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Stilbenes are developmentally induced metabolites in Scots pine heartwood where they have important role in protecting wood from decaying fungi. The stilbene pathway is also stress inducible, and ultraviolet (UV)-C radiation was among the first discovered artificial stress activators of the pathway. Many conditions that activate the pathway are known, but the specific transcriptional regulators and the biosynthetic enzyme responsible for activating the stilbene precursor cinnamate in the pathway are unknown.Here we describe the first large-scale transcriptomic analysis of pine needles in response to UV-C exposure and treatment with translational inhibitor cycloheximide, both activating the transcription of stilbene pathway genes, to uncover the candidates for the transcriptional regulation of the pathway and the cinnamate activating CoA ligase. We show that the regulation of pine stilbene pathway has shared features with grapevine stilbene pathway. In both species, the stilbene pathway was transcriptionally activated after UV-C treatment, protein phosphatase inhibitors and plant hormones ethylene and jasmonate.The pine stilbene synthase promoter retains its inducibility when transformed in plants that normally do not synthesize stilbenes. Pine stilbene synthase promoter was able to activate the expression of a reporter gene in Arabidopsis in response to UV-C exposure and phosphatase inhibitors, but not as response to plant hormone treatment. This indicates that stilbene synthase gene regulation occurs both via ancient stress-response pathway(s) but also via species specific regulators. With transcriptomic approach we identified candidate enzymes for cinnamate acting CoA ligase and transcription factors regulating the pathway.

Plant polyketides are well-known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide synthases (PKSs) and their final functional diversity is determined by post-PKS tailoring enzymes. Gerbera hybrida is rich in two defense-related polyketides: gerberin and parasorboside. Their synthesis is known to be initiated by GERBERA 2-PYRONE SYNTHASE 1 (G2PS1), but the polyketide reductases (PKRs) that determine their final structure have not yet been identified. We identified two PKR candidates in the pathway, GERBERA REDUCTASE 1 (GRED1) and GRED2. Gene expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants, and in vitro enzyme assays, were performed for functional characterization of the enzymes. GRED1 and GRED2 catalyze the second reduction step in parasorboside biosynthesis. They reduce the proximal keto domain of the linear CoA bound intermediate before lactonization. We identified a crucial tailoring step in an important gerbera PKS pathway and show that plant polyketide biosynthesis shares processing strategies with fungi and bacteria. The two tailoring enzymes are recruited from the ancient sporopollenin biosynthetic pathway to a defense-related PKS pathway in gerbera. Our data provide an example of how plants recruit conserved genes to new functions in secondary metabolism that are important for environmental adaptation.

Transport of monolignols into the apoplastic space is a major event in woody stems. This work identifies candidate transporters in Norway spruce through large-scale LC-MS/MS membrane proteomics and gene co-expression analyses., Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications. Published version

Phyllotaxis, the distribution of organs such as leaves and flowers on their support, is a key attribute of plant architecture. The geometric regularity of phyllotaxis has attracted multidisciplinary interest for centuries, resulting in an understanding of the patterns in the model plants

We studied the stress response of five-year-old Scots pine xylem to mechanical wounding using RNA sequencing. In general, we observed a bimodal response in pine xylem after wounding. Transcripts associated with water deficit stress, defence, and cell wall modification were induced at the earliest time point of three hours; at the same time, growth-related processes were down-regulated. A second temporal wave was triggered either at the middle and/or at the late time points (one and four days). Secondary metabolism, such as stilbene and lignan biosynthesis started one day after wounding. Scots pine synthesises the stilbenes pinosylvin and its monomethyl ether both as constitutive and induced defence compounds. Stilbene biosynthesis is induced by wounding, pathogens and UV stress, but is also developmentally regulated when heartwood is formed. Comparison of wounding responses to heartwood formation shows that many induced processes (in addition to stilbene biosynthesis) are similar and relate to defence or desiccation stress, but often specific transcripts are up-regulated in the developmental and wounding induced contexts. Pine resin biosynthesis was not induced in response to wounding, at least not during the first four days.

