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.