Modulation of the endosomal pathway for optimized response to drought stress: from model to crop plants

To actively regulate the abundance of proteins at the PM, they need to be recognized by components of the CME machinery, specifically by adaptors. These distinguish the proteins to be internalized by motifs found in the cytosolic domains of such prospective cargo. Sorting motifs can be present in the protein sequence (intrinsic motifs) or can occur through covalently linked specific post-translational modifications like phosphorylation or ubiquitination. Importantly, several distinct sorting signals in the same cargo protein can participate in cargo recognition, adding some flexibility to subsequent sorting events (Traub, 2009; Traub and Bonifacino, 2013; Arora and Van Damme, 2021).

Linear sorting motifs found in the cytosolic domain of the proteins, like the di-leucine- and the tyrosine (YXXΦ)-based sorting motif (where Y is tyrosine, X is any amino acid, and Φ is a bulky hydrophobic residue), have also been identified in plant PM proteins (Arora and Van Damme, 2021). Nevertheless, only the tyrosine motifs have so far been linked to endocytic sorting from the PM (Geldner and Robatzek, 2008), where they were found to be important for the internalization of several PM proteins like the pin-formed (PIN) auxin efflux facilitators (Kleine-Vehn et al., 2011; Sancho-Andres et al., 2016), the brassinosteroid hormone receptor brassinosteroid insensitive 1 (BRI1) (Liu et al., 2020), the tomato pathogen-related receptor-like protein Lycopersicon esculentum ethylene-inducing xylanase receptor (LeEIX2) (Ron and Avni, 2004), and the borate exporter boron transporter 1 (BOR1) (Takano et al., 2010). Protein phosphorylation is a dynamic post-translational modification, which has a central regulatory function in the endocytosis of PM proteins in plants, albeit frequently coupled to and in combination with protein ubiquitination (Bonifacino and Traub, 2003; Traub and Bonifacino, 2013). A very elegant mechanism combining these two modifications was recently demonstrated for the PM-localized IRON-REGULATED TRANSPORTER 1 (IRT1), where phosphorylation is triggered by direct metal binding to a histidine-rich stretch in the cytosolic domain of the transporter. This in turn facilitates recruitment of the IRT1 DEGRADATION FACTOR1 E3 ubiquitin ligase IDF1, allowing for the conclusion that protein phosphorylation seems to be a prior requirement for ubiquitination and both are needed for efficient endosomal sorting of IRT1 (Dubeaux et al., 2018).

The best documented post-translational modification for endocytosis of PM proteins in plants is ubiquitination. This reversible protein modification serves as a signal triggering endocytosis and consequent sorting for degradation, and thus plays a key role in directing the entry of PM proteins into the endosomal system (Clague et al., 2012; Piper et al., 2014; Dubeaux and Vert, 2017). Protein ubiquitination is catalyzed by a series of highly conserved enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3), resulting in the formation of an isopeptide bond between the C-terminus of ubiquitin and the free amine of a lysine residue on the target protein (Vierstra, 2012; Callis, 2014; Piper et al., 2014). Ubiquitin conjugation is extraordinarily complex in plants, exemplified by more than 1500 E3 ubiquitin ligases, found in the model plant Arabidopsis, which confer target specificity for ubiquitination (Hua and Vierstra, 2011). Furthermore, different E3 ubiquitin ligases can target the same substrates, likely essential for the fine-tuning of protein function, as well as the same E3 ubiquitin ligase can ubiquitinate different proteins (Bueso et al., 2014; Irigoyen et al., 2014; Zhao et al., 2017; Fernandez et al., 2020)

