Light-dependent Expression Research Articles

Light-dependent expression and accumulation of miR408-encoded peptide, miPEP408, is regulated by HY5 in Arabidopsis

Recent studies propose that primary transcripts of miRNAs (pri-miRNAs) contain small Open Reading Frames (ORFs) capable of encoding miRNA-encoded peptides (miPEPs). These miPEPs can function as transcriptional regulators for their corresponding pri-miRNAs, ultimately enhancing mature miRNA accumulation. Notably, pri-miR408 encodes the functional peptide miPEP408, regulating expression of miR408 and its target genes, providing plant tolerance to stresses. While miPEPs are crucial regulators, the factors governing them are have not been studied in detail. Here, we explored the light-dependent regulation of miPEP408 in Arabidopsis. Expression analysis during dark-light transitions revealed light-induced transcription and accumulation of the miPEP408. As the promoter of miR408 contains cis-acting elements responsible for binding to the bZIP-type transcription factor ELONGATED HYPOCOTYL5 (HY5), known for light-mediated regulation in plants, we studied its involvement in the regulation of miR408. Analysis of HY5 mutant (hy5-215), complemented line (HY5OX/hy5), and CONSTITUTIVE PHOTOMORPHOGENIC 1 mutant (cop1-4) plants supported HY5's positive regulation of miPEP408. Grafting and GUS assays further suggested the role of HY5 as a shoot-root mobile signal inducing light-dependent miPEP408 expression. This study underscores the regulatory impact of light on small peptides, exemplified by miPEP408, mediated by the key transcription factor HY5.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

Light-Dependent Expression and Promoter Methylation of the Genes Encoding Succinate Dehydrogenase, Fumarase, and NAD-Malate Dehydrogenase in Maize (Zea mays L.) Leaves.

The expression and methylation of promoters of the genes encoding succinate dehydrogenase, fumarase, and NAD-malate dehydrogenase in maize (Zea mays L.) leaves depending on the light regime were studied. The genes encoding the catalytic subunits of succinate dehydrogenase showed suppression of expression upon irradiation by red light, which was abolished by far-red light. This was accompanied by an increase in promoter methylation of the gene Sdh1-2 encoding the flavoprotein subunit A, while methylation was low for Sdh2-3 encoding the iron-sulfur subunit B under all conditions. The expression of Sdh3-1 and Sdh4 encoding the anchoring subunits C and D was not affected by red light. The expression of Fum1 encoding the mitochondrial form of fumarase was regulated by red and far-red light via methylation of its promoter. Only one gene encoding the mitochondrial NAD-malate dehydrogenase gene (mMdh1) was regulated by red and far-red light, while the second gene (mMdh2) did not respond to irradiation, and neither gene was controlled by promoter methylation. It is concluded that the dicarboxylic branch of the tricarboxylic acid cycle is regulated by light via the phytochrome mechanism, and promoter methylation is involved with the flavoprotein subunit of succinate dehydrogenase and the mitochondrial fumarase.

Genome-Wide Identification, Characterization, and Expression Analysis of SPIRAL1 Family Genes in Legume Species.

The SPIRAL1 (SPR1) gene family encodes microtubule-associated proteins that are essential for the anisotropic growth of plant cells and abiotic stress resistance. Currently, little is known about the characteristics and roles of the gene family outside of Arabidopsis thaliana. This study intended to investigate the SPR1 gene family in legumes. In contrast to that of A. thaliana, the gene family has undergone shrinking in the model legume species Medicago truncatula and Glycine max. While the orthologues of SPR1 were lost, very few SPR1-Like (SP1L) genes were identified given the genome size of the two species. Specifically, the M. truncatula and G. max genomes only harbor two MtSP1L and eight GmSP1L genes, respectively. Multiple sequence alignment showed that all these members contain conserved N- and C-terminal regions. Phylogenetic analysis clustered the legume SP1L proteins into three clades. The SP1L genes showed similar exon-intron organizations and similar architectures in their conserved motifs. Many essential cis-elements are present in the promoter regions of the MtSP1L and GmSP1L genes associated with growth and development, plant hormones, light, and stress. The expression analysis revealed that clade 1 and clade 2 SP1L genes have relatively high expression in all tested tissues in Medicago and soybean, suggesting their function in plant growth and development. MtSP1L-2, as well as clade 1 and clade 2 GmSP1L genes, display a light-dependent expression pattern. The SP1L genes in clade 2 (MtSP1L-2, GmSP1L-3, and GmSP1L-4) were significantly induced by sodium chloride treatment, suggesting a potential role in the salt-stress response. Our research provides essential information for the functional studies of SP1L genes in legume species in the future.

