Biomedical subjects

E M Tobin

Publications and source records attributed to E M Tobin.

At least 19 recordsLinked to original sources

PubMed2001

shygrl1 is a mutant affected in multiple aspects of photomorphogenesis.

We have used a counter-selection strategy based on aberrant phytochrome regulation of an Lhcb gene to isolate an Arabidopsis mutant designated shygrl1 (shg1). shg1 seedlings have reduced phytochrome-mediated induction of the Lhcb gene family, but normal phytochrome-mediated induction of several other genes, including the rbcS1a gene. Additional phenotypes observed in shg1 plants include reduced chlorophyll in leaves and additional photomorphogenic abnormalities when the seedlings are grown on medium containing sucrose. Mutations in the TATA-proximal region of the Lhcb1*3 promoter that are known to be important for phytochrome regulation affected reporter gene expression in a manner similar to the shg1 mutation. Our results are consistent with the possibility that the mutation either leads to defective chloroplast development or to aberrant phytochrome regulation. They also add to the evidence of complex interactions between light- and sucrose-regulated pathways.

Arabidopsis↗

PubMed2000

All in good time: the Arabidopsis circadian clock.

Biological time-keeping mechanisms have fascinated researchers since the movement of leaves with a daily rhythm was first described >270 years ago. The circadian clock confers a approximately 24-hour rhythm on a range of processes including leaf movements and the expression of some genes. Molecular mechanisms and components underlying clock function have been described in recent years for several animal and prokaryotic organisms, and those of plants are beginning to be characterized. The emerging model of the Arabidopsis clock has mechanistic parallels with the clocks of other model organisms, which consist of positive and negative feedback loops, but the molecular components appear to be unique to plants.

Animals↗

PubMed1999

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis.

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis.

Amino Acid Sequence↗

PubMed1999

Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression.

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Arabidopsis↗

PubMed1998

Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein.

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (beta) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third beta-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 beta-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

Amino Acid Sequence↗

PubMed1998

Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression.

The CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene encodes a MYB-related transcription factor involved in the phytochrome induction of a light-harvesting chlorophyll a/b-protein (Lhcb) gene. Expression of the CCA1 gene is transiently induced by phytochrome and oscillates with a circadian rhythm. Constitutive expression of CCA1 protein in transgenic plants abolished the circadian rhythm of several genes with dramatically different phases. These plants also had longer hypocotyls and delayed flowering, developmental processes regulated by light and the circadian clock. Furthermore, the expression of both endogenous CCA1 and the related LHY gene was suppressed. Our results suggest that CCA1 is a part of a feedback loop that is closely associated with the circadian clock in Arabidopsis.

Arabidopsis↗

PubMed1998

The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels.

Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba. We previously demonstrated that phytochrome action mediates changes in ABA levels in L. gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system. We have now further characterized phytochrome- and ABA-mediated regulation of L. gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start. Linker scan (LS) analysis of the region from -156 to -70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between -143 to -135 (LS2) and -113 to -101 (LS5). Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA. This suggests that, unlike the L. gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of the L. gibba NPR1 gene can be attributed to alterations in ABA levels.

Abscisic Acid↗

PubMed1997

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis.

Amino Acid Sequence↗

PubMed1996

The interaction of light and abscisic acid in the regulation of plant gene expression.

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.

Abscisic Acid↗

PubMed1996

A DNA binding activity for one of two closely defined phytochrome regulatory elements in an Lhcb promoter is more abundant in etiolated than in green plants.

The Lhcb2*1 gene of Lemna gibba is regulated positively by phytochrome, and two separate, 10-bp regions of this promoter have been shown to be necessary for phytochrome regulation. We have now analyzed the effects of one and two base pair mutations to define exactly two cis elements within these regions that are necessary for phytochrome regulation. These elements, designated REalpha and REbeta, consist in part of sequences highly conserved among promoter of genes encoding light-harvesting chlorophyll a/b proteins of photosystem II (Lhcb genes). They are located -134 to -129 bp and -114 to -109 bp from the transcription start site, respectively. REalpha has the sequence AACCAA and was found to interact specifically in vitro with a DNA binding activity in whole-cell extracts of plants. This activity was high in etiolated plants but much lower in green plants. REbeta has the sequence CGGATA. A GATA sequence created at a position six nucleotides upstream could replace the function of REbeta. We conclude that the phytochrome regulation of Lhcb2*1 is mediated by at least two cis elements. These elements are likely to function by repression of the promoter activity in darkness, although the REbeta region also may be able to play a role in the activation of transcription.

