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Browsing by master's degree program "Erasmus-Mundus Master Program in Plant Breeding (emPlant)"

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  • Ayupov, Temurkhan (2020)
    Chloroplasts are essential plant photosynthetic organelles evolved from a prokaryotic endosymbiont many years ago. A vast majority of chloroplast proteins are encoded in the nucleus and then imported post-translationally by multiprotein translocases located in the membrane of the organelle. It was identified that outer envelope membrane (OEM) components are subject to ubiquitin-proteasomal degradation, governed by a recently established proteolytic system called CHLORAD (chloroplast-associated degradation). It has been suggested that this machinery is involved in regulation of plastid biogenesis and stress tolerance in plants by protein import regulation and remodelling of the organellar proteome. In this study, to further investigate factors involved in chloroplast protein import regulation, we aimed to characterize two putative regulators SKIP6, an F-box/kelch repeat protein, and ASK1, a component of CUL1-based SCF E3 ligase, identified by tandem affinity purification of TOC components and SP1. We performed physiological analyses on skip6-1 and ask1-1 single mutant Arabidopsis plants to identify whether these factors are required for degradation of OEM translocase components (TOC machinery). To identify an association of these factors with the TOC machinery and CHLORAD components, we employed subcellular localization and co-immunoprecipitation (IP) assays in protoplasts. Double mutant sp1 ppi1 and sp2 ppi1 plants were previously shown to specifically supress an atToc33 mutation (specific suppression of ppi1 chlorosis phenotype), resulting in greener and larger plants. Following, for second-site specific suppressor analyses of the atToc33 mutation, we generated ask1-1 ppi1 double mutant plants and provided their initial characterization. As the CHLORAD system was shown to be vital for plant development and to contribute to stress tolerance, therefore, in this study the involvement of SKIP6 in stress tolerance in mutant plants was analysed by implementing osmotic and salt stresses. Physiological analyses revealed an early-senescence phenotype in the skip6-1 single mutant plants, which could be attributed to degradation of TOC components and subsequent decrease in chlorophyll level. Interestingly, an opposite effect was observed after dark treatment, in which SKIP6 knockout mutants remained greener with higher abundance of TOC proteins and chlorophyll level in comparison to wild-type plants. Stress-induced experiments did not show the involvement of SKIP6 in stress tolerance at early developmental stages. Subcellular localization and co-IP experiments revealed cytosolic localization of SKIP6 and its physical interaction with the TOC machinery, respectively. Obtained double mutant ask1 ppi1 plants presented male sterility as well as growth suppression followed by greener leaves at late developmental stages. In summary, our results provide initial characterization of unknown SKIP6 protein suggesting its involvement as a component of SCF E3 ligase (CUL1-ASK1-SKIP6) in the reorganization of the TOC machinery and CHLORAD components at early and late developmental stages, respectively. These initial data represent one of the first steps towards broadening our knowledge on the regulatory network of chloroplast biogenesis in plants, as well as important advance in the development of new strategies for crop improvement.
  • Hani, Umama (2021)
    To determine the role of metalloprotease EGY1 and hormone signalling pathways in PSII repair cycle, a mutant named white because of early senescence was identified having 4bp deletion in EGY1. To further characterize the growth responses in white mutant, two suppressors (white suppressor 1 and white suppressor 2), mutated in STAY GREEN1 (SGR1) which prevents chlorophyll degradation, restored the normal white phenotype was identified upon suppressor mutant screens. This study investigated the effect of chloroplast translation inhibitors (lincomycin/chloramphenicol) and MV (methyl viologen) on photosynthesis in Arabidopsis thaliana single and double white mutants. Furthermore, a second goal was to verify the correct identification of the mutations in white suppressor 1 and white suppressor 2. Western blotting and pulse amplitude modulated fluorimeter (PAM) was used to quantify the D1 protein (reaction core of PSII) levels and photochemical efficiency (Fv/Fm) respectively. Immunoblotting revealed a pronounced decrease in D1 levels for both white and egy1. PAM results showed a high tolerance of white mutant towards lincomycin/chloramphenicol. The white suppressors complemented the lincomycin/chloramphenicol tolerance of white mutant. The white mutant was highly MV sensitive. This MV response was altered in white double mutants (white ein2-1, white sr1-4D and white rcd1-4), suggesting that hormone signalling was involved in the response to MV. The decreased abundance of D1 in the white mutant suggests a role for EGY1 in PSII assembly and D1 turnover under light stress. In all assays (immunoblotting and PAM), the white mutant and egy1-2 gave the same results, this confirms the correct identification of the white mutant as a new egy1 allele. The successful restoration of lincomycin /chloramphenicol tolerance by white suppressors (S1 and S2), implicates that chlorophyll breakdown impacts on correct photosynthesis function. The suppressors S1 and S2 were transformed with wildtype SGR1, which restored the white mutant phenotype. Thus, the suppressor phenotype was caused by mutations in SGR1.
