Plant proteostasis – shaping the proteome: a research community aiming to understand molecular mechanisms that control protein abundance

Abstract

One of the major challenges for plant science going forward will be to provide the stability and increase in crop yields required to mitigate against climate change and population growth. Understanding how plants cope with changes in the environment by altering their proteome composition, will be an important component of developing approaches to deliver sustainability. In recent years, the cellular processes that change the proteome landscape are beginning to be unravelled. Protein homeostasis (or proteostasis) integrates cellular pathways that mediate biogenesis, folding, trafficking and degradation of polypeptides, to maintain the required concentrations of all proteins that compose the proteome (Fig. 1). As an example, changes in protein concentration can be the result of the degradation of transcription factors to modulate a transcriptional response, or the endocytosis and transport into the vacuole for degradation of activated receptor kinases. Other proteins that are required in higher concentrations can be stabilized by inhibition of their degradation, induced biosynthesis. Proteostasis is the result of the constant interplay between protein degradation and biosynthetic pathways, which allows cells to modulate the concentration of each protein with exquisite precision. Advances in genomics and proteomic analyses have revealed that biological complexity is largely orchestrated not through gene number but by variation at the protein level (proteoforms; Aebersold et al., 2018). Post-translational modification (PTM) events create a plethora of proteoforms to proteome generate flexibility in almost every biological process. Furthermore, the dynamic nature of PTMs achieved through covalent attachment of ubiquitin and ubiquitin-like proteins allows organisms, particularly plants with their inherently sessile nature, to respond to even highly transient changes in the environment with precise fine-tuning. In response to environmental cues, these protein modifications provide a fast and easily reversible modulation of protein function, which can regulate the intensity and amplitude of cellular responses to stress. The Plant Proteostasis community encompasses scientists working on diverse aspects of plant proteostasis, such as protein translation, protein quality control, proteases, PTMs including ubiquitin and ubiquitin-like proteins, vesicular traffic, autophagy, as well as proteasomal and vacuolar degradation (Fig. 1). Protein degradation systems have taken centre stage in investigations of proteoform function, as they are pivotal to ensure proteostasis during stress. These include the proteasomal and vacuolar degradation pathways, both of which encompass a variety of processes including the endocytic degradatory route, as well as other pathways driven by PTMs such as ubiquitination and SUMOylation, and related modifications such as involved in autophagy. The Arabidopsis genome project identified more than a thousand genes encoding components of protein degradation pathways, potentially representing c. 8% of the entire plant protein-coding genome, far more than any other eukaryote group, indicating that control of protein stability is a key adaptive trait for plants. Mutations in components of these systems affect all aspects of plant development, abiotic stress tolerance and pathogen defence. To date, every single plant hormone signalling pathway has been shown to be regulated by protein ubiquitination. More intriguing is the fact that a number of phytohormones are directly perceived by components of the ubiquitination machinery. This could be due to the high level of substrate selectivity built within the ubiquitin E3 targeting system, illustrating the extent to which plants have evolved to rely on protein degradation as a central signalling mechanism. In the last two decades, research in the area of plant proteostasis has been intense, and resulted in many ground-breaking discoveries that have significantly enhanced our understanding of plant cellular signalling. However, many key questions remain unanswered, and in particular, the molecular interaction between the different areas of proteostasis and signalling consequences remains an important under-investigated area. Research in the past concentrated on loss-of-function genetic approaches, or constitutive or conditional induction of specific pathways such as autophagy. However, to obtain mechanistic insights of the pathways safeguarding proteostasis, novel approaches are required. In recent years the Plant Proteostasis research community has developed new tools, such as ratiometric