RESUMO
Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cloroplastos/metabolismo , Plantas/metabolismo , Organelas/metabolismo , Transporte Proteico , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Autofagia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.
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Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the â¼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.
Assuntos
Cloroplastos , Ubiquitina , Fotossíntese , Complexo de Endopeptidases do Proteassoma , Proteínas de Cloroplastos/genética , Transporte ProteicoRESUMO
Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle's envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100cue8 identified a second mutation within the same gene, tic100soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Mutação/genética , Transporte Proteico/fisiologiaRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/fisiologia , Autofagia/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagia/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Folhas de Planta/genética , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
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.
Assuntos
Cloroplastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , Ubiquitina/metabolismo , Proteínas de Cloroplastos/metabolismo , Citosol/metabolismo , Organelas/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas/anatomia & histologia , Plantas/metabolismo , Precursores de Proteínas/metabolismo , ProteóliseRESUMO
Plant cells are characterized by a unique group of interconvertible organelles called plastids, which are descended from prokaryotic endosymbionts. The most studied plastid type is the chloroplast, which carries out the ancestral plastid function of photosynthesis. During the course of evolution, plastid activities were increasingly integrated with cellular metabolism and functions, and plant developmental processes, and this led to the creation of new types of non-photosynthetic plastids. These include the chromoplast, a carotenoid-rich organelle typically found in flowers and fruits. Here, we provide an introduction to non-photosynthetic plastids, and then review the structures and functions of chromoplasts in detail. The role of chromoplast differentiation in fruit ripening in particular is explored, and the factors that govern plastid development are examined, including hormonal regulation, gene expression, and plastid protein import. In the latter process, nucleus-encoded preproteins must pass through two successive protein translocons in the outer and inner envelope membranes of the plastid; these are known as TOC and TIC (translocon at the outer/inner chloroplast envelope), respectively. The discovery of SP1 (suppressor of ppi1 locus1), which encodes a RING-type ubiquitin E3 ligase localized in the plastid outer envelope membrane, revealed that plastid protein import is regulated through the selective targeting of TOC complexes for degradation by the ubiquitin-proteasome system. This suggests the possibility of engineering plastid protein import in novel crop improvement strategies.
Assuntos
Cloroplastos/metabolismo , Plastídeos/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Organelas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos/genética , Plastídeos/genética , Transporte ProteicoRESUMO
The translocon at the outer chloroplast membrane (TOC) is the gateway for chloroplast protein import and so is vital for photosynthetic establishment and plant growth. Chloroplast-associated protein degradation (CHLORAD) is a ubiquitin-dependent proteolytic system that regulates TOC. In CHLORAD, cytosolic Cdc48 provides motive force for the retrotranslocation of ubiquitinated TOC proteins to the cytosol but how Cdc48 is recruited is unknown. Here, we identify plant UBX-domain protein PUX10 as a component of the CHLORAD machinery. We show that PUX10 is an integral chloroplast outer membrane protein that projects UBX and ubiquitin-associated domains into the cytosol. It interacts with Cdc48 via its UBX domain, bringing it to the chloroplast surface, and with ubiquitinated TOC proteins via its ubiquitin-associated domain. Genetic analyses in Arabidopsis revealed a requirement for PUX10 during CHLORAD-mediated regulation of TOC function and plant development. Thus, PUX10 coordinates ubiquitination and retrotranslocation activities of CHLORAD to enable efficient TOC turnover.
RESUMO
Chloroplasts are the defining plant organelles with responsibility for photosynthesis and other vital functions. To deliver these functions, they possess a complex proteome comprising thousands of largely nucleus-encoded proteins. Composition of the proteome is controlled by diverse processes affecting protein translocation and degradation-our focus here. Most chloroplast proteins are imported from the cytosol via multiprotein translocons in the outer and inner envelope membranes (the TOC and TIC complexes, respectively), or via one of several noncanonical pathways, and then sorted by different systems to organellar subcompartments. Chloroplast proteolysis is equally complex, involving the concerted action of internal proteases of prokaryotic origin and the nucleocytosolic ubiquitin-proteasome system (UPS). The UPS degrades unimported proteins in the cytosol and chloroplast-resident proteins via chloroplast-associated protein degradation (CHLORAD). The latter targets the TOC apparatus to regulate protein import, as well as numerous internal proteins directly, to reconfigure chloroplast functions in response to developmental and environmental signals.
