Mutation of the inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 alters lignin composition and improves saccharification

Abstract

ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production.

Figure 1.

General phenylpropanoid- and monolignol-specific pathways and metabolic map of the changes in phenolic metabolism in atr2 mutant stems. The main route toward lignin (i.e. the general phenylpropanoid- and monolignol-specific pathway) is marked with a gray background. Metabolites that accumulate in atr2 mutants are marked in red, and those that decrease in atr2 mutants are marked in blue. Metabolic routes that are active in stems of wild-type plants are given in black. Metabolic routes of which the metabolites are near or below the detection limit in wild-type plants but detected in atr2 mutants are given in gray. Metabolites that are below the detection limit in both wild-type and mutant plants are indicated with daggers. The three CYP450s are marked in orange. PAL, PHENYLALANINE AMMONIA LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE:COENZYME A LIGASE; HCT, p-HYDROXYCINNAMOYL-COENZYME A:QUINATE SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE; C3H, p-COUMARATE 3-HYDROXYLASE; CSE, CAFFEOYL SHIKIMATE ESTERASE, CCoAOMT:CAFFEOYL-COENZYME A O-METHYLTRANSFERASE; CCR, CINNAMOYL-COENZYME A REDUCTASE; F5H, FERULATE 5-HYDROXYLASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; CAD, CINNAMYL ALCOHOL DEHYDROGENASE; HCALDH, HYDROXYCINNAMALDEHYDE DEHYDROGENASE; SGT, SINAPATE 1-GLUCOSYLTRANSFERASE; SST, SINAPOYLGLUCOSE:SINAPOYLGLUCOSE SINAPOYLTRANSFERASE. For each enzymatic step of the general phenylpropanoid- and monolignol-specific pathways, specific gene family members are given that largely contribute to lignification in the inflorescence stem (Sibout et al., 2005; Vanholme et al., 2012b).

Figure 2.

Expression of ATR2 follows that of lignin biosynthetic genes (evaluation of data from Vanholme et al. [2012b]). A, Both ATR1 and ATR2 are coexpressed with lignin biosynthetic genes during inflorescence stem development (at an inflorescence stem height of 8, 16, 24, and 32 cm). The blue line, dark-blue area, and light-blue area indicate average, sd, and spread of the expression, respectively, of the 1,760 probes with a lignin biosynthesis-like expression profile over development (Vanholme et al., 2012b, figure 3, cluster C). The green line indicates expression of ATR1, and the red line indicates expression of ATR2. R² values are average correlation coefficients ± sd of ATR1 and ATR2 with each of the 1,760 probes with a lignin biosynthesis-like expression profile. B, ATR2, but not ATR1, is coexpressed with genes involved in lignin biosynthesis in lignin mutant backgrounds. The black line, dark gray area, and light gray area indicate average, sd, and spread of the expression, respectively, of the 62 genes with a lignin biosynthesis-like expression profile in the lignin mutants (Vanholme et al., 2012b, figure 9). The green line indicates expression of ATR1, and the red line indicates expression of ATR2. R² values are average correlation coefficients ± sd of ATR1 and ATR2 with each of the 62 genes with a lignin biosynthesis-like expression profile (Vanholme et al. 2012b).

Figure 3.

qRT-PCR analysis of wild-type seedlings either untreated or treated with 80 nm isoxaben for 6, 24, and 72 h. Data represent mean values of four biological pools of 20 seedlings each, and error bars represent se. The relative expression in mock conditions was set to 1 for each gene tested. *0.05 > P > 0.01, **P < 0.01.

Figure 4.

Characterization of atr2 T-DNA insertion mutants. A, Representation of the ATR2 genomic structure. Exons are represented as black boxes and introns as gray lines. The T-DNA insertion sites for the two alleles atr2-1 and atr2-2 are indicated by triangles. The orientation of each T-DNA is indicated by a black arrow pointing in the direction of the left border. The primers used to confirm the mutants are represented as gray arrows. The numbers of the primers correspond to those given in “Materials and Methods.” UTR, Untranslated region. B, Expression analysis of ATR1, ATR2, PAL1, PAL2, C4H, C3H1, and F5H1 in atr2-1 and atr2-2 mutants and the wild type as determined via qRT-PCR. Two primer pairs were used to determine the expression of ATR2. Primer sets 1 (i.e. primers 17 and 18) and 2 (i.e. primers 15 and 16) spanned the atr2-1 and atr2-2 T-DNA insertion positions, respectively. For each gene tested, the relative expression was normalized to the one of the wild type. nd, Signal below detection limit. *0.05 > P > 0.01, ***P < 0.001.

Figure 5.

Phenotypic characterization of atr2 mutants. A, Phenotype of fully grown plants after 8 weeks of short-day growth conditions followed by 5 weeks of long-day growth conditions. B, Growth curves. Height was monitored every 2 d. At the final stage, atr2-2 was significantly smaller compared with the wild type. C, The main inflorescence stem of atr2-1 and atr2-2 showed reduced weight compared with that of the wild type. For B and C, error bars represent se. *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001. D, Transverse stem sections of atr2 mutants and the wild type. Mäule and Wiesner staining and lignin autofluorescence are shown. If, Interfascicular fibers; Xy, xylem. Bars = 100 µm.

Figure 6.

NMR lignin characterization. A, Two-dimensional HSQC NMR spectra of the lignin aromatic region of senesced stem material from the wild type, atr2-1, and atr2-2. The volume integrals of the S2/6, G2, and H2/6 contour peaks, when scaled (by 2 for G), represents the quantity of each monomer. B, Overview of the relative amounts of the different monolignol-derived units and the S/G ratio as determined from the HSQC aromatic region. C, Overview of the relative amount (as determined by uncorrected volume integrals) of interunit linkage types (β-O-4, β-5, and β-β) as determined from the HSQC side chain region (for the spectra of the side chain regions, see Supplemental Fig. S4). **0.01 > P > 0.001, ***P < 0.001; n = 3.

Figure 7.

Saccharification data of the atr2 mutants. Samples were saccharified with either no pretreatment or an alkaline pretreatment (6.25 mm NaOH). The cellulose-to-Glc conversion was measured at 3, 7, 24, and 48 h (n = 8). Error bars represent se. *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001; n = 8.