Differential metabolism of sodium azide in maize callus and germinating embryos

Mutation Research, 213 (1989) 157-163 157 Elsevier MUT 04761 Differential metabolism of sodium azide in maize callus and germinating embryos Stanton B. Dotson * and David A. Somers Department of Agronomy and Plant Genetics, Plant Molecular Genetics Institute, 411 Borlaug Hall, 1991 Buford Circle, Unioersity of Minnesota, St. Paul MN 55108 (U.S.A.) (Received 8 August 1988) (Accepted 23 January 1989) Keywords: Azide metabolism; Azidoalanine; Corn; Tissue culture; Mutant selection Summary Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos. Sodium azide is a potent seed mutagen in bar- ley (Hordeum oulgare L.) (Nilan et al., 1973), maize (Zea mays L.) (Hibberd and Green, 1978; Correspondence: Dr. D.A. Somers, Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Buford Circle, St. Paul, MN 55108 (U.S.A.). * Present address: Monsanto Company, 700 Chesterfield Parkway, St. Louis, MO 63198 (U.S.A.). Briggs and Bettendorf, 1987) and other plant species (for a review see Kleinhofs et al., 1978). It is also mutagenic in Salmonella typhimurium, Escherichia coli and yeast. Azide enters the cell as hydrazoic acid and is converted by O-acetylserine (thiol)lyase (EC 4.2.99.8) to azidoalanine (Owais et al., 1983). Azidoalanine is likely further metabolized to form a hypothetical ultimate mutagen based on differential mutagenicity of L-, D- and racemic forms of azidoalanine and the absence of 14C-labelled azidoalanine binding to 0027-5107/89/$03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Division) 158 DNA of treated barley embryos (Owais et al., 1986; Mangold and Lavelle, 1986; Veleminsky et al., 1987; Owais and Kleinhofs, 1988). Although, the actual mechanism by which mutagenesis oc- curs upon azide treatment is still under investiga- tion, it appears that azide induces single base lesions similar to those induced by ultraviolet and ionizing radiations (Sander et al., 1978; Velemins- ky et al., 1985). Variation in azidoalanine production, detoxifi- cation and the activity of DNA repair mechanisms may determine the efficacy of sodium azide muta- genesis. Supporting this view are observations that the frequency of mutations induced by azide treat- ment to seeds depends on the metabolic state of the seeds (Owais and Kleinhofs, 1988). Frequency of azide-induced barley mutants increased from 22.5% to 46.3% when seeds were presoaked 4 h in water before azide treatment (Nilan et al., 1973). Azide induction of somatic mutations in maize (Conger and Carabia, 1977) and mutations in the M 3 progeny of azide-treated kernels (Hibberd and Green, 1978) also increased when kernels were presoaked. In contrast, azide is not mutagenic in Arabidopsis (Gichner and Veleminsky, 1977), Neurospora (Landner, 1971) and Drosophila (Kamara and Gollapudi, 1979), even though cell- free extracts from these organisms synthesize azidoalanine (Rosichan et al., 1983). These results suggest that either some biological systems can detoxify the mutagenic metabolite, cannot further metabolize azidoalanine into the ultimate muta- gen, or that induced DNA lesions may be effi- ciently repaired in resistant species. Azide has advantages as a potential mutagen for inducing biochemical mutants in plant tissue cultures because it is a potent point rnutagen, is only weakly mutagenic in mammals (Arenaz and Nilan, 1981; Clive et al., 1979; Jones et al., 1980; Slamenova and Gabelova, 1980) and is non- carcinogenic (McCann and Ames, 1976; Ulland et al., 1973). Because of the high frequency of tissue culture-induced mutations in maize cultures (Lee and Phillips, 1988), azide treatments to maize callus should increase mutation frequencies at least 10-fold to meet our expectations of an effective tissue culture mutagen. The objectives of this re- search were (1) to evaluate the mutagenicity of azide in maize callus cultures using lysine plus threonine resistance (Hibberd and Green, 1982) as a model selectable marker; and (2) to compare azidoalanine metabolism in callus cultures versus in germinating maize embryos treated with sodium azide. Materials and