The metabolism of one-carbon (C 1) units is vital to plants. It involves unique enzymes and takes... more The metabolism of one-carbon (C 1) units is vital to plants. It involves unique enzymes and takes place in four subcellular compartments. Plant C 1 biochemistry has remained relatively unexplored, partly because of the low abundance or the lability of many of its enzymes and intermediates. Fortunately, DNA sequence databases now make it easier to characterize known C 1 enzymes and to discover new ones, to identify pathways that might carry high C 1 fluxes, and to use engineering to redirect C 1 fluxes and to understand their control better.
We describe the nucleotide sequence and some structural characteristics of a single copy gene enc... more We describe the nucleotide sequence and some structural characteristics of a single copy gene encoding pathogenesis-related protein 2 (PR2) in parsley (Petroselinum crispum). Transcriptional activation of this gene in cultured parsley cells treated with fungal elicitor leads to a rapid, large and transient accumulation of PR2 mRNA. The deduced PR2 protein belongs to a novel class of evolutionarily conserved polypeptides which are closely related to disease resistance in plants. Functional analysis of a series of truncated PR2 promoter fusions with the ,B-glucuronidase reporter gene, using parsley protoplasts for transient expression studies, identified a 5' upstream element between positions-168 and-52 necessary for strong elicitor responsiveness. This small promoter fragment is active in conjunction with its own TATA box region as well as with the corresponding region from a heterologous promoter. The PRH regulatory region exhibits no sequence similarity to any other elicitorresponsive promoter known to date.
... Abteilung Biochemie. Carl-von-Linnk-Weg 10. 0-50829 Koln. ... 3 . The most rapid responses of... more ... Abteilung Biochemie. Carl-von-Linnk-Weg 10. 0-50829 Koln. ... 3 . The most rapid responses of parsley cells to elicitor treatment detected so far are changes in the H', K', C1-and Ca2+ fluxes across the plasma membrane and the formation of H 2 0 2(oxidative burst), which 94. ...
The functional, spatial and temporal complexity of pathogen defence in plants is becoming ever-mo... more The functional, spatial and temporal complexity of pathogen defence in plants is becoming ever-more apparent. Functional complexity begins with the exogenous signals perceived from the pathogen, continues with the mechanisms of signal perception and signal transduction, and results in extensive 'reprogramming' of cellular metabolism, involving large changes in gene activity. The spatial organization of these reactions is similarly complex and affects intracellular compartmentation, the fate of cells and, ultimately, the tissues that surround the infection site. The highly dynamic nature of the response adds temporal complexity. Thus, pathogen defence entails a major shift in metabolic activity, rather than altered expression of a few unique, defence-related genes. The observed complexity serves as a paradigm of the flexibility and plasticity of plant metabolism.
The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon¯ow into diverse br... more The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon¯ow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and speci®cities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classi®ed into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to¯avonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than¯avonoids.
Proceedings of the National Academy of Sciences of the United States of America, Apr 1, 1986
Administration of a cell-wall preparation from the fungus Phytophthora megasperma f. sp. glycinea... more Administration of a cell-wall preparation from the fungus Phytophthora megasperma f. sp. glycinea, which acts as an elicitor of phytoalexin production in cell suspension cultures of parsley (Petroselinum crispum), also results in a rapid and dramatic increase in the relative amounts of mRNAs coding for a number of small proteins having low isoelectric points. According to various operational criteria, the translation products are classified as "pathogenesis-related" (PR) proteins. Here we report that the cDNA inserts of two pBR322-derived plasmids, pcPRi and pcPR2, are homologous
Current plant science and biotechnology in agriculture, 1991
The interaction between Arabidopsis thaliana and various phytopathogenic Pseudomonas pathovars pr... more The interaction between Arabidopsis thaliana and various phytopathogenic Pseudomonas pathovars presents an outstanding model to genetically define plant and bacterial loci necessary for generation of a hypersensitive resistance response (HR). Certain isolates of Pseudomonas syringae pv. maculicola are virulent on Arabidopsis, while others are not. We also tested 17 P. cichorii isolates, and found that all are avirulent. Variation exists among Arabidopsis ecotypes with respect to resistance/ susceptibility to P.s. maculicola isolates. We also identified candidate Arabidopsis mutants showing altered interaction phenotypes after inoculation with a P.cichorii isolate. Segregation analysis and RFZP mapping of the loci conditioning these altered responses is in progress. We also have cloned Arabidopsis homologs to genes induced by pathogen in another system. The function of many of these genes is unknown. Phenocooy mutants of one, which is highly induced by pathogen infiltration, will be constructed to test the gene product’s involvement in the HR. Finally we isolated mutants in genes encoding bacterial membrane proteins unable to generate a normal response on Arabidodpsis. These mutants appear to evade the plant defense response, and the normal protein may be important in triggering the HR.
