Progress 10/01/06 to 06/29/11
Outputs
OUTPUTS: The general objective of the project was to relate the effects of environmental stress on plants to oxidative damage to cellular components, including proteins or nucleic acids. Work focused on chilling and water stress as they relate to oxidative stress. In conjunction with colleagues in Taiwan, we had earlier reported that a pretreatment with a hydrogen peroxide spray could induce resistance to chilling injury in mung bean (Vigna radiata (L.) R. Wilczek) seedlings. In this project, we tested the hypothesis that chilling injury involved oxidation, with the implication that the effect of hydrogen peroxide involved induction of anti-oxidants. We found that chilling injury to mung bean seedlings was related to their nutritional state i.e, concentration of nutrients in the soil. Chilling-induced wilting could be alleviated by eluting solutes from the soil. We further found that chilling-induced wilting seemed to be caused by decreased hydraulic conductivity in chilled roots in the presence of moderate nutrient concentrations. Hydraulic conductivity was presumably normal in chilled roots when the concentration of solutes was reduced. Finally, the presence of some non-chilled organ in a plant (either root or shoot) alleviated any wilting when the other organ was chilled. These findings were difficult to relate to direct action of reactive oxygen species on root membranes, since (1) the general effect of these agents on membrane lipids would be expected to increase, rather than decrease, membrane hydraulic conductivity; (2) it is not clear why reducing the concentration of solutes in soil would affect oxidative damage to membrane lipids; and (3) they do not explain the ameliorative effect of warming shoots on root conductivity. It is also difficult to relate the findings to direct oxidation of shoot membranes, since in that case it is not clear in that case why warming the roots would reduce wilting. The alternative hypothesis on the role of reactive oxygen species posits that these species act as signaling agents. Rather than causing damage to membranes or DNA, they stimulate cell defenses against damage (Yu et al., Funct. Plant Biol., 29:1-7, 2002; Funct Plant Biol., 30:1-3, 2003), and this may be true even if the "damage" involves low hydraulic conductivity in chilled roots. In a related experiment, our colleagues provided tobacco plants engineered to form higher endogenous concentrations of hydrogen peroxide through an introduced gene for glucose oxidase (Marutasalem et al., Plant Cell Rep (2010) 29:1035-1048). We tested these plants for sensitivity to UV radiation in the expectation that the resistance generated by exposure to hydrogen peroxide would also help protect against UV wavelengths that generate reactive oxygen radicals. However, no increase in resistance was observed. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The observation that chilling stress in seedlings of mung beans, and potentially other crops, may be alleviated by control of water stress (e.g. reducing fertilizer concentrations) may have agricultural implications, particularly for locations with generally mild climates but occasional cold snaps. This may not apply to California directly, since mung beans are usually used for bean sprouts and grown dark in controlled environments. However, in Taiwan, mung beans are an outdoor crop and sites at high elevations may have temperatures as low as 3oC. In the midwest of the U.S. where mung beans are also field-grown, late cold snaps could damage a crop. Other alleviation methods include preheating (40oC for 3 hr: Collins et al., Int. J. Exp. Bot. 46:795-802, 1995), but such a treatment is not appropriate for a field crop. Reports that mung beans are sensitive to salts (sodium and potassium chlorides and sulfates) may be related to these results, suggesting that calcium treatments, known to alleviate salt sensitivity, may have an effect on chilling sensitivity also.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The general objective of the project is to explain the effects of oxidative stress on plant cells, including their genetic material. Recent work has focused on the mechanisms by which environmental conditions lead to oxidative stress. In the last two years, we reported that injury to mung bean (Vigna radiata (L.) R. Wilczek) seedlings by chilling, an effect which we and our colleagues in Taiwan have attributed to the production of reactive oxygen species, is related to their nutritional state. Furthermore, cold-induced wilting seems to be caused by low hydraulic conductivity in chilled roots in the presence of moderate nutrient concentrations. This latter finding is more difficult to relate to the action of reactive oxygen species--superoxide, hydroxyl radical, or hydrogen peroxide-- on membranes, since the general effect of these agents on membrane lipids (or on the integrity of DNA and gene expression) would be expected to increase, rather than decrease, membrane hydraulic conductivity. An alternative hypothesis on the role of reactive oxygen species posits that these species act as signaling agents that, rather than causing damage to membranes or DNA, stimulate cell defenses against such damage (Yu et al., Funct. Plant Biol., 29:1-7, 2002; Funct Plant Biol., 30:1-3, 2003). We are currently testing stress sensitivity of plants engineered to form higher endogenous concentrations of hydrogen peroxide through an introduced gene for glucose oxidase. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The observation that chilling stress in seedlings of mung beans, and potentially other crops, may be alleviated by control of water stress (e.g. reducing fertilizer concentrations) may have agricultural implications, particularly for locations with generally mild climates but occasional cold snaps. This may not apply to California directly, since mung beans are usually used for bean sprouts and grown dark in controlled environments. However, in Taiwan, mung beans are an outdoor crop and sites at high elevations may have temperatures as low as 3oC. In the midwest of the U.S. where mung beans are also field-grown, late cold snaps could damage a crop. Other alleviation methods include preheating (40oC for 3 hr: Collins et al., Int. J. Exp. Bot. 46:795-802, 1995), but such a treatment is not appropriate for a field crop. Reports that mung beans are sensitive to salts (sodium and potassium chlorides and sulfates) may be related to these results, suggesting that calcium treatments, known to alleviate salt sensitivity, may have an effect on chilling sensitivity also.