The Arabidopsis NLP7-HB52/54-VAR2 pathway modulates energy utilization in diverse light and nitrogen conditions

In plants, nitrate is the dominant nitrogen (N)source and a critical nutrient signal regulating various physiological and developmental processes.1,2,3,4 Nitrate-responsive gene regulatory networks are widely believed to control growth, development, and life cycle in addition to N acquisition and utilization,1,2,3,4 and NIN-LIKE PROTEIN (NLP) transcriptional activators have been identified as the master regulators governing the networks.5,6,7 However, it remains to be elucidated how nitrate signaling regulates respective physiological and developmental processes. Here, we have identified a new nitrate-activated transcriptional cascade involved in chloroplast development and the maintenance of chloroplast function in Arabidopsis. This cascade consisting of NLP7 and two homeodomain-leucine zipper (HD-Zip) class I transcription factors, HOMEOBOX PROTEIN52 (HB52) and HB54,8,9 was responsible for nitrate- and light-dependent expression of VAR2 encoding the FtsH2 subunit of the chloroplast FtsH protease involved in the quality control of photodamaged thylakoid membrane proteins.10,11 Consistently, the nitrate-activated NLP7-HB52/54-VAR2 pathway underpinned photosynthetic light energy utilization, especially in high light environments. Furthermore, genetically enhancing the NLP7-HB52/54-VAR2 pathway resulted in improved light energy utilization under high light and low N conditions, a superior agronomic trait. These findings shed light on a new role of nitrate signaling and a novel mechanism for integrating information on N nutrient and light environments, providing a hint for enhancing the light energy utilization of plants in low N environments.

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives.

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

HY5 regulates light-dependent expression and accumulation of miR858a-encoded peptide, miPEP858a

microRNA encoded peptide (miPEP) has been shown to have potential to regulate corresponding miRNA and associated function. miPEP858a regulate phenylpropanoid pathway and plant development. Several studies have suggested that various factors like light, temperature, heavy metals etc. can regulate gene and their associated functions. However, what are the regulators of miPEP are not reported till date. In this study we have reported that light directly regulates miPEP858a accumulation in Arabidopsis thaliana. Peptide assay in light and dark clearly showed the essential requirement of light. Along with this, we have reported that HY5 a shoot-to-root mobile, light-mediated transcription factor plays a crucial role in the function of miPEP858a. The transcript and endogenous protein accumulation of miPEP858a in hy5-215, OXHY5/hy5, and cop1-4 suggested that the HY5 positively regulates miPEP858a. In addition to that this study also include grafting assay between shoot of different mutant and transgenic lines with root of miPEP858a promoter:reporter lines and promoter deletion construct experiment clearly suggested that HY5 a transcription factor regulates light-dependent expression and accumulation of miPEP858a.

Molecular characterization, cellular localization and light-dependent expression of dinoflagellate vacuolar-type H+-ATPase (VHA) subunit B in the colourful outer mantle of the giant clam, Tridacna squamosa, indicate the involvement of VHA in CO2 uptake in the photosynthesizing symbionts

Giant clams are animal-dinoflagellate associations found in Indo-Pacific reef ecosystems. The clam host obtains organic nutrients from phototrophic dinoflagellates of genera Symbiodinium, Cladocopium, and Durusdinium, which reside extracellularly as symbionts (alias zooxanthellae) in the luminal fluid of zooxanthellal tubules located mainly in the colourful outer mantle. The host also needs to supply the symbionts with inorganic carbon for photosynthesis. Symbiont photosynthesis can be impeded by inhibitors of vacuolar H+-ATPase (VHA) because the host possesses a carbon concentration mechanism consisting of VHA to facilitate the supply of CO2(aq) to the symbionts. Here, we report that VHA was also expressed in dinoflagellates residing in the outer mantle of the fluted giant clam, Tridacna squamosa. Three complete cDNA coding sequences of VHA subunit B (VHA-B), one for each genus of dinoflagellate, had been obtained, and each sequence comprised 1482 bp, encoding a protein of 493 amino acids (~55 kDa). As these three sequences were highly similar, we could only design real-time PCR primers to quantify comprehensively zooxanthellae-VHA-B (Zoox-VHA-B) that represented VHA-B of all three genera of dinoflagellates. The outer mantle had the highest transcript level of Zoox-VHA-B among the three organs studied, and illumination led to a significant increase in the protein abundance of Zoox-VHA-B therein. Zoox-VHA-B was immunolocalized to intracellular vesicles, which could apparently align and fuse with the plasma membrane, in the symbiotic dinoflagellates. Overall, these results indicate that photosynthesizing symbionts could increase the capacity of H+ secretion through VHA-containing vesicles to promote the dehydration of luminal HCO3− and the absorption of CO2(aq) during illumination.

Phosphorylation of NONPHOTOTROPIC HYPOCOTYL3 affects photosensory adaptation during the phototropic response.

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light (BL) photoreceptor phototropin 1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here, we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We generated unphosphorable and phosphomimetic NPH3 proteins on seven phosphorylation sites in the etiolated seedlings of Arabidopsis. Unphosphorable NPH3 showed a shortening of its retention time in the cytosol and caused an inability to adapt to very low fluence rates of BL (∼10-5 µmol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened retention time in the cytosol and could not enable the adaptation to BL at fluence rates of 10-3 µmol m-2 s-1 or more. Our results indicate that the activation level of phot1 and the corresponding phosphorylation level of NPH3 determine the dissociation rate and the reassociation rate of NPH3 on the plasma membrane, respectively. These mechanisms may moderately maintain the active state of phot1 signaling across a broad range of BL intensities and contribute to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls.

