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 postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

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.

The development and operation of chloroplasts (or other members of the plastid family of plant organelles) requires the participation of thousands of different organellar proteins. Most chloroplast proteins are nucleus-encoded and synthesized in the cytosol in precursor form. These preproteins pass through multiprotein import machines in the organelle's outer and inner envelope membranes – these are called the TOC and TIC translocons, respectively. Receptor components of the TOC complex exist in multiple isoforms, and genetic studies in the model plant Arabidopsis have indicated that the isoforms have distinct preprotein recognition specificities and function in different import pathways. Operation of such client-specific import pathways controls the organelle's proteome and functions, and plays a role in the differentiation of different plastid types (e.g., chromoplast formation in ripening tomato fruit). Our work has shown that the chloroplast protein import machinery is proteolytically regulated by direct action of the ubiquitin-proteasome system (UPS), in a process termed "chloroplast-associated protein degradation" (CHLORAD). The CHLORAD machinery has three key components: the SP1 ubiquitin E3 ligase, the SP2 β-barrel channel protein, and the CDC48 AAA+ ATPase. The SP1 and SP2 proteins form a complex in the outer envelope membrane, and respectively mediate the ubiquitination and extraction (or retrotranslocation) of TOC protein targets. The CDC48 protein, located in the cytosol, provides the energy that drives the retrotranslocation step, delivering the target proteins to the cytosolic 26S proteasome for degradation. Recent advances in our understanding of the mechanisms and functions of CHLORAD will be discussed. Our work is supported by UK Research and Innovation - Biotechnology and Biological Sciences Research Council (UKRI-BBSRC).

Regulation of chloroplast protein import by chloroplast-associated protein degradation (CHLORAD) is crucial for chloroplast biogenesis and plant development. This study identifies PUX10 as a CHLORAD component that functions as a membrane-bound scaffold to recruit cytosolic Cdc48 to the chloroplast surface and bring it into proximity with CHLORAD substrates.

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. Extraction of ubiquitinated proteins from chloroplasts in CHLORAD is driven by the cytosolic ATPase Cdc48. The UBX-domain protein PUX10 is shown to be a CHLORAD component that recruits Cdc48 to the chloroplast surface.

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 ∼3,000 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.

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.

ABSTRACT Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 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.

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.

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.

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 cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100 . The tic100 cue8 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 tic100 cue8 identified a second mutation within the same gene, tic100 soh1 , which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100 soh1 retains but rapidly exits virescence, and rescues the synthetic lethality of tic100 cue8 when retrograde signalling is impaired by the gun1 mutation. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signalling 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 non-photosynthetic proteins, a process which initiates retrograde signalling. One sentence summary Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signalling role.

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.

Plant transpiration links physiological responses of vegetation to water supply and demand with hydrological, energy, and carbon budgets at the land–atmosphere interface. However, despite being the main land evaporative flux at the global scale, transpiration and its response to environmental drivers are currently not well constrained by observations. Here we introduce the first global compilation of whole-plant transpiration data from sap flow measurements (SAPFLUXNET, https://sapfluxnet.creaf.cat/, last access: 8 June 2021). We harmonized and quality-controlled individual datasets supplied by contributors worldwide in a semi-automatic data workflow implemented in the R programming language. Datasets include sub-daily time series of sap flow and hydrometeorological drivers for one or more growing seasons, as well as metadata on the stand characteristics, plant attributes, and technical details of the measurements. SAPFLUXNET contains 202 globally distributed datasets with sap flow time series for 2714 plants, mostly trees, of 174 species. SAPFLUXNET has a broad bioclimatic coverage, with woodland/shrubland and temperate forest biomes especially well represented (80 % of the datasets). The measurements cover a wide variety of stand structural characteristics and plant sizes. The datasets encompass the period between 1995 and 2018, with 50 % of the datasets being at least 3 years long. Accompanying radiation and vapour pressure deficit data are available for most of the datasets, while on-site soil water content is available for 56 % of the datasets. Many datasets contain data for species that make up 90 % or more of the total stand basal area, allowing the estimation of stand transpiration in diverse ecological settings. SAPFLUXNET adds to existing plant trait datasets, ecosystem flux networks, and remote sensing products to help increase our understanding of plant water use, plant responses to drought, and ecohydrological processes. SAPFLUXNET version 0.1.5 is freely available from the Zenodo repository (https://doi.org/10.5281/zenodo.3971689; Poyatos et al., 2020a). The “sapfluxnetr” R package – designed to access, visualize, and process SAPFLUXNET data – is available from CRAN.

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.

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.

Defects in chloroplast development are ‘retrograde-signalled’ to the nucleus, reducing synthesis of photosynthetic or related proteins. The Arabidopsis cue8 mutant manifests virescence, a slow-greening phenotype, and is defective at an early stage in plastid development. Greening cotyledons or early leaf cells of cue8 exhibit immature chloroplasts which fail to fill the available cellular space. Such chloroplasts show reduced expression of genes of photosynthetic function, dependent on the plastid-encoded polymerase (PEP), while the expression of genes of housekeeping function driven by the nucleus-encoded polymerase (NEP) is elevated, a phenotype shared with mutants in plastid genetic functions. We attribute this phenotype to reduced expression of specific PEP-controlling sigma factors, elevated expression of RPOT (NEP) genes and maintained replication of plastid genomes (resulting in densely coalesced nucleoids in the mutant), i.e. it is due to an anterograde nucleus-to-chloroplast correction, analogous to retention of a juvenile plastid state. Mutants in plastid protein import components, particularly those involved in housekeeping protein import, also show this ‘retro-anterograde’ correction. Loss of CUE8 also causes changes in mRNA editing. The overall response has strong fitness value: loss of GUN1, an integrator of retrograde signalling, abolishes elements of it (albeit not others, including editing changes), causing bleaching and eventual seedling lethality upon cue8 gun1 . This highlights the adaptive significance of virescence and retrograde signalling. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

Chloroplast autophagy and chloroplast-associated ubiquitination independently contribute to chloroplast degradation, the management of ROS damage, and the adaptation to starvation. 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.

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 β-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.

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.