Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization,stomatal closure,and tolerance response to drought stress

Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H₂O₂ after ABA treatment. These findings indicated that JUL1 acts downstream of H₂O₂ and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H₂O₂, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.     

A novel tomato SUMO E3 ligase,SlSIZ1, confers drought tolerance in transgenic tobacco

SUMOylation is an important post‐translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS‐type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up‐regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H₂O₂) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1‐2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.     

Arabidopsis C3HC4‐RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress‐responsive abscisic acid signaling

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)‐insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4‐RING finger domain in its C‐terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T‐DNA insertion mutant atairp4 effectively recovered the ABA‐associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild‐type and atairp4 mutant plants. In addition, the expression levels of ABA‐ and drought‐induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild‐type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA‐mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.     

The E3 ubiquitin ligase SP1‐like 1 plays a positive role in peroxisome biogenesis in Arabidopsis

Peroxisomes are dynamic organelles crucial for a variety of metabolic processes during the development of eukaryotic organisms, and are functionally linked to other subcellular organelles, such as mitochondria and chloroplasts. Peroxisomal matrix proteins are imported by peroxins (PEX proteins), yet the modulation of peroxin functions is poorly understood. We previously reported that, besides its known function in chloroplast protein import, the Arabidopsis E3 ubiquitin ligase SP1 (suppressor of ppi1 locus1) also targets to peroxisomes and mitochondria, and promotes the destabilization of the peroxisomal receptor–cargo docking complex components PEX13 and PEX14. Here we present evidence that in Arabidopsis, SP1's closest homolog SP1‐like 1 (SPL1) plays an opposite role to SP1 in peroxisomes. In contrast to sp1, loss‐of‐function of SPL1 led to reduced peroxisomal β‐oxidation activity, and enhanced the physiological and growth defects of pex14 and pex13 mutants. Transient co‐expression of SPL1 and SP1 promoted each other's destabilization. SPL1 reduced the ability of SP1 to induce PEX13 turnover, and it is the N‐terminus of SP1 and SPL1 that determines whether the protein is able to promote PEX13 turnover. Finally, SPL1 showed prevalent targeting to mitochondria, but rather weak and partial localization to peroxisomes. Our data suggest that these two members of the same E3 protein family utilize distinct mechanisms to modulate peroxisome biogenesis, where SPL1 reduces the function of SP1. Plants and possibly other higher eukaryotes may employ this small family of E3 enzymes to differentially modulate the dynamics of several organelles essential to energy metabolism via the ubiquitin‐proteasome system.     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

A RING‐type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis

Suppression of expression of DAF [DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)‐Activating Factor], a gene that encodes a putative RING‐finger E3 ligase protein, causes non‐dehiscence of the anthers, alters pollen development and causes sterility in 35S:DAF RNAi/antisense Arabidopsis plants. This mutant phenotype correlates with the suppression of DAF but not with expression of the two most closely related genes, DAFL1/2. The expression of DAD1 was significantly reduced in 35S:DAF RNAi/antisense plants, and complementation with 35S:DAF did not rescue the dad1 mutant, indicating that DAF acts upstream of DAD1 in jasmonic acid biosynthesis. This assumption is supported by the finding that 35S:DAF RNAi/antisense plants showed a similar cellular basis for anther dehiscence to that found in dad1 mutants, and that external application of jasmonic acid rescued the anther non‐dehiscence and pollen defects in 35S:DAF antisense flowers. We further demonstrate that DAF is an E3 ubiquitin ligase and that its activity is abolished by C132S and H137Y mutations in its RING motif. Furthermore, ectopic expression of the dominant‐negative C132S or H137Y mutations causes similar indehiscence of anthers and reduction in DAD1 expression in transgenic Arabidopsis. This result not only confirms that DAF controls anther dehiscence by positively regulating the expression of DAD1 in the jasmonic acid biosynthesis pathway, but also supports the notion that DAF functions as an E3 ubiquitin ligase, and that the conserved RING‐finger region is required for its activity.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1‐mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA‐dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA‐dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA‐responsive genes, such as PR1 and PR2. Furthermore, other SA‐accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.     

Site‐specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site‐specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)‐induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF‐κB reporter relative to wild‐type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated Hoip^K778R/K778R knock‐in mice, which show no overt developmental phenotypes; however, in response to TNF, Hoip^K778R/K778R mouse embryonic fibroblasts display mildly suppressed NF‐κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF‐induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in Hoip^K778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site‐specific ubiquitination of HOIP regulates a LUBAC‐dependent switch between survival and apoptosis in TNF signaling.     

The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7

E3 ubiquitin ligases select specific proteins for ubiquitin conjugation, and the modified proteins are commonly degraded through the 26S proteasome. XBAT32 is a RING-type E3 ligase involved in maintaining appropriate levels of ethylene. Previous work has suggested that XBAT32 modulates ethylene production by ubiquitinating two ethylene biosynthesis enzymes, ACS4 (type-II isoform) and ACS7 (type-III isoform). In Arabidopsis, conserved sequences within the C-terminal tail of type-I and -II 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) isoforms influence ubiquitin-dependent proteolysis. ACS7, the sole Arabidopsis type-III ACS, contains a truncated C-terminal tail that lacks all known regulatory sequences, which suggests that this isoform may not be subject to ubiquitin-mediated proteasomal degradation. Here we demonstrate in planta that ACS7 is turned over in a 26S proteasome-dependent manner and that degradation of ACS7 requires the E3 ligase XBAT32. Furthermore, the ethylene-related phenotypes that result from overexpression of ACS7 in wild-type plants are greatly exaggerated in xbat32-1, suggesting that XBAT32 is required to attenuate the effect of overexpression of ACS7. This observation is consistent with a role for XBAT32 in the ubiquitin-mediated degradation of ACS7. The dark-grown phenotype of xbat32-1 seedlings overexpressing ACS7 can be effectively rescued by aminoethoxyvinylglycine, an inhibitor of ACS activity. The degradation rate of ACS4 is also significantly slower in the absence of XBAT32, further implicating XBAT32 in the ubiquitin-mediated degradation of ACS4. Altogether, these results demonstrate that XBAT32 targets ethylene biosynthetic enzymes for proteasomal degradation to maintain appropriate levels of hormone production.     

The SUMO‐E3 ligase PIAS3 targets pyruvate kinase M2

Pyruvate kinase M2 (M2‐PK) controls the rate‐limiting step at the end of the glycolytic pathway in normal proliferating and tumor cells. Other functions of M2‐PK in addition to its role in glycolysis are little understood. The aim of this study was to identify new cellular interaction partners of M2‐PK in order to discover novel links between M2‐PK and cellular functions. Here we show that the SUMO‐E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2‐PK and its isoenzyme M1‐PK. Moreover, we demonstrate that endogenous SUMO‐1‐M2‐PK conjugates exist in mammalian cells. Furthermore, we show that transient expression of PIAS3 but not the RING domain mutant PIAS3 (C299S, H301A) is consistent with nuclear localization of M2‐PK and PIAS3 and M2‐PK partially co‐localize in the nucleus of these cells. This study suggests a link between PIAS3 and nuclear pyruvate kinase. J. Cell. Biochem. 107: 293–302, 2009. © 2009 Wiley‐Liss, Inc.     

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.