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

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.

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.

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.

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.

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.

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.

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.

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.