Biomedical subjects

E M Tobin

Publications and source records attributed to E M Tobin.

At least 19 recordsLinked to original sources

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↗

PubMed1993

An Arabidopsis mutant with a reduced level of cab140 RNA is a result of cosuppression.

We analyzed a mutant of Arabidopsis with a severely reduced level of cab140 RNA. This mutant, named lct for low level of cab140 transcript, was obtained during a selection for phytochrome signal transduction mutants. The selection was based on reduced expression of the tumor morphology shoots gene (tms2), an introduced counter-selectable marker under the control of the cab140 promoter. Expression of the introduced cab140::tms2 gene was also greatly reduced in lct, but surprisingly, expression of other phytochrome-regulated genes was not comparably affected. Furthermore, the lct phenotype could not be separated genetically from the T-DNA insert; thus, we suggest that this phenotype was caused by cosuppression of the introduced construct and the endogenous cab140 gene, and that the mutation causing the cosuppression was located on the T-DNA insert. In vitro nuclear transcription experiments demonstrated that the suppression was occurring at the level of transcription. We also found that the suppressed cab140 genes were not significantly more methylated than the nonsuppressed cab140 genes.

Amidohydrolases↗

PubMed1992

Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlings.

We found a transient increase in the amount of mRNA for four nuclear genes encoding chloroplast proteins during early development of Arabidopsis thaliana. This increase began soon after germination as cotyledons emerged from the seed coat; it occurred in total darkness and was not affected by external factors, such as gibberellins or light treatments used to stimulate germination. Three members of the cab gene family and the rbcS-1A gene exhibited this expression pattern. Because timing of the increase coincided with cotyledon emergence and because it occurred independently of external stimuli, we suggest that this increase represents developmental regulation of these genes. Further, 1.34 kilobases of the cab1 promoter was sufficient to confer this expression pattern on a reporter gene in transgenic Arabidopsis seedlings. The ability of the cab genes to respond to phytochrome preceded this developmental increase, showing that these two types of regulation are independent.

Base Sequence↗

PubMed1991

Deletion analysis of a phytochrome-regulated monocot rbcS promoter in a transient assay system.

We have developed a transient gene expression assay system in the aquatic monocot Lemna gibba in which DNA was introduced into intact tissue by particle bombardment. Constructs based on the Lemna rbcS gene SSU5B, which is positively regulated by phytochrome in vivo, also showed phytochrome regulation in the transient assay system. Reporter gene expression increased 12-fold over dark levels in response to a single treatment with red light. This increase was not observed if far-red light was immediately followed by the red light. A 5' deletion analysis of the promoter defined a region from position -205 to position -83 relative to the start of transcription as necessary to observe the phytochrome response. This region contains the binding site for the light-induced binding activity (LRF-1) found in Lemna nuclear extracts. Upstream of position -205, we found evidence for the presence of at least two upstream activating sequences and a silencer.

Chloramphenicol O-Acetyltransferase↗

PubMed1991

Phytochrome control of the tms2 gene in transgenic Arabidopsis: a strategy for selecting mutants in the signal transduction pathway.

Introduction of the tms2 gene from Agrobacterium tumefaciens into Arabidopsis thaliana yields transgenic seedlings with a new selectable phenotype: the seedlings are strongly growth inhibited on micromolar concentrations of auxin amide substrates that do not significantly affect wild-type seedlings. The tms2 gene encodes an amidohydrolase that catalyzes the conversion of biologically inactive auxin amides into active auxins, which are toxic to plants at elevated concentrations. In the absence of exogenous substrate, tms2+ transgenic seedlings grow normally and are fertile. When grown on auxin amides, both etiolated and green tms2+ seedlings exhibit a variety of dose-dependent auxin toxicity effects. tms2 mRNA and the encoded amidohydrolase activity are both detectable in transgenic but not in wild-type seedlings, demonstrating that a cognate activity is lacking in wild-type Arabidopsis. Furthermore, when the introduced tms2 gene is fused to the Arabidopsis cab140 promoter, the tms2 RNA and its encoded amidohydrolase activity and, thus, the conditional lethal phenotype can be modulated by phytochrome action. The tms2 gene can, therefore, serve as a regulatable selectable marker in Arabidopsis that should be useful in isolation of trans-regulatory mutants that have lost the imposed regulation of tms2 gene activity.