The pseudanthial inflorescences of the sunflower family, Asteraceae, mimic a solitary flower but are composed of multiple flowers. Our studies in Gerbera hybrida indicate functional diversification for SEPALLATA (SEP)-like MADS box genes that often function redundantly in other core eudicots. We conducted phylogenetic and expression analysis for eight SEP-like GERBERA REGULATOR OF CAPITULUM DEVELOPMENT (GRCD) genes, including previously unstudied gene family members. Transgenic gerbera plants were used to infer gene functions. Adding to the previously identified stamen and carpel functions for GRCD1 and GRCD2, two partially redundant genes, GRCD4 and GRCD5, were found to be indispensable for petal development. Stepwise conversion of floral organs into leaves in the most severe RNA interference lines suggest redundant and additive GRCD activities in organ identity regulation. We show conserved and redundant functions for several GRCD genes in regulation of flower meristem maintenance, while functional diversification for three SEP1/2/4 clade genes in regulation of inflorescence meristem patterning was observed. GRCD genes show both specialized and pleiotropic functions contributing to organ differentiation and flower meristem fate, and uniquely, to patterning of the inflorescence meristem. Altogether, we provide an example of how plant reproductive evolution has used conserved genetic modules for regulating the elaborate inflorescence architecture in Asteraceae.

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization. (C) 2016 Elsevier Ltd. All rights reserved.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Both the establishment of sustainable forestry practices and the improvement of commercially grown trees require better understanding of mechanisms used by forest trees to combat microbial pathogens. We investigated the contribution of a gene encoding Scots pine (Pinus sylvestris L.) antimicrobial protein Sp-AMP2 (PR-19) to the host defenses to evaluate the potential of Sp-AMP genes as molecular markers for resistance breeding. We developed transgenic tobacco plants expressing the Sp-AMP2 gene. Transgenic plants showed a reduction in the size of lesions caused by the necrotrophic pathogen Botrytis cinerea. In order to investigate Sp-AMP2 gene expression level, four transgenic lines were tested in comparison to control and non-transgenic plants. No Sp-AMP2 transcripts were observed in any of the control and non-transgenic plants tested. The transcript of Sp-AMP2 was abundantly present in all transgenic lines. Sp-AMP2 was induced highly in response to the B. cinerea infection at 3 d.p.i. This study provides an insight into the role of Sp-AMP2 and its functional and ecological significance in the regulation of plant–pathogen interactions.

Gaining membrane vesicles from different plant species and tissue types is crucial for membrane studies. Membrane vesicles can be used for further purification of individual membrane types, and, for example, in studies of membrane enzyme activities, transport assays, and in proteomic analysis. Membrane isolation from some species, such as conifers, has proved to be more difficult than that of angiosperm species. In this paper, we describe steps for isolating cellular membranes from developing xylem, phloem, and lignin-forming tissue-cultured cells of Norway spruce, followed by partial enrichment of plasma membranes by aqueous polymer two-phase partitioning and purity analyses. The methods used are partially similar to the ones used for mono- and dicotyledonous plants, but some steps require discreet optimization, probably due to a high content of phenolic compounds present in the tissues and cultured cells of Norway spruce.

Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs.

Identification of distinct allelic versions for dihydroflavonol 4-reductase in gerbera cultivars reveals that gerbera DFR enzymes have strong substrate preference in vivo that is not reflected to the activity in vitro. Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet colour. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. ‘Ivory’, a cultivar with white petals, is a sport of the pelargonidin-containing pink cultivar ‘Estelle’, i.e. it originates from an acyanic branch of ‘Estelle’. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to ‘Estelle’ with the functional allele GDFR1-2, ‘Ivory’ carries a mutation in this gene that results in an inactive enzyme. Interestingly, ‘Ivory’ also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin-containing variety ‘President’. Pelargonidin-containing cultivars do not react to the flavonoid 3′-hydroxylase inhibitor tetcyclacis, but cyanidin-containing cultivars lose their colour, instead of starting to synthesise pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of ‘Ivory’ are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier thought.