  • Hadid, Feras (2021)
    The growing demand for crop products caused by population growth and climate change encourages plant scientists to investigate sustainable strategies to tackle such problems. Translating the knowledge of plant molecular mechanisms into applicable practices in the field would help in improving the plants' efficiency and productivity. The size of a plant's shoot apical meristem (SAM) is an important factor in biomass production and crop productivity. Besides, understanding the molecular regulation of the shoot apical meristem would help to know more about the 3D growth innovations of different plant families across the kingdom. This thesis aimed to study the regulation mechanisms of the meristem activity in Gerbera, the model organism of the Asteraceae family that has an informative phylogenetic position to understand evolutionary events. The inflorescence of Gerbera is highly resembling clv3 mutants in Arabidopsis. CLV3 peptide is involved in maintaining the meristem activity, these differences between the two species suggested an evolutionary modification for this pathway. GhCLV3-GhWUS signaling components in Gerbera hybrida were identified following bioinformatics approaches. Then sequence comparisons and phylogenetic analyses between Gerbera sequences and other species were performed. The coding sequence of GhCLV1 was cloned to expression vectors. The expression pattern analysis of the receptors was performed by RT-PCR and RNA-seq data. The results suggest that GhCLV1 is an important factor in maintaining the enlarged inflorescence meristem in Gerbera. Also, all the other receptors were expressed at different levels suggesting their contribution in the GhCLV3-GhWUS pathway and inflorescence termination.
  • Michel, Matthieu (2020)
    Hybrid wheat has been the focus of much research for its potential high yield, high protein content and better resistance to biotic and abiotic stresses. Nowadays, only CHA (Chemical Hybridizing Agent) method is used to induce male sterility at a commercial scale. However, this technique is hard to implement on a large production scale and other methods have been investigated for several years. CMS (Cytoplasmic Male Sterility) has been shown to be a promising way to develop hybrid wheat. However, one downside of the technique is the challenging breeding stage step and the associated conversion and restoration process. To fully express the potential gain in yield, the restoration of the cytoplasmic sterility must be complete for the F1 to be fully fertile. In this study, we investigated different methods to assess fertility restoration in nursery and compared the results with the trial notations. The collected data were also used to feed a genomic selection model to predict the behavior of untested hybrids. The results showed a high experimental error of the bagging method originated mostly from human manipulation. The visual scoring showed higher repeatability but was poorly correlated with trial score. A deeper study of the trial scoring revealed an interesting effect coming from the female and an expression of sterility for commercial lines and CHA hybrid checks. Good prediction accuracies were found for genomic selection on both methods, however deeper studies and cross prediction are needed. The multilocation trials remained the best option to score fertility restoration
  • Mosalam, Mohamed (2021)
    The objectives of the study were to assess the efficiency of transient expression of sgRNA/Cas9 construct in Petunia V26, where sgRNA targeted a cytosine deaminase gene (CodA) that converts 5-fluorocytosine into the toxic compound 5-fluorouracil. Disrupting CodA by transient expression of sgRNA/Cas9 introduced a conditional negative selection system that allowed plants with mutated CodA to regenerate on media containing 5-fluorocytosine. The single transcriptional unit vector pMOH2 was designed to carry two amplified sgRNAs guiding Cas9 targeting at HinfI cutting sites. The expression vector was transformed into Petunia V26 using Agrobacterium tumefaciens (pGV2260). Successful mutations were detected on 62.5 mg/L 5-fluorocytosine. Large numbers of in vitro shoots were regenerated from the transformed leaves on a modified MS-media containing 1 mg/L zeatin. The study revealed that transient expression of the sgRNA/Cas9 construct is efficient and can be used to target other genes in Petunia V26. pMOH2 targeted its sites successfully, and proved that CodA can be used as a conditional negative selection marker to detect cells with an edited genome.