A single‐copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecul... more A single‐copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecular weight of 90 500 has been characterized. The position of the gene was mapped to the distal part of chromosome 4. Expression of the gene differs in various vegetative and floral plant tissues and is positively influenced by the phytohormone auxin. In Arabidopsis plants, a complex pattern of prha promoter‐driven GUS expression is observed, often associated with regions of developing vascular tissue. Exogenously applied auxin strongly increased endogenous prha transcript levels. In addition, the prha promoter is highly responsive to the synthetic auxin, naphthalene acetic acid, in transient assays using tobacco protoplasts. The PRHA protein has the capacity to bind to TAATTG core sequence elements but requires additional adjacent bases for high‐affinity binding. These findings are discussed in relation to studies of other plant homeobox genes as well as possible in vivo target genes for PRHA.
Proceedings of the National Academy of Sciences of the United States of America, May 15, 1992
Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates... more Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates ftransription of many genes encoding specific steps in pathogen defenserelated pathways. We report evidence that three cDNAs corresponding to such genes represent two key enzymes of the activated methyl cycle. Two cDNAs are derived from distinct members of the S-adenosyl-L-methionine synthetase gene family, based on extensive similarity of the deduced polypeptides with authentic enzymes from Arabidopsis thaliana, rat, yeast, and Escherichia coi. The third cDNA exhibits large sim it with a functionally related gene, encoding S-adenosyl-Lhomocysteine hydrolase, from rat and a slime mold. Marked differences in the mRNA levels occurred in different organs of parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase enzyme activity. These results suggest a close metabolic link between pathogen defense and an increased turnover of activated methyl groups.
We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabi... more We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. EL13 mRNA accumulates in A.thalana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM] locus, ELI3 mRNA accumulates to high levels 5-10 h postinoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM) action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h postinfiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the EU3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm) locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM) using an assay generalizable to any pathogen induced plant defense phenomena.
Current Plant Science and Biotechnology in Agriculture, 1991
Plants respond to pathogen attack by rapid activation of defense genes. In selected experimental ... more Plants respond to pathogen attack by rapid activation of defense genes. In selected experimental systems, the transcription of these genes is also induced when cultured plant cells or protoplasts are treated with elicitors. We have purified an extracellular glycoprotein of 42 kDa from the culture filtrate of the soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg), which is a potent elicitor of phytoalexin accumulation and defense gene activation in parsley (1Petroselinum crispum) cells and protoplasts. Reversible binding of the 25I-labelled glycoprotein to microsomes and protoplasts indicates the presence of specific binding sites on the parsley plasma membrane. The transduction of the elicitor signal from the cell surface to the nucleus was found to involve a rapid and transient uptake of Ca2+, alkalization of the culture medium and effluxes of K+ and C1-. These ion fluxes probably result from opening of elicitor-responsive ion channels, as indicated by preliminary results from patch-clamp analysis of parsley protoplasts in the presence of pure glycoprotein elicitor. Omission of Ca2+ from the culture medium substantially reduced the elicitor-induced transcription rates of plant defense genes. Treatment of cultured parsley cells or protoplasts with amphotericin B resulted in ion fluxes and defense gene activation similar to those elicited by the crude Pmg elicitor. The callose elicitors chitosan and digitonin, however, induced ion fluxes of different intensities, activated only some defense genes, and did not stimulate the synthesis of phytoalexins at concentrations optimal for elicitation of callose formation.
