Browsing by discipline "Bioteknik (MAAT)"

Ubiquitin-mediated proteolysis regulates many basic cellular processes in plant development. Especially, in Arabidopsis thaliana over 1400 genes encode components of the ubiquitin/26S proteasome pathway. Approximately, 90% of the genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity. However, till now few E3 target proteins were known. Hence, identification and characterization of E3-substrate interaction is essential for understanding the role of ubiquitinylation in various plant developmental pathways. The main objectives of my Master’s thesis were derived from the Flower- specific Ubiquitin Proteasome System (FUPS) research project on the identification of proteins related to FUPS by the candidate and genomic approaches. The candidate approach involved molecular cloning of candidate ubiquitin E3 ligase gene RFI2 and its interaction substrates SLK1 and SLK2 (SLKs). The genomic approach includes genotypic and phenotypic characterization of the Salk T-DNA mutant lines corresponding to the selected FUPS E3 components. The cloning of candidate genes RFI2 and SLKs were done by Gateway cloning technology in order to generate overexpression and inducible expression gene constructs. For functional characterization of candidate proteins in vivo, these constructs were transformed into plants by floral dip Agrobacterium mediated transformation. The yeast two hybrid (Y2H) system was employed to study protein-protein interactions. Genotypic characterization of the selected T-DNA mutant lines was carried out by genetic screening through kanamycin selection. Molecular cloning of candidate genes RFI2 and SLKs into various expression vectors was accomplished. The RFI2 expression constructs were successfully transformed into Arabidopsis plants. The overexpression construct of SLK2 and the RFI2+SLK2 double construct were successfully transformed into root callus. However, the Y2H interaction assay was inconclusive about RFI2-SLK protein interaction, but RFI2 was shown to form homodimers. The transformed plants and the tagged protein constructs of RFI2 and SLK2 will be utilized for protein purification, biochemical assay and in vitro ubiquitination assay to study protein interaction, stability and modification. The genetic screening of T-DNA lines resulted in identification of 10 homozygous lines. In phenotypic analysis of these homozygous lines, early-stage growth and developmental phenotypes such as germination, juvenile leaf emergence, rosette size, colour and root growth were observed. The phenotypic analysis is crucial in identification of the informative phenotype changes in the E3 T-DNA mutant lines. This approach has been successfully employed in understanding of genetic and molecular basis for many biological traits in plants.

Splicing of introns and joining of exons to yield mature and functional mRNA molecules is carried out through two sequential trans-esterification reactions by two distinct spliceosomes; the major U2-dependent and the minor U12-dependent spliceosomes. Alternative splicing is common in eukaryotes and occurs mostly with U2 type introns. The level of minor spliceosomes specific proteins, U11-48K and U11/U12-65K, are regulated via alternative splicing where U11 snRNP binds to the USSE (U11 snRNP binding splicing enhancer), a tandem repeat of 5’ss of U12-type introns, and activates an upstream 3’ss of the U2 dependent spliceosome. Here, I carried forward the study done in Verbeeren et al, 2010, through manipulation of the distance between the two 5’ss within the USSE element, and the distance between the upstream 3’splice site and USSE. The mechanisms through which USSE recognition by the U11 snRNP is achieved, as well as the distance constraints the USSE element imposes on alternative splicing, were analyzed. RT-PCR analysis shows that (a) occurrence of the alternative splicing event drops with reduced distance between 3’ss-USSE, and is finally lost when the distance is reduced to zero, (b) both increased and decreased distance between the two 5’ss within the USSE resulted in reduced long isoform formation. The data set suggests that the simultaneous binding of two 5’ss within USSE by U11/U12 snRNPs and their interaction is necessary for USSE mediated alternative splicing activation. So, the evolutionary conserved sequence within USSE, and the distance to 3’ss are critical for USSE mediated alternative splicing.

