Browsing by master's degree program "Master's Programme in Integrative Plant Sciences"
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(2024)Plants have evolved mechanisms to cope with various environmental stresses, including abiotic factors like temperature extremes and biotic factors involving the interactions with pathogens and herbivores. Kale (Brassica oleracea var. acephala) is a superfood famous for containing many compounds that are beneficial in the human diet but are primarily produced as specialised metabolites to aid in plant defence. Amongst these are glucosinolates which are defence compounds characteristic of plants in the Brassicaceae family. The aim of this study was to investigate how the diverse metabolic profiles of kale cultivars contribute to postharvest resistance against herbivory and necrotrophy. To assess the resistance of each kale cultivar against herbivory, I used the larvae of the wood tiger moth Arctia plantaginis as a test subject. We used detached leaves from 30 kale cultivars in an overnight feeding experiment with the larvae. The same 30 cultivars were used in a postharvest infection experiment with a generalist necrotroph B. cinerea to investigate the resistance of each kale cultivar against necrotrophy. For a comparative experiment between necrotrophs, we selected 10 kale cultivars to assess the necrosis caused by B. cinerea and a specialist necrotroph A. brassicicola. The A. brassicicola-infected and mock-treated leaves were analysed for their metabolic profiles to observe how these were altered by the infection. The weight gain of the tiger moth larvae was not significantly affected by the kale cultivars or their sugar content. A correlation between sucrose and indole glucosinolates might have reduced the kales’ palatability and potentially deterred the herbivores. In the B. cinerea experiment, we observed a positive correlation between necrotic lesion area and protein, sucrose, and indole GSL contents in kale leaves, even though indole GSLs are generally considered defence molecules against necrotrophic pathogens. When comparing the necrotic damage caused by the two necrotrophs, the specialist A. brassicicola exhibited a statistically significantly more violent infection compared to B. cinerea. Chlorophyll became degraded in the infected leaves compared to the uninfected controls. Amino acid content was high in the aged control and infected leaves, indicating protein degradation either due to senescence or cell wall-degrading enzymes from the pathogen. There was a statistically significant positive correlation between necrotic damage and protein in the infected leaves potentially due to proteins being secreted by the pathogen during infection. Starch levels decreased in the infected leaves compared to the controls. The infected samples also showed decreased glucose amounts potentially being taken up by the necrotroph during infection. Altogether, the study showed that kale cultivars respond to biotic stress factors by triggering metabolic changes that can affect the disease resistance and postharvest quality of the leafy vegetables.
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(2024)Drought events will increase in the future due to climate change and thereby threaten agricultural production. The growing world population needs stable yields nonetheless thus, solutions need to be found. One way to do so is to explore mechanisms that allow plants to withstand harsh conditions and recover from them. Autophagy is such a mechanism. It is a bulk degradation pathwaythat allows plants to recycle cell components. During biotic and abiotic stress autophagy pathways are enhanced. Even though this mechanism is very useful and is proven to enhance abiotic stress tolerance, very little is known about it in plants. This thesis aimed at defining the informative time points for drought and autophagy response during drought and recovery in barley. To do so barley plants were subjected to gradual drought by withholding irrigation. They were kept at a level of severe drought for 4 days and were then rewatered. The tip of the third leaf was sampled for RNA extraction followed by qPCR. The genes this thesis focused on were GST and HSP17 as indicators of drought stress and ATG6 as indicator for autophagy. Additionally, phenotypic data was collected via RGB imaging for monitoring wilting and thermal imaging for stomata closure. GST was upregulated during the onset of drought, which seems to make it an early response gene, while HSP17 was upregulated during severe drought. ATG6 had a high transcription rate during all stages of drought and only decreased during recovery. However, because the primer efficiencies calculated in this experiment appear to be very low, and data points were missing due to pipetting practice, the results are not reliable and need to be repeated. Thus, no prediction about the informative time points can be made. The thermal data showed good results and the expected pattern of stomatal closing during drought could be observed. But because the greenhouse is a variable environment a reference would make the data even more precise.
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(2024)Secondary growth in plant vasculature tissues originate from meristematic tissue vascular cambium. Vascular cambium produces xylem inwards and phloem outwards in the plant root and stem. Vascular cambium regulatory mechanisms are not completely known, and while plants and especially trees are dependent on the mechanical support and transportation systems the vasculature provide, increased knowledge of the vascular cambium and especially xylem development is valuable. In this thesis, I explored Arabidopsis thaliana single-cell RNA-sequencing data from secondary growth stage via cloning transcriptional reporter lines, and later utilized the transcriptomics data to find potential xylem development regulators. By loss-of-function mutant and inducible overexpression line screening, I found two genes in a family of MYB transcription factors to be potential regulators in xylem development. Loss-of-function mutant did not show significant phenotype, but overexpression lines under constitutive 35S promoter showed extreme halt of growth, and a stain-filled vessel phenotype in a fixed and resin-embedded Arabidopsis cross-section. Under developing xylem -specific promoters, inducible overexpression lines showed the same phenotype, and while grown to the secondary growth stage, I detected some patterning defects. Thus, these results indicate, that these MYBs expressed in the xylem parenchyma in the single-cell transcriptomics data, might have a function in xylem development.
