Franklin Gregory "HyperiSyn - Unravelling the molecular/ genetic network of hyperycin biosynthesis by employing innovative tools" OPUS 2017-2020

Hypericin is an important natural product which renders antidepressant, neuroprotective, anticancer, wound healing and several other pharmacological properties of Hypericum species (Acar et al 2014, Straub et al 2014, Walbroel et al 2014, Galeotti et al 2014). Hypericeum extracts are equally effective as most advanced antidepressant drugs (Prozac®, Zoloft® and Paxil®), while it displays almost no to much less adverse effects than conventional antidepressants (Rahimi et al 2009, Linde et al 2015). Currently, hypericin is considered as an important multifunctional lead drug molecule in new therapies (Karioti and Bilia 2010; Zhu et al 2014). Additionally, being a light sensitive molecule, photoexcitation properties of hypericin are under exploitation for use as fluorescent diagnostic tool and to treat tumours via photodynamic therapy (Kleeman et al 2014, Straub et al 2014, Liu et al 2015, Laffers et al 2014). In spite of several applications in the pharmaceutical industry and medical field, the biosynthesis of hypericin is not understood yet, mainly due to the lack of information about the genes involved in this pathway. Screening of differentially expressed transcripts and identifying putative genes involved in hypericin biosynthesis is the primary task of this project. Genes that are differentially expressed between the Hypericum plant materials with and without the dark glands/hypericin production will be identified and isolated. The functions of these genes will be validated through functional genomics approaches such as gene silencing and overexpression. The phenotypes of these manipulations will be evaluated at the level of dark gland development and hypericin production in these plants. With this straightforward approach we are certain to elucidate hypericin biosynthesis pathway, which not only benefits the basic research but also the applied sciences. Since H. perforatum is one of the most consumed medicinal plants in the world and would contribute to the knowledge-based bio economy, it is worth investing in the characterization of its biosynthesis pathways.


Robert Malinowski NCN OPUS: The role of phloem transport in pea plants adaptation to water deficit

Drought has a detrimental effect on crop cultivation. Each year due to the prolonging periods of water deficit in soil significant loss in yield of economically important crops occurs. Pea is a significant component of our diet but its successful cultivation depends on water availability during the early growth stage and flowering period. Limited water availability results in yield decrease and lower consumption or processing quality of pea. In Poland pea is mainly consumed as canned or frozen form. Breeding strategies of such varieties require detailed understanding of mechanisms responsible for drought tolerance. Studies on drought response in legumes show that pea plants are using drought escape (early varieties having short vegetation period) as well as the drought avoidance strategy. The latter strategy is based on increased water uptake by growing roots or decrease in water transpiration by stomata closing or leaf size decrease (Tabori et al., 2011). Pea plants developed also drought tolerance strategies based on the ability to increase the concentration of osmotically active substances like aldose and ketose molecules being the product of mannitol metabolism. Mannitol and sucrose are major photosynthetic products transported from the sites of synthesis (leaves) to other regions of plant via the phloem tissue. In this project we are aiming to verify the exact role of the structural and functional changes occurring within the phloem tissue in response to drought stress. We hypothesize that the ability of plant to modify osmotic properties in response to drought correlates with cellular/developmental phloem adjustment and increase in phloem transport. Our work will focus on anatomical and functional changes within the phloem as well as studies on sucrose and mannitol transport. We believe that the involvement of particular sugar loading and unloading proteins is essential in this response. Due to the fact that phloem encompasses only a small fraction of the total number of cells major problem in studies on sugar metabolism and phloem transport is the collection of defined biological material. All analyses performed on organ fragments can’t give the true appraisal of the process occurring in phloem. Here we are planning to use the stylectomy method based on the use of aphids as the tool for precise phloem sap collection. These organisms are capable for precise infestation from phloem sieve tubes. Excised mouth apparatus works as a capillary drain and allows to collect phloem sap for further studies. Samples obtained this way will be used for metabolic profiling of sugars. In order to obtain larger volumes of phloem sap we will use alternative phloem exudate collection method. We are also going to test drought driven expression levels of genes whose products are involved in phloem loading or unloading as well as factors regulating phloem differentiation. For this we will use tissue fragments from various plant areas to understand dynamics of the phloem related processes across the plant. These studies will be supported by anatomical studies of phloem bundles and in situ detection of differentially expressed gene transcripts. Our project will bring the new evidence on the involvement of phloem in plant adaptive responses to drought. At present functional phloem changes during water deprivation are unknown. therefore our research will help to get true appraisal of drought stress response in plants. Gathered knowledge can help future work on the increase of drought tolerance in pea plants. Our project is an original and multidisciplinary attempt for the complex and holistic description of a basic biological process mediating in plant adaptation to adverse environmental conditions.