Publications
- Murphy TM, Guo Y-Y. 2010 Antimutagenic specificities of two plant glycosylases, oxoguanine glycosylase and formamidopyrimidine glycosylase, assayed in vitro. Biochemical and Biophysical Research Communications 392:335-339.
- Ben-Yehuda N, Murphy TM. 2010. Rope from the Christmas Cave: In search of the Talmudic hemp. Archaeological Textiles Newsletter 50: 14-19.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The general objective of the project is to explain the effects of oxidative stress on plant cells, including their genetic material. Recent work has focused on the mechanisms by which environmental conditions lead to oxidative stress. Last year, we reported that injury to mung bean (Vigna radiata (L.) R. Wilczek) seedlings by chilling, an effect which we and our colleagues in Taiwan have attributed to the production of reactive oxygen species, is related to their nutritional state. We hypothesized that the wilting - a clear sign of water stress-is caused by a combination of factors that reduce water potential in the apoplast. These could include a release of solutes from leaf cells into the apoplast (through oxidative damage) or increased uptake of solutes by roots (with reduced incorporation into leaf cells). This year we tested these two factors by separately chilling shoots (keeping roots warm) and chilling roots (keeping shoots warm), then measuring xylem hydrostatic tension and the concentration of osmotic solutes in pressure-expressed xylem solution. Our hypothesis predicted an increase in solute concentration of the xylem solution when shoots or roots were chilled. When whole plants were chilled for 3 days, there was an increased tension (more negative water potential) in the xylem, as measured 7 days later. When shoots alone were chilled for 1, 2, or 3 days, there was little increase in xylem tension. When roots of nutrient-grown plants were chilled for 1 or 2 days, there was almost as much increase in xylem tension as with whole-plant chilling, although this was alleviated by 3 days. Roots of water-eluted plants had increased levels of xylem tension, independent of chilling. This suggests that plants in water-eluted soil, previously shown to be less susceptible to chilling, were less susceptible because they had adapted to increased xylem tension. Concentrations of solute measured in xylem solution did not vary when shoots were chilled, but were reduced when nutrient-grown plants were chilled (although not when roots were chilled) and were reduced in water-eluted plants. These results did not confirm our hypothesis that wilting results from high solute concentration in the apoplast. Instead, wilting seems to be caused by low hydraulic conductivity in chilled roots in the presence of moderate nutrient concentrations. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The observation that chilling stress in seedlings of mung beans, and potentially other crops, may be alleviated by control of water stress (e.g. reducing fertilizer concentrations) may have agricultural implications, particularly for locations with generally mild climates but occasional cold snaps. This may not apply to California directly, since mung beans are usually used for bean sprouts and grown dark in controlled environments. However, in Taiwan, mung beans are an outdoor crop and sites at high elevations may have temperatures as low as 3oC. In the midwest of the U.S. where mung beans are also field-grown, late cold snaps could damage a crop. Other alleviation methods include preheating (40oC for 3 hr: Collins et al., Int. J. Exp. Bot. 46:795-802, 1995), but such a treatment is not appropriate for a field crop. Reports that mung beans are sensitive to salts (sodium and potassium chlorides and sulfates) may be related to these results, suggesting that calcium treatments, known to alleviate salt sensitivity, may have an effect on chilling sensitivity also.
Publications
- Murphy TM, Guo Y-Y. 2010. Antimutagenic specificities of two plant glycosylases oxoguanine glycoylase and formamidopyrimidine glycosylase, assayed in vivo. Biochem Biophys Res Commundoi:10.1016/j.bbrc.2010.01.020 (in press)
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The general objective of the project is to explain the effects of oxidative stress on plant cells, including their genetic material. This year, we followed up the observation made last year that the injury to mung bean (Vigna radiata (L.) R. Wilczek) seedlings by chilling, an effect which we and our colleagues in Taiwan have attributed to the production of reactive oxygen species, is related to their nutritional state. In a series of experiments, we found that the injury is strongly correlated with wilting during and after chilling. We tested the preliminary observation that the wilting can be alleviated in two ways-by eluting solutes from the soil and by keeping the chilled plants in high humidity. Four replicate experiments each tested the effects of chilling 1, 2, or 3 days at 5 degrees C and compared TN5 vs V3327 varieties, open vs closed containers (lower vs high humidity), and nutrient status (provided with nutrient medium vs leached 3-4 times with distilled water). As expected, there was a strong regression between the duration of chilling and damage (corr. coef. = 0.73); and there was a strong correlation between wilting, measured the day after the cessation of chilling, and the lack of ability to begin regrowth (P less than 0.001). Surprisingly, there was no difference between the two varieties (P less than 0.15, NS), although V3327 has been described as chilling resistant. The most striking effects involved water status, with plants in closed humid chambers surviving better (P less than 0.04) and plants in soil leached with distilled water surviving better (P less than 0.001). We hypothesize that the wilting-a clear sign of water stress-is caused by a combination of factors that reduce water potential in the apoplast-release of solutes into the apoplast (through oxidative damage), uptake of solutes into the xylem and apoplast, and water potential of the air. We intend to conduct a series of experiments to determine whether water stress is a cause or a result of oxidative stress. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The observation that alleviating chilling stress in seedlings of mung beans, and potentially other crops, by control of water stress (e.g. reducing fertilizer concentrations) may have agricultural implications, particularly for locations with generally mild climates but occasional cold snaps. This may not apply to California directly, since mung beans are usually used for bean sprouts and grown dark in controlled environments. However, in Taiwan, mung beans are an outdoor crop and sites at high elevations may have temperatures as low as 3 degrees C. In the midwest of the U.S. where mung beans are also field-grown, late cold snaps could damage a crop. Other alleviation methods include preheating (40 degrees C for 3-6 hr: Chucheep et al., Int. J. Food Agr. Env. 3:110-116, 2005), but it is not clear that such a treatment could be applied to a field crop. Reports that mung beans are sensitive to salts (sodium and potassium chlorides and sulfates) may be related to these results, suggesting that calcium treatments, known to alleviate salt sensitivity, may have an effect on chilling sensitivity also.
