ABSTRACT
Proteome composition is dynamic and influenced by many internal and external cues, including developmental signals, light availability, or environmental stresses. Protein degradation, in synergy with protein biosynthesis, allows cells to respond to various stimuli and adapt by reshaping the proteome. Protein degradation mediates the final and irreversible disassembly of proteins, which is important for protein quality control and to eliminate misfolded or damaged proteins, as well as entire organelles. Consequently, it contributes to cell resilience by buffering against protein or organellar damage caused by stresses. Moreover, protein degradation plays important roles in cell signaling, as well as transcriptional and translational events. The intricate task of recognizing specific proteins for degradation is achieved by specialized systems that are tailored to the substrate's physicochemical properties and subcellular localization. These systems recognize diverse substrate cues collectively referred to as "degrons", which can assume a range of structural configurations. They are molecular surfaces recognized by E3 ligases of the ubiquitin-proteasome system, but can also be considered as general features recognized by other degradation systems, including autophagy or even organellar proteases. Here we provide an overview of the newest developments in the field, delving into the intricate processes of protein recognition and elucidating the pathways through which they are recruited for degradation.
ABSTRACT
Chloroplast function is essential for growth, development, and plant adaptation to stress. Organelle stress and plant defence responses were examined here using noxy8 (nonresponding to oxylipins 8) from a series of Arabidopsis mutants. The noxy8 mutation was located at the CLPC2 gene, encoding a chloroplast chaperone of the protease complex CLP. Although its CLPC1 paralogue is considered to generate redundancy, our data reveal significant differences distinguishing CLPC2 and CLPC1 functions. As such, clpc1 mutants displayed a major defect in housekeeping chloroplast proteostasis, leading to a pronounced reduction in growth and pigment levels, enhanced accumulation of chloroplast and cytosol chaperones, and resistance to fosmidomycin. Conversely, clpc2 mutants showed severe susceptibility to lincomycin inhibition of chloroplast translation and resistance to Antimycin A inhibition of mitochondrial respiration. In the response to Pseudomonas syringae pv. tomato, clpc2 but not clpc1 mutants were resistant to bacterial infection, showing higher salicylic acid levels, defence gene expression and 9-LOX pathway activation. Our findings suggest CLPC2 and CLPC1 functional specificity, with a preferential involvement of CLPC1 in housekeeping processes and of CLPC2 in stress responses.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Chloroplasts , Mutation , Stress, Physiological , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Chloroplasts/metabolism , Gene Expression Regulation, Plant , Molecular Chaperones/metabolism , Molecular Chaperones/genetics , Pseudomonas syringae/physiology , Lincomycin/pharmacology , Plant Diseases/microbiology , Salicylic Acid/metabolism , Chloroplast Proteins/metabolism , Chloroplast Proteins/geneticsABSTRACT
In humans, aggregation of polyglutamine repeat (polyQ) proteins causes disorders such as Huntington's disease. Although plants express hundreds of polyQ-containing proteins, no pathologies arising from polyQ aggregation have been reported. To investigate this phenomenon, we expressed an aggregation-prone fragment of human huntingtin (HTT) with an expanded polyQ stretch (Q69) in Arabidopsis thaliana plants. In contrast to animal models, we find that Arabidopsis sp. suppresses Q69 aggregation through chloroplast proteostasis. Inhibition of chloroplast proteostasis diminishes the capacity of plants to prevent cytosolic Q69 aggregation. Moreover, endogenous polyQ-containing proteins also aggregate on chloroplast dysfunction. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Synthetic Arabidopsis SPP prevents polyQ-expanded HTT aggregation in human cells. Likewise, ectopic SPP expression in Caenorhabditis elegans reduces neuronal Q67 aggregation and subsequent neurotoxicity. Our findings suggest that synthetic plant proteins, such as SPP, hold therapeutic potential for polyQ disorders and other age-related diseases involving protein aggregation.
Subject(s)
Arabidopsis , Protein Aggregates , Animals , Humans , Arabidopsis/genetics , Peptides/genetics , Neurons/metabolism , Caenorhabditis elegans/geneticsABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0150539.].