Franklin Gregory NCN OPUS HyperNano: Understanding plant secondary metabolic changes in response to nanoparticles via an integrated omics approach in Hypericum perforatum.

Nanotechnology is a fast-growing field of science that uses the concept of miniaturization for solving chemical and biological problems. Nanoparticles (NP) are submicron size particles with less than 100 nm size, at least in one dimension, which are intensively used in energy, materials, computer chips, manufacturing, health care, medical diagnosis and consumer product industries. As per the National Science Foundation (NSF, USA) projection, the world market for nanotechnological products would account for about 3 trillion USD in 2020. Today, more than 1300 products incorporaing NPs are commercialy available. For instance, gold NPs are used as antibacterial agents in biocide coating, soap, toothpaste and shampoo, and is the most prevalent nanoparticle in over 25 consumer products. Silver (Ag) NPs are deployed in water purification, antifouling surfaces, and aseptic food packaging because of their antimicrobial potential. Medical applications of Ag NPs include wound dressings, surface sterilization of devices, implantations, masks, clothing, and bedding. ZnO NPs are in heavy use in personal care products such as sunscreens, cosmetics, textiles, paintings, and in industrial coatings, dye-sensitized solar cells, antibacterial agents, and optic and electronic materials. Copper oxide (CuO) NPs are used in gas sensing, optoelectronics, catalysis, solar cells, semiconductors, pigments and as fungicides. Titanium oxide (TiO2) NPs are found in paints, coatings, plastics, papers, inks, medicines, pharmaceuticals, food products, cosmetics, and toothpaste. NPs are released into the environment during their lifecycle as nanomaterial-containing wastes resulting in environmental pollution (Nanopollution). Although the presence of NPs affects largely the functioning of ecosystems, the exact impact is still unknown, as there is no technology available to detect the presence of NPs in the environment. As plants are intimately connected to water and soil, the two major accumulation sites of NP, they are at the most risk. Basically, NPs are considered as pollutants of unforeseen consequences reaching the plants in significant amounts. Studies conducted so far in several model plant species and crops have demonstrated that the NPs affect plants’ growth and development in a concentration dependent manner, depending also on the plant species and the physical properties of NPs. Upon interaction with NPs, disturbances of metabolic processes due to the formation of reactive oxygen species, damage to the structure and function of cell membranes, and a decrease in enzyme activities and DNA synthesis are observed in plants. In addition, clues emerging from the literature also suggests that plant secondary metabolism could be affected by the nanopollution. However, the impact of NPs on plant secondary metabolism has so far not been studied in any of the plant species comprehensively. Plant secondary metabolism is vital for plants survival as they play indespensable roles in plants as protectants against herbivores, pathogenic microbes, as signals for plant symbiotic interactions with beneficial microorganisms, as allelopathic agents in natural habitats to protect against competitors, as physical and chemical barriers to abiotic stressors such as UV and as endogenous regulators of plant growth regulators. In addition to their roles in plant survival, many secondary metabolites are economically important as drugs, flavor and fragrances, dye and pigments, pesticides, and food additives. Importantly, many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. In spite of their importance to plants’ survival and human well-being, only limited information is available in the literature in the context of nanopollution effect on plant secondary metabolism. In HyperNano, we will investigate how NPs might affect plant secondary metabolism using H. perforatum as a model. Executing HyperNano is a basic scientific priority to will fill the gap in the scientific literature and widen our current knowledge. Altogether, HyperNano will be of great interest to medicinal plant farmers, consumers, pharmaceutical industry, environmentalists and to the scientific community.


William Truman "Understanding the role of chitin related defence responses during Plasmodiophora brassicae infection" OPUS 2016-2019

Plants lack an adaptive immune response and must rely on innate recognition of pathogen markers in order to counter infections. The cell walls of microbial pathogens are one source of conserved molecular motifs that plants can identify; short chains of chitin from the walls of fungi being one such elicitor. Some protists lack chitin in their cell walls but resting spores of Plasmodiophora brassicae, the causal agent of clubroot disease in brassicas, contain 25% chitin. During the infection of Arabidopsis by P. brassicae many defence genes known to be induced by chitin are significantly suppressed, including the chitin receptor and associated signalling components. Our conjecture is that this suppression of chitin-associated signalling pathways is a critical aspect of P. brassicae virulence. The genome of P. brassicae encodes several putative secreted, chitin binding proteins; some may act to deacetylate chitin forming chitosan, a less potent elicitor of host defences, while others have no clear molecular function. Chitin-binding virulence factors in other plant pathogens can both disguise chitin from host detection and protect against hydrolysis by plant chitinases. We hypothesise that chitin binding proteins within the P. brassicae secretome contribute to the defeat of host surveillance mechanisms and the establishment of clubroot disease in Arabidopsis. Chitinase enzymes are produced by plants in response to infection; they can attack pathogens by degrading cell walls, releasing chitin oligomers that stimulate host chitin receptors and amplify danger signals. The aim of this project is to characterise the dynamics of surveillance and counter-surveillance, measures and counter-measures that pivot around this fundamental microbial structural component.

