Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne)

Abstract

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

Figure 1.

Developmental Stages of Perennial Ryegrass Plants. Three substages within each of the vegetative (V), elongation (E), and reproductive (R) growth stages are shown: V1, first leaf collared; V2, leaf collared; V3, third leaf collared; E1, first node visible; E2, two nodes visible; E3, three nodes visible; R1, inflorescence emerging; R2, spikelets fully emerged; R3, full anthesis. Positions of nodes are shown by arrowheads. Internodes are shown (following the dash) for stages E1-E3. Bars = 2 cm (vegetative), 3 cm (elongation), and 2 cm (reproduction).

Figure 2.

Mäule Staining of Lignin in Transverse Sections of Developing Perennial Ryegrass Stems. Transverse sections were prepared from different internodes of perennial ryegrass plants at the elongation (E) and reproductive (R) stages of development. Mäule staining was used to detect lignin rich in guaiacyl (G) and syringyl (S) monolignol subunits, which stain brown and red, respectively. Developmental stages are as for Figure 1. Micrographs are representative of at least six separate stems from different plants. vb, vascular bundle; sc, sclerenchyma; pc, parenchyma; ec, epidermal cells. Bars = 90 μm for all sections.

Figure 3.

Normalized Expression Levels of Perennial Ryegrass OMT1, OMT3, and CCR1 Genes in Different Internodes during Stem Development. Data are normalized using expression values of three internal control genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone (His), and tubulin (Tub) (see Supplemental Table 3 online). Average values and standard errors from three technical replicates derived from a single pooled sample of 10 tillers from the same stage of development are shown.

Figure 4.

Analysis of OMT1 Expression in Transgenic Perennial Ryegrass Lines Containing Hairpin Construct for Silencing of the OMT1 Gene. (A) RNA gel blot analysis of Lp OMT1 (top panel) and 18S rRNA (bottom panel) gene expression in nine transgenic and two wild-type control (no construct) lines at the R1 stage. (B) Quantitative RT-PCR analysis of Lp OMT1 expression in transgenic and wild-type plants at the R1 stage. Data are normalized using expression values of three control genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone (His), and tubulin (Tub) (see Supplemental Table 3 online). Average values and standard errors from three technical replicates derived from a single pooled sample of 10 tillers from the same stage of development are shown.

Figure 5.

Mäule Staining of Lignin in Transverse Sections of Stems from hpOMT1 and hpCCR1 and Control Plants. The three internodes (R1-1, R1-2, and R1-3) from stems of plants at the R1 stage were stained with Mäule reagent to detect lignins rich in guaiacyl (G) and syringyl (S) monolignol subunits, which stain brown and red, respectively. Bars = 90 μm for all sections.

Figure 6.

Analysis of CCR1 Gene Expression in Transgenic Perennial Ryegrass Lines Containing hpCCR1. (A) RNA gel blot analysis of CCR1 (top panel) and 18S rRNA (bottom panel) gene expression in nine transgenic and wild-type control lines at the R1 stage. (B) Quantitative RT-PCR analysis of Lp CCR1 expression in transgenic and wild-type plants at the R1 stage. Data are normalized using expression values of three control genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone (His), and tubulin (Tub) (see Supplemental Table 3 online). Average values and standard errors from three technical replicates derived from a single pooled sample of 10 tillers from the same stage of development are shown.

Figure 7.

NMR Analysis of Aqueous Soluble Metabolites from hpCCR1-1 and Control Plants. (A) PCA analysis of ¹H NMR data (spectra acquired in D₂O for maximum organic acid and carbohydrate solubility). Wild-type (circles) and CCR1-1 plants (triangles) separate on the PC1 axis. (B) PC1 loadings plot describing the most significantly different metabolites between control and hpCCR1-1 plants. Negative loadings are indicative of metabolites upregulated in CCR1-1 plants, while positive loadings indicate metabolites are more abundant in wild-type plants.

Figure 8.

Statistical Analysis of Electrospray Ionization Negative LC-MS Data. (A) PCA plot demonstrates excellent separation between the plants. Wild-type (circles) and CCR1-1 plants (triangles) separate on the PC1 axis, which describes 82% of the LC-MS detected metabolite variation between the plants. (B) Loadings plot for PC1 depicting the m/z values and retention times for significantly up- and downregulated metabolites.

Figure 9.

Phenylpropanoid and Monolignol Biosynthetic Pathways. The yellow route toward the production of monolignols is conserved in angiosperms. The orange route is found in some species, including perennial ryegrass (this article). The blue route is found in some species. CAD, cinnamyl alcohol dehydrogenase; 4CL, 4-coumarate:CoA ligase; C3H, p-coumarate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; HCT, p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltransferase; F5H, ferulate 5-hydroxylase; PAL, phenylalanine ammonia-lyase; SAD, sinapyl alcohol dehydrogenase. Compounds marked in red were downregulated in hpCCR1-1 lines. Those marked in blue were upregulated in hpCCR1-1 lines.