21 results on '"E. Lam"'
Search Results
2. Caspase-like protease involvement in the control of plant cell death
- Author
-
E, Lam and O, del Pozo
- Subjects
Caspases ,Plant Cells ,Apoptosis ,Plants ,Signal Transduction - Abstract
Cell death as a highly regulated process has now been recognized to be an important, if not essential, pathway that is ubiquitous in all multicellular eukaryotes. In addition to playing key roles in the morphogenesis and sculpting of the organs to give rise to highly specialized forms and shapes, cell death also participates in the programmed creation of specialized cell types for essential functions such as the selection of B cells in the immune system of mammals and the formation of tracheids in the xylem of vascular plants. Studies of apoptosis, the most well-characterized form of animal programmed cell death, have culminated in the identification of a central tripartite death switch the enzymatic component of which is a conserved family of cysteine proteases called caspases. Studies in invertebrates and other animal models suggest that caspases are conserved regulators of apoptotic cell death in all metazoans. In plant systems, the identities of the main executioners that orchestrate cell death remain elusive. Recent evidence from inhibitor studies and biochemical approaches suggests that caspase-like proteases may also be involved in cell death control in higher plants. Furthermore, the mitochondrion and reactive oxygen species may well constitute a common pathway for cell death activation in both animal and plant cells. Cloning of plant caspase-like proteases and elucidation of the mechanisms through which mitochondria may regulate cell death in both systems should shed light on the evolution of cell death control in eukaryotes and may help to identify essential components that are highly conserved in eukaryotes.
- Published
- 2001
3. Markers for hypersensitive response and senescence show distinct patterns of expression
- Author
-
D, Pontier, S, Gan, R M, Amasino, D, Roby, and E, Lam
- Subjects
Genetic Markers ,Transcriptional Activation ,Time Factors ,Apoptosis ,Genes, Plant ,Plants, Genetically Modified ,Plant Leaves ,Tobacco Mosaic Virus ,Plants, Toxic ,Gene Expression Regulation, Plant ,Genes, Reporter ,Pseudomonas ,Tobacco ,RNA, Messenger ,Transgenes ,Promoter Regions, Genetic ,Copper ,Plant Diseases ,Signal Transduction - Abstract
Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.
- Published
- 1999
4. Signals controlling the expression of cytosolic ascorbate peroxidase during pathogen-induced programmed cell death in tobacco
- Author
-
R, Mittler, E, Lam, V, Shulaev, and M, Cohen
- Subjects
Herbicides ,Apoptosis ,Glutathione ,Gene Expression Regulation, Enzymologic ,Oxygen ,Tobacco Mosaic Virus ,Plants, Toxic ,Ascorbate Peroxidases ,Cytosol ,Onium Compounds ,Anti-Infective Agents ,Peroxidases ,Gene Expression Regulation, Plant ,Diuron ,Enzyme Induction ,Tobacco ,Phosphoprotein Phosphatases ,Calcium ,Enzyme Inhibitors ,Reactive Oxygen Species ,Salicylic Acid ,Signal Transduction - Abstract
In plants ascorbate peroxidase (APX) is an important H2O2-detoxifying enzyme. The expression of APX is rapidly induced in response to stresses that result in the accumulation of reactive oxygen species (ROS). We have recently reported that the steady-state level of transcripts encoding cytosolic APX (cAPX) is dramatically induced during the hypersensitive response (HR) of tobacco plants infected with tobacco mosaic virus (TMV). Because cAPX expression is closely linked to the production of ROS in plant cells, studying the regulation cAPX mRNA can reveal some of the signal transduction events associated with ROS metabolism during the HR. Analysis of cAPX mRNA induction during the HR suggested that the expression of cAPX is under the control of the HR signal transduction pathway. The activation of cAPX expression followed signaling events such as changes in protein phosphorylation and induction of ion fluxes. Expression of cAPX was suppressed under conditions of low oxygen pressure, and could only be mimicked by enhancing the intracellular generation of ROS. Interestingly, salicylic acid (SA), which is thought to be involved in ROS metabolism during the HR, did not affect the induction of cAPX mRNA during TMV-induced HR. Using cAPX expression as a marker for the production of ROS, it is suggested that SA may not be involved in the formation of ROS during the HR of tobacco to TMV, and that ROS may not be involved in the induction of the pathogenesis-related protein, PR-1, during this process.
- Published
- 1999
5. DNA-binding properties, genomic organization and expression pattern of TGA6, a new member of the TGA family of bZIP transcription factors in Arabidopsis thaliana
- Author
-
C, Xiang, Z, Miao, and E, Lam
- Subjects
Leucine Zippers ,Base Sequence ,Sequence Homology, Amino Acid ,Arabidopsis Proteins ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Arabidopsis ,Genes, Plant ,Polymerase Chain Reaction ,DNA-Binding Proteins ,Basic-Leucine Zipper Transcription Factors ,Multigene Family ,Amino Acid Sequence ,Cloning, Molecular ,Sequence Alignment ,DNA Primers ,Transcription Factors - Abstract
The TGA genes encode a family of basic domain-leucine zipper (bZIP) transcription factors that are conserved in higher plants. We have continued to unravel the complexity of this gene family by using a polymerase chain reaction (PCR)-based approach. Taking advantage of the conserved amino acid sequence in the bZIP domain found in all members of this gene family, two degenerate oligonucleotides were synthesized based on the sequence of this region in order to amplify by PCR the analogous genomic fragments from the various TGA loci in Arabidopsis. This approach has led us to the finding of a new member of the TGA gene family, and subsequently the isolation of a gene designated as TGA6. Further characterization of the TGA6 locus confirmed our prediction that the gene structure of this family is remarkably conserved. Genomic Southern blot analysis revealed that TGA6 is a single-copy gene in Arabidopsis. Based on the genomic sequence information, gene-specific primers were synthesized for isolating the cDNA that corresponds to the coding region. Subsequently, the cDNA for TGA6 was cloned and sequenced. Gel mobility shift assays with in vitro translated TGA6 protein showed that TGA6 is more like TGA5 in terms of its in vitro DNA-binding properties. The expression of TGA6 in different tissues was estimated by using reverse transcriptase (RT)-PCR and further analyzed in transgenic Arabidopsis lines expressing a TGA6 promoter-GUS fusion. TGA6 promoter activity is found primarily in roots of young seedlings. As seedlings develop, TGA6 is expressed in aging cotyledons, mesophyll cells of hydathodes on leaf margins, vascular tissue and trichomes of senescing rosette leaves, but is very low in primary roots of mature plants. High levels of expression persist in young lateral roots and in regions of the primary root where lateral roots are emerging. In flowers, the activity is localized predominantly to mature pollen grains. The expression pattern of TGA6 reported here is strikingly similar to that of an Arabidopsis acidic chitinase gene. Possible biological significance of TGA6 in cellular defense against pathogens and abiotic stress is discussed.
- Published
- 1997
6. Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress
- Author
-
R, Mittler and E, Lam
- Subjects
Plant Leaves ,Tobacco Mosaic Virus ,Plants, Toxic ,Endodeoxyribonucleases ,Enzyme Induction ,Tobacco ,Apoptosis ,DNA Fragmentation ,Stress, Mechanical ,Plant Proteins - Abstract
Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.
- Published
- 1997
7. Sequence-guided approach to genotyping plant clones and species using polymorphic NB-ARC-related genes.
- Author
-
Chu P, Wilson GM, Michael TP, Vaiciunas J, Honig J, and Lam E
- Subjects
- DNA Fingerprinting, DNA, Plant genetics, Nucleic Acid Amplification Techniques, Plant Proteins genetics, Araceae genetics, Cloning, Organism, Gene Expression Regulation, Plant physiology, Genotype, Plant Proteins metabolism, Polymorphism, Single Nucleotide
- Abstract
Key Message: Leveraging the heightened levels of polymorphism in NB-ARC-related protein encoding genes in higher plants, a bioinformatic pipeline was created to identify regions in this gene family from sequenced plant genomes that exhibit fragment length or single nucleotide differences in different accessions of the same species. Testing this approach with the aquatic plant Spirodela polyrhiza demonstrated its superior performance in comparison with currently available genotyping technologies based on PCR amplification. Rapid and economical genotyping tools that can reliably distinguish species and intraspecific variations in plants can be powerful tools for biogeographical and ecological studies. Clones of the cosmopolitan duckweed species, Spirodela polyrhiza, are difficult to distinguish morphologically due to their highly abbreviated architecture and inherently low levels of sequence variation. The use of plastidic markers and generic Amplification Fragment Length Polymorphism approaches have met with limited success in resolving clones of S. polyrhiza from diverse geographical locales. Using whole genome sequencing data from nine S. polyrhiza clones as a training set, we created an informatic pipeline to identify and rank polymorphic regions from nuclear-encoded NB-ARC-related genes to design markers for PCR, Sanger sequencing (barcoding), and fragment length analysis. With seven primer sets, we found 21 unique fingerprints from a set of 23 S. polyrhiza clones. However, three of these clones share the same fingerprint and are indistinguishable by these markers. These primer sets can also be used as interspecific barcoding tools to rapidly resolve S. polyrhiza from the closely related S. intermedia species without the need for DNA sequencing. Our work demonstrates a general approach of using hyper-polymorphic loci within genomes as a resource to produce facile tools that can have high resolving power for genotyping applications.