methods Sodium azide mutagenesis and L T selection. A friable, embryogenic maize callus line was ini- tiated from an immature embryo of an advanced generation of a A188 × B73 cross and maintained on a modified N6 medium (Armstrong and Green, 1985). Samples of finely dispersed callus (20 g callus/25 ml) were mutagenized by incubation in 0.5 M citrate-buffered medium, pH 4.45, contain- ing 0.0, 0.2, 1.0 or 1.5 mM NaN 3 at 28°C for 1 h on a gyratory shaker at 150 rpm. The pH of the azide solution was increased from pH 3.0 used by Kleinhofs et al. (1978) to pH 4.45 because low pH was toxic to maize cells. Azide was removed by washing 3 times with fresh culture medium. Mutagenized tissue (0.5 g fresh wt.) was plated on a 7 cm Whatman No. 1 filter paper overlaying 50 ml of agar solidified non-selective medium (mod- ified N6 without casamino acids) in a 25 × 100 mm petri dish. To determine azide dose-response curves, cultures were incubated for 11 d and dry wt. determined. To select lysine plus threonine resistant culture lines, the filter papers with the mutagenized cells were transferred after 3 days on non-selective medium to selective medium con- taining 3 mM L-lysine and 3 mM L-threonine (LT) and no casamino acids, a lethal selection dose for the maize cell lines used. After 4 wk, growing colonies were picked from the filter papers and individually subcultured on LT medium. After an additional 6 wk on LT medium, surviving colonies were scored as LT-resistant variants. A 'Black Mexican Sweet' maize suspension cul- ture (BMS) previously initiated from stem tissue was maintained on modified MS medium (Green, 1977). BMS suspension cells were harvested at mid-log phase 5 days after sub-culture and two replicates of 5 g fresh wt. were treated with 0.2 or 0.6 mM NaN 3 as described above. After treat- ment, two sub-samples of 1.5 g cells were taken from each of the two replicates per azide treat- ment, resuspended in 80 ml MS medium in a 250-ml flask, and incubated at 150 rpm at 28°C with low light for an additional 5 days. 5-day fresh weight increases were measured and cells from each flask were plated (1.0 g fresh wt. per plate) on filter papers overlaying MS medium containing 3 mM LT. After plating on selective medium, screening for LT resistant variants was performed as with the friable, embryogenic culture. A188 × B73 and B73 × A188 F 1 kernels were treated with 1 mM NaN 3 (Briggs and Bettendorf, 1987). Kernels were presoaked for 8.5 h at 28°C in 200 ml distilled H20 in a 500-ml flask on a gyratory shaker at 160 rpm. Kernels (250 per treatment) were then treated for 1 h in 250 ml of 0.1 M phosphate buffer, pH 3.0, with 1 mM NaN 3. Following treatment, kernels were rinsed 2 times with 200 ml deionized H20. Kernels were then planted according to Briggs (1977) to check mutagenicity of the treatment. In a separate ex- periment, embryos were isolated at various time points and extracted for mutagenic metabolite as- says. Mutagenic metabolite (azidoalanine) assay. Two replicates each of friable embryogenic callus, BMS callus and A188 × B73 kernels were treated with 1.0, 1.0 and 0.6 mM NaN 3, respectively. Immediately after azide treatment, friable, em- bryogenic callus (6 g) was divided into sub-sam- ples (1 g fresh wt.) and plated on 7-cm filter papers overlaying N6 medium without casamino acids. Treated BMS callus (callus from BMS sus- pensions) was similarly plated on agar-solidified MS medium. The cultures were incubated under low light at 28 ° C. Azide-treated kernels equaling ca. 1 g of embryo tissue were placed on moistened blotting paper and incubated in the dark at 28 ° C. At 4, 20 and 40 h, samples from each azide-treat- ment replicate of each callus type and the embryos isolated from kernels were weighed and frozen at -70°C until extracted. Tissue samples (1-1.5 g fresh wt.) were extracted in 2 ml of deionized H20 at 23°C using a ground glass tissue homogenizer. Extracts were cleared by centrifugation at 17000 × g for 20 rain. Residual azide was removed from supernatants by adjusting to pH 2.0 with 0.5 ml cone. HC1 to convert azide to volatile hydrazoic acid and evaporating to