Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification

Abstract

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

Figure 1

Alternative pathways for the methylation of monolignols. The enzymes are: PAL, l-phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate 3-hydroxylase; COMT, bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase; F5H, ferulate 5-hydroxylase; CC3H, coumaroyl CoA 3-hydroxylase; CCOMT, bispecific caffeoyl/5-hydroxyferuolyl CoA O-methyltransferase; 4CL, coumarate (hydroxycinnamate) CoA ligase; CCR, cinnamoyl CoA reductase; and CAD, coniferyl alcohol dehydrogenase. Dashed arrows represent reactions that have yet to be clearly demonstrated in vitro. cDNA clones encoding enzymes marked in bold were obtained from alfalfa. In some species, 4CL has very little activity with sinapate as substrate.

Figure 2

Activities of OMTs in developing alfalfa stem internodes. Total activities of COMT (white bars) and CCOMT (shaded bars) are shown in individual alfalfa internodes in relation to the total amount of lignin (line) per internode.

Figure 7

OMT substrate preference in alfalfa stem internodes in relation to lignin composition. A, Lignin content in individual internodes (counting from the top). DM, Dry matter. B, Lignin methoxyl content. C, OMT activity against free acid substrates. ○, Activity against caffeic acid; •, activity against 5-OH ferulic acid. D, OMT activity against CoA esters. ○, Activity against caffeoyl CoA; •, activity against 5-hydroxyferuloyl CoA. Error bars represent sd above and below the mean (n = 3 individual plants).

Figure 3

Comparison of the deduced amino acid sequences of alfalfa (Ms) and parsley (Pc) CCOMT. Six additional amino acids were found in the N-terminal region of the alfalfa sequence. Asterisks indicate nonconservative differences.

Figure 4

Tissue distribution of CCOMT transcripts in developing alfalfa seedlings. Total RNA from various alfalfa organs harvested from plants grown for the number of weeks shown was subjected to northern-blot analysis, using the full-length alfalfa CCOMT sequence as probe. The numbers for stem segments indicate internode number from the top of the plant. The blot was reprobed with an rRNA sequence to check for loading and transfer efficiency. pt., Point.

Figure 5

Tissue print localization of COMT and CCOMT transcripts in alfalfa stem internodes. Pictures indicate anatomy and COMT and CCOMT transcript distribution in the second (A–C), third (D–F), and fifth (G–I) internodes from the top of the stem. A, D, and G show sections stained with safranin O. B, E, and H are corresponding tissue prints hybridized with a COMT antisense probe, and C, F, and I are prints hybridized with a CCOMT antisense probe. p, Phloem; and x, xylem. Bars = 1.0 mm.

Figure 6

O-Methylation of hydroxycinnamoyl CoA esters by alfalfa CCOMT expressed in E. coli. HPLC chromatogram of the OMT reaction mixture using 5-hydroxyferuloyl CoA as substrate. For conditions, see Methods. Each peak was identified by direct comparison with authentic standards (B and D) and/or its UV/visible spectrum. A, 5-Hydroxyferuloyl CoA; B, 5-hydroxyferulic acid; C, sinapoyl CoA; and D, sinapic acid. No free acids (B and D) or sinapoyl CoA (C) were detected when the assay mixture was incubated without protein solution (upper trace).