Ectopic Defense Gene Expression Is Associated with Growth Defects in Medicago truncatula Lignin Pathway Mutants

Abstract

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.

Figure 1.

Phenotypes of M. truncatula monolignol biosynthesis pathway mutants. A, cse mutant plants. B, ccr1 mutant plants. C and D, hct mutant plants. E, comt mutant plants. F, ccoaomt mutant plants. G and H, comt ccoaomt mutant plants. Photographs were taken from 72-d plants in A, B, E, and F and from 24-d plants in C, D, G, and H. D and H are magnified views of the red rectangles in C and G, respectively. cse and ccr1 mutant plants show dwarf phenotypes (A and B), and hct and comt ccoaomt mutants show growth arrest (C, D, G, and H). WT, Wild type. Bars = 10 cm (A, B, E, and F) and 10 mm (D and H).

Figure 2.

SEM of leaf surfaces of M. truncatula mutants. SEM images of adaxial leaf blades are from control (Ctrl; A and E), cse mutant (B and F), β-as mutant (C and G), cse β-as double mutant (D and H), ccr1 mutant (I and N), hct mutant (J and O), comt mutant (K and P), ccoaomt mutant (L and Q), and comt ccoaomt double mutant (M and R) plants. Photographs were taken from the second developing leaves of 15-d plants. Analyses were repeated on leaves from 10 individual plants of each genotype (five from each allele), and representative results are shown. Bars = 200 μm (A–D and I–M) and 50 μm (E–H and N–R).

Figure 3.

Lignin content and composition of M. truncatula monolignol pathway mutants. Cell wall material was prepared from aboveground whole tissue from 10d seedlings (A–C) and mature stem tissue (D–F) and analyzed by thioacidolysis. A and D, Total lignin thioacidolysis yields (H + G + S units). B and E, Lignin monomer compositions. C and F, Lignin S/G ratios. Data are means ± sd (n = 6) of three replicates of each of the two independent alleles. WT, Wild type. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; and ***, 0.001 > P, Student’s t test.

Figure 4.

Phytohormone levels in monolignol pathway mutants. Phytohormone levels are shown in lignin biosynthesis pathway mutants as determined by UHPLC. A, SA. B, ABA. C, JA. D, MeJA. Fresh samples were prepared from aboveground whole tissue from 10d seedlings (black bars) and mature stem tissue (gray bars). Data are means ± sd (n = 3 biological replicates) of results from one allele for each target gene. WT, Wild type. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; and ***, 0.001 > P, Student’s t test.

Figure 5.

GO enrichment analysis of DEGs in M. truncatula monolignol pathway mutants. The DEGs were identified with the criteria ±1.5-fold change and P < 0.05. A, GO enrichment analysis in 10d seedlings. Transcriptomes of cse, comt, ccoaomt, and comt ccoaomt mutants were determined by DNA microarray analysis, whereas RNA-seq analysis was performed on the hct and ccr1 mutants. B, GO enrichment analysis in stems. Transcriptomes were determined by RNA-seq analysis.

Figure 6.

PCC analysis based on the abundance of DEGs at 10 d postgermination in M. truncatula monolignol pathway mutants. A, Profiles of all DEGs. B to I, Profiles of DEGs in each GO class as indicated. All gene lists involved in this analysis are available in Supplemental Data Set S5. Red color indicates that a set of genes is expressed in the same direction (up-up, down-down), and blue color indicates that a set of genes is expressed in the opposite direction (up-down, down-up).

Figure 7.

PCC analysis based on the abundance of DEGs in mature stem tissue of M. truncatula monolignol pathway mutants. A, Profiles of all DEGs. B to I, Profiles of DEGs in each GO class as indicated. All gene lists involved in this analysis are available in Supplemental Data Set S6. Red color indicates that a set of genes is expressed in the same direction (up-up, down-down), and blue color indicates that a set of genes is expressed in the opposite direction (up-down, down-up).

Figure 8.

PCC analysis based on the levels of accumulation of metabolites determined by GC-MS analysis of monolignol pathway mutants of M. truncatula. A and B, Accumulation profiles of compounds from 10d seedlings. C and D, Accumulation profiles of compounds from mature stem tissue. A and C represent accumulation profiles of compounds including lignin pathway compounds, whereas B and D show accumulation profiles of compounds excluding lignin pathway compounds. All compound lists involved in this analysis are given in Supplemental Data Set S1. Red color means that accumulation or decrease of compounds in monolignol pathway mutants occurs in the same direction (up-up, down-down), and blue color indicates the opposite direction (up-down, down-up).

Figure 9.

Diagram showing the relationship between resource allocation and phenotype in monolignol pathway mutants of M. truncatula. In the phenotypic depictions, black circles represent reproductive development and gray circles represent defective reproductive development. hct and comt ccoaomt mutant plants have developmental arrest without bolting. The number of arrows in the resource allocation categories indicates the strength of the allotted resource.