ABSTRACT
Several protein homeostasis (proteostasis) pathways safeguard the integrity of thousands of proteins that localize in plant chloroplasts, the indispensable organelles that perform photosynthesis, produce metabolites, and sense environmental stimuli. In this review, we discuss the latest efforts directed to define the molecular process by which proteins are imported and sorted into the chloroplast. Moreover, we describe the recently elucidated protein folding and degradation pathways that modulate the levels and activities of chloroplast proteins. We also discuss the links between the accumulation of misfolded proteins and the activation of signalling pathways that cope with folding stress within the organelle. Finally, we propose new research directions that would help to elucidate novel molecular mechanisms to maintain chloroplast proteostasis.
Subject(s)
Proteome , Proteostasis , Chloroplasts/metabolism , Protein Folding , Proteome/metabolism , Proteostasis/physiologyABSTRACT
[This corrects the article DOI: 10.1371/journal.pgen.1005824.].
ABSTRACT
Abstract Colombia's duality between the relative strength of the central government and the broad process of decentralization towards subnational and local governments has shaped the country's response to the COVID-19 pandemic. Substantial tensions have arisen between the national and subnational governments in the handling of the crisis. Thus, blurred policy boundaries challenge multi-level government coordination. Yet, intergovernmental relations and multi-level governance have worked at some extent. Meanwhile, leadership styles, the level of political support, the local governance environment and the level of local institutional capacity have affected the strategies of local government leaders and their success to tackle the pandemic. Moreover, uneven healthcare capacity across the Colombian territory posits further challenges for a national response to the pandemic that ensures both effectiveness and equity.
Resumo A dualidade da Colômbia, entre a força relativa do governo central e o amplo processo de descentralização com fortalecimento de governos subnacionais e locais, estabeleceu os contornos da resposta do país à pandemia de COVID-19. Testemunhou-se uma substancial tensão entre os governos nacional e subnacionais no tratamento da crise. Os limites pouco claros das políticas desafiam a coordenação governamental em vários níveis. No entanto, as relações intergovernamentais e a governança em vários níveis funcionaram até certo ponto. Enquanto isso, os estilos de liderança, o nível de apoio político, o ambiente local de governança e o nível local de capacidade institucional, afetaram as estratégias das lideranças governamentais locais e seu sucesso no combate à pandemia. Além disso, as desigualdades na capacidade de prover serviços de saúde em todo o território colombiano apresentam desafios adicionais para uma resposta nacional à pandemia que garanta efetividade e equidade.
Resumen La dualidad del modelo unitario descentralizado de Colombia, entre la fuerza relativa del gobierno central y el amplio proceso de descentralización hacia los gobiernos subnacionales y locales ha influido en la respuesta del país frente a la pandemia de COVID-19. Algunas tensiones sustanciales han aparecido entre los gobiernos nacional y subnacionales en el manejo de la crisis. Un contexto con algunos límites jurisdiccionales borrosos impone un reto a la coordinación gubernamental multinivel. Sin embargo, las relaciones intergubernamentales y la gobernanza multinivel parecen haber funcionado en alguna medida. Por otro lado, los estilos de liderazgo, el nivel de apoyo político, el entorno local de gobernanza y el nivel local de capacidad institucional han afectado las estrategias de las autoridades locales y su éxito al enfrentar la pandemia. Más aun, las disparidades en la capacidad de protección en salud a lo largo del territorio colombiano plantean desafíos adicionales para una respuesta coordinada nacional a la pandemia que asegure tanto efectividad como equidad.
Subject(s)
Humans , Male , Female , Politics , Health Management , Health Governance , COVID-19 , Interinstitutional RelationsABSTRACT
The COVID-19 pandemic has created a crisis that is challenging national and local governments to innovate in their responses to novel problems. Despite similarities to the challenges confronted in developed countries, for Latin American governments, these problems are amplified by structural obstacles such as social inequalities. These countries must respond with capacities and resources that are often limited by spoils systems and by social and political polarization. This essay provides an overview of some innovative practices in Argentina, Brazil, Chile, Colombia, and Mexico. In particular, this essay concentrates on some salient collaborative efforts in the region. To draw lessons from these practices, the authors focus on the formal and informal institutions that facilitate or obstruct collaboration across jurisdictions. The findings are discussed in terms of the transaction costs of collaboration identified in these experiences.
