Functional characterization and structural modelling of Helianthus annuus (sunflower) ketoacyl-CoA synthases and their role in seed oil composition

Abstract

Main conclusion: The enzymes HaKCS1 and HaKCS2 are expressed in sunflower seeds and contribute to elongation of C18 fatty acids, resulting in the C20-C24 fatty acids in sunflower oil. Most plant fatty acids are produced by plastidial soluble fatty acid synthases that produce fatty acids of up to 18 carbon atoms. However, further acyl chain elongations can take place in the endoplasmic reticulum, catalysed by membrane-bound synthases that act on acyl-CoAs. The condensing enzymes of these complexes are the ketoacyl-CoA synthase (KCSs), responsible for the synthesis of very long chain fatty acids (VLCFAs) and their derivatives in plants, these including waxes and cuticle hydrocarbons, as well as fatty aldehydes. Sunflower seeds accumulate oil that contains around 2-3% of VLCFAs and studies of the fatty acid elongase activity in developing sunflower embryos indicate that two different KCS isoforms drive the synthesis of these fatty acids. Here, two cDNAs encoding distinct KCSs were amplified from RNAs extracted from developing sunflower embryos and named HaKCS1 and HaKCS2. These genes are expressed in developing seeds during the period of oil accumulation and they are clear candidates to condition sunflower oil synthesis. These two KCS cDNAs complement a yeast elongase null mutant and when expressed in yeast, they alter the host's fatty acid profile, proving the encoded KCSs are functional. The structure of these enzymes was modelled and their contribution to the presence of VLCFAs in sunflower oil is discussed based on the results obtained.

Centromere-Specific Retrotransposons and Very-Long-Chain Fatty Acid Biosynthesis in the Genome of Yellowhorn (Xanthoceras sorbifolium, Sapindaceae), an Oil-Producing Tree With Significant Drought Resistance

“…We identified the biosynthetic pathway of VLCFA in yellowhorn and revealed associated gene expression patterns. KCS enzymes catalyze the synthesis of several VLCFA, including nervonic acid ( Millar and Kunst, 1997 ; Guo et al, 2009 ; González-Mellado et al, 2019 ). We assayed the expression of the yellowhorn KCS genes by comparing different tissues at various developmental stages using RNA-Seq.…”

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.)

“…RNA extraction and cDNA synthesis were performed as described by Aznar-Moreno et al (2018). The cDNA samples were subjected to real time quantitative PCR (qRT-PCR) with specific pair of primers for the genes coding for HaLPCAT1, HaLPCAT2, and HaLPCAT3 ( Supplementary Table S1) as described by González-Mellado et al (2019). The method of Livak and Schmittgen (2001) was used to calculate the relative expression levels of the studied genes.…”

“…The heterologous expression of HaLPCATs was carried out in yeast, a host that has been demonstrated to be appropriate for the characterization of other ER-associated enzymes involved in lipid metabolism (Zheng et al, 2012;Rodríguez-Rodríguez et al, 2016;González-Mellado et al, 2019). The first step in characterizing the recombinant sunflower LPCATs, produced in yeast, was to assess their functionality in vivo.…”

Tracing Key Molecular Regulators of Lipid Biosynthesis in Tuber Development of Cyperus esculentus Using Transcriptomics and Lipidomics Profiling

“…Many enzymes and transcriptional factors involved in TAG biosynthetic pathways were identified and confirmed as positive regulators in various plant species. Among those, fatty acids synthesis (PUSH), FAD2, FAD3 [9], KCSs [10], or MYB1 [11] represent the key regulators of fatty acids composition alteration. For instance, FAX1 and FAX2 facilitate fatty acids transport from plastids to improve seed oil accumulation [12,13].…”