Publications
- Dunbar, M., Murphy, T.M. 2009. DNA analysis of natural fiber rope. Journal of Forensic Sciences 54:108-113.
- Murphy TM, Belmonte M, Shu S, Britt AB, Hatteroth J. 2009. Requirement for Abasic Endonuclease Gene Homologues in Arabidopsis Seed Development. PLoS ONE 4(1): e4297 doi:10.1371/journal.pone.0004297
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Progress 01/01/07 to 12/31/07
Outputs
The general objective of the project is to explain the conditions under which enzymes for the repair of oxidative stress to DNA are needed by plants and explain the need for multiple types of enzymes. This year, we completed the use of Escherichia coli as a model system for measuring DNA repair activities of Arabidopsis formamidopyrimidine glycosylase (FPG) and oxoguanine glycosylase (OGG), as described in last year's report. Working with strains that signal A->C, G->A, G->C, G->T, A->T, and A->G mutations (as well as their complements, that is, T->G for A->C), we constructed strains in which the E. coli Fpg gene was replaced separately by cDNAs of two alternative splicing variants of the Arabidopsis Fpg gene or by the Arabidopsis Ogg gene. We then compared mutation rates in the various strains. Both FPG-1 (major splicing variant) and OGG reduced the rate of G->T mutations, consistent with their activities in other organisms and the in-vitro activities of the extracted
and purified Arabidopsis enzymes. OGG also decreased the rate of A->C mutations, suggesting that it has an unusual ability to remove the product(s) of A base oxidation. Surprisingly, cells with FPG-1 showed an increased rate of A->C mutations. However, FPG-1 decreased the rate of A->T mutations. Finally, FPG-2 (minor splicing variant) showed no effect on the mutation rate of any strain. In a second project, we have begun to re-evaluate the injury of mung bean seedlings by chilling, an effect which we and our colleagues in Taiwan have attributed to the production of reactive oxygen species. In a series of experiments, we have found that the injury is strongly correlated with wilting during and after chilling. The wilting can be alleviated in two ways--by eluting solutes from the soil, and by keeping the chilled plants in high humidity. We hypothesize that the wilting-a clear sign of water stress-is caused by a combination of factors that reduce water potential in the apoplast-release
of solutes into the apoplast (through oxidative damage), uptake of solutes into the xylem and apoplast, and water potential of the air. We intend to conduct a series of experiments to determine whether oxidative stress is a cause or result of water stress.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. Reactive oxygen, by oxidizing guanines in DNA, leads to G->T base substitution mutations. Both heat and cold, as well as high salt, drought, heavy metals, chilling, and challenge by pathogens, stimulate the formation of reactive oxygen. An increase in repair activity may reduce the sensitivity of plants to stresses that produce reactive oxygen. Conversely, a reduction in repair activity may accelerate mutagenesis, useful in connection with a selection procedure such as employed in the TILLING project for obtaining changes in specific genes. The observation that alleviating chilling stress in mung beans, and potentially other crops, by control of water stress (e.g. reducing fertilizer concentrations) may have agricultural implications.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The general objective of the project is to explain the conditions under which enzymes for the repair of oxidative stress to DNA are needed by plants and explain the need for multiple types of enzymes. This year, we expanded the use of Escherichia coli as a model system for measuring DNA repair activities of Arabidopsis formamidopyrimidine glycosylase (FPG) and oxoguanine glycosylase (OGG)(Shu et al., 2006). We had previously concentrated on the E. coli strain that specifically indicated G->T mutations. We are now working with strains that signal A->C, G->A, G->C, A->T, and A->G mutations (as well as their complements, that is, T->G for A->C). We have constructed strains in which the E. coli Fpg gene is replaced separately by cDNAs of three alternative splicing variants of the Arabidopsis Fpg gene or by the Arabidopsis Ogg gene. The comparisons of mutation rates in the various strains are on-going. We also have identified strains of Arabidopsis that lack various
combinations of the three abasic endonuclease genes, Ape1L, Ape2, and Arp, genes that code for the enzymes that function after FPG and OGG in the base excision repair pathway. The loss of any one gene has no apparent effect on the plant. The loss of both Ape1L and Arp or of both Ape2 and Arp also has no effect. However, the loss of both Ape1L and Ape2 is lethal, apparently in an early stage of seed development. Because the lethality is apparently absolute--no homozygous double mutants for Ape1L and Ape2 have been found--it seems likely that the function of the enzymes coded by these genes goes beyond the repair of randomly damaged bases. Choi et al. (Cell 110:33, 2002) have provided evidence that another potential DNA glycosylase, MEDEA, is required for seed development. However, MEDEA functions in the female gametophyte, whereas Ape1L and Ape2 appear to function in the young diploid sporophyte. We are currently attempting to identify the particular stage at which embryogenesis is
blocked in the double mutants.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. An increase in repair activity may reduce the sensitivity of plants to stresses that produce reactive oxygen. Both heat and cold, especially cold in the presence of high light intensity, as well as high salt, drought, heavy metals, chilling, and challenge by pathogens, stimulate the formation of reactive oxygen. Reactive oxygen, by oxidizing guanines in DNA, leads to G->T base substitution mutations. A reduction in repair activity may accelerate mutagenesis, useful in connection with a selection procedure such as employed in the TILLING project for obtaining changes in specific genes.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The objectives of the project are to confirm the expectation that there are multiple enzymes in Arabidopsis that have similar specificities and activities for removing oxidized purines from DNA and to explain why such multiple enzymes are needed by the plant. Last year, we described the use of Escherichia coli as a model system for measuring the rate of formation and removal of oxoguanine (Go)(Shu et al., in press): the oxidation of a particular guanine in the lac gene results in a G -> T mutation, leading to the recovery of activity of the protein product, B-galactosidase. The removal of Go by formamidopyrimidine-DNA glycosylase (FPG) and the subsequent "base excision repair" process blocks the mutation. We have now constructed strains in which the E. coli fpg gene is replaced by cDNAs of three alternative splicing variants of the Arabidopsis fpg gene. On the basis of previously reported in vitro assays, we predicted that AtFPG-1 would have full activity, and
