Catecholamines are a class of small chemical molecules. They include important animal neurotransmitters and hormones such as dopamine, norepinephrine (noradrenaline) and epinephrine (adrenaline). The function and biosynthesis of catecholamines is well-known in animals, however they also occur in plants where their function and biosynthesis are much less understood. They likely act as defence molecules against other living things, such as by harming competing plants or predatory insects, and they may have a role in protection against environmental stresses, such as drought. They also may influence carbohydrate levels in the plant, a role reminiscent of the regulation of insulin by dopamine in animals. Overall, however, data on the function of catecholamines in plants is very limited. Catecholamines are also very interesting from an evolutionary biology perspective. They almost certainly arose independently in plants and animals and may also have arisen several times independently in different plant species. This prompts questions such as: Are there multiple ways to biosynthesise catecholamines? Are similar types of genes and enzymes involved in different species? Did catecholamines evolve to serve a similar function in different species? Currently, evolutionary and functional insights are limited by our lack of knowledge of how these compounds are made in plants. Therefore, this project aims to determine the biosynthetic pathway that produces catecholamines in plants.
To achieve the overall aim of the project, the catecholamine biosynthetic pathway will be determined in three plant species, representing at least two evolutionary origins of these compounds: Beta vulgaris (beet), Mucuna pruriens (velvet bean) and Vicia faba (faba bean). The scientific approaches used to do this will include using cutting-edge nucleic acid sequencing technologies combined with the chemical analysis of catecholamine levels in different tissues of the plants. Enzymes thought to be involved in catalysing the biochemical reactions will be validated by using biochemical approaches and by producing transgenic plants in which enzyme function has been perturbed. Those enzymes that are proven to be involved in catecholamine biosynthesis will then be compared between species in order to determine their evolutionary history and relationships.
This project will represent the first time that the catecholamine biosynthetic pathway has been determined in any plant species. Determining the pathway in multiple plant species will then provide the foundation from which to compare how catecholamines evolved in these species, as well as between plants and animals. The genetic resources developed during the project will also provide insights into the function of catechol amines in plants and will provide an indispensable basis for ongoing research in this area.

Funding: FWF - Austrian Science Fund

Project number: ESP 122 ESPRIT-Programm

Duration: 01.02.2023 – 01.01.2026

Contact: PhD Hester Sheehan

Dihydroflavonol 4-reductase (DFR) is a key enzyme in flavonoid biosynthesis in plants and catalyzes the formation of anthocyanin precursors. The natural substrates for the DFR are dihydrokaempferol, dihydroquercetin and dihydromyricetin, which differ in their chemical structure by a different number of hydroxyl groups (OH groups) in the so-called B ring. The substrate specificity of DFRs can vary, with some DFRs accepting all three substrates equally, while others have high specificity for particular substrates. Since the number of OH groups in the substrate determines the color of the anthocyanins, the substrate specificity of the DFR is a decisive factor in the different expression of flower or fruit colors. The substrate specificity is determined by slight differences in the amino acid sequence in the region of the substrate binding site – and thus slightly altered structure – of the enzyme. Although numerous studies are available on the substrate specificity of the DFR, there is still no systematic understanding of how precisely this specificity is determined at the molecular level of the amino acid sequence. The aim is to establish the structure-function relationship of the DFR in terms of its specificity to the three substrates.

In the project, various DFRs from plants are produced in bacterial cultures and their substrate specificity is examined using enzyme assays. The results are related to the respective amino acid sequences in the region of the substrate binding site. In particular, the DFR from the grapevine is of importance, since the crystal structure of the enzyme is known. This enables the complementary combination of "in silico" techniques (theoretical enzyme modeling using special software) with experimental data from the enzyme assays. In this way, the effects of changes in the amino acid sequence can be predicted and verified experimentally, and vice versa the corresponding substrate specificity can be derived in the model based on the amino acid sequences. This is supported by the generation of point mutations in the enzyme, where one or more amino acids in the region of the substrate binding site are specifically exchanged and the effect on the substrate specificity is determined. Ultimately, it should be possible to predict substrate specificity based on amino acid sequence information. The precise understanding of the substrate specificity at the level of the amino acid sequence contributes significantly to the targeted breeding of plants with new flower and fruit colors. Furthermore, the project deals with the evaluation of different DFR enzyme tests to enable robust and comparable results within the research community.