reports to quantify protein degradation, as presented by Freddie Theodooulou (Rothamsted Research, Harpenden, UK), the use of pathogen effectors, synthetic and computational biology and new methods of mass-spectrometric analysis (discussed in Kowarschik et al., 2017; Stephani et al., 2019). The Plant Proteostasis community did not have an on-going meeting structure to share data, tools and build research collaborations to move the field forward. The two-day New Phytologist Workshop at Durham University (UK) in July 2018 brought together key researchers in this community to develop a strategic meeting structure to focus on the role of proteostasis in diverse aspects of plant development and response to the environment. The success of the New Phytologist Workshop underpinned the organization of a larger conference in Freiburg in September 2019 with more diverse sponsorship and participants, as well as a significantly greater number of participants, including many early career researchers. An overview of the topics discussed at the Freiburg Proteostasis meeting is given below. Ubiquitin and the ubiquitin-like proteins, SUMO and NEDD8, form a family of small proteins that are covalently attached to substrates for the PTM of cellular proteins. Their covalent attachment to target proteins provides a fast and reversible modulation of protein function and turnover. The central nature of these modifiers meant that an important focus of the Freiburg Proteostasis meeting was on processes that regulate ubiquitin and ubiquitin-like pathways. While we are beginning to understand the effects of PTMs on target proteins, the control of the processes leading to the modification of target proteins is also an active field of research. Claus Schwechheimer (Technical University of Munich, Germany) showed that in neddylation, the autoneddylation of the E1 activating enzyme can determine the activity of the ubiquitin conjugating enzyme (E2), and this process is closely regulated by the activity of NEDD8 proteases (Mergner et al., 2017). Ari Sadanandom (Durham University, UK) presented evidence that the specificity of SUMOylation, for which only three types of E3 ligases have been described so far, is potentially dependent on SUMO proteases. In stark contrast, over 1500 E3 ligases provide specificity to the ubiquitin modification system. Cleaving SUMO from target proteins may thus be an important mechanism providing reversibility to the pathway and controlling the intensity of dependant cellular responses (Orosa et al., 2018; Srivastava et al., 2018, 2020). Ubiquitin can be conjugated to substrates as a monomer or as a chain of different lengths interlinked to any of its seven lysine residues, or to the methionine in position 1. The linkage-type of the ubiquitin chain determines the fate of the substrate protein, leading to either degradation or to non-proteolytic alteration. E2s catalyse the attachment of ubiquitin to target proteins, and they contribute to determining ubiquitin chain linkages. Marco Trujillo (University of Freiburg, Germany) reported that E2 and E3 ubiquitin ligase enzymes interact dynamically in vivo, and that an E3 ligase can interact with multiple E2 conjugating enzymes, which cooperate to build ubiquitin chains (Trujillo, 2017; Turek et al., 2018). Gregory Vert (University of Toulouse, France) discussed the characterization of two E2 enzymes responsible for building K63-linked chains, and revealed K63-polyubiquitin networks and the connection to multiple E3 ligases. Erika Isono (University Konstanz, Germany) presented evidence that K63-linked chains are also a key signal that is deciphered by ubiquitin adaptor proteins at the endosomal sorting complex in protein trafficking (Mosesso et al., 2019). Beatriz Orosa (University of Edinburgh, UK) reported that proteins with some of these ubiquitin chain linkages accumulate during plant immune responses and are associated with specific E3 ligases. Luz Irina A. Calderón Villalobos (Leibniz IPB, Halle (Saale), Germany) elaborated on structural proteomics advancements to capture E3–target protein ensembles, and on the role of intrinsically disordered degrons in ubiquitylation targets for hormone-driven recognition by Cullin RING-type E3s (Winkler et al., 2017; Niemeyer et al., 2019). Steven Spoel (University of Edinburgh, UK) reported that a family of E3 ligases physically associate with the proteasome, potentially constituting an additional layer of regulation for previously ubiquitinated proteins targeted for degradation. Concomitant to the understanding of their regulation, the specific biological roles of ubiquitination and ubiquitin-like modifications is starting to be deciphered. A main focus has been the interplay between these modifications