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.

Nonsense-mediated mRNA decay pathway in plants under stress: general gene regulatory mechanism and advances.

MAIN CONCLUSION: Nonsense-mediated mRNA decay in eukaryotes is vital to cellular homeostasis. Further knowledge of its putative role in plant RNA metabolism under stress is pivotal to developing fitness-optimizing strategies. Nonsense-mediated mRNA decay (NMD), part of the mRNA surveillance pathway, is an evolutionarily conserved form of gene regulation in all living organisms. Degradation of mRNA-bearing premature termination codons and regulation of physiological RNA levels highlight NMD's role in shaping the cellular transcriptome. Initially regarded as purely a tool for cellular RNA quality control, NMD is now considered to mediate various aspects of plant developmental processes and responses to environmental changes. Here we offer a basic understanding of NMD in eukaryotes by explaining the concept of premature termination codon recognition and NMD complex formation. We also provide a detailed overview of the NMD mechanism and its role in gene regulation. The potential role of effectors, including ABCE1, in ribosome recycling during the translation process is also explained. Recent reports of alternatively spliced variants of corresponding genes targeted by NMD in Arabidopsis thaliana are provided in tabular format. Detailed figures are also provided to clarify the NMD concept in plants. In particular, accumulating evidence shows that NMD can serve as a novel alternative strategy for genetic manipulation and can help design RNA-based therapies to combat stress in plants. A key point of emphasis is its function as a gene regulatory mechanism as well as its dynamic regulation by environmental and developmental factors. Overall, a detailed molecular understanding of the NMD mechanism can lead to further diverse applications, such as improving cellular homeostasis in living organisms.

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.

Polygalacturonase gene family analysis identifies FcPG12 as a key player in fig (Ficus carica L.) fruit softening.

BACKGROUND: The fig (Ficus carica L.) tree has high economic value. However, its fruit have a short shelf life due to rapid softening. Polygalacturonases (PGs) are essential hydrolases, responsible for the pectin degradation that plays a key role in fruit softening. However, fig PG genes and their regulators have not yet been characterized. RESULTS: In this study, 43 FcPGs were identified in the fig genome. They were non-uniformly distributed on 13 chromosomes, and tandem repeat PG gene clusters were found on chromosomes 4 and 5. Ka/Ks calculation and collinear analysis indicated negative selection as the main driver of FcPG family expansion. Fourteen FcPGs were found expressed in fig fruit with FPKM values > 10, of which seven were positively correlated, and three, negatively correlated with fruit softening. Eleven FcPGs were upregulated and two downregulated in response to ethephon treatment. FcPG12, a member of the tandem repeat cluster on chromosome 4, was selected for further analyses due to its sharp increment in transcript abundance during fruit softening and its response to ethephon treatment. Transient overexpression of FcPG12 led to decreased fig fruit firmness and increased PG enzyme activity in the tissue. Two ethylene response factor (ERF)-binding GCC-box sites were found on the FcPG12 promoter. Yeast one-hybrid and dual luciferase assays showed that FcERF5 binds directly to the FcPG12 promoter and upregulates its expression. Transient overexpression of FcERF5 upregulated FcPG12 expression, thereby increasing PG activity and fruit softening. CONCLUSIONS: Our study identified FcPG12 as a key PG gene in fig fruit softening, and its direct positive regulation by FcERF5. The results provide new information on the molecular regulation of fig fruit softening.

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.

A whiff of the future: functions of phenylalanine-derived aroma compounds and advances in their industrial production.

Plants produce myriad aroma compounds-odorous molecules that are key factors in countless aspects of the plant's life cycle, including pollinator attraction and communication within and between plants. For humans, aroma compounds convey accurate information on food type, and are vital for assessing the environment. The phenylpropanoid pathway is the origin of notable aroma compounds, such as raspberry ketone and vanillin. In the last decade, great strides have been made in elucidating this pathway with the identification of numerous aroma-related biosynthetic enzymes and factors regulating metabolic shunts. These scientific achievements, together with public acknowledgment of aroma compounds' medicinal benefits and growing consumer demand for natural products, are driving the development of novel biological sources for wide-scale, eco-friendly, and inexpensive production. Microbes and plants that are readily amenable to metabolic engineering are garnering attention as suitable platforms for achieving this goal. In this review, we discuss the importance of aroma compounds from the perspectives of humans, pollinators and plant-plant interactions. Focusing on vanillin and raspberry ketone, which are of high interest to the industry, we present key knowledge on the biosynthesis and regulation of phenylalanine-derived aroma compounds, describe advances in the adoption of microbes and plants as platforms for their production, and propose routes for improvement.

