det1, cop1, and cop9 mutations cause inappropriate expression of several gene sets.

Genetic studies using Arabidopsis offer a promising approach to investigate the mechanisms of light signal transduction during seedling development. Several mutants, called det/cop, have been isolated based on their deetiolated/constitutive photomorphogenic phenotypes in the dark. This study examines the specificity of the det/cop mutations with respect to their effects on genes regulated by other signal transduction pathways. Steady state mRNA levels of a number of differently regulated gene sets were compared between mutants and the wild type. We found that det2, cop2, cop3, and cop4 mutants displayed a gene expression pattern similar to that of the wild type. By contrast, det1, cop1, and cop9 mutations exhibited pleiotropic effects. In addition to light-responsive genes, genes normally inducible by plant pathogens, hypoxia, and developmental programs were inappropriately expressed in these mutants. Our data provide evidence that DET1, COP1, and COP9 most likely act as negative regulators of several sets of genes, not just those involved in light-regulated seedling development.     

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.     

COP9: a new genetic locus involved in light-regulated development and gene expression in arabidopsis.

We report here the identification and characterization of a new Arabidopsis light-regulatory locus, COP9, mutation that leads to a constitutive photomorphogenic phenotype. Dark-grown cop9 seedlings exhibit many morphological characteristics of light-grown seedlings, including short hypocotyls and open and enlarged cotyledons with cell-type and chloroplast differentiation. Furthermore, the cop9 mutation leads to high-level expression of light-inducible genes in the absence of light, probably by altering the promoter activities of these genes. These properties imply that the mutation in the COP9 locus uncouples the light/dark signals from morphogenesis and light-regulated gene expression. In addition, light-grown cop9 mutants are severely dwarfed and are unable to reach maturation and flowering. This adult-lethal phenotype indicates that the COP9 locus also plays a critical role for normal development of the light-grown plant. Similar to cop1 mutants, but not det1, the cop9 mutants show (1) no effect on the phytochrome control of seed germination and (2) deficiency in the dark-adaptive change of expression of light-regulated genes. Our results suggest that the cop9 and cop1 mutations result in the same range of phenotypes and therefore COP9 and COP1 loci may encode closely related components in the same regulatory pathway.     

Overexpression of AtMYB52 confers ABA hypersensitivity and drought tolerance

We carried out activation tagging screen to isolate genes regulating abscisic acid (ABA) response. From the screen of approximately 10,000 plants, we isolated ca 100 ABA response mutants. We characterized one of the mutants, designated ahs1, in this study. The mutant is ABA-hypersensitive, and AtMYB52 was found to be activated in the mutant. Overexpression analysis to recapitulate the mutant phenotypes demonstrated that ATMYB confers ABA-hypersensitivity during postgermination growth. Additionally, AtMYB52 overexpression lines were drought-tolerant and their seedlings were salt-sensitive. Changes in the expression levels of a few genes involved in ABA response or cell wall biosynthesis were also observed. Together, our data suggest that AtMYB52 is involved in ABA response. Others previously demonstrated that AtMYB52 regulates cell wall biosynthesis; thus, our results imply a possible connection between ABA response and cell wall biosynthesis.     

Arabidopsis COP8, COP10, and COP11 genes are involved in repression of photomorphogenic development in darkness.

Wild-type Arabidopsis seedlings are capable of following two developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. Screening of Arabidopsis mutants for constitutive photomorphogenic development in darkness resulted in the identification of three new loci designated COP8, COP10, and COP11. Detailed examination of the temporal morphological and cellular differentiation patterns of wild-type and mutant seedlings revealed that in darkness, seedlings homozygous for recessive mutations in COP8, COP10, and COP11 failed to suppress the photomorphogenic developmental pathway and were unable to initiate skotomorphogenesis. As a consequence, the mutant seedlings grown in the dark had short hypocotyls and open and expanded cotyledons, with characteristic photomorphogenic cellular differentiation patterns and elevated levels of light-inducible gene expression. In addition, plastids of dark-grown mutants were defective in etioplast differentiation. Similar to cop1 and cop9, and in contrast to det1 (deetiolated), these new mutants lacked dark-adaptive change of light-regulated gene expression and retained normal phytochrome control of seed germination. Epistatic analyses with the long hypocotyl hy1, hy2, hy3, hy4, and hy5 mutations suggested that these three loci, similar to COP1 and COP9, act downstream of both phytochromes and a blue light receptor, and probably HY5 as well. Further, cop8-1, cop10-1, and cop11-1 mutants accumulated higher levels of COP1, a feature similar to the cop9-1 mutant. These results suggested that COP8, COP10, and COP11, together with COP1, COP9, and DET1, function to suppress the photomorphogenic developmental program and to promote skotomorphogenesis in darkness. The identical phenotypes resulting from mutations in COP8, COP9, COP10, and COP11 imply that their encoded products function in close proximity, possibly with some of them as a complex, in the same signal transduction pathway.     

A complement of ten essential and pleiotropic arabidopsis COP/DET/FUS genes is necessary for repression of photomorphogenesis in darkness.

