Chloroplast stress signals: regulation of cellular degradation and chloroplast turnover

For 40 years, it has been known that chloroplasts signal to the nucleus and the cell to coordinate gene expression, maximize photosynthesis, and avoid stress. However, the signaling mechanisms have been challenging to uncover due to the complexity of these signals and the stresses that induce them. New research has shown that many signals are induced by singlet oxygen, a natural by-product of inefficient photosynthesis. Chloroplast singlet oxygen not only regulates nuclear gene expression, but also cellular degradation and cell death. Stressed chloroplasts also induce post-translational mechanisms, including autophagy, that allows individual chloroplasts to regulate their own degradation and turnover. Such chloroplast quality control pathways may allow cells to maintain healthy populations of chloroplasts and to avoid cumulative photo-oxidative stress in stressful environments.24 month embargo; available online 21 August 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Control of chloroplast degradation and cell death in response to stress

Chloroplasts are the sites of photosynthesis in plants and algae and, by extension, are essential for most life on Earth. Their maintenance is costly and complex due to the inherent photo-oxidative damage incurred by photosynthetic chemistry. Chloroplast degradation and cell death are mechanisms by which plants acclimate to such stress and serve a dual purpose: protecting cells and organs by removing reactive oxygen species–producing chloroplasts and redistributing nutrients to other tissues. Here I review recent progress in understanding the molecular mechanisms initiating and facilitating such degradation and show these are complex processes involving multiple pathways. Due to the links to photosynthesis and nitrogen metabolism, there is great potential to manipulate these pathways to increase crop yield and quality under stressful environments.12 month embargo; available online: 6 April 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

All in the timing: Epigenetic control of greening

This article is a Commentary on Islam et al. (2021), 231: 1023–1039 in the New Phytologist.12 month embargo; first published: 14 June 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Putting the brakes on chloroplast stress signaling

As sessile organisms, plants must be able to sense their surroundings and adjust. One way plants do this is by using their energy-producing organelles (chloroplasts and mitochondria). During environmental stress, these organelles experience metabolic changes that induce signals for acclimation. While many metabolites have been proposed as signaling factors, reactive oxygen species (ROS) are known to play prominent roles. In the chloroplast, the ROS singlet oxygen (1O2) is naturally produced during impaired photosynthesis and can lead to retrograde signaling to the nucleus (to control the expression of hundreds of genes), chloroplast degradation, and cell death. The mechanisms controlling these pathways have mostly remained obscure. Recently, Dogra et al., 2022 reported a new role for EXECUTER2 (EX2) in these chloroplast 1O2 signaling pathways, demonstrating that EX2 acts as a buffer to prevent premature activation of 1O2 signaling. These exciting findings reveal an unexpected complexity to chloroplast stress signaling, and identify a decoy mechanism to prevent early activation of cell death.12 month embargo; published 07 March 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Targeted for destruction: Degradation of singlet oxygen-damaged chloroplasts

Photosynthesis is an essential process that plants must regulate to survive in dynamic environments. Thus, chloroplasts (the sites of photosynthesis in plant and algae cells) use multiple signaling mechanisms to report their health to the cell. Such signals are poorly understood but often involve reactive oxygen species (ROS) produced from the photosynthetic light reactions. One ROS, singlet oxygen (1O2), can signal to initiate chloroplast degradation, but the cellular machinery involved in identifying and degrading damaged chloroplasts (i.e., chloroplast quality control pathways) is unknown. To provide mechanistic insight into these pathways, two recent studies have investigated degrading chloroplasts in the Arabidopsis thaliana 1O2 over-producing plastid ferrochelatase two (fc2) mutant. First, a structural analysis of degrading chloroplasts was performed with electron microscopy, which demonstrated that damaged chloroplasts can protrude into the central vacuole compartment with structures reminiscent of fission-type microautophagy. 1O2-stressed chloroplasts swelled before these interactions, which may be a mechanism for their selective degradation. Second, the roles of autophagosomes and canonical autophagy (macroautophagy) were shown to be dispensable for 1O2-initiated chloroplast degradation. Instead, putative fission-type microautophagy genes were induced by chloroplast 1O2. Here, we discuss how these studies implicate this poorly understood cellular degradation pathway in the dismantling of 1O2-damaged chloroplasts.12 month embargo; published online: 08 June 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in Salmonella enterica▿

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP:adenosylcobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a ΔcobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycfN+ allele into a ΔcobU ΔycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors

The cbiS Gene of the Archaeon Methanopyrus kandleri AV19 Encodes a Bifunctional Enzyme with Adenosylcobinamide Amidohydrolase and α-Ribazole-Phosphate Phosphatase Activities

