Deciphering the mechanism of E3 ubiquitin ligases in plant responses to abiotic and biotic stresses and perspectives on PROTACs for crop resistance

Summary

With global climate change, it is essential to find strategies to make crops more resistant to different stresses and guarantee food security worldwide. E3 ubiquitin ligases are critical regulatory elements that are gaining importance due to their role in selecting proteins for degradation in the ubiquitin‐proteasome proteolysis pathway. The role of E3 Ub ligases has been demonstrated in numerous cellular processes in plants responding to biotic and abiotic stresses. E3 Ub ligases are considered a class of proteins that are difficult to control by conventional inhibitors, as they lack a standard active site with pocket, and their biological activity is mainly due to protein–protein interactions with transient conformational changes. Proteolysis‐targeted chimeras (PROTACs) are a new class of heterobifunctional molecules that have emerged in recent years as relevant alternatives for incurable human diseases like cancer because they can target recalcitrant proteins for destruction. PROTACs interact with the ubiquitin‐proteasome system, principally the E3 Ub ligase in the cell, and facilitate proteasome turnover of the proteins of interest. PROTAC strategies harness the essential functions of E3 Ub ligases for proteasomal degradation of proteins involved in dysfunction. This review examines critical advances in E3 Ub ligase research in plant responses to biotic and abiotic stresses. It highlights how PROTACs can be applied to target proteins involved in plant stress response to mitigate pathogenic agents and environmental adversities.

Introduction

The 21st century is facing unprecedented challenges in the agricultural sector. World population growth and recent climate change are of particular concern. With over 8 billion inhabitants, a critical phase has been reached, with populations increasingly faced with the problem of access to natural resources such as food crops (Vollset et al., 2020). Meeting the food needs of every population is a problem today, as some regions of the world are confronting intense famines, while in others, arable land has shrunk considerably with galloping urbanisation (Dasgupta and Robinson, 2022; Satterthwaite et al., 2010). Worse, climate change makes agriculture unpredictable, and farmers need reliable indicators to program their cultivation campaigns. On top of this, natural disasters are spreading, including fires, droughts and floods, due to climate change. Climate change is associated with multiple stress factors for plants, making global food security a major challenge for the 21st century (Duchenne‐Moutien and Neetoo, 2021). To cope with this demographic and climatic challenge, the agriculture sector needs significant development, and the protection of crops against biotic and abiotic stresses will have to be improved (Dasgupta and Robinson, 2022).

Plants are subject to numerous biotic and abiotic stresses that threaten crop production. Abiotic stress in plants can be caused by various conditions, including meteorological conditions (drought or extreme cold) or physicochemical conditions (high or low salt and water levels) that exceed normal physiological conditions (Francini and Sebastiani, 2019; Oshunsanya et al., 2019). Abiotic stress is the leading cause of reduced crop productivity. Every year, between 51% and 82% of crop yields in global agriculture are lost due to abiotic stress (Oshunsanya et al., 2019). Biotic stress is characterised by the plant's response to invasion by parasitic organisms, including infection by microbial pathogens. The consequences of climate change include new types of stressors that affect the vulnerability of many crops (Duchenne‐Moutien and Neetoo, 2021). Pathogens are also adapting to climate change and evolving their strategies. For example, due to rising temperatures, pathogens move from one region to another, exposing plants to new pathogens and challenging the basic concept of crop resistance (Singh et al., 2023).

Alternatives to plant stress are numerous but remain limited in the face of the current challenge. Rapid breeding technologies can help crops withstand biotic and abiotic stresses. Techniques such as next‐generation technologies, including genotyping, high‐throughput phenotyping, genome editing, marker‐assisted selection and genomic selection, will play a key role. However, how rapid breeding can adapt to a changing environment has yet to be resolved (Hickey et al., 2019). Alternatives to plant stress involve also physical, chemical and biocontrol strategies. Physical control strategies have recently included UV, heat and cold applications. However, these require significant energy resources, limiting them in an energy crisis (Picart‐Palmade et al., 2019). In chemical methods, pesticides and fertilisers control pathogens and enrich soils with essential chemical elements. Nevertheless, this is a growing concern due to increasing residues and the promotion of strain resistance (Ahammed et al., 2021; Kumar et al., 2022). Finally, biological methods are becoming increasingly popular, in particular, the implementation of microbial antagonists in agricultural fields to improve plant health (Ayaz et al., 2023; Collinge et al., 2022) and enhance pathogen control (Godana et al., 2023), but their effectiveness still needs to be improved. Innovative strategies that take advantage of fundamental research into the cellular mechanisms of stress regulation should improve food preservation and safety.

Recent advances in biotechnology have uncovered the fundamental mechanisms of cells, highlighting the role of E3 Ub ligases in plant stress (Stone et al., 2005). E3 Ub ligases represent a regulatory element that can bind to proteins, driving them to the cellular ubiquitin‐proteasome system for their elimination (Vierstra, 2009). In this way, they are essential to reshaping cells' proteome and regulating cell function. Advances in research in this field are mainly due to new biotechnological investigation approaches such as yeast two‐hybrid (Y2H), protein microarrays, global protein stability (GPS) profiling, high‐throughput quantitative microscopy (HCA), differential expression (shotgun) proteomics, cell‐free degradation, immunodetection analyses (Selote et al., 2018), proteomics single and double knockdown, protein localisation, knockout technology, RNA silencing and immunolabelling microscopic methodology (Iconomou and Saunders, 2016). Thanks to this advantage, the study of single or multiple stress induction and cell machinery has highlighted the importance of E3 Ub ligase in stress management. Plant stress and its relevance to biotic and abiotic stress is now a significant research topic (Gingerich et al., 2021). Numerous transcriptomic studies of plants subjected to abiotic and biotic stresses have revealed that E3 Ub ubiquitin ligases are differentially expressed, indicating their prominent role. Moreover, knowledge of their crucial role can improve plant resistance to stress. Research strategies are now focused on harnessing and managing E3 Ub ligases to improve plant resistance to stress.

E3 Ub ligases are critical regulators as they control phytohormone biosynthesis and signalling pathways. Their biological activity is shaped by a specific protein–protein interaction with a short phase of transitions often associated with conformation change. This mechanism, although exceptional, classifies E3 Ub ligases among the so‐called undruggable proteins, as such proteins are challenging to control with conventional inhibitors. E3 Ub ligase activity is pocket‐independent. For this reason, much interest is focused on strategies that can target E3 Ub ligases or proteins that are difficult to destroy and that may be involved in abiotic and biotic stress in plants.

Proteolysis‐targeted chimeras (PROTACs) are a new class of heterobifunctional molecules that have emerged in recent years as relevant alternatives for uncurable human diseases like cancer (Khan et al., 2024) because they can target recalcitrant proteins for destruction. PROTACs can exploit the function of the E3 Ub ligase to select proteins for degradation by the proteasome. PROTACs mainly enhance ubiquitination and proteasomal degradation by facilitating association with an E3 Ub ligase and a protein involved in stress (Li et al., 2022; Raina et al., 2016). The research on PROTACs over the last 10 years available on Web of Science sites shows that the number of publications on PROTACs is increasing rapidly every year, as shown in Figure 1. Developing PROTACs in plants may require consolidating our understanding of E3 Ub ligases, their role, and regulation in plant development and stress control. A major review of E3 Ub ligases' progress and the perspective of applying PROTACs to agriculture is of great interest.

Figure 1.

Evolution of the number of articles related to PROTACs from the year 2003 to the date 2023. Retrieved from PubMed on 2023 12 24. Research term enter ‘Proteolysis‐targeting chimeras (PROTACs)’ https://pubmed.ncbi.nlm.nih.gov/?term=Proteolysis‐targeting%20chimeras%20(PROTACs)%20&timeline=expanded.

The present review will describe recent advances in E3 Ub ligases and include recent study in the proteolytic process of the ubiquitin‐proteasome system, the molecular classification, structure and role of E3 Ub ligases, as well as the importance of E3s in plant response to abiotic and biotic stresses. Finally, this study will prospect the PROTAC methodology in plants to improve crop stress resistance and thus enhance food security worldwide.

Ubiquitin‐proteasome system in plant

Ubiquitin (Ub) is a highly conserved eukaryotic polypeptide of 76 amino acids (Adams and Spoel, 2018; Langin et al., 2023; Stone, 2019). Ubiquitination is the central post‐translational modification of proteins (Sun et al., 2020). It involves modulating a variety of biological functions, including protein homeostasis regulation (Sun et al., 2020), signal transduction (Liu et al., 2020), autophagy and DNA damage response (Huang and Zhang, 2020). During ubiquitination, Ub is attached to a protein target in various forms, resulting in the binding of either single Ub's or complex Ub polymers, causing mono‐, multi‐ and branched poly‐ubiquitylation (Singh et al., 2021). The position of the amino acid on which the Ub is anchored also determines the cellular role of the target (Huang and Zhang, 2020).

The ubiquitination process requires a ‘teamwork’ of three classes of enzymes, namely the E1 Ub activating enzyme, the E2 Ub binding enzyme and the E3 Ub ligase (Brillada and Trujillo, 2022). The main processes are as follows: first, with the participation of ATP, free inactive Ub is activated in the E1 Ub complex, and a high‐energy thioester bond is created between the glycine (Gly) carboxyl terminus of Ub and the E1 cysteine (Cys) residue of the active site; the activated Ub is then moved to the active site Cys residue of E2 Ub‐binding enzyme, forming the complex E2‐Ub thioester (E2‐ Ub); finally, E2 then is recruited and can transfer Ub to the corresponding E3 Ub ligase (Wang et al., 2022). E3 Ub ligase recognises the target protein and catalyses, directly or through a transitional state, the binding of Ub to the target protein (Skieterska et al., 2017), connecting the Ub molecule to the lysine residues of the target protein by forming an isopeptide bond. Figure 2 illustrates the ubiquitination process and proteasome degradation.

Figure 2.

Schematic representation of the ubiquitination process and proteasome degradation. Three classes of enzymes exist – E1 Ub activating enzyme, E2 Ub binding enzyme and E3 Ub ligase. They work as a team to achieve the ubiquitination of a targeted protein. First, with ATP, free inactive Ub is activated in E1 Ub, creating a high‐energy thioester bond between the glycine (Gly) carboxyl terminus of Ub and the active site's E1 cysteine (Cys) residue. The activated Ub is then moved to the active site of the Cys residue of the E2 Ub‐binding enzyme, forming the complex E2‐Ub thioester (E2‐Ub). Finally, E2 is recruited to the corresponding E3 Ub ligase. E3 Ub ligase finds the target protein and either directly or indirectly binds Ub to it by creating an isopeptide bond at the lysine residues of the target protein.

Classification and structure of E3 Ub ligase in plant

E3 Ub ligases classification based on their frame can evolve with advanced research. Hence, E3 Ub ligases are classified according to their functional domain into three structural families: HECT, RING and U‐box (Ryu et al., 2019). Depending on the number of subunits, E3 Ub ligases can be classified into four main types: HECTs, RINGs and U‐boxes, which are single E3 Ub ligase polypeptides, and CRLs, which are made up of several subunits (Chen and Hellmann, 2013; Vierstra, 2009).