dryness at 35 °C either in an Evapomix with 72 mm vacuum or in a water 159 bath under a stream of dry air (Owais et al., 1978). Dried extracts were redissolved in 1 ml conc. HC1 and evaporated to dryness under a stream of dry air. Extracted azidoalanine was dissolved in de- ionized water and adjusted to neutral pH with 10 N NaOH. Extracts were normalized to have equiv- alent volumes per fresh wt. of azide-treated tissue. Tissue extracts were used to revert histidine auxotrophic bacteria in the Ames Mutagenicity Test (Ames, 1975). For each replicated time point after azide-treatment of the different tissues two sub-replicates of 2-100/~1 of each extract, depend- ing on the mutagenicity of the extracts determined in a preliminary assay, were combined with 0.1 ml of a 10 h Salmonella typhimurium TA1530 culture and 2.5 ml of 0.5 mM histidine/biotin top agar and then poured over 30 ml minimal medium. Plates were incubated at 37°C for 48 h. Re- vertants surviving after histidine was depleted from the top agar were counted and data were plotted to verify a linear dose-response curve. Strain TA1530 was used to be consistent with previous reports (Owais et al., 1983). Results and discussion Azide treatment of callus LT resistant variants were recovered from both the non-mutagenized and mutagenized treatment of each maize cell line (Tables 1 and 2). Recovery of variants resistant to LT selection decreased when callus was treated with azide even after corrections were made for the reduction of callus growth due to azide toxic- ity. Similar results were observed in another repli- cated experiment with a different friable, embryo- genic callus line (data not shown). Because correc- tion for azide toxicity did not result in the frequency of variants recovered from azide treat- ments being comparable to the level of variants in the control, a negative cooperativity may exist between azide toxicity and the lethal LT selection used in this study. Without any evidence we speculate that the cooperativity may have resulted from azide acting as a substrate for O-acetylserine sulfhydrylase in the cysteine biosynthesis path- way. This activity may reduce the amount of cysteine available for methionine biosynthesis, thus enhancing the toxicity of LT selection. Toxic ef- fects of LT selection are due to depletion of 160 TABLE 1 THE EFFECT OF SODIUM AZIDE TREATMENT ON THE NUMBERS OF LYSINE PLUS THREONINE (LT)- RESISTANT VARIANTS SELECTED FROM FRIABLE, EMBRYOGENIC MAIZE CALLUS Treated callus (0.5 g) was plated as a lawn on a Whatman No. filter paper overlaying selective medium and transferred every 2 weeks Azide Total LT- Predicted Adjusted treatment resistant ll-days growth variants/plate (mM) variants (% control) selected 0.0 68 a 100.0 b 3.2 c 0.2 17 95.8 1.1 1.0 27 76.4 1.8 1.5 8 66.3 0.6 a Taken after 10 wk on LT selection medium from 21, 16, 20 and 20 plates for 0, 0.2, 1.0 and 1.5 mM azide treatments, respectively. b Predicted values from regression of azide toxicity data de- termined from additional replications within this experiment. c Variants selected corrected for reduction in growth as a percent of control and then divided by the total number of plates in the treatment. cellular methionine (Green, 1977). Regardless of the apparent negative cooperativity between azide treatment and LT selection, these results show that azide did not greatly increase the frequency of selectable LT-resistant variants from either fria- ble, embryogenic or BMS maize callus, and we conclude that azide was not apparently mutagenic in the tissue cultures. An alternative explanation may be that selec- tion for LT-resistance in friable, embryogenic maize callus favored pre-existing LT variants that were present at an approximate frequency of 6.4 variants per g fresh wt. callus (Table 1) and azide- induced LT variants were not detected. Friable, embryogenic callus cells underwent 1-3 doublings between the time of azide mutagenesis and the time when LT began to inhibit callus growth (3 days after plating on LT medium) as determined by monitor ing callus growth in a separate experi- ment (data not shown). Azide-induced