Plant U-Box 4 regulates chloroplast stress signaling and programmed cell death via Salicylic acid modulation.

In response to environmental stress, chloroplasts generate reactive oxygen species, including singlet oxygen (1O2), which regulates nuclear gene expression (retrograde signaling), chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates 1O2 and involves Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, in propagating these signals. To gain insights into 1O2 signaling pathways, we compared transcriptomes of fc2 and fc2 pub4 mutants. The accumulation of 1O2 in fc2 plants broadly repressed genes involved in chloroplast function and photosynthesis, while 1O2 induced genes and transcription factors involved in abiotic and biotic stress, the biosynthesis of jasmonic acid (JA), and Salicylic acid (SA). Elevated JA and SA levels were observed in stressed fc2 plants, but were not responsible for PCD. pub4 reversed the majority of 1O2-induced gene expression in fc2 and reduced the JA content, but maintained elevated levels of SA even in the absence of 1O2 stress. Reducing SA levels in fc2 pub4 restored 1O2 signaling and light sensitivity. Together, this work demonstrates that SA plays a protective role during photo-oxidative stress and that PUB4 mediates 1O2 signaling by modulating its levels.

Investigating the mechanism of chloroplast singlet oxygen signaling in the Arabidopsis thaliana accelerated cell death 2 mutant.

As sessile organisms, plants have evolved complex signaling mechanisms to sense stress and acclimate. This includes the use of reactive oxygen species (ROS) generated during dysfunctional photosynthesis to initiate signaling. One such ROS, singlet oxygen (1O2), can trigger retrograde signaling, chloroplast degradation, and programmed cell death. However, the signaling mechanisms are largely unknown. Several proteins (e.g. PUB4, OXI1, EX1) are proposed to play signaling roles across three Arabidopsis thaliana mutants that conditionally accumulate chloroplast 1O2 (fluorescent in blue light (flu), chlorina 1 (ch1), and plastid ferrochelatase 2 (fc2)). We previously demonstrated that these mutants reveal at least two chloroplast 1O2 signaling pathways (represented by flu and fc2/ch1). Here, we test if the 1O2-accumulating lesion mimic mutant, accelerated cell death 2 (acd2), also utilizes these pathways. The pub4-6 allele delayed lesion formation in acd2 and restored photosynthetic efficiency and biomass. Conversely, an oxi1 mutation had no measurable effect on these phenotypes. acd2 mutants were not sensitive to excess light (EL) stress, yet pub4-6 and oxi1 both conferred EL tolerance within the acd2 background, suggesting that EL-induced 1O2 signaling pathways are independent from spontaneous lesion formation. Thus, 1O2 signaling in acd2 may represent a third (partially overlapping) pathway to control cellular degradation.

Multiple pathways mediate chloroplast singlet oxygen stress signaling.

KEY MESSAGE: Chloroplast singlet oxygen initiates multiple pathways to control chloroplast degradation, cell death, and nuclear gene expression. Chloroplasts can respond to stress and changes in the environment by producing reactive oxygen species (ROS). Aside from being cytotoxic, ROS also have signaling capabilities. For example, the ROS singlet oxygen (1O2) can initiate nuclear gene expression, chloroplast degradation, and cell death. To unveil the signaling mechanisms involved, researchers have used several 1O2-producing Arabidopsis thaliana mutants as genetic model systems, including plastid ferrochelatase two (fc2), fluorescent in blue light (flu), chlorina 1 (ch1), and accelerated cell death 2 (acd2). Here, we compare these 1O2-producing mutants to elucidate if they utilize one or more signaling pathways to control cell death and nuclear gene expression. Using publicly available transcriptomic data, we demonstrate fc2, flu, and ch1 share a core response to 1O2 accumulation, but maintain unique responses, potentially tailored to respond to their specific stresses. Subsequently, we used a genetic approach to determine if these mutants share 1O2 signaling pathways by testing the ability of genetic suppressors of one 1O2 producing mutant to suppress signaling in a different 1O2 producing mutant. Our genetic analyses revealed at least two different chloroplast 1O2 signaling pathways control cellular degradation: one specific to the flu mutant and one shared by fc2, ch1, and acd2 mutants, but with life-stage-specific (seedling vs. adult) features. Overall, this work reveals chloroplast stress signaling involving 1O2 is complex and may allow cells to finely tune their physiology to environmental inputs.

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

Introduction: Plants 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. Methods: The 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. Results: While 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. Discussion: Together, 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.

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 thaliana1O2 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.

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.

Singlet Oxygen Leads to Structural Changes to Chloroplasts during their Degradation in the Arabidopsis thaliana plastid ferrochelatase two Mutant.

During stress, chloroplasts produce large amounts of reactive oxygen species (ROS). Chloroplasts also contain many nutrients, including 80% of a leaf's nitrogen supply. Therefore, to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues, chloroplasts are prime targets for degradation. Multiple chloroplast degradation pathways are induced by photo-oxidative stress or nutrient starvation, but the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole and degraded are poorly defined. Here, we investigated the structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Under mild 1O2 stress, most fc2 chloroplasts appeared normal, but had reduced starch content. A subset of chloroplasts was degrading, and some protruded into the central vacuole via 'blebbing' structures. A 3D electron microscopy analysis demonstrated that up to 35% of degrading chloroplasts contained such structures. While the location of a chloroplast within a cell did not affect the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics, allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A correlation was observed among chloroplast swelling, 1O2 signaling and the state of degradation. Finally, plastoglobule (PG) enzymes involved in chloroplast disassembly were upregulated while PGs increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

The core autophagy machinery is not required for chloroplast singlet oxygen-mediated cell death in the Arabidopsis thaliana plastid ferrochelatase two mutant.