Target proteins can be monoubiquitinated or poly-monoubiquitinated, where individual ubiquitin molecules are conjugated to cargo proteins or additional ubiquitin molecules are linked to an already attached ubiquitin to form chains. Different types of such polyubiquitin chains can form, depending on which lysine residues of the ubiquitin molecule are conjugated (Husnjak and Dikic, 2012). Two common forms are K48- and K63-linked ubiquitin chains, where the E2/E3 pairing determines substrate specificity and the type of chain linkage, although the same E3 may form different linkage types together with different E2s (Tomanov et al., 2014; Stewart et al., 2016). Ubiquitin binding domain (UBD)-containing proteins, which noncovalently associate with ubiquitin, are responsible for the decoding and translation of the ubiquitination code (Husnjak and Dikic, 2012). These ubiquitin receptors typically contain short amino acid stretches, lacking a strict consensus sequence, that display a low binding affinity toward ubiquitin (Husnjak and Dikic, 2012). Importantly, protein ubiquitination is a reversible process with de-ubiquitinating enzymes remodeling the ubiquitin code, contributing to the highly dynamic and reversible characteristics of endocytic trafficking decisions (Isono and Nagel, 2014). Ubiquitination of PM proteins, especially the addition of K63-linked polyubiquitin chains, serves as a signal for endocytosis and further sorting to the vacuole (Husnjak and Dikic, 2012; Romero-Barrios and Vert, 2018). This was shown for several different PM proteins like the iron transporter IRT1 (Dubeaux et al., 2018), the abscisic acid (ABA) receptors PYL4 and PYR1 (Bueso et al., 2014), and the auxin efflux facilitator PIN2 (Leitner et al., 2012b) as well as the brassinosteroid receptor BRI1 (Di Rubbo et al., 2013; Martins et al., 2015). Endocytic vesicles co-localize with K63-linked polyubiquitinated cargo (Johnson and Vert, 2016), and the molecular mechanisms driving endocytosis of such cargos are essentially conserved in plants. Nevertheless, some key players involved in this process appear plant-specific (Dubeaux and Vert, 2017; Gao et al., 2017; Schwihla and Korbei, 2020).

The ESCRT machinery is a conserved, multi-subunit membrane remodeling complex. It functions in the formation of membrane invaginations, which bud away from the cytoplasm, followed by scission. This stepwise process, performed by protein complexes termed ESCRT-0 to ESCRT-III and accessory components, acts in the recognition, concentration, and sequestering of cargo into the ILVs of MVBs and membrane-deforming events (Henne et al., 2011; Paez Valencia et al., 2016; Gao et al., 2017; Isono and Kalinowska, 2017).

The ESCRT-0 complex is required for the initial targeting and concentration of ubiquitinated cargo and further recruits ESCRT-I, to which it passes on the cargo (Hurley, 2010). The ESCRT-I then recruits the ESCRT-II to the endosomes and the presence of both complexes induces invagination of the limiting membrane toward the endosomal lumen (Hurley and Hanson, 2010). ESCRT-I and -II complexes might also act in parallel to cluster ubiquitinated cargo for internalization (Hurley, 2008; Hurley and Ren, 2009). ESCRT-II then recruits and possibly activates the ESCRT-III complex, which consists of small soluble subunits that assemble into higher-ordered multimers on endosomal membranes (Teo et al., 2004). Once the ESCRTs are assembled, the ubiquitin molecule is removed from the cargo proteins by deubiquitinating enzymes, while an AAA ATPase recycles ESCRT-III back into its monomeric form (Babst et al., 1998). The recruitment and activity of the AAA ATPase is mediated by ESCRT-III–related proteins (Gatta and Carlton, 2019).

In yeast/mammals, the ESCRT-0 complex is made up of two subunits and found at the EEs and the PM (Raiborg and Stenmark, 2009; Henne et al., 2011), where it appears to preassemble with cargoes to enhance the sorting efficiency without affecting vesicle formation per se (Mayers et al., 2013). The ESCRT-0 associates preferentially with K63-linked ubiquitin chains (Nathan et al., 2013), and some subunits can recruit deubiquitinating enzymes, permitting for adjustments in cargo ubiquitination, which enforces the decision if the cargo is to be recycled or degraded (Raiborg and Stenmark, 2009). While the other ESCRT machinery complexes are ubiquitous in eukaryotes and presumably ancient in origin, the ESCRT-0 might represent a more recent addition, not found in plant genomes. The TOM1 protein family represents a family of proteins sharing the same tandem array of domains, the VHS (Vps27/Hrs/Stam) and GAT domains as the ESCRT-0 (Winter and Hauser, 2006; Mosesso et al., 2019). Furthermore, it functions either in parallel, as in mammals (Wang et al., 2010), or alternatively, as in amoeba (Blanc et al., 2009), to the ESCRT-0 complex. TOM1L1 has also been proposed to package ubiquitinated PM proteins into CCVs (Liu et al., 2009). TOM1 is widely conserved in eukaryotes, and from its phylogenetic distribution, it is likely to have been either replaced or perhaps supplemented by the ESCRT-0 in opisthokonts (Herman et al., 2011). As plants do not have canonical ESCRT-0 components, they rely on other proteins to initially recognize ubiquitinated cargo at the PM (Mosesso et al., 2019). Potential candidates that substitute for this complex are from the conserved target of myb1 (TOM1)-like (TOL) protein family (Winter and Hauser, 2006; Schwihla and Korbei, 2020).

The A. thaliana TOL protein family has a domain organization similar to the ESCRT-0 and consists of nine proteins (TOL1–9), several of which have been demonstrated to be essential for vacuolar targeting and subsequent degradation of ubiquitinated PM proteins (Korbei et al., 2013; Moulinier-Anzola et al., 2020; Figure 1). TOLs bind ubiquitin with a preference toward K63-linked ubiquitin chains and participate in the early endocytic trafficking of membrane(-associated) proteins destined for degradation (Moulinier-Anzola et al., 2020), such as the auxin efflux facilitator PIN2, the syntaxin KNOLLE (Korbei et al., 2013), and the borate transporter BOR1 (Yoshinari et al., 2018). A higher-order quintuple tol mutant plant line has a severe and pleiotropic phenotype involving not only mislocalization of specific PM proteins (Korbei et al., 2013), but also a general defect in the degradation of ubiquitinated proteins (Moulinier-Anzola et al., 2020). TOL proteins function as ubiquitin receptors in the initial steps of the ESCRT pathway as they interact with the ESCRTI component VACUOLAR PROTEIN SORTING 23A (VPS23A), thus connecting the TOLs to the ESCRT machinery (Moulinier-Anzola et al., 2020). Function and localization of TOL6 are influenced by ubiquitination, which could assist in the fine-tuning of the interplay between protein recycling and downregulation (Moulinier-Anzola et al., 2020). TOLs can therefore be considered as a plant-specific substitute of ESCRT-0, functioning as multivalent ubiquitin-binding complexes and promoting cargo delivery to the vacuole (Sauer and Friml, 2014; Mosesso et al., 2019; Schwihla and Korbei, 2020).

The ESCRT-I complex, which in yeast/mammals forms a hetero-tetrameric complex containing one copy of each of the subunits (Schuh and Audhya, 2014), is responsible for binding ubiquitinated cargo and ESCRT-0 subunits (Bache et al., 2003). Arabidopsis contains two isoforms of each of the subunits VPS23 (ELC/VPS23A and VPS23B), VPS28 (VPS28-1 and VPS28-2), and VPS37 (VPS37-1 and VPS37-2), but no Mvb12-like protein (Winter and Hauser, 2006; Leung et al., 2008). Arabidopsis VPS23A, which participates in cell division and trichome development, is able to bind to ubiquitin and associate with VPS37 and VPS28 to form a putatively intact plant ESCRT-I complex (Spitzer et al., 2006).

The ESCRT-II, which has a pivotal role in linking the upstream ubiquitin-binding ESCRT complexes to the downstream ESCRT-III complex, is a Y-shaped heteromer of the subunits Vps22, Vps25, and Vps36 (Hurley, 2010). In A. thaliana, all ESCRT-II subunits are present as single-copy genes (Winter and Hauser, 2006; Leung et al., 2008; Richardson et al., 2011). The rice OsVPS22 gene encodes a functional VPS22 homolog, where vps22 mutants exhibit seedling lethality. Nevertheless, potential defects in endosomal sorting remain to be determined (Zhang et al., 2013). VPS36, which may form a putative ESCRT-II complex together with VPS22 and VPS25, is critical for embryo and seedling development and regulates vacuole biogenesis as well as endosomal sorting to the vacuole of several PM proteins (Wang et al., 2017).

ESCRT-III does not contain any known UBDs, and thus is unlikely to recognize ubiquitinated cargoes. Instead, ESCRT-III is essential for membrane scission during sorting of the endocytic cargo into ILVs (Henne et al., 2012). In contrast to the models proposed for animal systems, plant ILVs were shown to form networks of concatenated vesicles instead of individual vesicles in the lumen of the MVB. These networks remain connected by narrow bridges, thus trapping the cargo. The potential role of the ESCRT-III is to act as a diffusion barrier to prevent escape of the cargos destined for degradation (Buono et al., 2017). Consistent with this key function, overexpression of dominant negative alleles of ESCRT-III core subunits results in defects in MVB biogenesis and vacuolar degradation of PM proteins (Cai et al., 2014). Ultimately VPS4/SKD1, an ortholog of yeast/mammalian AAA ATPase, hydrolyzes ATP, leading to the disassembly of the ESCRT-III complex, allowing for recycling of the subunits. This is an essential step for the completion of the membrane scission and the formation of ILVs and, furthermore, for the MVBs to fuse with the vacuole/lysosomes (Hurley, 2010; Schuh and Audhya, 2014). All core subunits of the conserved ESCRT-III machinery have two homologs in Arabidopsis, with the exception of VPS2, which has three (Winter and Hauser, 2006; Cai et al., 2014). Nevertheless, only VPS2.1, which binds deubiquitinating enzymes, appears to function as a typical ESCRT-III subunit (Katsiarimpa et al., 2011). There is only one Arabidopsis VPS4/SKD1 homolog named SKD1 (Haas et al., 2007).

