Periodic Report Summary - RONET (The Reactive Oxygen Signaling Network of Cells)
Work performed
Major research activities
1. Arabidopsis Zat12:Luciferaze - EMS mutants screen
Seeds of Arabidopsis thaliana transgenic plants expressing the luciferase (Luc) reporter gene under the control of the zat12 promoter were treated with EMS. The mutagenised seeds (M1 generation) were germinated on soil, mature plants were allowed to self-seed and M2 seeds were collected.
A genetic screen was carried out with the germination of M2 seeds on solid MS plates containing a low concentration of the ROS-generating herbicide - paraquat (PQ - 0.01µM) and were grown under constant low light (approximately 50 µE).
Luciferase activity was estimated in 10 to 12-day-old seedlings by spraying luciferin and capturing the luminescence under a charge-coupled device (CCD) camera. The seeds were germinated in a grid (100 seeds per plate) and the seedlings showing a weak or an absence-of luciferase activity were selected and transferred to soil containing pots for further examination. Excised leaves from the selected plants were examined for ROS-induced luciferase expression in response to PQ spraying (1 µM PQ in 0.05 % Tween-20 for 1 h). Of these, plants that maintained weak or an absence of luciferase activity were further selected. Seeds of these selected mutants (M3 generation) were tested again for maintaining abrogated luciferase activity on solid MS plates containing PQ as described above and seedlings were sampled for ribonucleic acid (RNA) extraction. The endogenous expression level of zat12 transcript was examined in RNA blots. Paraquat-induced oxidative stress experiments for RNA blot were repeated at least twice. Mutants confirmed for displaying a reduced level of zat12 expression were further selected for backcrossing. Overall, 26 000 M2 seeds were screened for luciferase activity on MS plates containing PQ. Out of the 442 mutants that were selected, 148 were verified for displaying weak or no luciferase expression. So far, 100 lines have been inspected for endogenous zat12 expression, out of which 15 lines showing weak zat12 expression were picked for backcrossing with the non-mutated Zat12::Luc transgenic line. The remaining 48 lines are now being screened in RNA blots and we expect an additional 10 lines to proceed for backcrossing. Currently, the first selected 15 lines are growing and will be soon go through the first of two backcrosses.
In parallel to the mutant selection process, the zat12::Luc transgenic Arabidosis (Colombia background) was backcrossed six times to a WT Len ecotype. This Arabidopsis cv. Len containing the Zat12:Luc cassette will be used for the final backcross with chosen mutants that have been genetically 'cleaned up' by the two backcrosses described above.
2. Reactive oxygen species mediate a rapid long-distance self-propagating signal in plants
Cell-to-cell communication and long distance signalling play a key role in the response of multicellular organisms to pathogens, pests, mechanical wounding and extreme environmental conditions 1-4. Here, we uncovered a rapid systemic signal in plants that travels at a rate of 8.4 cm min-1 and is dependent on the presence of the respiratory burst oxidase homolog D (RbohD) gene. Signal propagation is accompanied by the accumulation of reactive oxygen species (ROS) in the extracellular spaces between cells, and by rapid expression of ROS-responsive transcripts. Once initiated, the signal can be blocked by the suppression of ROS accumulation at locations distant from the initiation site. Rapid systemic signalling is independent on ethylene, jasmonic acid (JA) or salicylic acid (SA) signalling, but can be triggered by wounding, heat, cold, high light and salinity stresses. Our results reveal a profound and general role played by ROS in mediating rapid, self-propagating systemic signals in plants, and raise the possibility that a similar class of ROS-related signals function in other organisms' response to external stimuli.
