The ARC1 E3 Ligase Promotes Two Different Self-Pollen Avoidance Traits in Arabidopsis

Flowering plants have evolved various strategies for avoiding self-pollen to drive genetic diversity. These strategies include spatially separated sexual organs (herkogamy), timing differences between male pollen release and female pistil receptivity (dichogamy), and self-pollen rejection. Within the Brassicaceae, these outcrossing systems are the evolutionary default state, and many species display these traits, including Arabidopsis lyrata. In contrast to A. lyrata, closely related Arabidopsis thaliana has lost these self-pollen traits and thus represents an excellent system to test genes for reconstructing these evolutionary traits. We previously demonstrated that the ARC1 E3 ligase is required for self-incompatibility in two diverse Brassicaceae species, Brassica napus and A. lyrata, and is frequently deleted in self-compatible species, including A. thaliana. In this study, we examined ARC1’s requirement for reconstituting self-incompatibility in A. thaliana and uncovered an important role for ARC1 in promoting a strong and stable pollen rejection response when expressed with two other A. lyrata self-incompatibility factors. Furthermore, we discovered that ARC1 promoted an approach herkogamous phenotype in A. thaliana flowers. Thus, ARC1’s expression resulted in two different A. lyrata traits for self-pollen avoidance and highlights the key role that ARC1 plays in the evolution and retention of outcrossing systems.     

The ARC1 E3 Ligase Gene Is Frequently Deleted in Self-Compatible Brassicaceae Species and Has a Conserved Role in Arabidopsis lyrata Self-Pollen Rejection

Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.     

Robust Self-Incompatibility in the Absence of a Functional ARC1 Gene in Arabidopsis thaliana

Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificity-determining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A. thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A. thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A. thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A. thaliana to its fully self-incompatible ancestral state.It is well established that specific pollen recognition in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interactions that occur at the stigma surface between two highly polymorphic proteins encoded in the S locus: the S-locus receptor kinase SRK and its ligand, the S-locus cysteine-rich protein SCR. Arabidopsis thaliana lacks a functional SI system and harbors nonfunctional S-locus variants that contain defective alleles of the SRK and/or SCR genes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Tang et al., 2007; Shimizu et al., 2008; Boggs et al., 2009a; Tsuchimatsu et al., 2010; Dwyer et al., 2013). Despite being highly self-fertile, A. thaliana can be made to express SI upon transformation with functional SRK-SCR gene pairs isolated from its self-incompatible close relatives (Nasrallah et al., 2002, 2004; Boggs et al., 2009a, 2009b). The first transfer of the SI trait into A. thaliana was achieved using the SRKb-SCRb gene pair isolated from the Sb locus of Arabidopsis lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). Many of the subsequent studies that have been performed in the transgenic A. thaliana SRK-SCR system have used plants transformed with p548, a plasmid that we constructed by inserting the A. lyrata SRKb and SCRb genes with their 5′ and 3′ regulatory sequences into the pBIN+ binary vector (Nasrallah et al., 2004).Indriolo et al. (2014) recently used the p548 plasmid to generate SRKb-SCRb transformants and test the role of the ARM Repeat Containing1 (ARC1) gene in SI. ARC1 was originally identified as a Brassica napus protein that interacts with the SRK kinase domain in yeast (Gu et al., 1998), and it was subsequently inferred to be required for SI because downregulation of the ARC1 gene in B. napus (Stone et al., 1999) and A. lyrata (Indriolo et al., 2012), as well as overexpression of ARC1’s target, Exo70A1, in B. napus (Samuel et al., 2009), caused partial breakdown of the SI response. However, the involvement of the proposed SRK-ARC1-Exo70A1 pathway in SI has been questioned because the ARC1 gene was found to be deleted in all A. thaliana accessions analyzed to date (Kitashiba et al., 2011; Indriolo et al., 2012), including those in which the SRKb-SCRb transgenes confer a strong SI phenotype (Kitashiba et al., 2011). Additionally, overexpression of Exo70A1 did not cause weakening of the SI response in A. thaliana SRKb-SCRb plants (Kitashiba et al., 2011).Indriolo et al. (2014) reported on their characterization of the SI response in plants of the Sha and Columbia-0 (Col-0) accessions, which they either transformed with the p548 plasmid alone or cotransformed with p548 and a plasmid containing an ARC1 gene isolated from A. lyrata or B. napus. They concluded that, along with SRK and SCR, “ARC1 is the third component that is required to return A. thaliana to its ancestral self-incompatibility state.” However, this conclusion is inconsistent with results of previous studies of SI in transgenic A. thaliana SRK-SCR transformants, which have shown that several A. thaliana accessions are rendered fully self-incompatible by transformation with the p548 plasmid without the addition of a functional ARC1 gene. Contrary to Indriolo et al.’s assertion that in previous studies of A. thaliana SRK-SCR transformants, “the self-pollen rejection response was incomplete,” we reported that among 11 A. thaliana accessions tested by transformation with the p548 plasmid, five accessions (C24, Cvi-0, Hodja, Kas-2, and Sha) were converted to full SI by expression of the SRKb and SCRb genes alone (Nasrallah et al., 2004; Boggs et al., 2009a). Importantly, the SI phenotype of these self-incompatible SRKb-SCRb transformants faithfully recapitulates the SI phenotype of naturally self-incompatible Brassicaceae with respect to the four defining features of SI in this family: (1) site of pollen inhibition at the stigma surface, (2) intensity of the response, (3) developmental regulation over the course of stigma maturation, and (4) heritability. These features suggest that the inhibition of self pollen in self-incompatible A. thaliana SRK-SCR transformants is achieved via the same signaling pathway as that utilized by other self-incompatible Brassicaceae species.     

