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 -olor and scent. (New Phytologist (2015) 208: 708-4)

Increasing temperatures due to changing global climate are interfering with plant-llinator 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 xybrida 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. (Plant, Cell and Environment (2015) 38, 1333-46)

Many attempts have been made to convert plants into efficient factories. The ability to modify genome sequences in plant cells is fundamental to modern agriculture, but current methods - classical and moleculular - are extremely inefficient. To overcome the biotechnological barriers to precise genome editing, we developed MemoGene technology for the creation of commercially successful cultivars in leading high-value plants. MemoGene (commercially developed by Danziger Innovations Ltd.) is a viral-based tissue-culture-independent technology that bypasses traditional genetic engineering for precise plant-genome modification in all plants. It is based on highly efficient viral vectors for DNA delivery and targeted endonucleases for nuclear and plastid genome manipulations: site-specific genetic modifications can be applied to plants and cell cultures, to meet the rapidly shifting trends in the areas of field and vegetable crops, horticulture, woody crops, bio-fuels, etc.

Secondary metabolites determine color, flavor, fragrance and the health-beneficial nutritional/pharmacological value of foods, beverages, detergents, cosmetics and pharmaceutical products. Tools allowing efficient metabolic engineering of these products have a major impact on the almost limitless world bio-agriculture market. Our characterization of novel plant-regulatory factors allowed us to genetically engineer petunia flowers with ca. 10-fold higher scent production/emission, and to transform naturally white flowering gypsophila, sold worldwide, into a novel cut-flower crop with purple flowers (currently being commercially developed by Danziger "Dan" Flower Farm). Our studs in yeast of factors regulating production of the secondary metabolite artemisinin, the most important antimalarial drug today, aim to boost artemisinin production in host Artemisia annua plants, and to produce it in tobacco and aspen. The drug will be produced at high levels in planta by inducing the expression of five genes necessary for its synthesis in specific tissues, cell types and intracellular compartments at specific developmental stages. The development of yeast/plant-based approaches (supported by Isaac Kaye award) for this drug's cost-effective production will bring this much awaited remedy to developing nations.

Floral scent is a complex trait of biological and applied significance. To evaluate whether scent production originating from diverse metabolic pathways (e.g. phenylpropanoids and isoprenoids) can be affected by transcriptional regulators, Arabidopsis PAP1 transcription factor was introduced into Rosa hybrida.In addition to increased levels of phenylpropanoid-derived color and scent compounds as compared to control flowers, PAP1-transgenic rose lines also emitted up to 6.5 times higher levels of terpenoid scent compounds. Olfactory assay revealed that bees and humans could discriminate between the floral scents of PAP1-transgenic and control flowers. The increase in volatile production in PAP1 transgenes was not due solely to transcriptional activation of their respective biosynthetic genes but probably also resulted from enhanced metabolic flux in both the phenylpropanoid and isoprenoid pathways. (New Phytologist 195:xx ( 2012)

The emergence of multidrug-resistant strains of Plasmodium spp., the etiological agent of malaria, constitutes a major threat to controlling the disease. Artemisinin, a natural compound from Artemisia annua (sweet wormwood) plants, is highly effective against drug-resistant malaria. The World Health Organisation (WHO; Geneva) promotes the use of artemisinin as a first-line treatment for malaria, and it is heavily involved in facilitating the development of artemisinin-based anti-malaria drugs1.Even so, low-cost artemisinin-based drugs are lacking because of the high cost of obtaining natural or chemically synthesized artemisinin. Despite extensive effort invested in the past decade in metabolic engineering of artemisinin and its precursors in both microbial and heterologous plant systems, production of artemisinin itself has never been achieved. Here we report the metabolic engineering of tobacco to produce artemisinin, generating transgenic plants that express five plant- and yeast-derived genes involved in the mevalonate and artemisinin pathways, all expressed from a single vector. Our experiments demonstrate that artemisinin can be fully biosynthesized in a heterologous (that is, other than A. annua) plant system, such as tobacco. The developed experimental platform should lead to the design of new routes for the drug’s commercial production in heterologous plant systems. (Nature Biotechnology 29:1072 (2012)

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds. Metabolic Eng. 13:474 (2011)

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. Trends in Biotech. 26:363 (2011)

Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plantג€™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 ofEOBII 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. Plant Cell 22:1961 (2010)

Flower fragrance is a composite character determined by secondary metabolites of diverse biosynthetic origin. Together with other traits, such as flower color, it is used by plants to lure pollinators and seed dispersers, thus ensuring plant survival. Research into the regulatory mechanisms leading to floral scent production/emission is still in its infancy and even less is known regarding flow within and cross-talk between secondary metabolic pathways leading to floral scent production. Using transgenic plants modified in anthocyanin production, we revealed an intriguing interrelationship between the branches of the phenylpropanoid pathway leading to the production of anthocyanins and volatiles. Specifically, we recorded five- to sevenfold higher levels of the volatile phenylpropanoids methyl benzoate and 2-hydroxymethyl benzoate in flavanone 3-hydroxylase (F3h)-suppressed carnation flowers with dramatically reduced anthocyanin levels, as compared to control non-transgenic flowers. Furthermore, overexpression in petunia flowers of the transcriptional regulator Pap1 (production of anthocyanin pigment 1), which activates the phenylpropanoid pathway, led to increases in both anthocyanin accumulation and volatile phenylpropanoid emission. Using virus-induced gene silencing (VIGS) for large-scale identification of floral scent genes, we further characterized metabolic flow within the pathway. The advantages of VIGS and of petunia as a model plant create a solid infrastructure for the future isolation of regulatory factors involved in floral scent production/emission. Knowledge gained from an understanding of mechanisms leading to floral scent production/emission should provide us with better insight into nature's way of ensuring evolutionary success, as well as with advanced tools for the metabolic engineering of fragrance. Plant Physiol. 145:1241 (2007); Plant Biotech. Journal 8:403 (2008)