Assuntos
Proteoma , Proteostase , Proteólise , Proteoma/metabolismo , Cloroplastos/metabolismo , Ubiquitinação , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Transporte Proteico/fisiologia , Proteínas de Plantas/metabolismoRESUMO
The chloroplast is the most prominent member of a diverse group of plant organelles called the plastids, and it is characterized by its vital role in photosynthesis.1,2,3 Most of the â¼3,000 different proteins in chloroplasts are synthesized in the cytosol in precursor (preprotein) form, each with a cleavable transit peptide.4,5,6,7,8 Preproteins are imported via translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively.9,10,11,12,13 Discovery of the chloroplast-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) demonstrated that the nucleocytosolic ubiquitin-proteasome system (UPS) targets the TOC apparatus to dynamically control protein import and chloroplast biogenesis in response to developmental and environmental cues. The relevant UPS pathway is termed chloroplast-associated protein degradation (CHLORAD).14,15,16 Two homologs of SP1 exist, SP1-like1 (SPL1) and SPL2, but their roles have remained obscure. Here, we show that SP1 is ubiquitous in the Viridiplantae and that SPL2 and SPL1 appeared early during the evolution of the Viridiplantae and land plants, respectively. Through genetic and biochemical analysis, we reveal that SPL1 functions as a negative regulator of SP1, potentially by interfering with its ability to catalyze ubiquitination. In contrast, SPL2, the more distantly related SP1 homolog, displays partial functional redundancy with SP1. Both SPL1 and SPL2 modify the extent of leaf senescence, like SP1, but do so in diametrically opposite ways. Thus, SPL1 and SPL2 are bona fide CHLORAD system components with negative and positive regulatory functions that allow for nuanced control of this vital proteolytic pathway.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Proteínas/metabolismo , Cloroplastos/metabolismo , Plastídeos/metabolismo , Plantas/metabolismo , Transporte Proteico , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Photosynthesis is the energetic basis for most life on Earth, and in plants it operates inside double membrane-bound organelles called chloroplasts. The photosynthetic apparatus comprises numerous proteins encoded by the nuclear and organellar genomes. Maintenance of this apparatus requires the action of internal chloroplast proteases, but a role for the nucleocytosolic ubiquitin-proteasome system (UPS) was not expected, owing to the barrier presented by the double-membrane envelope. Here, we show that photosynthesis proteins (including those encoded internally by chloroplast genes) are ubiquitinated and processed via the CHLORAD pathway: They are degraded by the 26S proteasome following CDC48-dependent retrotranslocation to the cytosol. This demonstrates that the reach of the UPS extends to the interior of endosymbiotically derived chloroplasts, where it acts to regulate photosynthesis, arguably the most fundamental process of life.
RESUMO
The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated by the ubiquitin-dependent chloroplast-associated protein degradation pathway. Here, we demonstrate that the TOC complex is also regulated by the small ubiquitin-like modifier (SUMO) system. Arabidopsis mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of ppi1, a pale-yellow mutant lacking the Toc33 protein. This suppression is linked to increased abundance of TOC proteins and improvements in chloroplast development. Moreover, data from molecular and biochemical experiments support a model in which the SUMO system directly regulates TOC protein stability. Thus, we have identified a regulatory link between the SUMO system and the chloroplast protein import machinery.