ABSTRACT
Chloroplasts are photosynthetic plant organelles descended from a bacterial ancestor. The vast majority of chloroplast proteins are synthesized in the cytosol and then imported into the chloroplast post-translationally. Translocation complexes exist in the organelle's outer and inner envelope membranes (termed TOC and TIC, respectively) to facilitate protein import. These systems recognize chloroplast precursor proteins and mediate their import in an energy-dependent manner. However, many unanswered questions remain regarding mechanistic details of the import process and the participation and functions of individual components; for example, the cytosolic events that mediate protein delivery to chloroplasts, the composition of the TIC apparatus, and the nature of the protein import motor all require resolution. The flux of proteins through TOC and TIC varies greatly throughout development and in response to specific environmental cues. The import process is, therefore, tightly regulated, and it has emerged that the ubiquitin-proteasome system (UPS) plays a key role in this regard, acting at several different steps in the process. The UPS is involved in: the selective degradation of transcription factors that co-ordinate the expression of chloroplast precursor proteins; the removal of unimported chloroplast precursor proteins in the cytosol; the inhibition of chloroplast biogenesis pre-germination; and the reconfiguration of the TOC apparatus in response to developmental and environmental signals in a process termed chloroplast-associated protein degradation. In this review, we highlight recent advances in our understanding of protein import into chloroplasts and how this process is regulated by the UPS.
Subject(s)
Chloroplasts/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Transport/physiology , Ubiquitin/metabolism , Chloroplast Proteins/metabolism , Cytosol/metabolism , Organelles/metabolism , Photosynthesis , Plant Proteins/metabolism , Plants/anatomy & histology , Plants/metabolism , Protein Precursors/metabolism , ProteolysisABSTRACT
Plant metabolism is strongly dependent on plastids. Besides hosting the photosynthetic machinery, these endosymbiotic organelles synthesize starch, fatty acids, amino acids, nucleotides, tetrapyrroles, and isoprenoids. Virtually all enzymes involved in plastid-localized metabolic pathways are encoded by the nuclear genome and imported into plastids. Once there, protein quality control systems ensure proper folding of the mature forms and remove irreversibly damaged proteins. The Clp protease is the main machinery for protein degradation in the plastid stroma. Recent work has unveiled an increasing number of client proteins of this proteolytic complex in plants. Notably, a substantial proportion of these substrates are required for normal chloroplast metabolism, including enzymes involved in the production of essential tetrapyrroles and isoprenoids such as chlorophylls and carotenoids. The Clp protease complex acts in coordination with nuclear-encoded plastidial chaperones for the control of both enzyme levels and proper folding (i.e. activity). This communication involves a retrograde signaling pathway, similarly to the unfolded protein response previously characterized in mitochondria and endoplasmic reticulum. Coordinated Clp protease and chaperone activities appear to further influence other plastid processes, such as the differentiation of chloroplasts into carotenoid-accumulating chromoplasts during fruit ripening.
Subject(s)
Chloroplasts/metabolism , Endopeptidase Clp/metabolism , Plant Proteins/metabolism , Plants/metabolismABSTRACT
Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.
