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. 2001 Jul;13(7):1567-86.
doi: 10.1105/tpc.010111.

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase

L Li  1 , X F Cheng, J Leshkevich, T Umezawa, S A Harding, V L Chiang
Affiliations

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase

L Li et al. Plant Cell. 2001 Jul.

Abstract

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

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Figures

Figure 1.
Figure 1.
Amino Acid Sequence Alignment of Aspen SAD and Selected CAD Proteins. The deduced amino acid sequences of aspen SAD and CAD, Eucalyptus CAD (Eucalyptus globulus), tobacco CAD (Nicotiana tabacum), and lucerne CAD (Medicago sativa) were aligned using the OMIGA program of the GCG software package (Genetics Computer Group, Madison, WI). Identical amino acid sequences are shaded. Locations of Zn1, Zn2, and NADP binding domains are indicated.
Figure 2.
Figure 2.
Phylogenetic Analysis of Aspen SAD and Plant CADs. An unweighted pair-group method using arithmetic averages was used for phylogenetic tree analysis of aspen SAD (PtSAD) and other full-length plant CAD protein sequences available in the GenBank database.
Figure 3.
Figure 3.
Molecular Characterization of Aspen PtCAD and PtSAD. (A) and (B) Genomic DNA gel blot analysis. Aspen genomic DNA (10 μg/lane) was digested with restriction enzymes and hybridized with 32P-labeled full-length PtCAD (A) and PtSAD (B) cDNAs. (C) and (D) RNA gel blot analysis of PtCAD and PtSAD tissue-specific expression patterns. Total RNA (10 μg/lane) from each organ or tissue type was hybridized with 32P-labeled full-length PtCAD (C) and PtSAD (D) cDNAs. (E) and (F) Protein gel blot analysis of anti-PtCAD and anti-PtSAD antibody specificity and tissue-specific expression of PtCAD and PtSAD. Immunoblots of E. coli–expressed and affinity-purified PtCAD and PtSAD recombinant proteins (25 ng/lane) and plant protein extracts (10 μg/lane) with anti-PtCAD (E) and anti-PtSAD (F) antibodies.
Figure 4.
Figure 4.
HPLC-UV/MS Analysis of Recombinant PtCAD and PtSAD Reactions. (A) HPLC-MS (selected ion monitoring, 70 V; mass-to-charge ratio [m/z], 179.0) chromatogram showing the PtCAD reduction (see Methods) of coniferaldehyde (blue; retention time [Rt] = 13.58 min) into coniferyl alcohol (brown; Rt = 7.79 min). The inset shows the negative ion electrospray mass spectrum (scanning mode at 70 V) of coniferyl alcohol with properties (UV [HPLC mobile phase] λmax I, 262 nm, λmax II, 294 nm; MS [150 V] mass-to-charge ratio [%], 179.1 [100%], 164 [39%], 146 [38%], 161 [25%]) identical to the authentic standard. MW, molecular weight. (B) HPLC-MS (selected ion monitoring, 70 V; mass-to-charge ratio [m/z], 209.0) chromatogram showing the PtSAD-mediated sinapaldehyde (red; Rt = 12.09 min) reduction (see Methods) into sinapyl alcohol (green; Rt = 7.03 min). The inset shows the negative ion electrospray mass spectrum of sinapyl alcohol with properties (UV [HPLC mobile phase] λmax I, 222 nm, λmax II, 274; MS [150 V] mass-to-charge ratio [%], 209.1 [100%], 194 [41%], 176 [11%]) identical to the authentic compound. MW, molecular weight. (C) and (D) HPLC-MS (selected ion monitoring, 70 V; mass-to-charge ratio [m/z], 179.0 and 209.0) chromatograms of PtCAD and PtSAD reactions (see Methods) with a mixture of equal molar coniferaldehyde (blue; Rt = 13.58 min) and sinapaldehyde (red; Rt = 12.09 min). Coniferyl alcohol (brown; Rt = 7.79 min) is the exclusive product of the PtCAD reaction (C), and sinapyl alcohol (green; Rt = 7.03 min) is the only product of the PtSAD reaction (D). O-Coumaric acid was the internal standard (I.S.) in all reactions.
Figure 5.
Figure 5.
