Roles of ubiquitination-mediated protein degradation ...

Environmental and Experimental Botany 114 (2015) 92–103

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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Roles of ubiquitination-mediated protein degradation in plant responses to abiotic stresses Zeyong Zhang a,1 , Junhua Li b,1 , Huanhuan Liu a , Kang Chong a , Yunyuan Xu a,∗ a b

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China College of Life Sciences, Henan Normal University, Xinxiang 453007, Henan, China

a r t i c l e

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Article history: Available online 21 July 2014 Keywords: Ubiquitination E3 ligase Plants Abiotic stress Signaling

a b s t r a c t Ubiquitination is a major modifier of signaling in all eukaryotes that results in the conjugation of ubiquitin to the lysine residues of acceptor proteins. The targeted protein is then subjected to degradation by the 26S proteasome, the major protein degradation system in eukaryotes. The ubiquitin–proteasome system (UPS) greatly influences plant growth and development by modulating the activity, localization, and stability of proteins. Plants are frequently exposed to various abiotic stresses during their life cycles; they rely on proteomic plasticity achieved by the UPS to adapt to unfavorable environmental conditions. In stress signal pathways, a large number of components are modified by specific ubiquitination machinery. In this review, we highlight recent advances in understanding the roles of ubiquitination in plant responses to abiotic stresses, including salt and drought, temperature, ultraviolet (UV), and nutrient availability. The review focuses primarily on the roles of the UPS. In salt and/or drought stress signaling, a number of E3 ligases mediate the stress response in both abscisic acid (ABA)-dependent and ABA-independant pathways. The UPS-mediated regulation of several key ABA-regulated transcriptional factors, e.g. ABI3 and ABI5, has been well documented. In cold signaling, the transcription factor ICE1 is targeted by E3 ligase HOSI for proteosomal degradation. Under UV stress, CUL4-DDB1A-DDB2 E3 ligase participates in DNA excision repair, and COP1 interacts with the UVR8 mediated UV response. The UPS is also involved in the uptake, transport, and homeostasis of nutrients such as iron, phosphorus, and nitrogen. SIZ1mediated sumoylation, a ubiquitin-like modification, is necessary for a number of processes involved in plant responses to abiotic stresses. A challenge moving forward for researchers is to define more UPS components and to characterize their functions in plant responses to stress conditions; there is particular interest in identifying the ubiquitination targets that function in specific stress signaling pathways. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ubiquitin (Ub) is a highly conserved protein found in all eukaryotic species. This small protein is involved in the destruction of endogenous targets via the ubiquitin 26S proteasome system, the major proteolysis mechanism in eukaryotic cells (Hershko et al., 2000). Ubiquitination plays an important role in many processes in plants, including organ development, photomorphogenesis, hormone responses, as well as biotic and abiotic stress responses (Hershko et al., 2000; Lyzenga and Stone, 2012; Pokhilko et al., 2011; Santner and Estelle, 2010; Sonoda et al., 2009; Spoel and Dong, 2012). Abiotic stresses such as high salinity, drought, low temperature, UV radiation, and nutrient deprivation have adverse impacts

on plant growth, development, and reproduction. Plants rely on proteomic plasticity to remodel themselves to maximize their chances of survival under varying environmental conditions (Fujita et al., 2005; Hirayama and Shinozaki, 2010; Lee and Kim, 2011). A variety of abiotic stresses affect plant growth and development throughout their life cycles. As such, plants have needed to evolve diverse strategies to combat all these various forms of abiotic stress (Lyzenga and Stone, 2012). The ubiquitin system is one of the most important stress response systems, as it functions across numerous signaling pathways. In this review, we address recent advances to our understanding of the role of the ubiquitin–proteasome system (UPS) during plant abiotic stress signaling.

2. The UPS and UPS enzymes ∗ Corresponding author. Tel.: +86 10 6283 6213; fax: +86 10 8259 4821. E-mail address: [email protected] (Y. Xu). 1 Both authors contributed equally to the work. http://dx.doi.org/10.1016/j.envexpbot.2014.07.005 0098-8472/© 2014 Elsevier B.V. All rights reserved.

Ubiquitin is a highly conversed protein of 76 amino acids found in all eukaryotes. Ubiquitin modifies target proteins to alter various aspects of their regulation via the UPS (Jentsch and Pyrowolakis,

Z. Zhang et al. / Environmental and Experimental Botany 114 (2015) 92–103

2000; Sadanandom et al., 2012). It has been supposed that ubiquitin has a compact structure called a ‘Ub fold’ with a five-strand mixed ␤-sheet that forms a cavity into which a single ␣-helix fits diagonally (Vierstra, 1996). Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme, UBA), E2 (ubiquitin-conjugating enzyme, UBC), and E3 (ubiquitin ligase) (Kraft et al., 2005; Mukhopadhyay and Riezman, 2007; Smalle and Vierstra, 2004) (Fig. 1A). The E1 enzymes initiate the Ub conjugation cascade in an ATP-dependent reaction. Ubiquitin activated by an E1 enzyme forms what is known as an E1-ubiquitin intermediate. The activated ubiquitin is then transferred from the intermediate to the cysteine residue of the E2 enzyme. Finally, the E2–ubiquitin intermediate binds with E3 to deliver ubiquitin onto the substrate or E3 enzyme (Sadanandom et al., 2012; Smalle and Vierstra, 2004). The 3-step pathway creates an isopeptide bond between the C-terminal glycine of ubiquitin and a lysine residue on the target protein. A variety of substrate modifications are possible in the conjugation cascade, including the addition of a single ubiquitin molecule (monoubiquitination), the attachment of multiple ubiquitin molecules to different lysines on the same target protein (multiubiquitination), or the addition of different types of polyubiquitin chains (polyubiquitination). There are seven lysine residues (K6, K11, K27, K29, K31, K48, and K63) in ubiquitin, any of which are available for ubiquitin attachment to produce polyubiquitin chains (Kim et al., 2007; Kirkpatrick et al., 2006). The structure of the attached polyubiquitin chains seems to affect the fate of the target protein. K48-linked chains are the best-characterized type of polyubiquitin chains; they target substrate proteins for proteasomal degradation (Pickart and Fushman, 2004; Vierstra, 1996, 2009). Recent evidence indicates that K11 linked chains also commit target proteins for proteasomal degradation (Matsumoto et al., 2010). K63-linked ubiquitin chains are associated with both proteasome-independent cellular processes such as DNA repair, signal transduction, and receptor endocytosis

