Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Petal fusion in petunia (Petunia 3 hybrida) results from lateral expansion of the five initially separate petal primordia, forming a ring-like primordium that determines further development. Here, we show that MAEWEST (MAW) and CHORIPETALA SUZANNE (CHSU) are required for petal and carpel fusion, as well as for lateral outgrowth of the leaf blade. Morphological and molecular analysis of maw and maw chsu double mutants suggest that polarity defects along the adaxial/abaxial axis contribute to the observed reduced lateral outgrowth of organ primordia. We show that MAW encodes a member of the WOX (WUSCHEL-related homeobox) transcription factor family and that a partly similar function is redundantly encoded by WOX1 and PRESSED FLOWER (PRS) in Arabidopsis thaliana, indicating a conserved role for MAW/ WOX1/PRS genes in regulating lateral organ development. Comparison of petunia maw and Arabidopsis wox1 prs phenotypes suggests differential recruitment of WOX gene function depending on organ type and species. Our comparative data together with previous reports on WOX gene function in different species identify the WOX gene family as highly dynamic and, therefore, an attractive subject for future evo-devo studies.

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3-and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.

It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets 1,2 indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida 3 and the fistulata (fis) mutant of Antirrhinum majus 4,5 , which have similar homeotic phenotypes, are recessive alleles of two homologous miRNA-encoding genes. The BL and FIS genes control the spatial restriction of homeotic class C genes 6,7 to the inner floral whorls, but their ubiquitous early floral expression patterns are in contradiction with a potential role in patterning C gene expression. We provide genetic evidence for the unexpected function of the MIRFIS and MIRBL genes in the center of the flower and propose a dynamic mechanism underlying their regulatory role. Notably, Arabidopsis thaliana, a more distantly related species, also contains this miRNA module but does not seem to use it to confine early C gene expression to the center of the flower.The spatial partitioning of floral homeotic gene expression is crucial for wild-type flower development. Several transcription factors participate in this control, which aims at transcriptional silencing of the so-called 'C genes' outside their genuine expression domain in the inner two whorls of the flower, where they govern reproductive organ (stamen and carpel) development 6,7 . The functions of orthologous repressor genes, constituting the A function of the floral ABCs 6 , are, in part, comparable between different species 8 , as are some of the cis-acting regulatory regions within the large second intron of their structurally and functionally related target C genes AGAMOUS (AG) in Arabidopsis thaliana 7 , pMADS3 in P. hybrida 9 and PLENA and FARINELLI (PLE and FAR) in A. majus 10 . There are also exceptions to these overall similarities among species. For instance, orthologs of the A. thaliana APETALA2 (AP2) gene have no role in C gene regulation in P. hybrida 11 or A. majus 12 , raising the question of whether other genes fulfill this role. Candidates are the BL gene in P. hybrida and FIS in A. majus, which, when mutated, show markedly similar homeotically converted stamenoid petals in their second floral whorls 4,5 (Fig. 1).By a combination of transposon tagging and map-based cloning strategies, we cloned the BL and FIS genes and found that they encode homologous bona fide miRNAs (miRBL and miRFIS), related in their core sequences to members of the large miR169 family 13,14 (Fig. 2). The bl-1 and fis-1 alleles lie within large genomic deletions and thus represent null alleles; bl-2 and fis-2 are transposon induced and genetically unstable alleles (Fig. 2a). miRNA-encoding genes are relatively small targets for mutation, and therefore, recessive mutants are infrequent; bl and fis thus offer a rare opportunity to study and compare the function of potential orthologs in two plant species. miRNAs control gene expression by recognizing short complementary sequences in their transcripts (miRNA-recognition elements, or MREs), which are then post-transcrip...

SUMMARYSEPALLATA (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.