Subject(s)
Evolution, Molecular , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Animals , Disease , HSP70 Heat-Shock Proteins/classification , Humans , Protein Aggregates , Protein Domains , Protein RefoldingABSTRACT
Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Carrier Proteins/metabolism , Chloroplasts/metabolism , Cold-Shock Response/physiology , Protein Biosynthesis , Ribosome Subunits, Small/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Carrier Proteins/genetics , Chloroplasts/genetics , Cold-Shock Response/genetics , Embryophyta/genetics , Gene Expression Regulation, Plant , Immunoprecipitation , Plants, Genetically Modified , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , Ribosome Subunits, Small/geneticsABSTRACT
Classical DNAJ proteins are co-chaperones that together with HSP70s control protein homeostasis. All three classical types of DNAJ proteins (DNAJA, DNAJB and DNAJC types) possess the J-domain for interaction with HSP70. DNAJA proteins contain, in addition, both the zinc-finger motif and the C-terminal domain which are involved in substrate binding, while DNAJB retains only the latter and DNAJC comprises only the J-domain. There is increasing evidence that some of the activities of DNAJ proteins do not require the J-domain, highlighting the functional significance of the other two domains. Indeed, the so-called DNAJ-like proteins with a degenerate J-domain have been previously coined as DNAJD proteins, and also proteins containing only a DNAJ-like zinc-finger motif appear to be involved in protein homeostasis. Therefore, we propose to extend the classification of DNAJ-related proteins into three different groups. The DNAJD type comprises proteins with a J-like domain only, and has 15 members in Arabidopsis thaliana, whereas proteins of the DNAJE (33 Arabidopsis members) and DNAJF (three Arabidopsis members) types contain a DNAJA-like zinc-finger domain and DNAJA/B-like C-terminal domain, respectively. Here, we provide an overview of the entire repertoire of these proteins in A. thaliana with respect to their physiological function and possible evolutionary origin.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , HSP40 Heat-Shock Proteins/metabolism , Conserved Sequence , HSP40 Heat-Shock Proteins/chemistry , PhylogenyABSTRACT
Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts.
Subject(s)
Arabidopsis Proteins/genetics , Chloroplasts/genetics , DNA-Binding Proteins/genetics , Heat-Shock Proteins/genetics , Plant Proteins/genetics , Transcription Factors/genetics , Arabidopsis/genetics , Endopeptidase Clp/genetics , Gene Expression Regulation, Plant/drug effects , Genome, Plant , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/genetics , Heat Shock Transcription Factors , Lincomycin/pharmacology , Molecular Chaperones/genetics , Photosynthesis/genetics , Plants, Genetically Modified , Seedlings/genetics , Signal Transduction , Unfolded Protein Response/geneticsABSTRACT
Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 (SCO2) is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCB1. In Arabidopsis thaliana, SCO2 function was previously reported to be restricted to cotyledons. Here we show that disruption of SCO2 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale-green true leaves can also be observed in A. thaliana sco2 (atsco2) mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSII-LHCII complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant development and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSII assembly or repair and constitutes a novel factor involved in leaf variegation.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Chloroplasts/physiology , Lotus/growth & development , Photosystem II Protein Complex/physiology , Plant Leaves/physiology , Protein Disulfide-Isomerases/physiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , HSP40 Heat-Shock Proteins/chemistry , Lotus/genetics , Mutation , Photosynthesis , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plant Proteins/physiology , Protein Disulfide-Isomerases/geneticsABSTRACT
Environmental stress conditions such as high light, extreme temperatures, salinity or drought trigger oxidative stress and eventually protein misfolding in plants. In chloroplasts, chaperone systems refold proteins after stress, while proteases degrade misfolded and aggregated proteins that cannot be refolded. We observed that reduced activity of chloroplast Hsp70 chaperone or Clp protease systems both prevented growth of Arabidopsis thaliana seedlings after treatment with the oxidative agent methyl viologen. Besides showing a role for these particular protein quality control components on the protection against oxidative stress, we provide evidence supporting the existence of a yet undiscovered pathway for Clp-mediated degradation of the damaged proteins.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , HSP70 Heat-Shock Proteins/metabolism , Plastids/metabolism , Arabidopsis/drug effects , Arabidopsis Proteins/genetics , Chloroplasts/drug effects , Chloroplasts/metabolism , Gene Expression Regulation, Plant , HSP70 Heat-Shock Proteins/genetics , Oxidative Stress/genetics , Oxidative Stress/physiology , Paraquat/pharmacology , Plastids/drug effects , Seedlings/drug effects , Seedlings/metabolismABSTRACT
