The Gerbera cDNA Microarray: A Tool for Large-Scale Identification of Genes Involved in Flower Development

During the past ten years, large-scale transcript analysis using microarrays has become a powerful tool to identify and predict functions for new genes. It allows simultaneous monitoring of the expression of thousands of genes and has become a routinely used tool in laboratories worldwide. Microarray analysis will, together with other functional genomics tools, take us closer to understanding the functions of all genes in genomes of living organisms. Flower development is a genetically regulated process which has mostly been studied in the traditional model species Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. The molecular mechanisms behind flower development in them are partly applicable in other plant systems. However, not all biological phenomena can be approached with just a few model systems. In order to understand and apply the knowledge to ecologically and economically important plants, other species also need to be studied. Sequencing of 17 000 ESTs from nine different cDNA libraries of the ornamental plant Gerbera hybrida made it possible to construct a cDNA microarray with 9000 probes. The probes of the microarray represent all different ESTs in the database. From the gerbera ESTs 20% were unique to gerbera while 373 were specific to the Asteraceae family of flowering plants. Gerbera has composite inflorescences with three different types of flowers that vary from each other morphologically. The marginal ray flowers are large, often pigmented and female, while the central disc flowers are smaller and more radially symmetrical perfect flowers. Intermediate trans flowers are similar to ray flowers but smaller in size. This feature together with the molecular tools applied to gerbera, make gerbera a unique system in comparison to the common model plants with only a single kind of flowers in their inflorescence. In the first part of this thesis, conditions for gerbera microarray analysis were optimised including experimental design, sample preparation and hybridization, as well as data analysis and verification. Moreover, in the first study, the flower and flower organ-specific genes were identified. After the reliability and reproducibility of the method were confirmed, the microarrays were utilized to investigate transcriptional differences between ray and disc flowers. This study revealed novel information about the morphological development as well as the transcriptional regulation of early stages of development in various flower types of gerbera. The most interesting finding was differential expression of MADS-box genes, suggesting the existence of flower type-specific regulatory complexes in the specification of different types of flowers. The gerbera microarray was further used to profile changes in expression during petal development. Gerbera ray flower petals are large, which makes them an ideal model to study organogenesis. Six different stages were compared and specifically analysed. Expression profiles of genes related to cell structure and growth implied that during stage two, cells divide, a process which is marked by expression of histones, cyclins and tubulins. Stage 4 was found to be a transition stage between cell division and expansion and by stage 6 cells had stopped division and instead underwent expansion. Interestingly, at the last analysed stage, stage 9, when cells did not grow any more, the highest number of upregulated genes was detected. The gerbera microarray is a fully-functioning tool for large-scale studies of flower development and correlation with real-time RT-PCR results show that it is also highly sensitive and reliable. Gene expression data presented here will be a source for gene expression mining or marker gene discovery in the future studies that will be performed in the Gerbera Laboratory. The publicly available data will also serve the plant research community world-wide.

Secondary metabolites in Gerbera hybrida

Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.

