Light control of seedling morphogenetic pattern.
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Genes, Plant↗
Biomedical subjects
X W Deng
Publications and source records attributed to X W Deng.
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.
Allium↗
Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development.
Environmental light signals are sensed by multiple families of photoreceptors and transduced by largely unknown mechanisms to regulate plant development. In this report, genetic analysis suggested that light signals perceived by both phytochromes and a blue light receptor converge to repress the action of Arabidopsis COP9 in suppressing seedling photomorphogenesis. Molecular cloning of the gene revealed that COP9 encodes a novel protein of 197 amino acids whose expression is not regulated by light. COP9 functions as a large (> 560 kDa) complex(es) that is probably subjected to light modulation. In addition, COP8 and COP11 are required for either the COP9 complex formation or its stability. Therefore COP9, together with COP8 and COP11, defines a novel signaling step in mediating light control of plant development.
Amino Acid Sequence↗
Fresh view of light signal transduction in plants.
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Darkness↗
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.
Arabidopsis↗
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.
Alleles↗
Regulatory hierarchy of photomorphogenic loci: allele-specific and light-dependent interaction between the HY5 and COP1 loci.
Previous studies suggested that the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) gene product represses photomorphogenic development in darkness and that light signals reverse this action. In this report, we used genetic analysis to investigate the regulatory hierarchical relationship of COP1 and the loci encoding the photoreceptors and other signaling components. Our results showed that cop1 mutations are epistatic to the long hypocotyl mutations hy1, hy2, hy3, and hy4, suggesting that COP1 acts downstream of the phytochromes and a blue light receptor. Although epistasis of a putative null cop1-5 mutation over a hy5 mutation implied that COP1 acts downstream of HY5, the same hy5 mutation can suppress the dark photomorphogenic phenotypes (including hypocotyl elongation and cotyledon cellular differentiation) of the weak cop1-6 mutation. This, and other allele-specific interactions between COP1 and HY5, may suggest direct physical contact of their gene products. In addition, the synthetic lethality of the weak deetiolated1 (det1) and cop1 mutations and the fact that the cop1-6 mutation is epistatic to the det1-1 mutation with respect to light control of seed germination and dark-adaptative gene expression suggested that DET1 and COP1 may act in the same pathway, with COP1 being downstream. These results, together with previous epistasis studies, support models in which light signals, once perceived by different photoreceptors, converge downstream and act through a common cascade(s) of regulatory steps, as defined by DET1, HY5, COP1, and likely others, to derepress photomorphogenic development.
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.
Arabidopsis↗
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.
Amino Acid Sequence↗
COP1, an Arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G beta homologous domain.
Plant seedling development is capable of following 1 of 2 distinct morphogenic pathways: skotomorphogenesis in darkness and photomorphogenesis in light. Dark-grown Arabidopsis seedlings with recessive mutations at the constitutively photomorphogenic (COP1) locus indicate that the wild-type COP1 protein represses photomorphogenesis in darkness and that light reverses this repressive activity. Using a T-DNA-tagged mutant, we have cloned the COP1 locus. The amino-terminal half of the encoded protein contains a conserved zinc-binding motif, whereas the carboxyl-terminal half contains a domain homologous to the WD-40 repeat motif of G beta proteins. The presence of both a putative DNA-binding motif and a G protein-related domain in a single polypeptide suggests that COP1 may be the first of a new class of regulatory molecules. This novel structure could endow COP1 with the capacity to function as a negative transcriptional regulator capable of direct interaction with components of the G protein signaling pathway.
Amino Acid Sequence↗
Regulation of Plastid Gene Expression during Photooxidative Stress.