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(Hicke, 2001; Löfke et al., 2013; Pickart and Fushman, 2004), and proteasome-dependent pathways and serve as targeting signals for the 26S proteasome (Saeki et al., 2009). In Arabidopsis (Arabidopsis thaliana), there are 2 E1s, more than 37 E2s, and 1400 E3s. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123kDa proteins with 81% amino acid sequence identity; both contain a cysteine residue in the putative active site for forming the ubiquitin thio-ester intermediate (Hatfield et al., 1997). They transfer the activated ubiquitin to E2 with near equal specificity and are similarly expressed in almost all tissues (Hatfield et al., 1997). E2 enzymes contain a 140-amino-acid UBC domain with a conserved cysteinyl residue required for accepting the ubiquitin from E1 to form the E2–ubiquitin intermediate (Kraft et al., 2005; Wu et al., 2003). E2s interact with E3s via their UBC domains (Kraft et al., 2005; Wu et al., 2003). The vast number and diversity of E3 ubiquitin ligases in the Arabidopsis genome facilitate the identification of specific substrates by the UPS (Lee and Kim, 2011). There are seven known types of E3 ligases in plants which can be divided into two groups: the single-subunit E3 ligases and the multi-subunit E3 ligases (Lyzenga and Stone, 2012) (Fig. 1B and C). The singlesubunit E3 ligases can be further classified based on the presence of one of three domains: the Homology to E6AP C-terminus (HECT) domain, the U-box domain, or the Really Interesting New Gene (RING) domain (Fig. 1B). The HECT-type E3 enzymes comprise the smallest of the E3 subfamilies. In the Arabidopsis and rice (Oryza sativa) genomes, only seven and eight to nine HECT-type E3 genes, respectively, have been identified (Downes et al., 2003). HECT E3 ligases have a conserved C-terminal HECT domain with 350-residues that was initially identified in human E6AP (Huibregtse et al., 1995). HECT E3 ligases differ from other E3s during the Ub transfer processes. During Ub transfer, HECT E3 ligases generate an Ub–E3 intermediate on a conserved cysteine residue of the HECT domain, then the Ub–E3 intermediate

Fig. 1. The ubiquitination pathway and ubiquitin E3 ligases. (A) a ubiquitin activating enzyme (E1) activates ubiquitin in an ATP dependent manner. Ubiquitin is then tansferred to a ubiquitin conjugating enzyme (E2), forming an E2-ubiquitin intermediate. Ubiquitin ligase (E3) then recruits the substrate and attaches to the E2-ubiquitin intermediate, forming a complex. Ubiquitin is finally transferred from the E2–ubiquitin intermediate to an internal lysine of the substrate. After the initial ubiquitination of the substrate, additional ubiquitin can be added onto the already attached ubiquitin. (B) Single-subunit E3 ligases possess one of the three domains: Homology to E6-AP C-Terminus (HECT), U-box, or Really Interesting New Gene (RING). (C) Four types of cullin-based E3 ligases in plants. Cullins provide a platform around which the various complex subunits can assemble. ASK and DDB1 serve as adaptor proteins, attaching to the F-box and DWD substrate-recruiting proteins in CUL1/2- and CUL4-based E3s, respectively. APC11 provides the RING domain in the Anaphase Promoting Complex (APC) complex.

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transfers the Ub to the substrate protein (Fig. 1B). The N-terminal region of the HECT domain forms a stable binding pocket for the E2–Ub intermediate, and the C-terminal region contains the active site cysteine for Ub attachment (Huibregtse et al., 1995). Some motifs upstream of the HECT domains are thought to be responsible for substrate recognition and cellular localization; these include Armadillo, IQ calmodulin-binding, C-type lectin-binding, transmembrane, Ub-interacting motif (UIM), Ub-associated (UBA), and Ub-like (UBL) domains (Downes et al., 2003; Smalle and Vierstra, 2004). HECT E3 ligases are known to participate in plant development: AtULP3 is involved in trichome development and AtULP5 is a negative regulator in leaf senescence (Downes et al., 2003; Miao and Zentgraf, 2010). The structure of the U-box domain is similar to the RING domain, which is composed of 75 amino acids and forms a scaffold (Aravind and Koonin, 2000). Unlike the RING finger which is stabilized by Zn2+ ions, the U-box scaffold is assumed to be stabilized by saltbridges and hydrogen bonds. There are more U-box E3 genes in plant genomes than there are in other eukaryotic species. For example, there are 64 and 77 U-box genes in Arabidopsis and rice, respectively, as compared with eight in human and two in yeast (Yee and Goring, 2009). Like the HECT and RING domains, the U-box domain also plays an important role in determining the substrate specificity of the ubiquitination system in plants and these enzymes are known to function in various cellular processes such as hormone and abiotic stress signaling (Aravind and Koonin, 2000; Wiborg et al., 2008; Yee and Goring, 2009). However, U-box E3 proteins have a greater variety of protein-protein interaction domains than the HECT and RING proteins (Yee and Goring, 2009). The RING type E3 ligases comprise the largest subfamily of E3 single-subunit ligases. There are 469, 378, and 399 RING-type E3 ligase encoding genes in Arabidopsis, rice, and poplar (Populus trichocarpa), respectively (Du et al., 2009; Kraft et al., 2005). The representative structure of a RING domain contains a cross-brace structure that coordinates two zinc ions; this cross-brace is formed by eight metal ligand residues consisting of cysteine and histidine residues. This structure is required for ubiquitin ligase activity (Kosarev et al., 2002; Lorick et al., 1999). Spacing between the cysteine and histidine residues is usually conserved. In addition to functioning as single-subunit E3 ligases, the RING finger-containing proteins are also known to function as components of cullin-based multi-subunit E3 ligase complexes (Santner and Estelle, 2010). Multi-subunit E3s in plants are usually comprised of a RING finger protein RBX1 (RING-Box 1), a cullin protein, and a substraterecognition protein (Santner and Estelle, 2010). RBX1 is responsible for recruiting E2. Sometimes, RBX1 is replaced by APC11 in the anaphase-promoting complex (APC) E3s. Cullin, a scaffold-like protein, connects RING finger proteins to substrate-recognition proteins (Thomann et al., 2005). Multi-subunit E3s are known to function in a variety of complexes, including the Skp1-Cullin-F-box (SCF), CUL3-Bric a brac, Tramtrack and Broad complex/Pox virus and Zinc finger (CUL3-BTB), and CUL4-Damaged DNA Binding Protein 1 (CUL4-DDB1) complexs, as well as in APC (Lyzenga and Stone, 2012) (Fig. 1C). The SCF complex has four subunits: ASK (the Arabidopsis counterpart of SKP1 in animals), CUL1, the F-box protein, and RBX1. CUL1 is a scaffold protein complex with RBX1 attachment at the C-terminus that recruits E2 and an ASK adapter protein at the N-terminus to interact with the F-box motif. ASK serves as an adaptor protein that connects CUL1 with the F-box protein (Zheng et al., 2002). The F-box protein anchors to ASK via an N-terminal F-box motif (Kipreos and Pagano, 2000). The C-terminal protein-protein interaction motifs of the F-box protein target a specific substrate. Substrates include but not limited to leucine-rich repeats, lectin binding, Armadillo, and DEAD box proteins (Gagne et al., 2002; Sadanandom et al., 2012; Zheng et al., 2002). In Arabidopsis, CUL2 can also be a subunit of the SCF complex (Risseeuw et al., 2003).