Sireenit, floksit ja iirikset- Puu-Käpylän koristekasvit

Puu-Käpylä (“Wooden Käpylä”), a neighbourhood of Helsinki, is the earliest example of the Garden City Movement in Finland. The suburb of valuable wooden architecture was built between 1920 and 1925, with the aim to provide a healthy housing area for working-class families with many children. The houses were erected by a co-operative (Käpylän kansanasunnot, “People?s Dwellings”) and they are protected by the city plan since 1960?s. However, the historical value of the sheltered courtyards has not been investigated. The aim of this study was to survey the garden flora of Puu-Käpylä and to evaluate the authenticity of the courtyard gardens. The survey covered the area of one residential quarter (1.2 ha) with twelve 2-storey semi-detached timber houses arranged around a common yard, which was originally appointed for the tenants? vegetable gardens. The houses are still rented, and each flat is allowed a small lot of the courtyard for cultivation. A complete list was made of all perennial, ornamental plant taxa present in the quarter. Spring bulbs were missed due to the timing of the survey. Generally, the plants were recorded on species level, with the exception of common lilacs, shrub roses, irises and peonies that were thoroughly studied for cultivar identification. It was assumed that plants initially grown in the courtyard could be distinguished by studying Finnish garden magazines, books and nursery catalogues published in the 1920?s and by comparing the present vegetation to surviving documents from the quarter. The total number of ornamental plant taxa identified was 172, of which 17 were trees, 47 shrubs, 7 climbers and 101 herbaceous perennials. The results indicated that a major part of the shrubs, climbers and perennials presumably originated from the 1970?s or later, whereas ca. 70 % of the tree specimens were deemed as original. The survey disclosed a heritage variety of common lilac, resembling cultivar „Prince Notger?, a specific peony taxon, Paeonia humilis Retz., cultivated in Nordic countries since long ago, and a few historic iris varieties. Well-preserved design elements included front gardens on one side of the quarter, a maple alley on another side as well as trees at the garden gates. Old garden books and magazines did not shed much light on the Finnish garden flora commonly used in the period when Puu-Käpylä was built. However, they gave a valuable picture of contemporary planting design. Nursery catalogues offered insight into the assortment of ornamental plants traded in the 1920?s. Conclusions on the authenticity of the current flora were mainly drawn on the basis of old photographs and a vegetation survey map drawn in the 1970?s. This study revealed a need for standardization of syrvey methods applied when investigating garden floras. Uniform survey techniques would make the results comparable and enable a future compilation of data from e.g. historic gardens.

Havupuiden erikoismuotojen lisäysmenetelmät

Havupuiden erikoismuotoja on käytetty koristekasveina jo vuosisatoja ympäri maailmaa. Niitä on lisätty pääsääntöisesti pistokkaista ja varttamalla. Suomessa kotimaisten metsäpuidemme erikois-muotoja on kartoitettu ja kerätty kokoelmiin järjestelmällisemmin 1960-luvulta alkaen. Taimisto-viljelijät, puutarhasuunnittelijat ja kotipuutarhurit ovat olleet enenevässä määrin kiinnostuneita näistä kotimaisista kestävistä havukasveista. Yli 90 prosenttia markkinoillamme olevista havukas-veista tuodaan ulkomailta, joten on selvää, että niiden talvenkestävyydessä on ongelmia. Tämän tutkimuksen tavoitteena oli selvittää kotimaisille erikoismuodoille sopivia lisäysmene-telmiä ja siten edistää kotimaisen havukasvituotannon mahdollisuuksia. Aineistona kokeissa oli kotimaisia erikoismuotoja metsäkuusesta (Picea abies (L.) Karsten) ja kotikatajasta (Juniperus communis L.), tavallisia metsäkuusia sekä kahdeksan ulkomaista havupuutaksonia. Lisäysmene-telmistä tutkittiin varttamista ja pistokaslisäystä ja kokeet suoritettiin Metsäntutkimuslaitoksen toimipaikoissa Lopen Haapastensyrjässä sekä Punkaharjulla. Varttamiskokeessa vertailtiin koti-maisen kuusen erikoismuotokloonien varttamisen onnistumista. Pistokaskokeissa tutkittiin geno-tyypin, emopuun iän, pistokasoksan sijainnin sekä hormonikäsittelyn vaikutusta havukasvien pis-tokkaiden juurtumiseen. Tavalliset metsäkuuset toimivat kontrolleina. Tutkimus osoitti, että varttaminen onnistui erinomaisesti kaikilla erikoismuotoklooneilla. Ovat-ko vartteet esteettisesti katsottuna koristekäyttöön sopivia, jää vielä seurattavaksi. Pistokaskokeis-sa havaittiin, että juveniilisuus vaikutti pistokkaiden juurtumiseen, mutta iäkkäistäkin puista lisää-minen onnistuu, kunhan genotyyppi on sopiva. Keskimäärin alaoksat juurtuivat paremmin kuin latvuksen yläosista otetut pistokasoksat, mutta vain yhdellä kloonilla ero oli tilastollisesti merkit-sevä. Hormonikäsittely heikensi selvästi kotimaisen kuusen ja katajan pistokkaiden juurtumista, mutta ulkomaisiin havupuulajeihin käsittelyllä ei ollut vaikutusta. Kotimaisen havukasvituotannon pohjaksi pitäisi tehdä kloonivalintaa, jossa koristearvon lisäksi otettaisiin huomioon myös kloonin lisättävyys. Taimien tuottaminen pistokkaista on selvästi edul-lisempaa kuin vartteiden tuottaminen, joskin varte kasvaa myyntikuntoon nopeammin kuin pisto-kastaimi. Pistokastaimi on kuitenkin omajuurinen ja stabiilimpi kasvutavaltaan kuin varte. Tämä korostuu etenkin kääpiömuotoja tuotettaessa.