The main objectives of the project are:

  1. Characterising the cellular events involved in host chitin perception during clubroot infection of Arabidopsis and determining the contribution of chitin signalling pathways to resistance.
  2. Profiling Arabidopsis chitinase activity in response to P. brassicae and assessing clubroot pathogenicity in lines with altered chitinase activity.
  3. To survey the natural variation arising in different Arabidopsis accessions and P. brassicae pathotypes with regards to chitin signalling responses and chitinase activity during infection.
  4. Understanding the functions of P. brassicae chitin binding proteins and establishing whether they are implicated in pathogen virulence.


Karolina Stefanowicz "The importance of cell wall changes occuring within host plant for the Plasmodiophora brassicae infection progression" SONATA (2016-2019)

Similar to animals, plants are also being invaded by various pathogenic factors like viruses, bacteria or protists. Every year plant diseases cause severe crop yield losses reaching as much as 40%. In view of the importance of plants to humans, research is currently focused on finding the genetic sources of resistance and on developing new resistant varieties of crop plants. The main recurring problem is, however, that with time genetic resistance is broken down leading to a substantial yield loss. This major issue has been well recognized in the case of plant resistance against a pathogenic protist Plasmodiophora brassicae, responsible for causing a clubroot disease in agriculturally important Brassica species. The disease has been named after one of the symptoms characterized by the development of galls on the underground parts of the infected plants.

Clubroot infection constitutes a serious problem worldwide and in Poland. It mainly affects oilseed rape which is a considerable source of plant oil. Due to misconducted cultivation (too short crop rotation or no rotation at all) the clubroot disease is of particular concern and became an enormous issue in countries leading in oilseed rape cultivation. These countries include Poland and Canada, which were the first to introduce cultivation of oilseed rape for oil production. Until now it is known that some of the P. brassicae pathotypes have already overcome the resistance provided by genes present in most of the currently cultivated resistant high-yield varieties.

One of the major problem in the development of plants resistant to P. brassicae is that instead of fighting a single pathogen, in fact we are dealing with a group of genetically different pathogens. This results in an extreme threat to oilseed rape cultivation and constitutes a risk of high financial loss. One of the possible strategies which could contribute to alleviate the effect of breaking genetic resistance is the introduction of tolerant plants which would be only slightly affected or in which the pathogen could not proliferate. It seems that the best alternative is to introduce genetic resistance to the plants characterized by an increased tolerance towards the pathogen. Nevertheless, in order to do so, it is necessary to thoroughly investigate the disease progression in the plant as well as the molecular and physiological aspects of plant-pathogen interactions. Studies are available which precisely demonstrate the changes in gene expression and metabolic alterations in infected plants. Furthermore, developmental changes leading to the development of galls have been also well described. Recently it has been shown that hormones responsible for plant growth regulation (brassinosteroids) participate in the development of the giant cells in which the pathogen produces resting spores (Schuller et al., 2014). Apparently, preventing from formation of the giant cells could significantly reduce the number of spores produced or perhaps could even impede spore maturation. Clearly, this cannot be achieved by modification of the activity of plant hormones since they are implicated in many crucial biological processes in the plant and such alterations could result in pleiotropic effects. However, it is possible to modify the factors which allow the formation of the giant cells. These key factors certainly comprise proteins involved in cell wall remodeling. Cell wall constitutes the most outer layer of plant cell. Its major component is cellulose which forms structures called microfibrils. The remaining elements of the primary cell wall include water, pectins, hemicellulose and proteins. In the course of cell maturation or differentiation of cells which perform specialized functions, cell wall is subjected to secondary modifications and thereby may contain substances like lignin or suberin. In order for the cell to grow, the cell wall must undergo remodeling or modifications ensuring elasticity upon increasing size of the protoplast. The processes of cell wall remodeling are regulated by proteins belonging to the group of cellulases and endotansglikosylases of xyloglucan, while cell elasticity by expansins.