HECT family

The HECT family (homologous to the E6‐associated protein C‐terminus) is a highly conserved family of enzymes (Shah and Kumar, 2021). Genomes of various plant and animal species encode different numbers of HECT E3 Ub ligases gene family members, including 7 in Arabidopsis (Downes et al., 2003; Ying, 2018), 10 in Brassica rapa (Alam et al., 2019), 19 in Glycine max (Meng et al., 2015), 28 in Homo sapiens (Sluimer and Distel, 2018), 14 in Solanum lycopersicum (Sharma et al., 2021), 12 in Zea mays (Li et al., 2019) and 19 in Malus domestica (Xu et al., 2016). The C‐terminus of HECT has a HECT structural domain consisting of a N‐lobe and a C‐lobe of approximately 40 kDa. The N‐lobe consists of the E2‐binding domain, and the C‐lobe represents the active site of cysteine (Goto et al., 2021). This structural domain is an α/β structure and has a catalytic role, directly catalysing the binding of Ub to the substrate protein and mediating the covalent transfer of Ub from E2 to the target protein (Sharma et al., 2021b). The HECT‐type E3 Ub ligase first loads the activated Ub of the E2‐Ub intermediate onto its active cysteine residue and then transfers the Ub to the lysine residue of the substrate (Delvecchio et al., 2021). The HECT family is divided into three subfamilies: the NEDD4 (9 members), the HERC (6 members) and other HECT E3 Ub ligases (13 members) (Singh et al., 2021b). The NEDD4 subfamily includes a C2 structural domain and two to four tryptophan–tryptophan (WW) protein interaction domains before the HECT structural domain (Singh et al., 2021b). This WW NEDD4 domain is critical for subtract selection as it can bind to the PPxY motif of the target protein (Weber et al., 2019). The HERC subfamily has two characteristic structural domains: the HECT structural domain and the chromosome condensation regulator (RCC1)‐like structural domain (RLD) (Qian et al., 2020). The third subfamily includes enzymes that contain neither WW nor RLD structural domains with various structural domains at the N‐terminus (Delvecchio et al., 2021).

RING family

The RING family (really interesting new gene) is the most prominent family of E3 Ub ligases (Vander Kooi et al., 2006). The RING structural domain contains a C6HC zinc finger structure, and two RING zinc finger‐like structures exist on both sides of C6HC (Bueso et al., 2014). Depending on whether position 5 of the metal ligand contains a Cys or His residue, RING finger domains have been categorised into two types: RING‐HC (C3HC4) and RING‐H2 (C3H2C3) (FREEMONT, 1993). This structural domain mainly assists the transfer of Ub through the covalent bond formed by the chelation of eight amino acids with zinc ions (Weber et al., 2019). The RING structural domain determines the specific activity of most E3 Ub ligases. Specifically, it was found that while OsSAR1 has E3 Ub ligase activity, MBP‐OsSADR1C168A, whose Cys168 is replaced by Ala168 in the RING structural domain, has no E3 Ub ligase activity (Park et al., 2018). E3 RING ligases may function as allosteric activators of E2, which catalyses the sliding of Ub from its active site to the lysine residues of the target protein. E3 Ub ligases act primarily as scaffolding to bring E2 into proximity to the protein substrate (Weber et al., 2019). Genomes of various plant species encode different numbers of RING protein members, including 477 in Arabidopsis (Stone et al., 2005), 715 in Brassica rapa (Alam et al., 2017), 425 in rice (Oryza sativa) (Lim et al., 2010) and 663 in Malus domestica (Li et al., 2011). RING‐type E3s can form complex multi‐subunit E3s (Al‐Saharin et al., 2022), dividing them into two subtypes: single‐subunit and multi‐subunit families (Wang et al., 2021). Single‐subunit RING E3 Ub ligases specifically possess three RING structural domains in tandem, and RSL1 belongs to the single‐subunit RING E3 Ub ligases with a transmembrane structural domain at the C‐terminus (Bueso et al., 2014). The multiple units, Cullin‐RING Ub ligase (CRL) family consists of a Cullin protein acting as the central scaffold and the N‐terminus binding to the adapter protein‐substrate receptor complex, and the C‐terminus binds to RING proteins (Bueso et al., 2014; Wang et al., 2021).

U‐box family

U‐box protein contains a structural domain of about 70 amino acids and is a single protein widespread in yeast, plants and animals (Wang et al., 2020). Genomes of various plant species encode different numbers of U‐box gene, including 66 in Arabidopsis (Wiborg et al., 2008), 101 in Brassica rapa (Wang et al., 2015), 30 in Chlamydomonas reinhardtii (Luo et al., 2015) (Oryza sativa), 125 in Glycine max (Wang et al., 2016), 21 Homo sapiens (Sharma and Taganna, 2020), 77 in Oryza sativa (Mueller et al., 2005), 2 in Saccharomyces cerevisiae (Koegl et al., 1999), 56 in Vitis vinifera (Jiao et al., 2017) and 62 in Solanum lycopersicum (Sharma and Taganna, 2020). Compared to the RING, the U‐box (PUB) family is less diverse (Smalley and Hellmann, 2022). However, U‐box and RING proteins present both single polypeptides, and both interact non‐covalently with E2‐Ub through the conserved U‐box structural domain and RING structural domain respectively (Mao et al., 2022; Trenner et al., 2022). The primary role of U‐box is to bind to E2‐Ub, mainly involving salt bridges, ion chelation, and hydrogen bonding, and facilitate Ub transfer (Trenner et al., 2022) to the target proteins. The plant U‐box protein family was first identified in Arabidopsis thaliana with 64 members. AtPUB14 protein was the first PUB to be structurally identified and contains a U‐box structural domain and an ARM repeat structure that allows the formation of a U‐box‐mediated featureless structural dimer (Andersen et al., 2004). Both the U‐box structural domain and the RING structural domain have a similar disposition of a three‐stranded chain and a helix as well as a particular long loop, but the U‐box does not have the histidine and cysteine residues as well as two zinc ions (Aravind and Koonin, 2000; Vander Kooi et al., 2006). The U‐box involves electrostatic interactions rather than metal chelation for stabilising the ubiquitin‐E2‐binding pocket (Mudgil et al., 2004; Wiborg et al., 2008). Further classification of plant U‐box protein is in progress (Hu et al., 2018; Li et al., 2019; Wang et al., 2020). Figure 3 illustrates the E3 Ub ligase structure and mechanism.

Figure 3.

E3 ubiquitin ligase structure and mechanism. Each E3‐ligase in plants is composed of multiple subunits. The numbers indicated within each subunit represent the genes in the Arabidopsis thaliana genome that potentially encode the E3 Ub ligase or E3 Ub ligase component. RING (really interesting new gene) and U‐box E3 Ub ligases engage with an E2 and the target, thereby enabling the direct transfer of Ub to the target protein. HECT E3 Ub ligases, which are defined as homologous to the E6AP carboxyl terminus, as well as RBR E3 Ub ligases, known as RING‐in‐Between‐RING E3 Ub ligases, facilitate the transfer of Ub from the E2 to a cysteine residue in the E3. Subsequently, the Ub is transferred from the cysteine to the target protein. The CRLs, called Cullin‐RING Ligases, are complex structures of multiple subunits. These structures consist of a backbone protein called Cullin (CUL), which acts as a binding site for the E2 protein and a target adapter protein or complex. In SCF complexes, which stand for Skp1/CUL1/F‐box complexes, the target adapter proteins known as F‐box proteins bind to a protein called Skp1, called S‐phase Kinase‐associated Protein 1. This Skp1‐like protein then interacts with CUL1. In the CRL3 complexes, a protein containing the BTB domain (broad‐complex, tramtrack and bric‐à‐brac) binds to both the target and CUL3. Within the CRL4 complexes, DWD proteins that contain the WD40 domain (DDB1 binding WD40) bind to the targets and interact with CUL4 through a protein bridge called DDB1 (damaged DNA binding 1). The APC/C (anaphase‐promoting complex/cyclosome) comprises a minimum of 11 subunits. APC2 is a protein similar to Cullin, and APC11 shares similarities with RBX1 (ring box protein 1). Three parts – CDC20 (cell division cycle 20), CDH1 (CDC20 homologue 1) and APC10 – work together to recognise the target (Gingerich et al., 2021).

CRL family

CRLs are indeed the best‐recorded E3 Ub ligases, involved in plants' physiological, developmental and stress responses (Chen and Hellmann, 2013). CRLs are complexes in which the cullin protein acts as an elongated scaffold, linking RING‐FINGER PROTEIN RING BOX PROTEIN 1 (RBX1) and E2 to its carboxyl (C)‐terminal region while linking a substrate adaptor to its amine (N)‐terminal region. In plants, there are four CRL subtypes harbouring distinct cullins: CUL1, CUL3, CUL4 and the cullin‐like protein ANAPHASE‐PROMOTING COMPLEX 2 (APC2) (Ban and Estelle, 2021; Vierstra, 2009). E3 CUL1 ligases, also known as SCF complexes (S‐PHASE KINASE‐ASSOCIATED PROTEIN 1 (SKP1)‐CUL1‐F‐box), are one of the most extensively studied CRL complexes, with the substrate adaptor consisting of SKP1 and an F‐box protein. Genomes of various plant species encode different numbers of F‐box protein members, including 600–700 in Arabidopsis (Gagne et al., 2002; Kuroda et al., 2002), 687/779 in rice (Oryza sativa) (Jain et al., 2007; Xu et al., 2009), 359 in Zea mays (Jia et al., 2013), 517 in Malus domestica (Cui et al., 2015), 337 in poplar (Xu et al., 2009), 226 in Pyrus spp. (Wang et al., 2016), 285 in chickpeas (Gupta et al., 2015) and 972 in Medicago (Song et al., 2015). SCFs are also essential regulators of plant hormone responses (auxin, jasmonic acid (JA) and ethylene) and many other processes (Ban and Estelle, 2021). In CRL3 complexes, the BTB (broad‐complex, tramtrack and bric‐à‐brac) domain binds both the target and CUL3. Genomes of various plants encode different numbers of MATH–BTB gene family members, including 6 in Arabidopsis (Juranić and Dresselhaus, 2014), 10 in Brassica rapa (Zhao et al., 2013), 68 in Oryza sativa (Gingerich et al., 2007), 5 in Solanum lycopersicum (Li et al., 2018), 31 in Zea mays (Juranić et al., 2013) and 19 in Malus domestica (Xu et al., 2016). In CRL4 complexes, DWD proteins incorporate the WD40 (DDB1 binding WD40) domain, which binds targets and interacts with CUL4 via a protein bridge called DDB1 (damaged DNA binding 1). Finally, the APC/C (anaphase‐promoting complex/cyclosome) complex comprises a minimum of 11 subunits. APC2 is a protein similar to Cullin, and APC11 has similarities with RBX1 (ring box protein 1). Classically, three parts – CD20 (cell division cycle 20), CDH1 (CDC20 Homologue 1) and APC10 – work together to recognise the target (Gingerich et al., 2021; Vierstra, 2009).

Role of plant E3 Ub ligase in different stresses

Role of E3 Ub ligase in abiotic stress

Drought stress

Plant species under drought stress express E3 Ub ligases that modify the ABA signalling pathway, disrupt gene expression in specific pathways for bioactive compound synthesis, and contribute to the ubiquitination and degradation of transcription factors responsible for the drought stress response (Cheng et al., 2012; Joo et al., 2017; Lim et al., 2017b; Park et al., 2010; Sun et al., 2022; Wang et al., 2018; Yu et al., 2021). Silencing these genes leads to drought and salt stress resistance (Park et al., 2010).

