Cinnamate:CoA Ligase Initiates the Biosynthesis of a Benzoate-Derived Xanthone Phytoalexin in Hypericum calycinum Cell Cultures    

Abstract

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg2+ and K+ at optimum concentrations of 2.5 and 100 mm, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…in supplying precursors for phenylpropanoid derivatives such as lignin). Recently, cDNAs encoding cinnamate:CoA ligase from Petunia hybrida and Hypericum calycinum were cloned (Gaid et al, 2012;Klempien et al, 2012). This enzyme channels the metabolic flux from the general phenylpropanoid pathway to benzenoid metabolism and further to aucuparin biosynthesis.…”

“…An SSH library was constructed as described previously (Gaid et al, 2012), except that poly(A + ) RNA was isolated from chitosan-treated (9 h after the onset of elicitation) and control rowan cell cultures and cDNA synthesis was started with 1 mg of mRNA from each.…”

“…The B4H reaction product was analyzed on an HPLC system described previously (Gaid et al, 2012). The mobile phase consisted of water acidified with 1 mM trifluoroacetic acid (A) and acetonitrile (B).…”

“…Similar PCR conditions for 30 cycles were used for the amplification of SaActin transcripts. The PCR products were analyzed as described previously (Gaid et al, 2012). At least two biological and three technical repetitions were performed.…”

“…A construct expressing p19 protein from Tomato bushy stunt virus (Voinnet et al, 2003) was used to suppress gene silencing. All vectors were transferred by electroporation in the A. tumefaciens strain C58C1 and transiently expressed in leaves of Nicotiana benthamiana, as described previously (Gaid et al, 2012). Prior to infiltration, bacterial cultures were resuspended in activation medium (Bendahmane et al, 1999) and adjusted to optical density at 600 nm = 1.5 (optical density at 600 nm = 0.9 for p19).…”