CAGED

1. COMPOUNDS AND GENES FOR ENHANCED PROTEIN ASSIMILATION AND DIGESTIBILITY OF FORAGE LEGUMES (CAGED -FAIR CT 98-4068) CAGED Bloat is a digestive disorder occurring in ruminants fed with fresh forage of many cultivated legumes e.g. lucerne and clover. In extreme cases the rumen blows up and the animal dies. This build up of gas results from the degradation of dietary protein in the rumen by micro-organisms. It has been observed that legume forages which contain low levels of condensed tannins (CT), e.g. Lotus, protect against bloat. The CT complexes with dietary protein and decreases rates of degradation. However, it is possible to have too much CT. This is true for some forages, e.g. Coronilla, the high levels of CT in these species results in poor digestibility, palatability and acceptability by the animal. The aim of forage legume breeders is therefore to induce desirable levels of CT biosynthesis in CT negative forages and reduce CT levels to acceptable levels in high CT species. This goal has not been achieved by interspecific crosses and somatic hybridisation. The long-term aim of the CAGED project (http://caged.irmgpf.pg.cnr.it/) is to assess options for the genetic engineering of commercially grown forage legumes to express desirable levels of CT by genetic transformation with CT specific structural and regulatory genes. The CT biosynthetic pathway shares many steps with anthocyanin synthesis, which has been characterised extensively. Many genes are common to the two pathways and these have been isolated and characterised. There is little information however, on the CT pathway genes from the point where the pathways diverge. It is paramount to the project that these terminal genes are identified and isolated. Central to our strategies is the species Lotus corniculatus. This model crop is a bone fide forage legume that is polymorphic for CT and well adapted to in vitro manipulation and genetic transformation. It is intended to use L. corniculatus for the preliminary task of isolating CT genes characterising their regulation and precise role in CT synthesis. We are additionally analysing protein degradation and fibre digestion in several forage legumes. The three partners of the CAGED project each have special expertise in areas that are complementary to this pan-European project. IRMGPF has extensive experience of plant genetic manipulation for alterations in CT phenotype, transformation of Lotus and analysis of transgenic lines for CT biosynthesis. The Genetics and Breeding of Forage Legumes Group at INRA have extensive experience of forage digestibility and protein degradation analysis and plant breeding. IGER has a central role in CAGED that enables the interaction of these two research groups. This is made possible by a broad technical skill base which includes plant molecular and analytical chemistry and a broad knowledge of Lotus cultivation and cultivar development. Institute for Grassland and Environmental Research, Aberystwyth, UK (Email:mark.robbins@bbsrc.ac.uk: Phillip.morris@bbsrc.ac.uk gordon.allison@bbsrc.ac.uk) Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, Perugia, Italy (Email: F.Damiani@irmgpf.pg.cnr.it) Station d’Amélioration des Plantes Fourragères INRA, Lusignan, France (Email:huyghe@lusignan.inra.fr; julier@lusignan.inra.fr) It is the role of IRMGPF to isolate genes from the terminal steps of the CT pathway. This will be achieved by the differential screening of RNA isolated from Lotus plants with high and low levels of CT by the two techniques of cDNA-AFLP and differential PCR (PCR-select Clontech). The task of differential screening has been facilitated by the production of transgenic lines of L. corniculatus that have been transformed with the mycgene Sn from Zea mays. This regulatory gene is a transactivator of anthocyanin biosynthesis in maize. In Lotus, Sn acts to alter levels of CT in leaves. Figure 1 shows the vast difference in CT in a sample leaf taken from a control plant (A) and one from a line from a CT up-regulated line (B). IGER has achieved several tasks in the first half of the CAGED project. Firstly, the Sn transgenic lines produced at Perugia have been characterised by an extensive chemical analysis of secondary metabolites (see Fig 2). These data are strongly indicative that Sn acts specifically on CT synthesis in Lotus and does not alter levels of other major end products. These data confirm the appropriateness of these transgenic lines as source material for differential screening. The Genetics and Breeding of Forage Legumes Group has used in vivo and in vitro methods to obtain quantitative data on the effects of CT on protein degradation and fibre digestibility in the rumen. These experiments have used a range of freeze-dried Lotus leaf and stem materials with differing amounts of CT (supplied by IGER). Preliminary results show clearly that there is substantially less degradation of dietary protein in the rumen over the first 12 h after consumption in leaves with 4.9% percentage dry mass CT as compared with leaves with only 0.6% CT (Fig 3a). After this time the protective effects of CT seem to diminish. Figure 3b shows that CT also effects the rate of digestion of dry matter in the rumen; however, this effect is less pronounced than that on protein degradation and does not decrease with time. These data also demonstrate that leaf material is more readily digestible, and susceptible to deamination, than stem material (Fig 3 a &b). Figure 2 Figure 1 Figure 3 A B C Chemical analysis of metabolic end-products for Sn transgenic and control lines: A, condensed tannins, B, flavonols and C, TGA soluble lignin. A B Leaves of L. corniculatus linesstained for the presence of condensed tannins with DMACA: A, control line, B. Sn transgenic line Sn 11. A B In addition to these studies, several sequences have been isolated form CT specific structural genes. These are being used to construct transgenic lines with altered CT phenotypes. The precise effects on CT accumulation and composition of these genes, and those genes isolated in Perugia, will be assessed in transgenic Lotus lines and reported in the near future. in saco analysis of protein degradation (A) and dry matter digestibility (B) of freeze-dried Lotus leaf and stem samples. CT content: leaf I=4.87% dry matter, leaf B=0.56%dry matter, stem B=0.34% dry matter. For differential screening to be successful it is vital that there is no genetic variation between the transgenic lines other than that due to the specific transformation. This has been made possible by making these lines in the isogenic genotype S50 (cultivar Leo) supplied to IPMGPF by IGER. Several sequences have been isolated so far and these will be assessed functionally at IGER once their differentiality has been verified. Further analysis of other Lotus samples are now in progress. In addition, these methods are being applied to determine the digestibility and protein degradation of other forage legume species e.g. lucerne, clover and Coronilla. Finally, the exact effect of CT composition on these parameters will be assessed by adding purified CT (supplied and characterised by IGER) to the plant material. These findings will be reported in full when available. CAGED is a project funded by the European Union. The Statements made here do not necessarily reflect EU policy. Poster compiled by Gordon Allison, IGER