Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis

Abstract

Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.

Fig. 1.

CAD substrates and products.

Fig. 2.

Position of AtCAD6–AtCAD8 on chromosome 4 of A. thaliana.

Fig. 3.

Representative purification of CAD, using AtCAD5 for illustrative purposes. Lanes: 1, molecular mass ladder; 2, Escherichia coli crude extract; 3–8, fractions eluted from the metal chelate affinity column between 70 and 230 mM imidazole in Tris·HCl buffer contained pure AtCAD5. Proteins were visualized by silver staining.

Fig. 4.

Lineweaver–Burk plots of AtCAD5 for substrates 1–5.

Fig. 5.

Relative substrate 1–5 efficacy of Arabidopsis recombinant CAD homologues.

Fig. 6.

A stereoview showing the distribution of structural elements of AtCAD4. The two Zn atoms are shown in yellow together with three coordinating amino acids (C47, H69, and C163), Q130, NADP(H), and sinapyl aldehyde (5). This figure was prepared by using web viewer.

Fig. 7.

Time course of Arabidopsis (Columbia) growth and development versus lignin content/monomer composition ratios. (A) Acetyl bromide lignin contents. (B) S:G ratios by nitrobenzene oxidation. (C) S:G ratios by thioacidolysis. CWR, cell wall residue.