AtFPG-2, which lacks a C-terminal "clamp" sequence, would have considerably less activity. AtFPG-3, which lacks a putative active-site amino acid, we predicted would have no activity. In vivo in E. coli, we find that both AtFPG-1 and AtFPG-2 have strong and approximately equal ability to repress G -> T mutations, although they both have distinctly less activity than the E. coli FPG. The presence of AtFPG-3 results in more mutations than in control, which we interpret as meaning that AtFPG-3 lacks catalytic activity, but binds to Go sites in a way that interferes with other repair processes. The presence of the AtFPG-3 splicing variant would seem to promote mutations. We are considering the possibility that this might in fact be its function and be related to the reproductive history of Arabidopsis. We are currently analyzing the structure and expression of fpg genes in rice (Oriza sativa). Unlike Arabidopsis, rice has two fpg genes, both of which are expressed. Also unlike
Arabidopsis, rice has little or no alternative splicing of the RNA transcripts of these genes. We hypothesize that the difference is related to the number and size of seeds produced by a single plant. An Arabidopsis plant produces a large number of very small seeds very rapidly: selection may allow it to tolerate or even derive advantage from mutations. Rice produces fewer, larger seeds: the greater investment per seed may place a disadvantage on mutations. Current work in our laboratory is attempting to determine the localization of FPG and oxoguanine glycosylase (OGG) in Arabidopsis to test the possibility that different enzymes act on the DNA of different organelles. We are also analyzing the activities of the FPG enzymes from the two rice fpg gene and the three Arabidopsis abasic endonucleases (the enzymes after FPG and OGG in the base excision repair pathway). We continue to test mutant Arabidopsis that lack fpg and ogg genes for their susceptibility to stressful conditions that
are thought to produce reactive oxygen and promote the formation of Go.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. An increase in repair activity may reduce the sensitivity of plants to stresses that produce reactive oxygen. Both heat and cold, especially cold in the presence of high light intensity, as well as high salt, drought, heavy metals, chilling, and challenge by pathogens, stimulate the formation of reactive oxygen. A reduction in repair activity may accelerate mutagenesis, useful in connection with a selection procedure such as employed in the TILLING project for obtaining changes in specific genes.
Publications
- MURPHY, T.M., GEORGE A. 2005. A comparison of two DNA base excision repair glycosylases from Arabidopsis thaliana. Biochemical and Biophysical Research Communications 329:869-872.
- SHU J., SCHELLHORN, H.E., MURPHY, T.M. 2005. Stationary phase-induction of G --> T mutations in Escherichia coli. Mutation Research (in press).
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Progress 01/01/04 to 12/31/04
Outputs
The objectives of the project are to confirm the expectation that there are multiple enzymes in Arabidopsis that have similar specificities and activities for removing oxidized purines from DNA and to explain why such multiple enzymes are needed by the plant. Last year, we reported a comparison of the in vitro activities of the products, oxoguanine glycosylase (OGG) and formamidopyrimidine glycosylase (FPG). This year we focused on testing the importance of the enzymes in vivo. We had confirmed the lack of sensitivity of homozygous Ds insertion mutants of the Arabidopsis gene for OGG to a variety of conditions that were expected to increase the levels of reactive oxygen in plant cells: high visible light together with cold, UV-A and UV-B irradiation, and treatments with methyl viologen. We also identified T-DNA insertion mutants of the genes for FPG and obtained homozygous progeny: tests with the homozygous mutants showed that they also were no more sensitive than
wild-type to UV-A and UV-B treatments. We crossed the homozygous plants and from the F2 progeny isolated plants that are homozygous with respect to both genes: these double homozygous mutants also showed no more sensitivity than wild type in growth, development, or reproductive potential under either normal conditions or conditions known to induce the formation of reactive oxygen, including UV-A irradiation, spraying with a solution of H2O2, and gamma irradiation. It is possible that other DNA repair enzymes can substitute for OGG and FPG. Alternatively it is possible that oxidative damage to DNA in the germline of plants, and thus the importance of OGG and FPG, occurs at a very specific period in a plant's life cycle or under very specific conditions, ones that we have not yet tested in our stress treatments. To help us focus on the this latter possibility, we have turned to the model organism, Escherichia coli, to determine conditions that favor mutations involving oxidation of
guanine in DNA. Using strains that allow us to measure the rates of specific base substitution mutations, and specifically G -> T transversions, we have found that such mutations in the gene for B-galactosidase occur most frequently in the presence of lactose and in minimal media containing low concentrations of glucose. Complex media, high concentrations of glucose, and high concentrations of oxygen repress the incidence of these mutations. Repair of the mutations occurs in the non-growing population of bacteria. We are now in the process of searching for and developing systems that allow us to apply this information to Arabidopsis.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. An increase in repair activity may reduce the sensitivity of plants to stresses that produce reactive oxygen. Both heat and cold, especially cold in the presence of high light intensity, as well as high salt, drought, heavy metals, chilling, and challenge by pathogens, stimulate the formation of ROS. A reduction in repair activity may accelerate mutagenesis, useful in connection with a selection procedure such as employed in the TILLING project for obtaining changes in specific genes.
