A New Class of Arabidopsis Constitutive Photomorphogenic Genes Involved in Regulating Cotyledon Development.

Light signals have profound effects on morphogenesis of hypocotyls and cotyledons of Arabidopsis seedlings, but the mechanisms by which light signals are transduced and integrated to control these processes are poorly understood. We report here the identification of a new class of constitutive photomorphogenic (cop) mutants, cop2, cop3, and cop4, in which dark-grown seedlings have open and enlarged cotyledons resembling those of light-grown wild-type seedlings. The epistatic relationships of these three mutations to previously characterized phytochrome-deficient mutations suggest that COP2, COP3, and COP4 may act downstream of phytochrome in the light regulatory pathway. Mutations in each of the three loci alleviate the normal inhibition of cell-type differentiation, cell enlargement, and lateral cell division observed in cotyledons of dark-grown wild-type seedlings, but do not affect plastid differentiation. The cop4 mutation also leads to high-level dark expression of nuclear, but not plastid-encoded, light-inducible genes. We further show that for the nuclear cab1 gene encoding a chlorophyll a/b binding protein of the photosynthetic light-harvesting complex, activation in dark-grown cop4 mutants is achieved by modulation of promoter activity. Interestingly, COP4 modulates cab1 promoter activity through a pathway distinct from that of COP1 and COP9. Furthermore, cop4 mutants are defective in both root and shoot gravitropic responses, indicating that the COP4 locus may be involved in both light-signaling and gravity-sensing processes.

PCI complexes: pretty complex interactions in diverse signaling pathways.

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

A hitchhiker's guide to the proteasome.

Regulated degradation of proteins is essential for viability and is involved in the control of many signal transduction pathways. von Arnim discusses a new model for how substrates destined for degradation by the 26S proteasome may be presented to the proteasome through a physical interaction between the proteasome and a complex consisting of the substrate and a ubiquitin-ligase. The new model suggests that the SCF (Skp1/cullin/F-box) protein complex may physically associate with the proteasome and that this interaction may be regulated by posttranslational modifications, such as phosphorylation or the covalent attachment of the Nedd8 protein, called neddylation. Additionally, other proteins may compete with the SCF complexes for binding to the proteasome and thus present another layer of regulation for controlling substrate targeting for ubiquitin-mediated degradation.

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants.

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP-S65T and GFP-Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.

Determinants of tomato golden mosaic virus symptom development located on DNA B.

Infectious clones have been constructed from two strains of the bipartite geminivirus tomato golden mosaic virus. The common strain and the yellow vein strain show marked phenotypic differences in Nicotiana benthamiana which are reproduced following infection with the cloned viral genomes. Pseudorecombinants between the two strains, produced by exchange of genome components (DNAs A and B), established that the difference in symptoms in several species of the Solanaceae is determined by DNA B. Recombinants produced in vitro between the DNA B components showed that determinants of symptom development map to the common region and gene BL1. DNA B is known to carry functions necessary for spread of viral DNA through the host plant. Our results emphasize the link between symptom type and virus spread.

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.

A role for transcriptional repression during light control of plant development.

Light mediates plant development partly by orchestrating changes in gene expression, a process which involves a complex combination of positive and negative signaling cascades. Genetic investigations using the small crucifer Arabidopsis thaliana have demonstrated a fundamental role for the down-regulation of light-inducible genes in response to darkness, thus offering a suitable model system for investigating how plants repress gene expression in a developmental context. Rapid progress in eukaryotic gene repression mechanisms in general, and light control of plant gene expression in particular, sheds new light on how a class of ten pleiotropic COP/DET/FUS genes might function to down-regulate light-inducible genes in plants.

Isolation and characterization of a gene encoding a chlorophyll a/b-binding protein from mustard and the targeting of the encoded protein to the thylakoid membrane of pea chloroplasts in vitro.

