Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a Cinnamoyl CoA reductase gene

Abstract

C4 grasses are favoured as forage crops in warm, humid climates. The use of C4 grasses in pastures is expected to increase because the tropical belt is widening due to global climate change. While the forage quality of Paspalum dilatatum (dallisgrass) is higher than that of other C4 forage grass species, digestibility of warm-season grasses is, in general, poor compared with most temperate grasses. The presence of thick-walled parenchyma bundle-sheath cells around the vascular bundles found in the C4 forage grasses are associated with the deposition of lignin polymers in cell walls. High lignin content correlates negatively with digestibility, which is further reduced by a high ratio of syringyl (S) to guaiacyl (G) lignin subunits. Cinnamoyl-CoA reductase (CCR) catalyses the conversion of cinnamoyl CoA to cinnemaldehyde in the monolignol biosynthetic pathway and is considered to be the first step in the lignin-specific branch of the phenylpropanoid pathway. We have isolated three putative CCR1 cDNAs from P. dilatatum and demonstrated that their spatio-temporal expression pattern correlates with the developmental profile of lignin deposition. Further, transgenic P. dilatatum plants were produced in which a sense-suppression gene cassette, delivered free of vector backbone and integrated separately to the selectable marker, reduced CCR1 transcript levels. This resulted in the reduction of lignin, largely attributable to a decrease in G lignin.

Fig. 1

Southern hybridisation analysis of genomic DNA (10 μg per lane) purified from P. dilatatum plants digested with HindIII (1), EcoRI (2), SacI (3) and probed with a 337 bp DIG-labelled fragment of a CCR gene amplified from P. dilatatum. M DIG Marker III

Fig. 2

Maximum likelihood phylogeny of deduced CCR protein sequences from Viridaeplantae. Numbers at nodes correspond to ML bootstrap support. Moderate to strong support (>80) is shown in black, and weak support (>79) is shown in grey. The shaded box highlights CCR1 proteins from monocotyledons. The dashed line indicates a long branch

Fig. 3

a PdCCR1 expression and b lignin content of cell wall extracts as percentage of cell wall residue (% CWR) quantified using an acetyl bromide soluble lignin assay in P. dilatatum tissues through development. Values are average and standard errors of three biological replicates. V Vegetative stage (Ps Pseudostem, L leaf), R1 early reproductive stage, R2 late reproductive stage (I1 internode 1, I2 internode 2, I3 internode 3, L1 leaf 1, L2 leaf 2, L3 leaf 3). Asterisks indicate a significant difference (t test) relative to V stage pseudostems (P < 0.05)

Fig. 4

Composition of lignin in P. dilatatum stems and leaf blades through development as determined by a Mäule staining of transverse sections and b thioacidolysis of cell wall extracts. Asterisks indicate a significant difference (t test) relative to V stage pseudostems (P < 0.05). V Vegetative stage (Ps Pseudostem, L leaf), R1 early reproductive stage, R2 late reproductive stage (I1 internode 1, I2 internode 2, I3 internode 3, L1 leaf 1, L2 leaf 2, L3 leaf 3). Epidermal cells (E), Parenchyma cells (P), Sclerenchyma cells (S), Vascular tissue (VT), scale bar = 100 μm. mg/g CW = mg per gram of dry cell wall residues (mean and standard error of three biological replicates) G guaiacyl lignin, H hydroxyphenyl lignin, S syringyl lignin

Fig. 5

a Expression of CCR1 in pseudostems and leaf blades of transgenic lines of P. dilatatum normalised to the wild-type. Data shown is mean and standard error of three replicates of each line. PS pseudostem, L leaf blade b Mäule staining of transverse sections of pseudostems from wild-type and transgenic lines of P. dilatatum. All samples were taken from plants at the vegetative stage