Publications
- BERRY, A.M., T.M. MURPHY, P.A. OKUBARA, K.R. JACOBSEN, S. SWENSEN AND K. PAWLOWSKI. 2004. Novel expression pattern of cytosolic glutamine synthetase in nitrogen-fixing root nodules of the actinorhizal host, Datisca glomerata. Plant Physiology 135:1849-1862.
- KRASNOW MN, MURPHY TM 2004. Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera) Journal of Agricultural And Food Chemistry 52:3467-3472.
- MURPHY, T.M. 2004. What's base excision repair good for?: Knockout mutants for FPG and OGG glycosylase genes in Arabidopsis. Physiologia Plantarum (in press).
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Progress 01/01/03 to 12/31/03
Outputs
The objectives of the project are to confirm the expectation that there are multiple enzymes in Arabidopsis that have similar specificities and activities for removing oxidized purines from DNA and to explain why such multiple enzymes are needed by the plant. The procedures involve in vitro assays of the various enzymes and analysis of knock-out mutants lacking these enzymes. We have obtained oxoguanine glycosylase (OGG) and formamidopyrimidine DNA glycosylase (FPG-1) by expressing Arabidopsis cDNAs in E. coli. We have compared these proteins and FPG from E. coli in three different assays. Using as an assay the opening of supercoiled, depurinated plasmids, we have shown that the relative specific activities of Arabidopsis OGG, Arabidopsis FPG-1, and E. coli FPG are approximately 1:200:40, respectively. With plasmids treated with methylene blue and light to form oxoguanine, the relative specific activities were 1:1:60. Using as an assay the cleavage of an end-labeled
oligonucleotide containing a single oxoguanine, the highest specific activity was observed with Arabidopsis OGG (45 pmol cleaved 2.5 pmol of substrate); less activity was seen with Arabidopsis FPG-1 (1 pmol cleaved 1 fmol) and E. coli FPG (0.07 pmol cleaved 1 fmol), giving relative activities of 55:1:15. The three assays demonstrate that each enzyme has a unique substrate preference based on the altered site and/or the general physical properties of the nucleic acid. We have confirmed the lack of sensitivity of homozygous Ds insertion mutants of the Arabidopsis gene for OGG to a variety of conditions that are expected to increase the levels of reactive oxygen in plant cells: high visible light together with cold, UV-A and UV-B irradiation, and treatments with methyl viologen. We have also identified T-DNA insertion mutants of the genes for FPG and obtained homozygous progeny. Initial tests with the homozygous mutants show that they are no more sensitive than wild-type to UV-A and UV-B
treatments. In both cases, RT-PCR confirmed the lack of cDNA from the knocked-out gene. We have crossed the homozygous plants and from the F2 progeny isolated plants that are homozygous with respect to both genes. During the next year, we expect to confirm the lack of corresponding cDNAs and enzymes in the double homozygous mutant and test its phenotype under stress.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. An increase in repair activity may reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. A reduction in repair activity may accelerate mutagenesis, useful in connection with a selection procedure such as employed in the TILLING project for obtaining changes in specific genes.
Publications
- YU, C.-W., T.M. MURPHY, AND C.-H LIN. 2003. Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Functional Plant Biology 30:955-963.
