Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development 1 . GA induces degradation of DELLA proteins via the ubiquitin/ proteasome pathway 2 , but light promotes accumulation of DELLA proteins by reducing GA levels 3 . It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression 4,5 . 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.Light and GA interact during Arabidopsis thaliana seedling development, regulating hypocotyl elongation, cotyledon opening and light-responsive gene expression; their pathways seem to converge at regulation of the abundance of DELLA proteins (GA pathway repressors) 3,6 . Arabidopsis has five DELLA proteins-RGA, GAI, RGL1, RGL2 and RGL3-defined by their unique DELLA domain and a conserved GRAS domain 4 . To analyse them in vivo, we raised antibodies against endogenous RGA and generated transgenic Arabidopsis expressing each of the five DELLA proteins with tandem affinity purification (TAP) tags ( Supplementary Fig. 1). The response of DELLA protein levels to exogenously applied GA 3 (an active form of GA) or PAC (paclobutrazol, a GA biosynthesis inhibitor) was examined. We found that one-hour-long GA treatment eliminates the majority of DELLA proteins, and this GA effect can be largely prevented by 100 mM MG132 (a 26S proteasome-specific inhibitor). PAC, on the other hand, promotes over-accumulation of DELLA proteins (Fig. 1). These results show for the first time in Arabidopsis that all the DELLA proteins are under negative control by GA and the proteasome. Next, we generated lines expressing TAPtagged RGAD17 and GAID17, which lack a 17 amino acid motif within the DELLA domain that is required for GA-induced degradation 7,8 . As expected, TAP-RGAD17 and TAP-GAID17 are completely resistant to GA and accumulate at higher levels than wild-type proteins, which cannot be further increased by PAC (Fig. 1, and *These authors contributed equally to this work. WTAnti-RPN6Anti-RGA Immunoblot analysis of RGA (by anti-RGA antibody) and TAP-DELLA proteins (by anti-MYC antibody) in various light-grown Ara...

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.

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.

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.

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.

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SummaryTandem 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 lowtemperature 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 multiprotein 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.

SummaryThe 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.