H-lignin can be deposited independently of CINNAMYL ALCOHOL DEHYDROGENASE C and D in Arabidopsis

Abstract

Lignin contributes substantially to the recalcitrance of biomass toward saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcriptional regulatory complex Mediator, the stunted growth phenotype of mutants in p-coumaroyl shikimate 3'-hydroxylase, reduced epidermal fluorescence 8-1 (ref8-1), can be alleviated. Furthermore, these plants synthesize a lignin polymer almost entirely derived from p-coumaryl alcohol. Plants deficient in cinnamyl alcohol dehydrogenase (CAD) are notable in that they primarily incorporate coniferaldehyde and sinapaldehyde into their lignin. We tested the hypothesis that by stacking mutations in the genes encoding for the CAD paralogs C and D on an Arabidopsis (Arabidopsis thaliana) med5a/5b ref8-1 genetic background, the biosynthesis of p-coumaryl alcohol would be blocked, making p-coumaraldehyde available for polymerization into a novel kind of lignin. The med5a/5b ref8-1 cadc cadd plants are viable, but lignin analysis demonstrated that they continue to synthesize p-hydroxyphenyl lignin despite being mutated for the CADs typically considered to be required for monolignol biosynthesis. In addition, enzyme activity tests showed that even in the absence of CADC and CADD, there is high CAD activity in stems. We tested the potential involvement of other CADs in p-coumaraldehyde biosynthesis in the quintuple mutant by mutating them using the CRISPR/Cas9 system. Lignin analysis demonstrated that the resulting hextuple mutant plants continue to deposit p-coumaryl alcohol-derived lignin, demonstrating a route for the synthesis of p-hydroxyphenyl lignin in Arabidopsis independent of four CAD isoforms.

Figure 1

The phenylpropanoid pathway. PAL, PHENYLALANINE AMMONIA-LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE-CoA LIGASE; HCT, HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE; C3′H, p-COUMAROYL SHIKIMATE 3′-HYDROXYLASE; CSE, CAFFEOYL SHIKIMATE ESTERASE; CCoAOMT, CAFFEOYL-CoA O-METHYLTRANSFERASE; F5H, FERULATE 5-HYDROXYLASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; and CAD, CINNAMYL ALCOHOL DEHYDROGENASE.

Figure 2

Lignin monomer composition determined by gas chromatography using the DFRC method. Error bars indicate the standard deviation among three biological replicates. The asterisk represents a difference in H-lignin content between CAD mutants in the med5a/5b ref8-1 background and med5a/5b ref8-1 plants determined by one-way ANOVA and Dunnet’s test (P < 0.05).

Figure 3

2D ¹H–¹³C correlation (HSQC) NMR spectra of med5 ref8-1 and med5a/5b ref8-1 cadc cadd Arabidopsis transgenic lines. Resonance signals arising from H, G, and S lignin subunits and side-chain compositions, including aldehydes, are color-coded to match the structures shown on the right. Quantification is from correlation peak volume integration. A, Aromatics are on an S + G + X2G′ = 100% basis. A phenylalanine peak (Phe2/6) overlapped with the p-hydroxyphenyl group’s H2/6 correlation, preventing accurate quantification of H-units that may be over-estimated. B, Side-chain units in the aliphatic region are on an A + B + C = 100% basis with X1 expressed on the A + B + C basis. C, The aldehyde region shows aldehyde end-group components.

Figure 4

CAD enzyme activity in 4-week-old stems. CAD activity using p-coumaraldehyde (top) and coniferaldehyde (bottom) as substrates reported as picokatal per mcrogram of protein. Error bars represent standard deviation among biological replicates (n = 4). Letters represent difference between genotypes determined by one-way ANOVA and Tukey’s honest significant difference test (P < 0.05).

Figure 5

Expression of CAD genes in 4-week-old Arabidopsis stems. Relative expression measured by RT-qPCR, normalized to the reference gene ACTIN2 (At1g18780). Error bars represent standard deviation among biological replicates (n = 3). Transcript levels significantly different from wild-type (P < 0.05), determined by one-way ANOVA and Dunnet’s test (P < 0.05) are marked with (*).

Figure 6

Lignin monomer composition of CADA knockouts determined by gas chromatography using the DFRC method. Error bars indicate the standard deviation among three biological replicates (n = 3). Statistical differences in H-lignin content were determined by Student’s t test, not significant (n.s.).

Figure 7

Lignin monomer composition of CADG knockouts determined by gas chromatography using the DFRC method. Error bars indicate the standard deviation among three biological replicates (n = 3). Products not detected (n.d.). Statistical differences in H-lignin content were determined by Student’s t test, not significant (n.s.).

Figure 8

Stem cross-sections and expression of CADC and CADD in stems. A, Stem cross-sections of 7-week-old plants. Left, cadc cadd. Right, F1 of a cross between cadc cadd and med5a/5b ref8-1 cadc cadd. Scale bar = 100 µm. B, Expression of CADC and CADD genes in 4-week-old stems. Relative expression of CADC (top) and CADD (bottom) measured by RT-qPCR, normalized to the reference gene ACTIN2 (At1g18780). Error bars represent standard deviation among biological replicates (n = 3). The letters represent differences between genotypes determined by one-way ANOVA and Tukey’s honest significant difference test (P < 0.05).

Figure 9

Height and lignin composition of CRISPR-CADD knockouts. A, Height measurements of 5-week-old stems. Box plots depicting the median, first, and third quartiles. Dots represent biological replicates (n = 17) and (****) marks a significant difference between genotypes (P < 0.0001) by Student’s t test. B, Lignin monomer composition of CRISPR-CADD knockouts determined by gas chromatography using the DFRC method. Error bars indicate the standard deviation among three biological replicates (n = 3). Statistical differences in H-lignin content were determined by Student’s t test, not significant (n.s.).