We have used the carotenoid biosynthesis inhibitor norflurazon to study the relationship between chloroplast and nuclear gene expression and the mechanisms by which plastid mRNA accumulation is regulated in response to photooxidative stress. By treating 4-week-old hydroponic spinach plants (Spinacea oleracea), we were able to determine the response at two distinct stages of chloroplast development. For all parameters studied, differences were found between the norflurazon-treated young and mature leaves. Young leaves lost essentially all pigment content in the presence of norflurazon, whereas mature leaves retained more than 60% of their chlorophyll and carotenoids. The accumulation of plastid mRNA was determined for several genes, and we found a decrease in mRNA levels for all genes except psbA in herbicide-treated young leaves. For genes such as atpB, psbB, and psaA, there was a corresponding change in the relative level of transcription, but for psbA and rbcL, transcription and mRNA accumulation were uncoupled. In norflurazon-treated mature leaves, all plastid mRNAs except psaA accumulated to normal levels, and transcription levels were either normal or higher than corresponding controls. This led to the conclusion that plastid mRNA accumulation is regulated both transcriptionally and posttranscriptionally in response to photooxidative stress. Although direct photooxidative damage is confined to the plastid and peroxisome, there is a feedback of information controlling the transcription of nuclear-encoded plastid proteins. Considerable evidence has accumulated implicating a "plastid factor" in this control. Therefore, the expression of several nuclear-encoded plastid proteins and the corresponding mRNAs were determined. Although the levels of both the small subunit of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b-binding protein and corresponding mRNAs were reduced, a 28-kilodalton chloroplast RNA-binding protein and corresponding mRNA were at normal levels in norflurazon-treated plants. Changes in mRNA and protein levels were not the result of a general loss due to photooxidation but rather the result of selective stabilization of certain components. The response of both genomes to photooxidative stress is discussed in terms of the postulated plastid factor.
Journal Article↗
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.
Arabidopsis↗
A negatively acting DNA sequence element mediates phytochrome-directed repression of phyA gene transcription.
Phytochrome represses transcription of its own phyA genes within 5 min of light-triggered conversion to its active Pfr form. We have utilized microprojectile mediated gene transfer into etiolated rice seedlings to delineate sequence elements in the oat phyA3 promoter responsible for this regulation. Linker-scan mutagenesis of this promoter has identified two positive elements which together are necessary for maximal transcription in the absence of Pfr. These elements are designated PE1, centered at position -357 bp, and PE3, centered at position -96 bp. Sequence mutagenesis immediately downstream of PE3 results in maximal transcription in the presence of high Pfr levels, indicating that Pfr represses phyA3 transcription through a negatively acting sequence element. This element, designated RE1, with the sequence CATGGGCGCGG, encompasses a motif that is highly conserved in all monocot phyA promoters thus far characterized. DNase I protection analysis indicates that oat nuclear extracts contain multiple factors that bind to an array of sequence motifs, including PE1 and part of PE3, within 400 bp upstream of the oat phyA3 transcription start site. This DNA-binding pattern is not altered by Pfr. Weak binding to part of the RE1 motif is evident but also with no difference between high and low Pfr levels. We conclude that the signal transduction chain that mediates Pfr-directed repression of phyA3 transcription terminates with a negatively acting transcription factor that binds to the sequence element RE1.
Base Sequence↗
cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis.
Light signals from the environment are perceived by specific regulatory photoreceptors in plants and are transduced by unknown mechanisms to genes that control growth and development. We have identified a genetic locus in Arabidopsis thaliana, which appears to play a central role in this transduction process. Mutations in this locus, designated cop1 (constitutively photomorphogenic), result in dark-grown seedlings with the morphology of wild-type seedlings grown in the light. In addition, these mutations lead to constitutive expression of an array of normally light-regulated genes in dark-grown seedlings and in light-grown adult plants placed in darkness. Promoter-reporter fusion constructs of some of these genes are constitutively expressed in dark-grown transgenic cop1 seedlings, indicating that the aberrant behavior of these genes results primarily from aberrant modulation of their promoter activities in the mutant. In contrast, light control of seed germination and diurnal control of cab gene expression is normal in the cop1 mutants. Because these mutations are recessive, we conclude that in seedlings and adult plants, the wild-type cop1 gene product normally acts in darkness to repress the expression of genes involved in the dark-adaptive developmental and that regulatory photoreceptors act to reverse this action upon exposure to light. However, photocontrol of seed germination and diurnal rhythms is apparently exerted via one or more separate pathways not involving the cop1 product. one or more separate pathways not involving the cop1 product.