The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death

Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events.     

The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in Arabidopsis. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. AtRDUF1 expression is upregulated by salt treatment. Overexpression of AtRDUF1 in Arabidopsis results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of AtRDUF1 and its close homolog AtRDUF2 (atrduf1atrduf2) was hypersensitive to salt treatment. The expression levels of the stress-response genes RD29B, RD22, and KIN1 are more sensitive to salt treatment in AtRDUF1 overexpression plants. In summary, our data show that AtRDUF1 positively regulates responses to salt stress in Arabidopsis.     

The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.     

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.     

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

The timing of flowering is coordinated by a web of gene regulatory networks that integrates developmental and environmental cues in plants. Light and temperature are two major environmental determinants that regulate flowering time. Although prolonged treatment with low nonfreezing temperatures accelerates flowering by stable repression of FLOWERING LOCUS C (FLC), repeated brief cold treatments delay flowering. Here, we report that intermittent cold treatments trigger the degradation of CONSTANS (CO), a central activator of photoperiodic flowering; daily treatments caused suppression of the floral integrator FLOWERING LOCUS T (FT) and delayed flowering. Cold-induced CO degradation is mediated via a ubiquitin/proteasome pathway that involves the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). HOS1-mediated CO degradation occurs independently of the well established cold response pathways. It is also independent of the light signaling repressor CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase and light wavelengths. CO has been shown to play a key role in photoperiodic flowering. Here, we demonstrated that CO served as a molecular hub, integrating photoperiodic and cold stress signals into the flowering genetic pathways. We propose that the HOS1-CO module contributes to the fine-tuning of photoperiodic flowering under short term temperature fluctuations, which often occur during local weather disturbances.     

The Arabidopsis Kelch Repeat F-box E3 Ligase ARKP1 Plays a Positive Role for the Regulation of Abscisic Acid Signaling

Ubiquitination is one of the most common posttranslational modifications. A series of E3 ligases are implicated in plant abiotic stress signaling, regulating the degradation of multiple specific target proteins. Here, we showed that a novel gene ABA-RESPONSE KELCH PROTEIN 1 (AtARKP1), which encodes an F-box subunit of Skp-cullin-F-box (SCF) ubiquitin ligase complex, was localized in the nucleus and could be induced by phytohormone abscisic acid (ABA) in Arabidopsis. ARKP1 interacted with ASK1 and ASK2, which tethered the rest of the complex to an F-box protein, suggesting that they might form an SCF ubiquitin ligase complex. Further analysis revealed that ARKP1 was exclusively expressed in the seed, rosette leaf, and root. arkp1 T-DNA insertion mutant plants were insensitive to ABA, displaying reduced ABA-mediated inhibition of seed germination, root elongation, and water loss rate of detached leaves. In contrast, transgenic plants showed enhanced sensitivity to ABA and tolerance to water deficit. Accordingly, the expressions of ABA and drought responsive marker genes were markedly upregulated in ARKP1 overexpressing plants than the wild-type and arkp1 mutant plants. Taken together, our findings suggest that AtARKP1 plays a positive role in ABA signaling network.     