Here we report the initial biochemical characterization of the bifunctional α-ribazole-P (α-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal α-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and α-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established α-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed

DataSheet_2_A genetic screen for dominant chloroplast reactive oxygen species signaling mutants reveals life stage-specific singlet oxygen signaling networks.xlsx

IntroductionPlants employ intricate molecular mechanisms to respond to abiotic stresses, which often lead to the accumulation of reactive oxygen species (ROS) within organelles such as chloroplasts. Such ROS can produce stress signals that regulate cellular response mechanisms. One ROS, singlet oxygen (1O2), is predominantly produced in the chloroplast during photosynthesis and can trigger chloroplast degradation, programmed cell death (PCD), and retrograde (organelle-to-nucleus) signaling. However, little is known about the molecular mechanisms involved in these signaling pathways or how many different signaling 1O2 pathways may exist.MethodsThe Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates chloroplast 1O2, making fc2 a valuable genetic system for studying chloroplast 1O2-initiated signaling. Here, we have used activation tagging in a new forward genetic screen to identify eight dominant fc2 activation-tagged (fas) mutations that suppress chloroplast 1O2-initiated PCD.ResultsWhile 1O2-triggered PCD is blocked in all fc2 fas mutants in the adult stage, such cellular degradation in the seedling stage is blocked in only two mutants. This differential blocking of PCD suggests that life-stage-specific 1O2-response pathways exist. In addition to PCD, fas mutations generally reduce 1O2-induced retrograde signals. Furthermore, fas mutants have enhanced tolerance to excess light, a natural mechanism to produce chloroplast 1O2. However, general abiotic stress tolerance was only observed in one fc2 fas mutant (fc2 fas2). Together, this suggests that plants can employ general stress tolerance mechanisms to overcome 1O2 production but that this screen was mostly specific to 1O2 signaling. We also observed that salicylic acid (SA) and jasmonate (JA) stress hormone response marker genes were induced in 1O2-stressed fc2 and generally reduced by fas mutations, suggesting that SA and JA signaling is correlated with active 1O2 signaling and PCD.DiscussionTogether, this work highlights the complexity of 1O2 signaling by demonstrating that multiple pathways may exist and introduces a suite of new 1O2 signaling mutants to investigate the mechanisms controlling chloroplast-initiated degradation, PCD, and retrograde signaling.</p

DataSheet_1_A genetic screen for dominant chloroplast reactive oxygen species signaling mutants reveals life stage-specific singlet oxygen signaling networks.docx

IntroductionPlants employ intricate molecular mechanisms to respond to abiotic stresses, which often lead to the accumulation of reactive oxygen species (ROS) within organelles such as chloroplasts. Such ROS can produce stress signals that regulate cellular response mechanisms. One ROS, singlet oxygen (1O2), is predominantly produced in the chloroplast during photosynthesis and can trigger chloroplast degradation, programmed cell death (PCD), and retrograde (organelle-to-nucleus) signaling. However, little is known about the molecular mechanisms involved in these signaling pathways or how many different signaling 1O2 pathways may exist.MethodsThe Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates chloroplast 1O2, making fc2 a valuable genetic system for studying chloroplast 1O2-initiated signaling. Here, we have used activation tagging in a new forward genetic screen to identify eight dominant fc2 activation-tagged (fas) mutations that suppress chloroplast 1O2-initiated PCD.ResultsWhile 1O2-triggered PCD is blocked in all fc2 fas mutants in the adult stage, such cellular degradation in the seedling stage is blocked in only two mutants. This differential blocking of PCD suggests that life-stage-specific 1O2-response pathways exist. In addition to PCD, fas mutations generally reduce 1O2-induced retrograde signals. Furthermore, fas mutants have enhanced tolerance to excess light, a natural mechanism to produce chloroplast 1O2. However, general abiotic stress tolerance was only observed in one fc2 fas mutant (fc2 fas2). Together, this suggests that plants can employ general stress tolerance mechanisms to overcome 1O2 production but that this screen was mostly specific to 1O2 signaling. We also observed that salicylic acid (SA) and jasmonate (JA) stress hormone response marker genes were induced in 1O2-stressed fc2 and generally reduced by fas mutations, suggesting that SA and JA signaling is correlated with active 1O2 signaling and PCD.DiscussionTogether, this work highlights the complexity of 1O2 signaling by demonstrating that multiple pathways may exist and introduces a suite of new 1O2 signaling mutants to investigate the mechanisms controlling chloroplast-initiated degradation, PCD, and retrograde signaling.</p