- MURPHY, T.M. (2003). In vitro culture systems: their use for physiological and biochemical investigations and for production of secondary metabolites. In: Encyclopedia of Rose Science (A. ROBERTS, S. GUDIN, T. DEBENER, eds.) Academic Press, London
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Progress 01/01/02 to 12/31/02
Outputs
The objectives of the project are to confirm the expectation that there are multiple enzymes in Arabidopsis that have similar specificities and activities for removing oxidized purines from DNA and to explain why such multiple enzymes are needed by the plant. The procedures involve in vitro assays of the various enzymes and analysis of knock-out mutants lacking these enzymes. We have inserted the cDNA for oxoguanine glycosylase (OGG) from Arabidopsis into a bacterial expression vector (pET28c), transformed E. coli, and confirmed that the protein produced by the expression of this gene is active in cleaving depurinated supercoiled plasmid DNA in vitro. We are comparing this protein to expression products of the Arabidopsis gene for an analogous enzyme, formamidopyrimidine DNA glycosylase (FPG-1). Using the depurinated plasmid assay, we have investigated a suggestion that the tight binding of OGG (and FPG-1) to DNA reduces turnover so that cleavage is stoichiometric,
rather than enzymatic (i.e., no turnover under the conditions of the assay). Temperature-dependence curves show that the activation energy for the cleavage of depurinated DNA is ca 16 kcal/mol for OGG and 20 kcal/mol for FPG-1, higher than a typical enzyme reaction and possibly indicative of a low release rate of the enzymes from the DNA. A partially degraded preparation of FPG-1 has a lower activation energy (15 kcal/mol) and ten-fold higher specific activity. On the other hand, gel retardation experiments show binding of plasmid DNA only with very high amounts of FPG-1. More extensive analyses of purified OGG and FPG are in progress. We have identified knock-out mutants of the Arabidopsis gene for OGG and isolated homozygous mutant and wild type strains from the original heterozygote. We have not been able to identify any phenotypic differences between the mutants and wild types under a variety of conditions that are expected to increase the levels of reactive oxygen in plant cells:
high visible light together with cold, UV-A and UV-B irradiation, and treatments with methyl viologen. However, assays of their DNAs indicate that mutants accumulate a slightly higher number of FPG-reactive sites (probably oxoguanines) than wild types under normal growing conditions. We are currently testing the DNA from plants subjected to stresses. We have also identified T-DNA insertion mutants of the genes for FPG and a putative abasic endonuclease, identified by its partial sequence similarity to human Ape-1. We have isolated plants homozygous with respect to each gene. During the next year, we expect to confirm the lack of corresponding enzymes in these plants and test their phenotypes under stress.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. It is conceivable that an increase in repair activity could reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. Alternatively, a reduction in repair activity might allow mutagenesis, useful in genetic selections, with less toxic treatments.
Publications
- YU, C.-W., T.M. MURPHY, W.-W. SUNG AND C.-H LIN. 2002. H202 [Hydrogen peroxide] treatment induces glutathione accumulation and chilling tolerance in mung bean. Functional Plant Biology 29:1-7.
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Progress 01/01/01 to 12/31/01
Outputs
This year we continued our work on the repair of oxidative damage to purines in plant DNA. In plants, reactive oxygen species, including superoxide, hydrogen peroxide, and hydroxyl radical, are produced photochemically and metabolically. If mechanisms to remove these compounds fail, DNA bases may be oxidized, producing potentially mutagenic or fatal lesions. In bacteria, formamidopyrimidine-DNA glycosylase (FPG) removes oxidized A and G from deoxyribose, initiating the replacement of the damaged base with the complement to the corresponding base on the undamaged DNA chain. In yeast and human cells, an analogous enzyme, oxoguanine glycosylase (OGG), takes the place of FPG. Plants are the only organisms known to have both FPG and OGG genes. We previously reported the cloning of seven variant cDNAs for FPG from Arabidopsis thaliana, representing the products of alternative splicing of a common transcript, and one OGG cDNA. We hypothesize that these different proteins may
be localized in different locations in the cells, have different preferences for oxidized substrates, and/or recruit different proteins that guide the subsequent steps of base excision repair. We have confirmed that the gene products from both of the original FPG cDNAs, as expressed in E. coli, show anti-mutagenic activity, reducing the rate of reversion of a mutant lac gene through G to T base change. We have constructed mutant glucuronidase (uidA) genes, transfected them into Arabidopsis, and are developing them for use to demonstrate the anti-mutagenic activity of FPGs and OGG in plants. We have constructed vectors that will allow us to produce purified OGG for in vitro assays and to test OGG for anti-mutagenic activity in E. coli allowing a comparison of this enzyme to FPG.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. It is conceivable that an increase in repair activity could reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. Alternatively, a reduction in repair activity might allow mutagenesis, useful in genetic selections, with less toxic treatments.
Publications
- GAO, M.-J. AND T.M. MURPHY. 2001. Alternative forms of formamidopyrimidine-DNA glycosylase from Arabidopsis thaliana. Photochemistry and Photobiology 73:128-134.
- MURPHY, T.M. AND M.-J. GAO. 2000. Multiple forms of formamidopyrimidine-DNA glycosylase produced by alternative splicing in Arabidopsis thaliana. Journal of Photochemistry and Photobiology B: Biology 61:87-93.
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Progress 01/01/00 to 12/31/00
Outputs
This year we continued our work on enzymes responsble for the repair of oxidative damage to plant DNA. In plants, reactive oxygen species, including superoxide, hydrogen peroxide, and hydroxyl radical, are produced photochemically and metabolically under a variety of conditions. If mechanisms to remove these compounds fail, DNA bases may be oxidized, producing potentially mutagenic or fatal lesions. In bacteria, formamidopyrimidine-DNA glycosylase (FPG) removes oxidized A and G from deoxyribose, initiating the replacement of the damaged base with the complement to the corresponding base on the undamaged DNA chain. In yeast and human cells, an analogous enzyme, oxoguanine glycosylase (OGG), takes the place of FPG. Plants are the only organisms known to have both FPG and OGG genes. We previously reported the cloning of two variant cDNAs for FPG from Arabidopsis thaliana, representing the products of alternative splicing of a common transcript. Through a second search of
the cDNA library and the use of reverse transcriptase-polymerase chain reaction, we have now identified six additional alternative splicing variant cDNAs. These code for up to seven distinct proteins, which have identical N-terminal, but different C-terminal, amino-acid sequences. We also have obtained a clone for an OGG cDNA. We hypothesize that these different proteins may be localized in different locations in the cells, may have different preferences for oxidized substrates, and/or may recruit different proteins that guide the subsequent steps of base excision repair. We have confirmed that the gene products from both of the original cDNAs, as expressed in E. coli, have abasic endonuclease activity and both cleave oxidized DNA; however, only one specifically recognizes the oxidized purine 8-oxo-G in a DNA oligonucleotide. We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. It is
conceivable that an increase in repair activity could reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. Alternatively, a reduction in repair activity might allow mutagenesis, useful in genetic selections, with less toxic treatments.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop cpecies. It is conceivable that an increase in repair activity could reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. Alternatively, a reduction in repair activity might allow mutagenesis, useful in genetic selections, with less toxic treatments.
