Browsing by Subject "Arabidopsis thaliana"

Salicylic acid (SA) is a well-known phytohormone involved in pathogen defense, development and controlling the cellular redox balance. In response to stresses, Arabidopsis thaliana synthesizes SA in the chloroplasts mainly via the isochorismate (IC) pathway: IC synthase (ICS) uses chorismate to produce IC, which is in turn converted to SA. ICS1 is the rate limiting enzyme in SA biosynthesis. The ICS1 protein localizes in the chloroplasts and contains a chloroplast transit peptide sequence, which is the first 45 amino acids in the N-terminus. Under different light conditions, plants employ different stress defense strategies. In a previous study, wild type (WT) Arabidopsis thaliana ecotype Columbia-0 grown under different light conditions [short day (SD; 8h light/16h dark), long day (LD; 16h light/8h dark) and 12h light/12h dark] was exposed to ozone for 1 hour (350 ppb). In a phosphoproteomic approach to study signaling mechanisms, ICS1 has been found as an in vivo phosphoprotein in ozone-treated plants grown under SD condition by mass spectrometry (MS). To analyze the phosphorylation of ICS1 in vitro, WT and phospho-negative ICS1 proteins were produced recombinantly as GST-fusion proteins in E. coli and used as the substrates for targeted kinase assays. Phospho-negative ICS1 protein was generated by mutating the phospho-sites to alanines by site-directed mutagenesis. ICS1 could be phosphorylated either in the cytosol or in the chloroplasts. Therefore, different cytosolic kinases, which are involved in various stress signaling events, and a chloroplast protein kinase that functions as a dominant regulator of chloroplast processes, were selected as the kinases to be tested. In this study, GST-ICS1 could be successfully phosphorylated in vitro. To a similar extent, GST-phospho-negative ICS1 was phosphorylated, indicating that ICS1 was phosphorylated in vitro at sites that were different from the ones found by MS from plant material. In order to study the identified phospho-sites in vivo, transgenic Arabidopsis carrying WT, phospho-positive, and phospho-negative ICS1 proteins were generated. Transgenic plants were confirmed by genotyping. Western blotting was carried out to evaluate ECFP-WT ICS1 protein expression. However, none of the transgenic plants could be confirmed to carry ECFP-WT ICS1. Additionally, an antibody against AtICS1 produced by the company Agrisera was tested for its specificity against the endogenous ICS1 protein. While the antibody could detect recombinant GST-ICS1 protein, ICS1 could not be detected from a total protein extract.

Reactive oxygen species (ROS) are one of the prominent groups of signal compounds that are produced in stress conditions such as excess light. Nuclear protein RADICAL-INDUCED CELL DEAT (RCD1) is sensitive to ROS and controls the expression of organelle components, e.g. mitochondrial alternative oxidases (AOX), thus balancing the redox-status of a plant cell. Plants have fast responses to fluctuating light conditions that happen even before gene expression: i.e. readjusting the capability to receive light energy between the two photosystems by state transitions and increasing the capacity to remove excess energy by non-photochemical quenching (NPQ). Various small auxiliary proteins function in these fast acclimation events. However, many of them are identified on gene level only. The goal of this master’s thesis is to describe the role of a hypothetical protein, PPD8 in Arabidopsis thaliana. We evaluate how PPD8 is associated with RCD1 and a chloroplast thiol-regulator enzyme NTRC. We created double (rcd1 ppd8) and triple mutant plant lines (rcd1 ppd8 ntrc) by crossing single knockout lines ppd8, rcd1 and ntrc. Photosynthetic performance, NPQ and sensitivity to ROS were observed in each line by using two different chlorophyll fluorescence measurement methods: pulse-amplitude-modulation (PAM) and novel OJIP imaging fluorometry. The leaves were exposed to methyl viologen (MV), which accelerates the chloroplastic ROS production in light, and also to hypoxic conditions in order to study how the effect of MV is altered in low concentrations of oxygen. Additionally, we examined the amount of photosynthetic proteins and stoichiometry of photosystems in ppd8, rcd1 and rcd1 ppd8 by immunological methods. Finally, PPD8 gene with attached hemagglutinin encoding tags was generated by cloning and reintroduced back to the ppd8 knockout lines. Plants lacking RCD1 are very tolerant against MV and ROS, but when rcd1 was crossed with ppd8 the resistance was suppressed. Both rcd1 ppd8 and ppd8 exhibited elevated chlorophyll fluorescence and NPQ values. The removal of PPD8 gene had an impact on the abundance and the stoichiometry of photosynthetic proteins reducing the plants’ performance. When RCD1, PPD8 and NTRC were simultaneously absent the plants had major defects: their NPQ and fluorescence values were drastically increased. Furthermore, several results hinted towards possible issues in the function of ATP synthase in ppd8 background plants. It is also known that NTRC regulates ATP synthase: taken together, the results suggest that PPD8 is necessary for a fully operative ATP synthase and photosynthetic machinery. By reintroducing PPD8 to knockout line ppd8, the phenotype could be reverted back to wild type -like, thus confirming the significance of the PPD8 gene product in plant.

