Development of composite inflorescences in the plant family Asteraceae has features that cannot be studied in the traditional model plants for flower development. In Gerbera hybrida, inflorescences are composed of morphologically different types of flowers tightly packed into a flower head (capitulum). Individual floral organs such as pappus bristles (sepals) are developmentally specialized, stamens are aborted in marginal flowers, petals and anthers are fused structures, and ovaries are located inferior to other floral organs. These specific features have made gerbera a rewarding target of comparative studies. Here we report the analysis of a gerbera EST database containing 16,994 cDNA sequences. Comparison of the sequences with all plant peptide sequences revealed 1656 unique sequences for gerbera not identified elsewhere within the plant kingdom. Based on the EST database, we constructed a cDNA microarray containing 9000 probes and have utilized it in identification of flower-specific genes and abundantly expressed marker genes for flower scape, pappus, stamen, and petal development. Our analysis revealed several regulatory genes with putative functions in flower-organ development. We were also able to associate a number of abundantly and specifically expressed genes with flower-organ differentiation. Gerbera is an outcrossing species, for which genetic approaches to gene discovery are not readily amenable. However, reverse genetics with the help of gene transfer has been very informative. We demonstrate here the usability of the gerbera microarray as a reliable new tool for identifying novel genes related to specific biological questions and for large-scale gene expression analysis.

SummaryChalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida).The tissue-and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences.Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis.Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.

Genetic modification of the flavonoid pathway has been used to produce novel colours and colour patterns in ornamental plants as well as to modify the nutritional and pharmaceutical properties of food crops. It has been suggested that co-ordinate control of multiple steps of the pathway with the help of regulatory genes would lead to a more predictable control of metabolic flux. Regulation of anthocyanin biosynthesis has been studied in a common ornamental plant, Gerbera hybrida (Asteraceae). An R2R3-type MYB factor, GMYB10, shares high sequence similarity and is phylogenetically grouped together with previously characterized regulators of anthocyanin pigmentation. Ectopic expression of GMYB10 leads to strongly enhanced accumulation of anthocyanin pigments as well as to an altered pigmentation pattern in transgenic gerbera plants. Anthocyanin analysis indicates that GMYB10 specifically induces cyanidin biosynthesis in undifferentiated callus and in vegetative tissues. Furthermore, in floral tissues enhanced pelargonidin production is detected. Microarray analysis using the gerbera 9K cDNA array revealed a highly predicted set of putative target genes for GMYB10 including new gene family members of both early and late biosynthetic genes of the flavonoid pathway. However, completely new candidate targets, such as a serine carboxypeptidase-like gene as well, as two new MYB domain factors, GMYB11 and GMYB12, whose exact function in phenylpropanoid biosynthesis is not clear yet, were also identified.

A previously isolated cDNA molecule from Gerbera hybrida (Asteraceae) codes for a new chalcone synthase-like polyketide synthase, 2-pyrone synthase (2PS). 2PS is able to synthesise 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor for gerberin and parasorboside, two abundant glucosides in gerbera. In this study, we show that gerbera plants transformed with the gene for 2PS in an antisense orientation and unable to synthesise gerberin and parasorboside are susceptible to Botrytis cinerea infection. In addition to the preformed glucosides, the transgenic plants also lack several compounds that are induced in control plants when infected with the mould. Some of these induced substances are effective in inhibiting fungal growth both in vitro and in vivo. Two of the phytoalexins were identified as the aglycones of gerberin and trans-parasorboside. The third phytoalexin is a rare coumarin, 4-hydroxy-5-methylcoumarin; however, it is typical of many plants of the sunflower family Asteraceae. The coumarin cannot be structurally derived from either gerberin or parasorboside, but may be derived from a related polyketide intermediate.

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4-hydroxy-5-methylcoumarin (HMC). Unlike most coumarins, 5-methylcoumarins have been suggested to be derived through the acetate-malonate pathway. All of these polyketide-derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2-pyrone synthases (G2PS1-3). The enzymes are chalcone synthase-like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7-dihydroxy-5-methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site-directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.