Publications
- Murphy, T.M., H.D. Vu, T. Nguyen AND C.H. WOO. 2000. Diphenylene iodonium sensitivity of a solubilized membrane enzyme from rose cells. Protoplasma 213:228-234.
- Gao, M.J. AND T.M. Murphy. (in press) Alternative forms of formamidopyrimidine-DNA glycosylase from Arabidopsis thaliana. Photochemistry and Photobiology.2000
- Murphy, T.M. AND M.J. Gao. 2000. Multiple forms of formamidopyrimidine-DNA glycosylase produced by alternative splicing in Arabidopsis thaliana. Third Internet Photochemistry and Photobiology Conference (http://www.photobiology.com/photobiology2000/index.htm)
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Progress 01/01/99 to 12/31/99
Outputs
This year, our work turned to the repair of oxidative damage in plants: we cloned, purified, and began characterizing an enzyme from Arabidopsis thaliana that is postulated to initiate the repair of oxidized purines in DNA. In plants, reactive oxygen species, including superoxide, hydrogen peroxide, and hydroxyl radical, are produced photochemically and metabolically under a variety of conditions. If mechanisms to remove these compounds fail, DNA bases may be oxidized, producing potentially mutagenic or fatal lesions. In bacteria, formamidopyrimidine-DNA glycosylases (FPGs)remove these bases from deoxyribose, initiating the replacement of the damaged base with the complement to the corresponding base on the undamaged DNA chain. A search for DNA repair genes in plants identified two cDNAs from A. thaliana, both homologous to bacterial Fpg (MutM) genes. The two cDNAs represent the products of alternative splicing of a common transcript. They code for distinct proteins,
which have identical N-terminal, but different C-terminal, amino-acid sequences. We hypothesize that these two proteins have distinct roles in protecting plant cells from genetic damage, differing either in their enzymatic specificities or in their cell or tissue localization. We have expressed the cDNAs in E. coli, using a vector that allows their quick purification. We have confirmed previous reports that one of the gene products is highly toxic to the bacterial cells, possibly because of its high DNA-binding affinity. We have also found that the other gene product is not toxic but still retains at least one of the enzymatic activities (abasic endonuclease). We are checking its activity in other assays.
Impacts
We expect that the analysis of DNA repair processes in plants will contribute to the management of stress tolerance and mutagenesis in crop species. It is conceivable that an increase in repair activity could reduce the sensitivity of plants to stresses, like high light in cold, that produce reactive oxygen. Alternatively, a reduction in repair activity might allow mutagenesis, useful in genetic selections, with less toxic treatments.
Publications
- MURPHY, T.M., H. ASARD AND A.R. CROSS. 1998. Possible sources of reactive oxygen during the oxidative burst in plants. In: Plasma Membrane Redox Systems and their role in Biological Stress and Disease. H. Asard and A. Berczi, eds. Kluwer Academic Publishers.
- MURPHY, T.M. AND M.-J. GAO. 1998. Two cDNAs (Accession Nos. AF099970 and AF099971) encoding Arabidopsis thaliana homologs of bacterial formamidopyrimidine-DNA glycosylase genes are produced by alternative processing. Plant Physiology 118:1535.
- OKUBARA, P.A., K. PAWLOWSKI, T.M. MURPHY AND A.M. BERRY. 1999. Symbiotic root nodules of the actinorhizal plant Datisca glomerata express rubisco activase mRNA. Plant Physiology 120:411-420.
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Progress 01/01/98 to 12/31/98
Outputs
This year, our efforts to purify and characterize a plasma membrane NADH-superoxide synthase were focused on the solubilized enzyme from the phase partition step of the plasma membrane preparation (supernatant from 100,000 g centrifugation). We had found that this preparation gave the highest specific activity in the lucigenin assay, although in this assay, direct reduction of lucigenin probably contributed to the apparent synthesis of superoxide. This enzyme has the basic characteristics (stimulation by Triton X-100, Km for NADH, sensitivity to diphenylene iodonium) of the NADH-superoxide synthesizing enzyme from (unsolubilized) plasma membrane, and we suggest that the two enzymes are similar or identical, except for their association with membrane vesicles. Sensitivity to diphenylene iodonium is particularly significant, since the effect of that inhibitor on the stress-induced oxidative burst is a major basis for the hypothesis that the reactive oxygen species
produced during the oxidative burst are synthesized by a mammalian-type NADPH oxidase. Although we have not been able to obtain enough enzyme to establish its degree of purification on SDS-PAGE gels, we have obtained enough to show by fluorescence spectrometry that the major redox cofactor is not flavin (the cofactor of NADPH oxidase), but a pterin. This adds strength to the counter-hypothesis that the source of reactive oxygen in the plant oxidative burst is quite different from that in the mammalian oxidative burst. We are currently w.