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.

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.

Arabidopsis (Arabidopsis thaliana J. Thal) is a common model species in plant research. Although its genome is completely sequenced, the complete effects of some genes remain undetermined. The aim of this study was to investigate the role of several E3 Ubiquitin (Ub) ligases in Arabidopsis thaliana development through insertional mutant lines from the SALK collection. Sixteen mutant lines in a wild-type Columbia-0 (Col-0) background were subjected to genotyping, and the subsequent confirmed homozygous lines were analyzed for developmental phenotype. Greenhouse experiments were conducted on the wild type Columbia (Col) and sixteen mutant lines. Yield parameters such as days to bolting and silique surface area were studied to identify differences among the mutant lines relative to wild-type. After phenotyping, statistical analysis was performed to determine if there were any significant differences among the lines. The mean of both number of days to bolting and average silique surface area, among line 8, line 9, and line 13b plants, were shown by one way ANOVA to be significantly different than wild-type. These results suggest that those E3 ubiquitin ligases might be involved in the regulation of plant growth and development.

With the aggravation of global environmental problems and the reduction of finite reserved fossil fuels, seeking for alternative energy sources has become one of the priorities for the sustainable development of human society. Vascular plants save the biomass mainly as the form of xylem (also called as wood), which is now considered as an ideal environmental-friendly energy resource. Wood is now being used as renewable biofuels, Biomass composites to replace plastic and so on. The lateral meristem vascular cambium gives rise to xylem and phloem, contributes to the radial growth of plants. Dr. Mähönen’s group choose Arabidopsis root as a model to understand the growth dynamics of vascular cambium. Auxin is essential for various plant developmental processes. The transcription factor family AUXIN RESPONSE FACTOR (ARF) is an important component in auxin signaling pathway, among which AUXIN RESPONSE FACTOR5 (ARF5)/MONOPTEROS (MP) has been discovered to be essential in various plant developmental processes. The first part of my thesis work mainly focuses on analyzing the expression of ARFs during Arabidopsis root secondary development by using both histological reporter GUS and green fluorescent protein GFP. The second part is screening the secondary growth phenotype among the arf knock out mutants. As mp mutant fails to form primary root, artificial microRNA technique is applied to inhibit MP expression in transcriptional level, this construct was established in an XVE inducible system and driven by a broadly expressed promoter to specifically inhibit MP expression at the secondary development stage. I overexpressed amiMP in both wild type background and arf7,19 double mutant background. These three ARFs have strong expressions in cambium and they might function redundantly to regulate cambium activity. Our preliminary results suggest that ARFs function redundantly in regulating root secondary growth, ARF1 and ARF2 together are functional in regulating vascular pattern formation, and ARF16 can repress the root secondary growth and secondary xylem formation. MP is proved to regulate cambium activity and secondary xylem formation by controlling various auxin-response genes, ARF7 and ARF19 might also participate in this process.