and immunity. Ubiquitin and ubiquitin-like pathways regulate every layer of immunity from perception to plant reprogramming (Orosa et al., 2018; Turek et al., 2018; Skelly et al., 2019). Key regulators in immunity appear to be tightly regulated by one or more PTM(s). For instance, the cell surface-resident receptor Flagellin Sensitive 2 (FLS2) is modified by SUMO (Orosa et al., 2018), ubiquitin (Lu et al., 2011), phosphorylation (Cao et al., 2013) and S-acylation (Hurst et al., 2019). In fact, the interplay between these modifications is critical for cell signalling. Jacqueline Monaghan (Queen's University, Kingston, Canada) talked about the interplay between phosphorylation and ubiquitination, where a kinase regulates the activity of immune-related E3 ligases, enhancing its ability to ubiquitinate the key immune kinase BIK1 (Monaghan et al., 2015). Similarly, Libo Shan (Texas A&M University, College Station, TX, USA) focused on how ubiquitination regulates cell surface receptor-like kinase complexes involved in immunity and growth, such as BAK1 (Zhou et al., 2019). The relevance of PTMs in plant immune responses is highlighted by the fact that these mechanisms are targeted by pathogens to disable plant immune responses. Paul Birch (University of Dundee, UK) and Pascal Genschik (IMBP-CNRS, France) discussed the capacity of different pathogens, fungi and viruses respectively, to hijack plant E3 ligases to avoid plant immunity and establish infection (Michaeli et al., 2019). Ubiquitin and ubiquitin-like modifications are also involved in the regulation of many other cellular processes. Pedro Rodriguez (IMBCP, Valencia, Spain) revealed that cullin3 (CUL3)-RING-based E3 ligases (CRL3s), target PP2Cs for degradation in a mechanism that is complementary to inhibition of PP2Cs by the ABA receptors PYR/PYL/RCAR during drought (Julian et al., 2019). Judy Callis (University of California Davis, USA) reported on the function of a membrane associated RING domain E3 ligase that governs amino acid secretion in Arabidopsis (Pratelli et al., 2012). Daniel Gibbs (University of Birmingham, UK) described for the first time how a group of E3 ligases may be associated with the regulation of mRNA translation, while Pablo Pulido (University of Oxford, UK) showed the importance of E3s in chloroplast envelope-protein removal and their degradation by the cytosolic 26S proteasome (Ling et al., 2019). Ute Hoecker (University of Cologne, Germany) reported how the control of light signal transduction is connected to ubiquitination via the activity of specific E3 ligases in a light-dependent manner (Ordoñez-Herrera et al., 2018), Sandra Noir (IMBP-CNRS, France) revealed the role of the ubiquitin pathway in cell cycle control and DNA damage responses (Noir et al., 2015 and unpublished data), while Michael Holdsworth (University of Nottingham, UK) discussed novel functions of the plant N-degron pathways. The relevance of these pathways across evolution was highlighted by Roberto Solano (CNB-CSIC, Madrid, Spain), who reported during his EMBO keynote lecture the functional conservation of the E3 complex co-receptor COI1 across 450 million years of evolution (Monte et al., 2019). Maria Lois (CRAG, Barcelona, Spain) showed that the E2 binding region in E1 activating enzymes is conserved only across phylogenetically closely related species (Liu et al., 2019). In the last 10 years, numerous efforts have been made to unravel how autophagy functions at the molecular level, and which processes are tightly associated with this degradation mechanism. Autophagy is implicated in a plethora of cellular processes in plants including reproduction, development, hormone signalling, cellular homeostasis, senescence, abiotic and biotic stress responses. Autophagy relies on a core set of conserved autophagy-related (ATG) genes to form double membrane compartments, termed autophagosomes, that sequester and deliver cytoplasmic cargo to the lytic vacuole for breakdown and recycling (Marshall & Vierstra, 2018). ATG8 proteins are required for membrane expansion and found on autophagosomes until their lytic destruction. Similar to the proteasomal pathway, autophagy is a major degradation route implicated in safeguarding cellular homeostasis, implicated in stress tolerance and immunity in eukaryotic organisms (Marshall & Vierstra, 2018). For a long time, autophagy was regarded as a largely unspecific (‘bulk’) degradation mechanism mainly recycling unwanted cytoplasmic contents. However, new evidence indicates that autophagy acts as a highly selective mechanism to target various components including proteins, organelles, or viral proteins, under different stress conditions (Marshall & Vierstra, 2018). Specificity is driven by autophagic receptors that interact with both ATG8 and ubiquitin, which are attached to autophagosomes and cargo, respectively. This surfacing view was best reflected by Richard Vierstra (Washington University, St Louis, MO, USA), who posed the question during his keynote lecture: ‘Is there even bulk autophagy, or is everything selective autophagy?’ His laboratory recently identified a new class of autophagy adaptors that were previously classified as ubiquitin-interacting motifs (UIMs)-like sequences, rather than the classical ATG8-interacting motif (AIM) (Marshall et al., 2019). This study revealed a large collection of novel ATG8 interactors in plants, yeast, and humans, expanding the repertoire of possible selective autophagy adaptors and potential autophagy cargo. These findings strongly suggest that there is even more specificity to autophagic degradation than previously assumed. In addition to its role in cellular housekeeping and development, autophagy is also implicated in plant–pathogen interactions (Üstün et al., 2017, 2018; Leary et al., 2019). Recent efforts revealed that the hemi-biotrophic oomycete Phytophthora infestans evolved the effector protein PexRD54 to counteract selective autophagy-mediated pathogen restriction (Dagdas et al., 2016, 2018). Along this line, the bacterial pathogen Pseudomonas syringae pv. tomato activates autophagy to mediate proteasome degradation and enhance its virulence (Üstün et al., 2018). Both Alex Leary (Imperial College, London, UK) and Suayib Üstün (University of Tübingen, Germany) introduced pathogenic effector molecules as tools to dissect the autophagy pathway and its role during plant defence responses. Of note, both speakers emphasized the tight interconnection between vesicle trafficking and autophagy during plant immunity, which is an upcoming theme in autophagy research (Zeng et al., 2019). Accordingly, Xiaohong Zhuang (Chinese University of Hong Kong, China) reported on how the BAR-domain protein SH3P2 regulates autophagosome formation (Zhuang et al., 2013), while also being involved in intracellular trafficking (Mosesso et al., 2019) and cytokinesis (Ahn et al., 2017). SH3P2 might be a perfect example showcasing the implication of proteins in autophagy, as well as vesicular traffic, thus connecting different pathways to autophagy. It is therefore not surprising that a bacterial effector targets this multi-functional protein to perturb autophagy and other cellular trafficking processes, which was introduced by Suayib Üstün. Marisa Otegui (University of Wisconsin-Madison, USA) presented an electron tomography approach to look at membrane remodelling during endosomal sorting, which may prove to be a valuable addition to our current toolbox to study autophagy responses. In addition to studies of plant immunity, Daphne Goring provided evidence on the role of autophagy in Brassicaceae self-incompatibility. Their findings reveal that autophagy is upregulated during pollen rejection (Doucet et al., 2019; Jany et al., 2019). In parallel, vesicle trafficking seems to be inhibited during self-incompatibility, again suggesting an interplay between vesicle transport and autophagy. Markus Wirtz discussed how sulphur deficiency activates the autophagy pathway (Dong et al., 2017) and follow-up studies on tissue-specific autophagy responses. Yasin Dagdas (GMI, Vienna, Austria) emphasized their efforts to develop new tools to study autophagy to circumvent the problem of pleiotropic autophagy mutants. His group is studying the autophagy degradome and ATG8-related specificity (Zess et al., 2019). With these new tools they were able to reveal new insights about the unfolded protein response and autophagy pathway. With regard to abiotic stress, Venkatesh P. Thirumalaikumar (Max Planck Institute for Molecular Plant Physiology, Potsdam, Germany) introduced the role of autophagy in resetting cellular memory of heat stress (Sedaghatmehr et al., 2018), which may be mediated by the selective autophagy adaptor NBR1, previously involved in aggregate clearance during heat stress (Zhou et al., 2013). Nuria S. Coll (CRAG, Barcelona, Spain) also discussed the role and dynamics of stress-induced protein aggregates in plants and which potential mechanisms are required to degrade them. This meeting highlighted the emerging view that ubiquitin and ubiquitin-like pathways regulate a huge diversity of layers of plant growth, development and response to the environment, from perception to transcriptional reprogramming, and our understanding of their mechanisms is rapidly increasing. However, there are still many key questions to be answered: How are the distinct ubiquitin signals generated and transduced into specific responses? 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