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.

Phenotypic differentiation and diversifying selection in populations of Eruca sativa along an aridity gradient.

BACKGROUND: The aridity gradient in the eastern Mediterranean offers an opportunity to investigate intra-specific genetic differentiation and local adaptation in plant populations. Here we used genetic (FST) and quantitative trait (PST) differentiation to assess local adaptation among three natural populations of Eruca sativa (Brassicaceae) distributed along a climatic range representing desert, semi-arid and Mediterranean habitats. RESULTS: Amplified fragment length polymorphism (AFLP) analysis revealed high genetic diversity in each population, but low genetic differentiation between populations and relatively high gene flow. Further phenotypic evaluation in a common garden experiment (conduced in a Mediterranean habitat) showed clear differences in phenological traits among populations (day of flowering and duration of the reproductive stage), shoot and root biomass, as well as fitness-related traits (total number of fruits and total seed weight). FST-PST comparison showed that PST values of the phenological traits, as well as below- and above-ground biomass and fitness-related traits, were higher than the FST values. CONCLUSIONS: Overall, our results support the identification of genotypic and phenotypic differentiation among populations of E. sativa. Furthermore, the FST-PST comparison supports the hypothesis that these were subjected to past diversifying selection. Thus, the results clearly demonstrate adaptive divergence among populations along an aridity gradient, emphasize the ecological value of early flowering time in arid habitats, and contribute to our understanding of the possible impact of climate change on evolutionary processes in plant populations.

Ectopic Expression of PAP1 Leads to Anthocyanin Accumulation and Novel Floral Color in Genetically Engineered Goldenrod (Solidago canadensis L.).

Floral pigmentation is of major importance to the ornamental industry, which is constantly searching for cultivars with novel colors. Goldenrod (Solidago canadensis) has monochromatic yellow carotenoid-containing flowers that cannot be modified using classical breeding approaches due to a limited gene pool. To generate Solidago with novel colors through metabolic engineering, we first developed a procedure for its regeneration and transformation. Applicability of different cytokinins for adventitious regeneration was examined in the commercial cv. Tara, with zeatin yielding higher efficiency than 6-benzylaminopurine or thidiazuron. A comparison of regeneration of commercial cvs. Tara, Golden Glory and Ivory Glory revealed Tara to be the most potent, with an efficiency of 86% (number of shoots per 100 leaf explants). Agrobacterium-based transformation efficiency was highest for cv. Golden Glory (5 independent transgenic shoots per 100 explants) based on kanamycin selection and the GUS reporter gene. In an attempt to promote anthocyanin biosynthesis, we generated transgenic Solidago expressing snapdragon (Antirrhinum majus) Rosea1 and Delila, as well as Arabidopsis thaliana PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) transcription factors. Transgenic cv. Golden Glory expressing cauliflower mosaic virus 35S-driven PAP1 generated red flowers that accumulated delphinidin and its methylated derivatives, as compared to control yellow flowers in the GUS-expressing plants. The protocol described here allows efficient engineering of Solidago for novel coloration and improved agricultural traits.

Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds.

Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles ( Paris et al., 1996 ; Olbrich et al., 2007 ). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids ( Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).