ERAD-related E2 and E3 enzymes modulate the drought response by regulating the stability of PIP2 aquaporins

Endoplasmic reticulum-associated degradation (ERAD) is known to regulate plant responses to diverse stresses, yet its underlying molecular mechanisms and links to various stress signaling pathways are poorly understood. Here, we show that the ERAD component ubiquitin-conjugating enzyme UBC32 positively regulates drought tolerance in Arabidopsis thaliana by targeting the aquaporins PIP2;1 and PIP2;2 for degradation. Furthermore, we demonstrate that the RING-type ligase Rma1 acts together with UBC32 and that the E2 activity of UBC32 is essential for the ubiquitination of Rma1. This complex ubiquitinates a phosphorylated form of PIP2;1 at Lys276 to promote its degradation, thereby enhancing plant drought tolerance. Extending these molecular insights into crops, we show that overexpression of Arabidopsis UBC32 also improves drought tolerance in rice (Oryza sativa). Thus, beyond uncovering the molecular basis of an ERAD-regulated stress response, our study suggests multiple potential strategies for engineering crops with improved drought tolerance.

The ubiquitin-conjugating enzyme UBC32 enhances drought tolerance by cooperating with the RING-type E3 ligase Rma1 to ubiquitinate a phosphorylated form of PIP2;1 at Lys276 to promote its degradation.     

Apple RING finger E3 ubiquitin ligase MdMIEL1 negatively regulates salt and oxidative stresses tolerance

RING-finger-containing E3 ubiquitin ligases play important roles in plant response to biotic and abiotoc stresses. In this study, through homology analysis, a Malus× domestica MYB30-Interacting E3 Ligase 1 gene, MdMIEL1, was identified and subsequently cloned from apple ‘Gala’ (Malus×domestica). MdMIEL1 contained a zinc finger domain close to N-terminus and a RING finger domain close to Cterminus. Expression of MdMIEL1 was significantly induced by NaCl and H₂O₂ treatments. Further study demonstrated that the MdMIEL1-overexpressing Arabidopsis and apple calli were less tolerance to salt stress than wild-type control. In addition, transgenic plants had higher levels of reactive oxygen species (ROS) (H₂O₂ and O₂ ^–). And transgenic Arabidopsis and apple calli exhibited more sensitive phenotype to H₂O₂ treatment, which was associated with increased levels of ROS. These findings indicate MdMIEL1 is an important regulator involved in plant response to salt and oxidative stresses tolerance.     

PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b

Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance.     

Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 Ligase,Regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation

Abscisic acid (ABA) is a phytohormone that plays a fundamental role in plant development and stress response, especially in the regulation of stomatal closure in response to water deficit stress. The signal transduction that occurs in response to ABA and drought stress is mediated by protein phosphorylation and ubiquitination. This research identified Arabidopsis thaliana RING ZINC-FINGER PROTEIN34 (RZP34; renamed here as CHY ZINC-FINGER AND RING PROTEIN1 [CHYR1]) as an ubiquitin E3 ligase. CHYR1 expression was significantly induced by ABA and drought, and along with its corresponding protein, was expressed mainly in vascular tissues and stomata. Analysis of CHYR1 gain-of-function and loss-of-function plants revealed that CHYR1 promotes ABA-induced stomatal closure, reactive oxygen species production, and plant drought tolerance. Furthermore, CHYR1 interacted with SNF1-RELATED PROTEIN KINASE2 (SnRK2) kinases and could be phosphorylated by SnRK2.6 on the Thr-178 residue. Overexpression of CHYR1^T178A, a phosphorylation-deficient mutant, interfered with the proper function of CHYR1, whereas CHYR1^T178D phenocopied the gain of function of CHYR1. Thus, this study identified a RING-type ubiquitin E3 ligase that functions positively in ABA and drought responses and detailed how its ubiquitin E3 ligase activity is regulated by SnRK2.6-mediated protein phosphorylation.     

Molecular characterization of rice arsenic‐induced RING finger E3 ligase 2 (OsAIR2) and its heterogeneous overexpression in Arabidopsis thaliana

Arsenic (As) accumulation adversely affects the growth and productivity of plants and poses a serious threat to human health and food security. In this study, we identified one As‐responsive R eally I nteresting N ew G ene (RING) E3 ubiquitin ligase gene from rice root tissues during As stress. We named it Oryza sativa As‐Induced RING E3 ligase 2 (OsAIR2). Expression of OsAIR2 was induced under various abiotic stress conditions, including heat, salt, drought and As exposure. Results of an in vitro ubiquitination assay showed that OsAIR2 possesses an E3 ligase activity. Within the cell, OsAIR2 was found to be localized to the Golgi apparatus. Using yeast two‐hybrid (Y2H) assay, the 3‐ketoacyl‐CoA thiolase (KAT) protein was identified as an interaction partner. We found that the O. sativa KAT1 (OsKAT1) is localized to the cytosol and peroxisomes. Moreover, in vitro pull‐down assay verified the physical interaction between OsAIR2 and OsKAT1. Interestingly, in vitro ubiquitination assay and in vivo proteasomal degradation assay revealed that OsAIR2 ubiquitinates OsKAT1 and promotes the degradation of OsKAT1 via the 26S proteasome degradation pathway. Heterogeneous overexpression of OsAIR2 in Arabidopsis improved the seed germination and increased the root length under arsenate stress conditions. Therefore, these results suggest that OsAIR2 may be associated with the plant response to As stress and acts as a positive regulator of As stress tolerance.     

The Protein Stability of Axin,a Negative Regulator of Wnt Signaling,Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys⁵⁰⁵ of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.     

Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.     

IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase,Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis

Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.