All green plants grow by converting light energy into chemical energy. They do this using a process called photosynthesis, which happens inside compartments in plant cells called chloroplasts. Chloroplasts use thousands of different proteins to make chemical energy. Some of these proteins allow the chloroplasts to absorb light energy using chlorophyll, the pigment that makes leaves green. The vast majority of these proteins are transported into the chloroplasts through a protein machine called the TOC complex. When plants lack parts of the TOC complex, their chloroplasts develop abnormally, and their leaves turn yellow. Photosynthesis can make toxic by-products, so cells need a way to turn it off when they are under stress; for example, by lowering the number of TOC complexes on the chloroplasts. This is achieved by tagging TOC complexes with a molecule called ubiquitin, which will lead to their removal from chloroplasts, slowing photosynthesis down. It is unknown whether another, similar, molecular tag called SUMO aids in this destruction process. To find out, Watson et al. examined a mutant of the plant Arabidopsis thaliana. This mutant had low levels of the TOC complex, turning its leaves pale yellow. A combination of genetic, molecular, and biochemical experiments showed that SUMO molecular tags control the levels of TOC complex on chloroplasts. Increasing the amount of SUMO in the mutant plants made their leaves turn yellower, while interfering with the genes responsible for depositing SUMO tags turned the leaves green. This implies that in plants with less SUMO tags, cells stopped destroying their TOC complexes, allowing the chloroplasts to develop better, and changing the colour of the leaves. The SUMO tagging of TOC complexes shares a lot of genetic similarities with the ubiquitin tag system. It is possible that SUMO tags may help to control the CHLORAD pathway, which destroys TOC complexes marked with ubiquitin. Understanding this relationship, and how to influence it, could help to improve the performance of crops. The next step is to understand exactly how SUMO tags promote the destruction of the TOC complex.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Proteína SUMO-1/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteína SUMO-1/genéticaRESUMO
The maturation of green fleshy fruit to become colourful and flavoursome is an important strategy for plant reproduction and dispersal. In tomato (Solanum lycopersicum) and many other species, fruit ripening is intimately linked to the biogenesis of chromoplasts, the plastids that are abundant in ripe fruit and specialized for the accumulation of carotenoid pigments. Chromoplasts develop from pre-existing chloroplasts in the fruit, but the mechanisms underlying this transition are poorly understood. Here, we reveal a role for the chloroplast-associated protein degradation (CHLORAD) proteolytic pathway in chromoplast differentiation. Knockdown of the plastid ubiquitin E3 ligase SP1, or its homologue SPL2, delays tomato fruit ripening, whereas overexpression of SP1 accelerates ripening, as judged by colour changes. We demonstrate that SP1 triggers broader effects on fruit ripening, including fruit softening, and gene expression and metabolism changes, by promoting the chloroplast-to-chromoplast transition. Moreover, we show that tomato SP1 and SPL2 regulate leaf senescence, revealing conserved functions of CHLORAD in plants. We conclude that SP1 homologues control plastid transitions during fruit ripening and leaf senescence by enabling reconfiguration of the plastid protein import machinery to effect proteome reorganization. The work highlights the critical role of chromoplasts in fruit ripening, and provides a theoretical basis for engineering crop improvements.
Assuntos
Proteínas de Cloroplastos/metabolismo , Frutas/crescimento & desenvolvimento , Redes e Vias Metabólicas , Plastídeos/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Envelhecimento , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Cloroplastos/metabolismo , Frutas/metabolismo , Solanum lycopersicum/metabolismo , Redes e Vias Metabólicas/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type ß-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ciclo Celular/metabolismo , Cloroplastos/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteólise , Ubiquitina/metabolismo , Arabidopsis/genética , Proteínas de Cloroplastos , Proteínas de Membrana , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Communication between organelles and the nucleus is essential for fitness and survival. Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signalling pathways that collectively regulate the nuclear transcriptome. Here, we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild-type background and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by overaccumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype. Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.
Assuntos
Proteínas de Arabidopsis/fisiologia , Proteínas de Ligação a DNA/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transdução de Sinais/fisiologia , TranscriptomaRESUMO
Chloroplasts, as well as other, non-photosynthetic types of plastid, are characteristic structures within plant cells. They are relatively large organelles (typically 1-5 µm in diameter), and so can readily be analysed by electron microscopy. Chloroplast structure is remarkably complex, comprising at least six distinct sub-organellar compartments, and is sensitive to developmental changes, environmental effects, and genetic lesions. Transmission electron microscopy (TEM), therefore, represents a powerful technique for monitoring the effects of various changing parameters or treatments on the development and differentiation of these important organelles. We describe a method for the analysis of Arabidopsis plant material by TEM, primarily for the assessment of plastid ultrastructure.
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Arabidopsis/ultraestrutura , Cloroplastos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Cloroplastos/metabolismo , Microscopia Eletrônica de Transmissão/normas , Microtomia , Compostos Organometálicos/metabolismo , Padrões de Referência , Plântula/metabolismo , Plântula/ultraestrutura , Coloração e RotulagemRESUMO
In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that can be accessed by everyone due to its simplicity. Despite its rapidity and simplicity, the method yields highly pure chloroplasts, and in addition works well with mutant plants that exhibit pale or chlorotic phenotypes. The protocol is also optimized for work with material from young plants (10-14 days old), when protein import is believed to be at its peak, and so plant growth can be conducted in vitro on Murashige and Skoog medium. The isolation method has been used not only for protein import assays, but also for proteomic analysis and further subfractionation studies.