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(2024)As biodiversity is declining globally, there is a crucial need to understand the connections between diversity and ecosystem functioning. Research has shown that diversity tends to lead to higher productivity, but our understanding of the underlying reasons is still limited. For example, biotic interactions with mycorrhizal fungi could cause these diversity effects in plant communities, but few studies have assessed the subject. In this thesis, I studied the impacts of arbuscular mycorrhizal symbiosis on diversity effects with three research questions. First of all, I aimed to determine whether mycorrhizal symbiosis affects the diversity–productivity relationship across plant communities. Secondly, I divided diversity effects into two parts: complementarity and selection effects. These effects can be determined statistically. Finally, I studied how different species within the community respond to diversity. To answer these questions, I conducted a greenhouse study with varying plant communities. I manipulated the plant species diversity and used a fungicide treatment to suppress mycorrhizal fungi. After the growing period, I measured the biomasses of the plants as an indicator of productivity. I tested the effectiveness and side effects of the fungicide by estimating mycorrhizal colonization rates in plant roots and by testing its effects on non-mycorrhizal plant species. The fungicide treatment did not affect diversity–productivity relationship nor diversity effects significantly. However, fungicide treatment failed to reduce the overall mycorrhizal colonization rate and had varying effects on different plant species. One of the plant species benefitted from species diversity whereas other species experienced either no effect or a negative effect of increased diversity. No specific trait explained the varying responses on diversity. More research is needed to understand plants’ complex responses to decreasing biodiversity and biotic interactions, as well as to clarify whether fungicide is a fitting method for mycorrhizal suppression studies.
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(2024)Nuclei isolation is a method used e.g. as a part of chromatin structure research. DNA structure can be examined in its 3D form from isolated nuclei because DNA is still wrapped around the histone proteins. Examining the chromatin structure can offer information e.g. about gene expression and how it is regulated. Isolating nuclei from plant cells demand more optimization compared to animal cells because of the cell wall, chloroplasts and secondary metabolites. The presence of organellar DNA can hamper the later DNA analysis. Secondary metabolites can hinder the actual isolation process. Finding the suitable isolation protocol for species of interest may need careful optimization of different aspects. Different species can differ from each other based on the structural and biochemical characteristics and because of this the same protocol may not ensure as good results for them. Different tissue types of the same species may have also differences in their biochemical and structural characteristics. In this thesis work, three different isolation protocols were used for three plant species; Pinus sylvestris, Betula pendula and Arabidopsis thaliana. The purpose of the work was to compare the results from each nuclei isolation protocol. Optimization of isolation protocol for P. sylvestris and B. pendula would help the isolation process for later research. Thesis work was done to get guidance for this optimization. Samples handled with different protocols were different from each other based on the sample concentration (particles/mL) and the average size of isolated particles. Chloroplast contamination was tested with chloroplast specific primers with PCR. None of the samples were free from chloroplasts.
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Plant biodiversity and ecosystem functioning in a latitudinal gradient of heath forests in Finland (2024)In this thesis, I study plant biodiversity and ecosystem functioning in Finnish heath forests. To improve the understanding of how terrestrial understory plant biodiversity affects forest ecosystem functioning, I use structural equation modelling with information from climate and landscape land cover, using study areas along a 800 km latitudinal gradient. In addition, I describe the characteristics of the studied plant communities from taxonomical and functional point of view together with related environmental variables. I conducted a field survey during summer 2022. Study area consisted of five regions, ranging from southern Finland to near Arctic Circle: Tvärminne, Helsinki Metropolitan Area, Evo, Konnevesi and Kuusamo. I sampled and identified terrestrial vascular plants, lichens and bryophytes by coverage from 80 vegetation plots of 1 m2 in each region. In total, data consists of 400 vegetation plots and total amount of species was 134. We measured environmental variables in the field or draw them from remote sensing databases. To model the causal pathways among climate, landscape land cover, forest ecosystem functioning measured as Normalized Difference Vegetation Index (NDVI) and terrestrial understory plant biodiversity, I constructed six structural equation models (SEM) with different combinations of taxonomical biodiversity metrics and landscape land cover variables. Based on previous studies I hypothesized that biodiversity is positively related to ecosystem functioning. Models explained 17 – 21 % of observed variation in ecosystem functioning. The most important explanatory variable was the number of growing degree days and the second most was the taxonomical understory biodiversity. To provide functional summary of the studied plant communities, I categorized plant species according to their functional group as well as Grime’s CSR Triangle Theory’s strategy classes. The three strategy classes places species according to their high or low tolerance to stress and disturbance. Three classes are competitors (C), stress-tolerators (S) and ruderals (R). S-strategy was most common in all areas but variation between regions was observed. This emphasizes the importance of functional and compositional dimensions of biodiversity in addition to taxonomical biodiversity since loss or shift in them can lead to lost or altered ecosystem functioning. There is now a broad scientific consensus that biodiversity influences ecosystem functioning and thus the ongoing biodiversity loss is an existential threat to humanity as we depend on functioning ecosystems and their ability to produce ecosystem services. This study is important since it provides indicative empirical evidence from complex real-world ecosystems that has been limited as most biodiversity-ecosystem functioning (BEF) -studies have been experimental. Biodiversity information data also creates benchmarks for future comparison and latitudinal gradients describe how biodiversity varies across space.
