The role of WHIRLY1 in chloroplast-based perception of stress and in chloroplast-to-nucleus signaling

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Plant development and productivity require an efficient adaptation of the photosynthetic apparatus to the environment and are limited by stress factors. Stress-induced disturbances in the photosynthetic apparatus are sensed in chloroplasts and are transmitted to the nucleus to adjust gene expression leading to an acclimation of the plants. Among the well-studied signals produced during stress in chloroplasts are reactive oxygen species (ROS) and redox changes in the photosynthetic electron chain. So far, the mechanisms of signal transference from chloroplasts to the nucleus are not well understood.In this project it will be investigated whether the RNA/DNA binding protein WHIRLY1, being dually located in chloroplasts and the nucIeus of the same cell is involved in chloroplast-based perception of stress and in stress-induced chloroplast-to-nucleus signaling. Preliminary studies showed that WHIRLY1 is located in chloroplast nucleoids as well as at the thylakoid membranes, making it an ideal candidate adaptor perceiving signals from photosynthesis and adjusting plastid gene expression. Accordingly, during high light stress chloroplast development is disturbed in transgenic barley plants with reduced amount of WHIRLY1. WHIRLY1 can form oligomers or can be present as a monomer. The monomeric form might be formed in response to redox changes in chloroplasts of stress exposed plants. The ratio of oligomers to monomers will be analysed biochemically with barley plants exposed to different stress conditions. Additional investigations will be done with isolated chloroplasts, in which the redox state and/or the production of ROS will be altered by application of inhibitors of photosynthesis. Transgenic barley lines with altered amounts of WHIRLY1 will be used to investigate the impact of WHIRLY1 on acclimation towards various stress conditions, i.e. drought and high irradiance in combination with cold. Measurements of photosynthesis and nuclear gene expression are envisaged. The distribution of WHIRLY1 between chloroplasts and nucleus will be immunologically analysed in barley. For investigations on the mechanisms of putative stress-induced changes in the subcellular distribution of WHIRLY1, transgenic Arabidopsis plants overexpressing mutated sequences of WHIRLY1 are available. Besides the lysine residue K91 responsible for oligomerization of the protein the conserved cysteine residue C159 possibly involved in redox dependent changes of the WHIRLY1 conformation has been replaced by alanine. Additionally, transplastomic tobacco plants expressing the mutated sequences inside the plastids have been prepared. Visualization of the subcellular distribution of WHIRLY1 will be done with Arabidopsis plants accumulating fluorescing fusion proteins (WHIRLY1:iLOV).