Estudios recientes en América Latina corroboraron la prevalencia de la hipertensión arterial, el colesterol elevado en plasma, los trastornos metabólicos y otros factores de riesgo cardiovascular (FRCV), todo esto sumado a la falta de control de la enfermedad, la escasa adhesión al tratamiento y el uso de pocas medidas preventivas, lo que derivó en la necesidad de educar tanto al médico como al paciente. Se han realizado estudios limitados para demostrar la eficacia del desarrollo profesional continuo y su capacidad de influir positivamente en los indicadores de salud del paciente. En Venezuela se llevó a cabo un estudio para tratar de medir la efectividad de un curso combinado basado en actividades online y presenciales sobre el manejo de los FRCV, dirigido a médicos que trabajan en atención primaria. Un grupo piloto de 37 médicos de atención primaria del área metropolitana de Caracas completó una intervención educativa de 12 semanas, la cual consistió en sesiones semanales en línea, utilizando la estrategia educativa basada en problemas sobre el manejo de los FRCV, así como talleres presenciales relacionados con los cambios de comportamiento del médico y del paciente, utilizando los principios de la educación terapéutica. Se trabajó con una cohorte de 303 pacientes con hipertensión arterial de grado I-II no controlada y/o colesterol plasmático elevado, y se llevó a cabo un seguimiento durante seis meses. Los resultados evidenciaron cambios significativos en los médicos respecto a los aspectos cognitivos y al estilo de su práctica profesional en el manejo de los FRCV. Los participantes se mostraron muy satisfechos con el contenido y el formato de la intervención educativa. De igual manera, encontramos un resultado positivo en la inducción de cambios en toda la cohorte de pacientes, sobre todo en el perfil lipídico, la presión arterial y la modificación del estilo de vida. Sería interesante su aplicación a mayor escala para medir su efectividad a largo plazo
Recent studies in Latin-America corroborated the prevalence of hypertension, elevated plasma cholesterol, metabolic disorders and other cardiovascular risk factors (CVRF) along with lack of disease awareness. Treatment and preventive measures indicated the urgent need for patient and physician education. Limited studies have been conducted to demonstrate the effectiveness of continual professional development (CPD) and its ability to positively improve health care outcomes. A study was done in Venezuela to measure the effectiveness of a blended web-based education in the management of CVRF by primary health care physicians (PCP), working in primary care services. A pilot group of 37 PCP, from the Caracas metropolitan area, completed a 12-week educational intervention consisting of weekly online problem based sessions on CVRF management and face-to-face workshops on physician and patient behavioural changes, using principles of therapeutic education. A cohort of 303 of their patients with uncontrolled grade I-II arterial hypertension and/or elevated plasma cholesterol were included and followed up for a 6-month period. Significant changes in the cognitive aspects and attitudes about the management of CVRF occurred among the PCP, who were highly satisfied with the content and format of the educational intervention. We also found a positive role in inducing changes in the entire patient cohort, particularly on the plasma lipid profile, blood pressure, and lifestyle changes. Results of this pilot study reflect the effective use of CPD on patient care and PCP interactions in creating synergy and understanding with participating institutions. There is a need of large scale implementation and more extensive comparison with other educational approaches
Subject(s)
Humans , Cardiovascular Diseases/prevention & control , Education, Medical, Continuing/organization & administration , Family Practice/education , Risk Factors , Primary Health Care/organization & administration , Educational Measurement , Professional Training , Evaluation of the Efficacy-Effectiveness of Interventions , VenezuelaABSTRACT
Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Chloroplasts/enzymology , Terpenes/metabolism , Agrobacterium tumefaciens/genetics , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , Chloroplasts/ultrastructure , Genes, Reporter , Genetic Vectors/genetics , Plant Leaves/enzymology , Plant Leaves/ultrastructure , Plants, Genetically Modified , Protein Aggregates , Protein Transport , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/enzymology , Thylakoid Membrane Proteins/genetics , Thylakoid Membrane Proteins/metabolism , Thylakoids/metabolism , Nicotiana , Transferases/genetics , Transferases/metabolismABSTRACT
The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid.
Subject(s)
Arabidopsis Proteins/genetics , Chloroplasts/genetics , Endopeptidase Clp/genetics , Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Transferases/genetics , Arabidopsis , Chloroplasts/metabolism , Endopeptidase Clp/metabolism , Heat-Shock Proteins/metabolism , Metabolic Networks and Pathways/genetics , Plastids/genetics , Plastids/metabolism , Protein Folding , Proteolysis , Terpenes/metabolismABSTRACT
Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.