The following research topics will be pursued in our group:

Singlet oxygen-mediated and tetrapyrrole-dependent plastid-to-nucleus signaling

A major consequence of ¹O₂-generation in the flu mutant is a rapid and drastic change in nuclear gene expression that reveals the rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of ¹O₂ generation in the flu mutant is not the control of chloroplast biogenesis and activity, but the activation of a broad range of stress-related signaling pathways. So far, the existence of different retrograde signaling pathways has been inferred from correlations between a particular disturbance of plastid homeostasis and a change in nuclear gene activities. Similarities and differences between retrograde signaling pathways will be assessed by comparing tetrapyrrole-dependent and ¹O₂-mediated retrograde signaling and identifying components engaged in the transmission of plastid-derived signals to the nucleus. We'll tackle the following specific aims:

Establish a transgenic flu line with a ¹O₂-responsive and -specific reporter gene that is suitable for an extensive second-site mutant screen for ¹O₂-specific signaling mutants.

Isolate trans-acting mutations that abrogate up-regulation of ¹O₂-responsive genes.

Establish conditions that reveal the genetic basis of tetrapyrrole-mediated retrograde signaling in norflurazon (NF)-treated seedlings.

Identify mutations that suppress the bleaching and restore chloroplast activity of NF-treated seedlings.

Probe the consequences of mutations affecting ¹O₂-mediated and tetrapyrrole-dependent retrograde signaling reciprocally in NF-treated and flu seedlings, respectively.

Identification of a singlet oxygen-responsive transcriptional network and its integrative role during stress responses of plants

¹O₂-mediated stress responses such as seedling lethality and growth inhibition and cell death of mature plants are not caused by oxidative damage, but result from genetic programs that are activated after the release of ¹O₂ has been perceived by the plant. Among the genes that are up-regulated immediately after the release of ¹O₂ those encoding transcription factors (TF) reach almost one fifth, whereas the overall portion of these genes makes up only 6% of the Arabidopsis genome. The overrepresentation of TF genes among the early ¹O₂-responsive genes implicates an as yet largely unexplored transcriptional regulatory network with triggering ¹O₂-mediated responses that closely resemble reactions activated by various abiotic and biotic stress factors. In the present research project we will exploit the unique properties of the flu mutant to reach the following goals:

Distinguish between primary and secondary nuclear target genes of ¹O₂-mediated signaling.

Identify ¹O₂-responsive regulatory modules consisting of cis regulatory elements and transcription factors acting on them.

Determine the impact of genetic perturbations on ¹O₂-mediated gene expression. Such perturbation experiments will help a) to elaborate the circuitry of primary and secondary target genes of ¹O₂-signaling, b) to define modules within the ¹O₂-responsive regulatory network and c) to reveal how this network interacts with other stress-related signaling routes of plants.

The role of ¹O₂-mediated peroxidation of polyunsaturated fatty acids during cell death and acclimation

By varying the length of the dark period the level of the photosensitizer protochlorphyllide can be modulated and conditions can be established that either endorse the cytotoxicity of ¹O₂ or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of ¹O₂: The impact of the EXECUTER  mutations and the prevalence of either non-enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings toxic effects of ¹O₂ prevail and non-enzymatic lipid peroxidation proceeds rapidly. In contrast, in light-grown flu plants that were subjected to an 8 h dark / light treatment lipid peroxidation happened almost exclusively enzymatically. The resulting oxidation product, 13-hydroperoxy octadecatrienoic acid (13-HPOT) serves as a substrate for the synthesis of 12-oxo phytodienoic acid (OPDA) and jasmonic acid (JA) both known to control various metabolic and developmental processes in plants. Inactivation of the two EXECUTER proteins abrogates not only ¹O₂-mediated cell death and growth inhibition of flu plants but also enzymatic  lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect ¹O₂-mediated growth inhibition and cell death. Hence, JA and OPDA do not act as a second messenger during ¹O₂ signaling, but form an integral part of a stress-related signaling cascade activated by ¹O₂ that encompasses several signaling pathways known to be activated by abiotic and biotic stressors. The role of these signaling pathways during cell death and acclimation will be analyzed.

The role of EXECUTER proteins during initiation of ¹O₂-mediated signaling within the plastid of the flu mutant

Attempts to characterize ¹O₂-mediated signaling have aimed so far at identifying constituents of the ¹O₂-dependent signal transduction pathway and characterizing ¹O₂-mediated acclimation and cell death. What is still missing is a detailed analysis of the initial events that lead to the activation of ¹O₂ signaling. Such an analysis has not yet been carried out for any organism known to generate ¹O₂. It is expected to reveal novel insights into how a highly reactive ROS may initiate signaling rather than oxidative damage.  Inactivation of the two plastid proteins EX1 and EX2 abolish cellular responses to ¹O₂, suggesting that the primary reactions of this ROS must be confined to the plastid compartment. ¹O₂ could directly interact with EXECUTER  proteins and change their functions or it may affect the EXECUTER proteins more indirectly by first interacting with a different plastid component that subsequently may alter the functional state of EX1 and EX2. In this part of our research we will characterize EX1 and EX2 biochemically and determine possible ¹O₂-mediated modifications of these proteins.