Ring ligases can positively regulate tolerance to drought stress by interfering with cellular mechanisms to reduce water loss and ion leakage. For example, OsCTR1 can inhibit the trafficking of OsCP12 and OsRP147 to chloroplasts (Lim et al., 2014). Overexpression of some E3 Ub ligases can make crops particularly resistant, such as the pepper E3 Ub ligase CaAIRF1, which regulates ABA and enhances the drought response via degradation of the protein phosphatase CaADIP1 (Brugière et al., 2017; Lim et al., 2017a). E3 Ub ligases regulate pathways in a multi‐level fashion, controlling the regulator‐specific cellular network and potentially producing bioactive compound synthesis. Some genes up‐regulated by drought resistance may also regulate elevated levels during salt stress (Chen et al., 2020; Cho et al., 2022; Joo et al., 2019, 2020; Yang et al., 2016).

PUB proteins play negative and positive roles in abiotic stress resistance (Ryu et al., 2019). Negative regulators, such as PUB11 signalling kinase, alter signal transduction during drought stress (Chen et al., 2021). They also modify the cell's hormone signalling system, particularly in ABA signalling pathways. Positive regulators, like OsPUB67, enhance cell protection against stress by improving reactive oxygen scavenging (Qin et al., 2020).

PUBs can interact with multiple regulators to positively regulate tolerance to drought stress. Water resources are crucial in drought management, and PUBs manage them by facilitating water channels and stomatal closure. Through the molecular mechanism of proteasomal degradation of aquaporins (AQPs), OsRINGzf1, an E3 Ub ligase, acts on the turnover of OsPIP2;1 (Chen et al., 2022). Still, in the strategic action of minimising water waste, PUBs also act on stomatal closure (Xiao et al., 2022). Figure 4 shows the role of E3 Ub in drought stress.

Figure 4.

Regulatory mechanism of E3 Ub in response to drought stress in the plant. E3 Ub ligases during drought stress are involved in (1) activating the response to drought stress and (2) attenuating the response to drought stress. In (1) response to drought stress, the ABA (abscisic acid) pathway is activated by CaAIRE1 (Capsicum annuum ABA induced RING‐type E3 Ub ligase 1), which mediates the elimination of CaAITP1 (protein phosphatase), a stimulator of the ABA pathway. CaASRF1 (E3 Ub ligase containing a C3H2C3‐type RING finger domain) mediates the accumulation of CaATBZ1 (pepper bZIP transcription factor), which can promote ABA pathways. After drought stress is abolished (2), the E3 Ub ligase domain mediates attenuation of the response to drought stress. CaAIRF1 (Capsicum annuum RING‐type E3 Ub ligase) and BRUTUS (iron‐binding E3 Ub ligase containing three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain) mediate destabilisation of CaADIP1 (Capsicum annuum type 2C protein phosphatase) and VOZ1/2 (vascular plant one‐zinc finger ½), respectively, which are two positive regulators of the ABA pathway. The E3 Ub ligase CaATIR1 (Capsicum annuum E3 Ub ligase ATBZ1 interacting RING finger protein 1) mediates destabilisation of CaATBZ1 (pepper bZIP transcription factor), RGLG2 (RING domain E3 Ub ligase 2) and AtAIRP1 (cytosolic protein containing a single C3H2C3‐type RING motif with in vitro E3 Ub ligase activity) mediate destabilisation of MAPKKK18 (mitogen‐activated protein kinase kinase kinase 18), OsPUB67 (U‐box E3 Ub ligase) mediates destabilisation of OsRZFP34 (RING zinc‐finger protein (RZFP))/OsDIS1, AtAIRP1 (cytosolic protein containing a single C3H2C3‐type RING motif with in vitro E3 Ub ligase activity) mediates destabilisation of AtKPNB1 (Arabidopsis importin‐β protein), RGLG2 mediates destabilisation of AtERF53 (ETHYLENE RESPONSE FACTOR 53) and TaDIS1 (Triticum aestivum L RING E3 Ub ligase) mediates destabilisation of TaSTP (Triticum aestivum L sugar transporter protein (STP)). CaATBZ, MAPKKK18, OsRZFP34/OsDIS1, AtKPNB1, AtERF53 and TaSTP are all critical for the continuation of the drought stress response.

Salt stress

Salt stress is one of the most damaging stresses for plants. It can cause osmotic, nutritional and metabolic dysfunctions. Salt stress affects plant primary growth and reduces productivity (Ahmad et al., 2023; Fu and Yang, 2023). Ring E3 Ub ligases play a crucial role in plant salt stress tolerance. Some ligases, such as AtPPRT1, are involved in hormone deregulation and activate organelles in ion trafficking (Pei et al., 2019). Others, like IbATL38, positively regulate salt stress tolerance (Du et al., 2021). Specific E3 Ub ligases, like the SpRing gene and SARS1, are inducible and improve tolerance (Qi et al., 2016). Inducible ligases, like CaAIP1 in pepper and OsHKT1;1 in rice, interact with ion transporters and can induce resistance (Xiao et al., 2022). Inhibition of specific E3 Ub ligases can result in opposition, while others, like SKP1‐Cullin/CDC53‐F‐box, are involved in salt and drought stress responses in Arabidopsis (Li et al., 2022). Understanding the complete mechanisms of these E3 Ub ligases is essential for understanding plant growth and stress responses.

PUB ligases play a crucial role in regulating salt stress tolerance in plants. They can affect salt stress by interfering with cell permeability, such as the E3 GmPUB21 ligase, which can enhance stomatal aperture density (Yang et al., 2022). A single PUB gene can regulate various abiotic stresses, such as osmotic and salt stress sensitivity and drought stress tolerance. PUBs also positively regulate phosphate stress tolerance, such as PRU2 enhancing phosphate uptake. Further studies are needed to understand their roles in stress responses by interacting with a CK2α1 kinase and a ribosomal protein RPL10 for degradation (Sun et al., 2022). Figure 5 shows the role of E3 Ub in salt stress.

Figure 5.

Regulatory mechanism of E3 Ub in response to plant salt stress. E3 Ub ligases during salt stress are involved in (1) activation of the salt stress response and (2) attenuation of the salt stress response. In (1), salt stress tolerance is activated by OsSIRP2 (rice gene encoding the RING Ub E3 Ub ligase), which mediates the elimination of OsTKL1 (transketolase), a negative regulator of salt stress tolerance. After removing salt stress (2), the E3 Ub ligase domain mediates the attenuation of the salt stress response. SINAT (RING‐domain containing E3 Ub ligase) mediates the destabilisation of ACS (1‐aminocyclopropane‐1‐carboxylic (ACC) synthase), which positively regulates the ethylene pathway. SRAS1.1 (salt‐responsive alternatively spliced gene one encoding a RING‐Type E3 Ub ligase, generates two splicing variants: SRAS1.1 and SRAS1.2) mediates the destabilisation of CSN5A (COP9 signalosome 5A), a positive regulator of stress response. OsMSRFP (MYBc stress‐related RING finger protein) mediates the elimination of OsMYBc (MYB transcription factor), the latter involved in the activation of HKT (OsHKkT1;1) (member of the high‐affinity K+ transporter (HKT) family in rice (Oryza sativa)). OsSIRP4 (E3 Ub ligase, the Oryza sativa salt‐induced RING finger) mediates the destabilisation of OsPEX11‐1 (Oryza sativa peroxisomal biogenesis factor 11–1), the latter promoting the stress response. XBAT35.2 (RING‐type E3 Ub ligase) mediates the destabilisation of ACD11 (Arabidopsis accelerated cell death 11). PRU2 (a ubiquitin E3 Ub ligase) mediates the destabilisation of RPL10 (ribosomal protein) and KINASE CK2α1 (serine–threonine protein kinase). KINASE CK2α1 is a positive regulator of the MAKP pathway, which is essential for stress tolerance. PQT3 (E3 Ub ligase PARAQUAT TOLERANCE 3) mediates the elimination of PRMT4b (protein arginine methyltransferases), which later activate APX1 (ascorbate peroxidase 1) and GPX1 (glutathione peroxidase‐1) both play essential role intolerance to salt stress. ACD11 is a positive regulator of tolerance to abiotic and biotic stresses.

Cold stress

E3s are crucial in disrupting cold stress tolerance in plants, with inhibitory actions centralised in the nucleus. In Arabidopsis, HOS1, an E3 Ub‐protein ligase, negatively modulates cold stress tolerance by reducing the expression of genes essential to the attenuation of cold (Shkryl et al., 2021). Ring type E3 Ub ligases, such as BBX37 (B‐box protein), also interact with critical hormones, reducing tolerance (An et al., 2021). PUB ligases, such as VpPUB24, positively regulate cold stress tolerance, with overexpression leading to cold stress resistance (Zhang et al., 2017). In A. thaliana, PUB25 and PUB26 operate by poly‐ubiquitination of MYB15, promoting CBF expression under cold conditions (Wang et al., 2019). PUB expression changes according to stress type, with up‐regulation under low‐temperature and up‐regulation under drought stress (Zhou et al., 2021). HECT‐type E3 Ub ligases are thought to affect abiotic stress by modulating the ABA pathway in plants (Sharma and Taganna, 2020). Table 1 shows the E3 Ub ligase genes in response to abiotic stresses in plants. Figure 6 shows the role of E3 Ub in abiotic stresses.

Table 1.