  • Xhelilaj, Kaltra (2021)
    Potyviruses are positive-sense single-stranded RNA viruses that can alter several functions of their host plants and consequently, cause significant economic losses in the infected crop plants. During the viral infection, the host transcriptome changes. Stress related genes are triggered, and genes allowing for susceptibility are target for viral-induced modifications. Therefore, in this study, we investigated whether the expression of potential proviral genes SUO1, AGO1, and the major antiviral player AGO2 change in Nicotiana benthamiana (N. benthamiana) in response to potato virus A (PVA; genus Potyvirus) infection. Moreover, we aimed to determine whether helper component protease (HCPro) and active replication have a role in the transcriptional regulation of these genes. Leaves infected with PVA tagged with Renilla luciferase were collected at 3, 6, and 9 days postinoculation, and the viral gene expression was quantified with a dual-luciferase assay. Total RNA was isolated, cDNA was synthesized, and samples were analyzed through qPCR. BLAST hit results revealed that N. benthamiana has three homologs of the SUO1 gene. qPCR data showed no significant change in neither the expression of SUO1 homologs nor the expression of AGO1 during wild-type PVA infection. Moreover, the lack of HCPro or viral replication did not affect the expression of these genes. On the other hand, the expression of AGO2 was approximately 6 and 5 fold up-regulated at 6 and 9 days post-inoculation, respectively. In contrast with the wild-type PVA infection, the mutated viruses had a pronounced effect on AGO2 transcripts at 3 days post-inoculation. Replication-deficient viral RNA increased AGO2 expression circa four-fold, followed by the HCPro-deficient viral RNA increasing expression circa two-fold. AGO2, the major player involved in antiviral defense, was up-regulated during the wild-type infection. Active viral replication and functional HCPro played a role in AGO2 regulation. However, Agrobacterium infiltration can be accounted for interfering with the interpretation of the AGO2 results. Although SUO1 and AGO1 may be potential genes allowing for susceptibility, this study revealed that the PVA infection does not affect the mRNA expression of these genes. Furthermore, it is concluded that active HCPro and viral replication do not have a role in the expression of these genes on mRNA level. To have a clearer view, integrating small RNA, mRNA, and protein quantification analysis of SUO1 homologs will be necessary. Keywords
  • Gauranvi (2021)
    It is important to study the factors which inhibit the cultivation of major crops which serve as a source of food and feed, with various other medicinal values as well. One of these factors is soil degradation and infertility which could be due to high amounts of toxic elements or unfavourable pH conditions. Faba bean is one such crop and is widely affected by the acidity and aluminium toxicity in soil. In this study, an effort has been made to observe the varying tolerance of faba bean accessions and understand the underlying mechanisms used by them under stress conditions. The accessions selected were Aurora, Babylon and Kassa. Each accession was subjected to three treatments and were grown in pH 7 (control), pH 4.5 (acid treatment) and pH 4.5 + Al3+ (aluminium treatment). The pH of peat for acidic treatment was reduced to 4.5 using Sulphuric acid (H2SO4) and for aluminium treatment, Aluminium sulphate (Al2(SO4)3) was added in addition to the acid. At 16 Day after Sowing (DAS) and 30 DAS the physiological data was collected which comprised of chlorophyll concentration (SPAD value), stomatal conductance, leaf temperature and photosynthesis rate. At 35 DAS, the experiment terminated and the shoot data (fresh and dry weights of leaves and stem; and leaf area) of each plant was recorded. Then the root data (tap root length, quality and quantity of nodules and photographs of roots) was taken for each plant. ICP samples for peat, shoot and shoot were also analysed. The data collected were subjected to analysis of variance using R version 4.0.3. (means separated by 5% significance level). From the plant data, Aurora was found to be tolerant. Kassa was sensitive (especially the roots) and Babylon was sensitive to both acid and aluminium treatments. The ICP results provided the reason for this tolerance pattern and a higher concentration of elements needed for plant growth such as P and S were found to be higher in aluminium and acid treatments.