Light-induced local gene expression in primary chick cell culture system.

The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light‐dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light‐dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.

Aconitate isomerase from maize leaves: Light-dependent expression and kinetic properties

Aconitate isomerase (EC 5.3.3.7) interconverts cis- and trans-isomers of aconitic acid. Expression of the gene encoding this enzyme was studied in maize (Zea mays L.) leaves depending on light regime. Aconitate isomerase was induced by white and by red light indicating the involvement of phytochrome in the regulation of gene expression. The enzyme was partially purified from maize leaves. The value of Km was 0.75 mM with cis-aconitate and 0.92 mM with trans-aconitate, pH optimum was 8.0–8.2 with both substrates, citrate and malate suppressed its activity. It is concluded that aconitate isomerase actively participates in the interconversion of cis- and trans-aconitate in the light providing a possibility of using the pool of trans-aconitate for the regulation of the tricarboxylic acid cycle activity and mediating citrate/isocitrate supply for the biosynthetic and signaling purposes in photosynthetic cells.

A negative regulatory element required for light-dependent pinopsin gene expression.

In vertebrates, a variety of light-stimulated genes are distributed in the retina, the pineal gland, and the suprachiasmatic nucleus, but a cis-element(s) responsible for the light-dependent transcriptional regulation is left unexplored. Focusing on the pinopsin gene, a light-stimulated gene in the chick pineal gland, we performed a transcriptional analysis in the primary culture of the chick pineal cells that were transiently transfected with a luciferase reporter gene fused with various lengths of the 5' upstream region of the pinopsin gene. Light-dependent enhancer activity was detectable in the construct with the upstream region between -1156 and +31. Introduction of mutations within the 18 bp sequence at positions -1103 to -1086 (TGGCACGTGGGGTTCCTC), including a CACGTG E-box sequence, elevated the transcriptional activity in the dark and thereby abrogated the light dependency, suggesting that the 18 bp sequence is essential for a reduction of the transcriptional activity in the dark. In an electrophoretic mobility-shift assay, we identified a pineal nuclear factor(s) capable of binding to the 18 bp element in a sequence-specific manner. When a 49 bp fragment (-1122 to -1074) including the 18 bp sequence was placed upstream of the simian virus 40 promoter, the transcriptional activity was dramatically suppressed regardless of light conditions in the chick pineal cells, and a more pronounced repression was observed in nonpineal/nonphotosensory LMH and NIH 3T3 cells. These results suggest that the 18 bp element in the pinopsin promoter constitutes the binding site of a ubiquitous factor that serves for the transcriptional repression that is required, although not sufficient, for the light-dependent expression of pinopsin gene in the chick pinealocytes.

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively ‘pumps’ H+ out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H+ produced during light-enhanced calcification. Additionally, the excreted H+ would augment HCO3– dehydration in the external medium and facilitate the uptake of CO2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H+ for the dehydration of HCO3– to CO2 during insolation to benefit the photosynthesizing zooxanthellae.

Light-dependent expression of a Na+/H+ exchanger 3-like transporter in the ctenidium of the giant clam, Tridacna squamosa, can be related to increased H+ excretion during light-enhanced calcification.

Na+/H+ exchangers (NHEs) regulate intracellular pH and ionic balance by mediating H+ efflux in exchange for Na+ uptake in a 1:1 stoichiometry. This study aimed to obtain from the ctenidium of the giant clam Tridacna squamosa (TS) the complete cDNA sequence of a NHE3‐like transporter (TSNHE3), and to determine the effect of light exposure on its mRNA expression level and protein abundance therein. The coding sequence of TSNHE3 comprised 2886 bp, encoding 961 amino acids with an estimated molecular mass of 105.7 kDa. Immunofluorescence microscopy revealed that TSNHE3 was localized to the apical membrane of epithelial cells of the ctenidial filaments and the tertiary water channels. Particularly, the apical immunofluorescence of the ctenidial filaments was consistently stronger in the ctenidium of clams exposed to 12 h of light than those of the control kept in darkness. Indeed, light induced significant increases in the transcript level and protein abundance of TSNHE3/TSNHE3 in the ctenidium, indicating that the transcription and translation of TSNHE3/TSNHE3 were light‐dependent. As light‐enhanced calcification generates H+, the increased expression of TSNHE3/TSNHE3 in the ctenidium could be a response to augment H+ excretion in pursuance of whole‐body acid‐base balance during light exposure. These results signify that shell formation in giant clams requires the collaboration between the ctenidium, which is a respiratory and iono‐regulatory organ, and the inner mantle, which is directly involved in the calcification process, and provide new insights into the mechanisms of light‐enhanced calcification in giant clams.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BackgroundCyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.ResultsBy combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.ConclusionsThis investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.

Light-dependent expression of flg22-induced defense genes in Arabidopsis.

Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes.

Time-of-day- and light-dependent expression of ubiquitin protein ligase E3 component N-recognin 4 (UBR4) in the suprachiasmatic nucleus circadian clock.

Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN), which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4), a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP)-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.

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Light-dependent expression of four cryptic archaeal circadian gene homologs

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. As an initial investigation of this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during balanced growth. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities.