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E M Tobin

Publications and source records attributed to E M Tobin.

At least 37 records · Page 2Linked to original sources
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

Isolation and Characterization of Three Genes Negatively Regulated by Phytochrome Action in Lemna gibba.

We have isolated three distinct cDNA clones from Lemna gibba representing mRNAs that increase in abundance during dark treatment. All three mRNAs showed reduced expression in response to red or white light. These mRNAs range from approximately 680 to 800 nucleotides in length and thus encode relatively small proteins (maximum relative molecular weight 17,000 to 19,000). The genes corresponding to these dark-abundant mRNAs are designated NPR (negatively phytochrome regulated) 1, 2, and 3. Differences in the rapidity of mRNA accumulation during dark treatment were observed for each of the genes in both mature green plants and in etiolated plants. Differences in accumulation pattern were also observed in etiolated plants, depending on whether the plants received a far-red light treatment prior to darkness. Transcription of all three genes, assayed in nuclei isolated from either green or etiolated plants, increased during dark treatment. In etiolated plants, a single 2 minute red light treatment caused a detectable decrease in the transcription of the genes after the dark treatment, and 10 minutes of far-red light given immediately after the red light resulted in a reversal of the effect of red light. Additionally, treatment of the plants with far-red light prior to darkness resulted in greater rates of transcription of the NPR genes. Therefore, we conclude that phytochrome action results in decreased transcription of these NPR genes. Each of the NPR mRNAs are encoded by one to two genes.

Journal Article↗
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↗
PubMed1990

Phytochrome-regulated expression of genes encoding light-harvesting chlorophyll a/b-protein in two long hypocotyl mutants and wild type plants of Arabidopsis thaliana.

The cab genes which encode the light-harvesting chlorophyll a/b-protein (LHCP) are expressed normally with respect to phytochrome regulation in the hy-3 and hy-5 long hypocotyl mutants of Arabidopsis thaliana. In etiolated seedlings of these mutants as well as of the wild type, 1 min of red light elevates cab mRNA levels substantially within 2 h; this increase is reversed if far-red light is given immediately after the red light treatment. We conclude that the genetic defects in these mutants do not affect steps in the signal transduction pathway leading to the regulated expression of cab genes. Additionally, the mRNA from one of the three known A. thaliana cab genes, AB140, is similar in quantity to the mRNAs from the other two, AB165 and AB180, in dark-grown seedlings of hy-3 and hy-5 as well as the parent A. thaliana (Landsberg) after a brief red light treatment. This aspect of cab gene expression differs from the strain Columbia of A. thaliana in which AB140 mRNA is the predominant message. In mature white light-grown plants of the strain Columbia, AB140 as well as a combination of AB165 and AB180 mRNAs are expressed at high levels, suggesting that AB165 and/or AB180 may be developmentally regulated.

Base Sequence↗
PubMed1989

A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes.

Proteins synthesized as soluble precursors in the cytoplasm of eukaryotic cells often cross organellar membrane barriers and then insert into lipid bilayers. One such polypeptide, the light-harvesting chlorophyll a/b-binding protein (LHCP), must also associate with pigment molecules and be assembled into the photosystem II light-harvesting complex in the chloroplast thylakoid membrane. A study of the import of mutant LHCPs into isolated chloroplasts has shown that a putative alpha-helical membrane-spanning domain near the carboxy terminus (helix 3) is essential for the stable insertion of LHCP in the thylakoid. Protease digestion experiments are consistent with the carboxy terminus of the protein being in the lumen. This report also shows that helix 3, when fused to a soluble protein, can target it to the thylakoids of isolated, intact chloroplasts. Although helix 3 is required for the insertion of LHCP and mutant derivatives into the thylakoid, the full insertion of helix 3 itself requires additionally the presence of other regions of LHCP. Thus, LHCP targeting and integration into thylakoid membranes requires a complex interaction involving a number of different domains of the LHCP polypeptide.

Base Sequence↗
PubMed1988

Expression of Light-Harvesting Chlorophyll a/b-Protein Genes Is Phytochrome-Regulated in Etiolated Arabidopsis thaliana Seedlings.

Phytochrome action results in a large and rapid increase in the light-harvesting chlorophyll a/b-protein (LHCP) mRNA level in etiolated seedlings of Arabidopsis thaliana: the RNA increase is detectable within 1 hour after 1 minute red illumination, reaches a maximum 30-fold higher than the dark level at ca. 2 hours, and decays back to dark levels by about 8 hours after the brief red illumination. S1 nuclease analysis distinguishes two kinds of mRNAs transcribed from the three members of the LHCP gene family previously characterized for Arabidopsis (LS Leutwiler, EM Meyerowitz, EM Tobin, 1986 Nucleic Acids Res 14: 4051-4064). One of these arises from the AB140 gene, while the other represents the product(s) of the AB165 and/or AB180 gene(s) (AB165/AB180 mRNA). In mature, white light-grown plants, the two kinds of mRNAs are present in nearly equal amounts. In contrast, in etiolated seedlings, 1 minute red light causes a sixfold greater increase in the level of AB140 mRNA than in the level of AB165/AB180 mRNA, although both levels are regulated by phytochrome action. The kinetics of the responses to 1 minute red light are similar for both kinds of transcripts. Additional evidence suggests that this differential expression is developmentally regulated. Because the AB140 gene offers an attractive target for further analysis of phytochrome-regulated gene expression in Arabidopsis, we have further characterized this gene by mapping its 5' and 3' transcript termini.

