Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.04

Expression of flavonoid genes in Lotus corniculatus mutants under different light treatments

Paolocci F., Tosti N., Bovone T., Damiani F.

IRMGPF- CNR, Perugia

condensed tannins, light, real time RT- PCR, forage legumes, anthocyanins

The importance of Condensed Tannins (CT) in forage legumes has been well documented in several studies. The research goal in this field is the acquisition of the know-how in order modulate the synthesis of CT in the leaves. In fact moderate amount of CT in the forages (2-5 % of dry matter) are welcome since they protect plant protein from microbial degradation occurring in the rumen and in the silage. Conversely, excessive amounts of CT (> of 10% of dry matter) have a negative impact on forage digestibility and palatability. This is the reason why several high productive species have null agronomic value.

In previous experiments we produced Lotus corniculatus transgenic individuals polymorphic for leaf CT accumulation through the transformation of a single genotype with a bHLH mais gene under a constitutive promoter. High variability in the level of CT accumulation is also induced by environmental conditions. Light, temperature and availability of storage compounds are demonstrated to interfere profoundly with CT accumulation. With the present work we wanted to analyse the expression profile of some key genes of the anthocyanin/CT pathway in CT polymorphic transgenic lines and control plants, grown under weak, low and high light conditions.

Cuttings of the each plant were grown replicated in the cabinet at 150, 450 and 1000 µE/cm light in the PAR. Leaves were stained for CT and total RNA was extracted and analysed through real time RT-PCR with primers designed on the sequence of PAL (phenylalanine ammonia liase) CHS (chalcone synthase), DFR (di-hydro flavonol reductase) ANS (anthocyanin synthase) genes. Repeated experiments were performed and data were elaborated as quantitative traits. Therefore, the analysis of variance for the expression of each gene was calculated, using different calibration systems: expression of house keeping genes (1-a Elongation Factor and b tubulin), total cDNA and quantitative amplification of DNA.

A strong interaction between genotype and light treatment occurred. Transgenic plants over-expressing CT showed higher CT levels at increasing light treatment on the contrary low CT transgenic plants did not show a consistent light effect on CT levels. Also for gene expression a significant value of the interaction (light treatment x genotype) was reported. A significant correlation between DFR and ANS expression and CT levels was observed. On the contrary, PAL and CHS did not show significant differences in their steady state level among genotypes, suggesting that they are not intimately involved in the polymorphism observed for CT accumulation in transgenic lines. In addition, the DFR and ANS genes were transactivated by the exogenous bHLH gene, whereas PAL and CHS are not.

Interestingly, it was observed , that low CT phenotypes show an increased polymorphisms relative to the dissociation profile of CHS and DFR cDNA amplicons. It is therefore likely that in non permissive conditions other loci were activated to compensate the reduced expression of the canonical ones.