In the CUL3-BTB and CUL4-DDB1 E3 complexes, CUL3 and CUL4 serve as scaffold proteins like CUL1 does in the SCF complex. BTB and CUL4-DDB1 are used in substrate recognition in the CUL3-BTB complex and the CUL4-DDB1 complex, respectively. In CUL4-based E3s, other interacting factors such as WD40-domain containing proteins DDB1-BINDING WD40 Protein (DWD) may be required for substrate recognition (Sadanandom et al., 2012; Santner and Estelle, 2010). The APC ligase complex contains 11 subunits including the cullin-like protein APC2, the RBX1-like protein APC11, and substrate-recruiting subunits including APC10, CDC20 (cell division cycle protein 20), and CDH1 (CDC20-homology 1) (Wang and Deng, 2011).

3. Roles of ubiquitination-mediated protein degradation in plant responses to abiotic stresses 3.1. High salinity and drought stress signaling is regulated by the UPS High salinity and drought are some of the major stresses that adversely affect plant growth and productivity. There are two signal transduction pathways triggered by salt and drought stresses based on their dependence on abscisic acid (ABA) (Takahashi et al., 2004) (Fig. 2A). Examples of ABA-dependent signal transduction pathways include the basic leucine zipper (bZIP)/ABA-responsive element (ABRE) system and the MYC/MYB transcription factors system (Abe et al., 1997; Fujita et al., 2005; Nakashima et al., 2009). It has been shown that the biosynthesis and accumulation of ABA increases under high salinity and drought stress conditions (Lyzenga and Stone, 2012; Nakashima et al., 2009). The bZIP and the MYC/MYB ABA-responsive transcription factors mediate the expression of downstream genes in this pathway (Hoth et al., 2002). In Arabidopsis, the bZIP family transcription factors bind to the ABRE cis-element to mediate the expression of ABA-responsive genes. For example, the expression of RD29B, AIL1, and RAB18 are known to be regulated by AREB1, AREB2 and ABF3 under salt and drought stress; the expression of these genes enhances salt and drought stresses tolerance (Fujita et al., 2005). MYC and MYB transcription factors activate the expression of the stress tolerance RD22 gene by binding its MYC- and MYB-recognition sequences (Abe et al., 1997). In addition to the ABA-dependent pathway, there is an ABA-independent pathway that functions in plant response to drought and high salt stresses. This pathway is controlled by the transcription factor DREB2A, which binds to the DRE cis-element in the promoter of downstream genes (Liu et al., 1998). A surprising number of E3s have been shown to be involved in the mediation of ABA signaling. The actions of several key transcription factors of ABA signaling such as ABI3/4/5, ABF1/3, and HB6, are controlled by the UPS. ABI5, a bZIP transcription factor, can be induced by ABA (Lopez-Molina et al., 2001). Under stress conditions, plant growth is delayed by the over accumulation of ABI5 (Lopez-Molina et al., 2003). The abundance of ABI5 is modulated by four E3 ligases: the RING E3 ligase named KEEP ON GOING (KEG) (Liu and Stone, 2013, 2014; Stone et al., 2006), the ABA-hypersensitive DCAF1 (ABD1) (Seo et al., 2014), and the two CUL4-DDB1 E3 ligases DWD hypersensitive to ABA1 (DWD1) and DWD2 (Lee et al., 2010). As a negative regulator of ABA signaling, KEG is responsible for maintaining low levels of ABI5 within the cytoplasm under normal conditions (Liu and Stone, 2013; Stone et al., 2006). Under stress conditions, ABA reduces KEG protein levels by inducing ubiquitination and proteasomal degradation of KEG, which inversely promote the accumulation of ABI5 to eventually delay plant growth and enhance stress tolerance (Liu and Stone, 2010). Further studies have suggested that KEG interacts directly with ABI5 in the cytoplasm and in the trans-Golgi network via its


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Fig. 2. The ubiquitin–proteasome system (UPS) and sumoylation regulated drought, salt, and cold signaling pathways in Arabidopsis. (A) UPS regulates drought and saltsignaling pathways. ABA-regulated transcription factors including ABI3/4/5, ABF1/3, and HB6, are modulated by the UPS. The ABA-independent DREB2A is also regulated by an E3 ligase. (B) The UPS-mediated cold signaling pathway. ICE1, a critical transcription factor in a cold signaling pathway, is modified by the RING E3 ligase HOS1 and the SUMO E3 ligase SIZ1.

conversed C3 region (Liu and Stone, 2013). Lysine 344 is critical for protein turnover of ABI5. Deletion of a nuclear localization signal (NLS) of ABI5 caused the accumulation of ABI5 in cytoplasm in the K344 mutant (Liu and Stone, 2013). ABD1, a DCAF protein, functions as a CUL4-DDB1 E3 ligase substrate receptor that negatively regulates ABA signaling (Seo et al., 2014). ABD1 is induced by ABA and interacts with DDB1 and ABI5 both in vitro and in vivo. Mutation in ABD1 leads to hypersensitivity to ABA and drought tolerance, increased accumulation of ABI5, and reduced water loss. Two Arabidopsis DWD protein components of CUL4-based E3 ligases, DWA1 and DWA2, as negative regulators of ABA signaling, attenuate ABA signaling by degrading ABI5 and help in reestablishing plant growth once environmental conditions have improved (Lee et al., 2010). DWA1 and DWA2 can interact with each other, and dwa1 dwa2 mutants exhibit drought tolerance in adult plants (Lee et al., 2010). DWA3 is also a negative regulator of ABA signaling; loss of DWA3 increases the ABA sensitivity of the mutant plant (Lee et al., 2011). In dwa3 mutant, ABI5 and AtMYC2 are accumulated and the expression of RD29A, RD29B, and RD22 is hyper-induced. However, ABI5 and AtMYC2 may not be the direct targets of CUL4-DDB1-DWA3 in ABA responses, and DWA3 cannot interact with DWA1 or DWA2 (Lee et al., 2011). Additionally, the ABI five binding protein (AFP) was reported to promote the degradation of ABI5 under ABA treatment (Lopez-Molina et al., 2003). KEG is also involved in regulating ABF1 and ABF3 (Chen et al., 2013). The degradation of ABF1 and ABF3 are affected in keg mutant. The ubiquitination of ABF1 and ABF3 by KEG was observed in vitro, and their direct interaction has been demonstrated. The loss-of-function mutants of ABF1 or ABF3 in the keg background display a similar phenotype to the mutant of abi5. These results suggest that ABF1 and ABF3 are substrates of KEG. In keg seedlings, the degradation of ABFs still occurs, which indicates that additional E3s take part in ABF1 and ABF3 proteolysis (Chen et al., 2013). ABI3, a B3-type transcription factor which is an unstable protein, disappears after seed germination and is known to participate in the establishment of desiccation tolerance and dormancy during zygotic embryogenesis (Lopez-Molina et al., 2001; LopezMolina et al., 2002). ABI3 functions upstream of ABI5 to mediate

