The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

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.

Anthocyanic Vacuolar Inclusions: From Biosynthesis to Storage and Possible Applications.

The ability of plants to accumulate specific metabolites in concentrations beyond their solubility in both aqueous and lipid environments remains a key question in plant biology. Natural Deep Eutectic Solvents (NADES) are mixtures of natural compounds in specific molar ratios, which interact through hydrogen bonding. This results in a viscous liquid that can solubilize high amounts of natural products while maintaining a negligible vapor pressure to prevent release of volatile compounds. While all the components are presents in plant cells, identifying experimental evidence for the occurrence of NADES phases remains a challenging quest. Accumulation of anthocyanin flavonoids in highly concentrated inclusions have been speculated to involve NADES as an inert solvent. The inherent pigment properties of anthocyanins provide an ideal system for studying the formation of NADES in a cellular environment. In this mini-review we discuss the biosynthesis of modified anthocyanins that facilitate their organization in condensates, their transport and storage as a specific type of phase separated inclusions in the vacuole, and the presence of NADES constituents as a natural solution for storing high amounts of flavonoids and other natural products. Finally, we highlight how the knowledge gathered from studying the discussed processes could be used for specific applications within synthetic biology to utilize NADES derived compartments for the production of valuable compounds where the production is challenged by poor solubility, toxic intermediates or unstable and volatile products.

Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles.

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography-mass spectrometry (GC-MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger ß-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC-MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites.

GA as a regulatory link between the showy floral traits color and scent.

Emission of volatiles at advanced stages of flower development is a strategy used by plants to lure pollinators to the flower. We reveal that GA negatively regulates floral scent production in petunia. We used Agrobacterium-mediated transient expression of GA-20ox in petunia flowers and a virus-induced gene silencing approach to knock down DELLA expression, measured volatile emission, internal pool sizes and GA levels by GC-MS or LC-MS/MS, and analyzed transcript levels of scent-related phenylpropanoid-pathway genes. We show that GA has a negative effect on the concentrations of accumulated and emitted phenylpropanoid volatiles in petunia flowers; this effect is exerted through transcriptional/post-transcriptional downregulation of regulatory and biosynthetic scent-related genes. Both overexpression of GA20-ox, a GA-biosynthesis gene, and suppression of DELLA, a repressor of GA-signal transduction, corroborated GA's negative regulation of floral scent. We present a model in which GA-dependent timing of the sequential activation of different branches of the phenylpropanoid pathway during flower development may represent a link between the showy traits controlling pollinator attraction, namely color and scent.

Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers.

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent.