Protein degrons and degradation: Exploring substrate recognition and pathway selection in plants.

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.

Correction: Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1005824.].

Mutant noxy8 exposes functional specificities between the chloroplast chaperones CLPC1 and CLPC2 in the response to organelle stress and plant defence.

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.

Protein import into chloroplasts and its regulation by the ubiquitin-proteasome system.

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.

Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis.

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.

CHLOROPLAST RIBOSOME ASSOCIATED Supports Translation under Stress and Interacts with the Ribosomal 30S Subunit.

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.

Control of plastidial metabolism by the Clp protease complex.

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.

Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

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.

Novel DNAJ-related proteins in Arabidopsis thaliana.

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.

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism.

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.

Arabidopsis J-protein J20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control.

Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis.

Isoprenoid molecules are essential elements of plant metabolism. Many important plant isoprenoids, such as chlorophylls, carotenoids, tocopherols, prenylated quinones and hormones are synthesised in chloroplasts via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Here we develop a mathematical model of diurnal regulation of the MEP pathway in Arabidopsis thaliana. We used both experimental and theoretical approaches to integrate mechanisms potentially involved in the diurnal control of the pathway. Our data show that flux through the MEP pathway is accelerated in light due to the photosynthesis-dependent supply of metabolic substrates of the pathway and the transcriptional regulation of key biosynthetic genes by the circadian clock. We also demonstrate that feedback regulation of both the activity and the abundance of the first enzyme of the MEP pathway (1-deoxy-D-xylulose 5-phosphate synthase, DXS) by pathway products stabilizes the flux against changes in substrate supply and adjusts the flux according to product demand under normal growth conditions. These data illustrate the central relevance of photosynthesis, the circadian clock and feedback control of DXS for the diurnal regulation of the MEP pathway.

Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene.

The plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus×canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-D-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. ß-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.

In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation.

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.

A proteostasis network safeguards the chloroplast proteome.

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.

Correction: Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

[This corrects the article DOI: 10.1371/journal.pone.0150539.].

An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress.

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli. Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo. Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.

Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts.

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx chi is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx chi knock-out mutant has been identified and a double mutant (denoted Delta 2cp) with <5% of 2-Cys Prx content has been generated. The phenotypes of the three mutants, ntrc, trxx, and Delta 2cp, were compared under standard growth conditions and in response to continuous light or prolonged darkness and oxidative stress. Though all mutants showed altered redox homeostasis, no difference was observed in response to oxidative stress treatment. Moreover, the redox status of the 2-Cys Prx was imbalanced in the ntrc mutant but not in the trxx mutant. These results show that NTRC is the most relevant pathway for chloroplast 2-Cys Prx reduction in vivo, but the antioxidant function of this system is not essential. The deficiency of NTRC caused a more severe phenotype than the deficiency of Trx chi or 2-Cys Prxs as determined by growth, pigment content, CO(2) fixation, and F(v)/F(m), indicating additional functions of NTRC.

The Transaction Costs of Government Responses to the COVID-19 Emergency in Latin America.

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.