Three 4‐coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms

Abstract

SummaryThe enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon¯ow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and speci®cities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classi®ed into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to¯avonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than¯avonoids.

Differential Substrate Inhibition Couples Kinetically Distinct 4-Coumarate:Coenzyme A Ligases with Spatially Distinct Metabolic Roles in Quaking Aspen

“…Further kinetic analysis revealed that Pt4CL1 activated CA and PA with similar catalytic efficiency (V max /K m ; Table I). This is consistent with characteristics of lignin-related 4CLs in other species with multiple, kinetically distinct isoforms (Knobloch and Hahlbrock, 1975;Ehlting et al, 1999). Pt4CL2 exhibited a much higher catalytic efficiency for PA than for CA, due primarily to a very low K m for PA (Table I).…”

“…Wounding, UV light, and elicitors increase transcript abundance of different 4CL isoforms (Uhlmann and Ebel, 1993;Ehlting et al, 1999), reinforcing the model that there are distinct associations between isoforms and specific metabolic activities. However, all 4CLs subjected to in vitro kinetic analysis so far exhibit similar broad substrate specificity, generally favoring PA, followed by FA and CA (Ranjeva et al, 1976;Grand et al, 1983;Uhlmann and Ebel, 1993;Hu et al, 1998;Ehlting et al, 1999). Site-directed mutagenesis has recently been applied to elucidate the mechanism and evolution of 4CL catalysis (Stuible et al, 2000).…”

“…; Hu et al, 1998), and these isoforms have been proposed to control the relative abundance of flavonoids and various lignin precursors (monolignols) during structural, protective, and reproductive development (Ranjeva et al, 1976;Knobloch and Hahlbrock, 1977;Grand et al, 1983). Wounding, UV light, and elicitors increase transcript abundance of different 4CL isoforms (Uhlmann and Ebel, 1993;Ehlting et al, 1999), reinforcing the model that there are distinct associations between isoforms and specific metabolic activities. However, all 4CLs subjected to in vitro kinetic analysis so far exhibit similar broad substrate specificity, generally favoring PA, followed by FA and CA (Ranjeva et al, 1976;Grand et al, 1983;Uhlmann and Ebel, 1993;Hu et al, 1998;Ehlting et al, 1999).…”

“…4-Coumarate:coenzyme A (CoA) ligase (4CL) mediates activation of hydroxycinnamic acids 4-(p)-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA), 5-hydroxyferulic acid (5HFA), and sinapic acid (SA) into the high-energy intermediates used for biosynthesis of lignin, flavonoids, and various other protective, attractant, and signaling metabolites (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995;Higuchi, 1997;Whetten et al, 1998). Multiple 4CL isoforms with differential in vitro substrate specificities have been reported in several species (Knobloch and Hahlbrock, 1975;Ehlting et al, 1999), including aspen (Populus tremuloides Michx. ; Hu et al, 1998), and these isoforms have been proposed to control the relative abundance of flavonoids and various lignin precursors (monolignols) during structural, protective, and reproductive development (Ranjeva et al, 1976;Knobloch and Hahlbrock, 1977;Grand et al, 1983).…”

“…Multiple 4CL isoforms with differential in vitro substrate specificities have been reported in several species (Knobloch and Hahlbrock, 1975;Ehlting et al, 1999), including aspen (Populus tremuloides Michx. ; Hu et al, 1998), and these isoforms have been proposed to control the relative abundance of flavonoids and various lignin precursors (monolignols) during structural, protective, and reproductive development (Ranjeva et al, 1976;Knobloch and Hahlbrock, 1977;Grand et al, 1983).…”