- Published
- 2018
- Full Text
- View/download PDF
8. Resurgence of duckweed research and applications: report from the 3rd International Duckweed Conference.
- Author
-
Appenroth KJ, Sree KS, Fakhoorian T, and Lam E
- Subjects
- Biodegradation, Environmental, Genome, Plant, Araceae genetics, Araceae microbiology, Araceae physiology
- Abstract
Duckweed, flowering plants in the Lemnaceae family, comprises the smallest angiosperms in the plant kingdom. They have some of the fastest biomass accumulation rates reported to date for plants and have the demonstrated ability to thrive on wastewater rich in dissolved organic compounds and thus could help to remediated polluted water resources and prevents eutrophication. With a high quality genome sequence now available and increased commercial interest worldwide to develop duckweed biomass for renewables such as protein and fuel, the 3rd International Duckweed Conference convened at Kyoto, Japan, in July of 2015, to update the community of duckweed researchers and developers on the progress in the field. In addition to sharing results and ideas, the conference also provided ample opportunities for new-comers as well as established workers in the field to network and create new aliances. We hope this meeting summary will also help to disseminate the key advances and observations that have been presented in this conference to the broader plant biology community in order to encourage increased cross-fertilization of ideas and technologies.
- Published
- 2015
- Full Text
- View/download PDF
9. Duckweed in bloom: the 2nd International Conference on Duckweed Research and Applications heralds the return of a plant model for plant biology.
- Author
-
Lam E, Appenroth KJ, Michael T, Mori K, and Fakhoorian T
- Subjects
- Bacteria metabolism, Biodegradation, Environmental, Biodiversity, Biomass, Genomics, Microbiota, Plant Growth Regulators pharmacology, Research, Transformation, Genetic, Wastewater, Water Purification, Araceae drug effects, Araceae genetics, Araceae growth & development, Araceae physiology, Models, Biological
- Abstract
More than 50 participants from around the world congregated at Rutgers University for 4 days to discuss the latest advances in duckweed research and applications. Among other developments in the field, exciting new information related to duckweed including genome sequencing, improved genetic transformation, and the identification of a novel plant growth promoting substance from bacteria were reported.
- Published
- 2014
- Full Text
- View/download PDF
10. Duckweed rising at Chengdu: summary of the 1st International Conference on Duckweed Application and Research.
- Author
-
Zhao H, Appenroth K, Landesman L, Salmeán AA, and Lam E
- Subjects
- Biodegradation, Environmental, Biomass, China, Conservation of Natural Resources, Waste Management, Araceae genetics, Araceae growth & development, Araceae physiology
- Abstract
Duckweeds, plants of the Lemnaceae family, have the distinction of being the smallest angiosperms in the world with the fastest doubling time. Together with its naturally ability to thrive on abundant anthropogenic wastewater, these plants hold tremendous potential to helping solve critical water, climate and fuel issues facing our planet this century. With the conviction that rapid deployment and optimization of the duckweed platform for biomass production will depend on close integration between basic and applied research of these aquatic plants, the first International Conference on Duckweed Research and Applications (ICDRA) was organized and took place in Chengdu, China, from October 7th to 10th of 2011. Co-organized with Rutgers University of New Jersey (USA), this Conference attracted participants from Germany, Denmark, Japan, Australia, in addition to those from the US and China. The following are concise summaries of the various oral presentations and final discussions over the 2.5 day conference that serve to highlight current research interests and applied research that are paving the way for the imminent deployment of this novel aquatic crop. We believe the sharing of this information with the broad Plant Biology community is an important step toward the renaissance of this excellent plant model that will have important impact on our quest for sustainable development of the world.
