Light-induced Gene Research Articles

Light-Induced Carotenogenesis inStreptomyces coelicolorA3(2): Identification of an Extracytoplasmic Function Sigma Factor That Directs Photodependent Transcription of the Carotenoid Biosynthesis Gene Cluster

Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found that Streptomyces coelicolor A3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and beta-carotene. The carotenoid biosynthesis gene cluster of S. coelicolor consists of two convergent operons, crtEIBV and crtYTU, as previously shown for Streptomyces griseus. The crtEIBV null mutant completely lost its ability to produce carotenoids. The crt gene cluster is flanked by a regulatory region that consists of two divergent operons, litRQ and litSAB. The lit (light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor (sigmaLitS), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters preceding crtE (PcrtE), crtY (PcrtY), litR (PlitR), and litS (PlitS) are activated upon illumination. A litS mutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that sigmaLitS directs light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified sigmaLitS recombinant protein generated specific PcrtE and PcrtY transcripts in an in vitro runoff transcriptional assay. A litR mutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression of litS resulted in constitutive carotenoid production in both the wild type and the litR mutant. These results indicate that sigmaLitS acts as a light-induced sigma factor that directs transcription of the crt biosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression in Streptomyces.

Entrainment in a Model of the Mammalian Circadian Oscillator

To adapt the timing of processes regulated by the circadian clock to seasonally varying photoperiods, the phase relation between the circadian clock and dusk or dawn ("phase of entrainment") must be tightly adjusted. The authors use a mathematical model of the molecular mammalian circadian oscillator to investigate the influence of the free-running period (tau) and the shape of the PRC on the phase of entrainment. They find that a phase-dependent sensitivity ("gating") of light-induced period gene transcription enables a constant phase relation to dusk or dawn under different photoperiods. Depending on the freerunning period tau and on the shaping of the PRC by gating, the model circadian oscillator tracks either light onset or light offset under different photoperiods. The study indicates that the phase of entrainment of oscillating cells can be systematically controlled by regulating both gating and the free-running period tau.

UBE2V2 (MMS2) is not required for effective immunoglobulin gene conversion or DNA damage tolerance in DT40

The RAD6/RAD18 heterodimer promotes translesion synthesis via the monoubiquitination of the DNA sliding clamp, PCNA. In S. cerevisiae, a second complex, UBC13/MMS2/RAD5, can extend this single ubiquitin with a non-canonical lysine 63-linked chain. This polyubiquitination step is required for an error-free mode of bypass, possibly template switching by the stalled replication complex. Evidence of a role for the human homologue of MMS2, UBE2V2, in such a process has been inferred from the abrogation of ultraviolet light-induced gene conversion following antisense knockdown of the transcript in human fibroblasts. To ask whether a similar mechanism contributes to abasic site-induced immunoglobulin gene conversion, and to ascertain the role of UBE2V2 in the vertebrate DNA damage response we created a ube2v2 mutant of the chicken cell line DT40. Unlike budding yeast mms2, ube2v2 DT40 does not exhibit significant hypersensitivity to DNA damage, nor the elevated sister chromatid exchange seen in vertebrate rad18 mutants suggesting that UBE2V2 plays a minor or redundant role in RAD18 dependent DNA damage tolerance. In addition, both ube2v2 and rad18 DT40 display more or less normal levels of immunoglobulin gene conversion and, despite the important role played by RAD18 in DNA damage induced translesion synthesis, rad18 DT40, unlike rev1 DT40, does not show a defect in non-templated immunoglobulin gene mutation. Together these data suggest that signalling through PCNA ubiquitination is not required for immunoglobulin diversification in DT40.