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8–4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 μM; however, the laccase has a lower catalytic efficiency (Vmax/Km) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

Gaining membrane vesicles from different plant species and tissue types is crucial for membrane studies. Membrane vesicles can be used for further purification of individual membrane types, and, for example, in studies of membrane enzyme activities, transport assays, and in proteomic analysis. Membrane isolation from some species, such as conifers, has proved to be more difficult than that of angiosperm species. In this paper, we describe steps for isolating cellular membranes from developing xylem, phloem, and lignin-forming tissue-cultured cells of Norway spruce, followed by partial enrichment of plasma membranes by aqueous polymer two-phase partitioning and purity analyses. The methods used are partially similar to the ones used for mono- and dicotyledonous plants, but some steps require discreet optimization, probably due to a high content of phenolic compounds present in the tissues and cultured cells of Norway spruce.

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A., published version, peerReviewed

Main conclusion Unauthorized genetically engineered orange petunias were found on the market. Genetic engineering of petunia was shown to lead to novel flower color some 20 years ago. Here we show that petunia lines with orange flowers, generated for scientific purposes, apparently found their way to petunia breeding programmes, intentionally or unintentionally. Today they are widely available, but have not been registered for commerce. Electronic supplementary material The online version of this article (doi:10.1007/s00425-017-2722-8) contains supplementary material, which is available to authorized users.

The principles for the safety assessment of genetically modified (GM) organisms (GMOs) are harmonised worldwide to a large extent. There are, however, still differences between the European GMO regulations and the GMO regulations as they have been formulated in other parts of the world. One of these differences relates to the so-called 'stacked GM events', that is, GMOs, plants so far, where new traits are combined by conventional crossing of different GM plants. This paper advocates rethinking the current food/feed safety assessment of stacked GM events in Europe based on an analysis of different aspects that currently form the rationale for the safety assessment of stacked GM events.

Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1.

Lignin, an important component of plant cell walls, is a polymer of monolignols derived from the phenylpropanoid pathway. Monolignols are oxidized in the cell wall by oxidative enzymes (peroxidases and/or laccases) to radicals, which then couple with the growing lignin polymer. We have investigated the characteristics of the polymerization reaction by producing lignin polymers in vitro using different oxidative enzymes and analyzing the structures formed with NMR. The ability of the enzymes to oxidize a high-molecular-weight compounds was tested using cytochrome c as a substrate. The results support an idea that lignin structure is largely determined by the concentration ratios of the monolignol (coniferyl alcohol) and polymer radicals involved in the coupling reaction. High rate of the lignin polymer oxidation compared to monolignol oxidation leads to a natural-like structure. The high relative rate can be achieved by an open active site of the oxidative enzyme, close proximity of the enzyme with the polymeric substrate or simply by high enzymatic activity that consumes monolignols rapidly. Monolignols, which are oxidized efficiently, can be seen as competitive inhibitors of polymer oxidation. Our results indicate that, at least in a Norway spruce (Picea abies L. Karst.) cell culture, a group of apoplastic, polymer-oxidizing peroxidases bind to the lignin polymer and are responsible for production of natural-like lignin in cell suspension cultures in vivo, and also in vitro. The peroxidases bound to the extracellular lignin had the highest ability to bind to various cell wall polymers in vitro. Extracellular lignin contains pectin-type sugars, making them possible attachment points for these cationic peroxidases.

