Cross-talk between redox, metabolic and hormonal retrograde chloroplast signals in the expressional regulation of nuclear encoded plastid proteins
The project is going to identify and characterize cis-elements and trans-active regulators that control nuclear genes of chloroplast proteins. The aim is to reconstruct retrograde signaling pathways that through employment of chloroplast-derived signals control nuclear encoded chloroplast proteins. In target-up approaches promoters of nuclear genes encoding chloroplast antioxidant enzymes will be dissected for the action primarily of redox signals, but also their integration with abscisic acid- and carbohydrate-linked signalling. The trans-active proteins will be characterized and analyzed for upstream interacting protein factors. T-DNA insertion mutants of Arabidopsis thaliana that lack these gene products will be analyzed for altered regulation of gene expression and impaired stress acclimation. Specifically, the project combines bioinformatic analysis of promoters for cis elements, yeast-1-hybrid-screens and protein-DNA-affinity purification with the aim to identify trans-regulators. Selected candidates will be investigated for interacting partners and their regulatory properties using a combination of in vitro experiments, such as EMSA, analysis of thiol-disulfide regulation, e.g. linkage to thioredoxin system, site-directed mutagenesis, FRET, determination of the redox potential, characterization of structural and conformational dynamics, and in vivo analysis of subcellular distribution in transfected protoplasts.
Figure: Summary of the questions addressed in the project on the protein level
Methods of protein chemistry, biochemistry and cell biology will be employed to understand the redox-dependent regulatory cycle of redox regulated transcription factors.
- Structural/ conformational requirements for DNA binding of the dimer,
- thiol/ disulfide-dependent transition from dimer to oligomer,
- subcellular partitioning between cytosol and nucleus,
- binding to other proteins,
- monomerisation and
- nature of reductive regenerant