Three independent clones carrying a mustard gene coding for the chlorophyll a/b-binding protein were isolated by screening a genomic library of mustard with a heterologous cDNA probe from pea. All of them encode the same CAB gene, which, as shown by sequence analysis and comparison with published CAB sequences, belongs to the family of type I PSII CAB genes, encoding a precursor protein of 266 amino acids. Several conserved sequence motifs are observed in the 5' and 3' non-coding region of the gene. The putative transcription start site could be localized to 60 bp upstream of SA-CAB1 initiator codon by S1 mapping. Plasmids were constructed which allow in vitro transcription and translation of the whole chlorophyll a/b-binding protein and of truncated species which lack increasing portions of the C-terminus. Whereas the in vitro import into pea chloroplasts is not affected by these C-terminal deletions, targeting to the thylakoid membrane is abolished by the removal of the C-terminal helical domain. Accordingly, the 54 amino acids which contain the C-terminal membrane-spanning helix and flanking regions is an essential component of the thylakoid targeting signal.

Light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning.

Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate this suppressive action. COP1, when fused to beta-glucuronidase (GUS), is enriched in the nucleus in darkness, but not in the light, in hypocotyl cells of Arabidopsis seedlings and epidermal cells of onion bulbs. In Arabidopsis hypocotyl cells, the nuclear GUS-COP1 level changes in response to dark-light transitions and quantitatively correlates with the extent of repression of photomorphogenic development. In root cells, GUS-COP1 is constitutively nuclear, consistent with an established role of COP1 in suppressing root chloroplast development in both light and darkness. We conclude that COP1 acts inside the nucleus to suppress photomorphogenesis and that light inactivation of COP1 involves a cell type-specific control of its nucleocytoplasmic partitioning.

Specificity of bipartite geminivirus movement proteins.

Pseudorecombinants produced by exchanging genome components (DNAs A and B) of the geminiviruses African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV), ACMV, and tomato golden mosaic virus (TGMV), and TGMV and abutilon mosaic virus (AbMV) are not infectious in their common host Nicotiana benthamiana. In each case, DNA A was unable to trans-replicate the heterogenomic DNA B component in a N. benthamiana leaf disc assay. The non-viability of the pseudorecombinants has been exploited to investigate the specificity of geminivirus movement proteins, encoded by DNA B, by co-inoculating N. benthamiana with both genome components of one virus and DNA A of a second virus. We demonstrate that ACMV can mediate the systemic movement of ICMV, TGMV and AbMV DNA A components. In reciprocal experiments, neither TGMV nor AbMV can mediate the systemic movement of ACMV DNA A although they can support the movement of each other's DNA A. The variation in movement protein specificity suggests evolutionary divergence of New and Old World geminiviruses. Co-inoculation of combinations of ACMV and ICMV genome components into discriminating hosts and comparison of their behavior in N. benthamiana and N. tabacum leaf disc assays suggests that the host range of ICMV, a subset of that of ACMV, is restricted by impaired viral DNA replication rather than the inability of the virus to spread in non-host backgrounds.

Ring finger motif of Arabidopsis thaliana COP1 defines a new class of zinc-binding domain.

The COP1 gene of Arabidopsis thaliana encodes a protein mediating the switch between the two developmental pathways utilized in light and darkness. A cysteine-rich motif identified the COP1 protein as a member of a group of regulatory proteins which share the amino acid motif Cys-X-X-Cys-loop I-Cys-X-His-X-X-Cys-X-X-Cys-loop II-Cys-X-X-Cys (ring finger). Although this new class of cysteine-rich motifs has been proposed to bind metal ions, no direct evidence supporting this has been presented. By analyzing the COP1 protein expressed in Escherichia coli, we demonstrate here that each COP1 molecule can bind up to two zinc atoms. The two zinc ions are bound with different affinities. One is tightly bound and resistant to urea and EDTA, whereas the other one is labile under those conditions. It is further shown that deletion of the ring finger motif abolishes the metal-binding capacity of COP1. We conclude that the ring finger motif constitutes a zinc-coordinating element distinct from previously characterized zinc-binding domains.

Detection and possible functions of African cassava mosaic virus DNA B gene products.

