Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides)

Abstract

4-Coumarate:CoA ligases (4CLs, EC 6.2.1.12) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants. So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics. However, we have discovered two functionally and structurally distinct 4CL genes, Pt4CL1 and Pt4CL2 (63% protein sequence identity), that are differentially expressed in aspen (Populus tremuloides). The Escherichia coli-expressed and purified Pt4CL1 and Pt4CL2 proteins exhibited highly divergent substrate preference as well as specificity that reveal the association of Pt4CL1 with the biosynthesis of guaiacyl-syringyl lignin and the involvement of Pt4CL2 with other phenylpropanoid formation. Northern hybridization analysis demonstrated that Pt4CL1 mRNA is specifically expressed in lignifying xylem tissues and Pt4CL2 mRNA is specifically expressed in epidermal layers in the stem and the leaf, consistent with the promoter activities of Pt4CL1 and Pt4CL2 genes based on the heterologous promoter-beta-glucouronidase fusion analysis. Thus, the expression of Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen; Pt4CL1 is devoted to lignin biosynthesis in developing xylem tissues, whereas Pt4CL2 is involved in the biosynthesis of other phenolics, such as flavonoids, in epidermal cells.

Figure 1

Biosynthetic pathways of hydroxylated cinnamic acids and cinnamoyl CoA thioesters for the formation of lignin and other phenylpropanoids. C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; OMT, O-methyltransferase; F5H, ferulate 5-hydroxylase; Pt4CL1 and 2, P. tremuloides 4CL1 and 2; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase.

Figure 2

Alignment of the deduced amino acid sequences of aspen Pt4CL1 (GenBank accession no. AF041049) and Pt4CL2 (GenBank accession no. AF041050). Symbols denote identical (*) and similar (•) amino acids and conserved Cys residues (shaded). Boxes I and II are the two putative AMP-binding motifs (19, 20). Locations of primers used for RT-PCR are indicated by arrows. Sequence alignment was performed using the clustlaw (1.60) program through the European Molecular Biology Laboratory world wide web server.

Figure 3

(A) Southern blot analysis of aspen genomic DNA (10 μg per lane) digested with BamHI (lane B) and XbaI (lane X), probed with ³²P-labeled Pt4CL1 and Pt4CL2 cDNAs, respectively. λ DNA digested with HindIII was used as size markers, and values are given in kb. (B) Northern blot analysis of total RNA (10 μg per lane) from young leaf (lane L), top internodes (TI), lower internodes (LI), and secondary developing xylem (X) of aspen, probed with ³²P-labeled Pt4CL1 and Pt4CL2 cDNAs, respectively.

Figure 4

Histochemical analysis of GUS gene expression in transgenic tobacco. Transverse sections of the stem (A, bar = 100 μm) and leaf (B, bar = 100 μm) excised from Pt4CL1p-GUS transgenic tobacco, and transverse sections of the stem (C, bar = 250 μm) and leaf (D, bar = 100 μm) from Pt4CL2p-GUS transgenic tobacco were stained for GUS activity as described (17).

Figure 5

Northern blot analysis of total RNA (20 μg per lane) from epidermal layers (lane E) and developing xylem (X) of aspen stem (one to 25 internodes), probed with ³²P-labeled Pt4CL1 and Pt4CL2 cDNAs, respectively.