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.

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 (ß-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.

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.

Quorum-sensing quenching by rhizobacterial volatiles.

We show that volatile organic compounds (VOCs) produced by rhizospheric strains Pseudomonas fluorescens B-4117 and Serratia plymuthica IC1270 may act as inhibitors of the cell-cell communication quorum-sensing (QS) network mediated by N-acyl homoserine lactone (AHL) signal molecules produced by various bacteria, including strains of Agrobacterium, Chromobacterium, Pectobacterium and Pseudomonas. This quorum-quenching effect was observed when AHL-producing bacteria were treated with VOCs emitted by strains B-4117 and IC1270 or with dimethyl disulfide (DMDS), the major volatile produced by strain IC1270. LC-MS/MS analysis revealed that treatment of strains Pseudomonas chlororaphis 449, Pseudomonas aeruginosa PAO1 or Ps. fluorescens 2-79 with VOCs emitted by strain IC1270 or DMDS drastically decreases the amount of AHLs produced by these bacteria. Volatile organic compounds produced by Ps. chlororaphis 449 were able to suppress its own QS-induction activity, suggesting a negative interaction between VOCs and AHL molecules in the same strain. Quantitative RT-PCR analysis showed that treatment of Ps. chlororaphis 449 with VOCs emitted by cells of IC1270, B-4117 or 449 itself, or with DMDS, leads to significant suppression of transcription of AHL synthase genes phzI and csaI. Thus, along with AHLs, bacterial volatiles might be considered another type of signal molecule involved in microbial communication in the rhizosphere.

Nontransgenic genome modification in plant cells.

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.

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.

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.

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.

Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica.

One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth medium.

Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress.

Chromoplastogenesis during flower development and fruit ripening involves the dramatic overaccumulation of carotenoids sequestered into structures containing lipids and proteins called plastid lipid-associated proteins (PAPs). CHRC, a cucumber (Cucumis sativus) PAP, has been suggested to be transcriptionally activated in carotenoid-accumulating flowers by gibberellin (GA). Mybys, a MYB-like trans-activator identified here, 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 tissues similar to other PAPs by various biotic and abiotic stresses, we employed a tomato (Lycopersicon esculentum) plant system and generated RNAi-transgenic lines with suppressed LeCHRC. Transgenic flowers accumulated approximately 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.

CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis.

Studies on the carotenoid-overaccumulating structures in chromoplasts have led to the characterization of proteins termed plastid lipid-associated proteins (PAPs), involved in the sequestration of hydrophobic compounds. Here we characterize 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 show 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 30-50% of their 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.

The global regulator genes from biocontrol strain Serratia plymuthica IC1270: cloning, sequencing, and functional studies.

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than Pseudomonas spp., gacA/gacS and rpoS homologues were cloned from biocontrol strain IC1270 of Serratia plymuthica, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned gacA/gacS-like genes-tentatively designated grrA (global response regulation activator) and grrS (global response regulation sensor) and of the cloned rpoS gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria Escherichia coli, Pectobacterium carotovora subsp. carotovora, and Serratia marcescens. grrA, grrS, and rpoS gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and N-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only grrA and grrS mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the grrA, grrS, and rpoS mutants were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.