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

Cell walls (CWs) are the safeguards of plant cells and have a crucial role in controlling cell integrity and plant-environment interactions. Perception of cell wall damage (CWD) results in activation of signaling pathways leading to activation of plant defiance responses. Previous research utilizing murus1 GDP-L-fucose deficient mutant has shown the role of GDP-L-fucose biosynthesis in controlling the structure and integrity of the CWs. Here, we investigated whether phenotypical consequences provoked by the lack of GDP-L-fucose, observed in mutants lacking MURUS1 can be suppressed by blocking signal transduction pathways involving THESEUS1 (THE1) receptor-like kinase or WALL-ASSOCIATED KINASE 2 (WAK2)- MAP KINASE 6 (MPK6) signaling module involved in CWD responses. For this, mur1 the1 and mur1 mpk6 double mutants were generated and analyzed. We found that the phenotypes of double mutants closely resemble that of mur1 mutants, indicating that the morphological consequences of GDP-L-fucose deficiency develop independently of THE1 and MPK6 signaling. Further, we aimed to investigate whether GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductases GER1 and GER2 controlling the final step of GDP-L-fucose synthesis are functionally redundant. Due to the inability of generating ger1 ger2 double mutants, we have applied the artificial microRNA (amiRNA) strategy to generate ger1 ger2 amiRNA knockdown plants. We found that these knockdown plants exhibit severe developmental growth defects similar to those observed in plants impaired in import of GDP-L-fucose into the Golgi apparatus and these growth defects exceeded those observed in plants lacking MUR1. Therefore, we have concluded that GER1 and GER2 are functionally redundant.

Late blight, caused by Phytophthora infestans (Mont.) de Bary, is considered the most devastating disease in potato (Solanum tuberosum L.) production worldwide. Control methods involve mostly the use of fungicides, which are costly and are under political pressure for reduction in Europe. Potatoes from the major potato cultivar in Sweden, King Edward, previously stacked with three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1) were tested in a local Swedish field, with spontaneous P. infestans infection over three seasons to evaluate the effectiveness and stability of the resistance on leaves. In addition, testing of resistance was done in both in leaves and tubers. Field results demonstrated that the 3R stacked into the cultivar King Edward, showed practically full resistance to infections of P. infestans, with no difference to fungicide use. Moreover, the resistance was effective in both leaves and tubers. The results reveal the 3R potatoes offer a functional field resistance, that could, alone, reduce the total use of fungicides in agriculture by several percent in Sweden, in an event of modifications in the EU legislation.

Strawberry breeding in Finland began 60 years ago and has largely relied on traditional breeding methods. The geography of Finland, as well as local consumer preferences for darker-coloured strawberries, have been the main focus of selection in all implemented Finnish breeding programs. With several successful and popular cultivars have been released, the current focus is on increasing yield and disease resistance while maintaining the excellent fruit quality of breeders' selections. A panel of 175 garden strawberry accessions was created and trialled at the Natural Resources Institute Finland (Luke) as part of a Nordic-Baltic pre-breeding collaboration between the institute and the Norwegian breeding company Graminor AS. A medium-density, genome-wide scan for 50K SNPs has been performed on each individual of the panel. This panel represents the genetic diversity present and typical for cultivated strawberry in the Nordic-Baltic region, and it is expected to provide Nordic breeders with markers tailored to their plant material diversity, supporting their future selection decisions and accelerating their breeding cycles. In this study, symptom severities of two strawberry leaf diseases (leaf spot caused by Mycosphaerella fragariae and leaf scorch caused by Diplocarpon earlianum) and seven fruit quality traits (berry weight, skin colour, flesh colour, evenness of flesh colour, basket appearance, predominant berry shape and tip type) were scored in the panel. GAPIT and statgenGWAS statistical packages in R were used to run five different GWAS models: Significant SNP-trait associations were found using single-locus (GLM and MLM) and multi-locus (FarmCPU and BLINK) association mapping analyses. The study had shed a light on the importance of considering several statistical models and parameters for a maximum benefit of association mapping studies. Among significant SNP-trait associations for fruit weight, fruit skin colour and fruit flesh colour, four were partially characterized by inspecting their allelic effects. On chromosome Fvb6-1, appearance of consensus, significant signals from the flesh colour trait of secondary-position berries is discussed. Nine significant SNP-marker associations were detected for berry weight.

Aluminum (Al3+) toxicity is a major limiting factor in acidic soils when pH<5.5 and faba bean experiences yield decreases in these conditions. The multidrug and toxic compound extrusion (MATE) family plays a vital role in Al3+ tolerance across species. This study searched for an ortholog of MtMATE66, a gene involved in Al3+ resistance in barrel medic, in faba bean and quantified the ortholog’s relative expression in 4 faba bean genotypes (GPID_0022, GPID_0153, GPID_0178 and GPID_0191). pBLAST of MtMATE66 in the faba bean unpublished genome identified the ortholog jg20333.t1, with 88.6 % identity, e-value 0.0 and bit score 880. InterPro Scan and NCBI CDD conserved domain queries classified jg20333.t1 as a MATE in the DinF subfamily. MEME Suite identified the 50-amino acid citrate exuding motif characteristic to MATEs exuding citrate in Al3+ tolerance while a multiple sequence alignment and phylogenetic analysis clustered jg20333.t1 with Al3+ -tolerant citrate exuding MATEs. The SWISS Model 3D structure and DeepTMHMM predicted an α-helical, twelve-transmembrane protein topology. The plasma membrane was predicted as the subcellular localisation of jg20333.t1 by ProtComp, WoLFSPORT and YLoc, however SignalP identified no signal peptides. The molecular weight 54.57kDa, theoretical isoelectric point 8.60 and grand average of hydropathicity 0.67 of jg20333.t1 were calculated by ProtParam. EMBOSS Needle and GSDS aligned 13 exons. Four biological replicates of faba bean plants were set up for 3 different treatments: acidic of pH 3.81 (Ac), acidic of pH 3.81 plus Al3+ (Al) and neutral of pH 6.01 (N) in a greenhouse and root tip samples were collected 45 days after transplanting in peat media for RNA extraction. The relative expression of jg20333.t1 was determined by RT-qPCR of jg20333.t1 as target gene, Vfactin as reference gene and N as internal calibrator. The Cq values generated were analysed using the 2-ΔΔCq method and showed high relative fold change in both Ac and Al. The upregulation in Al confirmed the implication of jg20333.t1 in faba bean tolerance to Al3+. The upregulation in Ac suggests upstream regulation by STOP transcription factor. The four genotypes had no significant difference in fold change. Based on these results, it is concluded that jg20333.t1 is a faba bean MATE gene, VfMATE, implicated in Al3+ tolerance by citrate exudation.

