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Molecular characterization of two chloroplast biogenesis regulators in Arabidopsis thaliana
Molecular characterization of two chloroplast biogenesis regulators in Arabidopsis thaliana
Plastid ribosomes are derived from ancestral endosymbiontic cyanobacteria and are composed of a large (50S) and a small (30S) subunit. Each subunit contains ribosomal proteins and ribosomal RNAs. The majority of these ribosomal proteins are also involved with ribosome biogenesis and functioning and are encoded in the nucleus. However only a small percentage of ribosomal proteins are chloroplast encoded. The first part of this doctoral thesis reports the identification and characterization of the Arabidopsis thaliana nuclear encoded CHLOROPLAST RIBOSOME ASSOCIATED (CRASS) protein. This protein has emerged during embryophyta evolution and resides in the chloroplast stroma of land plants but not in green algae or cyanobacteria. Under optimal growth conditions CRASS is not required for plant survival and crass mutants show minor defects in photosynthesis and plant fitness. On the other hand, translation inhibitors (lincomycin and chloramphenicol) and cold stress exacerbate the mutant plant phenotype. In co-immuno-precipitation experiments, CRASS is pulled down with 16S RNA and with the small ribosomal subunits PRPS1 and PRPS5. CRASS interacts with ribosomal proteins independently of ribosomal RNAs, suggesting a protein-protein interaction with other subunits or structural components of the ribosome. Double mutants have a synergistic mutant phenotype confirming that CRASS plays a role in the stability of the chloroplast and becomes crucial when stress conditions interfere with ribosome biogenesis and activity. An additional crucial role in the development of the chloroplasts is played by the assembly factors which allow correct formation of thylakoid membrane complexes needed to sustain phototrophic growth. The second part of this dissertation focuses on a DNAJ related protein, SNOWY COTYLEDON2 (SCO2), required for thylakoid complex assembly and protein interaction with the light-harvesting chlorophyll-binding protein LHCB1. Its role in chloroplast biogenesis in true leaves of Arabidopsis thaliana and Lotus japonicus, previously thought to be a protein acting exclusively in cotyledon greening, is here analysed. The lack of SCO2 in Arabidopsis results in a drastic decrease in plant growth and photosynthesis efficiency under short-day conditions, while SCO2 disruption in Lotus induces white and green variegated leaves and stunted growth. In this case, inhibition of translation rates does not decrease the variegation phenotype as in other variegated mutants. Furthermore, in Arabidopsis, the combined absence of SCO2 and ClpR1 causes a severe variegated phenotype. These effects suggest that SCO2 can be considered a new component able to suppress leaf variegation. Taken together, the results of this thesis highlight the possibility to discover new gene functions, such as CRASS and SCO2. These genes have been functionally characterized by inducing stresses that enhanced otherwise undetected phenotypes.
sco2, arabidopsis, crass, chloroplast
Zagari, Nicola
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Zagari, Nicola (2019): Molecular characterization of two chloroplast biogenesis regulators in Arabidopsis thaliana. Dissertation, LMU München: Faculty of Biology
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Abstract

Plastid ribosomes are derived from ancestral endosymbiontic cyanobacteria and are composed of a large (50S) and a small (30S) subunit. Each subunit contains ribosomal proteins and ribosomal RNAs. The majority of these ribosomal proteins are also involved with ribosome biogenesis and functioning and are encoded in the nucleus. However only a small percentage of ribosomal proteins are chloroplast encoded. The first part of this doctoral thesis reports the identification and characterization of the Arabidopsis thaliana nuclear encoded CHLOROPLAST RIBOSOME ASSOCIATED (CRASS) protein. This protein has emerged during embryophyta evolution and resides in the chloroplast stroma of land plants but not in green algae or cyanobacteria. Under optimal growth conditions CRASS is not required for plant survival and crass mutants show minor defects in photosynthesis and plant fitness. On the other hand, translation inhibitors (lincomycin and chloramphenicol) and cold stress exacerbate the mutant plant phenotype. In co-immuno-precipitation experiments, CRASS is pulled down with 16S RNA and with the small ribosomal subunits PRPS1 and PRPS5. CRASS interacts with ribosomal proteins independently of ribosomal RNAs, suggesting a protein-protein interaction with other subunits or structural components of the ribosome. Double mutants have a synergistic mutant phenotype confirming that CRASS plays a role in the stability of the chloroplast and becomes crucial when stress conditions interfere with ribosome biogenesis and activity. An additional crucial role in the development of the chloroplasts is played by the assembly factors which allow correct formation of thylakoid membrane complexes needed to sustain phototrophic growth. The second part of this dissertation focuses on a DNAJ related protein, SNOWY COTYLEDON2 (SCO2), required for thylakoid complex assembly and protein interaction with the light-harvesting chlorophyll-binding protein LHCB1. Its role in chloroplast biogenesis in true leaves of Arabidopsis thaliana and Lotus japonicus, previously thought to be a protein acting exclusively in cotyledon greening, is here analysed. The lack of SCO2 in Arabidopsis results in a drastic decrease in plant growth and photosynthesis efficiency under short-day conditions, while SCO2 disruption in Lotus induces white and green variegated leaves and stunted growth. In this case, inhibition of translation rates does not decrease the variegation phenotype as in other variegated mutants. Furthermore, in Arabidopsis, the combined absence of SCO2 and ClpR1 causes a severe variegated phenotype. These effects suggest that SCO2 can be considered a new component able to suppress leaf variegation. Taken together, the results of this thesis highlight the possibility to discover new gene functions, such as CRASS and SCO2. These genes have been functionally characterized by inducing stresses that enhanced otherwise undetected phenotypes.