The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Interactions and CCAAT-binding of Arabidopsis thaliana NF-Y subunits.

BACKGROUND: NF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer containing histone fold domains (HFDs). We previously identified a large NF-Y gene family in Arabidopsis thaliana, composed of 29 members, and characterized their expression patterns in various plant tissues. METHODS: We used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) in vitro experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs. RESULTS: Consistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for embryo development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits. CONCLUSIONS: Our results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.

Many jobs for one good cop - the COP9 signalosome guards development and defense.

The COP9 signalosome (CSN) is a multiprotein complex that regulates the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate substrate proteins and thus target them for proteasomal degradation. This post-translational modification of proteins is arguably as important as reversible protein phosphorylation. The number of putative CRLs that recognize specific substrate proteins is vast, and known CRL substrates are involved in many cellular plant processes such as hormone signaling, the cell cycle, and regulation of growth, development, and defenses. By controlling the activity of CRLs, the CSN may integrate and fine-tune all of these processes. Recent research has unraveled in great mechanistic detail how the two multiprotein complexes CSN and CRL interact. As a consequence of CSN pleiotropy, complete loss of CSN function results in seedling lethality. However, recent work on plants that exhibit a partial loss of CSN function, has uncovered a role of the CSN during later life stages in processes such as development and defenses against pathogens and herbivorous insects. Not all aspects of development and defense are affected equally by CSN silencing, probably due to the differential participation and importance of CSN-regulated CRLs in these processes. This review will provide an overview of the highly complex regulation of CRL activity by CSN, and the many roles of the CSN in plant development and defense.

Geminiviruses subvert ubiquitination by altering CSN-mediated derubylation of SCF E3 ligase complexes and inhibit jasmonate signaling in Arabidopsis thaliana.

Viruses must create a suitable cell environment and elude defense mechanisms, which likely involves interactions with host proteins and subsequent interference with or usurpation of cellular machinery. Here, we describe a novel strategy used by plant DNA viruses (Geminiviruses) to redirect ubiquitination by interfering with the activity of the CSN (COP9 signalosome) complex. We show that geminiviral C2 protein interacts with CSN5, and its expression in transgenic plants compromises CSN activity on CUL1. Several responses regulated by the CUL1-based SCF ubiquitin E3 ligases (including responses to jasmonates, auxins, gibberellins, ethylene, and abscisic acid) are altered in these plants. Impairment of SCF function is confirmed by stabilization of yellow fluorescent protein-GAI, a substrate of the SCF(SLY1). Transcriptomic analysis of these transgenic plants highlights the response to jasmonates as the main SCF-dependent process affected by C2. Exogenous jasmonate treatment of Arabidopsis thaliana plants disrupts geminivirus infection, suggesting that the suppression of the jasmonate response might be crucial for infection. Our findings suggest that C2 affects the activity of SCFs, most likely through interference with the CSN. As SCFs are key regulators of many cellular processes, the capability of viruses to selectively interfere with or hijack the activity of these complexes might define a novel and powerful strategy in viral infections.

Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day-grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time.

Coordinated regulation of Arabidopsis thaliana development by light and gibberellins.

Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development. GA induces degradation of DELLA proteins via the ubiquitin/proteasome pathway, but light promotes accumulation of DELLA proteins by reducing GA levels. It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression. However, the precise mechanism of their function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor. In the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor) and prevent PIF3 from binding to its target gene promoters and regulating gene expression, and therefore abrogate PIF3-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) elevate their direct interaction with DELLA proteins in the nucleus, trigger DELLA protein's ubiquitination and proteasome-mediated degradation, and thus release PIF3 from the negative effect of DELLA proteins.

Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases.