Scots pine (Pinus sylvestris L.) wood is desired in woodworking industries due to its favorable timber characteristics and natural durability that is contributed by heartwood extractives. It has been discussed whether the Scots pine heartwood extractives (mainly stilbenes and resin acids) are synthesized in the cells of the transition zone between sapwood and heartwood, or if they are transported from the sapwood. Timing of heartwood formation during the yearly cycle has also not been unambiguously defined. We measured steady-state mRNA levels in Scots pine transition zone and sapwood using RNA sequencing. Year-round expression profiles of selected transcripts were further investigated by quantitative RT-PCR. Differentially accumulating transcripts suggest that, of the Scots pine heartwood extractives, stilbenes are synthesized in situ in the transition zone and gain their carbon-skeletons from Suc and triglycerides. Resin acids, on the other hand, are synthesized early in the spring mainly in the sapwood, meaning that they must be transported to the heartwood transition zone. Heartwood formation is marked by programmed cell death that occurs during the summer months in the transition zone.

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.

Plant-specific TCP domain transcription factors have been shown to regulate morphological novelties during plant evolution, including the complex architecture of the Asteraceae inflorescence that involves different types of flowers. We conducted comparative analysis of the CYCLOIDEA/TEOSINTE BRANCHED1 (CYC/TB1) gene family in Gerbera hybrida (gerbera) and Helianthus annuus (sunflower), two species that represent distant tribes within Asteraceae. Our data confirm that the CYC/TB1 gene family has expanded in Asteraceae, a condition that appears to be connected with the increased developmental complexity and evolutionary success of this large plant family. Phylogenetic analysis of the CYC/TB1 gene family revealed both shared and lineage-specific duplications in gerbera and sunflower, corresponding to the three gene lineages previously identified as specific to core eudicots: CYC1, CYC2, and CYC3. Expression analyses of early stages of flower primordia development indicated that especially within the CYC2 clade, with the greatest number of secondary gene duplications, gene expression patterns are conserved between the species and associated with flower and inflorescence development. All sunflower and gerbera CYC2 clade genes showed differential expression between developing flower types, being upregulated in marginal ray (and trans) flowers. One gene in gerbera (GhCYC3) and two in sunflower (HaCYC2d and HaCYC2c) were indicated to be strong candidates as regulators of ray flower identity, a function that is specific for Asteraceae. Our data further showed that other CYC2 clade genes are likely to have more specialized functions at the level of single flowers, including the late functions in floral reproductive organs that may be more conserved across plant families. The expression patterns of CYC1 and CYC3 clade genes showed more differences between the two species but still pointed to possible conserved functions during vegetative plant development. Pairwise protein– protein interaction assays gave the first molecular evidence that CYC/TB1-like proteins function in complexes. Compared with sunflower, the gerbera proteins showed higher capacity for dimerization, between as well as within CYC clades. Our data from two distant species within the Asteraceae suggest that the expansion and the apparent conservation of especially the CYC2 clade CYC/TB1-like genes are associated with the evolution of the increased complexity of the Asteraceae inflorescence architecture.

The family of MADS box genes is involved in a number of processes besides controlling floral development. In addition to supplying homeotic functions defined by the ABC model, they influence flowering time and transformation of vegetative meristem into inflorescence meristem, and have functions in roots and leaves. Three Gerbera hybrida At-SOC1-like genes (Gh-SOC1-Gh-SOC3) were identified among gerbera expressed sequence tags.Evolutionary relationships between SOC1-like genes from gerbera and other plants were studied by phylogenetic analysis. The function of the gerbera gene Gh-SOC1 in gerbera floral development was studied using expression analysis, protein-protein interaction assays and reverse genetics. Transgenic gerbera lines over-expressing or downregulated for Gh-SOC1 were obtained using Agrobacterium transformation and investigated for their floral phenotype.Phylogenetic analysis revealed that the closest paralogues of At-SOC1 are Gh-SOC2 and Gh-SOC3. Gh-SOC1 is a more distantly related paralogue, grouping together with a number of other At-SOC1 paralogues from arabidopsis and other plant species. Gh-SOC1 is inflorescence abundant and no expression was seen in vegetative parts of the plant. Ectopic expression of Gh-SOC1 did not promote flowering, but disturbed the development of floral organs. The epidermal cells of ray flower petals appeared shorter and their shape was altered. The colour of ray flower petals differed from that of the wild-type petals by being darker red on the adaxial side and greenish on the abaxial surface. Several protein-protein interactions with other gerbera MADS domain proteins were identified.The At-SOC1 paralogue in gerbera shows a floral abundant expression pattern. A late petal expression might indicate a role in the final stages of flower development. Over-expression of Gh-SOC1 led to partial loss of floral identity, but did not affect flowering time. Lines where Gh-SOC1 was downregulated did not show a phenotype. Several gerbera MADS domain proteins interacted with Gh-SOC1.