E3 ubiquitin ligase genes involved in plant response to abiotic stresses

Species	Type of E3 Ub ligase	Year	Gene	Cellular localisation	Type of stress	Cellular target	Role in stress	References
Arabidopsis thaliana	RING	2020	AtAIRP1	Cytosol	Drought	AtKPNB1	Inhibit	Oh et al. (2020)
Arabidopsis thaliana	RING	2011	AtAIRP2	Cytosol	ABA‐dependent drought stress	Depending on SnRK protein kinase activities	Inhibit	Cho et al. (2011)
Arabidopsis thaliana	RING	2022	AtAIRP5/GARU	–	Drought	AtSCPL1	Inhibit	Cho et al. (2022)
Arabidopsis thaliana	RING	2013	AtATL78	–	Cold	–	Promote	Kim and Kim (2013)
Arabidopsis thaliana	RING	2016	AtATL78	Plasma membrane	Drought	Mediating ABA‐dependent ROS signalling	Promote	Suh et al. (2016)
Arabidopsis thaliana	RING	2015	AtATL80	Plasma membrane	Phosphate/Cold	–	Promote	Suh and Kim (2015)
Arabidopsis thaliana	RING	2013	AtRDUF1	Cytosol and nuclei	Salt	–	Inhibit	Li et al. (2013)
Arabidopsis thaliana	RING	2021	AtRZF1	Nucleus	Drought	Interacts with AtUAP1	Inhibit	Min et al. (2021)
Arabidopsis thaliana	RING	2011	AtSAP5	Primarily in nuclei of root cells	Abiotic stress	–	Inhibit	Kang et al. (2011)
Arabidopsis thaliana	RING	2018	BRUTUS	Nucleus	Drought/Cold	VOZ1/2 transcription factors	Inhibit	Selote et al. (2018)
Arabidopsis thaliana	RING	2016	COP1	Nucleus	Salt/cold/UV‐B exposure	AtSIZ1	Inhibit	Kim et al. (2016)
Arabidopsis thaliana	RING	2006	HOS1	–	Cold	ICE1	Promote	Dong et al. (2006)
Arabidopsis thaliana	RING	2020	JUN1	Nucleus	Drought	–	Inhibit	Yu et al. (2020)
Arabidopsis thaliana	RING	2022	PRU2	–	Pi stress	Kinase CK2α1 and a ribosomal protein RPL10	Inhibit	Sun et al. (2022)
Arabidopsis thaliana	RING	2021	RGLG1 and RGLG2	–	Drought	Modulate MAPKKK18	Promote	Yu et al. (2021)
Arabidopsis thaliana	RING	2012	RGLG2	Nucleus	Drought	AtERF53	Promote	Cheng et al. (2012)
Arabidopsis thaliana	RING	2015	RZFP34/CHYR1	Cytoplasm and nucleus	Drought	SNF1‐RELATED PROTEIN KINASE2 (SnRK2) kinases	Inhibit	Ding et al. (2015)
Arabidopsis thaliana	RING	2008	SDIR1	–	Drought	–	Inhibit	Zhang et al. (2008)
Arabidopsis thaliana	RING	2015	SDIR1	ER membrane	Salt	SDIR1‐INTERACTING PROTEIN1	Promote	Zhang et al. (2015)
Arabidopsis thaliana	RING	2021	SRAS1.1	Cytoplasm and nucleus	Salt	COP9 signalosome subunit 5A	Inhibit	Zhou et al. (2021)
Arabidopsis thaliana	RING	2015	STRF1	Plasma membrane and at the intracellular endosomes	Salt	Involved in membrane trafficking	Promote	Tian et al. (2015)
Arabidopsis thaliana	RING	2020	XBAT35.2	–	Salt	ACD11	Promote	Li et al. (2020)
Arabidopsis thaliana	F‐box	2022	AtSDR	Nucleus	Salt/drought	–	Inhibit	Li et al. (2022)
Arabidopsis thaliana	U‐box	2003	AtCHIP	–	Temperature	–	Promote	Yan et al. (2003)
Arabidopsis thaliana	U‐box	2021	AtPUB11	–	Drought	Degrading the receptor‐like protein kinases LRR1 and KIN7	Promote	Chen et al. (2021)
Arabidopsis thaliana	U‐box	2012, 2016, 2017	AtPUB18/19	Punctate bodies/membrane‐associated protein	ABA‐mediated drought stress	Exocyst subunit Exo70B1	Promote	Liu et al. (2017), Seo et al. (2012), Seo et al. (2016)
Arabidopsis thaliana	U‐box	2023	AtPUB2	Nucleus	Oxidative stress	–	Inhibit	Saini et al. (2023)
Arabidopsis thaliana	U‐box	2008, 2015, 2017	AtPUB22/23	Cytosol	Drought	RPN12a; RPN6; targeting ABA receptor PYL9 for degradation	Promote	Ahn et al. (2022), Cho et al. (2008), Zhao et al. (2017)
Arabidopsis thaliana	U‐box	2019, 2023	AtPUB25/26	–	Cold	Targeting MYB15 and ICE1	Inhibit	Wang et al. (2019, 2023)
Arabidopsis thaliana	U‐box	2017	AtPUB30	Cytoplasm, nucleus, and plasma membrane	Salt	Mediating BKI1 protein degradation	Promote	Zhang et al. (2017)
Arabidopsis thaliana	U‐box	2017	AtPUB46	–	Drought and oxidative stress	–	Inhibit	Adler et al. (2018, 2017)
Arabidopsis thaliana	U‐box	2017	AtPUB48	Nucleus	Drought/heat	–	Inhibit	Adler et al. (2017), Peng et al. (2019)
Arabidopsis thaliana	U‐box	2016	PARAQUAT TOLERANCE3(PQT3)	Nucleus	Oxidative stress	PRMT4b	Promote	Luo et al. (2016)
Arabidopsis thaliana	C3HC4 Zinc‐Finger	2020	AtPPRT1	–	Salt		Promote	Liu et al. (2020)
Arabidopsis thaliana	Putative C3HC4 Zinc‐finger	2019	AtPPRT1	Mitochondria	Drought	Negative Regulator in ABA	Promote	Pei et al. (2019)
Apple (Malus domestica)	RING	2024	MdATL16	Cell membrane and nucleus	Salt	Promoting ROS scavenging	Inhibit	Yuan et al. (2024)
Apple (Malus domestica)	RING	2017	MdMIEL1	–	Salt/oxidative	–	Inhibit	An et al. (2017)
Apple (Malus domestica)	RING	2022	MdMIEL1	–	Drought	MdBBX7/MdCOL9	Inhibit	Chen et al. (2022)
Apple (Malus domestica)	RING	2022	MdMIEL1	–	Cold	BBX37	Inhibit	An et al. (2021)
Apple (Malus domestica)	U‐box	2022	MdPUB23	Nucleus	Cold	MdICE1	Promote	Wang et al. (2022)
Apple (Malus domestica)	U‐box	2022	MdPUB29		Salt	–	Inhibit	Han et al. (2019)
Brassica napus	Cullin‐RING	2023	CRL3 BPM	–	Salt	–	Promote	Corbridge et al. (2023)
Brassica napus	RING	2014	BnaJUL1	–	Drought	BnaTBCC1	Inhibit	Hu et al. (2024)
Brassica napus	RING	2014	BnTR1	Plasma membrane	Heat	Ca2+ dynamics by regulating the activity of calcium channels	Inhibit	Liu et al. (2014)
Chinese grapevine (Vitis pseudoreticulata)	U‐box	2017	VpPUB24	All grapevine protoplasts	Cold	VpICE1	Inhibit	Yao et al. (2017)
Coptis chinensis	RING	2020	CcSDIR1	–	Drought	Increasing antioxidant enzyme activities and reducing oxidative damage	Inhibit	Chen et al. (2020)
Cowpea (Vigna unguiculata L. Walp)	RING	2014	VuDRIP	–	Abiotic stress	TF VuDREB2A	Regulate	Sadhukhan et al. (2014)
Cucumber (Cucumis sativus)	RING	2023	CsCHYR1	Nucleus and membrane	Drought	CsATAF1	Inhibit	Guo et al. (2023)
Cucumber (Cucumis sativus)	F‐box	2021	CsTLP8	Plasma membrane and nucleus	Osmotic stresses	ABA signalling pathway	Promote	Li et al. (2021)
Grapevine (Vitis vinifera)	RING	2014	VvHOS1	–	Cold/drought/high salt	–	Promote	Li et al. (2014)
Grapevine (Vitis vinifera)	U‐box	2023	VviPUB19	–	Drought/salt	VviExo70B	Promote	Wang et al. (2023)
Grimmia pilifera	Cullin‐RING	2016	GpDSR7	ER membrane	Drought	–	Inhibit	Wang et al. (2023)
Maize (Zea mays)	RING	2018	ZmAIRP4	Cytoplasm and nucleus	Drought	Enhances the expression level of drought responsive genes	Promote	Yang et al. (2018)
Maize (Zea mays)	RING	2012	ZmRFP1	Plasma membrane	Drought	ABA‐dependent manner	Inhibit	Xia et al. (2012)
Maize (Zea mays)	RING	2013	ZmRFP1	–	Drought	Regulating stomatal aperture and antioxidants	Inhibit	Liu et al. (2013)
Maize (Zea mays)	RING	2017	ZmXerico1	Endoplasmic reticulum	Drought	Regulation of ABA homeostasis	Inhibit	Brugière et al. (2017)
Nicotiana tabacum	RING	2013	NtRHF1	–	Drought	Regulating several stress‐responsive genes	Inhibit	Xia et al. (2013)
Pepper (Capsicum annuum)	RING	2012	CaRma1H1	Endoplasmic reticulum	Drought/salt	–	Inhibit	Seo et al. (2012)
Pepper (Capsicum annuum)	RING	2016	CaAIP1	Nucleus and cytoplasm	Drought/osmotic	Drought stress: via changes in the stomatal aperture and leaf temperature; osmotic stresses: via ABA‐mediated signalling	Inhibit	Park et al. (2016)
Pepper (Capsicum annuum)	RING	2015	CaAIR1	Nucleus	Drought	Controlling the transpirational rate via stomatal opening/closure	Promote	Park et al. (2016)
Pepper (Capsicum annuum)	RING	2021	CaAIRE1	Nucleus	Drought	Degradation of protein phosphatase CaAITP1	Inhibit	Baek et al. (2021)
Pepper (Capsicum annuum)	RING	2019	CaASRF1	Nucleus and cytoplasm	Drought	CaAIBZ1	Inhibit	Joo et al. (2019)
Pepper (Capsicum annuum)	RING	2020	CaATIR1	Nucleus	Drought	CaATBZ1	Inhibit	Joo et al. (2020)
Pepper (Capsicum annuum)	RING	2017	CaDIR1	Nucleus	Drought	Via ABA‐mediated signalling	Promote	Joo et al. (2017)
Pepper (Capsicum annuum)	RING	2018	CaDSR1	Nucleus	Drought	CaDILZ1	Inhibit	Lim et al. (2018)
Pepper (Capsicum annuum)	RING	2016	CaDTR1	Nucleus	Drought	Via ABA‐mediated signalling	Inhibit	Joo et al. (2016)
Pepper (Capsicum annuum)	RING	2024	CaFIRF1	Cytoplasm and nucleus	High salt	CaFAF1	Promote	Bae et al. (2024)
Pepper (Capsicum annuum)	RING	2017	CaREL1	Cytoplasm and nucleus	Drought	Via the ABA‐signalling pathway	Promote	Lim et al. (2017b)
Pepper (Capsicum annuum)	U‐box	2016	CaPUB1	–	Cold/drought stress	–	Inhibit cold; promote drought stress	Min et al. (2016)
Pepper (Capsicum annuum)	U‐box	2024	CaPUB24	Nucleus and cytoplasm	Drought	ABA‐dependent manner	Promote	Bae et al. (2024)
Pohlia nutans	U‐box	2019	PnSAG1	Cytoplasm	Salt	–	Promote	Wang et al. (2019)
Poplar (Populus trichocarpa)	RING	2020	PtXERICO	–	Drought	Through the increase of cellular ABA level	Inhibit	Kim et al. (2020)
Populus (Populus alba var. pyramidalis)	U‐box	2021	PalPUB79	Cytoplasm	Drought	PalWRKY77	Inhibit	Tong et al. (2021)
Populus (Populus euphratica)	RING	2018	PeCHYR1	Nucleus and ER	Drought	Increased sensitivity to exogenous ABA, enhanced ROS production and reduced stomatal aperture	Promote	He et al. (2018)
Potato (Solanum tuberosum)	RING	2020	SbRFP1	–	Salt	Regulating the expression of stress‐related genes	Promote	Zhao et al. (2020)
Potato (Solanum tuberosum)	RING	2022	StATL2‐like	Endoplasmic reticulum	Cold	Interacting with StCBF1/4	Promote	Song et al. (2022)
Potato (Solanum tuberosum)	RING	2020	StRFP2	Cell membrane and cytoplasm	Drought	–	Promote	Qi et al. (2020)
Potato (Solanum tuberosum)	U‐box	2010	StPUB17	–	NaCl	–	Inhibit	Ni et al. (2010)
Potato (Solanum tuberosum)	U‐box	2024	StPUB25	–	Drought	Increased peroxidase (POD) activity	Inhibit	Liu et al. (2024)
Potato (Solanum tuberosum)	U‐box	2020	StPUB27	–	Drought	Mediating stomatal conductance	Promote	Tang et al. (2020)
Rice (Oryza sativa)	RING	2018	OsSAR1	Cytosol and nucleus	Salt	OsSNAC2 and OsGRAS44	Promote	Park et al. (2018)
Rice (Oryza sativa)	RING	2019	H2Bub1	–	Drought	OsbZIP46	Inhibit	Ma et al. (2019)
Rice (Oryza sativa)	RING	2017	OsAIR2	Golgi apparatus	Arsenic	OsKAT1	Inhibit	Hwang et al. (2017)
Rice (Oryza sativa)	RING	2022	OsATL38	Plasma membrane	Cold	Via mono‐ubiquitination of OsGF14d 14–3‐3 protein	Promote	Cui et al. (2022)
Rice (Oryza sativa)	RING	2019	OsCLR1	Cytosol	Salt/drought	ABA‐dependent manner	Inhibit	Park et al. (2019)
Rice (Oryza sativa)	RING	2014	OsCTR1	Chloroplasts and cytosol	Drought	Via ABA‐dependent regulation and related systems	Inhibit	Lim et al. (2014)
Rice (Oryza sativa)	RING	2020	OsDHSRP1	Microtubule cytoskeleton	Drought/heat/NaCl	O. sativa glyoxalase (OsGLYI‐11.2) and O. sativa abiotic stress‐induced cysteine proteinase 1 (OsACP1)	Promote	Kim et al. (2020)
Rice (Oryza sativa)	RING	2018	OsDIRP1	Nucleus	Drought/salt/cold	–	Promote	Cui et al. (2018)
Rice (Oryza sativa)	RING	2011	OsDIS1	Nucleus	Drought	OsNek6	Promote	Ning et al. (2011)
Rice (Oryza sativa)	RING	2010	OsDSG1	–	Salt/drought	–	Promote	Park et al. (2010)
Rice (Oryza sativa)	RING	2014	OsHIR1	Plasma membrane and nucleus	Arsenic and cadmium uptakes	Degraded the protein level of OsTIP4;1	Inhibit	Lim et al. (2014)
Rice (Oryza sativa)	RING	2019	OsHIRP1	Nucleus	Heat	OsARK4 and OsHRK1	Inhibit	Kim et al. (2019)
Rice (Oryza sativa)	RING	2016	OsHTAS	Nucleus and cytoplasm	Heat	Promote H2O2‐induced stomatal closure	Inhibit	Liu et al. (2016)
Rice (Oryza sativa)	RING	2018	OsMAR1	Microtubules	Salt	Via the regulation of the OCPI2	Inhibit	Xiao et al. (2022)
Rice (Oryza sativa)	RING	2022	OsMSRFP	–	Salt	Through ubiquitination and proteasomal degradation of OsMYBc	Promote	Kim et al. (2022)
Rice (Oryza sativa)	RING	2022	OsRF1	Endoplasmic reticulum membrane	Drought/salt	Targets OsPP2C09 for Degradation	Inhibit	Kim et al. (2022)
Rice (Oryza sativa)	RING	2023	OsRFPHC‐13	Chloroplasts and cytoplasm	Salt	Via an ABA‐dependent mechanism	Inhibit	Kim et al. (2023b)
Rice (Oryza sativa)	RING	2023	OsRFPHC‐4	Plasma membrane	Salt	Via the regulation of changes in Na+/K+ transporters	Inhibit	Kim et al. (2023a), Kim et al. (2023)
Rice (Oryza sativa)	RING	2021	OsRFPv6	Plasma membrane and cytosol	Salt	Via Na+ uptake	Inhibit	Kim et al. (2021)
Rice (Oryza sativa)	RING	2014	OsRHP1	–	Drought/Salt	Through increased ABA level	Inhibit	Zeng et al. (2014)
Rice (Oryza sativa)	RING	2022	OsRINGzf1	Endoplasmic reticulum and plasma membrane	Drought	Degradation of OsPIP2;1	Inhibit	Chen et al. (2022)
Rice (Oryza sativa)	RING	2015	OsRMT1	Nucleus and microtubules	Salt	Interacted with OsSalT, OsCPA1, OsbZIP60, OsFKBP12, OsEDA16, OsDH1, OsPUB53 and OsPB1	Inhibit	Lim et al. (2015)
Rice (Oryza sativa)	RING	2011	OsSDIR1	Plasma membrane and other intracellular membranes	Drought	–	Inhibit	Gao et al. (2011)
Rice (Oryza sativa)	RING	2023	OsSIRHC‐2	Cytosol	Salt	Through the low absorption of Na+	Inhibit	Choi and Jang (2023)
Rice (Oryza sativa)	RING	2016	OsSIRP1	Cytoplasm	Salt	–	Promote	Hwang et al. (2016)
Rice (Oryza sativa)	RING	2018	OsSIRP2	Nucleus	Salt/Osmotic stress	O. sativa transketolase 1 (OsTKL1)	Inhibit	Chapagain et al. (2018)
Rice (Oryza sativa)	RING	2018	OsSIRP3	Cytoplasm	Salt	OsMADS70 and OsABC1P11	Promote	Park et al. (2018)
Rice (Oryza sativa)	RING	2021	OsSIRP4	Cytosol and plasma membrane	Salt	OsPEX11‐1	Promote	Kim and Jang (2021)
Rice (Oryza sativa)	RING	2015	OsSRFP1	Nucleus	Salt/Cold/Oxidative	Regulation of stress‐related genes	Promote	Fang et al. (2015)
Rice (Oryza sativa)	U‐box	2020	OsPQT3	–	Oxidative/Salt	Elevated expression of OsGPX1, OsAPX1andOsSOD1	Promote	Alfatih et al. (2020)
Rice (Oryza sativa)	U‐box	2017	OsPUB2/3	OsPUB2: exocyst positive organelle (EXPO)‐like punctate bodies and nuclei, OsPUB3: predominantly EXPO‐like structure	Cold	–	Inhibit	Byun et al. (2017)
Rice (Oryza sativa)	U‐box	2023	OsPUB7	–	Drought/salt	–	Inhibit	Kim et al. (2023)
Rice (Oryza sativa)	U‐box	2011	OsPUB15	Cytoplasm	Oxidative stress	–	Inhibit	Park et al. (2011)
Rice (Oryza sativa)	U‐box	2011	OsPUB16	Nucleus and cytoplasm	Water‐deficit	Attenuates the ABA and JA response	Promote	Lv et al. (2022)
Rice (Oryza sativa)	U‐box	2021	OsPUB41	Cytosol and nucleus	Drought	OsCLC6	Promote	Seo et al. (2021)
Rice (Oryza sativa)	U‐box	2020	OsPUB67	Nuclei, cytoplasm, and membrane	Drought	OsRZFP34 and OsDIS1	Inhibit	Qin et al. (2020)
Rice (Oryza sativa)	Cullin4‐based	2021	OsCBE1	–	Abiotic	OsC3H32 a CCCH‐type transcription factor	Inhibit	Choi et al. (2021)
Rice (Oryza sativa)	Zinc‐finger domain	2015	OsiSAP7	Nucleus or sub‐nuclear speckles	Water‐deficit stress	Regulates ABA stress signalling	Promote	Sharma et al. (2015)
Sickle medick (Medicago falcata)	RING	2019	MfSTMIR	Endoplasmic reticulum membrane	Salt	Interacting with MtUBC32 and MtSec61 gamma	Inhibit	Zhang et al. (2019)
Sorghum	RING	2020	SbHCI1	Cytosol/golgi apparatus under heat stress	Heat	OsHCI1	Inhibit	Lim et al. (2020)
Soybean (Glycine max)	U‐box	2020	GmPUB6	Peroxisome	Drought	ABA‐dependent	Promote	Wang et al. (2016)
Soybean (Glycine max)	U‐box	2016	GmPUB8	Post‐Golgi compartments	Drought/osmotic stress/salt	–	Promote	Wang et al. (2016)
Soybean (Glycine max)	U‐box	2022	GmPUB21	Cytoplasm, nucleus, and plasma membrane	Drought/salt	Via the ABA signalling pathway	Promote	Yang et al. (2022)
Sweet potato (Ipomoea batatas)	RING	2021	IbATL38	Nucleus and plasm membrane	Salt	Inducible expression of a series of stress‐responsive genes and prominently decrease of H2O2 content	Inhibit	Du et al. (2021)
Sweet potato (Ipomoea batatas)	RING	2021	IbMREL57	–	Combined salt and drought stress	WAVE‐DAM‐PENED2‐LIKE7	Inhibit	Dou et al. (2021)
Sweet potato (Ipomoea batatas)	RING	2024	IbNIEL	–	Salt/drought	IbNAC087	Promote	Li et al. (2024)
Tomato (Solanum lycopersicum)	RING	2017	SlRING1	Plasma membrane and nucleus	Cadmium	Stimulate both ROS and metal detoxification mechanisms	Inhibit	Ahammed et al. (2021)
Tomato (Solanum lycopersicum)	U box	2021	SlCHIP	–	Heat	Targeting degradation of misfolded proteins	Inhibit	Zhang et al. (2021)
Tomato (Solanum pimpinellifolium)	RING	2016	SpRing	Endoplasmic reticulum	Salt	–	Inhibit	Qi et al. (2016)
Wheat (Triticum aestivum)	RING	2022	TaSADR1	Nucleus	Drought	Regulating the expression of stress‐related genes	Promote	Zhang et al. (2017)
Wheat (Triticum aestivum)	RING	2017	TaSAP5	Cytosol and nucleus	Drought	TaDRIP, as well as AtDRIP1 and AtDRIP2	Promote	Zhang et al. (2017)
Wheat (Triticum aestivum)	U‐box	2021	TaPUB1	–	Cadmium	Degradation of TaIRT1 and TaIAA17	Inhibit	Zhang et al. (2021)
Wheat (Triticum aestivum)	U‐box	2020	TaPUB1	–	Salt	Interacted with TaMP (Triticum aestivssum α‐mannosidase protein)	Inhibit	Wang et al. (2020)
Wheat (Triticum aestivum)	U‐box	2021	TaPUB2/3	Cytoplasm and Golgi apparatus	Drought	–	Inhibit	Kim et al. (2021)
Wheat (Triticum aestivum)	U‐box	2022	TaPUB2/3	–	Salt	ABA‐dependent	Inhibit	Kim et al. (2022)
Wheat (Triticum aestivum)	U‐box	2023	TaPUB4	Nucleus	Drought	–	Inhibit	Kim et al. (2023)
Wheat (Triticum aestivum)	U‐box	2020	TaPUB26	Nucleus	Salt	Interacted with TaRPT2a	Promote	Wu et al. (2020)