  • Taniwan, Steven (2020)
    Norway spruce is commonly cultivated throughout Europe, Russia, and Japan. Cultivation of Norway spruce often faces the issue of fungal diseases, one of which is cherry rust disease caused by Thekopsora areolata. The gene model MA_10g0010 encoding an uncharacterized peroxidase (PabPrx86) has previously been associated with the presence of this pathogen. The aim of this study was to describe and assay the protein produced from this gene model, observe its localization in the cell, and determine its relative expression level in different tissues of Norway spruce. Experiments were performed by isolating the full length cDNA for PabPrx86 and cloning the cDNA into destination vectors pEAQ-HT-DEST1 and pK7FWG2 leading to a hypertranslatable transcript and a C-terminal GFP fusion, respectively. The plasmid constructs were transformed to Agrobacterium tumefaciens and agro-infiltrated to Nicotiana benthamiana. In addition, the relative expression level of this gene in different spruce tissues at different times of the year was determined using the qRT-PCR method. Sequencing showed that there were two allelic variants of this gene in the spruce individual sampled for RNA. Results showed that both alleles code for a peroxidase with basic pI. Subcellular localization with the GFP tag detected that PabPrx86 protein was located out of cytoplasm, indicating that the protein was translated in the ER-ribosomes, whereas relative expression level analysis revealed that PabPrx86 was highest expressed in the bud and lateral bud in June. Peroxidases are known to relate with plant defense, but further experiments are required to determine the role of PabPrx86 in Norway spruce and what the association with T. areolata means.
  • Rehman, Attiq ur (2020)
    Wheat (Triticum aestivum L.) is one of the major crops in the world and an important agricultural commodity in Finland with various uses. Fusarium head blight (FHB) is a deadly disease of cereal crops and with the gradual increase in temperature and precipitation, it is becoming alarming to Finnish agriculture. Deoxynivalenol (DON) is a vomitoxin produced by Fusarium graminearum species during the FHB infection and is hazardous to health if taken in larger quantities by humans and animals. European Union has legalized the maximum allowed DON content in wheat flour for human consumption at 1.75 ppm. Various types of resistance against FHB are known till date, including tolerance and escape from the disease. Anther extrusion (AE) is a highly heritable trait in wheat and is mechanistically involved in resistance against FHB by preventing the availability of nutrients for the fungus. Other traits such as heading, maturity, and height have shown correlations with FHB incidence and severity in previous studies. Genomic information is crucial to identify markers to accelerate wheat breeding programs against FHB. This experiment was conducted at Boreal Plant Breeding Ltd. Finland using 198 spring wheat breeding lines in a row-and-column design with three replications in an artificially spawn-inoculated F. graminearum field. The goal of the project was to evaluate the genetic diversity for various agronomic and FHB-resistance traits and to estimate correlations among them. A genome-wide association study was also performed by using 11,987 SNP markers to investigate any marker-trait association(s) in the spring wheat breeding germplasm. Larger phenotypic variability was observed in both agronomic and FHB-resistance related traits. Many spurious associations were found with general linear models (Naïve and Q model). No marker-trait associations were observed among the traits in mixed linear model (K) after including kinship as a covariate. Cryptic relatedness among breeding lines has shown a significant role during association mapping. An unexpected negative correlation was found between DON and Fusarium severity indicating inaccuracies in phenotyping. A negative phenotypic and genotypic correlation was found between AE and DON. Future studies on the validation of AE as a phenotypic marker against DON accumulation is recommended. Repeating the experiment with the inclusion of more lines with Fhb1 gene in homozygous state might be helpful in finding reliable associations for FHB-resistance related traits.