Tomatoes expressing thaumatin II retain their sweet taste after salting and pickling processing.

BACKGROUND: Thaumatin II, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), shows promise as a zero-calorie sweetener for use in the food and pharmaceutical industries and for improving the taste of fruit. RESULTS: We report on the stability of thaumatin in salted and pickled tomatoes, as well as on the effect of thaumatin on the taste quality of processed tomatoes. Fruit of tomato cv. Yalf, transformed with the thaumatin II gene were salted and pickled and then stored for 6 months. Western blot analysis showed relative thaumatin II stability at salting; its content in processed fruits was 62-83% of the initial level depending in the studied line. In pickled tomatoes, thaumatin II content was decreased by up to 25% of the initial amount. Both salted and pickled tomatoes had a sweet taste with a typical thaumatin aftertaste. Salted tomatoes were characterized as being sweeter than pickled tomatoes. The overall taste of pickled tomatoes was rated by panellists as significantly better compared to that of salted or non-processed ones. CONCLUSION: In the present study, we have shown that tomatoes expressing supersweet protein thaumatin II can be used for processing under mild conditions, including salting and pickling. © 2021 Society of Chemical Industry.

Proteome and transcriptome analyses reveal key molecular differences between quality parameters of commercial-ripe and tree-ripe fig (Ficus carica L.).

BACKGROUND: Fig fruit are highly perishable at the tree-ripe (TR) stage. Commercial-ripe (CR) fruit, which are harvested before the TR stage for their postharvest transportability and shelf-life advantage, are inferior to TR fruit in size, color and sugar content. The succulent urn-shaped receptacle, serving as the protective structure and edible part of the fruit, determines fruit quality. Quantitative iTRAQ and RNA-Seq were performed to reveal the differential proteomic and transcriptomic traits of the receptacle at the two harvest stages. RESULTS: We identified 1226 proteins, of which 84 differentially abundant proteins (DAPs) were recruited by criteria of abundance fold-change (FC) ≥1.3 and p < 0.05 in the TR/CR receptacle proteomic analysis. In addition, 2087 differentially expressed genes (DEGs) were screened by ≥2-fold expression change: 1274 were upregulated and 813 were downregulated in the TR vs. CR transcriptomic analysis. Ficin was the most abundant soluble protein in the fig receptacle. Sucrose synthase, sucrose-phosphate synthase and hexokinase were all actively upregulated at both the protein and transcriptional levels. Endoglucanase, expansin, beta-galactosidase, pectin esterase and aquaporins were upregulated from the CR to TR stage at the protein level. In hormonal synthesis and signaling pathways, high protein and transcriptional levels of aminocyclopropane-1-carboxylate oxidase were identified, together with a few diversely expressed ethylene-response factors, indicating the potential leading role of ethylene in the ripening process of fig receptacle, which has been recently reported as a non-climacteric tissue. CONCLUSIONS: We present the first delineation of intra- and inter-omic changes in the expression of specific proteins and genes of TR vs. CR fig receptacle, providing valuable candidates for further study of fruit-quality formation control and fig cultivar innovation to accommodate market demand.

Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

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.

Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit.

Thaumatin, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), is a promising zero-calorie sweetener for use in the food and pharmaceutical industries. Due to limited natural sources of thaumatin, its production using transgenic plants is an advantageous alternative. We report a simple protocol for purification of recombinant thaumatin II from transgenic tomato. Thaumatin was extracted from ripe tomato fruit in a low-salt buffer and purified on an SP-Sephacryl column. Recombinant thaumatin yield averaged 50 mg/kg fresh fruit. MALDI-MS analysis showed correct processing of thaumatin in tomato plants. The recombinant thaumatin was indistinguishable from the native protein in a taste test. The purified tomato-derived thaumatin had an intrinsic sweetness with a threshold value in taste tests of around 50 nM. These results demonstrate the potential of an expression system based on transgenic tomato plants for production of recombinant thaumatin for the food and pharmaceutical industries.