ABA-dependent processes; it interacts with the RING-type E3 ABI3-interacting Protein 2 (AIP2) (Kurup et al., 2000). ABA promotes the expression of AIP2 which reduces ABI3 protein levels and attenuates ABA signaling (Zhang et al., 2005). AIP2 can ubiquitinate ABI3 in vitro. Transgenic plants overexpressing AIP2 displayed a similar phenotype to abi3: a lower level of ABI3 protein than wild type, and greater resistance to ABA. This result indicates that AIP2 functions in keeping ABI3 levels in check (Zhang et al., 2005). ABI4 participates in ABA signaling in maturing seeds as well as in seedling responses to both ABA and salt (Finkelstein et al., 2002; Finkelstein et al., 1998). Under salt stress, ABI4 downregulates the expression of the sodium transporter HKT1;1 by directing binding to its promoter (Shkolnik-Inbar et al., 2013). The abi4 mutant contained lower levels of sodium ions and higher levels of proline than wild-type plants and displayed salt tolerance. On the contrary, overexpression of ABI4 caused hypersensitivity to salt stress (Shkolnik-Inbar et al., 2013). Although there is no direct evidence to suggest that ABI4 is degraded by the UPS, a recent study showed accumulation of ABI4 after treatment with proteasome inhibitors (Finkelstein et al., 2011). HB6, an ABA induced transcription factor and a negative regulator of ABA signaling, is also modulated by CUL3BPMs , a group of CUL3-based E3 ligases containing MATH-BTB substrate-binding adaptors (Lechner et al., 2011). CUL3BPM silencing by artificial microRNAs leads to HB6 protein accumulation. Reduced expression of CUL3BPMs and HB6 overexpression led to ABA insensitivity as well as increased water loss and enhanced stomatal opening. MATH-BTB proteins directly target HB6 for proteasomal degradation (Lechner et al., 2011). In addition to the aforementioned E3 ligases which participate in degrading the transcription factors of ABA signaling, there are many other E3s that have been implicated in ABA signaling, though the direct substrates have been identified for only few of these. The Arabidopsis XERICO gene encodes a RING-H2 zinc finger protein that interacts with an E2 ubiquitin-conjugating enzyme AtUBC8 and an ASK1-interacting F-box protein AtTLP9 and positively regulates ABA biosynthesis and drought tolerance in Arabidopsis (Ko et al., 2006). Heterologous overexpression of XERICO enhanced drought

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and salt stress tolerance in rice. Under stress conditions, rice plants overexpressing XERICO had increased ABA content and increased expression levels of the ABA biosynthesis or ABA-response genes OsNCED, OsABA3, OsABI5, and OsLEA3-1 (Zeng et al., 2013). The RING-type E3 ligase SALT AND DROUGHT-INDUCED RING FINGER1 (SDIR1) is a positive regulator of stress-responsive ABA signaling in Arabidopsis (Zhang et al., 2007). OsSDIR1, a rice ortholog of SDIR1, enhances drought tolerance in transgenic rice via less water loss and hypersensitivity to ABA (Gao et al., 2011). The homolog of SDIR1 in Zea mays, ZmRFP1, also functions as a positive regulator of ABA signaling during drought stress. Overexpression of ZmRFP1 enhanced drought tolerance in tobacco with phenotypes that included enhanced stomatal closure and an increase in proline accumulation, though it had reduced levels of malondialdehyde (MDA) (Liu et al., 2013). The tabacco ortholog of SDIR1, NtRHF1, also participates positively in drought stress response in tobacco (Xia et al., 2013). The Arabidopsis ABA-insensitive RING protein 1 (AtAIRP1), a C3H2C3-type RING E3 ligase, is induced by ABA and drought stress. Plants with overexpression of AtAIRP1exhibit drought tolerance and are hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure (Ryu et al., 2010). Another study suggests that the Arabidopsis RING E3 ligase AtAIRP2 plays complementary roles with AtAIRP1 in ABA-mediated drought stress responses (Cho et al., 2011). AtAIRP2 also functions as a positive regulator of ABA and drought stress. Plants overexpressing AtAIRP2 are highly tolerant to severe drought stress and are hyposensitive to ABA in terms of seed germination and stomatal closure (Cho et al., 2011). The transcription of AtAIRP3/LOG2, a RING E3 ubiquitin ligase, is induced by ABA and by drought and salt stress. It is involved not only in the amino acid export system but also in the ABA mediated stress response in Arabidopsis through degrading RD21 (Kim and Kim, 2013a). The Arabidopsis MBP-1-like protein (AtMBP-1) is a positive regulator of ABA responses; the proteasome-dependent degradation of AtMBP-1 is mediated by AtSAP5, a zinc finger protein with E3 ligase activity (Kang et al., 2013). The Arabidopsis RING-H2 E3 ligase RHA2a and its close homolog RHA2b, function redundantly in positively regulating ABA responses. The actions of RHA2a and RHA2b in ABA signaling occurs downstream of ABI2, and is independent of ABI3/4/5 action (Bu et al., 2009; Li et al., 2011). Our previous work implicated SKP1 in an ABA signaling pathway. The Arabidopsis SKP1 homologues ASK1 and ASK2 positively regulate ABA signaling downsteam of ABI5. Overexpression of Triticum aestivum SKP1-like 1 (TSK1) in Arabidopsis resulted in delayed seed germination and hypersensitivity to ABA (Li et al., 2012). Two RINGDUF1117 containing E3 ubiquitin ligases, AtRDUF1 and AtRDUF2, were identified; they participate in the positive regulation of ABAdependent drought and salt stress responses in Arabidopsis (Kim et al., 2012; Li et al., 2013). AtRDUF1 and AtRDUF2 are upregulated by ABA and dehydration. Plants with double knock-out mutation of the AtRDUFs displayed hyposensitivity to ABA and had reduced tolerance to drought and salt stresses. AtRDUF1 overexpressing plants had enhanced tolerance to salt stress and increased expression levels of RD29B, RD22, and KIN1 (Li et al., 2013). Some E3 ligases are known to be negative regulators of ABA signaling. Arabidopsis thaliana PUB18 (AtPUB18) and AtPUB19 are U-Box E3 ligases that function as negative regulators of ABA signaling. pub18 and pub19 mutant plants showed hypersensitivity to ABA, enhanced stomatal closing and enhanced drought tolerance; these proteins are known to affect the ABA signaling pathway downstream of hydrogen peroxide and upstream of calcium (Liu et al., 2011; Seo et al., 2012). AtPUB22 and AtPUB23 are U-box E3 ligases that negatively regulate drought responses. AtPUB18 has an agonistic function with AtPUB19 in the negative regulation of ABAmediated drought stress responses, but is independent of AtPUB22 and AtPUB23 (Seo et al., 2012). The Drought Tolerance Repressor