  • Delemme, Romain (2021)
    Fusarium Head Blight (FHB) is an important cereal disease worldwide and has become an essential breeding target in wheat. FHB generates considerable losses in terms of grain yield and quality of the seeds in cereal crops. The mycotoxins produced by some Fusarium species, such as deoxynivalenol (DON), directly impact the farmers. In fact, DON accumulation results in unmarketable harvest due to the associated health hazards (vomiting, diarrhea, fever etc). Facing DON risks, the European Commission had to establish a maximum concentration of the mycotoxin in unprocessed cereals. Avoidance mechanisms of the plants against disease infections were identified in diverse studies and are known as the passive resistance. These mechanisms related to phenotypic traits such as variations in plant height (PH), heading date (HD) or the presence of awns could possibly reduce the FHB infection of the wheat. On another hand, the active resistance is determined by genetic factors so called quantitative trait loci (QTL). QTL identification via population mapping was established to be a useful tool to find loci regions associated with FHB resistance. Therefore, in this study we aimed to find phenotypic and genotypic correlations with Fusarium Head Blight resistance among 108 winter bread wheat genotypes. By estimating the heritability of the agronomic traits, we wanted to determine if it would be efficient to breed for those traits. Furthermore, we had the objective to detect FHB resistance QTL from our winter bread wheat genotypes and finally, to observe the overlapping QTL’s regions between FHB resistance and the QTL of the HD or of the PH. It was found that the HD had strong negative phenotypic and genotypic coefficients of correlation with FHB severity and DON concentration. The HD had also an important heritability and direct effect on FHB severity. By performing a GWAS analysis, QTL associated to FHB resistance were found on the chromosomes 1B, 2B, 3A, 3B, 5A, 5B, 5D, 6A, 6B ,7B among the studied genotypes. Overlapping QTL were observed between FHB resistance and HD on the chromosomes 1B, 2B, 3B, 5A, 5B, 6B but also between FHB resistance and PH on the chromosomes 2B, 3A, 3B, 5A, 6A. In conclusion, the HD was considered as an escape mechanism against FHB. It seems to be feasible to select chromosomes fragments with favorable QTL for FHB genetic resistance. Those traits could be involved in marker assisted or genomic selection programmes after the approval of the observed QTL detected to develop FHB resistant cultivars.
  • Kafle, Madan (2020)
    Oat (Avena sativa L.) is the second largest cereal crop (in terms of production) in Finland and the prevalence of Fusarium graminearum in Nordic region is increasing. Infection by F. graminearum causes fusarium head blight (FHB) leading to accumulation of mycotoxin (deoxynivalenol) in addition to the reduction of yield. European union has set the limit for deoxynivalenol for unprocessed oat as 1.75 ppm. Therefore, it is a challenge for the production and marketing of oat to stay within the limit. Being aware of these problems and lack of much understanding in this area, this study was carried out at Boreal plant breeding company located in Jokioinen, Finland. It was aimed to check for associations between traits and markers in an oat field artificially inoculated with F. graminearum. The traits under consideration were plant height, maturity class, heading date, anther extrusion, deoxynivalenol content and germination capacity of kernels. To check the correlation between the traits and explore the variation existing in our germplasm was also the focus of this study. The experimental field was sown as row plots and after one month of sowing, it was inoculated with oat seeds infected with F. graminearum. The phenotypic data were recorded on suitable time and the genome-wide association study was carried out using genome association and prediction integrated tool in R software. Among the traits under consideration, maturity class showed an association with the markers and showed significant positive correlation with deoxynivalenol accumulation and can be considered for further study.
