Characterization of Chloroplast Quality Mechanisms during Singlet Oxygen Stress

Abstract

As sessile organisms, plants cannot escape an unfavorable environment. Instead, they must rely on sensory mechanisms that perceive environmental conditions in real-time to acclimate accordingly. One such way plants assess the environment is through changes in metabolism of their energy-producing organelles, such as the powerhouse of the cell – mitochondria – and the photosynthetic solar battery of the cell – chloroplast. As these organelles are sites of primary metabolism, changes in the environment can alter their biochemical reactions and lead to unwanted side reactions. Therefore, these organelles monitor their metabolism, and when environmental conditions are unfavorable, they signal to the rest of the cell indicating there is a problem. Since the world relies heavily on the ability of plants to perform photosynthesis, not only for the oxygen we breathe but for the crops we consume, studying how the chloroplast perceives and reports on the environment is essential for maintaining plants for our growing population. Chloroplast signaling to the rest of the cell (referred to as retrograde signaling) is not entirely understood. Currently, researchers know environmental stresses can negatively impact photosynthesis, leading to the production of cytotoxic molecules called reactive oxygen species (ROS). These ROS are highly reactive. They can react with nearby molecules, such as DNA, RNA, proteins, and lipids, damaging not only the chloroplast but the rest of the cell. However, in lower concentrations, these ROS can cause signaling events within the cell that lead to acclimation to the environment, and thus, efficient photosynthesis. To understand this process, researchers employ genetic mutants that produce ROS conditionally to simulate a stressful environment. Through using these genetic mutants, we can isolate a specific type of ROS and unravel cellular components involved with perception, signaling, and acclimation. In Chapter 2 of my dissertation, I performed a comprehensive analysis of genetic mutants that produce the specific ROS singlet oxygen (1O2) to show how similarly they signaled. These mutants experience photobleaching and cell death due to high levels of 1O2. For my experiments, I focused on four mutants that produce 1O2 in different ways. I hypothesized these mutants might reveal if 1O2 signaling uses one specific pathway or if multiple might exist. Two of the mutants, plastid ferrochelatase II (fc2) and fluorescence in blue light (flu), accumulate 1O2 due to overaccumulation of chlorophyll precursors that absorb light energy and pass it to nearby ground-state molecular oxygen (3O2). Another mutant, chlorina 1 (ch1), cannot convert chlorophyll a to chlorophyll b, preventing the formation of the antenna complex located in photosystem II (PSII). The reaction center in ch1 mutants is unprotected, easily reduced, and passes excess energy to 3O2. Finally, the accelerated cell death 2 (acd2) mutant accumulates chlorophyll breakdown products from when chlorophyll is no longer needed. Like chlorophyll precursors, these molecules have potential to absorb light energy and pass it to 3O2. From my research, I showed 1O2-signaling is complex, and that there are at least two separate pathways that exist regarding 1O2 originating within the chloroplast. One such pathway illustrated by the flu mutant shows signaling dependent on the EXECUTER 1 (EX1) protein whereas fc2, ch1, and acd2 do not signal using EX1. Instead, they employ OXIDATIVE INDUCIBLE KINASE 1 (OXI1) and PLANT U-BOX 4 (PUB4) in response to their corresponding 1O2 production. In Chapter 3 of my dissertation, I performed a reverse genetics approach to identify genetic components involved with 1O2 signaling in the fc2 mutant. Based on prior research with the flu mutant, scientists demonstrated blue light and blue light photoreceptors are required to transduce the intracellular signal originating within the chloroplast due to 1O2. I introduced mutant alleles of both cryptochromes (cry1 and cry2) and two red-light sensing phytochromes (phyA and phyB) into the fc2 mutant background to determine if these secondary mutations might suppress the fc2 cell death phenotype. I showed CRY2 is involved with in the fc2 mutant but in a novel way. The fc2;cry2 double mutant experiences significantly less cell death. However, the double mutant still induces some stress marker genes. We have not observed this uncoupling of cell death and stress gene induction in the fc2 mutant. Most suppressors either lower chlorophyll biosynthesis, prevent 1O2 production, or block a signal to prevent stress gene induction. Through using the fc2;cry2 double mutant, we could potentially tease out how this mutant appears stressed genetically but grows healthily. In Chapter 4 of my dissertation, I aimed to understand how the central carbon metabolic enzyme PHOSPHOENOLPYRUVATE CARBOXYLASE 2 (PPC2) may protect stressed/damaged chloroplasts. In the fc2 mutant, PPC2 localizes to the chloroplast under stress conditions. Furthermore, the isolated PPC2 was monoubiquitinated. I made several over-expression lines of PPC2 to determine how it might localize to or protect the chloroplasts, and if localization and protection are caused by different attributes of PPC2. For one of the constructs, I substituted the monoubiquitinated lysine for arginine to prevent ubiquitination of PPC2 at this amino acid site to determine the effect of this posttranslational modification. Also, I created two constructs that are predicted as catalytically dead to determine if PPC2 must maintain its enzymatic activity for chloroplast localization and/or protection. My fourth chapter focuses on the characterization of these lines, most notably that they are expressing consistently and if they prevent cell death in the fc2 mutant. Not only will this research potentially identify another component in chloroplast stress signaling, but the over-expression of PPC2 has previously been documented to increase plant biomass. We can use these findings to begin work on making more resilient crops without sacrificing yield. My dissertation illustrates different ways to understand how chloroplasts are involved with sensing the environment and eliciting an appropriate response. One day, these findings might influence how we genetically modify crops to grow better in our everchanging climate.