Identification of novel genes involved in Petunia flower development using transcript profiling and reverse genetics

Resumo

Petals are a key element on plant life cycle as, in many species, they attract pollinators, thus aiding to reproduction. Furthermore, they have economic importance in ornamental crops. In the present study, petal transcriptional patterns were compared within the flower organs in Arabidopsis thaliana. It was found that catalytic molecular functions were overrepresented in petals. A shortlist comprising the top ten differentially expressed genes in petals were mapped to the model species with industrial value Petunia hybrida, and further downregulated by RNAi. The silencing phenotypes found permitted to assign functions in petal development to seven novel genes: when silenced, they triggered alterations on flower size and shape (PhCYP76, PhNPH3, PhFeSOD, PhXTH, PhCYP96 and PhWAK ), petal smoothness (PhPRA), color (PhNPH3 and PhWAK ) and symmetry (PhCYP76 ). Pleiotropic phenotypes were found, such as changes in root morphology and leaf color (PhCYP76 ), flower number, capsule and seed morphology (PhCYP96 ) and plant height (PhCYP76 and PhCYP96 ). To accomplish the experimental design, three methods were developed. First, the ¿pESTle¿ management system that assembles, annotates, stores and serves expressed sequence tag data. Second, a reference gene selection for real time PCR experiments that includes a new method for stability estimation based on rank aggregation of published algorithms, and concludes that a normalization factor with two members of EF1¿, SAND, CYP or RAN1 is stable enough under most conditions. And third, a PCR efficiency estimator based on amplicon characteristics which allows efficiency-driven primer design in a Web tool.