Molecular analysis of petal development by X-ChIP and two-hybrid technology

Flowers of eudicots like Antirrhinum and Arabidopsis are composed of four concentric whorls, where four different kinds of organs develop: sepal and petals in the two outermost whorls and the sexual organs, stamens and carpels, in the centre of the flower. Organ development is determined by homeotic genes that interact according to the ABCDE model. The homeotic B class genes, represented by DEFICIENS (DEF) and GLOBOSA (GLO) in Antirrhinum and APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis, participate together with class A and E factors in regulating petals development and together with class C and E in stamens development. In the work here presented, class B target genes are investigated in Antirrhinum and Arabidopsis by Crosslinked Chromatin ImmunoPrecipitation (X-ChIP) experiments, to prove the direct protein-DNA interaction by an immunoprecipitation. In this study a new approach is presented and discussed for the X-ChIP data analysis that includes more controls with respect to the standard procedure reported in literature, and that allows the discrimination of false positive results. This new approach showed that the interaction of DEF protein with the promoter of the putative targets GLOBOSA, DEFICIENS and EXTENSIN is not demonstrable at these conditions and revealed its interaction with the promoter of DEFH125. Protein-protein and protein-DNA interactions that involve Class B factors have been studied in yeast: new interactors of AP3 and PI were isolated by a three-hybrid screening. Class B transcription factors determine the fate of petal primordia, but not their initiation, which is controlled by other genes, like ROXY1, which belongs to the plant glutaredoxin (GRX) family. roxy1 mutant phenotype presents a reduced number of petals and abnormalities during further petal development. GRXs are small proteins involved in posttranslational modifications of target proteins via redox reactions. In order to shed light on its activity, ROXY1 was used as bait in a two-hybrid screening. Among many isolated clones, the transcription factors TCP3, TCP4 and TCP21 were chosen for a further investigation. TCP proteins have been shown to participate in the control of cell proliferation and can be functionally linked to ROXY1 because, at early stages of roxy1 developing flower, misregulation of cell proliferation leads to phenotypic abnormalities. In order to support an in vivo interaction, in situ hybridizations were conducted, revealing that the expression domains of TCP3, TCP4 and TCP21 overlap with that of ROXY1 at early stages of floral development