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PubMed2005

Light-induced carotenogenesis in Streptomyces coelicolor A3(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.

Amino Acid Sequence↗

PubMed1987

Differential expression of C4 pathway genes in mesophyll and bundle sheath cells of greening maize leaves.

Pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase, and NADP-malate dehydrogenase function in a series of reactions for fixing CO2 in mesophyll cells and NADP-malic enzyme (ME) catalyzes the production of CO2 and NADPH in bundle sheath cells of maize which is a NADP-ME type C4 plant. Northern blot analyses with cDNA clones for pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase and in vitro translation-immunoprecipitation experiments with antiserum to NADP-malate dehydrogenase showed that pools of transcripts of these three genes grow and shrink coordinately in mesophyll cells but not in bundle sheath cells upon illumination of dark-grown maize seedlings. Western blot analyses indicated that the protein levels of phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase are low in dark-grown maize seedlings and increase progressively following light-induced transient accumulation of their mRNAs in mesophyll cells. These proteins continue to accumulate and plateau in late-greening and green leaves in spite of a rapid drop in the sizes of their mRNA pools. Surprisingly, relatively large amounts of NADP-malate dehydrogenase are present in mesophyll cells of etiolated leaves despite the low level of the corresponding mRNA. No phosphoenolpyruvate carboxylase or NADP-malate dehydrogenase were detected in bundle sheath cells. On the other hand, the ME gene responds to light induction at both the transcriptional and translational levels only in bundle sheath cells. Moreover, the steady-state level of ME mRNA stays high in late-greening and green leaves in contrast to the rapid decline of mRNA levels of three other C4 pathway genes in mesophyll cells. In addition, low levels of both the mRNA and protein encoded by the PPDK gene were detected in bundle sheath cells. These levels were not influenced by light as distinguished from the patterns observed in mesophyll cells.

Centrifugation, Density Gradient↗

PubMed1995

The promoter from the rice nuclear gene encoding chloroplast aldolase confers mesophyll-specific and light-regulated expression in transgenic tobacco.

The rice genome contains at least four separate loci that encode aldolase isozymes. Among these, the aldolase P (AldP) gene, a nuclear gene coding for chloroplast aldolase, is expressed predominantly in the leaf blade mesophyll cells in rice. To dissect promoter elements that regulate such tissue- or cell type-specific expression, we constructed various AldP promoter-beta-glucuronidase (GUS) fusion genes and transferred them into Nicotiana tabacum (tobacco) plants. Analysis of GUS activities in the transgenic tobacco revealed the presence of at least two elements within 2.0 kb AldP promoter region. One is located within the segment from position -2.0 kb to -1.2 kb and acts as a negative element. The other is a positive element located between -1.2 kb and -0.31 kb that confers developmentally regulated, mesophyll cell-specific expression. In addition, the 1.2 kb rice promoter segment flanking the transcription start site contains an element(s) that serves as target for light induction in tobacco. The results suggest that the AldP gene promoter of rice, a monocot promoter, can function in an essentially physiological manner in the dicot tobacco plant.

Blotting, Northern↗

PubMed2004

The light-responsive promoter of cyanobacterial ORF76 gene overlaps with the htpG terminator.

A Synechococcus PCC7942 gene, encoding 76 amino acids of an unknown protein (designated ORF76), is located at the same orientation and downstream of the htpG gene. The processed site of ORF76 transcripts is located at position +39 with respect to the true transcription initiation site. The non-E. colisigma70-like basal promoter of ORF76 (-51 to -9) is controlled by three cis-acting elements: positively acting element (-160 to -86), negative regulatory element (-86 to -51) and light-responsive element (-51 to +63) that, together, respond to high light induction at transcriptional level. The ORF76 gene is expressed as monocistronic transcript. The promoter elements of ORF76 gene overlap with the coding sequence and 3' end formation signal of htpG gene.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. Here we report the characterization of the photoinducible promoter of the albino-3 (al-3) gene, encoding GGPP synthase. We have modified the 5' non-coding sequence of the cloned al-3 gene by deletion and site-directed mutagenesis, and we have tested the residual photoinducibility of the different constructs by transformation and subsequent analysis of gene expression in dark-grown and light-induced mycelia. The results indicate that a promoter region between positions -226 and -55 contains all the necessary information for blue light photoinduction. Multiple regulatory elements are involved in the regulated expression of the al-3 gene. One (termed the APE element) is important for the light-induction switch on of the gene and a second belongs to the CCAAT boxes family. The putative APE element is also found in the promoter of other N. crassa photoinducible genes.

Alkyl and Aryl Transferases↗

PubMed2004

Circadian and light-induced transcription of clock gene Per1 depends on histone acetylation and deacetylation.

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. Here, we show that the state of acetylated histones fluctuated in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) repressed transcription with HDACs and mSin3B, which was relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induced endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which interacted with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulated rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. We also showed that TSA administration into the lateral ventricle induced mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation.

Acetylation↗

PubMed2006

The GUS reporter-aided analysis of the promoter activities of a rice metallothionein gene reveals different regulatory regions responsible for tissue-specific and inducible expression in transgenic Arabidopsis.