Arabidopsis HAF2 Gene Encoding TATA-binding Protein (TBP)-associated Factor TAF1, Is Required to Integrate Light Signals to Regulate Gene Expression and Growth

Plant growth and development are sensitive to light. Light-responsive DNA-binding transcription factors have been functionally identified. However, how transcription initiation complex integrates light signals from enhancer-bound transcription factors remains unknown. In this work, we characterized mutations within the Arabidopsis HAF2 gene encoding TATA-binding protein-associated factor TAF1 (or TAF(II)250). The mutation of HAF2 induced decreases on chlorophyll accumulation, light-induced mRNA levels, and promoter activity. Genetic analysis indicated that HAF2 is involved in the pathways of both red/far-red and blue light signals. Double mutants between haf2-1 and hy5-1, a mutation of a light signaling positive DNA-binding transcription factor gene, had a synergistic effect on photomorphogenic traits and light-activated gene expression under different light wavelengths, suggesting that HAF2 is required for interaction with additional light-responsive DNA-binding transcription factors to fully respond to light induction. Chromatin immunoprecipitation assays showed that the mutation of HAF2 reduced acetylation of histone H3 in light-responsive promoters. In addition, transcriptome analysis showed that the mutation altered the expression of about 9% of genes in young leaves. These data indicate that TAF1 encoded by the Arabidopsis HAF2 gene functions as a coactivator capable of integrating light signals and acetylating histones to activate light-induced gene transcription.

Alterations of gene expression in skin and lung of mice exposed to light and cigarette smoke.

We previously showed that sunlight-mimicking light induces genotoxic damage not only in skin but also even in lung, bone marrow, and peripheral blood of hairless mice. Moreover, light and smoke acted synergically in the respiratory tract. To clarify the mechanisms involved, we investigated by cDNA-arrays the expression of 746 toxicologically relevant genes in skin and lungs of mice exposed for 28 days to light and/or environmental cigarette smoke. Glutathione-S-transferase-Pi and catalase were overexpressed in the lungs of mice exposed to light only. Moreover, the light induced in skin the expression of genes involved in carcinogenesis, photoaging, and production of genotoxic and oxidizing derivatives traveling at a distance. Smoke induced the expression of multiple genes in both skin and lung, which reflect adaptive responses and mechanisms related to cancer and, possibly, to emphysema and stroke. As shown in mice exposed to both light and smoke, the light tended to increase smoke-induced gene expression in lungs, while smoke tended to attenuate light-induced gene expression in skin. The oral administration of the nonsteroidal anti-inflammatory drug sulindac inhibited the light-induced overexpression of cyclooxygenase-2 and oxidative stress-related genes in skin, and down-regulated smoke-induced genes involved in oxidative stress, removal of damaged proteins, inflammation, and immune response in lung. These results provide a mechanistic insight explaining the systemic alterations induced by both light and smoke in mouse skin and lungs.

Negative Control of Circadian Clock Regulator E4BP4 by Casein Kinase Iϵ-Mediated Phosphorylation

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock [1]. E4bp4 is a light-inducible gene in the chick pineal gland [2], and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene [2, 3]. Here, we demonstrate that prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 was phosphorylated progressively and then disappeared in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1ϵ (CK1ϵ). CK1ϵ physically associated with E4BP4 and phosphorylated it. CK1ϵ-catalyzed phosphorylation of E4BP4 resulted in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation was attenuated by CK1ϵ in a kinase activity-independent manner. CK1ϵ-mediated posttranslational regulation was accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1ϵ as a negative regulator for E4BP4-mediated repression of cPer2.

A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17β-hydroxysteroid dehydrogenase (17β-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed.

669. Light-Induced Adenovirus Gene Transfer

The development of methods for efficient and specific delivery of therapeutic genes into target tissues is an important issue for further development of in vivo gene therapy. In the present study the physical targeting technique, photochemical internalization (PCI), has been used in combination with adenovirus. PCI is based on the use of photosensitizing compounds that localize in endocytic membranes of cells. Upon light exposure the photosensitizer induce damage to the membrane, facilitating release of internalized particles from the endocytic vesicles into the cytosol. The combination of PCI and adenoviral transduction has previously been shown to be favorable compared to adenovirus alone, and enables up to 20 times increase in gene transfer in a variety of cell lines. The observed enhancement in gene transduction efficacy may prove important in the development of a viable gene therapeutic strategy, which is dependent on an efficient, targeted and safe methodology for gene delivery. The aim of this study was to increase the understanding of the molecular mechanism behind photochemical enhancement of adenoviral transduction. K44A Hela cells, mutated in dynamin, were treated with photosensitizer before infection with adenovirus and exposure to light. Cells expressing normal dynamin showed an enhanced gene expression after light treatment, while exposure to light did not have a pronounced effect in cells expressing negative mutated dynamin. Dynamin is involved in both clathrin and caveolar mediated endocytosis. To investigate if both pathways were involved during photochemical transduction, cells were treated with chlorpromazine and filipinIII before infection and light treatment. Chlorpromazine inhibits clathrin-mediated endocytosis, while filipinIII inhibits the caveolar mediated uptake. Treating the cells with chlorpromazine induced a dose dependent reduction of gene transfer in photochemically treated cells. Furthermore, the cellular localization of both viral particles and photosensitizer, after treatment with inhibitor agent, were investigated using fluorescence microscope.