mutants would probably exist as 2 -8 cell colonies at the time LT medium exerted selection pressure. Pre- existing, spontaneous LT resistant variants pre- sumably generated by somaclonal variation would exist as larger cell aggregates at the time of LT selection. The smaller azide-induced LT resistant colonies may not have been selected because of decreased selection efficiency for small cell col- onies compared to larger, pre-existing variant col- onies (Horsch and Jones, 1980). To avoid this potential problem, azide-treated BMS suspensions were given 5 days of non-selective growth in sus- pensions before plating and LT selection. The rapid growth rate of BMS suspensions (doubling time of 25-30 h) should have allowed azide- induced mutants to form 16-32 cell colonies dur- ing the 5 days on non-selective medium and the 3 days on LT medium before selection became ef- fective. Selection efficiency of LT-resistant BMS cell colonies was expected to be similar to the selection efficiency of pre-existing LT variants in the BMS culture. BMS suspension cultures are composed of ca. 1.7 x 107 ce l l s /g fresh wt. (Smith, 1982), therefore, approx. 10 8 maize cells per azide treatment were selected for LT variants. However, the number of LT-resistant variants isolated from BMS after correction for azide toxicity also de- TABLE 2 THE EFFECT OF SODIUM AZIDE TREATMENT OF BMS MAIZE SUSPENSION CELLS ON THE NUMBERS OF LYSINE PLUS THREONINE (LT)-RESISTANT VARIANTS SELECTED Azide treated callus was grown 5 days as suspensions in non-selective medium to allow newly induced mutants to form small colonies before initiating LT selection by spreading callus (1.0 g) as a lawn on a filter papers overlaying selective medium. The data is presented as the total of two treatment replications each divided into sub-replications after the initial azide treatment. Azide Total LT- 5 days growth Adjusted treatment resistant (% control) variants/plate (raM) variants selected 0.0 10 a 100.0% b 0.50 c 0.2 4 68.0% 0.29 0.6 1 41.0% 0.17 a Taken after 8 wks on selective medium from 20, 20, and 15 plates for 0, 0.2, 0.6 mM azide treatments, respectively. b 5-day suspension callus fresh wt. increases expressed as a percent of control growth and averaged across two treatment replications each with two sub-replications. c Variants selected corrected for reduction in growth as a percent of control then divided by the total number of plates in a treatment. creased with increasing azide treatment (Table 2) indicating that azide was not apparently muta- genic in maize cell cultures. More than 100 plants were regenerated from the control cultures and azide-treated LT-resistant cell lines. There was no observable increase in visible somatic mutations in the plants regen- erated from azide-treated callus, thus providing evidence that azide did not mutate other loci in maize callus. These results agree with the report of Wang et al. (1986) which showed no difference in mutation frequency between progeny of plants regenerated from maize callus treated with sodium azide in phosphate buffer, pH = 3.0, and plants regenerated from callus treated with the phos- phate buffer without azide. Furthermore, three LT-resistant mutants have been selected in M 2 barley populations after seed treatment with azide (Bright et al., 1982) indicating that the LT-resis- tance loci are not recalcitrant to azide mutagene- sis. Azide was a potent mutagen in maize kernels. Light green, albino, brown-spotted or yellow-gold stripes were observed in 56% of the M 1 plants grown from kernels treated with 1 mM NaN 3. The azide-treated kernels were from the same genetic background as the friable, embryogenic callus, i.e. A188 × B73. Although it is likely that somatic nuclear mutations induced by azide accounted for only a portion of the sectored leaves, M 1 sector frequency and mutation frequency in M 2 genera- tions are correlated in azide-treated barley (A. Kleinhofs, personal communication) and in ethyl- eneimine-treated Pisum sativa L. (Blixt et al., 1963) Conger and Carabia also reported somatic muta- tions in M1 seedlings of Yg/yg maize (1977). Briggs and