thuillier, Irene, Venegas-Calerón, Mónica, Garcés, Rafael, von Wettstein-Knowles, Penny, Martínez-Force, Enrique
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.01.2015; Springer Berlin Heidelberg; Springer Nature B.V
• Subjects: acyl carrier protein; Agriculture; Amino Acid Sequence; biochemical pathways; Biomedical and Life Sciences; Biosynthesis; Biosynthetic Pathways - genetics; Blotting, Western; cotyledons; DNA; E coli; Ecology; Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) - chemistry; Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) - genetics; Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) - metabolism; Escherichia coli; fatty acid composition; Fatty Acid Synthases - chemistry; Fatty Acid Synthases - genetics; Fatty Acid Synthases - metabolism; Fatty acids; Fatty Acids - biosynthesis; fatty-acid synthase; Forestry; gels; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; genes; Helianthus; Helianthus - genetics; Helianthus - metabolism; Helianthus annuus; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; leaves; Life Sciences; Liliopsida; Models, Molecular; Molecular Sequence Data; NAD (coenzyme); NADP (coenzyme); NADP - metabolism; Original Article; plant fats and oils; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Sciences; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; roots; seed development; Seeds; Sequence Homology, Amino Acid; stems; Substrate Specificity; Fatty acid synthase (FAS); Enoyl-[acyl carrier protein]-reductase (ENR); Oil biosynthesis; Substrate specificity
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-014-2162-7

MAIN CONCLUSION : Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different Vₘₐₓ.