669. Light-Induced Adenovirus Gene Transfer

669. Light-Induced Adenovirus Gene Transfer

Regulation of ultraviolet light-induced gene expression by gene size.

UV light induces the expression of a wide variety of genes. At present, it is unclear how cells sense the extent of DNA damage and alter the expression of UV-induced genes appropriately. UV light induces DNA damage that blocks transcription, and the probability that a gene sustains transcription-blocking DNA damage is proportional to locus size and dose of UV light. Using colon carcinoma cells that express a temperature-sensitive variant of p53 and undergo p53-dependent apoptosis after UV irradiation, we found that the number of p53-induced genes identified by oligonucleotide microarray analysis decreased in a UV dose-dependent manner. This was associated with a statistically significant shift in the spectrum of p53-induced genes toward compact genes with fewer and smaller introns. Genes encoding proapoptotic proteins involved in the initiation of the mitochondrial apoptotic cascade were prominent among the compact p53 target genes, whereas genes encoding negative regulators of p53 and the mitochondrial apoptotic pathway were significantly larger. We propose that the shift in spectrum of UV-responsive gene expression caused by passive effects of UV lesions on transcription acts as a molecular dosimeter, ensuring the elimination of cells sustaining irreparable transcription-blocking DNA damage.

Singlet oxygen signaling: from intimate to global.

Reactive oxidizing species (ROS), such as hydrogen peroxide, nitric oxide, superoxide anion, and singlet oxygen, can damage cells as well as initiate responses such as new gene expression. The cell response evoked is strongly dependent on several factors. The subcellular location for formation of an ROS may be especially important for a highly reactive ROS, because it diffuses only a very short distance before reacting with a cellular molecule. How does a short-lived, locally acting ROS trigger responses at distant subcellular sites? This issue is discussed in light of a study of gene expression initiated by singlet oxygen, a ROS that exists for less than 100 ns in cells. Singlet oxygen is generated after a photosensitizer absorbs light energy and transfers the energy to molecular oxygen. To assess the role of singlet oxygen in light-induced gene expression in Arabidopsis, a mutant was used that accumulates the photosensitizer protochlorphyllide in chloroplasts. Three mechanisms are discussed that may connect the site of singlet oxygen formation to the signal transduction components: (i) direct oxidation of a signaling component by singlet oxygen, (ii) formation of oxidation products near the sites of singlet oxygen formation that diffuse to and react with signaling components, and (iii) alteration of the redox balance of the cell to a more oxidized state such that a greater proportion of a signaling pathway component is oxidized.

The photomorphogenesis-related mutant red1 is defective in CYP83B1, a red light-induced gene encoding a cytochrome P450 required for normal auxin homeostasis.

Previous genetic analysis identified a component, RED1, that is required for normal de-etiolation of Arabidopsis thaliana (L.) Heynh. seedlings in continuous red light (Rc). red1 mutant seedlings exhibit elongated hypocotyls and reduced cotyledon size specifically in Rc and not in continuous far-red light (FRc). Here, we show that red1 is allelic to sur2 and atr4, and is defective in the cytochrome P450 CYP83B1, an enzyme required for normal auxin homeostasis. Two alleles of atr4, like red1, exhibit increased hypocotyl elongation and reduced cotyledon expansion in Rc but not in FRc. We further show that CYP83B1 transcript levels are elevated specifically in Rc-grown seedlings when compared with seedlings grown in darkness or FRc. Hence, the Rc-specific phenotype of the red1 mutant may indicate that Rc-induction of the CYP83B1 transcript is necessary for normal seedling de-etiolation in the wild type.