The metabolism of one-carbon (C 1) units is vital to plants. It involves unique enzymes and takes... more The metabolism of one-carbon (C 1) units is vital to plants. It involves unique enzymes and takes place in four subcellular compartments. Plant C 1 biochemistry has remained relatively unexplored, partly because of the low abundance or the lability of many of its enzymes and intermediates. Fortunately, DNA sequence databases now make it easier to characterize known C 1 enzymes and to discover new ones, to identify pathways that might carry high C 1 fluxes, and to use engineering to redirect C 1 fluxes and to understand their control better.
We describe the nucleotide sequence and some structural characteristics of a single copy gene enc... more We describe the nucleotide sequence and some structural characteristics of a single copy gene encoding pathogenesis-related protein 2 (PR2) in parsley (Petroselinum crispum). Transcriptional activation of this gene in cultured parsley cells treated with fungal elicitor leads to a rapid, large and transient accumulation of PR2 mRNA. The deduced PR2 protein belongs to a novel class of evolutionarily conserved polypeptides which are closely related to disease resistance in plants. Functional analysis of a series of truncated PR2 promoter fusions with the ,B-glucuronidase reporter gene, using parsley protoplasts for transient expression studies, identified a 5' upstream element between positions-168 and-52 necessary for strong elicitor responsiveness. This small promoter fragment is active in conjunction with its own TATA box region as well as with the corresponding region from a heterologous promoter. The PRH regulatory region exhibits no sequence similarity to any other elicitorresponsive promoter known to date.
... Abteilung Biochemie. Carl-von-Linnk-Weg 10. 0-50829 Koln. ... 3 . The most rapid responses of... more ... Abteilung Biochemie. Carl-von-Linnk-Weg 10. 0-50829 Koln. ... 3 . The most rapid responses of parsley cells to elicitor treatment detected so far are changes in the H', K', C1-and Ca2+ fluxes across the plasma membrane and the formation of H 2 0 2(oxidative burst), which 94. ...
The functional, spatial and temporal complexity of pathogen defence in plants is becoming ever-mo... more The functional, spatial and temporal complexity of pathogen defence in plants is becoming ever-more apparent. Functional complexity begins with the exogenous signals perceived from the pathogen, continues with the mechanisms of signal perception and signal transduction, and results in extensive 'reprogramming' of cellular metabolism, involving large changes in gene activity. The spatial organization of these reactions is similarly complex and affects intracellular compartmentation, the fate of cells and, ultimately, the tissues that surround the infection site. The highly dynamic nature of the response adds temporal complexity. Thus, pathogen defence entails a major shift in metabolic activity, rather than altered expression of a few unique, defence-related genes. The observed complexity serves as a paradigm of the flexibility and plasticity of plant metabolism.
The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon¯ow into diverse br... more The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon¯ow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and speci®cities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classi®ed into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to¯avonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than¯avonoids.
Proceedings of the National Academy of Sciences of the United States of America, Apr 1, 1986
Administration of a cell-wall preparation from the fungus Phytophthora megasperma f. sp. glycinea... more Administration of a cell-wall preparation from the fungus Phytophthora megasperma f. sp. glycinea, which acts as an elicitor of phytoalexin production in cell suspension cultures of parsley (Petroselinum crispum), also results in a rapid and dramatic increase in the relative amounts of mRNAs coding for a number of small proteins having low isoelectric points. According to various operational criteria, the translation products are classified as "pathogenesis-related" (PR) proteins. Here we report that the cDNA inserts of two pBR322-derived plasmids, pcPRi and pcPR2, are homologous
Current plant science and biotechnology in agriculture, 1991
The interaction between Arabidopsis thaliana and various phytopathogenic Pseudomonas pathovars pr... more The interaction between Arabidopsis thaliana and various phytopathogenic Pseudomonas pathovars presents an outstanding model to genetically define plant and bacterial loci necessary for generation of a hypersensitive resistance response (HR). Certain isolates of Pseudomonas syringae pv. maculicola are virulent on Arabidopsis, while others are not. We also tested 17 P. cichorii isolates, and found that all are avirulent. Variation exists among Arabidopsis ecotypes with respect to resistance/ susceptibility to P.s. maculicola isolates. We also identified candidate Arabidopsis mutants showing altered interaction phenotypes after inoculation with a P.cichorii isolate. Segregation analysis and RFZP mapping of the loci conditioning these altered responses is in progress. We also have cloned Arabidopsis homologs to genes induced by pathogen in another system. The function of many of these genes is unknown. Phenocooy mutants of one, which is highly induced by pathogen infiltration, will be constructed to test the gene product’s involvement in the HR. Finally we isolated mutants in genes encoding bacterial membrane proteins unable to generate a normal response on Arabidodpsis. These mutants appear to evade the plant defense response, and the normal protein may be important in triggering the HR.