Gerbera hybrida (sädelatva) on suosittu koristekasvi. Terälehtien värit ovat peräisin vesiliukoisistaantosyaaneista, jotka ovat sekundäärimetabolian tuotteita. Tutkiessamme tätä metaboliareittiä, löysimmesen läheltä haaran, joka johtaa karvaan makuisiin yhdisteisiin, gerberiiniin ja parasorbosidiin.Näiden karvasaineiden merkitys gerberalle on ollut tuntematon.Gerberan karvasaineiden biosynteesiin vaikutettiin geenitekniikan keinoin hiljentämällä keskeisen2PS-geenin toiminta. Anti-2PS-linjojen siirtogeenisiin gerberoihin ei gerberiiniä ja parasorbosidiasynny, vaan ne maistuvat tavanomaiseen gerberaan verrattuna makealta. Epäily näiden makealta maistuvienkasvien herkkyydestä hyönteisherbivoriaan heräsi kasvihuonehavaintojemme perusteella.Teimme erilaisia syöttö- ja munintakokeita, joissa hyönteiset saivat valita karvasaineettoman jakarvasainetta sisältävän gerberan välillä. Karvasaineilla oli voimakas estävä vaikutus täplätupsukkaan(Orgyia antiqua) ja krysanteemiyökkösen (Spodoptera littoralis) herbivoriaa vastaan. Ansarijauhiasen(Trialeurodes vaporariorum) toukille karvasaineilla ei ollut merkitystä.Tutkimuksemme perusteella karvasaineilla on hyönteisherbivoriaa ehkäisevä vaikutus. Eri lajikkeillaon vaihtelua karvasaineiden määrän suhteen ja yhdisteiden pitoisuuksien mittaaminen onyksinkertainen keino uusien lajikkeiden hyönteiskestävyyden arvioimiseen jalostustyössä. Pitkällätähtäyksellä on myös mahdollista molekyylibiologisin keinoin siirtää koko karvasainereitti uusiinhyötykasvilajeihin, käytännössä koristekasveihin.

According to the classical ABC model, B-function genes are involved in determining petal and stamen development. Most core eudicot species have B class genes belonging to three different lineages: the PI ,e uAP3, and TM6 lineages, although both Arabidopsis and Antirrhinum appear to have lost their TM6-like gene. Functional studies were performed for three gerbera (Gerbera hybrida) B class MADS-box genes—PI/GLO-like GGLO1 ,e uAP3 class GDEF2, and TM6-like GDEF1—and data are shown for a second euAP3-like gene, GDEF3. In phylogenetic analysis, GDEF3 is a closely related paralogue of GDEF2, and apparently stems from a duplication common to all Asteraceae. Expression analysis and transgenic phenotypes confirm that GGLO1 and GDEF2 mediate the classical B-function since they determine petal and stamen identities. However, based on assays in yeast, three B class heterodimer combinations are possible in gerbera. In addition to the interaction of GGLO1 and GDEF2 proteins, GGLO1 also pairs with GDEF1 and GDEF3. This analysis of GDEF1 represents the first functional characterization of a TM6-like gene in a core eudicot species outside Solanaceae. Similarly to its relatives in petunia and tomato, the expression pattern and transgenic phenotypes indicate that GDEF1 is not involved in determination of petal identity, but has a redundant role in regulating stamen development.