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.     

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.     

Targeting the Ubiquitin E3 Ligase MuRF1 to Inhibit Muscle Atrophy

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.     

SUGAR-INSENSITIVE3, a RING E3 Ligase,Is a New Player in Plant Sugar Response

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development.Almost all living organisms rely on the products of plant photosynthesis for sustenance, either directly or indirectly. Carbohydrates, the major photosynthates, provide both energy and carbon skeletons for fungi, plants, and animals. In addition, sugars, such as Suc and Glc, function as signaling molecules to regulate plant growth, development, gene expression, and metabolic processes. Sugar response pathways are integrated with other signaling pathways, such as those for light, phytohormones, stress, and nitrogen (Dijkwel et al., 1997; Zhou et al., 1998; Roitsch, 1999; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Coruzzi and Zhou, 2001; Rook et al., 2001; Rolland et al., 2006).Several components of plant sugar response pathways have been identified based on the conservation of sugar-sensing mechanisms among eukaryotic cells (Rolland et al., 2001, 2006) or by mutant screens. Yeast HEXOKINASE2 functions in the Glc-mediated catabolite repression pathway (Entian, 1980). In Arabidopsis (Arabidopsis thaliana), mutations in HEXOKINASE1 (HXK1) cause a Glc-insensitive phenotype, and HXK1 demonstrates dual functions in Glc sensing and metabolism (Moore et al., 2003; Cho et al., 2006). Recent studies revealed the involvement of G-protein-coupled receptor systems in sugar response in yeast and Arabidopsis (Chen et al., 2003; Lemaire et al., 2004). Arabidopsis regulator of G-protein signaling1 (rgs1) mutant seedlings are insensitive to 6% Glc (Chen and Jones, 2004), whereas G-protein α-subunit (gpa1) null mutant seedlings are hypersensitive to Glc (Chen et al., 2003). The SNF1/AMPK/SnRK1 protein kinases are postulated to be global regulators of energy control (Polge and Thomas, 2007). Studies conducted on two members of the Arabidopsis SnRK1 (for SNF1-Related Protein Kinases1) family, AKIN10 and AKIN11, have revealed their pivotal roles in stress and sugar signaling (Baena-González et al., 2007). A genetic screen for reduced seedling growth on 175 mm Suc identified the pleiotropic regulatory locus1 (prl1) mutant, which encodes a nuclear WD protein. Further analyses revealed that PRL1 functions in Glc and phytohormone responses (Németh et al., 1998). Interestingly, PRL1 negatively regulates the Arabidopsis SnRK1s AKIN10 and AKIN11 in vitro (Bhalerao et al., 1999).Isolation of additional mutants defective in sugar response has revealed cross talk between sugar and phytohormone response pathways. For example, abscisic acid (ABA) biosynthesis and signaling mutants have been isolated by several genetic screens for seedlings with reduced responses to the inhibitory effects of high levels of Suc or Glc on seedling development. These mutants include abscisic acid-deficient1 (aba1), aba2, aba3, salt-tolerant1/nine-cis-epoxycarotenoid dioxygenase3, abscisic acid-insensitive3 (abi3), and abi4 (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001; Cheng et al., 2002; Rolland et al., 2002; Huang et al., 2008), indicating interplay between ABA- and sugar-mediated signaling. Ethylene also exhibits interactions with sugars in controlling seedling development. Both the ethylene overproduction mutant eto1 and the constitutive ethylene response mutant ctr1 exhibit Glc (Zhou et al., 1998) and Suc (Gibson et al., 2001) insensitivity, whereas the ethylene-insensitive mutants etr1, ein2, and ein4 show sugar hypersensitivity (Zhou et al., 1998; Gibson et al., 2001; Cheng et al., 2002).Further characterization of sugar response factors has suggested that ubiquitin-mediated protein degradation may play a role in sugar response. In particular, the PRL1-binding domains of SnRK1s