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

MAIN CONCLUSION: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.

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.

Petunia × hybrida floral scent production is negatively affected by high-temperature growth conditions.

Increasing temperatures due to changing global climate are interfering with plant-pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography-mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid-based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down-regulation of scent-related structural gene expression from both phenylpropanoid and shikimate pathways, and up-regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic 'Blue Spark' plants overexpressing CaMV 35S-driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long-term high-temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up-regulation of transcription might negate the adverse effects of temperature on scent production.

The R2R3-MYB-like regulatory factor EOBI, acting downstream of EOBII, regulates scent production by activating ODO1 and structural scent-related genes in petunia.

Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI's wide-ranging involvement in the production of floral volatiles.

Tomato fruits expressing a bacterial feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the shikimate pathway possess enhanced levels of multiple specialized metabolites and upgraded aroma.

Tomato (Solanum lycopersicum) fruit contains significant amounts of bioactive compounds, particularly multiple classes of specialized metabolites. Enhancing the synthesis and accumulation of these substances, specifically in fruits, are central for improving tomato fruit quality (e.g. flavour and aroma) and could aid in elucidate pathways of specialized metabolism. To promote the production of specialized metabolites in tomato fruit, this work expressed under a fruit ripening-specific promoter, E8, a bacterial AroG gene encoding a 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is feedback-insensitive to phenylalanine inhibition. DAHPS, the first enzyme of the shikimate pathway, links between the primary and specialized metabolism derived from aromatic amino acids. AroG expression influenced the levels of number of primary metabolites, such as shikimic acid and aromatic amino acids, as well as multiple volatile and non-volatile phenylpropanoids specialized metabolites and carotenoids. An organoleptic test, performed by trained panellists, suggested that the ripe AroG-expressing tomato fruits had a preferred floral aroma compare with fruits of the wild-type line. These results imply that fruit-specific manipulation of the conversion of primary to specialized metabolism is an attractive approach for improving fruit aroma and flavour qualities as well as discovering novel fruit-specialized metabolites.

EOBII, a gene encoding a flower-specific regulator of phenylpropanoid volatiles' biosynthesis in petunia.

Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plant's life cycle. Phenylpropanoid volatiles are the main determinants of floral scent in petunia (Petunia hybrida). A screen using virus-induced gene silencing for regulators of scent production in petunia flowers yielded a novel R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis, EMISSION OF BENZENOIDS II (EOBII). This factor was localized to the nucleus and its expression was found to be flower specific and temporally and spatially associated with scent production/emission. Suppression of EOBII expression led to significant reduction in the levels of volatiles accumulating in and emitted by flowers, such as benzaldehyde, phenylethyl alcohol, benzylbenzoate, and isoeugenol. Up/downregulation of EOBII affected transcript levels of several biosynthetic floral scent-related genes encoding enzymes from the phenylpropanoid pathway that are directly involved in the production of these volatiles and enzymes from the shikimate pathway that determine substrate availability. Due to its coordinated wide-ranging effect on the production of floral volatiles, and its lack of effect on anthocyanin production, a central regulatory role is proposed for EOBII in the biosynthesis of phenylpropanoid volatiles.

Metabolic engineering of plants for artemisinin synthesis.

Artemisinin, a natural compound from Artemisia annua, is highly effective in treating drug-resistant malaria. Because chemical synthesis of this natural terpenoid is not economically feasible, its only source remains as the native plant which produces only small quantities of it, resulting in a supply that is far short of demand. Extensive efforts have been invested in metabolic engineering for the biosynthesis of artemisinin precursors in microbes. However, the production of artemisinin itself has only been achieved in plants. Since, A. annua possesses only poorly developed genetic resources for traditional breeders, molecular breeding is the best alternative. In this review, we describe the efforts taken to enhance artemisinin production in A. annua via transgenesis and advocate metabolic engineering of the complete functional artemisinin metabolic pathway in heterologous plants. In both cases, we emphasize the need to apply state-of-the-art synthetic biology approaches to ensure successful biosynthesis of the drug.

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.