To gain a better understanding of the regulatory mechanism of plant metallothionein (MT) genes, a chimeric expression unit consisting of the beta-glucuronidase (gusA) reporter gene under the control of a 1,324 bp fragment of the rice MT (ricMT) promoter was introduced into Arabidopsis via Agrobacterium tumefaciens. The strongest histochemical staining for GUS activity was observed in the cotyledons and hypocotyls of the transgenic seedlings and in the stigma, filaments and anthers of young and mature flowers, and especially in the wounded tissues of transgenic plants. In contrast, a relatively low level of reporter gene expression was seen in the young roots of transgenic seedlings and no GUS activity was detected in the stems, seeds and leaves, but GUS activity was observed in cotyledons and the first two true leaves. Promoter analysis of 5' deletions further identified several important regions responsible for organ-specific expression including roots, flowers and wound induction, light and ABA, Cu and Zn responses. These findings demonstrate that a 1,324 bp fragment of the rice MT promoter performs a complicated transcriptional regulation with clearly functional regions in a model plant, and provide an important insight into the transcriptional regulation mechanisms that operate the temporal- and spatial-specific expression and stress responses of the rice MT gene. These results suggest that the ricMT promoter and its functional regions are potentially useful in genetic engineering of plants to express the desired genes whose products are preferentially needed in roots, flowers and wound induction.

Arabidopsis↗

PubMed1986

A morphogen for the sporulation of Physarum polycephalum detected by cell fusion experiments.

The light stimulus, which under conditions of starvation induces the development of sporangia in the slime mold Physarum polycephalum, can be transferred from the light-exposed part to the unexposed part of a plasmodium by means of plasma circulation. A small quantity of protoplasm from a sporulating donor plasmodium, which had passed through the premorphogenetic phase, was transferred by a short period fusion with a briefly starved, light-induction-incompetent acceptor plasmodium. This led to sporulation and even to a reduction of the premorphogenetic phase from 9 down to 3 h in the acceptor plasmodium. After fusion with a sporulating plasmodium, a highly starved plasmodium from a non-sporogenic culture line or a growing plasmodium from a normal line prevents further morphogenesis of sporangia in the sporulating partner.

Cell Fusion↗

PubMed1992

Repair of UV-induced (6-4)photoproducts measured in individual genes in the Drosophila embryonic Kc cell line.

The nucleotide excision repair (NER; dark-repair) of (6-4)photoproducts ((6-4)PPs) was assayed in cells from a permanent Drosophila melanogaster embryonic cell line, Kc, after exposure to 20 or 40 J/m2 ultraviolet (UV) light. Induction rates in the transcriptionally active genes Gart and Notch as well as in the inactive white locus is similar. They are formed with a frequency of about one-third of that of cyclobutane pyrimidine dimers (CPDs). In all three genes, (6-4)PPs are repaired with the same rate and to the same extent: 31% of the (6-4)PPs are removed in 4 hours post-irradiation and after 16 hours repair is nearly complete. In none of the three genes strand-specific repair was found. Exposure of cells that were irradiated with 40 J/m2 UV to photoreactivating light for 1 hour prior to dark-repair incubation, resulted in enhanced repair of (6-4)PPs.

Animals↗

PubMed1989

Fast light-regulated genes of Neurospora crassa.

Several physiological reactions including the sexual differentiation of the ascomycete Neurospora crassa are triggered by blue light. Mutants in the white-collar genes wc-1 and wc-2 are blind for all the blue light effects tested so far. We have previously shown that blue light induces some translatable mRNAs at different times after beginning the illumination. Here we report the cDNA cloning of four genes that are induced by blue light. Induction of these transcripts is temporally ordered (lag times from 2 to 45 min). Analysis of run-on transcripts show that the increases in mRNA levels are due to de novo transcription. None of these transcripts is inducible in white-collar mutants.

Blotting, Northern↗

PubMed1997

Cell type- and positionally specific regulation of the aldolase P gene expression in rice seedlings.

We describe here different regulation of the AldP gene, a nuclear gene encoding chloroplast aldolase, in different tissues and growth ages of rice seedlings. Expression of the AldP gene is mesophyll cell-specific, and increases from the basal to the upper region in each leaf. The gene expression is repressed in the dark-grown leaf blade, but is induced by a short-term-exposure to light, to a level higher than that seen in the normal leaf blade. However, the light-inducibility differs among the tissues, and shows different patterns among leaf positions; i.e., the extent of light-induction is higher in the third leaf blade as compared with the earlier developed second leaf blade. Such positional differences in the regulation are also seen in the leaf sheath. These responses are not accompanied by changes of the cell type specificity in the expression.

Fructose-Bisphosphate Aldolase↗

PubMed1996

Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene.

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is positively regulated by light and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analyses of the eas (ccg-2) promoter were carried out to localize the cis-acting elements mediating clock, light, and developmental control. The primary sequence determinants of a positive activating clock element (ACE) were found to reside in a 45-bp region, just upstream from the TATA box. Using a novel unregulated promoter/reporter system developed for this study, we show that a 68-bp sequence encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N. crassa protein extracts that recognize and bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmental control are identified and shown not to be required for clock-regulated expression of eas (ccg-2). The distinct nature of the ACE validates its use as a tool for the identification of upstream regulatory factors involved in clock control of gene expression.