- Published
- 2012
- Full Text
- View/download PDF
11. Caspase-like protease involvement in the control of plant cell death.
- Author
-
Lam E and del Pozo O
- Subjects
- Plant Cells, Signal Transduction, Apoptosis, Caspases physiology, Plants enzymology
- Abstract
Cell death as a highly regulated process has now been recognized to be an important, if not essential, pathway that is ubiquitous in all multicellular eukaryotes. In addition to playing key roles in the morphogenesis and sculpting of the organs to give rise to highly specialized forms and shapes, cell death also participates in the programmed creation of specialized cell types for essential functions such as the selection of B cells in the immune system of mammals and the formation of tracheids in the xylem of vascular plants. Studies of apoptosis, the most well-characterized form of animal programmed cell death, have culminated in the identification of a central tripartite death switch the enzymatic component of which is a conserved family of cysteine proteases called caspases. Studies in invertebrates and other animal models suggest that caspases are conserved regulators of apoptotic cell death in all metazoans. In plant systems, the identities of the main executioners that orchestrate cell death remain elusive. Recent evidence from inhibitor studies and biochemical approaches suggests that caspase-like proteases may also be involved in cell death control in higher plants. Furthermore, the mitochondrion and reactive oxygen species may well constitute a common pathway for cell death activation in both animal and plant cells. Cloning of plant caspase-like proteases and elucidation of the mechanisms through which mitochondria may regulate cell death in both systems should shed light on the evolution of cell death control in eukaryotes and may help to identify essential components that are highly conserved in eukaryotes.
- Published
- 2000
- Full Text
- View/download PDF
12. Cell death: the 'Yin' path in the balancing act of the life cycle.
- Author
-
Lam E and Greenberg J
- Subjects
- Plants genetics, Apoptosis, Plant Cells
- Published
- 2000
- Full Text
- View/download PDF
13. Markers for hypersensitive response and senescence show distinct patterns of expression.
- Author
-
Pontier D, Gan S, Amasino RM, Roby D, and Lam E
- Subjects
- Apoptosis drug effects, Apoptosis genetics, Copper pharmacology, Genes, Reporter genetics, Genetic Markers genetics, Plant Diseases, Plant Leaves drug effects, Plant Leaves genetics, Plant Leaves microbiology, Plant Leaves physiology, Plants, Genetically Modified, Promoter Regions, Genetic genetics, Pseudomonas physiology, RNA, Messenger analysis, RNA, Messenger genetics, RNA, Messenger metabolism, Time Factors, Nicotiana drug effects, Nicotiana genetics, Nicotiana microbiology, Transcriptional Activation drug effects, Transgenes genetics, Apoptosis physiology, Gene Expression Regulation, Plant drug effects, Genes, Plant genetics, Plants, Toxic, Signal Transduction drug effects, Nicotiana physiology, Tobacco Mosaic Virus physiology
- Abstract
Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.
- Published
- 1999
- Full Text
- View/download PDF
14. Signals controlling the expression of cytosolic ascorbate peroxidase during pathogen-induced programmed cell death in tobacco.
