Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

Abstract

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Figure 1.

The Angiosperm Phenylpropanoid Pathway. The highlighted gray region outlines the strategy for lignin-incorporated coniferaldehyde enrichment that is achieved by blocking at F5H and CAD. The strategy for lignin-incorporated sinapaldehyde enrichment expands on the coniferaldehyde enrichment strategy (gray, and the additional area outlined in green) and includes the combination of F5H overexpression and CAD disruption.

Figure 2.

Representative Pictures of F5H- and CAD-Manipulated Plants at 2 Months of Age. Bars = 1 cm.

Figure 3.

The Dwarf Phenotype of cadd C4H-F5H Does Not Appear to Be Caused Solely by a Decrease in Total Lignin Content. (A) Height of the primary inflorescence was measured in 2-month-old plants. (B) Lignin content of 8-week-old cell wall-extracted tissue from inflorescence stems was determined by the acetyl bromide method. Error bars represent the sd of biological triplicates. Asterisks indicate the significant difference between the plants with modified lignin composition compared with the wild type (*P < 0.05).

Figure 4.

HSQC Spectra Showing the Impact That CAD and F5H Manipulation Have on Lignin Subunit Composition. Expanded aromatic (A) and aldehyde (B) subregions of HSQC NMR spectra of ball-milled whole cell walls from Arabidopsis manipulated lines.

Figure 5.

Soluble Metabolites Are Modestly Affected by CAD Disruption, Whereas Cell Wall-Bound Metabolite Levels Are Increased. Soluble (A) and cell wall-bound (B) metabolites of 3-week-old whole rosettes and 2-month-old inflorescence stems, respectively. Error bars represent the sd of biological triplicates. Asterisks indicate the significant difference between the plants with modified lignin composition compared with either the wild type or fah1 (*P < 0.05).

Figure 6.

Histochemical Staining of 2-Month-Old Inflorescence Stem Sections from F5H- and CAD-Disrupted Plants. Sections were either left unstained or stained with phloroglucinol-HCl or Mäule. Bars = 100 μm.

Figure 7.

Confocal Laser Scanning Microscopy of Vascular Bundles of 2-Month-Old Inflorescence Sections Stained Using the Fluorescent Dye Acriflavine. Bars = 20 μm.

Figure 8.

Transmission Electron Microscopy Imaging of 2-Month-Old Inflorescence Tissue Showing Cell Wall Ultrastructure. Bars = 1 μm.

Figure 9.

Cellulose Conversion of Ground Inflorescence Tissue from Aldehyde-Enriched Plants. Error bars represent the sd of biological triplicates. Asterisks indicate the significant difference between the plants with modified cellulose conversion compared with the wild type (*P < 0.05).