Base Sequence↗

PubMed2003

Regulation of root ion transporters by photosynthesis: functional importance and relation with hexokinase.

Coordination between the activity of ion transport systems in the root and photosynthesis in the shoot is a main feature of the integration of ion uptake in the whole plant. However, the mechanisms that ensure this coordination are largely unknown at the molecular level. Here, we show that the expression of five genes that encode root NO(3)(-), NH(4)(+), and SO(4)(2-) transporters in Arabidopsis is regulated diurnally and stimulated by sugar supply. We also provide evidence that one Pi and one K(+) transporter also are sugar inducible. Sucrose, glucose, and fructose are able to induce expression of the ion transporter genes but not of the carboxylic acids malate and 2-oxoglutarate. For most genes investigated, induction by light and induction by sucrose are strongly correlated, indicating that they reflect the same regulatory mechanism (i.e., stimulation by photosynthates). The functional importance of this control is highlighted by the phenotype of the atnrt2 mutant of Arabidopsis. In this mutant, the deletion of the sugar-inducible NO(3)(-) transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO(3)(-) influx by light and sugar. None of the sugar analogs used (3-O-methylglucose, 2-deoxyglucose, and mannose) is able to mimic the inducing effect of sugars. In addition, none of the sugar-sensing mutants investigated (rsr1-1, sun6, and gin1-1) is altered in the regulation of AtNrt2.1 expression. These results indicate that the induction of AtNrt2.1 expression by sugars is unrelated to the main signaling mechanisms documented for sugar sensing in plants, such as regulation by sucrose, hexose transport, and hexokinase (HXK) sensing activity. However, the stimulation of AtNrt2.1 transcript accumulation by sucrose and glucose is abolished in an antisense AtHXK1 line, suggesting that HXK catalytic activity and carbon metabolism downstream of the HXK step are crucial for the sugar regulation of AtNrt2.1 expression.

Anion Transport Proteins↗

PubMed2002

WHITE COLLAR-1, a multifunctional neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2.

WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression of wc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series of wc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains.

Circadian Rhythm↗

PubMed2005

[Specific expression of the foreign gene regulated by the rice rbcS promoter in transgenic rice].

To use different types of promoters in transgenic rice research, the 5'-upstream regulation region of rice Rubisco small subunit gene (rbcS) was cloned from a Chinese cultivar Wuyunjing 8, and its sequences were confirmed by comparison with the known genome sequences of both japonica and indica rice. The cloned rbcS promoter was fused to the 5'-upstream of GUS (beta-glucuronidase) coding region in a binary vector, and introduced into rice by Agrogacterium-mediated transformation. The integration of the rbcS-GUS fusion gene in transgenic rice was confirmed by PCR analysis. The results of both histochemical staining and quantitative analysis of GUS activity showed that the expression level of GUS fusion gene was significantly stronger in leaf blade and sheath than in other organs of transgenic rice plants, and the GUS activity was restricted to the mesophyll cells of leaf tissue, which showed that the rice rbcS promoter could control not only the tissue- but also the cell-specific expression of foreign genes in transgenic rice. The present results also demonstrated that light induction had a significant effect on the enhancement of transgene's expression when regulated by the rice rbcS promoter in transgenic rice. Our results showed that the rice rbcS promoter might be very useful for the expression of target genes in transgenic rice, with particularly high efficiency in leaf tissues.

Gene Expression Regulation, Plant↗

PubMed2005

Extension of a genetic network model by iterative experimentation and mathematical analysis.

Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening-expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.

Arabidopsis↗

PubMed2002

Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco.

Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.

Arabidopsis↗

PubMed1979

Temperature-sensitive Responses of Red Light-dependent Betacyanin Synthesis.

Amaranthus tricolor half-seedlings show a greater accumulation of betacyanin in response to a defined period of red light if given a pretreatment at an elevated temperature (40 C). Red light given before the shift to 40 C is ineffective. The maximum response is achieved after a 2-hour shift to 40 C and if the red light is given 1 hour after return to the germination temperature (25 C). The effect on red light induction of betacyanin synthesis and on fusicoccin induction by these conditions is similar, whereas the increase in cytokinin-dependent synthesis is greater. Both phytochrome-cytokinin synergism and fusicoccin-cytokinin synergism are changed in the same manner by this treatment. Phytochrome and fusicoccin responses are inhibited similarly by carbonyl-cyanide-p-trifluoromethoxy-phenylhydrazone.If the seedlings are again returned to 40 C after the 25 C shift there is an inhibition of betacyanin synthesis. The interpretation of these results is that a shift to 40 C induces the formation of some component necessary for the full expression of phytochrome potential, that for the maximum formation or action of this component it is necessary for the seedlings to spend 1 to 2 hours at a lower temperature (for example 25 C) before red light is given and that for the subsequent processes in betacyanin synthesis an elevated temperature is inhibitory.

Journal Article↗