3. MitoNEET, a protein with a potential function in regulating iron and ROS
The grant directly supported a graduate student (Yael Harir) and two undergraduate students (Dan Roizman-Sade and Shlumit Kangiser) who worked at the lab in the Hebrew University of Jerusalem. We have been focusing on one ROS-response gene that we have identified as a gene of unknown function with homology to a human gene (mitoNEET), which is thought to be involved in iron cluster transfer across the mitochondrial membrane and was shown in humans to bind a key anti-diabetic drug (Pioglitazone, a member of the thiazolidinedione, TZD, class). The Arabidopsis gene (At5g51720) with unknown function shows sequence homology to the human gene. Moreover, structural mapping of the Arabidopsis protein on the solved structure of the human protein demonstrates a high degree of structural similarity. To test the function of At5g51720 and human mitoNEET, we have expressed these proteins in transgenic plants with and without the addition of a GFP tag. Human mitoNEET is localised to mitochondria and At5g51720 is localised to chloroplasts. These results are in agreement with previous findings that iron cluster biogenesis in plants can occur via two different pathways: one in the mitochondria and one in the chloroplast, and suggest that At5g51720 is involved in the chloroplastic pathway.
We are currently conducting biochemical analysis of At5g51720, generating RNAi lines for it and testing the tolerance of At5g51720 mutants and over-expressors to different abiotic stresses and iron deficiency.
4. Biochemical characterisation: Cluster (2Fe-2S) transfer activity
Two methods were used in order to determine the 2Fe-2S cluster transfer ability of At5g51720 to an apo-acceptor protein, apo-mFd; a biochemical determination using native gels and following changes by absorbance spectroscopy. The At5g51720 absorbance peaks at 458nm and holo-mFd absorbance peaks at 423nm.
The At5g51720 protein was purified after over-expression in bacterial cells and incubated with colourless apo-acceptor protein (apo-mFd); the absorption spectra were recorded at different time points along the incubation period.
In the presence of 5mM MgCl2 in the incubation media, after four hours of incubation, the non-existing absorbance of the apo-mFd is changing into an absorbance almost identical to the absorbance of the holo-mFd.
The absorbance changes strongly suggest that the 2Fe-2S cluster of At-mitoNEET was transferred to the apo-mFd to create the holo-mFd. In order to prove this hypothesis, samples obtained during the incubation, were loaded on non-denaturing gel. Ten minutes after incubating At-mitoNEET with apo-mFd, a red band at the position of holo-mFd was detected.
We plan to devote the upcoming year of the research to identifying the protein(s) partner(s) of At-mitoNEET with whom it associates to perform its biological function.
Major research activities
1. Arabidopsis Zat12:Luciferaze - EMS mutants screen
Seeds of Arabidopsis thaliana transgenic plants expressing the luciferase (Luc) reporter gene under the control of the zat12 promoter were treated with EMS. The mutagenised seeds (M1 generation) were germinated on soil, mature plants were allowed to self-seed and M2 seeds were collected.
A genetic screen was carried out with the germination of M2 seeds on solid MS plates containing a low concentration of the ROS-generating herbicide - paraquat (PQ - 0.01µM) and were grown under constant low light (approximately 50 µE).
Luciferase activity was estimated in 10 to 12-day-old seedlings by spraying luciferin and capturing the luminescence under a charge-coupled device (CCD) camera. The seeds were germinated in a grid (100 seeds per plate) and the seedlings showing a weak or an absence-of luciferase activity were selected and transferred to soil containing pots for further examination. Excised leaves from the selected plants were examined for ROS-induced luciferase expression in response to PQ spraying (1 µM PQ in 0.05 % Tween-20 for 1 h). Of these, plants that maintained weak or an absence of luciferase activity were further selected. Seeds of these selected mutants (M3 generation) were tested again for maintaining abrogated luciferase activity on solid MS plates containing PQ as described above and seedlings were sampled for ribonucleic acid (RNA) extraction. The endogenous expression level of zat12 transcript was examined in RNA blots. Paraquat-induced oxidative stress experiments for RNA blot were repeated at least twice. Mutants confirmed for displaying a reduced level of zat12 expression were further selected for backcrossing. Overall, 26 000 M2 seeds were screened for luciferase activity on MS plates containing PQ. Out of the 442 mutants that were selected, 148 were verified for displaying weak or no luciferase expression. So far, 100 lines have been inspected for endogenous zat12 expression, out of which 15 lines showing weak zat12 expression were picked for backcrossing with the non-mutated Zat12::Luc transgenic line. The remaining 48 lines are now being screened in RNA blots and we expect an additional 10 lines to proceed for backcrossing. Currently, the first selected 15 lines are growing and will be soon go through the first of two backcrosses.