Amidohydrolases↗

PubMed1990

Differential expression of individual genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in Lemna gibba.

The gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase in the monocot Lemna gibba contains approximately twelve members. We have isolated six of these genes from a genomic library, and sequenced five of the coding regions. The transit peptide nucleotide sequences are conserved, but less highly than the mature polypeptide coding sequence. The mature polypeptide amino acid sequences are identical to each other and to the sequence deduced from a cDNA clone derived from a seventh gene. Each of the five fully characterized genomic sequences contains a single intron in precisely the same position as the second intron of several dicots. The intron sequences differ in length and are less conserved than the coding sequences. The 3'-untranslated regions of the different genes have been sequenced and used to prepare gene-specific probes. These probes have been used to study the expression levels of individual rbcS sequences. Expression of six of the seven genes can be detected in total RNA isolated from plants grown in continuous light. The levels of RNA encoded by each expressed gene are regulated by the action of phytochrome, but there is variability in the amount of expression of each RNA.

Amino Acid Sequence↗

PubMed1990

Post-transcriptional regulation of organ-specific expression of individual rbcS mRNAs in Lemna gibba.

Many studies of nuclear genes encoding chloroplast proteins have focused on the transcriptional regulation of their expression. The genes (rbcS) encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase, a major stromal protein, comprise one such group. We have examined the role played by post-transcriptional events in determining the relative levels of individual rbcS mRNAs in different organs of the aquatic monocot Lemna gibba. L. gibba is unusual among angiosperms in that its roots are normally exposed to light during growth and contain chloroplasts. We have found that such roots transcribe rbcS genes and contain rbcS mRNA. We have used sequence-specific probes from the 3'-untranslated region of six rbcS genes from L. gibba to analyze the expression of the individual genes in different organs. All six genes were expressed in steady-state mRNA in fronds grown in constant white light. However, only five of these were easily detectable in steady-state mRNA isolated from roots of the same plants, and the relative expression of each gene varied between the roots and the fronds. In steady-state mRNA, SSU1 was found to be highly expressed in both roots and fronds, whereas SSU40B was expressed at low levels in the roots as compared with the fronds, and SSU5B RNA was barely detectable in the roots. The extremely low level of SSU5B RNA in steady-state root mRNA is likely to be a consequence of post-transcriptional events because this gene was transcribed at comparable rates in vitro in nuclei isolated from either roots or fronds. Localization of individual gene transcripts by in situ hybridization showed that SSU1 and SSU5B are expressed in the same cells in the fronds. Thus, the mechanism of differential expression is likely to involve an organ-specific post-transcriptional mechanism.

Gene Expression Regulation↗

PubMed1990

A light-regulated DNA-binding activity interacts with a conserved region of a Lemna gibba rbcS promoter.

We have characterized a DNA-binding activity, designated light-regulated nuclear factor (LRF-1), which interacted with a specific sequence located 150 nucleotides upstream from the transcription start site of a phytochrome-regulated Lemna gibba rbcS gene (SSU5B). There was a higher level of LRF-1 activity recovered from nuclei of light-grown plants than from dark-treated plants. In light-grown plants given a 1-day dark treatment, either white light or a single 2-min red illumination caused a rapid twofold to threefold increase in this activity, suggesting that the phytochrome system is probably involved in its regulation. The nuclear extracts also contained an activity that bound specifically to Box II sequences from a pea rbcS gene [Green, P.J., Yong, M.H., Cuozzo, M., Kano-Murakami, Y., Silverstein, P., and Chua, N.-H. (1988). EMBO J. 7, 4035-4044], but this activity was not higher in the light-grown compared with the dark-treated plants. Comparison of about 700 base pairs upstream from the SSU5B transcription start site with the upstream sequences of two other Lemna rbcS genes revealed several conserved regions. One of these regions is found upstream of rbcS genes in other species and is contained in the sequence which was shown to interact with LRF-1.

Base Sequence↗