Abstract Nuclear bodies (NB) have been studied for their importance of being one of the sites for gene regulation activities. RADICAL INDUCED CELL DEATH1 (RCD1) has proven to be a potential nuclear protein in A.thaliana that localizes to NB. It can interact with different transcription regulators responsible for many physiological functions. One of which is light signaling. Hence, it shares mutual functions with some phytochrome photoreceptors (PHYs), e.g., PHYB. It also contains intrinsically disordered regions (IDRs) that stabilize RCD1 protein upon phosphorylation. This study aims to examine the colocalization events of RCD1 that coexpressed with PHYB in full-length RCD1 complementation line and domain deletion lines, given the fact that both RCD1 and PHYB have previously shown mutual interaction with some of PHYTOCHROME INTERACTING FACTORS (PIFs). To achieve this aim, the colocalization of Venus-tagged RCD1 and Green fluorescent protein (GFP)-tagged PHYB using confocal Microscopy was performed. Another objective is to study the phosphorylation effect of one of the IDRs between WWE and PARP-like domain -IDR2- on RCD1 NB localization. Two phosphomutants -non-phosphorylatable and phosphomimetic- constructs were transiently and stably expressed in the rcd1-4 background. Furthermore, they were screened using Confocal Microscopy. We were able to see the colocalization events in 2 domain deletion lines, RCD1-∆WWE-3xVenus and RCD1 ∆PARP 3xVenus. On the other hand, we could not see any colocalization in the RCD1 ∆RST 3xVenus, which indicates the importance of the RST domain in the colocalization. In addition, phosphorylation was found to affect the abundance of RCD1 protein in both transiently and stably expressed lines. Our study showed non-phosphorylatable forms of IDR2 having a higher abundance of RCD1 NB than the control line RCD1-3xVenus, whereas phosphomimetic IDR2 showed no signal. Collectively, Our experiments showed the effect of phosphorylation on RCD1 NB localization and the importance of the RST domain in the colocalization of RCD1 with PHYB.

Abstract Background. Faba bean (Vicia faba L.,) is a protein-rich grain crop and a good source of many nutrients. However, the productivity and composition of this crop can be limited by several biotic and abiotic stresses. Acid soil leads to a reduction in water uptake, and influences on major biological and chemical processes. However, some plants can manage stress. Mostly, low pH reduces plant vegetative organs and a has a negative effect on physiological processes such as gas exchange, photosynthesis, chlorophyll concentration, and transpiration. The aim of this study was to find homolog genes in close spp. and o test selected gene response as a factor in resilience to rootzone acidity in Vicia faba L. Methods. Four faba bean genotypes were selected for our experiment based on germplasm screening in aquaponic conditions from a previous study. According to obtained results of morpho-physiological measures from a previous experiment, IG11204 and Bourdon showed tolerance to acid only. DULUC is tolerant to acid and Al treatments. In contrast, Nora38 demonstrated susceptibility to both stresses. Selected genotypes were grown under the same experimental conditions in order to proceed with samples for further analysis (total RNA extraction, cDNA synthesis, RT-PCR, gel-electrophoresis, validation of selected gene by RT-qPCR). Results. Selected genes have been identified as hub genes in A. thaliana (alternative oxidase 1A, glutamate dehydrogenase 1, zinc fingers superfamily protein), which demonstrated an increase in expression levels under acidic pH stress. We have found homolog genes in the Hedin cultivar. Selected homolog gene were used to check its expression in four genotypes under Al3+ and acidic stresses using RT-qPCR. Finally, up-regulation of the GDH1 was detected by acid and also occurred by Al3+ treatments. Discussion. Our analysis of gene expression revealed that GDH1 was expressed differently between control and treated groups. However, its activity was detected for both stresses. The expression of GDH1 transcripts in this study, also concurs with findings where GDH1 in A. thaliana were expressed under Al3+ stress. Furthermore, it was confirmed that they are novel Al3+ resistance genes, which are regulated by the transcription factor STOP1 (sensitive-to-proton-rhizotoxicity1). GDHs are involved in stress tolerance, and pH-regulated metabolic pathways – GABA shunt. It participates in the regulation of cytosolic pH, carbon fluxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling in plant cells under different stress conditions. The GABA shunt is enhanced by stresses, which cause cytosol acidification. Genes expressed in conditions of low pH partly overlap with those expressed in plants exposed to Al3+ stress. Generally, acidic soil is associated with the co-occurrence of Al3+ and H+ rhizotoxicities.