Vernalization downregulates Flowering Locus C in Cichorium intybus

Since proper timing of flowering is critical for the survival of plant species, plants have evolved a complex genetic network to regulate their transition to flowering in response to endogenous signals and environmental cues. In winter annuals ecotypes of Arabidopsis, a flowering repressor, FLOWERING LOCUS C (FLC), a MADS box transcription factor, is expressed at such level as to inhibit flowering in the first growing season. FLC expression is enhanced by FRIGIDA (FRI) to levels that inhibit the transition to flowering by repressing the expression of the genes often referred to as Floral Pathways Integrators. The main process promoting flowering by the repression of FLC is the vernalization and the duration of cold has been shown to be proportional to the degree of down-regulation of FLC; such repression is maintained for the rest of the plant life even after cold exposure ends, but is restored after meiosis. The repression involves epigenetically stable modifications in FLC chromatin that include a H3 Lys27 trimethylation (H3K27me3) and a H3 Lys9 trimethylation, (Sung et al, 2006). Interestingly, for the light-dependent, autonomous and GA integration and meristematic pathways, comparative genetic approaches show that flowering time genes are conserved between Arabidopsis and a large range of crop species, including legumes and cereals. By contrast, the vernalization pathway seems to be only partially conserved, since FLC and FRI were not characterized in dicots other than Brassicaceae, and recently in sugar beet, vitis and tomato. Wild chicory (Cichorium intybus L.) is a biennial species which requires vernalization to flower. In Italy different types of chicory (the so called Italian red and variegate types) have been selected by farmers as leafy vegetable. These types show quite different classes of precocity in relation to flowering. Given the high heterogeneity, in regard to flowering, manifested by plants belonging to the same variety, the "control" of the switch by agronomical procedures results difficult. The knowledge about the genetic control of flowering time in chicory could be useful to enhance the vegetative phase and then, increase the productivity of the crop. In our study, we are investigating the molecular basis that regulate the switch to flower in chicory by vernalization, to verify whether such mechanism is the same that controls flowering in Arabidopsis, and, finally, to address the diversity of the classes of precocity to one of the cases known for this model plant. We isolated FLC homologues from chicory and characterized their expression patterns in plant tissues and in response to vernalization. We also studied the pattern of cytosine methylation in chicory genomic DNA in response to vernalization. In addition, the vernalization-mediated decrease of FLC transcript was related with changes in SAM morphology. Biological function of CiFLC has been studied by AtFRIflc3 complementation. Up to now our result indicate that arabidopsis and chicory share homologies in regulating FLC expression in the vernalization response, but the absence of complementation of the mutant suggest a disagree in biological function of CiFLC or a loss of function of the transgene in Arabidopsis genetic background. Further analysis will be conducted to define if the machinery in FLC regulation and its biological function is shared between the two species. For this purpose, chicory mutants will be generated. Other aim of this work has been the identification of FLC genomic sequences in chicory. For this purpose, genome walking technique was used. Knowledge of the genomic sequence of CiFLC will allow comparing the regulative regions with those of AtFLC and performing experiments of chromosome hybridization (i.e. FISH). The goal is identify the number of copies of the gene and characterize its position within the chromosomes. With these results we will be able to formulate hypothesis about the evolution of FLC in Cichorium intybus.