Protein-protein interactions (PPIs) regulate many different cellular processes including transcription, translation, cell division, signal transduction, and oncogenic transformation. It is therefore important to develop sensitive and versatile techniques for the detection of these protein-protein interactions in order to fully understand protein functions. The most commonly used and traditional technique, the yeast two-/three hybrid (Y2H/Y3H) method, often results in false positives and false negatives, and other widely used techniques, such as bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC) require extensive instrumentation. When compared with other PPI detection methods, the luciferase-based complementation assay specially split luciferase is believed to deliver the most sensitive and highest dynamic range, making it ideal for large-scale analysis. Therefore, for testing PPIs in planta, split Renilla luciferase complementation assay was chosen. In order to conduct this experiment, a series of plasmid constructs were made to enable the transient expression of fusion proteins. A well known protein pair, Arabidopsis nuclear Histone 2A and 2B, was tested initially as a proof of concept, and then three more proteins of the Gerbera MADS-box B class were investigated. For Arabidopsis Histone 2A and 2B, the intensity in all combinations was on average 9.4-fold higher in Relative Luminescence Units (RLUs) than the mock treated protoplasts. Moreover, in the case of Gerbera MADS-box B class proteins, the protein pairs GDEF1-GDEF2, GDEF1-GGLO1, and GDEF2-GGLO1 showed 8.4-19.4, 9.5-15.8, and 8.3-9.1-fold higher signals than the mock treated protoplasts. These results suggest that various complexes formed from different combinations of these three B class MADS-box proteins may increase the complexity of their regulatory functions, thus specifying the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in Gerbera. Finally, it was concluded that split Renilla luciferase can be a simple, reliable, fast, and effective method for examining PPIs in planta.

Production of biofuels from non-food-based materials, such as lignocellulose, provides a good alternative for the traditional burning of fossil fuels. Some of the researched and existing biofuel applications are based on the utilization of enzymes. There are multiple cellulolytic enzymes required in the efficient hydrolysis of lignocellulose, and one of the key enzyme group is β-glucosidases. These enzymatic systems are mainly adopted from wood-decaying fungi. The overall enzymatic system consists of different types of cellulases that first degrade the crystalline cellulose to oligosaccharides and cellobiose. In the final step, β-glucosidases hydrolyse the oligosaccharides to glucose (a fermentable sugar). In fact, β-glucosidases are one of the limiting enzyme classes in this process, due to phenomena such as end-product inhibition. β-Glucosidases belong to Glycoside hydrolases (GH), that can be classified into different protein families. In an industrial perspective, the main interest resides in GH1 and GH3 family enzymes. Many industrially relevant extracellular β-glucosidases belong to GH3 family. However, intracellular GH1 β-glucosidases often exhibit higher tolerance to harsh conditions such as high substrate and product concentrations, high temperatures and low pH. The goal of this MSc thesis work was to purify and characterize a novel GH1 β-glucosidase, named NBG. Both GH1 and GH3 family enzymes were used as references for the characterization work. The GH3 reference enzyme was a β-glucosidase from Aspergillus niger (An Cel3A), derived from the commercial enzyme preparation Novozym 188. The used GH1 reference was a β-glucosidase from termite Nasutitermes takasagoensis (Nt GH1). The applicability of NBG β-glucosidase in biomass hydrolysis was also examined, together with possible considerations for applicability by other type of applications. The purification of An Cel3 reference enzyme was performed as described previously in literature. A novel protocol combining thermal treatment and low resolution IEX purification was developed for the NBG enzyme in this study. The enzyme’s activity on various pNP-substrates was determined, followed by pH stability, thermostability and inhibition studies. According to the result, NBG is a potential candidate for industrial use. The enzyme was found to be thermostable and active in a wide pH range when compared to the reference enzymes (stable up to 20 h at +60 ˚C and in pH 3.5 – 6.0). NBG also exhibited wider activity on pNP-substrates than the reference enzymes, highest specific activity being on pNPG, followed by moderate activity on pNPFuc and low activities on pNPGal and pNPXyl. Furthermore, NBG exhibited higher tolerance to inhibitors such as glucose and ethanol. Glucose inhibition was not observed until concentration of 200 mM for NBG, while in the same concentration the reference enzymes were almost completely inhibited. A Clear activation (of +16 %) by 100 mM glucose was observed with NBG. This enzyme also outperformed the An Cel3A-reference in ethanol tolerance, retaining activity better in 15 and 20 % ethanol. Activation by ethanol was also observed for both of the fungal enzymes, the most pronounced effect being observed for NBG in 15 % ethanol (+21 % of initial activity). The hydrolysis of insoluble cellulosic substrate (Avicel) was investigated using a commercial cellulase mixture (Celluclast 1.5L), where a semi-pure β-glucosidase preparation was added: novel β-glucosidase preparation (NBG (2-S2)) or the reference preparation An Cel3A (Nz188). According to the results, the NBG (2S-2) was outperformed by An Cel3A (Nz188) in Avicel 4 – 72 h hydrolysis experiments. The amount of reducing sugars released from Avicel was approximately 18–19 % higher with the commercial Nz188 preparation when compared to the 2S-2 preparation. Further analyses of samples revealed accumulation of cello-oligosacchardes, which may accumulate due to two possible reasons: Either the NBG enzyme does not possess high enough cellobiase activity (needed in biomass hydrolysis to glucose), or accumulation of cellobiose is due to transglycosylation activity of NBG. According to activity (and 3D modelling) data, NBG may not be a true β-glucosidase belonging to the EC 3.2.1.21 (and having cellobiase activity). Further investigation of the possible substrate specificity and transglycosylation activity of the NBG will be needed in assessing its applicability in other types of biotechnical applications.