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(2024)The many symbiotic microbes associated with plants can represent the first line of defence against viral pathogens, which can have profound impacts on plant health and productivity. Unfortunately, although countless studies have investigated variations in the composition of microbial communities associated with crops, showing benefits in the plants’ nutrient acquisition, stress tolerance or growth promotion, much less is known about how viruses might affect the composition of the microbiota associated with plants, and especially non-crop plants. Furthermore, we also often lack an understanding of how plant viruses are transmitted across their environment. Addressing these knowledge gaps is bringing us closer to better describe and manage the complex relationships between plant microbiota and viral pathogens in their natural environment. The main objective of my Master’s study is to examine variations in the bacterial community associated with a plant in the agricultural landscape, and to identify potential players in microbial community changes. With this in mind, I chose to work with the ribwort plantain, Plantago lanceolata, because it has a widespread distribution, it is present around cultivars, and there is extensive knowledge of its biology and genetics. Thus, P. lanceolata, represents an excellent system for investigating the causes of variation in the plant-associated microbiota. More precisely, I worked with the P. lanceolata population evolving in the Åland Islands, in Southwestern Finland, which has been the target of long-term ecological and evolutionary metapopulation studies. The population is also known for co-evolving with several viral pathogens, and with diverse Hemiptera insects (plant sucking-insects) that have been suggested as possible vectors of the viruses between plants. I collected P. lanceolata leaves, and insects found on P. lanceolata specimens from 28 habitat patches, or meadows, from five localities across the Åland Islands (Geta, Sund, Lemland, Finström, Eckerö). I extracted DNA from a total of 10 leaves per patch, as well as 60 Hemiptera specimens (of several uncharacterized species). I screened for viral infection in all samples by amplifying the viral loci for reverse transcriptase form Plantago latent caulimovirus (cauV) and the replication associated protein gene from Plantago lanceolata latent virus (PlLV). In parallel, I characterized the bacterial communities associated with each plant and insect through metabarcoding of the highly variable V5-V6 region of the 16S rRNA bacterial gene. My MSc's project shows that bacterial species richness (alpha-diversity metric) and community composition (beta-diversity metric) vary between plant and insects. But that they do not vary between the virus-infected and uninfected plants tested, and neither between geographical locations within the Åland Islands. This is the first study investigating the effects of two virus infections on the microbiota of a common weed from the Åland Islands. It provides preliminary data for the study of how the bacterial microbiota of P. lanceolata might respond to virus infections, and how viruses might be transmitted between individual plants.
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(2024)Gerbera hybrida, a common ornamental plant, has natural resistance to fungal diseases. While there may be several bioactive compounds behind this trait, this master’s thesis focuses on two of them: gerberin and parasorboside. The gerberin/parasorboside biosynthesis has been profoundly investigated by Gerbera Laboratory at University of Helsinki. Gerberin and parasorboside are polyketide derivatives, a vast group of bioactive metabolites. The pathway that produces these compounds involves several enzymes: a polyketide synthase (PKS), two polyketide reductases (PKRs) and a glycosyl transferase. For gerberin to be synthesized, it requires presence of three enzymes: (1) G2PS1 (Gerbera 2-pyrone synthase 1) which initiates the chain by synthesizing the carbon backbone, and (2) an unknown first acting reductase, that continues the chain by reduction, lactonization, and (3) addition of a sugar molecule. Parasorboside production requires also a fourth enzyme, a reductase described by Zhu et al. (2022). This master’s thesis delves into post-modification of gerberin and parasorboside intermediates by the PKRs, which have not yet been verified. Until now, there has only been PKR-candidates for this position, derived from gerbera transcriptome library. The genes behind the proposed reductases were known from previous research. The objective of this master’s thesis is to demonstrate which of the proposed reductases coexpressed along with the G2PS1-reductase will produce the desired compounds (gerberin and parasorboside) in a model plant tobacco (Nicotiana tabacum in stable transformation and Nicotiana benthamiana in transient expression). The gene combinations were constructed into plasmids via PCR and Golden Gate cloning. Enzyme production was analysed by western blot and the secondary metabolites by HPLC. The hypothesis for this work was to produce the aglycones of gerbera’s natural products gerberin and parasorboside in the model plant tobacco via constructing the plasmids carrying our genes of interest. This master's thesis documents the successful construction of double and triple fusion plasmids, their integration into agrobacteria, and subsequent transformation into stably transgenic tobacco. Gerberin production in tobacco was observed with certain gene combinations, parasorboside production was not analysed yet in this work. Through meticulous work, the desired combination of genes for gerberin synthesis was identified in both agroinfiltrated plants with co-expressed genes and stable transgenic tobacco lines expressing genes from a single transcript cleaved by 2A-peptides.
