Stomatal guard cells are controlled by a modified circadian oscillator to ensure correct daylength-dependent regulation of stomatal aperture.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)—a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography–MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

In petunia (Petunia × hybrida), MADS-box homeotic genes dictate floral organ identity. For instance, DEFICIENS (PhDEF), GLOBOSA1, and GLOBOSA2 (PhGLO1/2) are responsible for petal and stamen identity. However, whether homeotic genes, particularly PhDEF, have a function at the later stages of flower development remains elusive. In petunia flowers, scent production initiates at anthesis, when the flower is ready for pollination, and is triggered by activation of EMISSION OF BENZENOIDS I (EOBI) and EOBII, MYB transcriptional regulators of scent-related genes. Here, we revealed the role of PhDEF in mature flowers, showing that it activates scent production. PhDEF suppression using a transient viral system in petunia flowers led to a significant reduction in volatile emission and pool levels, and in the transcript levels of scent-related transcriptional regulators and enzymes. Promoter activity assays demonstrated that PhDEF activates EOBI, EOBII, and the phenylpropanoid biosynthesis genes L-PHENYLALANINE AMMONIA LYASE and PHENYLACETALDEHYDE SYNTHASE. Our findings underscore the importance of PhDEF in petunia flower development from initiation to maturation and in coordinating petal specification and the establishment of showy pollination-related traits.

Studies are increasingly showing that metabolic and circadian (~24 h) pathways are strongly interconnected, with the circadian system regulating the metabolic state of the cell, and metabolic products feeding back to entrain the oscillator. In plants, probably the most significant impact of the circadian system on metabolism is in its reciprocal regulation of photosynthesis; however, the pathways by which this occurs are still poorly understood. We have previously shown that members of the basic helix-loop-helix (bHLH) transcription factor PHYTOCHROME INTERACTING FACTOR (PIF) family are involved in the photosynthate entrainment of the circadian oscillator. In this paper, using Arabidopsis mutants and overexpression lines, we examine how temperature and light quality affect PIF-mediated sucrose signaling to the oscillator and examine the contributions of individual PIF members. Our results also show that the quality of light is important for PIF signaling, with red and blue lights having the opposite effects, and that temperature affects PIF-mediated sucrose signaling. We propose the light sensitivity of PIF-mediated sucrose entrainment of the oscillator may be important in enabling plants to distinguish between sucrose produced de novo from photosynthesis during the day and the sucrose products of starch degradation at the end of the night.