Polyclonal antisera raised against synthetic oligopeptides have been used to detect the DNA B gene products BV1 and BC1 of the geminivirus African cassava mosaic virus following SDS-PAGE fractionation of Nicotiana benthamiana extracts. BV1 antiserum detected a soluble protein of 29 kDa, consistent with the size predicted from sequence data. BC1 antiserum detected proteins of 37, 39, and 42 kDa in addition to variable, less abundant species, all of which are larger than the predicted size of 34 kDa. BC1 antiserum detected a single protein of 35-36 kDa following in vitro translation in reticulocyte lysate, suggesting that BC1 is post-translationally modified in plants. The nature of the modification was not resolved, although neither glycosylation nor association with nucleic acids is involved. In common with putative spread proteins of several other plants viruses, BC1 co-fractionated with the cell wall. The replication of both genomic components in N. tabacum protoplasts was unaffected by the introduction of frameshift mutations into BV1 and BC1 coding regions. In inoculated N. benthamiana leaves, however, the accumulation of a BV1 mutant was significantly reduced compared to the levels attained by co-inoculated, complementing BV1 and BC1 mutants. In contrast, the accumulation of a BC1 mutant was unaffected, although symptom induction in inoculated leaves and systemic infection occurred only in the presence of both BV1 and BC1. The results are consistent with a role for BV1 in localized cell-to-cell spread and for BC1, possibly together with BV1, in long-distance vascular spread of the virus.

A novel motif mediates the targeting of the Arabidopsis COP1 protein to subnuclear foci.

The constitutive photomorphogenesis 1 (COP1) protein of Arabidopsis thaliana accumulates in discrete subnuclear foci. To better understand the role of subnuclear architecture in COP1-mediated gene expression, we investigated the structural motifs of COP1 that mediate its localization to subnuclear foci using mutational analysis with green fluorescent protein as a reporter. In a transient expression assay, a subnuclear localization signal consisting of 58 residues between amino acids 120 and 177 of COP1 was able to confer speckled localization onto the heterologous nuclear NIa protein from tobacco etch virus. The subnuclear localization signal overlaps two previously characterized motifs, a cytoplasmic localization signal and a putative alpha-helical coiled-coil domain that has been implicated in COP1 dimerization. Moreover, phenotypically lethal mutations in the carboxyl-terminal WD-40 repeats inhibited localization to subnuclear foci, consistent with a functional role for the accumulation of COP1 at subnuclear sites.

Arabidopsis COP1 protein specifically interacts in vitro with a cytoskeleton-associated protein, CIP1.

Arabidopsis COP1 acts inside the nucleus to suppress photomorphogenic cellular development, and light inactivation of COP1 may involve a specific control of its nuclear activity in hypocotyls and cotyledons, but not in roots, of developing seedlings. To understand the molecular mechanisms of COP1 action during light-mediated development, we initiated a screen for Arabidopsis cDNAs encoding proteins which interact directly with COP1 in vitro as a step to identify the cellular components involved. We report here the isolation and characterization of a cDNA clone encoding a protein designated CIP1 (COP1-interactive protein 1). CIP1 is predominantly alpha-helical and most likely involved in coiled-coil formation. It interacts specifically with the putative coiled-coil region of COP1 in vitro. Further, CIP1 is encoded by a single gene in Arabidopsis, and its mRNA and protein levels are not regulated by light. Immunofluorescent labeling of CIP1 in Arabidopsis seedling protoplasts demonstrated that CIP1 is part of, or associated with, a cytoskeletal structure in hypocotyl and cotyledon cells, but not in roots. Our results are consistent with a possible role of CIP1 in mediating light control of COP1 nuclear activity by regulating its nucleocytoplasmic partitioning.

Genetic and developmental control of nuclear accumulation of COP1, a repressor of photomorphogenesis in Arabidopsis.