Base Sequence↗

PubMed1995

A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway.

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.

Arabidopsis↗

PubMed1995

Isolation of new promoter-mediated co-suppressed lines in Arabidopsis thaliana.

Four new independent lines that exhibit co-suppression of an introduced cab140::tms2 gene and the native cab140 gene have been isolated in Arabidopsis thaliana. These lines are of particular interest because the homology shared between the introduced and native genes is 1.3 kb of promoter DNA that only contains 14 bp of transcribed region. Most other reported examples of co-suppression involve homologies between transcribed portions of genes. A similar line, lct, had been isolated previously from EMS-mutagenized seeds, and we concluded that this example of co-suppression was probably due to a mutation that mapped at or near the introduced cab140::tms2 gene [Brusslan JA, Karlin-Neumann GA, Huang L, Tobin EM: Plant Cell 5: 667-677 (1993)]. Our observations with these four new lines, however, suggest that an epigenetic event(s) rather than a mutation might be the cause of co-suppression in these and the lct line.

Arabidopsis↗

PubMed1995

A region of the Arabidopis Lhcb1*3 promoter that binds to CA-1 activity is essential for high expression and phytochrome regulation.

We have previously characterized a protein from Arabidopsis thaliana, called CA-1, that bound to a specific region of the Lhcb1*3 promoter. This binding activity was of interest because the sequence to which it bound is included in a portion of the promoter that is sufficient for phytochrome regulation and because the activity was absent in photomorphogenic mutant det1 seedlings (L. Sun, R.A. Doxsee, E. Harel, E.M. Tobin [1993] Plant Cell 5: 109-121). We have now directly tested whether the nucleotide sequence to which CA-1 binds is required for regulation of the transcription of this gene by phytochrome. A mutation that abolished CA-1 binding in vitro was introduced into a 1.15-kb segment of the Lhcb1*3 promoter, and both the wild-type and mutant promoter fragments were fused to a uidA reporter gene and used to stably transform A. thaliana. Ten different homozygous lines were examined for phytochrome responsiveness for each of the two constructs by assaying beta-glucuronidase activity. The wild-type construct showed normal phytochrome responsiveness. The mutant construct showed no phytochrome response, and the overall level of beta-glucuronidase activity in etiolated seedlings was decreased by about 2 orders of magnitude. We did not detect a response to a B photoreceptor other than phytochrome itself for either the wild-type or mutant construct. We conclude that information essential for both a high level of expression and phytochrome responsiveness is contained in a 27-bp region to which the CA-1 activity binds.

Arabidopsis↗

PubMed1994

Phytochrome regulated gene expression.

Light is used by plants as a signal for many physiological and developmental processes. Phytochrome is the most extensively studied family of photoreceptors that plants use to perceive the presence and quality of light in their environment. While the initial action of the phytochrome molecule is not yet known, one important kind of response, changes in the expression of specific nuclear genes, has been intensively investigated. Although phytochrome-regulated promoters are complex and can also respond to other signals, specific DNA elements that are involved in conferring phytochrome responsiveness have been identified. Potential signal transduction pathway components include G proteins, cyclic GMP and Ca2+/calmodulin. In addition, the study of transcription factors involved in phytochrome-regulated gene expression has yielded insights into some of the final steps of transcriptional regulation by phytochrome.

Base Sequence↗

PubMed1994

NPR genes, which are negatively regulated by phytochrome action in Lemna gibba L. G-3, can also be positively regulated by abscisic acid.

We have found that NPR1 and NPR2, two genes from Lemna gibba L. G-3 that can be negatively regulated by phytochrome action, can also be positively regulated by the plant hormone abscisic acid (ABA). Both genes were responsive to low concentrations of exogenous ABA; an increase in NPR1 RNA could be detected in response to concentrations as low as 10 nM. We have also tested phytochrome responsiveness of 5' promoter-deletion constructs of one of these genes, NPR1, in transient assays utilizing particle bombardment. This analysis demonstrated that DNA sequences important for phytochrome regulation are present downstream of -198 from the transcription start site. A response to ABA treatment could also be observed in the transient assay system. When intact plants were placed in darkness, there was an increase in ABA levels as well as increased levels of NPR1 and NPR2 RNA.