  • Adunola, Paul Motunrayo (2021)
    Lipoxygenase enzymes, which contribute significantly to storage protein in legume seeds have been reported to cause the emission of volatile compounds associated with the generation of off-flavours. This is an are important factor limiting the acceptance of faba bean (Vicia faba) I foods. This study aimed at using bioinformatic tools to identify seed-borne lipoxygenase (LOX) genes and to design a biological tool using molecular techniques to find changes in sequence in faba bean lines. LOX gene mining by Exonerate sequence comparison on the whole genome sequence of faba bean was used to identify six LOX genes containing Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) and/or LH2 LOX domains. Their sequence properties, evolutionary relationships, important conserved LOX motifs and subcellular location were analysed. The LOX gene proteins identified contained 272 – 853 amino acids (aa). The molecular weight ranged from 23.67 kDa in Gene 6 to 96.45 kDA in Gene 1. All the proteins had isoelectric points in the acidic range except Genes 6 and 7 which were alkaline. Only one gene had both LOX conserved domains with aa sequence length similar with that found in soybean and pea LOX genes and isoelectric properties with soybean LOX3. Phylogenetic analysis indicated that the genes were clustered into 9S LOX and 13S LOX types alongside other seed LOX genes in some legumes. Five motifs were found, and sequence analysis showed that three genes (Gene 1, 2 and 3) contained the 38-aa residue motif that includes five histidine residues [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His]. The subcellular localization of the lipoxygenase proteins was predicted to be primarily the cytoplasm and chloroplast. Primers covering ~1.2 kb were designed, based on the conserved region of Genes 1, 2 and 3 nucleotide sequences. Gel electrophoresis showed the PCR amplification of the seed LOX gene at the expected region for twelve faba bean lines. Phylogenetic analysis showed evolutionary divergence among faba bean lines for sequenced and amplified region of their respective seed LOX alleles.
  • Shehbala, Eyerusalem Sija (2021)
    Genus Vicia comprises more than 190 species that chiefly grow in the temperate area of Europe and Asia as well as some parts of tropical Africa, North America, and South America. Based on inflorescence length and the presence of nectariferous spots on the stipules, the genus is divided into two subgenera Cracca (Vicilla) and Vicia, consist of 17 and 9 sections, respectively. The most widely known species is faba bean (V. faba), also referred to as poor man’s meat. However, the close relatedness of V. faba from section Faba with section Narbonensis is still uncertain while V. hirsuta placed in section Cracca although morphological and biochemical evidence support its separation from Cracca. Moreover, there is also the issue of further dividing the genus by separating Ervum as the third subgenus. Therefore, this work aimed to examine the evolutional relationship among 24 selected species of genus Vicia by using housekeeping genes, nuclear ribosomal internal transcribed spacer (ITS2) and mitochondrial cytochrome c oxidase subunit I (Cox1). The plant DNA was extracted and amplified with gene-specific primers followed by PCR product clean up by using GeneJET. The cleaned DNA fragments were cloned with a pGEM-T vector and sequenced. The sequence alignment was performed using CLUSTAL W and MAGA-X to construct maximum likelihood phylogenetic trees. The result showed that most of the species were monophyletic and there were few paraphyletic groupings. Species in section Narbonensis were in the last part of the tree whereas V. faba was placed in the upper part far from those species. V. hirsuta formed a clade with section Narbonensis rather than with section Cracca. Similarly, section Ervum grouped with species of section Cassubicae and Panduralae instead of being separated from the rest of the species. In conclusion, this work showed the molecular phylogenetic tree of 24 selected species of genus Vicia which are able to provide more acceptable details to classify as compared to previous Vicia morphological phylogenetic tree. ITS2 was a more informative tool than Cox1 for dealing with phylogenetic relationships.
  • Palermo, Ronald (2021)
    Soil acidity limits the growth and development of crops. Yield of plants decreases due to not only low pH conditions in acid soils but also dissolution of detrimental elements affecting the availability of essential nutrients. Aluminum toxicity have been one major cause of decline in yield due to the stunting of the primary root and inhibition of lateral root formation. Plants have ways to mitigate aluminum toxicity which includes Al exclusion mechanisms as a first defense to external Al ions and Al detoxifying mechanisms where Al ions are transported and sequestered inside the plant cells. Vicia faba, commonly known as faba or broad bean is one of the oldest crops grown for being a cheap but excellent foundation of protein in human diets, a good fodder and a forage crop for animals, and one of the most powerful nitrogen fixers. The crop is well adapted to low temperatures but moderately susceptible to hostile subsoils and has very low exchangeable Aluminum tolerance. Responsive varieties were previously identified but mechanisms to counteract with the conditions are still to be realized hence the study aimed to understanding the molecular mechanisms of rootzone aluminum toxicity tolerance in faba bean which was realized by using RNA-Seq technique to analyze the transcriptome of roots of faba bean exposed to acidic and aluminum stressed environment and by then, Al responsive genes were identified. The administration of stress was done in a aquaponics set-up. Results revealed that although gene directly interacting with Al was not identified, faba bean have a variety of well-adapted mechanisms to encounter with the Al stressed condition and mitigate the adverse effects to the plant growth and development which includes (1)transporter activity, (2)signaling(ROS), and (3)ubiquitination. The findings will provide valuable information for designing future crop breeding programs.
