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.

Petunia PHYTOCHROME INTERACTING FACTOR 4/5 transcriptionally activates key regulators of floral scent.

Floral scent emission of petunia flowers is regulated by light conditions, circadian rhythms, ambient temperature and the phytohormones GA and ethylene, but the mechanisms underlying sensitivity to these factors remain obscure. PHYTOCHROME INTERACTING FACTORs (PIFs) have been well studied as components of the regulatory machinery for numerous physiological processes. Acting redundantly, they serve as transmitters of light, circadian, metabolic, thermal and hormonal signals. Here we identified and characterized the phylogenetics of petunia PIF family members (PhPIFs). PhPIF4/5 was revealed as a positive regulator of floral scent: TRV-based transient suppression of PhPIF4/5 in petunia petals reduced emission of volatiles, whereas transient overexpression increased scent emission. The mechanism of PhPIF4/5-mediated regulation of volatile production includes activation of the expression of genes encoding biosynthetic enzymes and a key positive regulator of the pathway, EMISSION OF BENZENOIDS II (EOBII). The PIF-binding motif on the EOBII promoter (G-box) was shown to be needed for this activation. As PhPIF4/5 homologues are sensors of dawn and expression of EOBII also peaks at dawn, the prior is proposed to be part of the diurnal control of the volatile biosynthetic machinery. PhPIF4/5 was also found to transcriptionally activate PhDELLAs; a similar positive effect of PIFs on DELLA expression was further confirmed in Arabidopsis seedlings. The PhPIF4/5-PhDELLAs feedback is proposed to fine-tune GA signaling for regulation of floral scent production.

SCARECROW-like GRAS protein PES positively regulates petunia floral scent production.

Emission of scent volatiles by flowers is important for successful pollination and consequently, reproduction. Petunia (Petunia hybrida) floral scent is formed mainly by volatile products of the phenylpropanoid pathway. We identified and characterized a regulator of petunia scent production: the GRAS protein PHENYLPROPANOID EMISSION-REGULATING SCARECROW-LIKE (PES). Its expression increased in petals during bud development and was highest in open flowers. Overexpression of PES increased the production of floral volatiles, while its suppression resulted in scent reduction. We showed that PES upregulates the expression of genes encoding enzymes of the phenylpropanoid and shikimate pathways in petals, and of the core regulator of volatile biosynthesis ODORANT1 by activating its promoter. PES is an ortholog of Arabidopsis (Arabidopsis thaliana) PHYTOCHROME A SIGNAL TRANSDUCTION 1, involved in physiological responses to far-red (FR) light. Analyses of the effect of nonphotosynthetic irradiation (low-intensity FR light) on petunia floral volatiles revealed FR light as a scent-activating factor. While PHYTOCHROME A regulated scent-related gene expression and floral scent production under FR light, the influence of PES on volatile production was not limited by FR light conditions.

Spatial patterning of scent in petunia corolla is discriminated by bees and involves the ABCG1 transporter.

Floral guides are patterned cues that direct the pollinator to the plant reproductive organs. The spatial distribution of showy visual and olfactory traits allows efficient plant-pollinator interactions. Data on the mechanisms underlying floral volatile patterns or their interactions with pollinators are lacking. Here we characterize the spatial emission patterns of volatiles from the corolla of the model plant Petunia × hybrida and reveal the ability of honeybees to distinguish these patterns. Along the adaxial epidermis, in correlation with cell density, the petal base adjacent to reproductive organs emitted significantly higher levels of volatiles than the distal petal rim. Volatile emission could also be differentiated between the two epidermal surfaces: emission from the adaxial side was significantly higher than that from the abaxial side. Similar emission patterns were also observed in other petunias, Dianthus caryophyllus (carnation) and Argyranthemum frutescens (Marguerite daisy). Analyses of transcripts involved in volatile production/emission revealed lower levels of the plasma-membrane transporter ABCG1 in the abaxial versus adaxial epidermis. Transient overexpression of ABCG1 enhanced emission from the abaxial epidermis to the level of the adaxial epidermis, suggesting its involvement in spatial emission patterns in the epidermal layers. Proboscis extension response experiments showed that differences in emission levels along the adaxial epidermis, that is, petal base versus rim, detected by GC-MS are also discernible by honeybees.