Funding: FWF - Austrian Science Fund

Project number: I 5161

Duration: 01.05.2023 – 31.10.2026

Contact: Senior Scientist Dipl.-Ing. Dr.rer.nat. Christian Haselmair-Gosch

This project studies the key step in the biosynthesis of phloridzin, which is the prevalent polyphenolic compound in apple. Phloridzin represents more than 90% of the soluble phenolic compounds in apple leaves. The presence of such high amounts of phloridzin makes apple unique since other species accumulate only very low amounts and many closely related species like pear are not able to form phloretin or its glucosylated relative phloridzin. The last decade has seen an explosion of research on the beneficial effects of phloretin and phloridzin for human health but the physiological relevance for apple is still unclear. A possible involvement in disease resistance is discussed. Previously we have shown with apple leaf extracts that phloridzin formation is based on three biosynthetic steps: (1) the formation of dihydro-p-coumaroyl-CoA from p-coumaroyl-CoA by a dehydrogenase, (2) further formation of phloretin by the common chalcone synthase and (3) the glucosylation of phloretin in position 2’. Whereas the last two steps were already intensively studied, the knowledge of the first step is limited. The enzyme is crucial, because it seems to be the key point making the phloridzin-hoarding apple unique in comparison to other plants. In our previous FWF project (P25399-B16) we successfully completed a challenging purification process and were able to purify for the first time a candidate enzyme from apple leaves, which exhibits strong enzyme activity with p-coumaroyl-CoA to form dihydro-p-coumaroyl-CoA. The planned follow-up project will now target the detailed characterization of this important enzyme from apple leaves for the first time. Structural studies will resolve the enzymatic mechanism, such as protein crystallization and effects of substrates, inhibitors/effectors or other factors. The DNA sequence of the dehydrogenase will be isolated from apple and transferred into bacteria to produce large amounts of the enzyme for detailed characterization. It will be tested, in which tissue and developmental stage the dehydrogenase gene is switched on or off.  Functional activity of the gene products will be tested with genetically modified plants where phloretin formation will be enabled by the dehydrogenase in thale cress (Arabidopsis) or disabled in apple. Comparison of the DNA and protein sequence of the dehydrogenase from different plant species will give insight to structure-activity relationship of the enzyme on the molecular level. The project members consist of three teams which provide complementary know-how and resources: One of the Austrian teams offers knowledge in phloridzin biosynthesis, molecular biology and enzymatic evaluation, the other Austrian team has profound experience in protein characterization and crystallisation, whereas the German team provides the infrastructure and long-term experience in the creation of transgenic plants. An external team from New Zealand, which is funded by own sources will additionally contribute its know-how on pear transformation.

Funding: FWF - Austrian Science Fund and ANR - Agence Nationale de la Recherche

Project number: I 4296

Duration: 01.05.2020 – 30.04.2024

Contact: Senior Scientist Dipl.-Ing. Dr.rer.nat. Christian Haselmair-Gosch

The proposal "B-Ring hydroxylation in the flavonoid pathway" studies key enzymes in the biosynthesis of flavonoids and anthochlor pigments (chalcones and aurones), which provide yellow, red and blue flower colour in a number of ornamental plants, form UV-honey guides in certain Asteraceae species and also show health-beneficial effects in humans. Furthermore, chalcones are the immediate precursors for the formation of flavonoids, which are important compounds in plant-derived human diets and of isoflavonoid-based plant defence compounds.
This is a follow up proposal to the expiring P29552-B29 project, that started the establishment of the first crystal structure of a cytochrome P450 dependent enzyme of the flavonoid pathway using chalcone 3-hydroxylase (CH3H) as model. Funding for this follow up project will be crucial to allowing the team to capitalize on the foundation work already performed during the previous project. CH3H catalyzes the introduction of a hydroxyl group in position 3 of chalcones. The reaction shows high similarity to the hydroxylation of flavonoids in position 3' but cannot be catalyzed by the prominent flavonoid 3'-hydroxylase (F3'H) despite its broad substrate specificity. F3'5'H, in contrast, catalyze the introduction of two hydroxyl groups in positions 3' and 5', thereby shifting the colour of resulting pigments from red to blue and violet. Previously, we successfully established methods for the large-scale production and purification of sufficient amounts of CH3H for crystallization experiments. The elucidated three-dimensional structure of CH3H is essential to fully understand the structural differences determining the divergent functionality of CH3H and F3'H and of the reaction mechanism of F3'5'H. In future, this will feed into the breeding of plants showing increased disease tolerance and/or improved attractiveness of plants and enhanced health beneficial effects to consumers.

