Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants

Abstract

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis. De novo fatty acid synthesis in plants takes place in the plastid, with acetyl-coenzyme A (acetyl-CoA) the main precursor. This metabolite is primarily generated from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO 2 and NADH 1. The PDH complex contains 3 subunits: E1 or pyruvate dehydrogenase, E2 or dihydrolipoyl acetyltransferase, and E3 or dihydrolipoyl transhydrogenase. Of these, E2 requires a lipoic acid (LA; 6,8-dithiooctanoic acid or 1,2-dithiolane-3-pentanoic acid) prosthetic group to be functional, a sulphur-containing enzyme co-factor that is essential for the catalytic activity of several key enzyme complexes involved in fundamental metabolic processes in most prokaryotic and eukaryotic organisms. Besides PDH, this prosthetic group is also required by α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, acetoin dehydrogenase, and the glycine cleavage complex (glycine decarboxylase) 2-5 .

Characterization of the acyl-ACP thioesterases from Koelreuteria paniculata reveals a new type of FatB thioesterase

“…Sequence analysis and phylogenetic tree constructions K. paniculata thioesterase proteins were confirmed and they were identified as acyl-ACP thioesterases through alignment to the homologous sequences from other plant species using the alignment search tool BLASTp (Camacho et al, 2009). Alignments and phylogenetic tree were performed as described previously (Martins-Noguerol et al, 2020).…”

Data_Sheet_1.CSV

“…In this model, the β strands remained in the core of the protein while the α helices had an outward disposition. This was a structure very similar to the two plastidial isoforms of sunflower lipoyl synthase (HaLIP1p1 and HaLIP1p2) characterized previously by our group (Martins-Noguerol et al, 2020).…”

“…LCelectrospray ionization (ESI)-HRMS2 analysis was performed by coupling the LC system to a hybrid quadrupole time-of-flight high definition (QToF) mass spectrometer (Agilent 6538, Agilent Technologies) equipped with an ESI dual source. MassHunter B.07 software was used to control the parameters and the chromatogram was built as indicated (Martins-Noguerol et al, 2020). The peaks were annotated using two different databases: lipid Match (Koelmel et al, 2017) and lipid Blast (Kind et al, 2013).…”

“…In mitochondria, most of the de novo fatty acid synthesis is directed toward the synthesis of LA (Wada et al, 1997). Plastidial lipoyl synthases from sunflower have been characterized (Martins-Noguerol et al, 2020) and a set of plastidial octanoyltransferases has also been evaluated in this species. Here, a different system for LA synthesis in sunflower was found, comprised of a lipoyl synthase (HaLIP1m) and an octanoyltransferase (HaLIP2m) that differed in their sequence and predicted location from the aforementioned plastidial forms.…”

Characterization of Helianthus annuus Lipoic Acid Biosynthesis: The Mitochondrial Octanoyltransferase and Lipoyl Synthase Enzyme System

“…In mitochondria, most of the de novo fatty acid synthesis is directed toward the synthesis of LA ( Wada et al, 1997 ). Plastidial lipoyl synthases from sunflower have been characterized ( Martins-Noguerol et al, 2020 ) and a set of plastidial octanoyltransferases has also been evaluated in this species. Here, a different system for LA synthesis in sunflower was found, comprised of a lipoyl synthase ( Ha LIP1m) and an octanoyltransferase ( Ha LIP2m) that differed in their sequence and predicted location from the aforementioned plastidial forms.…”