A subset of WD40 proteins that contain a DWD motif (for DDB1 binding WD40) is reported to act as substrate receptors for DDB1-CUL4-ROC1 (for Damaged DNA Binding 1-Cullin 4-Regulator of Cullins 1) based E3 ubiquitin ligases in humans. Here, we report 85 Arabidopsis thaliana and 78 rice (Oryza sativa) proteins containing the conserved 16-amino acid DWD motif. We show by yeast two-hybrid and in vivo coimmunoprecipitation that 11 Arabidopsis DWD proteins directly interact with DDB1 and thus may serve as substrate receptors for the DDB1-CUL4 machinery. We further examine whether the DWD protein PRL1 (for Pleiotropic Regulatory Locus 1) may act as part of a CUL4-based E3 ligase. PRL1 directly interacts with DDB1, and prl1 and cul4cs mutants exhibited similar phenotypes, including altered responses to a variety of stimuli. Moreover, AKIN10 (for Arabidopsis SNF1 Kinase Homolog 10) was degraded more slowly in cell extracts of prl1 and cul4cs than in cell extracts of the wild type. Thus, both genetic and biochemical analyses support the conclusion that PRL1 is the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10. This work adds a large new family to the current portfolio of plant E3 ubiquitin ligases.

Over-expression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability.

The Arabidopsis AtMYB41 gene encodes an R2R3-MYB transcription factor whose expression is not detectable under normal growth conditions in any organ or at any developmental stage analysed. It is expressed at high levels in response to drought, ABA and salt treatments, suggesting a possible role in stress responses. Transgenic lines over-expressing this transcription factor showed a pleiotropic phenotype similar to that exhibited by some mutants that affect cuticle biosynthesis. This includes a dwarf appearance, dependent on smaller cells with abnormal morphology, enhanced sensitivity to desiccation, and enhanced permeability of leaf surfaces, suggesting discontinuity in the cuticle. The expression of genes involved in lipid metabolism and transport, in cell-wall modifications and cell expansion, genes coding for membrane-associated proteins and genes specifically involved in cuticle metabolism was differentially modulated between wild-type and transgenic plants, suggesting a direct or indirect role of AtMYB41 in the regulation of their transcription. Taken together, our results suggest that AtMYB41 is part of a complex network of transcription factors controlling cell expansion and cuticle deposition in response to abiotic stress.

Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana.

In this work we analysed, at the transcript level, the response of Arabidopsis anthocyanin regulatory genes of the MYB (PAP1 and PAP2), bHLH (TT8, EGL3 and GL3) and WD40 (TTG1) families to white light in seedlings and to different light qualities in rosette leaves. Our experiments showed strong light induction of the MYB genes PAP1 and PAP2. In particular, the kinetics of PAP1 expression preceded those of PAP2 and all of the structural genes (CHS, DFR, F3H, LDOX), consistent with the hypothesis that it has a key role in light induction of anthocyanin biosynthesis. All bHLH genes analysed showed light induction, and in seedlings their expression preceded that of the late structural genes, suggesting their possible role in light regulation of these structural genes. TTG1 expression is essentially constitutive in both systems. Experiments with transgenic lines over-expressing the MYB factors show that PAP1, but not PAP2, strongly stimulates expression of the anthocyanin structural gene encoding dihydroflavonol reductase, but neither factor affected expression of the early flavonoid biosynthesis gene encoding chalcone synthase. Consistent with these findings, PAP1, but not PAP2, stimulated light induction of anthocyanin biosynthesis in seedlings. We conclude that specific members of the MYB and bHLH families play important roles in regulating anthocyanin biosynthesis in response to different light qualities in Arabidopsis.

Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis Cullin3-based E3 ligases.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex that regulates a variety of biological processes. Among its eight subunits, CSN5 and CSN6 contain a characteristic MPN (for Mpr1p and Pad1p N-terminal) domain and, in Arabidopsis thaliana, are each encoded by two genes: CSN5A, CSN5B and CSN6A, CSN6B, respectively. We characterized both MPN subunits using a series of single and double mutants within each gene family. Our results indicate that although CSN6A and CSN6B retain mostly redundant functions, CSN5A and CSN5B play unequal roles in the regulation of plant development. Complete depletion of either of the two MPN members results in CSN instability and the decay of various CSN components, along with the complete loss of CUL1, CUL3, and CUL4 derubylation. Furthermore, we demonstrate that CSN interacts with CUL3, in addition to CUL1 and CUL4, and that the lack of CSN activity differentially affects the stability of those three cullins. Interestingly, we also show that optimal CUL3 activity is required to maintain the cellular pool of CSN5, through a posttranscriptional mechanism. Our data suggest the existence of reciprocal regulation between CUL3 and CSN5 accumulation. This study thus completes the genetic analysis of all CSN subunits and confirms the structural interdependence between PCI and MPN subunits in functional CSN complex formation.