Using a beta-glucuronidase (GUS) reporter-COP1 fusion transgene, it was shown previously that Arabidopsis COP1 acts within the nucleus as a repressor of seedling photomorphogenic development and that high inactivation of COP1 was accompanied by a reduction of COP1 nuclear abundance (A.G. von Arnim, X.-W. Deng [1994] Cell 79: 1035-1045). Here we report that the GUS-COP1 fusion transgene can completely rescue the defect of cop1 mutations and thus is fully functional during seedling development. The kinetics of GUS-COP1 relocalization in a cop1 null mutant background during dark/light transitions imply that the regulation of the functional nuclear COP1 level plays a role in stably maintaining a committed seedling's developmental fate rather than in causing such a commitment. Analysis of GUS-COP1 cellular localization in mutant hypocotyls of all pleiotropic COP/DET/FUS loci revealed that nuclear localization of GUS-COP1 was diminished under both dark and light conditions in all mutants tested, whereas nuclear localization was not affected in the less pleiotropic cop4 mutant. Using both the brassinosteroid-deficient mutant det2 and brassinosteroid treatment of wild-type seedlings, we have demonstrated that brassinosteroid does not control the hypocotyl cell elongation through regulation nuclear localization of COP1. The growth regulator cytokinin, which also dramatically reduced hypocotyl cell elongation in the absence of light, did not prevent GUS-COP1 nuclear localization in dark-grown seedlings. Our results suggest that all of the previously characterized pleiotropic COP/DET/FUS loci are required for the proper nuclear localization of the COP1 protein in the dark, whereas the less pleiotropic COP/DET loci or plant regulators tested are likely to act either downstream of COP1 or by independent pathways.

Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis.

Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings. In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development. Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation. Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene. Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels. This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1. It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.

Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta.

The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness. Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis. The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function. In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression. The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1. Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1. Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.

Discrete domains mediate the light-responsive nuclear and cytoplasmic localization of Arabidopsis COP1.

The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) protein plays a critical role in the repression of photomorphogenesis during Arabidopsis seedling development. We investigated the control of COP1 partitioning between nucleus and cytoplasm, which has been implicated in the regulation of COP1 activity, by using fusion proteins between COP1 and beta-glucuronidase or the green fluorescent protein. Transient expression assays using onion epidermal cells and data from hypocotyl cells of stably transformed Arabidopsis demonstrated that COP1 carries a single, bipartite nuclear localization signal that functions independently of light. Nuclear exclusion was mediated by a novel and distinct signal, bordering the zinc-finger and coiled-coil motifs, that was able to redirect a heterologous nuclear protein to the cytoplasm. The cytoplasmic localization signal functioned in a light-independent manner. Light regulation of nuclear localization was reconstituted by combining the individual domains containing the nuclear localization signal and the cytoplasmic localization signal; the WD-40 repeat domain of COP1 was not required. However, phenotypic analysis of transgenic seedlings suggested that the constitutively nuclear-localized WD-40 repeat domain was able to mimic aspects of COP1 function, as indicated by exaggerated hypocotyl elongation under light conditions.

Arabidopsis eIF3e (INT-6) associates with both eIF3c and the COP9 signalosome subunit CSN7.

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.

The Arabidopsis protein COP1, encoded by the constitutive photomorphogenic locus 1, is an essential regulatory molecule that plays a role in the repression of photomorphogenic development in darkness and in the ability of light-grown plants to respond to photoperiod, end-of-day far-red treatment, and ratio of red/far-red light. The COP1 protein contains three recognizable structural domains: starting from the N terminus, they are the zinc binding motif, the putative coiled-coil region, and the domain with multiple WD-40 repeats homologous to the beta subunit of trimeric G-proteins (G beta). To understand the functional implications of these structural motifs, 17 recessive mutations of the COP1 gene have been isolated based on their constitutive photomorphogenic seedling development in darkness. These mutations define three phenotypic classes: weak, strong, and lethal. The mutations that fall into the lethal class are possible null mutations of COP1. Molecular analysis of the nine mutant alleles that accumulated mutated forms of COP1 protein revealed that disruption of the G beta-protein homology domain or removal of the very C-terminal 56 amino acids are both deleterious to COP1 function. In-frame deletions or insertions of short amino acid stretches between the putative coiled-coil and G beta-protein homology domains strongly compromised COP1 function. However, a mutation resulting in a COP1 protein with only the N-terminal 282 amino acids, including both the zinc binding and the coiled-coil domains, produced a weak phenotypic defect. These results indicated that the N-terminal half of COP1 alone retains some activity and a disrupted C-terminal domain masks this remaining activity.