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability. Plant Mol. Biol. 72:235 (2010)

Flowering is a unique and highly programmed process, but hardly anything is known about the developmentally regulated proteome changes in petals. Here, we employed proteomic technologies to study petal development in rose (Rosa hybrida). Using two-dimensional polyacrylamide gel electrophoresis, we generated stage-specific (closed bud, mature flower and flower at anthesis) petal-protein maps with ca. 1,000 unique protein spots. Expression analyses of all resolved protein spots revealed that almost 30% of them were stage-specific, with ca. 90 protein spots for each stage. Most of the proteins exhibited differential expression during petal development, whereas only ca. 6% were constitutively expressed. Eighty-two of the resolved proteins were identified by mass spectrometry and annotated. Classification of the annotated proteins into functional groups revealed energy, cell rescue, unknown function (including novel sequences) and metabolism to be the largest classes, together comprising ca. 90% of all identified proteins. Interestingly, a large number of stress-related proteins were identified in developing petals. Analyses of the expression patterns of annotated proteins and their corresponding RNAs confirmed the importance of proteome characterization. Planta 222:37 (2005)

Chromoplastogenesis during flower development and fruit ripening involves the dramatic overaccumulation of carotenoids sequestered into structures containing lipids and proteins, termed PAPs (plastid lipid-associated proteins). CHRC, a cucumber (Cucumis sativus) PAP, has been suggested to be transcriptionally activated in carotenoid-accumulating flowers by gibberellic acid (GA). Mybys, a MYB-like trans-activator identified in this study, may represent a chromoplastogenesis-related factor: its expression is flower-specific and parallels that of ChrC during flower development; moreover, as revealed by stable ectopic and transient-expression assays, it specifically trans-activates ChrC promoter in flowers accumulating carotenoids and flavonoids. A detailed dissection of ChrC promoter revealed a GA-responsive element, gacCTCcaa, the mutation of which abolished ChrC activation by GA. This cis-element is different from the GARE motif and is involved in ChrC activation, probably via negative regulation, similar to other GA-responsive systems. The GA responsiveness and MYBYS floral activation of the ChrC promoter do not overlap with respect to cis-elements. To study the functionality of CHRC, which is activated in vegetative tissuessimilar to other PAPsby various biotic and abiotic stresses, we employed a tomato plant system and generated RNAi-transgenic lines with suppressed LeCHRC. Transgenic flowers accumulated ca. 30% less carotenoids per unit protein than controls, indicating an interrelationship between PAPs and flower-specific carotenoid accumulation in chromoplasts. Moreover, the transgenic LeCHRC-suppressed plants were significantly more susceptible to Botrytis cinerea infection, suggesting CHRC's involvement in plant protection under stress conditions and supporting the general, evolutionarily preserved role of PAPs. Plant Physiol. 142:233 (2008). Plant Physiol. 104:321 (1994).

As noted above, studies on the carotenoid-overaccumulating structures in chromoplasts led to the characterization of PAPs, involved in the sequestration of hydrophobic compounds. Here we characterized the PAP CHRD, which, based on sequence homology, belongs to a highly conserved group of proteins, YER057c/YjgF/UK114, involved in the regulation of basic and vital cellular processes in bacteria, yeast and animals. Two nuclear genes were characterized in tomato plants: one (LeChrDc) is constitutively expressed in various tissues and the other (LeChrDi) is induced by stress in leaves and is upregulated by developmental cues in floral tissues. Using RNAi and antisense approaches, we showed their involvement in biologically significant processes such as photosynthesis. The quantum yield of photosynthetic electron flow in transgenic tomato leaves with suppressed LeChrDi/c expression was 30 to 50% of that in control, non-transgenic counterparts and was ascribed to lower PSI activity. Transgenic flowers with suppressed LeChrDi/c also accumulated up to 30% less carotenoids per unit protein as compared to control plants, indicating an interrelationship between PAPs and floral-specific carotenoid accumulation in chromoplasts. We suggest that CHRD's role in the angiosperm reproductive unit may be a rather recent evolutionary development; its original function may have been to protect the plant under stress conditions by preserving plastid functionality. Planta 225:89 (2006)

Current means of production for plant-derived aroma compounds include chemical synthesis and extraction from plant material. Both methods are environmentally detrimental and relatively expensive: plant material is only seasonally available and only a small subset of the plant biomass produces the desired aroma compounds, while organic synthesis inevitably involves waste byproducts with a negative ecological impact. Benzenoids are a class of plant metabolites that includes a number of aroma compounds. This research explores, for the first time, the feasibility of producing benzenoids in yeast. We elucidated a method for the production of the phenylpropanoid methyl benzoate in Saccharomyces cerevisiae using benzoic acid as the substrate, via heterologous expression ofAntirrhinum majus benzoic acid methyl transferase. Production was pH-dependent with a maximal yield of approximately 50 micrograms of methyl benzoate per liter of culture per hour, and with linear kinetics for at least 24 h. In addition, we analyzed two alternative expression vectors for the production of benzoic acid methyl transferase in S. cerevisiae: a constitutive triosephosphate isomerase promoter-based system was compared with a copper-inducible CUP1 promoter system. We found major differences in the amounts of methyl benzoate produced by these respective systems. Journal of Biotech. 122:307 (2006)