Light-induced Gene Expression Research Articles

Neuropeptide Y differentially suppresses per1 and per2 mRNA induced by light in the suprachiasmatic nuclei of the golden hamster.

Neuropeptide Y (NPY), present in an input pathway to the suprachiasmatic nuclei (SCN), can block the effects of light on circadian rhythms. The authors have studied this interaction using an in vitro brain slice technique. Effects of NPY on light-induced period1 and period2 mRNA in the SCN were examined in vitro following a light pulse during early subjective night. Golden hamsters (n = 91) were housed under a 14:10 LD cycle and then moved to constant dim red light for 3 days. Hamsters were exposed to a 5-min light pulse previously shown to induce phase shifts and prepared for in vitro application of NPY. Hypothalamic slices containing the SCN were maintained in vitro for 40 min to 4 h after the light pulse, then quick-frozen. Sections were evaluated by in situ hybridization with [35S]-labeled cRNA probes for per mRNA. Rapid light induction of both per1 and per2 by 40 min and 1 h after the light pulse, respectively, was apparent, with NPY inhibition of this response significant by at least these same time points. However, although striking suppression of per2 mRNA by the NPY continued through the peak for per2 at 2 h, per1 mRNA levels rebounded quickly to equal the per1 induction peak at 1 h and mirrored the control light induction pattern for per1 thereafter. Delaying NPY to 30 min after slice preparation demonstrated that NPY is capable of suppressing peak per1 levels. These results confirm the feasibility of measuring light-induced gene expression in the SCN in vitro. A differential regulation of per1 and per2 transcription might be of critical importance for the modulation of circadian responses to light.

Dissociation between Light-Induced Phase Shift of the Circadian Rhythm and Clock Gene Expression in Mice Lacking the Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1(-)/-) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1(-)/- mice. However, in contrast to the phase advance seen in wild-type mice, PAC1(-)/- mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.

Primary structure and expression of a 24-kD vacuolar protein (VP24) precursor in anthocyanin-producing cells of sweet potato in suspension culture.

A 24-kD vacuolar protein (VP24) accumulates abundantly in intravacuolar pigmented globules in anthocyanin-containing sweet potato (Ipomoea batatas) cells in suspension culture. A cDNA clone encoding VP24 was isolated from a cDNA library constructed from light-irradiated suspension-cultured cells. Sequence analysis revealed that a 2.9-kbp VP24 cDNA encodes a protein of 893 amino acid residues with a molecular mass of 96.3 kD. According to the deduced amino acid sequence of VP24 cDNA, VP24 is probably synthesized as a large precursor protein with an N-terminal extension composed of a signal peptide and a propeptide, plus the polypeptide of the mature VP24 and its C-terminal propeptide, which contains the multiple transmembrane domains. A search in the ProDom database revealed the mature VP24 domain belongs to the zinc metalloprotease family. Northern analysis revealed that the single 2.9-kb VP24 mRNA increases rapidly after light irradiation, whereas VP24 mRNA was undetectable in the dark-cultured cells or in the presence of a high concentration of 2,4-dichlorophenoxyacetic acid. Light-induced VP24 gene expression closely correlated with the accumulation of anthocyanin in the vacuoles. These results suggested that proteins derived from the VP24 precursor protein may be involved in vacuolar transport and/or accumulation of anthocyanin synthesized in the cytosol.

Proteome analysis of light-induced proteins in Synechocystis sp. PCC 6803: identification of proteins separated by 2D-PAGE using N-terminal sequencing and MALDI-TOF MS.

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

Role of a white collar-1-white collar-2 complex in blue-light signal transduction

Mutations in either white collar-1 (wc-1) or white collar-2 (wc-2) lead to a loss of most blue-light-induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that WC-1 and WC-2 are transcription factors regulating the expression of light-induced genes. The WC proteins form homo- and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that WC-1 undergoes a change in mobility due to light-induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized WC-1. WC-2 is unmodified and also does not change quantitatively in the time frame examined. Light-dependent phosphorylation of WC-1 also occurs in a wc-2 mutant, suggesting that a functional WC-2 is dispensable for this light-specific event. These results suggest that light-induced phosphorylation and degradation of WC-1 could play a role in the transient expression of blue-light-regulated genes. Our findings suggest a mechanism by which WC-1 and WC-2 mediate light responses in Neurospora.