(DOR) F-box protein interacts with ASK14 and CUL1 and functions in the negative regulation of ABA signaling. Knock-out of DOR led to a hypersensitive ABA response and enhanced drought tolerance (Zhang et al., 2008). The expression level of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1), a RING E3 ligase, is known to be reduced by drought stress (Ju et al., 2013). AtRZF1-overexpressing plants displayed greater sensitivity to drought with increased water loss and membrane ion leakage. Contrastingly, an atrzf1 mutant enhanced drought tolerance, suggesting that AtRZF1 participates negatively in drought responses (Ju et al., 2013). DET1-DDB1-ASSOCIATED1 (DDA1) provides substrate specificity for CUL4-RING E3 ligases by binding to the ABA receptors PYL8, PYL4, and PYL9 to regulate their stability through the proteasomal degradation pathway and thus negatively regulates ABA-mediated developmental responses (Irigoyen et al., 2014). DREB2A interacts with the RING E3 ligases DREB2AINTERACTING PROTEIN1 (DRIP1) and DRIP2 in the ABA-independent drought and high salt stress response pathways. Loss of DRIP1 and DRIP2 results in the accumulation of DREB2A; the mutant plants showed higher tolerance to drought than did wild type plants (Qin et al., 2008). OsDIS1 (Oryza sativa drought-induced SINA protein 1), a SINA type E3 ligase, also plays a negative role in drought stress tolerance by interacting with OsNek6 and OsSKIPa. Under drought conditions, a large number of drought-responsive genes are repressed in the OsDIS1 overexpression plants; the rice transgenic lines exhibited a reduced drought tolerance phenotype (Ning et al., 2011a, 2011b). Currently, not much is known about the involvement of E1 and E2 proteins during plant abiotic stress tolerance. Recent studies have indicated that several E2 ubiquitin conjugating enzymes participate in the regulation of salt and drought stress tolerance. Arabidopsis plants overexpressing soybean GmUBC2 had increased tolerance to salinity and drought stresses (Zhou et al., 2010). The expression of the peanut ubiquitin-conjugating enzyme gene AhUBC2 is up-regulated by salt, ABA, and low temperature, and constitutive expression of AhUBC2 in Arabidopsis confers improved drought stress (Wan et al., 2011). Over-expression of the mung bean gene VrUBC1 in Arabidopsis enhanced plant osmotic stress tolerance via enhancing ABA- or salt-induced stomatal closing (Chung et al., 2013). AtUBC32 is a stress-induced E2 localized to the ER membrane; genetic and physiological data have shown that it plays a role in brassinosteroid-mediated salt stress tolerance (Cui et al., 2012).

3.2. Temperature stress signaling is mediated by the UPS Temperature stress induced by excessive heat or cold can affect plant growth, cause injury or death, and seriously reduce crop productivity (Sanghera et al., 2011). The UPS signal pathway is involved in plant responses to temperature stress (Fig. 2B). In Arabidopsis, C-repeat (CRT)-binding factors (CBFs) belonging to the AP2/ERF superfamily (including CBF1, CBF2, and CBF3), bind to cis-element CRT/DRE (CCGAC) and regulate the expression of cold responsive genes (Liu et al., 1998; Stockinger et al., 1997). ICE1 (Inducer of CBF expression 1) is a MYC transcription factor that belongs to the bHLH superfamily. It activates the expression of CBF3 in response to low temperature through binding to the MYC elements in the CBF3 promoter (Chinnusamy et al., 2003). The UPS also regulates low temperature signaling by modifying ICE1 (Dong et al., 2006; Lourenço et al., 2013). HOS1 (high expression of osmotically responsive gene 1), a RING E3 ligase, physically interacts with ICE1 and mediates the ubiquitination and degradation of ICE1. CBFs and downstream low temperature signaling genes had higher expression in the Arabidopsis hos1 mutant than in wild-type plants (Dong et al., 2006). In rice, OsHOS1 interacts with OsICE1. OsDREB1A is


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Fig. 3. A working model for the role of CUL4-DDB1A-DDB2 in DNA repair via the nucleotide excision repair (NER) pathway. (1) Upon UV-stress, DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner to recruit DDB2. (2) Recognition and binding of DNA lesions by DDB2. (3) DDB2 ubiquitination. (4) NER machinery recruitment and degradation of DET1 and DDB2. Proteolysis of DDB2 and DET1 may allow their eviction from chromatin to enable the excision repair process to occur efficiently.

more highly expressed in RNAi::OsHOS1 plants than in wild type plants (Lourenço et al., 2013). In addition to HOS1, several other UPS members are known to take part in the regulation of temperature stress signaling. These include AtATL78, AtCHIP, OsHCI1, TaUb2, and BnTR1. AtATL78, an Arabidopsis RING E3 ligase plays opposing roles in cold and drought stress responses. AtATL78 expression is induced by cold but suppressed by drought. Suppression of AtATL78 enhanced cold tolerance but reduced drought tolerance (Kim and Kim, 2013b). AtCHIP, a U-box-type E3 ligase similar to the animal CHIP protein, is induced by both low and high temperature and targets non-native or damaged proteins for degradation by the 26S proteasome. Overexpression of AtCHIP resulted in high sensitivity to both low- and high-temperature treatments and had increased electrolyte leakage under chilling treatment (Yan et al., 2003). Further research showed that AtCHIP interacts with a subunit of protein phosphatase 2A (PP2A), which appeared to be a substrate of AtCHIP (Luo et al., 2006). Two chip knockout mutant plants displayed tolerance phenotypes to heat, oxidative and salt stresses with increased accumulation of insoluble proteins under heat stress. These results are similar to those observed in the nbr1 mutant. It has also been suggested that CHIP and NBR1 function in distinct but complementary pathways to protect plant against proteotoxicity via selective autophagy (Zhou et al., 2014). Oryza sativa heat and cold induced 1 (OsHCI1) function as a RING E3 ligase that is induced by heat and cold stresses treatments but not by salinity or dehydration. At room temperature, OsHCl1 is mainly localized in the Golgi apparatus. Under heat treatment, OsHCI1 accumulates in the nucleus and interacts with OsbHLH065, OsGRP1, and OsPGLU1, mediating their inactivation through nuclear-cytoplasmic trafficking (Lim et al., 2013). Heterologous overexpression of the wheat monoubiquitin gene TaUb2 in tobacco confers chilling tolerance with lower levels of MDA, electrolyte leakage, and less accumulation of reactive oxygen species (ROS) in sense plants. The sense plants also exhibited increased accumulation of proline and solute sugar and greater antioxidant enzyme activity (Feng et al., 2014). BnTR1, a membrane-bound RINGv (C4 HC3 ) E3 ligase identified from Brassica napus, enhances plant heat stress responses, likely via regulation of Ca2+ dynamics by regulating the activity of calcium channels (Liu et al., 2014).

