To Live and Let Die: Genetic Analyses of Singlet Oxygen-Induced Chloroplast Quality Control and Programmed Cell Death Pathways

Abstract

Plants have evolved complex signaling mechanisms to sense and respond to abiotic stresses. These stresses can lead to the excess production of reactive oxygen species (ROS) that damage cellular components and can lead to cellular dysfunction and, ultimately, cell death. Chloroplasts generate large amounts of ROS, including singlet oxygen (1O2), during photosynthesis. This 1O2 can act as a retrograde signal for the selective degradation of damaged chloroplasts and the activation of programmed cell death (PCD). While such processes allow plants to maintain efficient energy production and avoid toxic ROS accumulation, the signaling mechanisms and genes involved are largely unknown. This raises two intriguing questions: How do plants sense chloroplast dysfunction and selectively degrade damaged chloroplasts to sustain healthy chloroplast populations? -and- What mechanisms do plants use to activate PCD when their chloroplasts accumulate too much 1O2 to manage via selective degradation? To answer these questions, my dissertation research has utilized both forward and reverse genetic approaches to identify and characterize genes and pathways involved in 1O2-mediated chloroplast retrograde signaling (CRS), chloroplast quality control (CQC), and PCD. The Arabidopsis plastid ferrochelatase 2 (fc2) mutant has proven to be useful as a system in which to study 1O2-induced CRS, CQC, and PCD. These mutants conditionally accumulate 1O2 under diurnal (cycling) light conditions, leading to enhancements of chloroplast degradation and cell death in stressed fc2 plants. As these fc2 mutants have a clear conditional phenotype that correlates with chloroplast degradation, they are ideal for genetic suppressor screens to identify genes that play a role in 1O2-induced CRS, CQC, and PCD. In such screens, fc2 mutants are further mutagenized to screen for secondary mutations that lead to an fc2 suppressor (fts) phenotype. Such loss-of-function screens have already yielded many suppressor mutants. Our model suggests that 1O2 acts as an initial signal of chloroplast dysfunction, leading to the activation of CRS to signal stress, followed by CQC to selectively degrade damaged chloroplasts, and eventually PCD to prevent accumulation of cytotoxic elements and allow for nutrient distribution to healthy cells should the preceding mechanisms be insufficient to save the cell. My dissertation research focuses on the identification of genes and pathways involved in chloroplast 1O2-induced CRS, CQC, and PCD and the characterization of the molecular mechanisms involved in these processes. My first chapter focuses on the mechanism of 1O2-induced CQC in the fc2 mutant. Here, we provide evidence that 1O2-induced CQC is not dependent on autophagosome formation, making it a distinct process from other forms of chloroplast degradation that do depend on autophagosome formation. We also provide evidence that points toward autophagosome-independent microautophagy as a plausible mechanism for this phenomenon. Chapter two of my dissertation provides further support for the proposal that multiple 1O2 signaling mechanisms and pathways can be employed in CRS and PCD. Here, we show that oxi1, a mutation shown to block PCD in fc2, does not block PCD in acd2 (another 1O2 accumulating mutant), indicating that the mechanisms of PCD between fc2 and acd2 are not entirely dependent on the same mechanism. In my third chapter, I report the results of a forward genetic screen to identify dominant alleles in negative regulators of 1O2-induced CRS and PCD and provide evidence that life-stage-specific 1O2 signaling mechanisms exist. In this study, we isolated eight gain-of-function fc2 activation tagged suppressor (fas) mutants that block PCD, show that the majority of these mutants are specific to 1O2-induced CRS, and demonstrate that these mutations affect genes that are primarily active in the adult stage. One mutant isolated in our activation tagging screen, however, was found to be generally tolerant to a wide range of stresses. In the fourth and final chapter of my dissertation, I report the mapping and characterization of this mutant, named fc2 fas2. Here, the causative T-DNA insertion in fc2 fas2 was mapped, and the gene of interest (DDF1) was confirmed. DDF1 encodes an AP2/ERF transcription factor (TF). Exogenous overexpression of DDF1 in fc2 leads to suppression of PCD in cycling light, confirming its contribution to the suppressor phenotype of fc2 fas2. As DDF1 is a documented TF, we performed RNAseq to assess the effects of DDF1 overexpression on the transcriptome of fc2 and found that it affects multiple stress response mechanisms. This is intriguing, as 1O2 stress is very specific, and there is little evidence to suggest a clear overlap of this form of stress with other forms of abiotic stress responses (freezing, heat, carbon starvation, etc.). Furthermore, the overexpression of DDF1 appears to block 1O2-induced CRS and PCD by preventing the accumulation of 1O2, suggesting that the mechanism of suppression in fc2 fas2 is acting upstream of 1O2 production, possibly by priming the chloroplast to block 1O2 production or by enhancing the 1O2 quenching capacities of the chloroplast. Together, the work contained in this dissertation furthers our understanding of chloroplast 1O2-induced CRS, CQC, and PCD. With the work reported herein, we provide mechanistic insight into CQC and the vacuolar transport of 1O2-damaged chloroplasts. We also provide support to the notion that multiple 1O2 pathways exist through the introduction of eight new 1O2 signaling mutants that highlight the stage-specific nature of negative regulators of 1O2-induced CRS and PCD. Finally, we provide evidence that shows that plants can activate general stress response mechanisms to protect against and overcome chloroplast 1O2 accumulation.