In parallel to the mutant selection process, the zat12::Luc transgenic Arabidosis (Colombia background) was backcrossed six times to a WT Len ecotype. This Arabidopsis cv. Len containing the Zat12:Luc cassette will be used for the final backcross with chosen mutants that have been genetically 'cleaned up' by the two backcrosses described above.
2. Reactive oxygen species mediate a rapid long-distance self-propagating signal in plants
Cell-to-cell communication and long distance signalling play a key role in the response of multicellular organisms to pathogens, pests, mechanical wounding and extreme environmental conditions 1-4. Here, we uncovered a rapid systemic signal in plants that travels at a rate of 8.4 cm min-1 and is dependent on the presence of the respiratory burst oxidase homolog D (RbohD) gene. Signal propagation is accompanied by the accumulation of reactive oxygen species (ROS) in the extracellular spaces between cells, and by rapid expression of ROS-responsive transcripts. Once initiated, the signal can be blocked by the suppression of ROS accumulation at locations distant from the initiation site. Rapid systemic signalling is independent on ethylene, jasmonic acid (JA) or salicylic acid (SA) signalling, but can be triggered by wounding, heat, cold, high light and salinity stresses. Our results reveal a profound and general role played by ROS in mediating rapid, self-propagating systemic signals in plants, and raise the possibility that a similar class of ROS-related signals function in other organisms' response to external stimuli.
3. MitoNEET, a protein with a potential function in regulating iron and ROS
The grant directly supported a graduate student (Yael Harir) and two undergraduate students (Dan Roizman-Sade and Shlumit Kangiser) who worked at the lab in the Hebrew University of Jerusalem. We have been focusing on one ROS-response gene that we have identified as a gene of unknown function with homology to a human gene (mitoNEET), which is thought to be involved in iron cluster transfer across the mitochondrial membrane and was shown in humans to bind a key anti-diabetic drug (Pioglitazone, a member of the thiazolidinedione, TZD, class). The Arabidopsis gene (At5g51720) with unknown function shows sequence homology to the human gene. Moreover, structural mapping of the Arabidopsis protein on the solved structure of the human protein demonstrates a high degree of structural similarity. To test the function of At5g51720 and human mitoNEET, we have expressed these proteins in transgenic plants with and without the addition of a GFP tag. Human mitoNEET is localised to mitochondria and At5g51720 is localised to chloroplasts. These results are in agreement with previous findings that iron cluster biogenesis in plants can occur via two different pathways: one in the mitochondria and one in the chloroplast, and suggest that At5g51720 is involved in the chloroplastic pathway.
We are currently conducting biochemical analysis of At5g51720, generating RNAi lines for it and testing the tolerance of At5g51720 mutants and over-expressors to different abiotic stresses and iron deficiency.
4. Biochemical characterisation: Cluster (2Fe-2S) transfer activity
Two methods were used in order to determine the 2Fe-2S cluster transfer ability of At5g51720 to an apo-acceptor protein, apo-mFd; a biochemical determination using native gels and following changes by absorbance spectroscopy. The At5g51720 absorbance peaks at 458nm and holo-mFd absorbance peaks at 423nm.
The At5g51720 protein was purified after over-expression in bacterial cells and incubated with colourless apo-acceptor protein (apo-mFd); the absorption spectra were recorded at different time points along the incubation period.
In the presence of 5mM MgCl2 in the incubation media, after four hours of incubation, the non-existing absorbance of the apo-mFd is changing into an absorbance almost identical to the absorbance of the holo-mFd.
The absorbance changes strongly suggest that the 2Fe-2S cluster of At-mitoNEET was transferred to the apo-mFd to create the holo-mFd. In order to prove this hypothesis, samples obtained during the incubation, were loaded on non-denaturing gel. Ten minutes after incubating At-mitoNEET with apo-mFd, a red band at the position of holo-mFd was detected.
We plan to devote the upcoming year of the research to identifying the protein(s) partner(s) of At-mitoNEET with whom it associates to perform its biological function.