have been shown to recruit SKP1/ASK1, a conserved SCF ubiquitin ligase subunit, as well as the α4/PAD1 proteasomal subunit, indicating a role for SnRK1s in mediating proteasomal binding of SCF ubiquitin ligases (Farrás et al., 2001). In addition, recent studies indicate that PRL1 is part of a CUL4-based E3 ligase and that AKIN10 exhibits decreased rates of degradation in prl1 than in wild-type extracts (Lee et al., 2008). The ubiquitin/26S proteasome pathway plays important roles in many cellular processes and signal transduction pathways in yeast, animals, and plants (Hochstrasser, 1996; Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). The key task of the pathway is to selectively ubiquitinate substrate proteins and target them for degradation by the 26S proteasome. In short, the multistep ubiquitination process starts with the formation of a thiol-ester linkage between ubiquitin and a ubiquitin-activating enzyme (E1). The activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) then mediates the covalent attachment of ubiquitin to the substrate protein. The specificity of the pathway is largely realized by the E3s, which recognize the substrates that should be ubiquitinated. In Arabidopsis, more than 1,300 genes encode putative E3 subunits and the E3 ligases can be grouped into defined families based upon the presence of HECT (for Homology to E6-AP C Terminus), RING (for Really Interesting New Gene), or U-box domains (Smalle and Vierstra, 2004). The RING-type E3s can be subdivided into single-subunit E3s, which contain the substrate recognition and RING finger domains on the same protein, and multisubunit E3s, which include the SCF (for Skp1-Cullin-F-box), CUL3-BTB (for Broad-complex, Tramtrack, Bric-a-Brac), and APC (for Anaphase-Promoting Complex) complexes (Weissman, 2001; Moon et al., 2004).The Cys-rich RING finger was first described in the early 1990s (Freemont et al., 1991). It is defined as a linear series of conserved Cys and His residues (C3HC/HC3) that bind two zinc atoms in a cross-brace arrangement. RING fingers can be divided into two types, C3HC4 (RING-HC) and C3H2C3 (RING-H2), depending on the presence of either a Cys or a His residue in the fifth position of the motif (Lovering et al., 1993; Freemont, 2000). A recent study of the RING finger ubiquitin ligase family encoded by the Arabidopsis genome resulted in the identification of 469 predicted proteins containing one or more RING domains (Stone et al., 2005). However, the in vivo biological functions of all but a few of the RING proteins remain unknown. Recent studies have implicated several Arabidopsis RING proteins in a variety biological processes, including COP1 and CIP8 (photomorphogenesis; Hardtke et al., 2002; Seo et al., 2004), SINAT5 (auxin signaling; Xie et al., 2002), ATL2 (defense signaling; Serrano and Guzman, 2004), BRH1 (brassinosteroid response; Molnár et al., 2002), RIE1 (seed development; Xu and Li, 2003), NLA (nitrogen limitation adaptation; Peng et al., 2007), HOS1 (cold response; Dong et al., 2006), AIP2 (ABA signaling; Zhang et al., 2005), KEG (ABA signaling; Stone et al., 2006), and SDIR1 (ABA signaling; Zhang et al., 2007).Here, we report the isolation, identification, and characterization of an Arabidopsis mutant, sugar-insensitive3 (sis3), which is resistant to the early seedling developmental arrest caused by high exogenous sugar levels. The responsible locus, SIS3, was identified through a map-based cloning approach and confirmed with additional T-DNA insertional mutants and complementation tests. The SIS3 gene encodes a protein with a RING-H2 domain and three putative transmembrane domains. Glutathione S-transferase (GST)-SIS3 recombinant proteins exhibit in vitro ubiquitin E3 ligase activity. Together, these results indicate that a ubiquitination pathway involving the SIS3 RING protein is required to mediate the sugar response during early seedling development.     

The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.