The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes

Abstract

4-Coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role in the biosynthesis of plant secondary compounds at the divergence point from general phenylpropanoid metabolism to several major branch pathways. In Arabidopsis thaliana, we have identified a previously undetected, fourth and final member of the At4CL gene family. The encoded enzyme, At4CL4, exhibits the rare property of efficiently activating sinapate, besides the usual 4CL substrates (4-coumarate, caffeate, and ferulate), indicating a distinct metabolic function. Phylogenetic analysis suggests an early evolutionary and functional divergence of three of the four gene family members, At4CL2-4, whereas At4CL1 appears to have originated much later by duplication of its structurally and functionally closest relative, At4CL2. Various characteristics shared by all known plant 4CL genes, as well as by the encoded proteins, define and delimit the At4CL gene family and distinguish it from the closely related family of "At4CL-like" genes.

Fig. 1.

Chemical structures of the four naturally occurring 4CL substrates investigated in this study.

Fig. 2.

Comparison of exon/intron structures of At4CL1-4.

Fig. 3.

Relative positions of sequence-related genes on inversely duplicated segments of A. thaliana chromosomes I and III. Bold letters indicate crosswise functional as well as close sequence relationship. Ubi-spec., ubiquitin-specific.

Fig. 4.

Unrooted phylogenetic tree of all presently known plant 4CL isoenzymes. The putative root position is marked by an arrow. Contiguous graduated lines indicate class membership (dark gray = sinapate-activating isoenzymes). At, Arabidopsis thaliana; Ca, Capsicum annum; Gm, Glycine max; Le, Lithospermum erythrorhizon; Lp, Lolium perenne; Nt, Nicotiana tabacum; Os, Oryza sativa; Pb, Populus balsamifera subsp. trichocarpa × Populus deltoides; Pc, Petroselinum crispum; PtPi, Pinus taeda; PtPo Populus tomentosa; PtQa, Populus tremuloides; Ri, Rubus idaeus; St, Solanum tuberosum; Vp, Vanilla planiforia. Not shown are “Pc4CL3” and “Nt4CL3” (see text), both of which were deduced from partial nucleotide sequences and thus insufficiently characterized. Dotted circles enclose monocotyledonous representatives.

Fig. 5.

Proposed scheme for the evolutionary divergence of At4CL1-4.