A single‐copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecul... more A single‐copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecular weight of 90 500 has been characterized. The position of the gene was mapped to the distal part of chromosome 4. Expression of the gene differs in various vegetative and floral plant tissues and is positively influenced by the phytohormone auxin. In Arabidopsis plants, a complex pattern of prha promoter‐driven GUS expression is observed, often associated with regions of developing vascular tissue. Exogenously applied auxin strongly increased endogenous prha transcript levels. In addition, the prha promoter is highly responsive to the synthetic auxin, naphthalene acetic acid, in transient assays using tobacco protoplasts. The PRHA protein has the capacity to bind to TAATTG core sequence elements but requires additional adjacent bases for high‐affinity binding. These findings are discussed in relation to studies of other plant homeobox genes as well as possible in vivo target genes for PRHA.
Proceedings of the National Academy of Sciences of the United States of America, May 15, 1992
Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates... more Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates ftransription of many genes encoding specific steps in pathogen defenserelated pathways. We report evidence that three cDNAs corresponding to such genes represent two key enzymes of the activated methyl cycle. Two cDNAs are derived from distinct members of the S-adenosyl-L-methionine synthetase gene family, based on extensive similarity of the deduced polypeptides with authentic enzymes from Arabidopsis thaliana, rat, yeast, and Escherichia coi. The third cDNA exhibits large sim it with a functionally related gene, encoding S-adenosyl-Lhomocysteine hydrolase, from rat and a slime mold. Marked differences in the mRNA levels occurred in different organs of parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase enzyme activity. These results suggest a close metabolic link between pathogen defense and an increased turnover of activated methyl groups.
We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabi... more We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. EL13 mRNA accumulates in A.thalana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM] locus, ELI3 mRNA accumulates to high levels 5-10 h postinoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM) action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h postinfiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the EU3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm) locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM) using an assay generalizable to any pathogen induced plant defense phenomena.
Current Plant Science and Biotechnology in Agriculture, 1991
Plants respond to pathogen attack by rapid activation of defense genes. In selected experimental ... more Plants respond to pathogen attack by rapid activation of defense genes. In selected experimental systems, the transcription of these genes is also induced when cultured plant cells or protoplasts are treated with elicitors. We have purified an extracellular glycoprotein of 42 kDa from the culture filtrate of the soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg), which is a potent elicitor of phytoalexin accumulation and defense gene activation in parsley (1Petroselinum crispum) cells and protoplasts. Reversible binding of the 25I-labelled glycoprotein to microsomes and protoplasts indicates the presence of specific binding sites on the parsley plasma membrane. The transduction of the elicitor signal from the cell surface to the nucleus was found to involve a rapid and transient uptake of Ca2+, alkalization of the culture medium and effluxes of K+ and C1-. These ion fluxes probably result from opening of elicitor-responsive ion channels, as indicated by preliminary results from patch-clamp analysis of parsley protoplasts in the presence of pure glycoprotein elicitor. Omission of Ca2+ from the culture medium substantially reduced the elicitor-induced transcription rates of plant defense genes. Treatment of cultured parsley cells or protoplasts with amphotericin B resulted in ion fluxes and defense gene activation similar to those elicited by the crude Pmg elicitor. The callose elicitors chitosan and digitonin, however, induced ion fluxes of different intensities, activated only some defense genes, and did not stimulate the synthesis of phytoalexins at concentrations optimal for elicitation of callose formation.
Uploads
Papers by Imre Somssich