The phosphatase/kinase balance affects phytochrome A and its native pools, phyA' and phyA″, in etiolated maize roots: evidence from the induction of phyA' destruction by a protein phosphatase inhibitor sodium fluoride.

Phytochrome A (phyA) comprises two native types, phyA' and phyA″, with distinct spectroscopic, photochemical, and functional properties, differing at the N-terminal extension, probably, by the state of phosphorylation. To find out if and how protein phosphatases (PP) affect the state of the phyA species in planta, we studied the effect of the non-specific phosphatase inhibitor NaF on etiolated maize seedlings with the use of low-temperature fluorescence spectroscopy and photochemistry. In roots, phosphatase inhibition facilitated photoreceptor destruction in its labile phyA' form and shifted the phyA'/phyA″ ratio towards the more stable phyA″. The effect of NaF was not observed in stems. It was similar, though less pronounced, in comparison to the effects of the serine/threonine PP inhibitors, okadaic and cantharidic acids (OA and CA), which likewise facilitate the destruction of phyA' in etiolated maize stems, not, however, in roots (Sineshchekov et al., Photochem. Photobiol 89:83-96, 2013). The phyA'/phyA″ balance thus depends on the kinase/phosphatase equilibrium in the root cells. The relatively low effect of NaF on phyA in roots, together with the lack of the effect of OA and CA in them, may imply that the mechanism controlling the phyA'/phyA″ balance in roots can be different from that in shoots.

CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and the Circadian Control of Stomatal Aperture.

The endogenous circadian (∼24 h) system allows plants to anticipate and adapt to daily environmental changes. Stomatal aperture is one of the many processes under circadian control; stomatal opening and closing occurs under constant conditions, even in the absence of environmental cues. To understand the significance of circadian-mediated anticipation in stomatal opening, we have generated SGC (specifically guard cell) Arabidopsis (Arabidopsis thaliana) plants in which the oscillator gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) was overexpressed under the control of the guard-cell-specific promoter, GC1. The SGC plants showed a loss of ability to open stomata in anticipation of daily dark-to-light changes and of circadian-mediated stomatal opening in constant light. We observed that under fully watered and mild drought conditions, SGC plants outperform wild type with larger leaf area and biomass. To investigate the molecular basis for circadian control of guard cell aperture, we used large-scale qRT-PCR to compare circadian oscillator gene expression in guard cells compared with the "average" whole-leaf oscillator and examined gene expression and stomatal aperture in several lines of plants with misexpressed CCA1 Our results show that the guard cell oscillator is different from the average plant oscillator. Moreover, the differences in guard cell oscillator function may be important for the correct regulation of photoperiod pathway genes that have previously been reported to control stomatal aperture. We conclude by showing that CONSTANS and FLOWERING LOCUS T, components of the photoperiod pathway that regulate flowering time, also control stomatal aperture in a daylength-dependent manner.

PHYTOCHROME INTERACTING FACTORS mediate metabolic control of the circadian system in Arabidopsis.

The circadian (c. 24 h) system has a central role in regulating the timing and coordination of photosynthesis, and in turn photosynthesis and photosynthetic products which are controlled by the circadian clock feedback to affect the circadian oscillator that generates rhythms. However, little is known about the mechanism(s) by which this feedback occurs. One group of likely candidates for signal transduction to the circadian clock are the PHYTOCHROME INTERACTING FACTOR (PIF) family of transcription factors which have been shown to be involved in numerous signaling pathways in Arabidopsis. Yet despite evidence that some PIF genes are under circadian control and bind promoter motifs present in circadian genes, until now PIFs have not been shown to affect the circadian system. Using a range of techniques, we have examined how circadian rhythms are affected in higher order pif mutants and the mechanisms by which PIFs regulate signaling to the circadian clock. We show that PIFs mediate metabolic signals to the circadian oscillator and that sucrose directly affects PIF binding to the promoters of key circadian oscillator genes in vivo that may entrain the oscillator. Our results provide a basis for understanding the mechanism for metabolic signaling to the circadian system in Arabidopsis.