Impacts
(N/A)
Publications
- BOLWELL, G.P., D.R. DAVIES, C. GERRISH, C-K. AUH AND T.M. MURPHY. 1998. Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms. Plant
- MURPHY, T.M., H.D. VU AND T. NGUYEN. 1998. The superoxide synthases of rose cells: comparison of assays. Plant Physiology 117:1301-1305.
- MURPHY, T.M., H. ASARD AND A.R. CROSS. 1998. Possible sources of reactive oxygen during the oxidative burst in plants. In: Plasma Membrane Redox Systems and their role in Biological Stress and Disease. H. Asard and A. Berczi, eds. Kluwer Academic Publishers (in
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Progress 01/01/98 to 12/01/98
Outputs
This year, our efforts to purify and characterize a plasma membrane NADH-superoxide synthase were focused on the solubilized enzyme from the phase partition step of the plasma membrane preparation (supernatant from 100,000 g centrifugation). We had found that this preparation gave the highest specific activity in the lucigenin assay, although in this assay, direct reduction of lucigenin probably contributed to the apparent synthesis of superoxide. This enzyme has the basic characteristics (stimulation by Triton X-100, Km for NADH, sensitivity to diphenylene iodonium) of the NADH-superoxide synthesizing enzyme from (unsolubilized) plasma membrane, and we suggest that the two enzymes are similar or identical, except for their association with membrane vesicles. Sensitivity to diphenylene iodonium is particularly significant, since the effect of that inhibitor on the stress-induced oxidative burst is a major basis for the hypothesis that the reactive oxygen species
produced during the oxidative burst are synthesized by a mammalian-type NADPH oxidase. Although we have not been able to obtain enough enzyme to establish its degree of purification on SDS-PAGE gels, we have obtained enough to show by fluorescence spectrometry that the major redox cofactor is not flavin (the cofactor of NADPH oxidase), but a pterin. This adds strength to the counter-hypothesis that the source of reactive oxygen in the plant oxidative burst is quite different from that in the mammalian oxidative burst. We are currently w.
Impacts
(N/A)
Publications
- BOLWELL, G.P., D.R. DAVIES, C. GERRISH, C-K. AUH and T.M. MURPHY. 1998. Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms. Plant
- MURPHY, T.M., H.D. VU and T. NGUYEN. 1998. The superoxide syntheses of rose cells: comparison of assays. Plant Physiology 117:1301-1305.
- MURPHY, T.M., H. ASARD and A.R. CROSS. 1998. Possible sources of reactive oxygen during the oxidative burst in plants. In: Plasma Membrane Redox Systems and their role in Biological Stress and
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Progress 01/01/97 to 12/01/97
Outputs
Our efforts to purify and characterize the plasma membrane NADH-superoxide synthase have been made more complex by recent reports that cast doubt on the validity of the lucigenin assay for detection of superoxide. We have measured the activity of three preparations from cultured rose cells (purified plasma membrane, cytoplasmic enzymes (supernatant from 100,000 g centrifugation), and a partially solubilized preparation from the plasma membrane purification procedure) using three different assay techniques (reduction of cytochrome c, formation of monoformazan from nitroblue tetrazolium, and lucigenin chemiluminescence). We found that each assay ascribed the highest activity to a different preparation: the cytochrome c assay to plasma membrane, the nitroblue tetrazolium assay to cytoplasmic enzymes, and the lucigenin assay to the partially solubilized plasma membrane proteins, suggesting that no two assays measured the same set of enzymes. With the plasma membrane
preparation, the presence of superoxide-dismutase insensitive cytochrome c reductase confounded attempts to use cytochrome c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the apparent synthesis of superoxide by the chemiluminescence assay. In addition, a review of the literature suggested that the oxidative burst, stimulated in plant cells by fungal elicitors, may be produced by different mechanisms in different plant/elicitor systems. This year, e.
Impacts
(N/A)
Publications
- BOLWELL, G.P., DAVIES, D.R., GERRISH, C., AUH, C-K. and MURPHY, T.M. 1998. Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms. Plant
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Progress 01/01/96 to 12/30/96
Outputs
Our efforts at the characterization of the plasma membrane NADH-superoxide synthase have progressed. Solubilizing the supernatant from the final ultracentrifugation of the final (third) PEG-dextran partition with dodecylmaltoside, we were able to obtain a single peak of activity by chromatography on a MonoQ anion exchange column. In non-ionic detergents (dodecylmaltoside or Triton X-100), the peak may be complexed with other membrane components, since it elutes in a wide, heterogenous band on Superose. However, the activity peak is correlated with the presence of one polypeptide band as visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining. Although the identity of the band is not yet known, there is a good chance that it represents the enzyme or a major component of the enzyme. This band does not show any cross-reactivity with an antiserum to the p67phox component of the mammalian NADPH oxidase, suggesting that its enzymatic
activity does not depend on the presence of a polypeptide homologous to p67phox.
Impacts
(N/A)
Publications
- MURPHY, T.M. and AUH, C.-K. 1996. The Superoxide Synthases of Rose Cell Plasma Membrane. Plant Physiology 110: 621-629.
- HU, P. and MURPHY, T.M. 1996. Ferricyanide Reductase of Rose Plasma Membranes isRegulated by Nitrogen Supply. Plant Cell Reports 15:833-835.
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