Abscisic Acid↗

PubMed1994

Two 10-bp regions are critical for phytochrome regulation of a Lemna gibba Lhcb gene promoter.

Two small regions of the promoter of an Lhcb gene encoding a light-harvesting chlorophyll a/b protein were identified as essential in conferring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, showed that sequences within the region from -174 to -104 relative to the start of transcription were necessary for phytochrome regulation. Internal deletion and substitution mutants were used to demonstrate that no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending from -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. Specific binding of Lemna proteins to the region necessary for phytochrome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mutations in two separate 10-bp sequences, from -134 to -125 and from -114 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of this gene. One of these regions contains a CCAAT motif and the other a GATA motif. These motifs are conserved in the promoters of many Lhcb genes and may be important elements in the phytochrome responsiveness of this gene family.

Base Sequence↗

PubMed1993

Analysis of genes negatively regulated by phytochrome action in Lemna gibba and identification of a promoter region required for phytochrome responsiveness.

As a step to understanding how the photoreceptor phytochrome acts to change the transcription of specific nuclear genes in Lemna gibba, we wish to compare promoter elements involved in negative regulation by phytochrome with those involved in positive regulation. We have isolated three genes negatively regulated by phytochrome, designated NR (negatively phytochrome regulated) genes (P.A. Okubara, E.M. Tobin [1991] Plant Physiol 96:1237-1245), and we have now sequenced two of these. The promoters of both contain some sequence motifs that are identical with motifs from other genes. We used a transient assay in L. gibba to demonstrate that approximately 1.7 kb pairs of the NPR1 promoter and 1.1 kb pairs of the NPR2 promoter could confer negative phytochrome regulation to a luciferase reporter gene. Deletion analysis of the NPR2 promoter showed that sequences between -208 and -82 from the transcription start were necessary for negative phytochrome regulation. However, this region was not sufficient to confer negative regulation by phytochrome to another promoter. Additionally, we noted that this region showed no similarity to a region identified as important for the negative regulation of the oat phyA promoter (W.B. Bruce, X.-W. Deng, P.H. Quail [1991] EMBO J 10:3015-3024), but it does contain a sequence element found in several other kinds of genes, including ones positively regulated by phytochrome. The deduced amino acid sequences of NPR1 and NPR2 were found to share similarities with many abscisic acid-induced or seed-abundant proteins. Thus, these genes, like other phytochrome-regulated genes, might respond to multiple regulatory signals.

Amino Acid Sequence↗

PubMed1993

CA-1, a novel phosphoprotein, interacts with the promoter of the cab140 gene in Arabidopsis and is undetectable in det1 mutant seedlings.

We have identified and partially purified a DNA binding protein from Arabidopsis that interacts specifically with the phytochrome-responsive promoter of the Arabidopsis cab140 gene. Promoter deletion analyses in transgenic tobacco showed that, if a region that includes the sequence interacting with this protein was deleted, both expression and phytochrome responsiveness were lost. The protein protected a cytosine- and adenine-rich region from DNase I digestion, and therefore it has been called Ca-1. CA-1 was shown to be a phosphoprotein, and dephosphorylation changed the migration of the protein-DNA complex in DNA mobility shift assays. The data suggested that the protein has an apparent molecular weight of 70,000. The CA-1-protected region of the cab140 promoter included an ACGT motif that has been found in the target sequences of a number of bZIP transcription factors, but the binding behavior of CA-1 differed from those factors. CA-1 binding activity was present in plants grown in either white light or darkness, and no differences in the binding activity were detected in the dark-grown plants after short red or white light treatments. However, the CA-1 binding activity was not detectable in extracts of seedlings bearing the det1 mutation grown in the dark and given the same illumination treatments as wild type. In contrast to wild type, the mutant seedlings express cab RNA at a high level when grown in complete darkness, and we found no further increase in cab140 mRNA in response to brief red illumination. The lack of CA-1 activity in the det1 mutant suggests that it may function as a transcriptional repressor regulating the expression of the cab140 gene in Arabidopsis.

Arabidopsis↗