Browsing by Subject "S1"

Tiivistelmä – Referat – Abstract Plant lives and grows in variable environment and climate conditions. Everyday plants can be confronted with a variety of abiotic (temperature, light, salt, water availability) and biotic stress (pathogens, insects etc). These abiotic and biotic stress can halt plant growth and influence crop productivity. Plant has evolved signaling mechanism and different responses to adapt or respond with these unfavorable environmental conditions. Our group’s previous research identified a new mutant in the model plant Arabidopsis thaliana with a striking phenotype – when the plants ages it progressively becomes yellow and eventually the entire plant is white. The mutant was named “white” after its striking appearance. The phenotype is associated with increased accumulation of mRNA transcript for stress and senescence regulated genes. Mapping of the mutation identified a 4 bp deletion in a gene EGY1 that encodes a metalloprotease located in the chloroplast. To identify molecular mechanisms that regulate this unusual type of premature senescence, a suppressor mutants screen was performed in the white mutant, and three suppressors that restore normal appearance to the plant was identified. Mapping of one of these suppressors, identified a mutation in STAY GREEN1 (SGR1) as a likely candidate. SGR1 encodes the protein that catalyze the first step in chlorophyll breakdown, removal of Mg2+ from chlorophyll. The overall aim of my master thesis was to understand the molecular mechanisms behind the development of the age and chlorophyll related phenotypes in the white mutant and its two suppressors S1 and S2. Furthermore, with gene expression analysis, plant stress and senescence responses were studied in white, S1 and S2. By complementation method I proved that mutations in SGR1 gene caused the development of suppressor mutant phenotype and restoration of wild type allele of SGR1 gene restore white phenotype in suppressor mutant. Measurements of chlorophyll concentration provided further evidence that the mutation in SGR1 stabilizes the suppressor mutant phenotype, stops chlorophyll breakdown and keep the leaves green. Gene expression study using qPCR with marker genes provided insight of molecular changes within these phenotypes.

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.