Circadian rhythms and light responsiveness of mammalian clock gene, Clock and BMAL1, transcripts in the rat retina

Circadian expression and light-responsiveness of the mammalian clock genes, Clock and BMAL1, in the rat retina were examined by in situ hydbribization under constant darkness. A small but significant daily variation was detected in the Clock transcript level, but not in BMAL1. Light increased the Clock and BMAL1 expressions significantly when examined 60 min after exposure. The light-induced gene expression was phase-dependent for Clock and peaked at ZT2, while rather constant throughout the day for BMAL1. These findings suggest that Clock and BMAL1 play different roles in the generation of circadian rhytm in the retina from those in the suprachiasmatic nucleus. Different roles are also suggested between the two genes in the photic signal transduction in the retina.

COP1 and HY5 interact to mediate light-induced gene expression

The plant photomorphogenic mutants hy5 and cop1 have reciprocal phenotypes: hy5 has the long hypocotyl morphology of a dark-grown seedling when grown in the light, whereas cop1 mutants undergo many processes associated with photomorphogenesis, even in the absence of light. The opposing phenotypes of these two mutants extend into general growth characteristics such as lateral root development. The demonstration of a direct molecular interaction between COP1, a repressor of photomorphogenesis localized to the nucleus during the dark, and HY5,1 a bZIP transcription factor2that binds sequence specifically to the light-regulated CHS promoter,1 provides a possible mechanism by which COP1 transduces light signals to gene expression in Arabidopsis. BioEssays 20:445–448, 1998. © 1998 John Wiley & Sons Inc.

Light is associated with a 34 kDa ribonucleoprotein in spinach chloroplasts.

Chloroplast ribonucleoproteins (RNPs) encoded by the nuclear genome may play an important role in the post-transcriptional steps of light-induced chloroplast gene expression. To study the relationship between the synthesis of a 34 kDa ribonucleoprotein (p34) and light in spinach chloroplasts, a polyclonal antibody was raised against p34. Immunoblotting revealed that p34 synthesis was induced in a light dependent manner. In addition, levels of the large subunit of ribulose-1,5-bis phosphate carboxylase/oxygenase (Rubisco) encoded by the chloroplast genome fluctuated in relation to those of p34. These results suggest that the synthesis of p34 in response to light induces of chloroplast gene expression.

Role of a COP1 interactive protein in mediating light-regulated gene expression in arabidopsis.

Arabidopsis seedlings display distinct patterns of gene expression and morphogenesis according to the ambient light condition. An Arabidopsis nuclear protein, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), acts to repress photomorphogenesis in the absence of light. The Arabidopsis CIP7 protein was identified by its capability to interact with COP1. CIP7 is a novel nuclear protein that contains transcriptional activation activity without a recognizable DNA binding motif. CIP7 requires light for its high level of expression, and COP1 seems to play a role in repressing its expression in darkness. Decreasing CIP7 expression by introducing antisense CIP7 RNA resulted in defects in light-dependent anthocyanin and chlorophyll accumulation. Antisense plants also displayed reduced expression of light-inducible genes for anthocyanin biosynthesis and photosynthesis. However, no defect was observed in light-dependent inhibition of hypocotyl elongation. Taken together, our data indicate that CIP7 acts as a positive regulator of light-regulated genes and is a potential direct downstream target of COP1 for mediating light control of gene expression.

A Serum Shock Induces Circadian Gene Expression in Mammalian Tissue Culture Cells

The treatment of cultured rat-1 fibroblasts or H35 hepatoma cells with high concentrations of serum induces the circadian expression of various genes whose transcription also oscillates in living animals. Oscillating genes include rper1 and rper2(rat homologs of the Drosophila clock gene period), and the genes encoding the transcription factors Rev-Erbα, DBP, and TEF. In rat-1 fibroblasts, up to three consecutive daily oscillations with an average period length of 22.5 hr could be recorded. The temporal sequence of the various mRNA accumulation cycles is the same in cultured cells and in vivo. The serum shock of rat-1 fibroblasts also results in a transient stimulation of c- fos and rper expression and thus mimics light-induced immediate-early gene expression in the suprachiasmatic nucleus.

CREB-induced transcriptional activation depends on mGluR6 in rod bipolar cells

To investigate the molecular mechanisms of stimuli-induced transcriptional activation in neuronal cells, we have investigated the light-induced gene expression in the neural retina of rats. The immunoreactivity for phosphorylated cAMP responsive element binding protein (PCREB-IR) was expressed in the outer half of the inner nuclear layer (INL) and the ganglion cell layer (GCL) after 5 min exposure to steady light also in mice. In addition to these cells, PCREB-IR was also detected in the inner border of the INL after 5 min exposure to flashing light. Both steady and flashing lights induced c- fos mRNA in the same types of cells as the PCREB-IR-positive cells. Majority of PCREB immunoreactive nuclei in the outer half of the INL were also immunopositive for anti-protein kinase C α (PKC α), a marker of rod bipolar cells, while CaM kinase IV immunoreactivity was not detected in these cells. PCREB-IR and c- fos gene expression in the PKC α positive rod bipolar cells were lost in mice lacking metabotropic glutamate receptor 6 (mGluR6). Thus, we propose that the transcriptional response of CREB to light stimulation in rod bipolar cells is regulated via mGluR6.