Cell wall remodeling studies rely on the isolation of cell wall proteins following the removal of other cell components. Based on the fact that proteins differ in their mass and charge, it is possible to separate them by electrophoresis and after isolation determine their amino acid composition using mass spectrometry. In this project we aim to isolate and identify cell wall proteins which either emerge or disappear at the time of giant cell formation as well as upon cell disintegration during development of galls after P. brassicae infection. For better understanding of the role of regulation of these identified proteins in pathogenesis, we will use genetic engineering techniques allowing to modify the activity level of genes encoding the proteins of interest. The effects of these alterations will be then observed microscopically as well as in tests of infection capacity of developing resting spores of P. brassicae.


Jorge Almiro Pinto Paiva "PurpleWalls - Unraveling genome regulation to modulate wood formation in Salix purpurea: an integrative approach" SONATA BIS 2016 - 2019

The use of bioenergetic plantations of sources of renewable energy is one of the main directives of the energetic policy in Europe. Being part of the natural composition of Polish vegetation, willow has been recommended as bioenergy crop for Poland. Indeed, Poland has favourable conditions for production of willow biomass, being estimated that 1.5-to 2.1 Mha the surface that could be dedicated for short rotation coppice plants. Willows plantations are being used primarily for energy and phytoremediation. In 2009, willows plantations for bioenergy surface (6,160 ha) represented 60.5% of total energy crop surface and 95% of all perennial energetic plantations. Willow plantations are expected to play a significant role amongst the energy-dedicated lignocellulosic biomass crops to reach the goal of 14% of energy derived from biomass in Europe in 2020). Understand the molecular mechanisms underlying wood formation and the modulation of the cell wall composition can lead to new breeding strategies of willows for bioenergy purposes. PurpleWalls will address new and relevant insight genetic and molecular mechanisms controlling phenotype determination, and phenotypic adaptation to growing conditions in Salix purpurea as model for other Salicaceae and perennial species used for bioenergy. Until now, most of the research developed on identification of candidate genes and key regulatory gene networks in developing xylem, did not consider the interplay between different levels of regulation in woody plants. PurpleWalls will use an Integrative Biology approach to reveal new key genes and regulatory gene networks that modulate xylogenesis and the secondary cell walls biosynthesis. Data generated within the project will be made available through dedicated and open access databases for scientific community. Publication of the results will be directed towards the top ranked peer-reviewed journals, due the novelty and importance of this research theme. Preferentially, it will be chosen open-access journals in order to facilitate article dissemination. Results will be also presented (posters and oral communications) at renown national and international conferences This highly innovative and appealing were able to attract new national and international collaborations, and together with advanced formation (master and PhD thesis) will contribute to reinforce the transfer of knowledge and technology and the capacity building of the new team –Cell Wall by Design- to be created in the frame of this project. At long-term, it is expected that the tools and knowledge gathered in the frame of PurpleWalls project will contribute to reduce production inputs, and to increase productivity of planted forest and quality of the forest raw products, thus contributing to a sustainable mitigation of human pressures on native forests, associated biodiversity, and of the use of marginal land, and to the empowerment of local populations. Moreover, the PurpleWalls potentially able to patentable applications, to be transformed into practical breeding applications with high commercial impact.


Franklin Gregory “HyperAgro: Understanding the relevance of pathogenesis-related defence mechanisms in plant recalcitrance against Agrobacterium mediated transformation” OPUS 2018-2021

Agrobacterium tumefaciens mediated plant transformation is an indispensable tool for crop improvement, plant functional genomics, genome editing and synthetic biology (Hwang et al., 2015; Nester, 2015; Sainsbury and Lomonossoff, 2014; Xu et al., 2014). Unfortunately, several economically important plant species (example: members of legumes, cereals, biofuel crops, fruit trees, medicinal plants) remain difficult to transform using this bacterium (plant recalcitrance), a phenomenon yet to be understood. Moreover, the widespread of plant recalcitrance phenomenon in the plant kingdom makes the use of such contemporary tools as CRISPR / Cas9 or RNAi limited to only a few plant species. Although the mechanism of plant recalcitrance is still unknown, successful activation of plant defense response against A. tumefaciens is the prevailing cause (Hou et al 2016). We have shown that an incompatible interaction between A. tumefaciens and a recalcitrant plant leads to the death of A. tumefaciens and prevents T-DNA transfer (Franklin et al 2008, 2009, Hou et al 2016). Although many studies enriched the wealth of our knowledge regarding plant- A. tumefaciens interaction (Veena et al 2003, Ditt et al 2006, Yuan et al 2007, Anand et al 2008), the molecular determinants of plant recalcitrance and how recalcitrant plant species effectively avoid gene transfer into their cells are yet to be uncovered. Therefore, the discovery of this incompatible Plant- A. tumefaciens interaction will provide us with the exciting possibility to utilize this system as a model to understand how recalcitrant plants avoid gene transfer into their cells. In this way, successful completion of the project would revolutionize functional genomics of several economically important crops.