RESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oxigênio Singlete/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mutação , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Oxigênio/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ferroquelatase , Regulação da Expressão Gênica de Plantas , Mutação/genética , Plastídeos/metabolismo , Oxigênio Singlete/metabolismoRESUMO
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.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Fatores de Terminação de Peptídeos/genética , Fenótipo , Plantas Geneticamente Modificadas , Plastídeos/genética , Fator sigma/genética , Fator sigma/metabolismo , Oxigênio Singlete/metabolismo , Tetrapirróis/biossínteseRESUMO
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.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagia/genética , Morte Celular , Cloroplastos/metabolismo , Ferroquelatase/genética , Oxigênio Singlete/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Morte Celular/genética , Ferroquelatase/metabolismo , Genes de Plantas , Mutação , Plastídeos/metabolismo , Plântula , Estresse Fisiológico , TranscriptomaRESUMO
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.
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Proteínas de Arabidopsis , Regulação da Expressão Gênica de Plantas , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Citidina Trifosfato , DNA de Cloroplastos/genética , Mutação , Nucleotídeos/genéticaRESUMO
Current theories attribute aging to a failure of selection, due to either pleiotropic constraints or declining strength of selection after the onset of reproduction. These theories implicitly leave open the possibility that if senescence-causing alleles could be identified, or if antagonistic pleiotropy could be broken, the effects of aging might be ameliorated or delayed indefinitely. These theories are built on models of selection between multicellular organisms, but a full understanding of aging also requires examining the role of somatic selection within an organism. Selection between somatic cells (i.e., intercellular competition) can delay aging by purging nonfunctioning cells. However, the fitness of a multicellular organism depends not just on how functional its individual cells are but also on how well cells work together. While intercellular competition weeds out nonfunctional cells, it may also select for cells that do not cooperate. Thus, intercellular competition creates an inescapable double bind that makes aging inevitable in multicellular organisms.
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Envelhecimento , Algoritmos , Animais , Sobrevivência Celular , Senescência Celular , Humanos , Modelos Biológicos , Neoplasias/patologiaRESUMO
During wound healing, human mesenchymal stem cells (hMSCs) migrate to injuries to regulate inflammation and coordinate tissue regeneration. To enable migration, hMSCs re-engineer the extracellular matrix rheology. Our work determines the correlation between cell engineered rheology and motility. We encapsulate hMSCs in a cell-degradable peptide-polymeric hydrogel and characterize the change in rheological properties in the pericellular region using multiple particle tracking microrheology. Previous studies determined that pericellular rheology is correlated with motility. Additionally, hMSCs re-engineer their microenvironment by regulating cell-secreted enzyme, matrix metallopro-teinases (MMPs), activity by also secreting their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We independently inhibit TIMPs and measure two different degradation profiles, reaction-diffusion and reverse reaction-diffusion. These profiles are correlated with cell spreading, speed and motility type. We model scaffold degradation using Michaelis-Menten kinetics, finding a decrease in kinetics between joint and independent TIMP inhibition. hMSCs ability to regulate microenvironmental remodeling and motility could be exploited in design of new materials that deliver hMSCs to wounds to enhance healing.
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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.
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Cloroplastos/metabolismo , Metabolismo Energético , Estresse Fisiológico , Núcleo Celular/metabolismo , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
All cells and intracellular components are remodeled and recycled in order to replace the old and damaged cells. Autophagy is a process by which damaged, and unwanted cells are degraded in the lysosomes. There are three different types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Autophagy has an effect on adaptive and innate immunity, suppression of any tumour, and the elimination of various microbial pathogens. The process of autophagy has both positive and negative effects, and this pertains to any specific disease or its stage of progression. Autophagy involves various processes which are controlled by various signaling pathways, such as Jun N-terminal kinase, GSK3, ERK1, Leucine-rich repeat kinase 2, and PTEN-induced putative kinase 1 and parkin RBR E3. Protein kinases are also important for the regulation of autophagy as they regulate the process of autophagy either by activation or inhibition. The present review discusses the kinase catalyzed phosphorylated reactions, the kinase inhibitors, types of protein kinase inhibitors and their binding properties to protein kinase domains, the structures of active and inactive kinases, and the hydrophobic spine structures in active and inactive protein kinase domains. The intervention of autophagy by targeting specific kinases may form the mainstay of treatment of many diseases and lead the road to future drug discovery.
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Autofagia , Inibidores de Proteínas Quinases , Humanos , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Imunidade Inata , Transdução de Sinais , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The assessment of the biodegradability of nanomaterials is of pragmatic importance for understanding the interactions between nanomaterials and biological systems and for the determination of ultimate fate of these materials as well as their potential use. We recently developed carbon-based biconcave nanodisks (CBBNs) serving as a versatile nanocarrier for enhanced accumulation in tumors and combined photothermal-chemotherapy. Here, we investigate both the enzymatic and cellular degradation of CBBNs by monitoring their cellular response with electron microscopy, near-infrared absorbance spectroscopy, and cell viability and oxidative stress assessments. Our results show that CBBNs underwent significant degradation in solutions catalyzed by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), or in the presence of macrophage cells. The ability of CBBNs to be degraded in biological systems provides suitability for their future biomedical applications.
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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.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/metabolismoRESUMO
Amorphous calcium phosphate (ACP) has been widely found during bone and tooth biomineralization, but the meta-stability and labile nature limit further biomedical applications. The present study found that the chelation of polyacrylic acid (PAA) molecules with Ca2+ ions in Mg-ACP clusters (~2.1 ± 0.5 nm) using a biomineralization strategy produced inorganic-organic Mg-ACP/PAA hybrid nanoparticles with better thermal stability. Mg-ACP/PAA hybrid nanoparticles (~24.0 ± 4.8 nm) were pH-responsive and could be efficiently digested under weak acidic conditions (pH 5.0-5.5). The internalization of assembled Mg-ACP/PAA nanoparticles by MC3T3-E1 cells occurred through endocytosis, indicated by laser scanning confocal microscopy and cryo-soft X-ray tomography. Our results showed that cellular lipid membranes remained intact without pore formation after Mg-ACP/PAA particle penetration. The assembled Mg-ACP/PAA particles could be digested in cell lysosomes within 24 h under weak acidic conditions, thereby indicating the potential to efficiently deliver encapsulated functional molecules. Both the in vitro and in vivo results preliminarily demonstrated good biosafety of the inorganic-organic Mg-ACP/PAA hybrid nanoparticles, which may have potential for biomedical applications.