For the second time since banana is commercially grown on large scale, banana is severely threatened by extinction by a fungal pathogen called Fusarium odoratissimum. Phenylphenalenones (PP) provide a promising dual protective strategy by acting as lead compounds for the synthesis of efficient plant protectants on the one hand and by mediating native defence mechanisms to banana species. The project will elucidate unknown steps in the biosynthesis of PPs to lay the foundation for the breeding of naturally resistant banana varieties. Therefore, a multidisciplinary approach is chosen using chemical and biological processes: The project investigates important parts of the interaction of bananas with the fungus Fusarium odoratissimum Tropical Race 4 (TR4). The cultivation of two different banana varieties with different degrees of resistance and the TR4 inoculation of the two banana varieties allows the determination of the phytochemical profiles and of the distribution of Cu and S at a cellular level. The location where these processes take place will be determined by a combination of nuclear magnetic resonance (NMR) spectroscopy, as well as different mass spectrometry- (MS) and microscopy- techniques. High-resolution transcriptome mapping of the Musa-TR4-interaction using fluorescent reporter Fusarium TR4 will be performed to generate spatially resolved gene expression maps. Single molecule fluorescent in-situ hybridization (smFISH) will be carried out to confirm spatiotemporal gene expression of selected PP/ML biosynthesis genes (polyphenol oxidases (PPOs)) in cross sections. The fundamental basic of our project are hypothetical detailed mechanistic considerations of the reaction sequences of the biosynthesis of phenylphenalenones (PPs) and musellarins (MLs), and an evaluation of the significant genetic information of the Musa genome and Musa transcriptomes. especially enabling the assignment of our key research enzymes, the PPOs. The formation of o-dihydroxy compounds and o-quinones, the subsequent ring closure reactions and the enzymatically catalyzed formation of aromatic systems will be investigated. PPOs are our key research enzyme candidates for these reaction steps. PPOs are to be processed by molecular biology and characterized by biochemical and X-ray crystal structure analysis. Finally, possible antifungal effects of PPs and MLs and their Cu complexes are to be investigated in bioassays.

Funding: FWF - Austrian Science Fund

Project number: I 6939

Duration: 01.06.2024 – 31.05.2027

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

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 and ANR - Agence Nationale de la Recherche

Project number: I 6151

Duration: 01.05.2023 – 31.10.2026

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

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

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