Population genetics of the invasive plant species Impatiens glandulifera in Southern Finland

Biological invasions affect biodiversity worldwide, and, consequently, the invaded ecosystems may suffer from significant losses in economic and cultural values. Impatiens glandulifera Royle (Balsaminaceae) is an invasive annual herb, native to the western Himalayas and introduced into Europe in the 19th century as a garden ornamental plant. The massive invasion of I. glandulifera is due to its high reproductive output, rapid growth and its ability to outcompete native species. In Finland, the first observations regarding the presence of I. glandulifera date from the year 1947, and today it is considered a serious problem in riparian habitats. The aim of this master’s thesis research is to reveal the population genetic structure of I. glandulifera in Finland and to find out whether there have been one or multiple invasions in Finland. The study focuses on investigating the origin of I. glandulifera in Southern Finland, by comparing plant samples from the Helsinki region with those from its native region and other regions of invasion. Samples from four populations in Helsinki and from the United Kingdom, Canada, India and Pakistan were collected and genotyped using 11 microsatellite markers. The genetic analyses were evaluated using the programs Arlequin and Structure. The results of the genetic analyses suggested that I. glandulifera has been introduced to Finland more than once. Multiple introductions are supported by the higher level of genetic diversity detected within and among Finnish populations than would be expected for a single introduction. Results of the Bayesian Structure analysis divided the four Finnish populations into four clusters. This geographical structure was further supported by pairwise Fst values among populations. The causes and potential consequences of such multiple introductions of I. glandulifera in Finland and further perspectives are discussed.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Phytophthora in Finnish nurseries

Human-mediated movement of plants and plant products is now generally accepted to be the primary mode of introduction of plant pathogens. Species of the genus Phytophthora are commonly spread in this way and have caused severe epidemics in silviculture, horticulture as well as natural systems all over the world. The aims of the study were to gather information on the occurrence of Phytophthora spp. in Finnish nurseries, to produce information for risk assessments for these Phytophthora spp. by determining their host ranges and tolerance of cold temperatures, and to establish molecular means for their detection. Phytophthora cactorum was found to persist in natural waterbodies and results suggest that irrigation water might be a source of inoculum in nurseries. In addition to P. cactorum, isolates from ornamental nursery Rhododendron yielded three species new to Finland: P. ramorum, P. plurivora and P. pini. The only species with quarantine status, P. ramorum, was most adapted to growth in cold temperatures and able to persist in the nursery in spite of an annual sanitation protocol. Phytophthora plurivora and the closely related P. pini had more hosts among Nordic tree and plant species than P. ramorum and P. cactorum, and also had higher infectivity rates. All four species survived two weeks in -5 °C , and thus soil survival of these Phytophthoras in Finland is likely under current climatic conditions. The most common tree species in Finnish nurseries, Picea abies, was highly susceptible to P. plurivora and P. pini in pathogenicity trials. In a histological examination of P. plurivora in P. abies shoot tissues, fast necrotrophic growth was observed in nearly all tissues. The production of propagules in P. abies shoot tissue was only weakly indicated. In this study, a PCR DGGE technique was developed for simultaneous detection and identification of Phytophthora spp. It reliably detected Phytophthora in plant tissues and could discriminate most test species as well as indicate instances of multiple-species infections. It proved to be a useful detection and identification tool either applied alone or in concert with traditional isolation culture techniques. All of the introduced species of Phytophthora had properties that promote a high risk of establishment and spread in Finland. It is probable that more pathogens of this genus will be introduced and become established in Finland and other Nordic countries unless efficient phytosanitary control becomes standard practice in the international plant trade.