Circadian Rhythm↗
The chloroplast genome exists in multimeric forms.
Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. We found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density. DNA gyrase decatenation analysis demonstrates that the majority of the DNA molecules are oligomers rather than catenanes. The relative amounts of monomer, dimer, trimer, and tetramer forms, quantitated by molecular hybridization, are 1, 1/3, 1/9, and 1/27, respectively, and do not change during leaf maturation. The possible mechanisms of chloroplast DNA oligomer formation are discussed.
Journal Article↗
Post-transcriptional control of plastid mRNA accumulation during adaptation of chloroplasts to different light quality environments.
The adaptation of germinating spinach seedlings to yellow and red light was studied and compared with plants grown in white light. Spinach chloroplasts isolated from cotyledons and leaves of yellow and white light-grown plants showed similar membrane structures and compositions, while chloroplasts from plants grown in red light have significant adaptive changes. Based on an equal amount of chlorophyll, these changes include a reduction in the number of photosystem I complexes, an increase of photosystem II antenna size, and an increased ratio of stacked to unstacked membranes in red light-adapted chloroplasts. The decrease in the number of photosystem I complexes per unit of chlorophyll in these chloroplasts was qualitatively correlated with an approximately 10-fold decrease in the level of the psaA mRNA encoding the photosystem I 65-kilodalton to 70-kilodalton chlorophyll apoprotein, as well as with a differential decrease in mRNA levels of other photosynthetic proteins. Light quality adaptations do not significantly affect the plastid to nuclear DNA ratio or the overall chloroplast transcription activity. The relative transcriptional activities of 10 plastid genes, as determined by run-on transcription assays, are similar in chloroplasts from cotyledons and leaves of plants grown under the three light qualities. Only the psaA gene shows a 30% to 40% decrease in transcription activity in chloroplasts of plants adapted to red light. This decrease in psaA transcription activity, however, cannot fully account for the decrease of its mRNA level. We conclude, therefore, that post-transcriptional mechanisms are primarily responsible for the control of differential chloroplast mRNA accumulation in light quality adaptations.
Adaptation, Physiological↗
Constitutive transcription and regulation of gene expression in non-photosynthetic plastids of higher plants.
The plastid genome in higher plants contains >50 genes for rRNAs, tRNAs and proteins for transcriptional and translational functions, besides the genes encoding photosynthetic proteins. Considering the totipotency of most higher plant cells and the differentiation capacity of plastids, it can be inferred that at least the genes for genetic functions must be constitutively expressed in all plant organs, including non-photosynthetic roots, to maintain a basal level of transcriptional and translational activities. To test this hypothesis, transcription, RNA accumulation and polysome formation were analyzed in root amyloplasts, and in plastids from hypocotyls and cotyledons of dark-grown spinach seedlings. The results for 10 representative genes show that they are constitutively transcribed at relative rates which are similar in root amyloplasts and leaf chloroplasts. The differential accumulation of their mRNAs in roots and other non-photosynthetic plant organs is controlled at the post-transcriptional level by a developmental program. Although mRNAs for photosynthetic proteins are detectable in root amyloplasts, some of them are specifically depleted from polysomes relative to mRNAs for ribosomal proteins. This translational discrimination does not result from modifications in splicing or 5'- and 3' -end processing of mRNAs for photosynthetic proteins, since processing is identical in root amyloplasts and leaf chloroplasts. The results support the model of constitutive transcription of the plastid genome, and indicate that the expression of most plastid genes in spinach plants is controlled primarily by post-transcriptional and translational mechanisms.
Journal Article↗
Transcriptional and post-transcriptional control of plastid mRNA levels in higher plants.
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Chloroplasts↗