Figure 6.

Regulation of E3 Ub in response to plant cold stress. E3 Ub ligases during cold stress are involved in (1) activation of the cold stress response and (2) attenuation of the cold stress response. In (1), cold stress CBF‐dependent cold signalling pathway is activated by the action of PUB25 (U‐box‐type E3 Ub ligase 25) and PUB26 (U‐box‐type E3 Ub ligase 26). PUB25 and PUB26 mediate the destabilisation of MYB15 (transcriptional repressor of the CBF‐dependent cold signalling pathway); the latter regulates the CBF‐dependent cold signalling pathway. BRUTUS (iron‐binding E3 Ub ligase containing three hemerythrin (HHE) domains and a really interesting new gene (RING) domain) mediates the destabilisation of VOZ1/2 (vascular plant one‐zinc finger ½); the latter regulates negative tolerance to cold stress. After removing cold stress (2), the E3 Ub ligase domain mediates the attenuation of the cold stress response. SINAT (RING‐domain containing E3 Ub ligase) mediates the destabilisation of ACS (1‐aminocyclopropane‐1‐carboxylic (ACC) synthase); the latter activates ethylene synthesis, and the last promotes tolerance to cold stress. OsCBE1 (substrate receptor of cullin4‐based E3) mediates destabilisation of OsC3H32 (CCCH‐type transcription factor), the latter contributing to tolerance to cold stress. PQT3 (E3 Ub ligase PARAQUAT TOLERANCE 3) intervenes in the elimination of PRMT4b (protein arginine methyltransferases), which later activate APX1 (ascorbate peroxidase 1) and GPX1 (glutathione peroxidase‐1) both play essential role intolerance to cold stress.