In this investigation, a GT1-7 cell-based cytotoxicity screening assay in 96-well microplates was set up. The assay, using propidium iodide fluorescence, was proven to be reliable, with good quality (Z' = 0.51) and low plate-to-plate and day-to-day variations. Further on, a library containing extracts from 227 genetic modification (GM) Gerbera hybrida and 42 Gerbera varieties was screened; however, no differences between them were found. Based on these findings, we propose the use of the current assay within the first-tier screening studies of large collections. Also, these results provide valuable information for GM Gerbera risk-assessment purposes and offer a model for the toxicity cell-based screening of GM crops.

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.

Several key processes in plant development are regulated by TCP transcription factors. CYCLOIDEA-like (CYC-like) TCP domain proteins have been shown to control flower symmetry in distantly related plant lineages. Gerbera hybrida , a member of one of the largest clades of angiosperms, the sunflower family (Asteraceae), is an interesting model for developmental studies because its elaborate inflorescence comprises different types of flowers that have specialized structures and functions. The morphological differentiation of flower types involves gradual changes in flower size and symmetry that follow the radial organization of the densely packed inflorescence. Differences in the degree of petal fusion further define the distinct shapes of the Gerbera flower types. To study the role of TCP transcription factors during specification of this complex inflorescence organization, we characterized the CYC-like homolog GhCYC2 from Gerbera . The expression of GhCYC2 follows a gradient along the radial axis of the inflorescence. GhCYC2 is expressed in the marginal, bilaterally symmetrical ray flowers but not in the centermost disk flowers, which are nearly radially symmetrical and have significantly less fused petals. Overexpression of GhCYC2 causes disk flowers to obtain morphologies similar to ray flowers. Both expression patterns and transgenic phenotypes suggest that GhCYC2 is involved in differentiation among Gerbera flower types, providing the first molecular evidence that CYC-like TCP factors take part in defining the complex inflorescence structure of the Asteraceae, a major determinant of the family's evolutionary success.

The effects of the transformation of a 4-coumarate:coenzyme A ligase (4CL) gene involved in lignin biosynthesis on the interactions between transgenic plants and soil microbiota were studied in silver birch (Betula pendula Roth). Although the genetic transformation did not lead to expected changes in wood chemistry, it had significant influence on root biomass and morphology. The transgenic lines had less root biomass and lower numbers of root tips than the wild-type birch. However, the transgenic lines formed normal ectomycorrhizas with Paxillus involutus (Batsch) Fr. and no differences between the transgenic lines and the wild-type birch were detected in the colonization by mycorrhizas in vitro. In a field trial, the decomposition of the transgenic and wild-type birch litter was equally rapid, and no marked differences were found between the transgenic lines and wild-type birch in total microbial biomass (SIR) or activity (basal respiration). The effects of the genetic transformation on the ergosterol content of the leaf litter were contradictory; the litter from one transgenic line had significantly higher ergosterol content and the litters from two other transgenic lines had significantly lower ergosterol content than the litter from wild-type birch. This study shows that the 4CL antisense transformation and the pleiotropic effects of the transformation on growth characteristics of birch did not disturb the formation of ectomycorrhizas with P. involutus and had no clear influence on the biodegradability of the leaf litter.

Background Transient gene expression utilizing syringe agroinfiltration offers a simple and efficient technique for different transgenic applications. Leaves of Nicotiana benthamiana show reliable and high transformation efficiency, but in quantitative assays also a certain degree of variation. We used a nested design in our agroinfiltration experiments to dissect the sources of this variation. Results An intron containing firefly luciferase gene was used as a reporter for agroinfiltration. A number of 6 week old tobacco plants were infiltrated for their top leaves, several samples were punched from the leaves after 2 days of transient expression, and protein extracts from the samples were repeatedly measured for luciferase activity. Interestingly, most of the variation was due to differences between the sampling spots in the leaves, the next important source being the different leaves on each plant. Variation between similar experiments, between plants and between repetitive measurements of the extracts could be easily minimized. Conclusions Efforts and expenditure of agroinfiltration experiments can be optimized when sources of variation are known. In summary, infiltrate more plants but less leaves, sample more positions on the leaf but run only few technical replicates. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0091-5) contains supplementary material, which is available to authorized users.