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(2024)Lichens are symbioses between fungi, photosynthetic algae and other organisms. The fact that several different fungi may occur in the same lichen thallus adds a further dimension to the diversity of these miniature ecosystems. Calicioids are a polyphyletic group of predominantly lichenised fungi which includes many species with stalked ascomata, topped with a persistent spore mass (mazaedium). One such species is Chaenotheca chrysocephala, a relatively common crustose lichen with a wide distribution. The lichenicolous fungus Chaenothecopsis consociata grows on the thallus of C. chrysocephala and is generally considered to be a parasite. This study aims to map the temporal and geographical distribution of C. chrysocephala and C. consociata in Europe in order to find out whether the parasite was present there before the year 1942, when the species was described, and to identify possible patterns which may affect the population dynamics of these two species. This study found that C. consociata was indeed present in Europe before 1942, at least in Norway, Sweden and Finland. Additionally, the percentage of infected C. chrysocephala thalli seems to have increased in Europe since the parasite was first found. The GBIF dataset showed a significant increase in infected C. chrysocephala thalli in northern Europe (N-EU) compared to central-southern Europe (CS-EU). Although the exact reason is unclear, this increase may be related to differences in forest structure across Europe. Firstly, the host lichen seems to prefer Picea and Pinus as its substrate, these being typical trees of needleleaf forests in Europe. Furthermore, the decrease of old-growth forest stands in Sweden and Finland may have reduced suitable habitat for the host, while the concurrent increase of disturbed stands may have favoured the dispersal of the parasite.
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(2024)For a better understanding of global climate change we need evidence allowing us to track changes in the environment. Pollen is geologically stable, making it a key option as a potential proxy for tracing historic environmental changes. To quantify past environmental changes, it is necessary to test proxies under today’s climate. The amount of UV-B radiation reaching the surface of the Earth has varied throughout the Earth’s history. These variations are ecologically important because changes in UV-B radiation impact plant regulation, growth, defense, and decomposition. The availability of fossil pollen and spores has resulted in significant interest in the potential of using the relationship between UV-B radiation and the accumulation of phenolic sunscreens as a proxy to trace past changes in UV-B radiation. Fossil pollen from Pinus sylvestris is readily available and proven techniques exist to quantify levels of UV-B absorbing compounds from both fossil and extant pollen. We investigated how levels of UV-B-absorbing compounds in Pinus sylvestris pollen change after strobili developed under UV attenuating filters. Fifteen Pinus sylvestris trees were selected from a seed orchard of trees in Nurmijärvi, Finland. The treatments used were Rosco 226 film – filtering solar UV-A and UV-B light, polyester film – filtering solar UV-B, polyethylene film – acting as a transparent control, and an open control condition with no filter. The filters were installed in April 2022 and 2023 and remained in place each year beyond dehiscence towards the end of May. The pollen was analysed using Fourier-transform infrared spectroscopy. Principal component analyses and linear regression models were used to simplify the multivariate data and then describe the levels of UV-B absorbing compounds in the different treatment groups. A sample of needles from underneath the filters was used to verify the effectiveness of UV treatments across the experimental design by analysing their epidermal flavonol content. Our results found no clear link between UV-B exposure and accumulation of UV-B absorbing compounds in Pinus sylvestris pollen. However, we did find statistically significant differences in concentrations of UV-B absorbing compounds in pollen between the different trees. The needle analyses verified that the experimental design had the potential to affect the biochemistry of these branches by revealing significant differences in relative absorbance by epidermal flavonols due to UV treatment type. Multifactorial drivers affect the concentrations of UV-B-absorbing compounds in plants, and viewing the response of these compounds to a single driver may be an oversimplification complicating their use as a proxy. We argue that methodologies used in previous research have inconsistencies which fail to account for environmental factors that either covary with UV radiation or diverge from it. This may explain why our results go against the trend. Finally, we examine our own research experimental design and suggest improvements and avenues by which this research can move forward.
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