Role of E3 Ub ligase on biotic stress

Biotic stress can have a significant impact on agriculture, reducing, in some cases, average plant productivity by 65%–87% (Francini and Sebastiani, 2019; Sharma et al., 2023). Its factors commonly include harmful microbes, insects, nematodes, weeds and pathogens (Mohapatra et al., 2021). Biotic stress influences plant cellular mechanisms and can increase reactive oxygen species and disrupt physiological and molecular functions (Chaudhary et al., 2022). Different E3 Ub ligases are crucial for molecular regulation and can influence biotic stress tolerance, either positively or negatively.

Plant immunity involves molecular interactions enabling plants to recognise and respond to pathogens, involving systemic acquired and induced resistance. Understanding plant immunity is crucial for disease management and global food security (Bjornson and Zipfel, 2021). HECT‐type E3 Ub ligases are essential for plant immune responses. Most of them, including UPL1, UPL3 and UPL5, interact with the hormone salicylic acid (SA). Microbial pathogens and insects that invade plants can cause plants to activate numerous RING‐type E3 Ub ligases as positive or negative regulators to control biotic stress response (Furniss et al., 2018). For example, Arabidopsis ATL9 is a chitin‐inducible E3 Ub ligase that shapes basal resistance to Golovinomyces cichoracearum (Deng et al., 2017). Recognition of chitin present in the structural membrane of these pathogens will trigger a plant protection response. It can be activated when plants recognise chitin present in the structural membrane of these pathogens. For example, RING‐type E3 Ub ligase MIEL interacts with MYB96, a regulator of ABA signalling, and limits its accumulation. MIEL1 is critical in biotic stress in conjunction with the ABA pathway (Lee and Seo, 2016). Some PUB E3 Ub ligases exhibit adverse regulatory effects on plant disease resistance, contributing to cell death. PTI and PAMPs‐induced signalling are the core defensive mechanisms of plant systems against pathogen infection (Lu et al., 2011). PUB12 and PUB13 were reported to negatively regulate FLS2‐mediated PTI immune response (Lu et al., 2011). Some plant U‐box proteins, such as CMPG1, PUB44 and PUB17, have positive regulatory roles in plant disease resistance. Some pathogens have evolved with plants and manipulated E3 Ub ligase machinery for their benefit (Zhu et al., 2015). Figure 7 shows the role of E3 Ub ligase in biotic stress. Table 2 shows the E3 Ub ligase genes in response to biotic stresses in plants.

Figure 7.

Regulation of E3 Ub in response to plant biotic stress. E3 Ub ligases during biotic stress are involved in (1) activation of the biotic stress response and (2) attenuation of the biotic stress response. In (1), the biotic stress PTI (PAMP‐triggered immunity) is activated by the action of PUB44 (U‐box‐type E3 Ub ligase 44) and PUB4 (U‐box‐type E3 Ub ligase 4). PUB4 mediates the accumulation of BIK1 (receptor‐like cytoplasmic kinase), which is directly involved in the activation of PTI. ATL9 (RING zinc finger protein with E3 Ub ligase) positively regulates PAMPs (pathogen‐ or microbe‐associated molecular patterns) that promote PTI and tolerance to biotic stress. MDPUB29 (apple U‐box E3 Ub ligase) promotes the activation of the SA pathway for biotic stress tolerance. After removing biotic stress (2), the E3 Ub ligase domain mediates the attenuation of the biotic stress response. PUB22 (U‐box‐type E3 Ub ligase 22) mediates the destabilisation of Exo70B2 (exocyst subunit), which activates PAMP. PUB12 (U‐box‐type E3 Ub ligase 12) and PUB13 (U‐box‐type E3 Ub ligase 13) regulate negatively the FLS2 (pattern recognition receptor FLAGELLIN‐SENSING 2), the latter activating PTI. MIEL1 (RING‐type E3 Ub ligase MYB30‐INTERACTING E3 Ub LIGASE 1) directly mediates the elimination of MYB30 (transcription factor), the latter activating the MYB30 responsive genes that activate PTI. MIEL1 also mediates the destabilisation of MYB96 (transcription factor); the latter activates the ABA pathway. The ABA pathway also promotes MYB30‐responsive genes that positively regulate PTI. MIEL1 links abiotic and biotic stress. UPL1/3/5 (HECT domain‐containing family of ubiquitin‐protein ligases 1, 3, 5) mediate the destabilisation of the proteasome, the latter being involved in the continuation of the stress response. PUB26 (U‐box‐type E3 Ub ligase 26) and PUB25 (U‐box‐type E3 Ub ligase 25) mediate the destabilisation of MYB6 (R2R3‐MYB transcription factor), which is involved in the activation of the gene for the response to abiotic stress. PQT3 (E3 Ub ligase PARAQUAT TOLERANCE 3) mediates the elimination of PRMT4b (protein arginine methyltransferases), which later activate APX1 (ascorbate peroxidase 1) and GPX1 (glutathione peroxidase‐1), both essential in tolerance to biotic stress. XBAT35.2 (RING‐type E3 Ub ligase) mediates the destabilisation of ACD11 (arabidopsis accelerated cell death 11), the latter being involved in maintaining tolerance to biotic stress. XB3 mediates the destabilisation of XA21 (receptor‐type protein kinase), which responds to biotic stress.

Table 2.

E3 ubiquitin ligase genes involved in plant response to biotic stresses

Species	Type of E3 Ub ligase	Year	Gene	Cellular localisation	Type of stress	Cellular target	Role in stress	References
Arabidopsis thaliana	HECT	2018	UPL3	–	Biotic stress	Interacts with the regulatory particle of the proteasome and with other ubiquitin‐26S proteasome pathway components	Inhibit	Furniss et al. (2018)
Arabidopsis thaliana	RING	2014	ATL1	Endomembrane structures	Powdery mildew	Interacts with EDR1	Promote	Serrano et al. (2014)
Arabidopsis thaliana	RING	2010, 2017	ATL9	Endoplasmic Reticulum	Biotic stress	Interacting partner of the PEST domain and the RING domain	Inhibit	Berrocal‐Lobo et al. (2010), Deng et al. (2017)
Arabidopsis thaliana	RING	2022	AtRDUF1/2	–	Pseudomonas syringae pv. tomato DC3000	Involved in SA‐mediated PR1 gene expression	Inhibit	Yi et al. (2022)
Arabidopsis thaliana	RING	2018	Keep on Going (KEG)	–	Biotic and abiotic stress	Degrade FDH	Inhibit	McNeilly et al. (2018)
Arabidopsis thaliana	RING	2013	MIEL1	–	Biotic stress	This leads to MYB30 proteasomal degradation	Promote	Marino et al. (2013)
Arabidopsis thaliana	RING	2012	RGLG3 and RGLG4	–	Pseudomonas syringae pv. tomato DC3000	Controls the accessibility of JA‐Ile (COR) to the coreceptor COI1	Inhibit	Zhang et al. (2012)
Arabidopsis thaliana	RING	2021	SDIR1	ER membrane	Pseudomonas syringae pv. tomato DC3000	–	Promote	Ramu et al. (2021)
Arabidopsis thaliana	RING	2020	SINAT4	–	Pseudomonas syringae pv. tomato DC3000	Interacts with RD21A	Promote	Liu et al. (2020)
Arabidopsis thaliana	RING	2017	XBAT35.2	Golgi	Pseudomonas syringae pv. tomato DC3000	ACD11	Inhibit	Liu et al. (2017)
Arabidopsis thaliana	U‐box	2016	AtCHIP	–	Virulent pathogens	Interacts with HSP Chaperones	Inhibit	Copeland et al. (2016)
Arabidopsis thaliana	U‐box	2022	AtPUB2/4	AtPUB2: Plasma membrane; AtPUB4: cytoplasm and plasma membrane	Pseudomonas syringae	Interacts with PTI signalling components, including FLS2, BIK1, PBL27, and RbohD	Inhibit	Wang et al. (2022)
Arabidopsis thaliana	U‐box	2022	AtPUB4	–	Biotic stress	BIK1 (targeted by bacterial type‐III effector)	Inhibit	Yu et al. (2022)
Arabidopsis thaliana	U‐box	2011	AtPUB12/13	–	Pseudomonas syringae pv. tomato DC3000 and Pseudomonas syringae maculicola ES4326	FLS2	Promote	Lu et al. (2011)
Arabidopsis thaliana	U‐box	2024	AtPUB20/21	–	Pseudomonas syringae pv. tomato DC3000		Promote	Yi et al. (2024)
Arabidopsis thaliana	U‐box	2012	AtPUB22	–	Biotic stress	Mediates the ubiquitination and degradation of Exo70B2	Promote	Stegmann et al. (2012)
Arabidopsis thaliana	U‐box	2008	PUB22/23/24	–	Pseudomonas syringe pv tomato DC3000 and Ha isolate Emco5	–	Promote	Trujillo et al. (2008)
Arabidopsis thaliana	U‐box	2021	PUB25/PUB26	–	Verticillium dahliae	MYB6	Promote	Ma et al. (2021)
Arabidopsis thaliana	U‐box	2018	PUB25/PUB26	–	Botrytis cinerea and Pseudomonas syringae pv tomato DC3000	BIK1	Promote	Wang et al. (2018)
Arabidopsis thaliana	U‐box	2017	SAUL1	Plasma membrane	Pseudomonas syringae pv. tomato DC3000	Monitored by the TNL SOC3	Inhibit	Tong et al. (2017)
Apple (Malus domestica)	U‐box	2019	MdPOB1	–	Botryosphaeria dothidea	MdPUB29	Promote	Han et al. (2019)
Apple (Malus domestica)	U‐box	2019	MdPUB29	–	Botryosphaeria dothidea	Regulates the SA pathway	Inhibit	Han et al. (2019)
Chinese wild grapevine (Vitis yeshanensis)	U‐box	2024	VyPUB21	–	Powdery mildew	Targets VyNIMIN protein hydrolysis through the 26S proteasome system	Inhibit	Wang et al. (2024)
Cotton (Gossypium hirsutum)	U‐box	2021	GhXB3s (GhXB32A, GhXB35A and GhXB37A)	–	Verticillium dahliae	–	Inhibit	Ge et al. (2021)
Grapevine (Vitis vinifera)	U‐box	2021	VvPUB17	–	Powdery mildew	Via the salicylic acid signalling pathway	Inhibit	Li et al. (2021)
Kiwifruit (Actinidia chinensis)	U‐box	2024	PUB23	–	Pseudomonas syringae pv. actinidiae	GT1	Promote	Wang et al. (2024)
Medicago truncatula	U‐box	2010	PUB1	Plasma membrane	Infection and nodulation	PUB1 is phosphorylated by LYK3	Inhibit	Mbengue et al. (2010)
Nicotiana benthamiana	U‐box	2015, 2021	NbPUB17	Nucleus	Phytophthora infestans	Promotes specific PTI and PCD pathways	Inhibit	He et al. (2015)
Potato (Solanum tuberosum)	U‐box	2020	StPUB17	Nucleus	Phytophthora infestans	Targeting a Negative Regulator, KH17	Inhibit	McLellan et al. (2020)
Rice (Oryza sativa)	U‐box	2015	OsPUB15	Cytosol	Biotic stress	Interacts with the receptor‐like kinase PID2	Inhibit	Wang et al. (2015)
Rice (Oryza sativa)	U‐box	2022	OsPUB33/34/39	–	Magnaporthe grisea	–	Inhibit/Promote	Zhang et al. (2022)
Rice (Oryza sativa)	U‐box	2014, 2022	OsPUB44	Entire region	Xanthomonas oryzae pv. oryzae	PBI1	Inhibit	Ichimaru et al. (2022)
Rice (Oryza sativa)	U‐box	2012, 2015	OsSPL11	–	Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae	SPIN6	Inhibit/Promote	Liu et al. (2012, 2015)
Tobacco (Nicotiana tabacum)	U‐box	2010	CMPG1	–	Phytophthora infestans	AVR3a	Inhibit	Bos et al. (2010)
Tobacco (Nicotiana tabacum)	U‐box	2006	NtCMPG1	–	Biotic stress	Involved in Pto/AvrPto and Inf1‐mediated cell death	Inhibit	González‐Lamothe et al. (2006)
Tomato (Solanum lycopersicum)	U‐box	2018	SlPUB13	–	Biotic stress	Works with group III E2s to ubiquitinate FLS2	Inhibit	Zhou and Zeng (2018)
Tomato (Solanum lycopersicum)	U‐box	2021	SlPUB24	Cytoplasm, plasma membrane and nucleus	Xanthomonas euvesicatoria pv. perforans	SlCWP	Inhibit	Liu et al. (2021)
Wheat (Triticum aestivum)	U‐box	2015	CMPG1‐V	Nucleus, endoplasmic reticulum, plasma membrane and partially in trans‐Golgi network/early endosome vesicles	Blumeria graminis f. sp. tritici	Reactive oxidative species and the phytohormone pathway	Inhibit	Zhu et al. (2015)