3.3. Cullin-RING ligases (CRLs) participate in plant responses to UV stress Ultraviolet B (UV-B, 280–315 nm) is an intrinsic part of sunlight that induces significant biological effects. UV-B stress primarily causes unspecific damage responses in living organisms (Tilbrook et al., 2013). UV-B impairs DNA metabolism by forming cyclobutane pyrimidine dimmers (CPD) and pyrimidine–pyrimidone (6-4) photoproducts (6-4PP) (Cadet et al., 1992; Clingen et al., 1995). Plants repair DNA damage through two main mechanisms, photoreactivation and nucleotide excision repair (NER) (Tuteja et al., 2009). Plant tolerance to UV stress is positively regulated by two E3s: CUL4-DDB1A-DDB2 and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). CUL4-DDB1A-DDB2 participates in DNA repair through the NEB pathway (Fig. 3) (Castells et al., 2011; Molinier et al., 2008; Wittschieben et al., 2005). COP1 is responsible for regulating UV signaling by mediating the expression of HY5 which is strictly regulated by the UV RESPONSE LOCUS 8 (UVR8)-COP1 interaction (Fig. 4) (Favory et al., 2009; Yi and Deng, 2005). The CUL4-DDB1A-DDB2 CRL E3 is a protein complex comprised of the subunits DDB1A and DDB2. The DDB1A-DDB2 complex functions in the initial detection of UV lesions in vivo (Wittschieben et al., 2005). Upon UV stress, DDB1A shuttles from the cytoplasm to the nucleus to degrade DDB2 with the help of ATR kinase (Molinier et al., 2008). DDB2 is localized to the nucleus where it recognizes and binds DNA lesions. DE-ETIOLATED 1 (DET1) is required for DDB2’s degradation by CUL4-DDB1A, as DDB2 is not degraded in det1 mutant plants under UV treatment (Castells et al., 2011) (Fig. 3). Degradation of DDB2 and DET1 by the UPS may allow their eviction from chromatin to enable the excision repair process to occur efficiently (Castells et al., 2011; Molinier et al., 2008). The RING E3 ligase COP1 is a negative regulator of photomorphogenesis that is under the negative control of white light (WL)-activated phytochromes (PHY) and cryptochromes (CRY) (Yi and Deng, 2005). COP1 represses photomorphogenesis in the form of CUL4-DDB1-COP1-(SUPPRESSOR OF PHYA (SPA) E3 ligase complexes, which targeting HY5 for degradation (Chen et al., 2010; Osterlund et al., 2000). COP1 is a UV-B-inducible gene whose expression in response to UV-B is positively regulated by the FHY3

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3.4. The UPS plays essential roles in plant responses to nutrient stresses

Fig. 4. A model for the functional interaction of COP1, UVR8, RUP1, RUP2, and HY5 in ultraviolet B (UV-B) signaling. Under white light (WL) devoid of UV-B, COP1 represses photomorphogenesis by degrading HY5 in the form of CUL4-DDB1-based E3 ligase complexes, though COP1 is under the negative control of light-activated phytochromes (PHY) and cryptochromes (CRY). Under UV-B conditions, UVR8 monomers accumulate in the nucleus and interact with COP1, inhibiting degradation of HY5 and triggering downstream signaling. In addition to activating the transcription of downstream genes, HY5 also binds to the promoter of COP1 to activate COP1 transcription. RUP1 and RUP2 interact with the UVR8-COP1 complex to mediate redimerization of UVR8 and the release of COP1.

and HY5 transcription factors; these transcription factors directly bind to distinct regulatory elements within the COP1 promoter (Huang et al., 2012). Under WL conditions devoid of UV-B, UVR8 is present mainly as a homodimer. Upon UV-B irradiation, UVR8 monomerizes and interacts with the C-terminal WD40-repeat domain of COP1, while the CUL4-based COP1-SPA E3 apparatus tends to diminish, thus the downstream transcription factor HY5 is stabilized, and UV-B-responsive genes are activated (Favory et al., 2009; Heijde and Ulm, 2013; Huang et al., 2013). Signaling pathways usually encompass negative feedback loops that serve to balance the signaling response (Brandman and Meyer, 2008). REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 are transcriptionally activated by UV-B in a UVR8-, COP1- and HY5dependent manner. rup1 rup2 double mutant plants showed an enhanced UV-B response and elevated UV-B tolerance. Conversely, RUP2 overexpression confers reduced UV-B response and hypersensitivity to UV-B stress. RUP1 and RUP2 were shown to interact directly with UVR8. These results suggested that RUP1 and RUP2 act downstream of UVR8-COP1 in a negative feedback loop that impinges on UVR8 function and balances UV-B defense (Gruber et al., 2010).