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo.

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation.

Tandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis. In our newly generated TAPa tag we have replaced the tobacco etch virus (TEV) protease cleavage site with the more specific and low-temperature active rhinovirus 3C protease site. In addition, the second purification step can now be performed through two different affinity tags: a six His repeat or nine copies of a myc repeat. To examine our purification procedure we generated a C-terminal fusion between the TAPa tag and CSN3, a component of the multi-protein COP9 signalosome (CSN) complex. Subsequent analysis showed that CSN3-TAPa could rescue a csn3 mutant, and that the components of the CSN complex could be co-purified with CSN3-TAPa. As part of our long running interest in light signaling in Arabidopsis we have generated Arabidopsis transgenic lines harboring, both N-terminal and C-terminal TAPa fusions of many different light signaling pathway regulators. Molecular characterization of these transgenic lines showed fusion expression in 88% of the genes analyzed and that this expression is largely independent of the fusion orientation. Mutant complementation analysis showed that most of the TAPa fusions analyzed retained function of the wild-type proteins. Taken together, the data demonstrate the suitability of the TAPa system to allow efficient multi-protein complex isolation from stably transformed Arabidopsis.

The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome complexes, and mutations in their JAMM domains exhibit differential dominant negative effects on development.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex involved in a variety of signaling and developmental processes through the regulation of protein ubiquitination and degradation. A known biochemical role attributed to CSN is a metalloprotease activity responsible for the derubylation of cullins, core components for several types of ubiquitin E3 ligases. The CSN's derubylation catalytic center resides in its subunit 5, which in Arabidopsis thaliana is encoded by two homologous genes, CSN5A and CSN5B. Here, we show that CSN5A and CSN5B subunits are assembled into distinct CSN complexes in vivo, which are present in drastically different abundances, with CSN(CSN5A) appearing to be the dominant one. Transgenic CSN5A and CSN5B proteins carrying a collection of single mutations in or surrounding the metalloprotease catalytic center are properly assembled into CSN complexes, but only mutations in CSN5A result in a pleiotropic dominant negative phenotype. The extent of phenotypic effects caused by mutations in CSN5A is reflected at the molecular level by impairment in Cullin1 derubylation. These results reveal that three key metal binding residues as well as two other amino acids outside the catalytic center play important roles in CSN derubylation activity. Taken together, our data provide physiological evidence on a positive role of CSN in the regulation of Arabidopsis SCF (for Skp1-Cullin-F-box) E3 ligases through RUB (for Related to Ubiquitin) deconjugation and highlight the unequal role that CSN(CSN5A) and CSN(CSN5B) play in controlling the cellular derubylation of cullins. The initial characterization of CSN5A and CSN5B insertion mutants further supports these findings and provides genetic evidence on their unequal role in plant development.

Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes.

COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also shows enhancement of E2 activity (UEA) similar to that observed with COP10 itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the degradation of positive regulators of photomorphogenesis, such as the transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD complex may act as a ubiquitylation-promoting factor to regulate photomorphogenesis.

Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits.

Nuclear factor Y (NF-Y) is a highly conserved trimeric activator that recognizes with high specificity and affinity the widespread CCAAT box promoter element. We previously cloned the genes of 23 NF-Y genes of Arabidopsis thaliana (Gene 264 (2001) 173). Now that the Arabidopsis genome sequencing project is complete, we present the cloning, alignments and expression profiles of the remaining six genes coding for the three NF-Y subunits. Consistent with our previous reports, most of the new members of the three subunits show a unique tissue-specific pattern, while another AtNF-YC9 is rather ubiquitous.