Research efforts have made considerable progress in the structural characterisation of E3 Ub ligases and their importance in plant resistance to abiotic and biotic stresses. However, it is vital to capitalise on this advance to meet the food security challenge. The following section proposes the PROTAC methodology to improve plant resistance to stress.

PROTACs: Concept, mode of action, origin and development

PROTACs can target the E3 Ub ubiquitin ligase for improved plant stress resistance. This section introduces the concept of PROTACs, their mode of action, origin and development.

The concept of PROTACs

PROTACs are defined as proteolysis‐targeted chemotherapeutics or proteolysis‐targeted protein‐degrading chemotherapeutics. They are small molecules with dual functions. PROTACs are made up of a 01 linker and 02 moieties (Figure 8). One moiety binds the target protein (protein of interest, POI), and the other binds the E3 Ub ligase. By linking a POI to an E3 Ub ligase, PROTACs facilitate the ubiquitination of the POI and, consequently, its degradation by the proteasome. PROTAC class compounds offer a solid complementary alternative for gene silencing or targeted inhibition and represent a viable option for controlling proteins responsible for plant sensitivity to stress. PROTAC may ultimately target the plant E3 Ub ligases that regulate biotic and abiotic stresses in plants.

Figure 8.

The mechanism of PROTAC‐mediated targeted protein degradation. PROTACs act as a bridge between the target protein or protein of interest (POI) and the E3 Ub ligase, creating sufficient proximity to allow the transfer of ubiquitin from the E2 ligase to the target protein. This action will be repeated several times, and the POI will have a ubiquitin chain leading to its recognition and degradation by the proteasome. The PROTAC molecule can conserve its properties after several cycles and eliminate the POI.

The mode of action of PROTACs

The mode of action of PROTACs is innovative and relevant to eliminating E3 Ub ligases important in plant stress, as illustrated in Figure 8. Concisely, the PROTAC mechanism causes the elimination of the target protein from the cell and not just inhibits it, as the conventional chemical pesticides used in agriculture do. PROTACs may not be subject to the traditional stoichiometric ratio like common chemicals applied in crop protection because of their unique chemical architecture. As major heterobifunctional degraders, PROTACs are relevant for targeting the E3 Ub ligases responsible for plant stress. They offer a viable alternative to address pathological conditions of plants where the proteins involved in stress are complex to inhibit by conventional chemical strategies. More interestingly, PROTACs do not require the absolute presence of an active site on the targeted protein to act but instead can be designed to find affinity with different areas of the protein structure. PROTAC technologies are relevant to target molecules that are difficult to inhibit and could be used to treat so‐called incurable diseases involving proteins with smooth surfaces or huge active sites. The unique properties of PROTACs could pave the way for solving the problem of recalcitrant proteins, which account for around 80% of all cellular proteins in eukaryotic cells, including the E3 Ub ligases. According to this perspective, PROTACs have the potential to transform agriculture by controlling the regulation of E3 Ub ligase in plant stress response, which is of great importance in the agricultural sector.

The origin of PROTACs

Plants and viruses originally inspired the concept of exploiting cellular protein degradation systems for therapeutic purposes (Békés et al., 2022). Research into PROTACs dates back some 20 years (since 2001) but has already made a significant contribution, with numerous clinical trials in cancer treatment entering phase 2 (Békés et al., 2022). Today, PROTACs offer vast prospects in human medicine but are still limited in the plant field. PROTAC concept has risen to the challenge of moving from idea to real‐world application. Since humans and plants share a similar eukaryotic system, PROTACs' success story in human health can be projected to plants.

PROTACs development

PROTAC methodology holds great promise for agriculture. In the agricultural sector, PROTACs can be studied on large farms relatively quickly and would not undergo the tedious clinical steps required for human studies. The role of E3 Ub ligases is crucial in the plant kingdom, which possesses over 1400 of them (Mazzucotelli et al., 2006), compared with around 600 in humans (Békés et al., 2022). Recently, the perspective of applying PROTACs in plants has attracted significant biotech and pharmaceutical companies, including Bayer, Novartis, Pfizer, Amgen, GlaxoSmithKline, Merck, PROTAC® and others (Gao et al., 2020). Bayer, a leading pharmaceutical and agricultural company, drives the PROTAC investment for sustainable agriculture. Arvinas is a clinical‐stage biotechnology company innovating in the field of targeted protein degradation therapies. Bayer and Arvinas launched a separate joint venture to develop PROTAC® agricultural applications. Bayer contributes over $55 million to the financing, while Arvinas provides the technology and intellectual property. Nevertheless, the development of PROTACs requires multidisciplinary collaboration. Synthetic methods may evolve toward computational chemistry and can be relevant to designing new linkers in silico (Bemis et al., 2021). Furthermore, molecular prospecting, advances in biotechnology (including chemical biology), and bioinformatics (exploiting existing data from medical research) can also be used to make significant advances in the field of PROTACs in plants (Guedeney et al., 2023). Efforts will be made to overcome the limitations of designing chimeras targeting proteolysis (Leon and Bassham, 2024).

Role of E3 Ub ligases in new methodology for PROTACs development in plant

Exploration of E3 Ub ligases in designing PROTACs for agriculture is in its infancy. This section gives an overview of promising PROTAC methodologies that can be used in plants while considering current challenges in E3 Ub ligase research to improve crop stress resistance.

Characterisation of plant homologues of human E3 Ub ligases to convert medical PROTAC methodology into agricultural applications

Identifying plant homologues of human E3 Ub ligases is essential for developing PROTAC methodology in agriculture. Although PROTAC‐related research in plants may consider all stress‐promoting proteins in cells as potential targets, starting by directing research at plant homologues of the human E3 Ub ligases used by PROTAC methodology in medicine may facilitate PROTAC design for agriculture. For example, CRBN, F‐box and NPR1 are substrate recognition subunits of Cullin RING E3 Ub ligase complexes recruited by PROTACs in human cancer research (Cruz Walma et al., 2022). Homologue substrate recognition subunits of E3 Ub ligases can be found in plants and considered in the PROTAC strategy. In Figure 9, the superposition of the plant CRBN and the human CRBN shows a certain similarity in the folding of the central part representing the Lon protease. The SCF (SCF‐like cullin‐RING complexes), whose substrate receptor subunits are interchangeable, regulates a broad spectrum of cellular functions in plants by ubiquitinating various protein substrates and can be an essential asset for PROTAC strategies. Besides, NPR1 may act as a substrate‐specific adapter of an E3 ubiquitin‐protein ligase complex (CUL3‐RBX1‐BTB). NPR1 was reported to modulate defensive‐related transcription factors and crosstalk between salicylate‐ and jasmonate‐dependent defence pathways (Johnson et al., 2003; Spoel et al., 2003).

Figure 9.

Superposition of two protein cereblon (CRBN) from Arabidopsis thaliana and Human. These two structures are superimposed in the central part, which represents the Lon protease and N‐terminal domain. PROTACs that target this region can be studied in both humans and plants. CRBN_ARATH, in yellow, represents the structure of the protein cereblon from the organism Arabidopsis thaliana (mouse‐ear cress). CRBN_ARATH functions in the protein ubiquitination pathway. The Uniprot code is Q8VXV2. CRBN_HUMAN, in blue, represents the 3D structure of Protein cereblon from organism HUMAN (mouse‐ear cress), which functions as a Substrate recognition component of a DCX (DDB1‐CUL4‐X‐box) E3 protein ligase complex that mediates the ubiquitination and subsequent proteasomal degradation of target proteins, such as MEIS2 or ILF2 (Kim et al., 2019). The Uniprot code is Q96SW2. All the PDB formats were obtained from Alphafold Alignment and were done on PyMOL, PyMOL Molecular Graphics System, Version 2.5.7pre, Schrödinger, LLC.

Consideration in POIs selection and PROTACs design in plant

Proteins targeted by fungicides and herbicides may be relevant as POI in PROTAC strategies for stress resistance. A specific requirement in selecting POIs is that PROTACs that bind to POIs should not obstruct the binding of a partner E3 Ub ligase. Some essential regions to consider for PROTAC binding are unstructured regions since they do not interfere with the proteolytic action of the proteasome. Research into the structure of the linker or E3 Ub ligase moiety of PROTACs can also benefit from data derived from research into PROTAC therapy in cancer. As a partner, E3 Ub ligase for PROTACs can be a similar subunit recovered in plants and animals with conserved regions. Applicability and specific modality in agriculture versus medicine should consider key stress factors, environment, and plant cell structure. For example, E3 Ub ligases and POIs with favourable and cooperative protein–protein interactions (PPIs) between them must be regarded for developing PROTACs. Targeting complexes such as the scaffolding protein will be a difficult task, and an alternative may be to target the nearby or neighbouring protein (Békés et al., 2022).

Specificity of PROTAC methodology in plant

Under stress, the proportions and compositions of E3 Ub ligases depend on the plant organ and plant variety. Omic methods such as transcriptomics and proteomics could be important for characterising E3 Ub ligase expression under specific conditions. While these preliminary studies can help to tailor PROTAC methodology to specific conditions such as different plant varieties or particular plant organs, it is essential that further PROTAC development takes into account the diversity of E3 Ub ligases and may involve better identification of the role and structure of plant E3 Ub ligases (Tables 1 and 2).

