Elena Shklarman

Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene

Anthocyanins are the major pigments contributing to carnation flowercoloration. Most carnation varieties are sterile and hence molecular breedingis an attractive approach to creating novel colors in this commercially importantcrop. Characterization of anthocyanins in the flowers of the modern carnationcv. Eilat revealed that only the orange pelargonidin accumulates, due to a lackof both flavonoid 3′,5′-hydroxylase and flavonoid3′-hydroxylase activities. To modify flower color in cv. Eilat, we usedantisense suppression to block the expression of a gene encoding flavanone3-hydroxylase, a key step in the anthocyanin pathway. The transgenic plantsexhibited flower color modifications ranging from attenuation to complete lossof their original orange/reddish color. In the latter, only traces ofpelargonidin were detected. Dramatic suppression of flavanone 3-hydroxylaselevel/activity in these transgenes was confirmed by northern blot, RT-PCR andenzymatic assays. The new phenotype has been stable for over 4 years ofvegetative propagation. Moreover, transgenic plants with severe colormodification were more fragrant than control plants. GC-MS headspace analysesrevealed that transgenic anti-f3h flowers emit higherlevels of methyl benzoate. The possible interrelation between pathways leadingto anthocyanin and fragrance production is discussed.

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 and color play major roles in the plants life cycle. Using petunia as a model system, a MYB-like factor was identified that transcriptionally regulates floral scent but not pigmentation. The multilayered regulation allows efficient control of metabolic flux in the phenylpropanoid pathway. Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plants 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.

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.

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 CHRCs 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.

Generation of Transgenic Carnation Plants with Novel Characteristics by Combining Microprojectile Bombardment with Agrobacterium Tumefaciens Transformation

As one of the major contributors to the cut-flower market and a commercial leader in terms of number of stems sold worldwide (Jensen, Malter 1995), carnation (Dianthus caryophyllus L.) has been an important target for the breeding of new varieties with novel characteristics. Although new carnation varieties are continuously being produced through classical breeding, their high heterozygosity and limited gene pool, and a lack of knowledge regarding their genetic makeup, severely restrict such breeding programs (Woodson 1991). Thus, the possibility of genetically transforming carnation, as well as other cut-flower species, via direct gene transfer is quite attractive (reviewed by Zuker et al. 1998).

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.

Transgenic Dianthus spp. (Carnation)

Carnation, native to the Mediterranean coastal region, is one of the world’s major cut-flower crops (VBN 1995). It is a member of the family Caryophyllaceae and belongs to the genus Dianthus, which contains over 300 species. Commercial carnations, grouped into the phenotypical categories standard and spray, result from crosses within Dianthus caryophyllus. Pot carnations, resulting from crosses involving D. chinensis and D. barbatus, are also becoming popular among consumers. As one of the major contributors to the cut-flower market and a commercial leader in terms of the number of stems sold worldwide (Jensen and Malter 1995), carnation has been an important target for the breeding of new varieties with novel characteristics. To date, new carnation varieties have been produced mainly via classical breeding, and are propagated vegetatively. However, high heterozygosity, a limited gene pool, and almost no knowledge of carnation’s genetic makeup severely restrict such breeding programs (Woodson 1991).