Light-induced CREB phosphorylation and gene expression in rat retinal cells.

The signal pathway for light-induced expression of c-fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c-fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c-fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca2+ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c-fos/SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser133 and CaM kinase II in the neural retina.

The rat suprachiasmatic nucleus is a clock for all seasons.

Seasonal changes of daylength (photoperiod) affect the expression of hormonal and behavioral circadian rhythms in a variety of organisms. In mammals, such effects might reflect photoperiodic changes in the circadian pace-making system [located in the suprachiasmatic nucleus (SCN) of the hypothalamus] that governs these rhythms, but to date no functionally relevant, intrinsic property of the SCN has been shown to be photoperiod dependent. We have analyzed the temporal regulation of light-induced c-fos gene expression in the SCN of rats maintained in long or short photoperiods. Both in situ hybridization and immunohistochemical assays show that the endogenous circadian rhythm of light responsiveness in the SCN is altered by photoperiod, with the duration of the photosensitive subjective night under the short photoperiod 5-6 h longer than under the long photoperiod. Our results provide evidence that a functional property of the SCN is altered by photoperiod and suggest that the nucleus is involved in photoperiodic time measurement.

Regulation of Early Light-Inducible Protein Gene Expression by Blue and Red Light in Etiolated Seedlings Involves Nuclear and Plastid Factors.

Early light-inducible proteins (ELIPs) are nuclear-encoded chloroplast proteins whose genes are transiently transcribed during the greening process of etiolated plants. In the present work the regulation of ELIP gene expression by blue and red light has been investigated in plumulas of etiolated pea plants (Pisum sativum). The results show that the steady-state level of ELIP transcripts is controlled by a combined action of phytochrome and blue light receptor systems and, in addition, depends on the age of the seedlings. Both a low-light fluence system of blue and a very-low-fluence system of red light are involved in ELIP induction. The threshold for accumulation of ELIP transcripts was as low as 10-5 [mu]E m-2 s-1 for both light qualities but a different pattern of accumulation was obtained in blue and in red light. Blue light not only acts at the level of transcription but also regulates the stability of the ELIP transcripts in a light intensity-dependent manner. Moreover, it is shown that product(s) of nuclear gene(s) negatively regulate the steady-state level of ELIP transcripts during the 1st h of illumination with red light. Preillumination of seedlings with white light abolishes this repression. Accumulation of ELIP transcripts requires "plastid factors" in both blue and red light qualities.

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.

Protein phosphatase activity is required for light-inducible gene expression in maize : EMBO J. 12, 3497–3505

Protein phosphatase activity is required for light-inducible gene expression in maize : EMBO J. 12, 3497–3505

Light-induced gene expression in the suprachiasmatic nucleus of young and aging rats

We investigated age-related changes in molecular mechanism associated with synchronization of circadian rhythms to the environment. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker that regulates a variety of physiological and behavioral rhythms. Recent studies have demonstrated photic induction of immediate early genes (IEGs) such as c- fos in the SCN in a circadian-phase dependent manner, suggesting that IEGs may be part of the pathway for entrainment of circadian rhythms. We find that there is a decreased response of the IEGs c-fos, and NGFI-A but not NGFI-B in the SCN of old animals after photic stimulation. Changes in gene transcription indicated by IEGs may provide insights into the molecular machinery of the biological clock and ultimately elucidate mechanisms underlying the age-dependent decay of circadian organization.

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.

Light-inducible and tissue-specific pea lhcp gene expression involves an upstream element combining enhancer- and silencer-like properties

Studies in viral and mammalian cell systems have identified regulatory elements, collectively termed enhancers, which are cis-acting elements effective in either orientation and at both 5′ and 3′ ends of a gene, and which increase transcription when linked to either their own or a heterologous promoter gene system1–5. Certain upstream elements in yeast have also been shown to exert a negative effect on the rate of transcription of cell-cycle-specific genes and these have been called silencers6. Two of the best characterized plant gene families are those of the small subunit of the ribulose 1,5-bisphosphate carboxylase (rbcS) genes7 and the lightharvesting chlorophyll a/b-bind ing protein (lhcp) genes8. The first enhancer element in plants has recently been described showing that an upstream element of an rbcS gene could, in both orientations, confer light-inducible expression on a heterologous promoter/gene system9. We report here that a 247-base pair (bp) element from an lhcp gene acts not only as a light-inducible enhancer but also as a tissue-specific ‘silencer’.