Nutrient availability is one of the most important factors influencing plant metabolism and development. It is clear that the UPS is involved in the uptake, transport, and balancing of many nutrients. Investigation into the roles of the UPS in nutrient utilization is crucial for efforts to improve crop production efficiency. Iron (Fe) is a plant micronutrient and a limiting factor for plant growth that is required for many cellular processes including photosynthesis, hormone synthesis, respiration, and DNA synthesis (Curie and Briat, 2003). Iron homeostasis needs to be tightly regulated, as high iron levels are highly toxic to cells (Briat and Lebrun, 1999; Curie and Briat, 2003). IRON-REGULATED TRANSPORTER1 (IRT1), a critical protein for uptake of iron, plays an essential role in maintaining iron homeostasis in Arabidopsis. The loss-of-function irt1 mutant displays chlorotic symptoms and a growth arrest phenotype (Vert et al., 2002). IRT1 is induced by Fe deficiency. It has been shown that IRT1 is monoubiquitinated before being sent for vacuolar degradation, which is a type of proteasome-independent protein turnover (Barberon et al., 2011). IRT1 DEGRADATION FACTOR1 (IDF1), a RING E3 ligase, mediates the degradation of IRT1 by binding IRT1 in the plasma membrane. The UPS mediated degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe uptake (Shin et al., 2013). It has been shown that SORTING NEXIN1 (SNX1) modulates the trafficking and stability of IRT1, which is colocalized with IRT1. SNX1 loss-of-function plants display a hypersensitive response to iron deficiency with reduced iron import efficiency into the root (Ivanov et al., 2014). IRT1 is the target of transcription factors FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT), AtbHLH38, and AtbHLH39, and is directly regulated by FIT/AtbHLH38 or FIT/AtbHLH39 complexes (Yuan et al., 2008) (Fig. 5A). The iron-deficiency-inducible bHLH transcription factor Popeye (PYE) interacts with its homolog IAA-Leu Resistant3 (ILR3), and both are known to be involved in maintaining iron homeostasis (Long et al., 2010; Rampey et al., 2006). Loss of PYE function causes

Fig. 5. Working models for the roles of UPS and sumoylation in iron (Fe)- and phosphorus (Pi)-deficiency-signaling pathways. (A) Under Fe deficiency conditions, FIT actives the expression of IRT1 by forming a complex with the bHLH protein. Overexpression of IRT1 enhances iron uptake. IRT1 is degraded by E3 ligase IDF1 and stabilized by SNX1. PYE and BTS interact with ILR3 and have opposite effects on the regulation of the PYE target genes ZIF1, FRO3, and NAS4. (B) Under Inorganic Pi deficiency, PHR1, which is itself sumoylated by SIZ1, modulates phosphate starvation-induced (PSI) gene expression to enhance Pi uptake and transport; this process is regulated by the E2 enzyme PHO2. PHO2 is targeted by miR399, which is induced by PHR1. The PSI gene AtIPS1 and At4 repress miR399 through a target mimicry mechanism, thus affecting the mRNA level of PHO2. PHO2 modulates the degradation of PHO1.


increased and prolonged expression of FRO3, NAS4, and ZIF1, which encode proteins involved in metal ion homeostasis, and are direct targets of PYE (Long et al., 2010). ILR3 interacts with a putative E3 ligase BRUTUS (BTS), which negatively regulates plant responses to iron deficiency (Long et al., 2010). As such, it has been proposed that BTS influences the stability of ILR3 and indirectly affects PYE during iron deficiency (Long et al., 2010) (Fig. 5A). The Fe deficiency response is also mediated by E2. Ectopic expression of the cucumber UBC13 gene in Arabidopsis led to a more pronounced and Fe-responsive formation of branched root hairs (Li and Schmidt, 2010). Phosphorus is indispensable for photosynthesis, energy metabolism, and carbon assimilation in plants. Organic phosphate is abundant in soil but is less soluble and less available to plants. Inorganic phosphate (Pi) is accessible to plants but natural soils generally contain low concentrations of Pi. Pi deficiency deeply affects plant growth, development, and metabolism (Ticconi and Abel, 2004). pho2 is a Pi-overaccumulator mutant with enhanced uptake and root-to-root translocation of Pi. pho2 is defective in UBC24, which encodes a putative E2 enzyme (Aung et al., 2006; Bari et al., 2006). In Pi deficiency conditions, the transcription factor PHR1 upregulates miR399, which targets the 5 UTR of PHO2 for cleavage. PHO2 represses the expression of a subset of phosphate starvation-induced (PSI) genes including AtIPS1, At4, and Pi transporters Pht1;8 and Pht1;9 (Bari et al., 2006). AtIPS1 and At4 contain a motif that is partially complementary to miRNA399. The imperfect pairing of AtIPS1/At4 with miRNA399 results in the sequestration of miRNA399, thus affecting miR399-guided PHO2 cleavage (Franco-Zorrilla et al., 2007) (Fig. 5B). It was recently discovered that PHO2 modulates the degradation of PHOSPHATE1 (PHO1), which is involved in Pi loading to the xylem (Liu et al., 2012) (Fig. 5B). Nitrogen, an essential macronutrient, plays a critical role in plant growth and development. NITROGEN LIMITATION ADAPTATION (NLA), a RING-type E3 ligase is a regulator of plant adaptive response to nitrogen limitation. The loss-of-function nla mutant displayed an early senescence phenotype when supplied with insufficient inorganic nitrogen (Peng et al., 2007). A detailed analysis showed that the early senescence phenotype in the nla mutant was caused by Pi toxicity. Upon Pi deficiency, NLA is posttranscriptionally downregulated by miR827 (Kant et al., 2011). The results demonstrated that NLA mediated-ubiquitination plays an important role in the regulation of both Pi and nitrogen metabolism. Plants are able to respond to the balance between carbon and nitrogen availability; this is referred to as the C/N response (Coruzzi and Zhou, 2001). The C/N response is regulated by the RING-type E3 ligase CN1/ATL31 and its homologs, ATL6 and ATL2 (Sato et al., 2009). The post-germinative growth of ATL31 knock-out mutant was arrested under conditions with a high C/N ratio, while ATL31 overexpression plants had a suppressed response to high C/N conditions (Sato et al., 2009). Boron is an indispensable nutrient in the formation of plant cell wall components (O’Neill et al., 2001). A boric acid/borate exporter BORON TRANSPORTER1 (BOR1), and a boric acid channel protein NIP5 have been shown to be indispensable for efficient boron uptake and subsequent transport in roots (Takano et al., 2002; Takano et al., 2006). In low boron conditions, NIP5 and BOR1 are localized to the outer and the inner plasma membrane domains, respectively, of root cells, and facilitate the transport of boron (Takano et al., 2010). In high boron conditions, BOR1 is selectively transported to the endosomes for degradation, preventing boron toxicity (Takano et al., 2010). There are hints of the involvement of the UPS in the transport and homeostasis of boron, although the precise degradation mechanism of BOR1 remains uncharacterized.