Inhibition Kinetics of PtCAD and PtSAD. Lineweaver-Burk plots of 1/v versus 1/[S] in the presence of different levels of inhibitor concentrations as indicated. The insets show replots of apparent Km′ versus the corresponding inhibitor concentration, used to calculated the Ki. (A) Competitive inhibition effects of coniferaldehyde on PtCAD reduction of sinapaldehyde in mixed substrate assays. (B) Competitive inhibition effects of sinapaldehyde on PtSAD reduction of coniferaldehyde in mixed substrate assays.
Figure 6.
Figure 6.
Detection of Guaiacyl and Syringyl Lignins in Aspen Stem. Cross/Bevan histochemical analysis of transverse sections of stem internodes showing the exclusive presence of guaiacyl lignin (brown) in primary xylem tissues ([A] to [C]) and the deposition of guaiacyl-syringyl lignin (red) in secondary growth tissues ([E] to [G] and [I]). The differential deposition of these lignins along the stem was confirmed by thioacidolysis analysis of the stem lignin ([D] and [H]). (A) Internode 3. (B) Internode 4. (C) A magnified section of the image in (B). (D) and (H) Gas chromatograms of trithioethylated monomeric lignin products after thioacidolysis, demonstrating the exclusive presence of guaiacyl lignin in internodes 1 to 4 (D) and the presence of guaiacyl and syringyl lignins in internodes 5 to 20 (H). Typical erythro (e) and threo (t) isomers (1:1 ratio) of guaiacyl and syringyl monomers were present. The internal standard (I.S.) was hexacosane. (E) Internode 8. The primary xylem is the only vascular tissue having the pure guaiacyl lignin. (F) Internode 10. The primary xylem is the only vascular tissue having the pure guaiacyl lignin. (G) Internode 8 revealing the sequential deposition of guaiacyl (light brown) followed by syringyl (pink to red) lignins in secondary xylem elements. Note the deposition of only the guaiacyl lignin in metaxylem vessels. (I) Internode 6 showing the onset of syringyl lignin (pink) deposition in primary phloem fibers. Dsxf, developing secondary xylem fibers; Dsxv, developing secondary xylem vessels; Mxv, metaxylem vessels; Ppc, protophloem parenchyma cells; Ppf, primary phloem fibers; Px, primary xylem; Pxv, protoxylem vessels; Rp, ray parenchyma cells; Sx, secondary xylem. Bars in (A), (B), (E), and (F) = 100 μm; bars in (C), (G), and (I) = 30 μm.
Figure 7.
Figure 7.
Immunolocalization of PtCAD, PtCAld5H, and PtSAD Proteins in Aspen Stem. Light micrographs of stem transverse sections showing localizations of PtCAD (red; [A], [D], [F], and [H]), PtCAld5H (blue; [C]), and PtSAD (blue; [B], [E], [G], and [I]). (A) to (C) Internode 3. PtCAD was localized exclusively to primary xylem elements (A), whereas PtSAD (B) and PtCAld5H (C) were not detected in these primary xylem elements but were abundant in protophloem parenchyma cells and the medullary sheath. (D) and (E) Primary phloem fibers in internode 8. (F) and (G) Internode 8. Note the strong PtCAD signals in developing secondary xylem vessels (F), but PtSAD signals were nearly absent from these cells (G). (H) and (I) Internode 12. The appearance of PtSAD (I) lagged behind that of PtCAD (H) in fusiform initials. Ap, axial ray parenchyma cells; Ffi, fusiform initials; Ms, medullary sheath. Other abbreviations are as given in Figure 6. Bars in (A) to (C), (H), and (I) = 50 μm; bars in (D) to (G) = 30 μm.
Figure 8.
Figure 8.
Immunoblot Detection of CAD and SAD Proteins in Various Plants. CAD was detected by immunoblotting in developing xylem of all plants analyzed (top), but SAD was found only in angiosperm species (bottom). Seventy-five nanograms of recombinant protein per lane was used, and other lanes were loaded with 10 μg of plant xylem crude protein extracts.
Figure 9.
Figure 9.
Proposed Principal Biosynthetic Pathway for the Formation of Monolignols in Angiosperms. C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; 4CL, 4-coumarate:CoA ligase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase. Inconclusive pathways are shown in gray.
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