Funding: FWF - Austrian Science Fund

Project number: P 32901

Duration: 01.01.2020 – 31.12.2023

Contact: Associate Prof. Dipl.-Ing. Dr.in techn. Heidi Halbwirth

Establishing a strong and lasting international training network for innovation in food and juice industries: a 4D-research approach for fruit juice processing.

HiStabJuice is a European intersectoral and interdisciplinary network offering research training to 11 ESRs (early stage researchers), which is relevant to the entire food industry worldwide. Initiated by the International Fruit and Vegetable Juice Association (IFU), the European training network (ETN) combines the scientific expertise of 5 universities and 2 research institutions with the technological experience of 10 industry partners from7 EU countries. This ETN stands out by its exceptionally high industrial involvement that ensures training in real world ability and in solving key problems with exceptional analytical and technological skills combined with training in key transferable skills for public and private sector employment. The ESRs will work together on the evaluation of various factors influencing colour stability in fruit juices, focussing on raw materials and preservation techniques, as well as associated effects, deleterious to the health benefits of the final products. Pioneering aspects include the first empirical analysis of the contribution to colour stability and nutritional value of thermostable enzymes, fruit variety, ripeness, harvest time, traditional and modern preserving techniques (pasteurization, freezing, pulsed electric field, ohmic heating, high pressure processing), which will be evaluated in a 4D approach (microbes, enzymes, nutrients and chemical-physical parameters). Upon conclusion of the action, the ESRs will have established a universal, empirical system for deciding which fruit types to harvest at which times, at which stage of the ripening process, and which preservation method will give the best colour and nutrient stability. In line with the Horizon2020 strategic priority of Open Science this knowledge will be freely accessible. This has the potential to revolutionise the fruit juice industry and will fortify the European industry for decades to come. The participation of IFU guarantees unsurpassed intercommunication between the ETN and industry stakeholders across the EU.

Funding: European Commission, Horizon2020

Project number: ITN-ETN-956257

Duration: 01.11.2020 – 28.02.2025

Contact: Projektass. Dipl.-Chem. Dr.rer.nat. Christian Molitor

Earth laughs in flowers: Research training in horticultural precision breeding addressing flower colour as established model.

The COLORnamental team comprises a promising Experienced Researcher (ER) in the field of horticulture and molecular breeding and her future supervising team, i.e. an academic beneficiary in Austria, an associated partner from the commercial horticultural industry in Germany and an academic associated partner in Germany hosting a 6 month secondment. Together they have defined an innovative and challenging research project that offers a well-balanced mixture of research at the interface of fundamental and applied research addressing promising opportunities in the non-academic sector and scientific hot-spots in plant research simultaneously. An innovative breeding approach will exploit flower colour as an established model to implement for the first time the use of MAD7 nucleases in ornamental breeding, and protoplast transformation to modify the bract colour of poinsettia. The genome editing approach addresses the substrate specificity of dihydrolflavonol 4-reductase (DFR) and will promote the accumulation of orange pelargonidin based pigments. Flavonoids, including the colourful anthocyanins, are the most important secondary metabolites, contributing a broad range of physiological functions in plants and humans, the latter when consumed as plant derived food. This makes them an attractive topic for industry and academia across many research fields. The pathway has always served as an important model to establish fundamental scientific knowledge of enzymes, gene regulation in plants and a variety of evolutionary processes. This will allow the ER to augment her expertise with knowledge that is seen as the future of food security, while herself imparting a new perspective to her hosts. A tailor-made training plan, composed of scientific key transferable skills elements for public and private sector employment (research management, presentation and language skills) was designed to empower the ER to move up the career ladder.

Funding: European Commission, Horizon Europe

Project number: 101065228

Duration: 01.04.2023 – 30.09.2025

Contact: Associate Prof. Dipl.-Ing. Dr.in techn. Heidi Halbwirth