Induction of early light-inducible protein gene expression in Pisum sativum after exposure to low levels of UV-B irradiation and other environmental stresses.

Plants are constantly subjected to environmental changes and have developed various defence mechanisms to facilitate their continued existence. Pisum sativum plants were exposed to low levels of UV-B radiation and ELIP (early light-inducible proteins) mRNA, with a probable protective function, was rapidly and strongly induced during this type of stress. To our knowledge, this is the only photosynthetic gene that is up-regulated following exposure to UV-B, and this result has to be compared with studies predominantly reporting down-regulation by UV-B of genes encoding proteins localised in the plastid. The expression pattern of ELIP mRNA in pea was also investigated during salt, wounding and ozone stress. The transcript levels of ELIP were induced after the salt and wounding treatments but not during ozone fumigation.

The suprachiasmatic nucleus: a clock of multiple components.

Although impressive progress has been made in understanding the molecular basis of pacemaker function in the suprachiasmatic nucleus (SCN), fundamental questions about cellular and regional heterogeneity within the SCN, and how this heterogeneity might contribute to SCN pacemaker function at a tissue level, have remained unresolved. To reexamine cellular and regional heterogeneity within the SCN, the authors have focused on two key questions: which SCN cells are endogenously rhythmic and/or directly light responsive? Observations of endogenous rhythms of electrical activity, gene/protein expression, and protein phosphorylation suggest that the SCN in mammals examined to date is composed of anatomically distinct rhythmic and nonrhythmic components. Endogenously rhythmic neurons are primarily found in rostral, dorsomedial, and ventromedial portions of the nucleus; at mid and caudal levels, the distribution of endogenously rhythmic cells in the SCN has the appearance of a "shell." The majority of nonrhythmic cells, by contrast, are located in a central "core" region of the SCN, which is complementary to the shell. The location of light-responsive cells, defined by direct retinohypothalamic input and light-induced gene expression, largely overlaps the location of nonrhythmic cells in the SCN core, although, in hamsters and mice light-responsive cells are also present in the ventral portion of the rhythmic shell. While the relative positions of rhythmic and light-responsive components of the SCN are similar between species, the precise boundaries of these components, and neurochemical phenotype of cells within them, are variable. Intercellular communication between these components may be a key feature responsible for the unique pacemaker properties of the SCN observed at a tissue and whole animal level.

Light directed gene transfer by photochemical internalisation.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

The ERK/MAP kinase pathway couples light to immediate-early gene expression in the suprachiasmatic nucleus.

Signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been identified as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Given this observation, it was of interest to determine where within the entrainment process the MAPK pathway was functioning. In this study, we examined the role of the MAPK pathway as a regulator of light-induced gene expression in the SCN. Towards this end, we characterized the effect pharmacological disruption of the MAPK cascade has on the expression of the immediate-early genes c-Fos, JunB and EGR-1. We report that uncoupling light from MAPK pathway activation attenuated the expression of all three gene products. In the absence of photic stimulation, inhibition of the MAPK pathway did not alter basal gene product expression levels. Light-induced activation of cAMP response element (CRE)-dependent transcription, as assessed using a CRE-LacZ transgenic mouse strain, was also disrupted by blocking MAPK pathway activation. These results reveal that the MAPK cascade functions as one of the first transduction steps leading from light to rapid transcriptional activation, an essential event in the entrainment process. MAPK pathway-dependent gene expression in the SCN may result, in part, from stimulation of CRE-dependent transcription.

