Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenyl... more Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenylalanine produces three isomeric tyrosines, o-tyrosine (2-hydroxyphenylalanine), m-tyrosine (3-hydroxyphenylalanine), and p-tyrosine (4-hydroxyphenylalanine). These may be separated by high-performance liquid chromatography and measured using an electrochemical detector. Since L-phenylalanine is relatively nontoxic, it is proposed that generation of these three tyrosines from phenylalanine can be used as an assay for .OH in biological systems. The use of the assay to measure .OH production by leghemoglobin (plus H2O2) and by activated human neutrophils is described. No .OH production by activated human neutrophils was observed unless a source of iron ions was added to the reaction mixture, which suggests that these cells do not release an iron "promoter" of .OH generation from superoxide and hydrogen peroxide.
The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationsh... more The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible ...
The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpre... more The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpressing the housekeeping catalase katB is delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H(2)O(2) is required for optimal progression of infection threads through the root hairs and plant cell layers.
Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixin... more Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti. An S. meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an S. meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that gamma-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibi...
The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the ... more The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti. Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its own expression in S. meliloti.
Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant develop... more Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.
The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acqui... more The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest. In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described. GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K(m) value of 13.8 microm. Cadmium was the only other metal tested able to inhibit zinc uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.
Research on legume nodule development has contributed greatly to our current understanding of pla... more Research on legume nodule development has contributed greatly to our current understanding of plantmicrobe interactions. However, the factors that orchestrate root nodule senescence have received relatively little attention. Accumulating evidence suggests that redox ...
The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur,... more The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H2O2 by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H2O2-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.
Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenyl... more Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenylalanine produces three isomeric tyrosines, o-tyrosine (2-hydroxyphenylalanine), m-tyrosine (3-hydroxyphenylalanine), and p-tyrosine (4-hydroxyphenylalanine). These may be separated by high-performance liquid chromatography and measured using an electrochemical detector. Since L-phenylalanine is relatively nontoxic, it is proposed that generation of these three tyrosines from phenylalanine can be used as an assay for .OH in biological systems. The use of the assay to measure .OH production by leghemoglobin (plus H2O2) and by activated human neutrophils is described. No .OH production by activated human neutrophils was observed unless a source of iron ions was added to the reaction mixture, which suggests that these cells do not release an iron "promoter" of .OH generation from superoxide and hydrogen peroxide.
The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationsh... more The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible ...
The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpre... more The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpressing the housekeeping catalase katB is delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H(2)O(2) is required for optimal progression of infection threads through the root hairs and plant cell layers.
Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixin... more Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti. An S. meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an S. meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that gamma-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibi...
The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the ... more The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti. Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its own expression in S. meliloti.
Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant develop... more Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.
The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acqui... more The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest. In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described. GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K(m) value of 13.8 microm. Cadmium was the only other metal tested able to inhibit zinc uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.
Research on legume nodule development has contributed greatly to our current understanding of pla... more Research on legume nodule development has contributed greatly to our current understanding of plantmicrobe interactions. However, the factors that orchestrate root nodule senescence have received relatively little attention. Accumulating evidence suggests that redox ...
The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur,... more The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H2O2 by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H2O2-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.
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