BACKGROUND: GREEN PETAL (GP) is thought to be a petunia class B floral homeotic gene, because the gp mutant flower displays a severe homeotic conversion of petals into sepals in the second whorl. However, since the third whorl stamens remain unaffected in the gp null mutant, gp is different from class B mutants in Arabidopsis and Antirrhinum, which also show a conversion of the third whorl stamens into the carpelloid tissue. BLIND (BL) is thought to be a petunia class A floral homeotic gene, because the bl mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures in the second whorl. RESULTS: A double mutant line homozygous for both bl and gp mutations was constructed. The bl gp double mutant flower displays homeotic conversions of sepals into the stigmatoid tissue in the first whorl and of the corolla limb into antheroid structures with stigmatoid tips in the second whorl. In the third and fourth whorls of the mutant flower, organs remained unchanged. In the gp flower, a petunia B-type gene FBP1 is expressed strongly in the third whorl organs, but much more weakly in the second whorl organs. In the bl gp flower, FBP1 was found to be expressed strongly in the second whorl organs as well as in the third whorl organs. CONCLUSIONS: Petunia has a class B gene other than GP that determines organ identities, both in the second and third whorls of the double mutant flower, and the action of the postulated class B gene (here called PhBX) is prevented by the BL gene in the second whorl of the gp flower. PhBX appears to be a gene that specifically interacts with the FBP1 gene, and is involved in the up-regulation of FBP1.

We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family.

Abstract SEPALLATA (SEP) MADS-box genes are required for the regulation of floral meristem determinacy and the specification of sepals, petals, stamens, carpels and ovules, specifically in angiosperms. The SEP subfamily is closely related to the AGAMOUS LIKE6 (AGL6) and SQUAMOSA (SQUA) subfamilies. So far, of these three groups only AGL6-like genes have been found in extant gymnosperms. AGL6 genes are more similar to SEP than to SQUA genes, both in sequence and in expression pattern. Despite the ancestry and wide distribution of AGL6-like MADS-box genes, not a single loss of function mutant exhibiting a clear phenotype has yet been reported; consequently the function of AGL6-like genes has remained elusive. Here, we characterize the Petunia hybrida AGL6 (PhAGL6, formerly called PETUNIA MADS BOX GENE4/pMADS4) gene, and show that it functions redundantly with the SEP genes FLORAL BINDING PROTEIN2 (FBP2) and FBP5 in petal and anther development. Moreover, expression analysis suggests a function for PhAGL6 in ovary and ovule development. The PhAGL6 and FBP2 proteins interact in in vitro experiments overall with the same partners, indicating that the two proteins are biochemically quite similar. It will be interesting to determine the functions of AGL6-like genes of other species, especially those of gymnosperms.