BACKGROUND: Chloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently, it was demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored. RESULTS: To further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations affecting the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1O2-mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. CONCLUSIONS: Our results support the hypothesis that 1O2-dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1O2-damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

Chloroplast quality control pathways are dependent on plastid DNA synthesis and nucleotides provided by cytidine triphosphate synthase two.

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways, but the mechanisms are largely unknown. Here we characterize one fc2 suppressor mutation and map it to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that provide nucleotides for chloroplast function, thereby allowing stress signaling to occur.

Roles for the chloroplast-localized pentatricopeptide repeat protein 30 and the 'mitochondrial' transcription termination factor 9 in chloroplast quality control.

Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS) which oxidize DNA, proteins and lipids. Reactive oxygen species can also act as signals to induce acclimation through chloroplast degradation, cell death and nuclear gene expression. To better understand the mechanisms behind ROS signaling from chloroplasts, we have used the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates the ROS singlet oxygen (1 O2 ) leading to chloroplast degradation and eventually cell death. Here we have mapped mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in post-transcriptional gene expression. These mutants exhibited broadly reduced chloroplast gene expression, impaired chloroplast development and reduced chloroplast stress signaling. Levels of 1 O2 , however, could be uncoupled from chloroplast degradation, suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild-type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. While broad inhibition of plastid transcription with rifampicin was also able to suppress cell death in fc2 mutants, specific reductions in plastid gene expression using other mutations was not always sufficient. Together these results suggest that plastid gene expression, or the expression of specific plastid genes by PPR30 and mTERF0, is a necessary prerequisite for chloroplasts to activate the 1 O2 signaling pathways to induce chloroplast quality control pathways and/or cell death.

Chloroplast Autophagy and Ubiquitination Combine to Manage Oxidative Damage and Starvation Responses.

Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis (Arabidopsis thaliana). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation.

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.

Chloroplast quality control - balancing energy production and stress.

Contents 36 I. 36 II. 37 III. 37 IV. 38 V. 39 VI. 40 VII. 40 40 References 40 SUMMARY: All organisms require the ability to sense their surroundings and adapt. Such capabilities allow them to thrive in a wide range of habitats. This is especially true for plants, which are sessile and have to be genetically equipped to withstand every change in their environment. Plants and other eukaryotes use their energy-producing organelles (i.e. mitochondria and chloroplasts) as such sensors. In response to a changing cellular or external environment, these organelles can emit 'retrograde' signals that alter gene expression and/or cell physiology. This signaling is important in plants, fungi, and animals and impacts diverse cellular functions including photosynthesis, energy production/storage, stress responses, growth, cell death, ageing, and tumor progression. Originally, chloroplast retrograde signals in plants were known to lead to the reprogramming of nuclear transcription. New research, however, has pointed to additional posttranslational mechanisms that lead to chloroplast regulation and turnover in response to stress. Such mechanisms involve singlet oxygen, ubiquitination, the 26S proteasome, and cellular degradation machinery.

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts.

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. Thus, we have identified a signal that leads to the targeted removal of ROS-overproducing chloroplasts.

Sigma factor-mediated plastid retrograde signals control nuclear gene expression.

Retrograde signalling from plastids to the nucleus is necessary to regulate the organelle's proteome during the establishment of photoautotrophy and fluctuating environmental conditions. Studies that used inhibitors of chloroplast biogenesis have revealed that hundreds of nuclear genes are regulated by retrograde signals emitted from plastids. Plastid gene expression is the source of at least one of these signals, but the number of signals and their mechanisms used to regulate nuclear gene expression are unknown. To further examine the effects of plastid gene expression on nuclear gene expression, we analyzed Arabidopsis mutants that were defective in each of the six sigma factor (SIG) genes that encode proteins utilized by plastid-encoded RNA polymerase to transcribe specific sets of plastid genes. We showed that SIG2 and SIG6 have partially redundant roles in plastid transcription and retrograde signalling to control nuclear gene expression. The loss of GUN1 (a plastid-localized pentatricopeptide repeat protein) is able to restore nuclear (but not plastid) gene expression in both sig2 and sig6, whereas an increase in heme synthesis is able to restore nuclear gene expression in sig2 mutants only. These results demonstrate that sigma factor function is the source of at least two retrograde signals to the nucleus; one likely to involve the transcription of tRNA(Glu) . A microarray analysis showed that these two signals accounted for at least one subset of the nuclear genes that are regulated by the plastid biogenesis inhibitors norflurazon and lincomycin. Together these data suggest that such inhibitors can induce retrograde signalling by affecting transcription in the plastid.

Organelle signaling: how stressed chloroplasts communicate with the nucleus.

Plastids are able to relay information to the nucleus to regulate stress responses. A new genetic screen has identified an isoprenoid intermediate that accumulates in stressed plastids and acts as a novel retrograde signal.

Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants.

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.