Angela Jane Lidgett

Isolation and characterization of a cinnamoyl-CoA reductase gene from perennial ryegrass (Lolium perenne)

Summary Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyses the conversion of hydroxycinnamoyl-CoA thioesters to cinnamaldehydes at the entry point to the monolignol-specific branch of the lignin biosynthetic pathway. A gene encoding CCR in perennial ryegrass ( LpCCR1 ) was isolated and fully sequenced. LpCCR1 contains five exons of similar sizes to other CCRs and four introns in conserved positions. The 5′ untranslated region contains motifs common to lignin biosynthetic genes. LpCCR1 is present as a single copy gene and maps to LG7 in perennial ryegrass. LpCCR1 is constitutively expressed, is stimulated by mechanical wounding, and transcript abundance in stems increases with maturity.

Isolation and characterisation of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.)

Summary Three cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologue cDNAs ( LpCAD1, LpCAD2 and LpCAD3 ) were isolated and characterised in perennial ryegrass ( Lolium perenne ). Sequence analysis revealed a partial LpCAD3 clone is highly homologous to typical monocot CAD cDNAs while full-length LpCAD1 and LpCAD2 cDNAs show lower homology to known CAD cDNAs. Northern hybridisation analysis revealed LpCAD1 and LpCAD3 transcripts in lignifying tissues while LpCAD2 was uniquely abundant in stem tissue. All three CAD cDNAs are upregulated in response to mechanical wounding stimulus. Southern hybridisation analysis suggested that LpCAD3 is a single copy gene within the Lolium genome while LpCAD1 and LpCAD2 each belong to a small gene family.

Isolation and characterisation of a fructosyltransferase gene from perennial ryegrass (Lolium perenne)

Summary A fructosyltransferase ( LpFT1 ) cDNA and gene from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an open reading frame of 1947 bp encoding for a protein of 648 amino acids. LpFT1 is 69 % identical to barley 6-SFT, and contains plant fructosyltransferase functional domains. LpFT1 is present as a single copy gene and maps to the distal region of LG7 in perennial ryegrass. The expression pattern analysis of LpFT1 revealed transcript accumulation in young roots and shoots and in mature sheaths.

Breeding Forage Plants in the Genome Era

Forage plant breeding has been largely based on phenotypic selection following sexual recombination of natural genetic variation found between and within ecotypes. Advances in plant genetic manipulation over the last 15 years have provided convincing evidence that these powerful technologies can complement and enhance plant breeding programs. Significant progress in the establishment of the methodologies required for the molecular breeding of forage plants has been made. Examples of current products and approaches for the application of these methodologies to forage grass and legume improvement are outlined. Large-scale genomic analysis of many organisms is under way with human, arabidopsis and rice genome sequences almost completed. Forage plant breeding is just now entering the genome era. The plethora of new technologies and tools now available for high-throughput gene discovery and genome-wide gene expression analysis have opened up opportunities for innovative applications in the identification, functional characterisation and use of genes of value in forage production systems and beyond. Examples of these opportunities, such as ‘molecular phenotyping’, ‘symbio-genomics’ and ‘xeno-genomics’ are introduced.

Isolation and characterisation of an invertase cDNA from perennial ryegrass (Lolium perenne)

An invertase (LpFT2) cDNA from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an ORF of 2016 bp encoding a protein of 671 amino acids. LpFT2 is 76% identical to sugarcane soluble acid invertase, and contains invertase and fructosyltransferase functional domains. LpFT2 is present as a single copy gene and maps to the distal region of LG6 in perennial ryegrass. The expression pattern analysis of LpFT2 revealed transcript accumulation in seedlings and in mature leaf sheaths. The LpFT2 recombinant protein expressed in yeast showed invertase and fructan exohydrolase-like activities with complete breakdown of sucrose, 1-kestose (DP3), 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) into glucose and fructose.

Isolation and characterisation of three 4-coumarate : CoA-ligase homologue cDNAs from perennial ryegrass (Lolium perenne)

Summary 4-Coumarate : CoA-ligase (4CL, EC 6.2.1.12) catalyses the conversion of hydroxycinnamic acids to hydroxycinnamoyl-CoA thioesters, precursors for a variety of phenylpropanoid biosynthetic derivatives including lignins, flavonoids and phytoalexins. Three 4CL homologue cDNAs were isolated from a perennial ryegrass cDNA library. Sequence analysis indicates that Lp4CL2 and Lp4CL3 correspond to class I type 4CLs, while Lp4CL1 is most related to class II type 4CLs. Southern hybridisation analysis suggests that Lp4CL1, Lp4CL2 and Lp4CL3 each belong to a small multigene family. Northern hybridisation analysis revealed constitutive expression of the Lp4CL genes in perennial ryegrass. Lp4CL1 and Lp4CL3 were mapped to perennial ryegrass linkage groups 2 and 6 respectively.

Transgenesis and Genomics in Molecular Breeding of Temperate Pasture Grasses and Legumes

Significant advances in the establishment of the methodologies required for the molecular breeding of temperate forage grasses (Lolium and Festuca species) and legumes (Trifolium and Medicago species) are reviewed. Examples of current products and approaches for the application of these methodologies to forage grass and legume improvement are outlined. The plethora of new technologies and tools now available for high-throughput gene discovery and genome-wide expression analysis have opened up opportunities for innovative applications in the identification, functional characterisation and use of genes of value in forage production systems and beyond. Selected examples of our current work in pasture plant genomics, xenogenomics, symbiogenomics and microarray-based molecular phenotyping are discussed.