R2R3-MYB EVER links emission of volatiles with epicuticular wax biosynthesis in petunia petal epidermis

The epidermal cells of petunia flowers are the main site of volatile emission. However, data on the mechanisms underlying the release of volatiles into the environment are lacking. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by VIGS and CRISPR, and metabolomics we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)—a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. Using a three-step viral-based CRISPR/Cas9 editing system, ever knockout lines were generated and 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 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. LC/GC-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. Thus, we reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a new regulatory layer in the scent-emission machinery in petunia flowers.

[1]  Y. Tabach,et al.  Developmental and temporal changes in petunia petal transcriptome reveal scent-repressing plant-specific RING–kinase–WD40 protein , 2023, Frontiers in Plant Science.

[2]  T. Masci,et al.  SCARECROW-like GRAS protein PES positively regulates petunia floral scent production. , 2023, Plant Physiology.

[3]  I. Maoz,et al.  Emission of floral volatiles is facilitated by cell-wall non-specific lipid transfer proteins , 2022, bioRxiv.

[4]  A. Aharoni,et al.  Tree tobacco (Nicotiana glauca) cuticular wax composition is essential for leaf retention during drought, facilitating a speedy recovery following rewatering. , 2022, The New phytologist.

[5]  A. Aharoni,et al.  AtMYB31 is a wax regulator associated with reproductive development in Arabidopsis , 2022, Planta.

[6]  B. Savoie,et al.  Diffusion of Volatile Organics and Water in the Epicuticular Waxes of Petunia Petal Epidermal Cells. , 2022, The Plant journal : for cell and molecular biology.

[7]  Lluis Montoliu,et al.  Genome Editing , 2018, Advances in Experimental Medicine and Biology.

[8]  Sangdun Choi,et al.  CRISPR/Cas System and Factors Affecting Its Precision and Efficiency , 2021, Frontiers in Cell and Developmental Biology.

[9]  P. Morel,et al.  Petal Cellular Identities , 2021, Frontiers in Plant Science.

[10]  A. Allan,et al.  Genomic analysis uncovers functional variation in the C-terminus of anthocyanin-activating MYB transcription factors , 2021, Horticulture research.

[11]  N. Dudareva,et al.  Dynamic histone acetylation in floral volatile synthesis and emission in petunia flowers , 2021, bioRxiv.

[12]  Jiayang Li,et al.  FIS1 encodes a GA2-oxidase that regulates fruit firmness in tomato , 2020, Nature Communications.

[13]  Silvio C. E. Tosatto,et al.  The InterPro protein families and domains database: 20 years on , 2020, Nucleic Acids Res..

[14]  P. Liao,et al.  Cuticle thickness affects dynamics of volatile emission from petunia flowers , 2020, Nature Chemical Biology.

[15]  N. Dudareva,et al.  Aromatic Amino Acids: A Complex Network Ripe for Future Exploration. , 2020, Trends in plant science.

[16]  B. Bakan,et al.  Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides. , 2019, The New phytologist.

[17]  M. Farhi,et al.  Ectopic Expression of PAP1 Leads to Anthocyanin Accumulation and Novel Floral Color in Genetically Engineered Goldenrod (Solidago canadensis L.) , 2019, Front. Plant Sci..

[18]  Takayuki Tohge,et al.  Mapping the Arabidopsis Metabolic Landscape by Untargeted Metabolomics at Different Environmental Conditions. , 2018, Molecular plant.

[19]  A. Cna’ani,et al.  Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles , 2017, Front. Plant Sci..

[20]  B. Spitzer-Rimon,et al.  GA as a regulatory link between the showy floral traits color and scent. , 2017, The New phytologist.

[21]  R. Schuurink,et al.  Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter , 2017, Science.

[22]  Kevin W. Eliceiri,et al.  ImageJ2: ImageJ for the next generation of scientific image data , 2017, BMC Bioinformatics.

[23]  Rémy Bruggmann,et al.  Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida , 2016, Nature Plants.

[24]  M. Bliek,et al.  Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis , 2016, Plant Cell.

[25]  C. Kuhlemeier,et al.  MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation , 2015, Nature Genetics.

[26]  J. Flexas,et al.  Differential tissue-specific expression of NtAQP1 in Arabidopsis thaliana reveals a role for this protein in stomatal and mesophyll conductance of CO2 under standard and salt-stress conditions , 2014, Planta.

[27]  S. Shafir,et al.  PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers. , 2012, The New phytologist.

[28]  J. Noel,et al.  The Rise of Chemodiversity in Plants , 2012, Science.

[29]  M. Haring,et al.  Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals , 2012, Journal of experimental botany.

[30]  T. Sun,et al.  The Molecular Mechanism and Evolution of the GA–GID1–DELLA Signaling Module in Plants , 2011, Current Biology.

[31]  Michael S. Barker,et al.  The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants , 2011, Science.

[32]  J. Y. Kim,et al.  PhMYB4 fine-tunes the floral volatile signature of Petunia×hybrida through PhC4H , 2010, Journal of experimental botany.

[33]  Jing-Ke Weng,et al.  The origin and evolution of lignin biosynthesis. , 2010, The New phytologist.

[34]  J. Vermeer,et al.  An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour , 2008, Nature Cell Biology.

[35]  A. Aharoni,et al.  Reverse Genetics of Floral Scent: Application of Tobacco Rattle Virus-Based Gene Silencing in Petunia1[OA] , 2007, Plant Physiology.

[36]  E. Pichersky,et al.  Plant Phenylacetaldehyde Synthase Is a Bifunctional Homotetrameric Enzyme That Catalyzes Phenylalanine Decarboxylation and Oxidation* , 2006, Journal of Biological Chemistry.

[37]  J. Mol,et al.  PH4 of Petunia Is an R2R3 MYB Protein That Activates Vacuolar Acidification through Interactions with Basic-Helix-Loop-Helix Transcription Factors of the Anthocyanin Pathway[W] , 2006, The Plant Cell Online.

[38]  C. Dumas,et al.  Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent1 , 2005, Plant Physiology.

[39]  J. Ohlrogge,et al.  Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis1[W] , 2005, Plant Physiology.

[40]  Wen-Hsiung Li,et al.  Transcription Factor Families Have Much Higher Expansion Rates in Plants than in Animals1 , 2005, Plant Physiology.

[41]  S. Goff,et al.  Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo , 1997, Molecular and cellular biology.

[42]  A. Vainstein,et al.  Biogenesis of petunia and carnation corolla chloroplasts: changes in the abundance of nuclear and plastid‐encoded photosynthesis‐specific gene products during flower development , 1993 .

[43]  M. Haring,et al.  ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers , 2005 .