Genetic Analysis of Redundant and Diverged MADS-box Genes Involved in Floral Development in Petunia hybrida

Contains fulltext : 29878.pdf (publisher's version ) (Open Access)Flowers play a central role in angiosperm biology. Genetic studies of floral mutants in Arabidopsis and Antirrhinum laid the basis for the ABC model. Most of these genes are MADS-box genes and their proteins act as transcription factors in binding DNA as dimers. In Petunia, B-class genes are duplicated. PhGLO1 and PhGLO2 belong to the GLO/PI lineage; PhDEF and PhTM6 belong to the DEF/AP3 lineage. Functional analysis has been performed by creating single, double and triple mutant combinations with available transposon insertion alleles of phglo1, phglo2, phtm6, and phdef (EMS). Single and double mutants were also crossed with the A-function mutant blind. Expression of B- and C-class genes in wild-type and various mutant flowers was determined by real-time PCR, a yeast two-hybrid assay was performed, in situ localization for the PhGLO2 and PhTM6 transcripts, and an RNAi strategy was applied to downregulate PhTM6. PhTM6 was also overexpressed in wild-type and phdef mutant backgrounds. Overall, petal development in Petunia is controlled by the concerted action of PHDEF/PHGLO1 and PHDEF/PHGLO2 heterodimers. Function, regulation and interaction of PHTM6 are different compared to the known euAP3-type DEF/AP3-like proteins. Our results indicate that PHDEF/PHGLO2, PHDEF/PHGLO1 and PHTM6/PHGLO2 heterodimers are sufficient to determine stamen identity. Expression maintenance of PhDEF, PhGLO1, and PhGLO2 is interdependent in the second whorl. Our results also indicate PHDEF and PHTM6 act redundantly in maintaining PhGLO1 and PhGLO2 expression in the third whorl. These results show that PHDEF and PHTM6 proteins have functionally diverged and are differently regulated. PHDEF functions as a classical euAP3 type protein in the determination of petals and stamens. PHTM6, as a B-type protein, is only involved in conferring stamen identity. Noteworthy, ectopic expression of PhTM6 is able to restore petal development in a phdef mutant background and it also induces development of ectopic petals in wild-type flowersRU Radboud Universiteit Nijmegen, 18 december 2006Promotor : Gerats, A.G.M.192 p

The duplicated B-class heterodimer model: Whorl-specific effects and complex genetic interactions in Petunia hybrida flower development

Item does not contain fulltextIn both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes