Michal Moyal Ben Zvi

Interlinking showy traits: co‐engineering of scent and colour biosynthesis in flowers

The phenylpropanoid pathway gives rise to metabolites that determine floral colour and fragrance. These metabolites are one of the main means used by plants to attract pollinators, thereby ensuring plant survival. A lack of knowledge about factors regulating scent production has prevented the successful enhancement of volatile phenylpropanoid production in flowers. In this study, the Production of Anthocyanin Pigment1 (Pap1) Myb transcription factor from Arabidopsis thaliana, known to regulate the production of non-volatile phenylpropanoids, including anthocyanins, was stably introduced into Petunia hybrida. In addition to an increase in pigmentation, Pap1-transgenic petunia flowers demonstrated an increase of up to tenfold in the production of volatile phenylpropanoid/benzenoid compounds. The dramatic increase in volatile production corresponded to the native nocturnal rhythms of volatile production in petunia. The application of phenylalanine to Pap1-transgenic flowers led to an increase in the otherwise negligible levels of volatiles emitted during the day to nocturnal levels. On the basis of gene expression profiling and the levels of pathway intermediates, it is proposed that both increased metabolic flux and transcriptional activation of scent and colour genes underlie the enhancement of petunia flower colour and scent production by Pap1. The co-ordinated regulation of metabolic steps within or between pathways involved in vital plant functions, as shown here for two showy traits determining plant-pollinator interactions, provides a clear advantage for plant survival. The use of a regulatory factor that activates scent production creates a new biotechnological strategy for the metabolic architecture of fragrance, leading to the creation of novel genetic variability for breeding purposes.

PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers

• 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 PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) transcription factor was introduced into Rosa hybrida. • Color and scent profiles of PAP1-transgenic and control (β-glucuronidase-expressing) rose flowers and the expression of key genes involved in the production of secondary metabolites were analyzed. To evaluate the significance of the scent modification, olfactory trials were conducted with both humans and honeybees. • In addition to increased levels of phenylpropanoid-derived color and scent compounds when compared with 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 caused solely by transcriptional activation of their respective biosynthetic genes, but probably also resulted from enhanced metabolic flux in both the phenylpropanoid and isoprenoid pathways. The mechanism(s) governing the interactions in these metabolic pathways that are responsible for the production of specialized metabolites remains to be elucidated.

Reverse Genetics of Floral Scent: Application of Tobacco Rattle Virus-Based Gene Silencing in Petunia

Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.

Expression of a bacterial feedback‐insensitive 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism

The shikimate pathway of plants mediates the conversion of primary carbon metabolites via chorismate into the three aromatic amino acids and to numerous secondary metabolites derived from them. However, the regulation of the shikimate pathway is still far from being understood. We hypothesized that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme regulating flux through the shikimate pathway. To test this hypothesis, we expressed a mutant bacterial AroG gene encoding a feedback-insensitive DAHPS in transgenic Arabidopsis plants. The plants were subjected to detailed analysis of primary metabolism, using GC-MS, as well as secondary metabolism, using LC-MS. Our results exposed a major effect of bacterial AroG expression on the levels of shikimate intermediate metabolites, phenylalanine, tryptophan and broad classes of secondary metabolite, such as phenylpropanoids, glucosinolates, auxin and other hormone conjugates. We propose that DAHPS is a key regulatory enzyme of the shikimate pathway. Moreover, our results shed light on additional potential metabolic bottlenecks bridging plant primary and secondary metabolism.

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.

Activation of the Phenylpropanoid pathway in Nicotiana tabacum improves the performance of the whitefly Bemisia tabaci via reduced jasmonate signaling.

Background Phloem-feeding insects can manipulate plant-induced resistance and are able to suppress effective jasmonic acid/ethylene (JA/ET) defenses by the induction of inefficient salicylic acid (SA) based responses. As a result, activation of the phenylpropanoid biosynthesis pathway in transgenic plants is anticipated to cause complex interactions between phloem-feeding insects and their host plants due to predicted contradiction between two defense forces: the toxicity of various phenylpropanoids and the accumulation of SA via a branch of the activated pathway. Methodology/Principal Findings Here, we investigated the effect of activating the phenylpropanoids pathway in Nicotiana tabacum, by over-expression of the PAP1 transcription factor, on the whitefly Bemisia tabaci, a phloem-feeding insect model. Our performance assays indicated that the over-expression made the transgenic plants a more suitable host for B. tabaci than wild-type (WT) plants, although these plants accumulated significantly higher levels of flavonoids. Transcription analyses of indicator genes in the SA (PR1a) and JA/ET (ERF1, COI1 and AOC) pathways followed by quantification of the SA and JA hormone levels, indicated that B. tabaci infestation periods longer than 8 hours, caused higher levels of activity of SA signaling in transgenic plants and higher levels of JA/ET signaling in WT plants. Conclusions/Significance Taken together, these results emphasize the important role JA/ET-induced defenses play in protecting plants from successful infestation by B. tabaci and likely other phloem-feeding insects. It also indicates the necessity of phloem feeders to suppress these defenses for efficient utilization of plant hosts. Our data also indicate that the defensive chemistry produced by the phenylpropanoids pathway has only a minor effect on the insect fitness.

LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues

BackgroundRapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium.FindingsWe report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids.ConclusionsThe proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.

Agrobacterium-mediated transformation of gypsophila (Gypsophila paniculata L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA3-pretreated mother plants and a two-step selection scheme. The GA3 treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenesrolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.