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.

Main conclusion Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5–5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC– MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.

Kasvien syntetisoimien kemiallisten yhdisteiden (sekundäärimetaboliittien) kirjo on laaja ja suhteessa tähän monimuotoisuuteen vain muutamien yhdisteiden biokemiallinen synteesireitti tunnetaan. Leikko- ja ruukkukasvina kasvatettava sädelatva eli gerbera (Gerbera hybrida, Asteraceae) tuottaa maanpäällisiin osiinsa kahta glukosidista karvasainetta, gerberiiniä ja parasorbosidia. Jälkimmäinen tunnetaan myös pihlajan karvasaineena. Glukosidit sellaisenaan torjuvat hyönteistuhoilta, ja sieniperäisten taudinaiheuttajien läsnäollessa ne hajotetaan aglykoneiksi joilla on sienitauteja torjuva vaikutus. Olemme tutkineet gerberan sekundääriaineenvaihdunnan tuotteita ja niistä vastaavia geenejä, ja löytäneet avaingeenin, joka johtaa gerberiinin ja parasorbosidin biosynteesiin. Yhdisteet muodostuvat asetyyli-CoA:sta ja malonyyli-CoA:sta reaktiossa, jonka ensimmäistä vaihetta katalysoi kasveille tyypillinen polyketidisyntaasiperheeseen kuuluva entsyymi. Vastaavanlaisia yhdisteitä tunnetaan sienistä ja bakteereista, mutta aiemmin ei tiedetty niitä esiintyvän myös kasveilla. Uutettavan gerberiiniaglykonin määrä eri gerberalajikkeissa korreloi harmaahomeen (Botrytis cinerea) kestävyyden kanssa. Gerberiini/parasorbosidireitin aktiivisuus toimii siten merkkiominaisuutena taudinkestävyydelle ja mahdollistaa kestävyystestauksen ilman taudinaiheuttajaa. Tutkimuksemme yhtenä tavoitteena on selvittää miten tämä merkkiominaisuus on helpoin mitata lajiketestauksessa. Vaihtoehdot ovat metaboliittien kemiallinen analyysi, lähetti-RNA:n analyysi tai DNA-testi. Tutkimuksemme laajempana tavoitteena on osoittaa gerberasta löydetyn gerberiini/parasorbosidireitin hyödynnettävyys pelto- ja puutarhakasvien kestävyysjalostuksessa. Mikäli reitti saadaan koottua toimivaksi uudessa kohdekasvissa, on odotettavissa että se toimii hyvin laajasti lähtömetaboliittien ollessa perusaineenvaihdunnan välituotteita. Esitetyllä metabolisella kestävyysjalostuksella on todennäköisesti laaja sovellettavuusala, joka viime kädessä riippuu kunkin hyötykasvin tautien ja tuholaisten herkkyydestä k.o. aineille. Ensimmäinen sovellusala olisi puutarhassa ja kasvihuoneella viljeltävät koristekasvit. Soveltaminen elintarvikkeiden raaka-aineisiin edellyttää toksikologisia tutkimuksia, mutta lähtökohtana on merkillepantavaa, että parasorbosidia esiintyy luonnostaan pihlajanmarjoissa, marja-aroniassa ja amerikkaisessa karpalossa.