- Author
-
Mittler R, Lam E, Shulaev V, and Cohen M
- Subjects
- Anti-Infective Agents pharmacology, Ascorbate Peroxidases, Calcium metabolism, Cytosol enzymology, Diuron pharmacology, Enzyme Induction drug effects, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Plant, Glutathione metabolism, Herbicides pharmacology, Onium Compounds pharmacology, Oxygen pharmacology, Peroxidases genetics, Phosphoprotein Phosphatases metabolism, Reactive Oxygen Species metabolism, Salicylic Acid pharmacology, Signal Transduction, Nicotiana enzymology, Nicotiana virology, Tobacco Mosaic Virus physiology, Apoptosis, Peroxidases metabolism, Plants, Toxic, Nicotiana genetics
- Abstract
In plants ascorbate peroxidase (APX) is an important H2O2-detoxifying enzyme. The expression of APX is rapidly induced in response to stresses that result in the accumulation of reactive oxygen species (ROS). We have recently reported that the steady-state level of transcripts encoding cytosolic APX (cAPX) is dramatically induced during the hypersensitive response (HR) of tobacco plants infected with tobacco mosaic virus (TMV). Because cAPX expression is closely linked to the production of ROS in plant cells, studying the regulation cAPX mRNA can reveal some of the signal transduction events associated with ROS metabolism during the HR. Analysis of cAPX mRNA induction during the HR suggested that the expression of cAPX is under the control of the HR signal transduction pathway. The activation of cAPX expression followed signaling events such as changes in protein phosphorylation and induction of ion fluxes. Expression of cAPX was suppressed under conditions of low oxygen pressure, and could only be mimicked by enhancing the intracellular generation of ROS. Interestingly, salicylic acid (SA), which is thought to be involved in ROS metabolism during the HR, did not affect the induction of cAPX mRNA during TMV-induced HR. Using cAPX expression as a marker for the production of ROS, it is suggested that SA may not be involved in the formation of ROS during the HR of tobacco to TMV, and that ROS may not be involved in the induction of the pathogenesis-related protein, PR-1, during this process.
- Published
- 1999
- Full Text
- View/download PDF
15. DNA-binding properties, genomic organization and expression pattern of TGA6, a new member of the TGA family of bZIP transcription factors in Arabidopsis thaliana.
- Author
-
Xiang C, Miao Z, and Lam E
- Subjects
- Amino Acid Sequence, Arabidopsis genetics, Base Sequence, Basic-Leucine Zipper Transcription Factors, Cloning, Molecular, DNA Primers, DNA-Binding Proteins biosynthesis, Leucine Zippers, Molecular Sequence Data, Multigene Family, Polymerase Chain Reaction, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Transcription Factors biosynthesis, Arabidopsis metabolism, Arabidopsis Proteins, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Genes, Plant, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
The TGA genes encode a family of basic domain-leucine zipper (bZIP) transcription factors that are conserved in higher plants. We have continued to unravel the complexity of this gene family by using a polymerase chain reaction (PCR)-based approach. Taking advantage of the conserved amino acid sequence in the bZIP domain found in all members of this gene family, two degenerate oligonucleotides were synthesized based on the sequence of this region in order to amplify by PCR the analogous genomic fragments from the various TGA loci in Arabidopsis. This approach has led us to the finding of a new member of the TGA gene family, and subsequently the isolation of a gene designated as TGA6. Further characterization of the TGA6 locus confirmed our prediction that the gene structure of this family is remarkably conserved. Genomic Southern blot analysis revealed that TGA6 is a single-copy gene in Arabidopsis. Based on the genomic sequence information, gene-specific primers were synthesized for isolating the cDNA that corresponds to the coding region. Subsequently, the cDNA for TGA6 was cloned and sequenced. Gel mobility shift assays with in vitro translated TGA6 protein showed that TGA6 is more like TGA5 in terms of its in vitro DNA-binding properties. The expression of TGA6 in different tissues was estimated by using reverse transcriptase (RT)-PCR and further analyzed in transgenic Arabidopsis lines expressing a TGA6 promoter-GUS fusion. TGA6 promoter activity is found primarily in roots of young seedlings. As seedlings develop, TGA6 is expressed in aging cotyledons, mesophyll cells of hydathodes on leaf margins, vascular tissue and trichomes of senescing rosette leaves, but is very low in primary roots of mature plants. High levels of expression persist in young lateral roots and in regions of the primary root where lateral roots are emerging. In flowers, the activity is localized predominantly to mature pollen grains. The expression pattern of TGA6 reported here is strikingly similar to that of an Arabidopsis acidic chitinase gene. Possible biological significance of TGA6 in cellular defense against pathogens and abiotic stress is discussed.
- Published
- 1997
- Full Text
- View/download PDF
16. Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress.
- Author
-
Mittler R and Lam E
- Subjects
- Endodeoxyribonucleases biosynthesis, Endodeoxyribonucleases genetics, Enzyme Induction genetics, Plant Leaves enzymology, Plant Leaves genetics, Plant Leaves virology, Plant Proteins biosynthesis, Plant Proteins genetics, Plants, Toxic, Stress, Mechanical, Nicotiana enzymology, Nicotiana genetics, Nicotiana virology, Tobacco Mosaic Virus genetics, Apoptosis, DNA Fragmentation, Endodeoxyribonucleases metabolism, Plant Proteins metabolism
- Abstract
Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.