Two genetic screens, one for mutations resulting in photomorphogenic development in darkness and the other for mutants with fusca phenotype, have thus far identified six pleiotropic Arabidopsis COP/DET/FUS genes. Here, we characterized representative mutants that define four additional pleiotropic photomorphogenic loci and a null mutant allele of the previously defined DET1 locus. Dark-grown seedlings homozygous for these recessive mutations exhibit short hypocotyls and expanded cotyledons and are lethal before reaching reproductive development. Dark-grown mutant seedlings also display characteristic photomorphogenic cellular differentiation and elevated expression of light-inducible genes. In addition, analyses of plastids from dark-grown mutants reveal partial chloroplast differentiation and absence of etioplast development. Root vascular bundle cells of light-grown mutant seedlings develop chloroplasts, suggesting that these FUS gene products are important for suppression of chloroplast differentiation in light-grown roots. Double-mutant analyses indicate that these pleiotropic cop/det/fus mutations are epistatic to mutations in phytochromes, a blue-light photoreceptor, and a downstream regulatory component, HY5. Therefore, there is a complement of at least 10 essential and pleiotropic Arabidopsis genes that are necessary for repression of photomorphogenic development.     

Genetic interactions between the chlorate-resistant mutant cr 8 8 and the photomorphogenic mutants cop1 and hy5

The chlorate-resistant mutant cr88 is defective in photomorphogenesis, as shown by the phenotypes of long hypocotyls in red light and yellow cotyledons under all light conditions. A subset of light-regulated genes is expressed at subnormal levels in cr88. To analyze further the role that CR88 plays in photomorphogenesis, we investigated the genetic interactions between cr88 and mutants of two other loci affecting photomorphogenesis, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and LONG HYPOCOTYL5 (HY5). COP1 represses the expression of light-regulated genes in the dark, and HY5 inhibits hypocotyl elongation in the light. Using morphological, cellular, and gene expression criteria for epistasis analyses to position CR88 in the genetic hierarchy of the photomorphogenesis pathway, we determined that CR88 acts downstream of COP1 but in a branch separate from HY5. In the course of our analysis, we discovered that light causes extensive destruction of plastids in dark-grown cop1 seedlings and that cr88 prevents this destruction.     

Analysis of the mutational effects of the COP/DET/FUS loci on genome expression profiles reveals their overlapping yet not identical roles in regulating Arabidopsis seedling development

Microarray gene expression profiling was used to examine the role of pleiotropic COP/DET/FUS loci as well as other partially photomorphogenic loci during Arabidopsis seedling development and genome expression regulation. Four types of lethal, pleiotropic cop/det/fus mutants exhibit qualitatively similar gene expression profiles, yet each has specific differences. Mutations in COP1 and DET1 show the most similar genome expression profiles, while the mutations in the COP9 signalosome (CSN) and COP10 exhibit increasingly diverged genome expression profiles in both darkness and light. The genome expression profiles of the viable mutants of COP1 and DET1 in darkness mimic those of the physiological light-regulated genome expression profiles, whereas the genome expression profiles of representative lethal mutants belong to another clade and significantly diverge from the normal light control of genome expression. Instead, these lethal pleiotropic mutants show genome expression profiles similar to those from seedlings growth under high light intensity stress. Distinct lethal pleiotropic cop/det/fus mutants also result in distinct expression profiles in the small portion of genes examined and exhibit similar relatedness in both light and darkness. The partial cop/det/fus mutants affected expression of both light regulated and non-light regulated genes. Our results suggest that pleiotropic COP/DET/FUS loci control is largely overlapping but also has separable roles in plant development. The partially photomorphogenic loci regulate a subset of photomorphogenic responses as well as other non-light regulated processes.     

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome.     

A gain-of-function phenotype conferred by over-expression of functional subunits of the COP9 signalosome in Arabidopsis

The COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild-type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6-1 mutant phenotype. Moreover, light-grown rFUS6 over-expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild-type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long-hypocotyl phenotype was also observed in transgenic seedlings over-expressing Arabidopsis COP9. This finding indicates that over-expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain-of-function phenotype relative to the complex. Human GPS1, when expressed in the fus6-1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low-abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.     

Flowering of Arabidopsis cop1 mutants in darkness

To elucidate the role of the COP1 gene in flowering, we analyzed flowering of cop1 mutant lines in darkness. When grown in the presence of 1% (w/v) sucrose, the cop1-6 mutant flowered in darkness, but cop1-1 and cop1-4 did not. However, cop1-1 and cop1-4 flowered in darkness when grown in the presence of 5% (w/v) sucrose. Therefore, the COP1 gene represses not only photomorphogenesis in seedlings but also flowering in darkness. Comparison of mRNAs levels of floral identity genes in cop1-6 and wild-type plants grown in darkness revealed increased mRNA levels of genes that act downstream of CO and reduced FLC mRNA level in cop1-6. Double mutants of cop1-6 and each of the late-flowering mutations cry2-1, gi-2, co-1, and ld-1 flowered in darkness. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The ld-1 cop1-6 double mutant flowered much earlier than the ld-1 mutant. The delay in flowering in the double mutants was not strongly influenced by the light conditions, whereas that of the gi-2 cop1-6 double mutant was reduced in darkness.     

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.     

The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in arabidopsis

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1-SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness.     

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.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.