A further protein interacting with the ESCRT-III is ALIX, which also interacts with ESCRT-I subunits, therefore having the potential to link ESCRT-I and ESCRT-III (Bissig and Gruenberg, 2014). ALIX binds ubiquitin, specifically K63-linked ubiquitin chains (Dowlatshahi et al., 2012), suggesting it may function as a ubiquitin receptor for protein sorting into MVBs (Pashkova et al., 2013). Similar functions have been attributed to Arabidopsis ALIX (Figure 1), which furthermore is indispensable for plant growth and development as well as vacuole and MVB biogenesis (Cardona-Lopez et al., 2015; Kalinowska et al., 2015).

Plant genomes encode unique ESCRT components with limited sequence similarities to mammalian/yeast subunits, which interact with known ESCRT subunits to regulate endocytic sorting processes. The SH3 DOMAIN-CONTAINING PROTEIN 2 (SH3P2; Figure 1) has been implicated in vacuolar trafficking of ubiquitinated cargoes as it localizes to CCVs and binds K63-linked ubiquitin chains, VPS23A, and deubiquitinating enzymes (Kolb et al., 2015; Nagel et al., 2017). FYVE 1 (Fab 1, YOTB, Vac 1, and EEA1)/FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1) localizes to MVBs and binds ubiquitin, interacts with SH3P2, both VPS23 subunits, and is incorporated into the ESCRT-III complex (Barberon et al., 2014; Gao et al., 2014; Kolb et al., 2015; Belda-Palazon et al., 2016; Figure 1). It is essential for ILV formation, vacuolar biogenesis, autophagic degradation, seedling development, and localization and degradation of PM proteins and cytosolic ABA receptors (Barberon et al., 2014; Gao et al., 2014; Kolb et al., 2015; Belda-Palazon et al., 2016).

The high number of plant-specific factors, next to the conserved canonical trafficking machinery responsible for transporting PM proteins destined for degradation, reflects the need of plants to precisely adjust the abundance of their PM proteins in order to be able to respond quickly and accurately to their surroundings. Furthermore, some ESCRT subunits, as, for example, the TOL proteins, have undergone drastic gene expansions (Gao et al., 2017; Cui et al., 2018; Mosesso et al., 2019; Vietri et al., 2020). Thus, to fully understand plant adaptation mechanisms, deciphering the molecular mechanisms underlying the regulation of ESCRT-dependent degradation of ubiquitinated PM proteins is crucial.

While it is evident that the ESCRT machinery participates in virtually every aspect of plant growth and development, its contribution to mediating adaptive responses to environmental stimuli appears remarkable. Specifically, a variety of loci associated with ESCRT-mediated sorting have been linked to often times highly defined aspects of higher plants’ responses to biotic as well as abiotic stress conditions. Prominent examples are the ESCRT-I subunits, where a double mutant line (vps28-2 vps37-1) displays reduced endosomal sorting of a pathogen-related receptor. This plant line thus shows impaired immune responses against bacterial pathogens, although its development is otherwise normal, suggesting functional redundancy with the other isoforms (Spallek et al., 2013). Apart from that, a Zea mays ortholog of the AAA ATPase SKD1 is not essential for plant survival, but was demonstrated to be upregulated by salt or drought stress (Xia et al., 2013), and appears to be critical for adequate responses to both biotic and abiotic stresses (Wang et al., 2014, 2015). Recently, several components of the ESCRT machinery have been shown to play a critical role in the ABA signaling pathway, which is also reflected in a higher drought tolerance of their respective loss-of-function mutants (Belda-Palazon et al., 2016; Yu et al., 2016; Garcia-Leon et al., 2019). The ESCRT-I subunit VPS23A, for example, influences the stability and subcellular localization of ABA receptors by affecting their endosomal trafficking to the vacuole for degradation (Yu et al., 2016). Thus, the core function of the ESCRT machinery is essential in plant development, but diversification and redundancies within the subunits might contribute to and play an important role in adaptation processes.