- Published
- 1997
- Full Text
- View/download PDF
17. Isolation and analysis of the soybean SGA2 gene (cDNA), encoding a new member of the plant G-protein family of signal transducers.
- Author
-
Gotor C, Lam E, Cejudo FJ, and Romero LC
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Conserved Sequence, DNA, Complementary genetics, GTP-Binding Proteins chemistry, GTP-Binding Proteins physiology, Gene Expression, Molecular Sequence Data, Molecular Weight, Phylogeny, Sequence Homology, Amino Acid, Glycine max chemistry, Glycine max physiology, GTP-Binding Proteins genetics, Genes, Plant, Monomeric GTP-Binding Proteins, Plant Proteins, Signal Transduction, Glycine max genetics
- Abstract
We have isolated a cDNA clone from Glycine max, named SGA2, coding for a G alpha-subunit protein. The encoded polypeptide, SG alpha2, shows a molecular mass of 45 kDa and contains most of the conserved regions involved in guanine nucleotide binding and hydrolysis. Comparison at the nucleotide and amino acid sequence levels with the other plant G alpha's shows a high degree of conservation (>85% similarity). Phylogenetic analysis of these plant genes with the other G alpha's from different species clearly indicate that those proteins represent a new member of the heterotrimeric G-protein family, named Gp. Tissue localization of SGA2 transcripts in root, stem and leaf organs shows that this gene is widely expressed throughout the plant although it is most abundant in the vascular tissues of all these organs. Furthermore, the transcript is more abundant in young tissues and organ primordia than mature tissues. The high degree of sequence conservation among the plant G alpha's and the differences to other species of other kingdoms, suggest that plant G proteins may function in specialized signalling processes.
- Published
- 1996
- Full Text
- View/download PDF
18. Coordinated activation of as-1-type elements and a tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxide.
- Author
-
Xiang C, Miao ZH, and Lam E
- Subjects
- Acetates pharmacology, Binding Sites, Cyclopentanes pharmacology, Genes, Reporter genetics, Glucuronidase genetics, Glutathione Transferase genetics, Hydrogen Peroxide pharmacology, Indoleacetic Acids pharmacology, Oxylipins, Plants, Genetically Modified, Promoter Regions, Genetic genetics, RNA, Messenger analysis, RNA, Plant analysis, Salicylates pharmacology, Salicylic Acid, Gene Expression Regulation, Plant drug effects, Plant Growth Regulators pharmacology, Plants, Toxic, Regulatory Sequences, Nucleic Acid genetics, Signal Transduction genetics, Nicotiana genetics
- Abstract
The molecular mechanism of signal transduction pathways which mediate the action of phytohormones are poorly understood. Recently, we and others have shown that the as -1 type cis-acting elements can respond to auxin and salicylic acid, two well-characterized signaling molecules in plants. In the present work, we have examined a comprehensive set of physiological and abiotic agents and found that auxin, salicylic acid and methyl-jasmonate are three effective inducers of the as-1-type elements in transgenic tobacco. Using a cell suspension culture containing a synthetic promoter-GUS fusion, we demonstrated rapid and sensitive induction of the as-1-type element by these phytohormones. Furthermore, a tobacco glutathione S-transferase gene, GNT35, that contains an as-1-type binding site in its promoter is also inducible by auxin, salicylic acid and methyl-jasmonate with similar kinetics. As Ulmasov et al. have recently reported, we found that the as-1-type elements can also respond to weak/inactive analogues of auxin and salicylic acid. In addition, we show that hydrogen peroxide can also effectively activate the expression of GNT35 as well as the as-1-type element in a cell suspension culture, but not with whole seedlings. These results are discussed with respect to the possible mechanism(s) through which a single cis element may respond to a diverse array of molecules.