Developmental and temporal changes in petunia petal transcriptome reveal scent-repressing plant-specific RING-kinase-WD40 protein.

In moth-pollinated petunias, production of floral volatiles initiates when the flower opens and occurs rhythmically during the day, for optimal flower-pollinator interaction. To characterize the developmental transcriptomic response to time of day, we generated RNA-Seq databases for corollas of floral buds and mature flowers in the morning and in the evening. Around 70% of transcripts accumulating in petals demonstrated significant changes in expression levels in response to the flowers' transition from a 4.5-cm bud to a flower 1 day postanthesis (1DPA). Overall, 44% of the petal transcripts were differentially expressed in the morning vs. evening. Morning/evening changes were affected by flower developmental stage, with a 2.5-fold larger transcriptomic response to daytime in 1DPA flowers compared to buds. Analyzed genes known to encode enzymes in volatile organic compound biosynthesis were upregulated in 1DPA flowers vs. buds-in parallel with the activation of scent production. Based on analysis of global changes in the petal transcriptome, PhWD2 was identified as a putative scent-related factor. PhWD2 is a protein that is uniquely present in plants and has a three-domain structure: RING-kinase-WD40. Suppression of PhWD2 (termed UPPER - Unique Plant PhEnylpropanoid Regulator) resulted in a significant increase in the levels of volatiles emitted from and accumulated in internal pools, suggesting that it is a negative regulator of petunia floral scent production.

PIF-mediated sucrose regulation of the circadian oscillator is light quality and temperature dependent.

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.

The Impact of Domestication on the Circadian Clock.

A recent publication shows that, during their domestication and spread from Equatorial South America, circadian rhythms of tomatoes have been modified. The modifications have resulted in tomato plants that are adapted to growing under the long day conditions characteristic of summers at higher latitudes.

The use of fluorescent proteins to analyze circadian rhythms.

Compared with luciferase which is widely used as a reporter for circadian rhythms in Arabidopsis thaliana, available fluorescent markers are generally too stable to allow circadian oscillations to be measured. However, we have developed a technique to use the nuclear localization of circadian-controlled transcription factors fused to a fluorescent reporter as a means of measuring circadian rhythms. This technique has the advantage of being suitable for analyzing rhythms at the level of individual cells and in living plants.

Protein phosphatase activity and acidic/alkaline balance as factors regulating the state of phytochrome A and its two native pools in the plant cell.

Phytochrome A (phyA), the most versatile plant phytochrome, exists in the two isoforms, phyA' and phyA'', differing by the character of its posttranslational modification, possibly, by phosphorylation at the N-terminal extension [Sineshchekov, V. (2010) J. Botany 2010, Article ID 358372]. This heterogeneity may explain the diverse modes of phyA action. We investigated possible roles of protein phosphatases activity and pH in regulation of the phyA pools' content in etiolated seedlings of maize and their extracts using fluorescence spectroscopy and photochemistry of the pigment. The phyA'/phyA'' ratio varied depending on the state of development of seedlings and the plant tissue/organ used. This ratio qualitatively correlated with the pH in maize root tips. In extracts, it reached a maximum at pH ≈ 7.5 characteristic for the cell cytoplasm. Inhibition of phosphatases of the PP1 and PP2A types with okadaic and cantharidic acids brought about phyA' decline and/or concomitant increase of phyA'' in coleoptiles and mesocotyls, but had no effect in roots, revealing a tissue/organ specificity. Thus, pH and phosphorylation status regulate the phyA'/phyA'' equilibrium and content in the etiolated (maize) cells and this regulation is connected with alteration of the processes of phyA' destruction and/or its transformation into the more stable phyA''.