In the future, PROTAC strategies and tailor‐made treatments may be developed for any plant susceptible to a pathogen. Targeting specific E3 Ub ligase protein will enable specific plant treatment for particular plant varieties. A worldwide bank of well‐known microbial effectors could be developed and studied for PROTACs. Future considerations involve characterising the complete list of plant and microbial effectors, constructing a ligand library, constructing a target library and studying the involvement of E3 ligands in plant specificities.

The prospect of combining several PROTACs to attenuate the stress response of crops

PROTACs in plants should consider interrupting the stress response after the stress has been abolished. The persistence of the stress response can be detrimental to plants, as it can represent a waste of vital resources, which is why the response must also be regulated after stress inhibition. As shown in Tables 1 and 2, multiple E3 Ub ligases play diverse roles in abiotic stress and are expressed differently during stress induction studies. The prospect of combining multiple PROTACs should be considered seriously, as E3 Ub ligases have numerous targets playing a role in induced stress. Considering the previous discussion on drought, salt, cold and biotic stress, applying PROTACs in plants should focus on developing strategies to activate stress tolerance in plants and deactivate stress tolerance when the pressure is alleviated. A relevant system can be achieved by developing PROTACs that facilitate the elimination of the POI responsible for impairing stress tolerance. Once the stress is alleviated, the inhibition of PROTACs can be lifted to halt the stress response, allowing the plant to revert to its normal physiological state.

Consideration in selecting E3 Ub ligase as a partner of PROTACs and as POIs

The role of E3 Ub ligase as a partner and E3 Ub ligase as a POI will be the two critical perspectives for applying PROTACs in agriculture. Tables 1 and 2 show essential E3 Ub ligases involved in stress response.

Selecting E3 Ub ligase as POI

As POIs, special attention can be given to this E3 Ub ligase, which is differentially regulated during stress conditions. However, research is limited in predicting the overall connection of a particular E3 Ub ligase in plant molecular mechanism, and the possibility of unexpected results or some dysfunction needs to be considered. More importantly, genome‐wide classification of all classes of E3 Ub ligase in various species needs to be completed.

Selecting E3 Ub ligase as a partner of PROTACs

For E3 Ub ligases to be used as partners, their frequency of appearance during stress responses, their quantity, and how easily the ligand can be designed for the PROTACs strategy must be taken into account. For example, SCFs (SCF‐like cullin‐RING complexes), whose substrate‐receiving subunits are interchangeable, regulate a broad spectrum of cellular functions in plants by ubiquitinating various protein substrates and can therefore be an essential asset for PROTAC strategies. Elucidating the interaction mechanism between hormones and E3 Ub ligases can help develop PROTACs adapted to the recruitment of E3 Ub ligases, thus enabling the elimination of proteins that increase sensitivity to stress.

The PROTAC design strategy can learn how hormones interact with the E3 Ub ligase F‐box substrate receptor to design an E3 Ub ligase moiety of the PROTAC essential for recruiting the E3 Ub ligase to degrade the target protein. For example, auxin is an essential hormone for the development of plants. The auxin response requires a proper interaction between the Aux/IAA protein and SCFTIR1. Advances in research have shown that the auxin molecule binds preliminarily and specifically in a restricted space to TIR1 – the F‐box protein acting as a substrate receptor for the SCF complex. Following auxin binding to TIR1, the affinity of the AUX/IAA protein to auxin‐SCFTIR1 is increased. The AUX/IAA protein then binds to the E3 Ub ligase (forming an AUX/IAA‐auxin‐SCFTIR1 complex). Once the AUX/IAA protein complex is bound to TIR1, ubiquitination occurs through the transfer of ubiquitin from the E2 ligase to the AUX/IAA protein; this final step continues until the AUX/IAA protein is attached to a ubiquitin chain. The AUX/IAA protein is then directed to the proteasome for degradation. Degradation of the AUX/IAA protein leads to the accumulation of the ARF/MYB2 transcription factor set, enabling the expression of an appropriate auxin response in the plant (Tan et al., 2007). A similar phenomenon was observed for jasmonic acid, which interacts with SCFCOI1 to eliminate JASMONATE‐ZIM DOMAIN (JAZ) proteins that inhibit MYB2, a transcription factor involved in responses to abiotic stress. MYB2 accumulation plays a regulatory role in salt, cold, and drought tolerance. A structural analogue of auxin or jasmonic acid or their derivatives can be developed and tested based on their superior affinity with F‐BOX TIR1 or F‐BOX COI1 to design a PROTAC capable of recruiting the E3 Ub ligase SCFTIR1 or SCFCOI1 for removal of the POI, probably an E3 Ub ligase known to promote stress in the plant. Eliminating stress‐promoting E3 Ub ligases is more likely to increase plant resistance to stress.

Importance of inducible E3 Ub ligase in strategy of PROTACs in plant

Targeting or recruiting stress‐inducible E3 Ub ligases as part of the PROTAC strategy in plants could help control stress as soon as it occurs (Qi et al., 2016). For example, an inducible E3 Ub ligase partner or POI can be used, including SARS1, CaAIP1 and OsHKT1;1. So, the PROTACs study can consider this reference for more models to control stress and improve tolerance (Qi et al., 2016; Xiao et al., 2022).

Considering multi‐stress E3 Ub ligases in PROTAC strategies

POIs of PROTACs will include various enzymes, membrane receptors such as the ABA receptor or the chloride channel, and transcription factors (Tables 1 and 2) (Zhao et al., 2017). The discovery of specific E3 Ub ligases acting with multiple targets may inspire research into the design of PROTAC chimeras capable of controlling one or more stress response factors simultaneously and allowing plants to be resistant to multiple stresses (Xiao et al., 2022). PROTACs should also consider targeting the elimination of specific E3 Ub ligases effector of multiple stress sensitivity as GmPUB8 (Wang et al., 2016). Some E3 Ub ligases are overexpressed under various stresses, for example, SDIR1 (salt‐ and drought‐induced ring finger1) (Alfatih et al., 2020). Using these E3 Ub ligases as PROTAC partners could be recommended to impact multiple stresses consistently.

PROTACs can target microbial effectors and mimic insects and microbes that hijack the plant ubiquitin system

Plant pathogens cause biotic stress to plants, resulting in significant activation and deactivation of E3 Ub ligases. PROTACs can improve plant sensibility to herbicides and plant resistance to microbial pathogens. PROTACs could mediate ubiquitination and the proteasomal elimination of any infectious effectors introduced in the plant by pathogens. This prospect is attractive as it will contribute to controlling plant pathogens specialised in hijacking the eukaryotic ubiquitin‐proteasome system (Zhao et al., 2019). Beyond triggering immunity during microbial infection, microbes' primary strategy is mimicking essential E3 Ub ligases to induce plant vulnerability. One of PROTAC's exciting prospects will be to target specific microbial E3 Ub ligases that mimic the ubiquitin function of the plant proteasomal pathway.

Pathogens stimulate the plant immune system and release effector molecules to bypass defences. The plant ubiquitin‐proteasome system regulates protein abundance and controls immunity. Pathogen effector molecules disrupt the UPS by hijacking its functions or affecting target degradation. Studying the interactions between microbial effectors and UPS can help develop PROTACs with greater affinity for POI and E3 Ub ligase (Hijacking protein degradation, 2020; Langin et al., 2023). The model of insects and microbes manipulating the E3 Ub ligase system to their advantage can be studied to develop innovative PROTAC ligands. Hijacking protein degradation (2020).

Delivering PROTACs in plant system

The large size (700–1000 Da) and unusual physicochemical properties of PROTACs can significantly limit their plant uptake (Martín‐Acosta and Xiao, 2021). However, PROTACs need to penetrate plant cells and reach their molecular targets to exert their action. The complexity of plant cells and cell walls will likely hinder the uptake of PROTACs (Kastl et al., 2021). Strategies to maximise the uptake and delivery of PROTACs into plant cells may be essential for any breakthrough. Delivery systems or structural modifications of PROTACs could improve solubility, stability and membrane permeability (An and Fu, 2018). Research will be needed to optimise the design and delivery of PROTACs for plant systems.

The challenge of PROTACs in improving plant resistance to stress and the role of E3 Ub ligases beyond PROTACs

PROTACs have a long way to go before being applied to plant stress resistance. Indeed, many challenges must be resolved first (Leon and Bassham, 2024). Among the plausible limitations of PROTACs, implementing PROTACs in agriculture will require the regulation of heterobifunctional molecules and a complete study of their toxicology for plants, animals, humans and the environment. Promoting bioPROTACs or peptide‐PROTACs that integrate biocompatible elements into the structure of PROTACs could reduce toxicity issues (Lim et al., 2020). Low permeability or low stability of PROTACs may be recurrent in the plant system and lead to ineffective PROTACs; development of resistance may also exist with PROTACs as they are based on E3 Ub ligase specificity, and mutation in the recognition site may lead to resistance (Zhang et al., 2019). A similar situation was observed when cancer cells developed resistance to PROTACs. The integrative strategies will remain relevant in the future field of PROTACs and may involve combining pesticides and PROTACs.

A key research challenge is controlling the chemical characteristics of PROTAC chimeras and the ligand identification strategy for screening POIs. Molecular glues are molecules known to stabilise PPIs. They show promise in E3 Ub ligases and their targets. Molecular glues comprise one component and are not heterobifunctional like PROTAC molecules. The molecular glue concept could be applied to PROTACs to achieve more significant target protein degradation. In addition to PROTACs, other heterobifunctional molecules are non‐UPS pathways for the degradation of a POI, such as autophagy‐targeted chimeras. Research in these areas holds great promise for the integral management of plant resistance in the face of increasing disease threats associated with climate change.

Conclusion

Ensuring access to a sufficient and reliable food supply is a global challenge. The world is currently confronted with major threats such as climate change, population growth and the proliferation of stressful factors, including drought, extreme cold, salinity and the unprecedented invasion of pathogens. Developing a sustainable and comprehensive food production strategy requires innovative advancements in the molecular mechanisms of plants. This paper demonstrates how advances in molecular research of E3 Ub ligase can provide opportunities for innovation in biotechnology approaches highly relevant to enhancing plant resistance to stress. The discussion focused on the concept and mechanism of the newly discovered class of heterobifunctional molecules known as PROTACs. The emphasis highlights its significance in enhancing control over plants' abiotic and biotic stress. Targeting E3 Ub Ligase, a crucial factor in stress response, as a therapeutic target and partner will significantly advance plant disease control. This potential will substantially enhance global food security. Hence, there are still crucial prerequisites for implementing PROTACs in crop production. These include overcoming the barriers of fully understanding the properties and stability of PROTAC chimeras, as well as determining the administration method for PROTACs in plants to ensure their entry into cells and contact with the molecular targets, taking into account their significantly larger size compared to pesticide molecules. Exciting breakthroughs in molecular basis E3 Ub ligase in plant research will certainly fasten progress opportunities in food security worldwide.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Author contributions

Y.S. and G.L.N.N. Contributed equally. Designing the framework of the article, Y.S. and G.L.N.N.; writing – original draft, Y.S., G.L.N.N., K.W., Y.L., E.A.G., and M.A.; writing – review & editing, Q.Y., Q.Z., and H.Z.

Data availability statement

The data that support the findings of this study are available in PubMed at https://pubmed.ncbi.nlm.nih.gov/?term=Proteolysis‐targeting%20chimeras%20(PROTACs)%20&timeline=expanded. These data were derived from the following resources available in the public domain: – PubMed, https://pubmed.ncbi.nlm.nih.gov/ – WebofKnowledge, https://www.webofknowledge.com – Google Scholar, https://scholar.google.com – Springer, https://www.springer.com.