Flowers present a complex display of signals to attract pollinators, including the emission of floral volatiles. Volatile emission is highly regulated, and many species restrict emissions to specific times of the day. This rhythmic emission of scent is regulated by the circadian clock; however, the mechanisms have remained unknown. In Petunia hybrida, volatile emissions are dominated by products of the floral volatile benzenoid/phenylpropanoid (FVBP) metabolic pathway. Here we demonstrate that the circadian clock gene P. hybrida LATE ELONGATED HYPOCOTYL (LHY; PhLHY) regulates the daily expression patterns of the FVBP pathway genes and floral volatile production. PhLHY expression peaks in the morning, antiphasic to the expression of P. hybrida GIGANTEA (PhGI), the master scent regulator ODORANT1 (ODO1), and many other evening-expressed FVBP genes. Overexpression phenotypes of PhLHY in Arabidopsis caused an arrhythmic clock phenotype, which resembles those of LHY overexpressors. In Petunia, constitutive expression of PhLHY depressed the expression levels of PhGI, ODO1, evening-expressed FVBP pathway genes, and FVBP emission in flowers. Additionally, in the Petunia lines in which PhLHY expression was reduced, the timing of peak expression of PhGI, ODO1, and the FVBP pathway genes advanced to the morning. Moreover, PhLHY protein binds to cis-regulatory elements called evening elements that exist in promoters of ODO1 and other FVBP genes. Thus, our results imply that PhLHY directly sets the timing of floral volatile emission by restricting the expression of ODO1 and other FVBP genes to the evening in Petunia.circadian rhythm | floral volatile | benzenoids | Petunia hybrida | LHY P lant development and physiology are extensively influenced by the circadian clock (1). The precise timing of a single plant behavioral output often requires a suite of internal mechanisms to occur in coincidence or in quick succession before the behavior taking place. Transcriptome analysis revealed that the circadian clock controls transcription of one third of genes in Arabidopsis (2). In this way, the clock can exert a holistic effect on a complex mechanism at a precise moment in time. The effectiveness of the clock's ability to coordinate complex behaviors has been used by many aspects of plant physiology, such as photosynthesis, stem and leaf growth, and flowering (3, 4).The precise timing of sexual reproductive events is critical, as plants are sessile and individuals are often spread over large distances. In addition to regulating the timing of flower formation, when they have opened, many flowers emit floral scents to lure pollinators. Attractive floral volatiles are often emitted in a rhythmic fashion, with peaks of emission coinciding with the primary pollinator's period of activity (5). Although studies have shown that rhythmic emission of scent requires the influence of a circadian clock (6-8), no study of which we are aware has shown a mechanistic link between clock function and floral volatile production.Research on floral vol...

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged.

Plant MADS-box genes have duplicated extensively, allegedly contributing to the immense diversity of floral form in angiosperms. In Arabidopsis thaliana (a core eudicot model plant), four SEPALLATA (SEP) genes comprise the E-class from the extended ABCE model of flower development. They are redundantly involved in the development of the four types of floral organs (sepals, petals, stamens and carpels) and in floral meristem determinacy. E-class genes have been examined in other core eudicots and monocots, but have been less investigated in non-core eudicots. Our goal was to functionally characterize the E-class genes in the early-diverging eudicot Thalictrum thalictroides (Ranunculaceae), whose flowers are apetalous. We identified four SEP orthologs, which when placed in a phylogenetic context, resulted from a major gene duplication event before the origin of angiosperms and a subsequent duplication at the origin of the Ranunculales. We used Virus-Induced Gene Silencing (VIGS) to down-regulate the three expressed paralogs individually and in combination to investigate their function and to determine the degree of conservation versus divergence of this important plant transcription factor. All loci were partially redundant in sepal and stamen identity and in promoting petaloidy of sepals, yet the SEP3 ortholog had a more pronounced role in carpel identity and development. The two other paralogs appear to have subfunctionalized in their cadastral roles to keep the boundaries between either sepal and stamen zones or stamen and carpel zones. Double knockdowns had enhanced phenotypes and the triple knockdown had an even more severe phenotype that included partial to complete homeotic conversion of stamens and carpels to sepaloid organs and green sepals, highlighting a role of E-class genes in petaloidy of sepals in this species. While no floral meristem determinacy defects were observed, this could be due to residual amounts of gene expression in the VIGS experiments being sufficient to perform this function or to the masking role of a redundant gene.

26During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as 27 mosses) were progressively transformed into indeterminate branching shoots with specialized 28 reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first 29 zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have 30 facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary 31 trajectory has been challenging, however, because there is no functional overlap between mosses and 32 flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis 33 in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the 34 apical stem cells of both haploid and diploid phases of the lifecycle. These results support an 35 evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was 36 progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged. 37 38 39 40 AUTHOR CONTRIBUTIONS 41 VSD cloned the CrLFY coding sequences and made the RNAi constructs during a sabbatical visit to the 42 University of Oxford; ARP and EHR performed transformations in Ceratopteris richardii and EHR 43 maintained T0 transgenic plants; ARP cloned the CrLFY1 promoter, made GUS reporter constructs, 44 validated transgenic reporter lines, conducted GUS staining, and performed gel blot analysis of CrLFY 45 copy number; SJC and KDHH screened, validated and characterized the RNAi lines; KDHH performed 46 ontogenetic gene expression analysis; VSD performed statistical analyses; SJC conducted in situ 47 localization experiments; JAL performed the phylogenetic analysis; JAL & ARP wrote the first draft of 48 the paper, all authors contributed to the final version.