Identification of the functions of 4-coumarate-CoA ligase/ acyl-CoA synthetase paralogs in potato

Background: The 4CL/ ACS protein family is well known for its 4-coumarate-CoA ligase (4CL) enzymes but there are many aspects of this family that are still unclear or generally known. Cytosolic class I and class II 4CL enzymes control the biosynthesis of lignin/ suberin and flavonoids, respectively. Many 4CL homologs have broad substrate permissiveness in vitro and have no clear cut function. However, it has been demonstrated unequivocally that a peroxisomal 4CL-like homolog from Arabidopsis efficiently uses p-coumarate for ubiquinone biosynthesis. Another homolog has been shown to act as a fatty acyl-CoA synthetase and yet another as OPDA-CoA ligase. Hence, despite this knowledge, most homologs remain annotated as 4CL-like whereas other researches study the ACS protein family. Results: We set out identify the specific functions of 4CL/ ACS homologs, specifically in order to study the 4CL family in Solanum tuberosum. An in depth phylogenetic analysis was done. Using clustering techniques, functional annotation and taxonomic signals, three major clades were depicted. Clade 1 is composed of class I from monocotyledons, class I from dicotyledons and class II canonical 4CL enzymes subclades. Specificity determining positions and 3D structure analysis shows that clade 2 cytosolic 4CL-like enzymes show a rather different binding cleft and presumably use medium- to long-chain fatty acids. Clade 3 is composed of five subclades, four of which have a broad taxonomic contribution and a similar binding cleft as 4CLs whereas a fifth, specific for dicotyledons shows a significantly different binding pocket. The potato 4CL family comprises four class I (St4CL-I(A-D)) and one class II (St4CL-II) members. Transcript levels of St4CLs and of marker genes of the flavonoid (chalcone synthase, CHS) and suberin (feruloyl-CoA transferase, FHT) pathways were determined by qRT-PCR in flesh and skin from Andean varieties. St4CL-IA was barely detected in the skin of some varieties whereas St4CL-IB did not show a clear pattern. St4CL-IC and St4CL-ID could not be detected. St4CL-II expression pattern was similar to CHS. St4CL-IA and St4CL-IB were induced by wounding as did FHT whereas St4CL-II and CHS expression was repressed. Constitutive and wound-induced expression suggests that St4CL-IA and St4CL-IB isoforms are likely involved in soluble and/ or suberin-bound phenolic compounds while St4CL-II appears to be involved in flavonoid biosynthesis.