Yeasts are significant plant pathogens, yet plant-yeast interactions are poorly understood. Mammals have immune receptors for yeast-specific MAMPs (microbe-associated molecular patterns), suggesting that plants might have similar receptors as well. Detection of MAMPs by PRRs (pattern recognition receptors) triggers the plant immune system, leading to the first phase of PTI (pattern-triggered immunity). The yeast cell wall is layered, so only some of the potential MAMP molecules, like mannans, are easily available for the plant receptors. In order to utilize yeasts and PRR-based yeast resistance in agriculture and forestry, it is crucial to identify these receptors and gain a better understanding of their functions in plant-yeast interactions. PRRs can be divided into two groups, RLPs (receptor-like proteins) and RLKs (receptor-like kinases). Some of the L-type lectin receptor kinases have already been shown to participate in the interactions between plants and fungi or fungal like pathogens, making them promising candidates for yeast receptors. G-type lectins remain less studied but have promising genes as well. In this thesis, we use forward and reverse genetics methods to show that lectin receptor kinases might include putative yeast pattern recognition receptors. We also establish the first protocol and genetic screen for the identification of plant PRRs participating in the recognition of yeast cell wall MAMPs. Only genes present in both silver birch (Betula pendula) and arabidopsis (Arabidopsis thaliana) were examined to enable following studies with an Arabidopsis model system of the interactions between silver birch and the dimorphic birch pathogen Taphrina betulina. This information can later be used in plant production with other plants and their yeasts as well.

Dr. Mähönen’s group is studying the cambium meristem. In their studies they have discovered that PLT/ANT genes are important especially for the protoxylem pericycle cell (ppx) development. Aim of my master’s thesis was to find out, in Arabidopsis thaliana, at which developmental stage AINTEGUNMENTA (ANT) is required for the proliferation of pericycle cells that are touching the protoxylem. In order to answer the question above I have successfully used artificial microRNA against ANT (amiANT) in an inducible system. By using the inducible system, I was able to silence ANT during various developmental stages. Besides this I also wanted to reproduce the ant,plethora3,plethora7 triple mutant phenotype, where protoxylem pericycle cells do not proliferate or differentiate, with the amiANT line. I induced amiANT at three different time points: after the activation stage (later stages), at the activation stage and continuously. Plants were analyzed by making cross sections and the amiANT efficiency was measured with qPCR. I reproduced the triple mutant phenotype with the activation stage induction. This tells that the system is working and suggests that ANT is needed for the protoxylem pericycle cell proliferation at the activation stage or before it. I could not reproduce this phenotype with the continuous induction, so I cannot be sure if ANT is needed already before the activation stage. At the later stages I could not reproduce the phenotype, so ANT is needed already before the later stages.