The activity-regulated cytoskeleton-associated ( arc) gene is a new light-inducible early gene in the mouse suprachiasmatic nucleus

The mammalian circadian system is entrained to the environmental light/dark cycle by shifting the phase of the master clock in the suprachiasmatic nucleus. Accompanying the light-induced phase-shift, a variety of immediate-early genes appears in suprachiasmatic nucleus clock cells, and here, we report the expression of a new immediate-early gene Arc (activity-regulated cytoskelton-associated gene) in mice. Arc messenger RNAs were strongly induced at 30-120 min after the light exposure at subjective night (CT12-CT20) in neurons of the retinorecipient area of the suprachiasmatic nucleus, although their spontaneous expression was absent in usual light-dark cycles and in constant dark conditions. At protein level, ARC appeared not only in the nucleus but also in the perikarya and their processes of the suprachiasmatic nucleus neurons. These findings indicate that Arc is an activity-regulated cytoskeletal gene possibly involved in the light-induced phase-shift of the circadian rhythm.

Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var. crispa

In Perilla frutescens, the expression of all structural anthocyanin genes is coordinately regulated by light in form-specific manner in aerial vegetative tissues. To find the regulatory factors for entire anthocyanin pathway in dicot plants, the red form specifically expressed genes were profiled by mRNA differential display. As a result, a novel bHLH factor gene Myc-F3G1 was isolated. The deduced amino acid sequence of MYC-F3G1 exhibited high homology with that of petunia an1, which directly regulates the expression of anthocyanin structural genes. The expression of Myc-F3G was detected specifically in red form but not in green form and induced by light induction. These expression patterns are similar to those of structural anthocyanin genes and Myb-P1, a light-inducible Myb gene. In yeast two-hybrid systems, MYC-F3G1 interacted with another bHLH factor MYC-RP and a WD40 repeat protein PFWD but not with a light-inducible Myb factor Myb-P1. These results suggest that a protein complex including MYC-F3G1, MYC-RP, Myb-P1 and PFWD regulates the expression of anthocyanin biosynthetic genes in P. frutescens. These findings shall provide valuable information for genetic engineering of entire anthocyanin pathway in dicot plants.

CigA, a light-inducible gene involved in vegetative growth in Mucor circinelloides is regulated by the carotenogenic repressor crgA

A strategy based on the differential-display technique has been applied to identify and to isolate potential target genes controlled by crgA, a negative regulator of the light-inducible carotenogenesis in the fungus Mucor circinelloides. This approach resulted in the identification and cloning of the gene cigA, whose expression is repressed by the crgA gene. After a light pulse, there is an immediate though transient accumulation of transcripts of cigA gene. A similar result was obtained when mycelia were exposed to continuous illumination, suggesting the existence of a photoadaptation mechanism in M. circinelloides. The protein sequence deduced from the nucleotide sequence of cigA shows homology to the mouse and human GDP-fucose protein O-fucosyltransferases. Null cigA mutants were generated by gene replacement. Lack of cigA function does not affect carotenogenesis but provokes a slight reduction in vegetative growth that is independent of light. Introduction of wild-type cigA alleles into a cigA − strain restored the wild-type phenotype for vegetative growth. These results suggest that cigA is involved in vegetative growth and, moreover, that crgA controls cellular processes other than carotenogenesis.

Aberrant expression of the light-inducible and circadian-regulated APRR9 gene belonging to the circadian-associated APRR1/TOC1 quintet results in the phenotype of early flowering in Arabidopsis thaliana.

Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member (APRR1/TOC1) of the family of pseudo-response regulators. We previously showed that transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and sequentially at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1, under the conditions of continuous light. Nevertheless, no evidence has been provided that each member of the APRR1/TOC1 quintet, except for APRR1/TOC1, is indeed relevant to the mechanisms underlying circadian rhythms. Here we attempt to provide such evidence by characterizing transgenic plants that aberrantly (or constitutively) express the APRR9 gene in a manner independent of circadian rhythms. The resulting APRR9-ox plants showed intriguing phenotypes with regard to circadian rhythms, in two aspects. First, the aberrant expression of APRR9 resulted in a characteristic phenotype with regard to transcriptional events, in which short-period rhythms were commonly observed for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, ELF3, and CAB2. With regard to biological consequences, such APRR9-ox plants flowered much earlier than wild-type plants, in a manner independent of photoperiodicity (or under short-day conditions). These results suggest that APRR9 (and perhaps other members of the APRR1/TOC1 quintet) must also be taken into consideration for a better understanding of the molecular mechanisms underlying circadian rhythms, and also underlying control of the flowering time through the photoperiodic long-day pathway.