- Published
- 1996
- Full Text
- View/download PDF
19. The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization.
- Author
-
Meisel L and Lam E
- Subjects
- Amino Acid Sequence, Base Sequence, Conserved Sequence, Histocytochemistry, Homeodomain Proteins genetics, Molecular Sequence Data, Nuclear Proteins genetics, Plant Proteins genetics, Plants, Genetically Modified, Plants, Toxic, Recombinant Fusion Proteins metabolism, Sequence Deletion, Sequence Homology, Amino Acid, Nicotiana genetics, Vegetables genetics, Zea mays genetics, Cell Compartmentation, Cell Nucleus metabolism, Homeodomain Proteins metabolism, Nuclear Proteins metabolism, Plant Proteins metabolism
- Abstract
Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals.
- Published
- 1996
- Full Text
- View/download PDF
20. TGA3 is a distinct member of the TGA family of bZIP transcription factors in Arabidopsis thaliana.
- Author
-
Miao ZH, Liu X, and Lam E
- Subjects
- Amino Acid Sequence, Arabidopsis chemistry, Base Sequence, Basic-Leucine Zipper Transcription Factors, Blotting, Northern, Blotting, Southern, Cloning, Molecular, DNA chemistry, DNA-Binding Proteins chemistry, DNA-Binding Proteins isolation & purification, Exons, Introns, Molecular Sequence Data, Plant Proteins chemistry, Plant Proteins isolation & purification, Polymerase Chain Reaction, RNA, Messenger analysis, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Transcription Factors chemistry, Transcription Factors isolation & purification, Arabidopsis genetics, Arabidopsis Proteins, DNA-Binding Proteins genetics, Plant Proteins genetics, Transcription Factors genetics
- Abstract
TGA1a is a well-characterized transcription factor that may mediate the root-specific and auxin-responsive expression of some plant genes. In tobacco, Southern blot and genomic cloning analyses have shown that TGA1a consists of at least four closely related genes. Since TGA1a belongs to the bZIP class of transcriptional factors, the protein products of the tobacco TGA1a family are likely to form hetero-dimers with each other in addition to the homo-dimers. In order to find a model plant system that may have less genomic complexity, we have now characterized a TGA1a-related gene (TGA3) from Arabidopsis thaliana. Southern blot analyses at high stringency suggest that Arabidopsis contains only one copy of TGA3 per haploid genome. However, low stringency Southern blot analyses with homologous and heterologous probes suggest that there is a multigene family of TGA1a-related genes present in Arabidopsis, of which TGA1, TGA2 and TGA3 are members. Although these gene members share a highly conserved bZIP region, they are not genes with high homologies at the nucleotide level. Similar to TGA1a of tobacco, TGA3 is most highly expressed in root tissues and recombinant TGA3 protein shows similar DNA-binding site specificity to that of TGA1a in vitro. Comparison of the genomic organization between TGA3 and the tobacco homologue PG13 reveals striking conservation in the sizes and positions of exons and introns in the region surrounding the bZIP domain.
- Published
- 1994
- Full Text
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21. Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin.
- Author
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Lam E and Chua NH
- Abstract
We have examined the effects of novobiocin and template topology on the transcription of two chloroplast genes encoding the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the chloroplast ATPase (atpB), in an in vitro transcription system. The template topology was monitored by agarose gel electrophoresis while the in vitro transcripts were determined by 5' S1 nuclease analysis under identical conditions. We discovered that our chloroplast transcription extracts contain a DNA gyrase activity and a chromatographically separable topoisomerase I activity. Incubation of a supercoiled template with the extracts under the same conditions in which transcription assays were carried out leads to a decrease in the supercoiled from and concomitant appearance of distinct topoisomers. More extensive relaxation of the supercoiled template occurs when nucleotide triphosphates are omitted from the reaction mixture or when a low concentration (25 μg/ml) of novobiocin is added. Higher concentrations (≥ 250 μg/ml) of the drug, however, also inhibit the topoisomerase I activity. The transcription of the atpB gene is inhibited by lower concentrations of novobiocin as compared to the rbcL gene in the same reaction mixture. Relaxed, closed circular template and linearized DNA are not substrates for chloroplast transcription extracts, although they are transcribed accurately by the E. coli RNA polymerase under our conditions. Control of in vitro transcription of the two chloroplast genes by template topology can also be demonstrated by modulating the relative activity for the topoisomerases in the transcription extract. Our results suggest that changes in template topology may be a mechanism by which chloroplast genes are differentially regulated and the chloroplast DNA gyrase and topoisomerase I are key enzymes for this mode of regulation in vivo.
- Published
- 1987
- Full Text
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