Bettendorf (1987) reported M 3 muta- t ion frequencies of 20% for maize seed treated with 1 mM NaN 3 and Hibberd and Green (1978) reported that 15% of the M 3 kernels or plants from a 1 mM azide treatment displayed visible mutations. Azidoalanine metabolism. To determine if maize callus synthesized a mutagenic metabolite, extracts of azide-treated callus and kernel em- bryos were assayed by the Ames Mutagenicity Test. Free azide was removed so that only non- volatile, acid-stable, mutagenic metabolites such 161 TABLE 3 MUTAGENIC METABOLITE (PRESUMABLY AZIDOALANINE) IN MAIZE TISSUE EXTRACTS SAM- PLED AT DIFFERENT TIMES AFTER SODIUM AZIDE TREATMENT DETECTED BY THE AMES MUTAGENIC- ITY TEST Tissues treated with azide were extracted, adjusted to equal volumes/g fresh wt. of treated tissue, and extracts assayed for mutagenicity. Data are the averages of two azide-treatment replication~ and two Ames plates per replication. The average spontaneous frequency of 44 revertants per plate was de- termined using 5 Ames plates and was subtracted from the treatment averages. Extracts of untreated embryos, friable, embryogenic callus and BMS callus, induced 1, 1 and 8 re- vertants above the spontaneous frequency respectively (average of 4 Ames plates each). Additional controls included adding azide directly to the assay which induced revertants and adding azide to callus extracts which did not induce revertants because the free azide was volatilized as HN 3 by the extraction proce- dure. Tissue Azide Hours after treatment azide treatment (mM) 4 20 40 (His + Revertants) Embryos 1.0 1480 a 1800 a 980 a Friable, embryo- genic callus 1.0 1 160 a 42 35 BMS callus 0.6 830 46 28 a Too many revertants to count ( > 1200 per plate) from 100/~1 of extract. Data are revertants from 10 /~1 of extract multi- plied by 10 based on the experimentally determined linear relationship between revertants and extract dose. as azidoalanine were detectable (Owais et al., 1983). Extracts of friable, embryogenic callus and isolated embryos 4 h after azide treatment ex- hibited similar His + reversion frequencies indi- cating similar initial tissue concentrations of mutagenic metabolite per g fresh wt. (Table 3). Extracts of azide-treated BMS callus had lower levels of mutagenic metabolite. Mutagenic metabolite levels in both callus tissues decreased to sfightly above control levels by 20 h after treatment which was before callus growth had resumed as measured by fresh weight increase. The decrease of mutagenic metabolite in friable, embryogenic callus 20 h after azide treatment also was observed in two additional experiments (data not shown). Mutagenic metabolite levels in em- bryo extracts remained high up to 40 h after azide 162 treatment, by which time germination had re- sumed as indicated by the appearance and elonga- tion of the radical. The lack of evidence for azide mutagenesis at LT-resistance loci in maize callus and the potent mutagenicity in maize kernels was associated with the rapid decrease of mutagenic metabolite ob- served in callus tissue compared with the higher levels of mutagenic metabolite persisting in em- bryos of treated kernels. Maize callus appeared to possess pathways which metabolized the acid-sta- ble mutagenic metabolite, azidoalanine. Presuma- bly, azidoalanine was detoxified rather than metabolized to the ult imate mutagen proposed by Owais et al. (1986) and Owais and Kleinhofs (1988) given the observed lack of mutagenicity in both maize callus tissues. Callus tissue may also have efficiently repaired any DNA lesions caused by transient levels of mutagen before respiratory arrested cells resumed growth and became mutag- enized. Detoxif ication pathways and repair mech- anisms were predicted to require little energy be- cause azide arrests oxidative respiration. In summary, our results indicate that tissue specific differences in mutagenic metabolite detoxif ication exist between maize embryos and two types of maize callus. Acknowledgements Scientific paper No. 16158. Minnesota Agri- cultural Experiment Station project No. 0302- 4813-56. 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