Functional Analysis of the D- and E- subunits of photosystem I in Arabidopsis thaliana

Abstract

Although photosynthesis has been intensively studied, many open questions remain, which still need to be answered. The aim of this thesis was to further investigate the PSI complex, which in the course of the light reaction of photosynthesis catalyzes the light-induced transfer of electrons from plastocyanin on the lumenal side to ferredoxin on the stromal side. In this thesis emphasis was put on the reducing side of PSI, so-called stromal ridge of PSI, which is composed of the subunits D, E and C. Functional analyses of Arabidopsis plants carrying disrupted genes for PSI-E and PSI-D subunits were performed. Analyzing PSI-D, it was shown that of the two genes coding for this subunit only a mutation in PSI-D1 led to a general alteration in the polypeptide composition of PSI and thus also in the photosynthetic electron flow. The characterization of psad1-1 psad2-1 double mutant indicated that PSI-D is necessary for the stability of PSI in Arabidopsis. A complete lack of the D subunit led to seedling lethality under photoautotrophic conditions. The instability of Arabidopsis PSI without PSI-D can be explained either by an increase in degradation of the incomplete PSI complex or by downregulation of the synthesis of PSI subunits. In contrast, Arabidopsis plants lacking the PSI-E subunit were able to grow under photoautotrophic conditions. However, they showed severe phenotype including a significant reduction in size and pale green pigmentation, which was turning more yellowish during development. The psae1-3 psae2-1 double mutant exhibited a high-chlorophyll fluorescence phenotype, which had already been observed in the psad1-1 psad2-1 double mutant. This indicates that photosynthetic electron flow is severely altered also in Arabidopsis plants lacking PSI-E subunit. Further spectroscopic, biochemical and physiological studies are in progress in order to understand the biological consequences of a complete lack of PSI-E subunit and its importance for photosynthesis in plants. An unexpected feature observed in both psad1-1 and psae1-3 single mutants, was a significantly increased level of thylakoid protein phosphorylation and therefore the presence of some new phosphopeptides that could not be detected in WT. A striking feature was that even the level of PSI phosphorylation was affected by these mutations. The only PSI phosphopeptide detected so far had been the PSI-D1 protein. Regarding the complementation analysis performed in this thesis it seems that phosphorylation of PSI-D1 does not play a key function in the PSI. Anyhow, it can not be excluded that this phosphorylation might play a role, if plants are grown under particular environmental conditions, not tested in this work. Mass spectrometry techniques and methods of proteomics allowed the successful identification and analysis of one previously unknown phosphoprotein Lhca4. This was the first light-harvesting protein outside of LHCII to be phosphorylated. However, a distinct role for Lhca4 phosphorylation remains unknown. Further studies are needed in order to elucidate the cause and consequences of Lhca4 phosphorylation and to get further insight into the implication of a high level of thylakoid protein phosphorylation as observed in psad1-1 and psae1-3 mutants