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 μM; however, the laccase has a lower catalytic efficiency (V(max)/K(m)) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

We have identified an R2R3-type MYB factor, GMYB10, from Gerbera hybrida (Asteraceae) that shares high sequence homology to and is phylogenetically grouped together with the previously characterized regulators of anthocyanin pigmentation in petunia (Petunia hybrida) and Arabidopsis. GMYB10 is able to induce anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum), especially in vegetative parts and anthers. In G. hybrida, GMYB10 is involved in activation of anthocyanin biosynthesis in leaves, floral stems, and flowers. In flowers, its expression is restricted to petal epidermal cell layers in correlation with the anthocyanin accumulation pattern. We have shown, using yeast (Saccharomyces cerevisiae) two-hybrid assay, that GMYB10 interacts with the previously isolated bHLH factor GMYC1. Particle bombardment analysis was used to show that GMYB10 is required for activation of a late anthocyanin biosynthetic gene promoter, PGDFR2. cis-Analysis of the target PGDFR2 revealed a sequence element with a key role in activation by GMYB10/GMYC1. This element shares high homology with the anthocyanin regulatory elements characterized in maize (Zea mays) anthocyanin promoters, suggesting that the regulatory mechanisms involved in activation of anthocyanin biosynthesis have been conserved for over 125 million years not only at the level of transcriptional regulators but also at the level of the biosynthetic gene promoters.

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

A method for the separation of parasorboside and gerberin from the ornamental plant Gerbera hybrida (Asteraceae) has been developed. The two closely related glucosides were extracted using an Extrachrom® instrument, a prototype multi-functional separation tool equipped with an extraction chamber. The rotation planar extraction procedure was compared with that of a medium pressure solid–liquid extraction system. The resulting extracts were pre-purified using rotation planar chromatography and the results compared with those obtained using medium pressure liquid chromatography with silica gel as the stationary phase and a mobile phase of methanol:ethyl acetate:tetrahydrofuran at selectivity point Ps = 111 with 1% formic acid as modifier. The title compounds were isolated from the purified extracts by TLC and their structures confirmed by 1H- and 13C-NMR spectroscopy. Copyright © 2002 John Wiley & Sons, Ltd.

Agrobacterium tumefaciens -mediated genetic transformation method of the carnivorous medicinal plant round leaved sundew ( Drosera rotundifolia L.) was developed. The micropropagation conditions of sundew aseptically germinated seeds were defined and the internal kanamycin resistance of sundew was tested. Transformation was made by cocultivation of micropropagated sundew leaves with A. tumefaciens strain C58C1 containing a cointegrate plasmid vector with neomycin phosphotransferase and luciferase genes. Transgenic sundews were selected for kanamycin resistance and viable, fully developed plantlets were further assayed by luciferase activity and by PCR and Southern analyses using luc -primers and a luc -probe. The transformation efficiency of D. rotundifolia L. was 17%. Sundew is an important source of pharmacologically active 1,4-naphthoquinones, and genetic transformation allows the engineering of its biochemical pathways.

The processing quality of cereals can be modified by altering the structural grain constituents or the enzyme activities that mobilize storage reserves of the seeds. In order to complement the malt enzyme spectrum, a gene encoding for a thermotolerant fungal endo-(1,4)-ß-glucanase was introduced into two barley cultivars, Kymppi and Golden Promise. The gene was expressed in the seeds during germination, thus providing a thermotolerant enzyme that is active under mashing conditions. The amount of thermotolerant ß-glucanase produced by the seeds (ca. 0.025% soluble seed protein) has been shown to be sufficient to reduce wort viscosity by decreasing the soluble ß-glucan content. For the safe commercial cultivation of transgenic plants risk assessment of their cultivation is needed. In our study experimental estimates of the transgene flow from transgenic barley by pollen dispersal were produced. Field trials were conducted during the summers of 1996 and 1997. A transgenic barley line homozygous for the gene encoding for neomycin phosphotransferase was used as a source of pollen and male-sterile barley lines as recipients. In order to be able to transform the cross-fertilization frequencies to corresponding values of normal male-fertile barley, plots of normal barley were also included in the experimental plan. On the basis of our study, cross-fertilization in male-sterile recipient barley is possible with very low frequency up to 50 meters from the donor area. However, the frequency dramatically decreases with distance and due to self-pollination the possibility of cross-fertilization remains very low in normal cultivated barley.