Browsing by Subject "NTRC"

Reactive oxygen species (ROS) are one of the prominent groups of signal compounds that are produced in stress conditions such as excess light. Nuclear protein RADICAL-INDUCED CELL DEAT (RCD1) is sensitive to ROS and controls the expression of organelle components, e.g. mitochondrial alternative oxidases (AOX), thus balancing the redox-status of a plant cell. Plants have fast responses to fluctuating light conditions that happen even before gene expression: i.e. readjusting the capability to receive light energy between the two photosystems by state transitions and increasing the capacity to remove excess energy by non-photochemical quenching (NPQ). Various small auxiliary proteins function in these fast acclimation events. However, many of them are identified on gene level only. The goal of this master’s thesis is to describe the role of a hypothetical protein, PPD8 in Arabidopsis thaliana. We evaluate how PPD8 is associated with RCD1 and a chloroplast thiol-regulator enzyme NTRC. We created double (rcd1 ppd8) and triple mutant plant lines (rcd1 ppd8 ntrc) by crossing single knockout lines ppd8, rcd1 and ntrc. Photosynthetic performance, NPQ and sensitivity to ROS were observed in each line by using two different chlorophyll fluorescence measurement methods: pulse-amplitude-modulation (PAM) and novel OJIP imaging fluorometry. The leaves were exposed to methyl viologen (MV), which accelerates the chloroplastic ROS production in light, and also to hypoxic conditions in order to study how the effect of MV is altered in low concentrations of oxygen. Additionally, we examined the amount of photosynthetic proteins and stoichiometry of photosystems in ppd8, rcd1 and rcd1 ppd8 by immunological methods. Finally, PPD8 gene with attached hemagglutinin encoding tags was generated by cloning and reintroduced back to the ppd8 knockout lines. Plants lacking RCD1 are very tolerant against MV and ROS, but when rcd1 was crossed with ppd8 the resistance was suppressed. Both rcd1 ppd8 and ppd8 exhibited elevated chlorophyll fluorescence and NPQ values. The removal of PPD8 gene had an impact on the abundance and the stoichiometry of photosynthetic proteins reducing the plants’ performance. When RCD1, PPD8 and NTRC were simultaneously absent the plants had major defects: their NPQ and fluorescence values were drastically increased. Furthermore, several results hinted towards possible issues in the function of ATP synthase in ppd8 background plants. It is also known that NTRC regulates ATP synthase: taken together, the results suggest that PPD8 is necessary for a fully operative ATP synthase and photosynthetic machinery. By reintroducing PPD8 to knockout line ppd8, the phenotype could be reverted back to wild type -like, thus confirming the significance of the PPD8 gene product in plant.