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.     

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.     

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.     

The COP/DET/FUS proteins-regulators of eukaryotic growth and development

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.     

Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.     

A mutation in the Arabidopsis DET3 gene uncouples photoregulated leaf development from gene expression and chloroplast biogenesis

The genetic and phenotypic characterization of a new Arabidopsis mutant, de-etiolated -3, ( det 3), involved in light-regulated seedling development is described. A recessive mutation in the DET 3 gene uncouples light signals from a subset of light-dependent processes. The det 3 mutation causes dark-grown Arabidopsis thaliana seedlings to have short hypocotyls, expanded cotyledons, and differentiated leaves, traits characteristic of light-grown seedlings. Despite these morphological changes, however, the det 3 mutant does not develop chloroplasts or show elevated expression of nuclear- and chloroplast-encoded light-regulated mRNAs. The det 3 mutation thus uncovers a downstream branch of the light transduction pathways that separates leaf development from chloroplast differentiation and light-regulated gene expression. In addition, light-grown det 3 plants have reduced stature and apical dominance, suggesting that DET3 functions during growth in normal light conditions as well. The genetic interactions between mutations in det 1, det 2, and det 3 are described. The phenotypes of doubly mutant strains suggest that there are at least two parallel pathways controlling light-mediated development in Arabidopsis .     

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.     

Photomorphogenic development of the Arabidopsisshy2–1D mutation and its interaction with phytochromes in darkness

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2–1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2–1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2–1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2–1D mutant with the phytochrome-deficient mutants hy2, hy3 (phyB-1) and fre1–1 (phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2–1D single mutant. These results showed that phytochromes function in darkness in the shy2–1D mutant background. The implications of these results are discussed.     

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.     

Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis.

The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC (COP), DEETIOLATED (DET), and FUSCA (FUS) loci are essential regulatory genes involved in the light control of seedling developmental patterns in Arabidopsis. Although COP1, DET1, COP9, and FUS6 (also called COP11) have been cloned, their biochemical activities and interactions remain elusive. We have recently suggested that multiple pleiotropic COP, DET, and FUS genes may encode subunits of a large regulatory complex. In this study, we generated specific antibodies against Arabidopsis FUS6 and show that accumulation of both COP9 and FUS6 is coordinated in the pleiotropic cop, det, and fus mutant backgrounds and in wild-type plants throughout development. Both COP9 and FUS6 cofractionated into identical high molecular mass fractions in an analytical gel filtration assay, and neither was found in its monomeric form. Moreover, antibodies raised against either COP9 or FUS6 selectively coimmunoprecipitated both proteins. We have also developed an Arabidopsis protoplast immunolocalization assay and demonstrated that the COP9 complex is localized in the nucleus and that its nuclear localization is not affected by light conditions or tissue types. The integrated genetic and biochemical results strongly support the conclusion that both COP9 and FUS6 are components of the nuclear-localized COP9 complex. Therefore, we have provided the strongest evidence for the conclusion that at least some of the pleiotropic COP, DET, and FUS loci act in the same signaling pathway.     

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.     

AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.     

The Signaling Mechanism of Arabidopsis CRY1 Involves Direct Interaction with COP1

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.     

Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors

The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1,5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response. Received: 22 April 1999 / Accepted: 6 June 1999     

Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings.

A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins)

When grown in the absence of light, Arabidopsis thaliana deetiolated (det) mutants develop many of the characteristics of light-grown plants, including the development of leaves and chloroplasts, the inhibition of hypocotyl growth elongation, and elevated expression levels of light-regulated genes. We show here that dark-grown wild-type seedlings exhibit similar phenotypic traits if any one of a variety of cytokinins are present in the growth medium. We further show that the striking phenotype of det mutants is unlikely to be caused by different levels of cytokinins in these mutants. The three major Arabidopsis cytokinins, zeatin, zeatin riboside, and isopentenyladenosine, accumulate to similar levels in wild-type seedlings grown in either the light or the dark. There is no consistently different pattern for the levels of these cytokinins in wild-type versus det1 or det2 mutants. However, det1 and det2 have an altered response to cytokinin in a detached leaf senescence assay and in tissue culture experiments. A model is proposed in which light and cytokinins act independently or sequentially through common signal transduction intermediates such as DET1 and DET2 to control the downstream light-regulated responses.     

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.     

Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.