  • Sultana, Dalia Mrs (2021)
    Anthocyanins are an important class of flavonoids under the class of phenolic compounds and contribute to flower color variation. Gerbera hybrida is a flowering plant of Asteraceae family having mainly two colors of flowers – orange and red. Dihydroflavonol 4-reductase (DFR) is a key enzyme catalyzing a reaction in anthocyanin biosynthesis, the reduction of dihydroflavonols to leucoanthocyanidins. GDFR1-2 and GDFR1-3 are two allelic forms of gerbera DFR differing in substrate specificity for the dihydroflavonols - dihydrokaempferol, dihydroquercetin and dihydromyricetin and also differ in 13 amino acids where eight are considered to be important for substrate specificity. GDFR1-2 has strong preference for dihydrokaempferol and GDFR1-3 doesn’t have any preference for the three substrates. In order to find out the amino acids responsible for substrate specificity, swap mutations were generated between GDFR1-2 and GDFR1-3 by two PCR methods– first, running separate PCR from the templates of GDFR1-2 and GDFR1-3, making a heteroduplex by mixing separate PCR where non-matching nucleotides are expected to be corrected by E. coli and, second, by running PCR from mixed templates with short extension time of PCR to make swaps by template switching. The second method was found more effective than the first method. 81 lines (named GDAT1-81) were sequenced and 35 unique swap mutants were found. In this work the DFR assay was done from six randomly picked GDAT lines where GDAT5 had a swap in one amino acid showing still a similar pattern of substrate specificity as the reference (GDFR1-3) indicating that the mutated amino acid doesn’t have any role in substrate specificity. GDAT14 had an extra mutation (S167P) along with 2 swaps showing incapability of reducing dihydrokaempferol, demonstrating that the mutated amino acids are important and other 4 lines were identical to either GDFR1-2 or to GDFR1-3. This was a preliminary test with 6 lines. In order to get more explanations about the roles of amino acids in substrate specificity, DFR assay was done for all the 81 lines in experiments outside of this thesis and five patterns of substrate specificity were identified indicating that substrate specificity of DFR can be altered by changing only three important amino acids. The amino acids at the position 85,135 and 181 in DFR coding sequence have been identified having important roles in substrate specificity. In addition, the amino acid at position 167 may have a function in making the gerbera DFR able to reduce dihydrokaempferol.
  • Rashid, Mamunur (2021)
    Secondary metabolites are bioactive compounds that help the plant to adapt in different adverse environmental conditions but are not an essential part of plant developmental processes and also secondary metabolites have pharmaceutical value because of their antioxidant, anticancer, antibacterial and antifungal properties. Type-III polyketide synthases (PKSs) are a group of polyketide synthases that produce secondary metabolites with diverse biological activities in plants. The main objective was to localize the PKSs G2PS1, G2PS2, FvCHS2-1, HlVPS into the peroxisomes for synthesizing secondary metabolites in plants. The experiment was performed by amplifying the genes with specific Px-targeting signal. Then the genes cloned into destination vector pEAQ-HT-DEST1. The plasmid constructs were transformed to Agrobacterium tumefaciens and agro-infiltrated to Nicotiana benthamiana and Petunia hybrida leaf tissue. Western blotting results revealed that all proteins were expressed in infiltrated leaves of both tobacco and petunia but HPLC chromatograms showed that only the protein FvCHS2-1 produced novel peaks for metabolites in tobacco.