Journal Article↗
PubMed1987

Amino acid charge distribution influences the assembly of apoprotein into light-harvesting complex II.

The light-harvesting complex II of thylakoid membranes channels light energy into the photosynthetic reaction center II. The major apoproteins of this complex are the nuclear encoded light-harvesting chlorophyll a/b-proteins (LHCP). A model for the arrangement of LHCP in the thylakoid membrane predicts three alpha-helical membrane-spanning regions. The first and third putative membrane-spanning regions include oppositely charged amino acid residues. When the first and third helices are altered to carry only positive charges, the in vitro accumulation of LHCP in the complex is reduced. This mutation is partially rescued by the introduction of a new negative charge in the third helix, an arrangement that is reversed from the wild type. An arginine in the first helix is also important in some aspect of the process leading to the successful accumulation of the LHCP in thylakoids.

Amino Acids↗
PubMed1986

Structure and expression of three light-harvesting chlorophyll a/b-binding protein genes in Arabidopsis thaliana.

The genome of Arabidopsis thaliana is exceedingly small, in part because it lacks the large middle repetitive DNA component characteristic of other plants. In this paper we have characterized a member of the low copy DNA component: the gene family for the light-harvesting chlorophyll a/b-protein. This gene family is unusual in that it contains far fewer members than the 7-16 coding sequences for this protein found in other plants. We used cross-hybridization with a Lemna gene encoding a light-harvesting chlorophyll a/b-protein to isolate 3 genes from Arabidopsis, all of which are clustered on an 11-kb genomic clone. Southern blot analysis suggests that there is a fourth related gene in Arabidopsis. Sequence analysis of the three genes demonstrates that within the translated region the nucleic acid sequence homology is 96%, the deduced amino acid sequence of the mature proteins is identical for the three genes, and two of the genes have a high degree of sequence homology in both their 5' and 3' immediate flanking regions. The genes have regulatory sequences typical of eukaryotic genes upstream of the translation start sites. However, not all of these genes are equally expressed in plants grown under normal light-dark conditions.

Amino Acid Sequence↗
PubMed1986

Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework.

We have identified three major blocks of amino acid homology shared by the transit peptides of two nuclear-encoded chloroplast proteins, the light-harvesting chlorophyll a/b-protein (LHCP) II of the thylakoid membrane and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of the stroma. These previously unrecognized homology blocks lie at the beginning, middle and end of both transit sequences, and are separated by differing lengths of unshared (interblock) sequence in the two proteins. These interblocks may be dispensible or they might confer a specific property on the individual proteins, such as facilitating proper compartmentalization within the chloroplast. We propose that these three shared sequence elements form a common framework in transit-bearing chloroplast precursors which mediates the common functions performed by each transit peptide. Ferredoxin, the only other such nuclear-encoded protein for which a published transit sequence exists, conforms to the predictions of this hypothesis. These findings stand in contrast to mitochondrial leader sequences and the well-studied signal peptides of secretory and certain integral membrane proteins in which no such framework has been observed.

Amino Acid Sequence↗
PubMed1986

Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes.

The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding region. We have studied the processing, the integration into thylakoid membranes, and the assembly into light-harvesting complexes of six of these deletions. Four different mutant LHCPs are found as processed proteins in the thylakoid membrane, but only one appears to have an orientation in the membrane that is similar to that of the wild type. No mutant LHCP appears in LHC II. The other two mutant LHCPs cannot be detected within the chloroplasts. We conclude that stable complex formation is not required for the processing and insertion of altered LHCPs into the thylakoid membrane. We discuss the results in light of our model.

Amino Acid Sequence↗
PubMed1986

Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts.

When the in vitro synthesized precursor of a light-harvesting chlorophyll a/b binding protein (LHCP) from Lemna gibba is imported into barley etiochloroplasts, it is processed to a single form. Both the processed form and the precursor are found in the thylakoid membranes, assembled into the light-harvesting complex of photosystem II. Neither form can be detected in the stromal fraction. The relative amounts of precursor and processed forms observed in the thylakoids are dependent on the developmental stage of the plastids used for uptake. The precursor as well as the processed form can also be detected in thylakoids of greening maize plastids used in similar uptake experiments. This detection of a precursor in the thylakoids, which has not been previously reported, could be a result of using rapidly developing plastids and/or using an heterologous system. Our results demonstrate that the extent of processing of LHCP precursor is not a prerequisite for its inclusion in the complex. They are also consistent with the possibility that the processing step can occur after insertion of the protein into the thylakoid membrane.

Biological Transport↗
PubMed1986

Chloroplast Import of Light-Harvesting Chlorophyll a/b-Proteins with Different Amino Termini and Transit Peptides.

We have previously isolated and sequenced two genes encoding light-harvesting chlorophyll a/b-proteins (LHCP) from Lemna gibba. One of these, AB30, encodes a protein that is highly homologous to LHCP sequences reported from other species, but the second, AB19, encodes a protein that has a transit peptide and first 12 amino-terminal residues of the mature protein that are substantially different. Despite these differences, we can demonstrate that AB19 encoded protein synthesized in vitro can be imported into isolated chloroplasts, and we provide evidence that at least some of the imported molecules are assembled into the light-harvesting complex of photosystem II. Thus, our results are consistent with the possibility that there are two functional forms of LHCP.

Journal Article↗