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4. Sumoylation-mediated plant responses to abiotic stresses SUMO is a small ubiquitin-like modifier that conjugates the SUMO superfamily of proteins to various substrates (sumoylation) (Johnson, 2004). Sumoylation affects both positive and negative regulation, acting via its roles in protein–protein interaction, protein subcellular localization, enzymatic activity, and protein stability. In Arabidopsis, several abiotic stress responses are mediated by SIZ1, a SUMO E3 ligase that transfers the SUMO peptide to the substrate. SIZ1 participates in cold acclimation by targeting ICE1 for sumoylation. Sumoylation leads to the activation and/or stabilization of ICE1, thereby facilitating the expression of CBF3 (which is downstream of ICE1) and thus improves low temperature tolerance (Fig. 2B) (Miura et al., 2007). The siz1 mutation enhanced the ABA-mediated inhibition of seed germination and primary root growth (Miura et al., 2009). SIZ1 was found to attenuate ABA signaling by facilitating the sumoylation of ABI5 (Fig. 2A). Sumoylated ABI5 is inactive and resistant to proteasome-dependent degradation (Miura et al., 2009). SIZ1 also mediates the sumoylation of MYB30, a transcription factor that functions in parallel pathway with ABI5 (Zheng et al., 2012). The SIZ1-dependent sumoylation of MYB30 and ABI5 may balance the gene expression required for regulation of ABA signaling during germination (Zheng et al., 2012). SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance (Miura et al., 2012). Data demonstrates that this is through the control of salicylic acid-induced accumulation of reactive oxygen species. Mutations in SIZ1 also reduce the uptake and accumulation of the toxic ion Na+ , leading to enhanced tolerance to salt stress (Miura et al., 2011). After being transported from the soil into cells, nitrate is reduced sequentially to ammonia in reactions that are catalysed by nitrate reductase (NR) (Solomonson and Barber, 1990; Tanaka et al., 1994). The assimilation of nitrogen is positively regulated by SIZ1 through promoting sumoylation of NRs (NIA1 and NIA2), which become active after sumoylated (Park et al., 2011). Decreased NR activity was observed in siz1-2 plants. The modulator of low Pi-induced responses, PHR1, is sumoylated and may be activated by SIZ1 (Fig. 5B). The sumoylation of PHR1 positively controls the expression of its regulon (AtIPS1 and AtRNS1) (Miura et al., 2005). Copper (Cu) is essential for plant growth but is needed in only small quantities. Excess Cu in the plant is toxic because of its high redox activity. Plants have evolved multiple mechanisms to avoid such toxicity. SIZ1-mediated sumoylation is involved in Cu homeostasis and tolerance in plants (Chen et al., 2011). siz1 mutant plants are hypersensitive to excess Cu and show an anomalous distribution of Cu. Under excess Cu stress, the siz1 mutant is unable to down-regulate the expression of the metal transporters YSL1 and YSL3. 5. Crosstalk between ubiquitination and other posttranslational modifications (PTMs) in the mediation of plant responses to abiotic stresses In addition to ubiquitination and sumoylation, many other types of reversible PTMs exist. Examples include phosphorylation, acetylation, methylation, and O-GlcNacylation. Crosstalk between PTMs can be either positive or negative (Hunter, 2007). The autoubiquitination of E3 ligases can inhibit their E3 ligase activity. For example, ABA negatively regulates the KEG protein level by inducing KEG autoubiquitination and subsequent proteolytic degradation, thus decreasing KEG E3 activity toward ABI5 (Liu and Stone, 2010). The inactive form of ABI5 is protected by sumoylation from degradation through the proteasome pathway (Miura et al., 2009). Phosphorylation can regulate ubiquitination by regulating the activity of E3 ligases, promoting recognition by an E3 ligase, and

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can influence ubiquitination by regulating substrate/ligase interactions at the level of cellular compartmentalization (Hunter, 2007). KEG contains a kinase domain, its phophorylation (probably selfphosphorylation) is required for ABA-induced autoubiquitination and degradation (Liu and Stone, 2010). ATL31 targets 14-3-3 proteins for ubiquitination and modulates protein abundances in response to C/N-nutrient status (Sato et al., 2011). Phosphorylation at 14-3-3 binding sites on the E3 ligase ATL31 is required for interaction with the 14-3-3 proteins (Yasuda et al., 2014). MPK1 is a key regulator of plant responses to UV-B stress (Gonzalez Besteiro et al., 2011). MPK1 is continuously turned over under non-stress conditions, but is phosphorylated and stabilized in response to UVB stress (Besteiro and Ulm, 2013). In addition, phosphorylation may integrate with sumoylation in controlling NR activity (Park et al., 2011). While phosphorylation is known to regulate ubiquitination, ubiquitination is known to regulate protein kinase activity (thus affecting phosphorylation). Calcineurin B-like Interacting Protein Kinase (CIPK) 26 is involved in ABA signaling; it is targeted by KEG for degradation (Lyzenga et al., 2013). CIPK26 is capable of phosphorylating ABI5 in vitro (Lyzenga et al., 2013). 6. Conclusion and future perspectives Plants face a variety of abiotic stress conditions throughout their life cycles. Abiotic stresses impair plant growth and development and seriously reduce crop productivity (Fujita et al., 2005; Hirayama and Shinozaki, 2010). To date, many stress signal pathways have been revealed through the use of multiple genetic and biochemical approaches, though more details about the mechanisms of plant proteomic plasticity need to be uncovered. There has been remarkable progress in defining the components and functions of the UPS in plant responses to abiotic stress. The Arabidopsis genome contains more than 1300 genes predicted to encode E3 ligases (Smalle and Vierstra, 2004), and many of the E3 ligases involved in plant responses to abiotic stresses have been identified, but only limited information about their target proteins is known. Discovering the substrates of E3 ligases is challenging, since the Ub conjugates are transient and present at extremely low levels. By affinity purification of ubiquitinated proteins using GST tagged ubiquitin binding domains, potential ubiquitination targets were isolated; a total of 294 proteins were identified using a multidimensional protein identification technology system (Maor et al., 2007). In another study performed by Elrouby and Coupland (2010), 230 putative SUMO substrates were identified by yeast two-hybrid screening using SUMO-conjugating enzyme and SUMO protease as bait. A two-step affinity approach was developed for target identification, in which a transgenic Arabidopsis line expressing 6His-UBQ was employed (Saracco et al., 2009). The Ub conjugates were initially enriched using the Ub-binding region of human HHR23A, followed by nickel-chelate affinity chromatography. The efficiency of this method was improved with the use of high-sensitive mass spectrometry and an improved two-step affinity purification; these methods led to the identification of 950 ubiquitination targets (Kim et al., 2013). Another strategy detects the thioester bonds between E2 and a subclass of E3, or E3 and its substrate, thus allowing investigation of E2/E3 or E3/substrate specification (Zhao et al., 2012). These methods now make target identification more straight forward. Studying the UPS targets and their functions will offer broader insight into how plants maintain and regulate the proteomic plasticity that is necessary for proper responses to unfavorable conditions. Acknowledgments We are grateful to the editor and the reviewer for their critical comments on the manuscript. This work has been funded by the

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