Expression analysis in response to low temperature stress in blood oranges: implication of the flavonoid biosynthetic pathway.

The productivity and the geographical distribution of most commercially important Citrus varieties are markedly affected by environmental low temperatures. As gene engineering has been shown to be a favourable alternative to produce germplasm with improved cold tolerance, a broad group of cold regulated genes have been previously identified from several Citrus spp. By contrast, little information regarding the cold stress response of pigmented sweet orange varieties is available although they might provide a pivotal contribution to define the whole events occurring in cold exposed Citrus fruits. In our work, the transcriptome analysis based on subtractive hybridisation was performed in order to emphasise the overall induction in gene expression after the exposure of blood oranges [(Citrus sinensis) L. Osbeck Tarocco Sciara] to low temperature. The cold induction of several gene expressions was then validated by real-time RT-PCR. Overall, we observed the enhancement of transcripts involved in the defence mechanisms against oxidative damage, osmoregulating processes, lipid desaturation as well as many ESTs implicated in the primary and secondary metabolisms. In particular, the results show that cold stress induces transcriptomic modifications directed towards the increase of flavonoid biosynthesis, including those reactions involved in anthocyanin biosynthesis, as well as of the metabolic pathways supplying it. By comparing the blood orange response to cold stress with those of other plant sources, such as grapefruit, it seems to be similar to that of the chilling acclimated species. Interestingly, among the genes encoding for the regulatory proteins, several transcription factors have been identified for the first time as cold responsive genes in plants, indicating novel investigation lanes which should receive special attention in the future.