(2001), that were immediately frozen in liquid nitrogen. when each compound was applied singly, although inhibition was still not total (Fig. 1b). We also tested the effects of these compounds alone at the above concentrations on soybean root growth: there were no negative effects ( 0.05). Confocal microscopy using the NO indicator dye diaminofluorescein diacetate (DAF-2DA) demonstrated that NO accumulation was asymmetric, with Rabbit Polyclonal to Thyroid Hormone Receptor beta NO being detected predominantly in the lower cells (Fig. 1c). Moreover, NO accumulation appeared to begin in the most apical region of the root (Fig. 1). Gravity-induced increases in fluorescence were not observed when 4-aminofluorescein diacetate (4-AF DA, the negative control for DAF-2DA) was used MEK inhibitor (data not shown). Pretreatment with NaN3, l-NNA, cPTIO, or NPA all reduced NO fluorescence (Fig. 1c). At low concentrations (1 and 5 axis: C, Control; G, gravistimulated. Values are the mean se for five independent experiments. Data analyzed by one-way ANOVA followed by Tukey’s test. Different symbols indicate significant differences between treatments ( 0.05). c, Gravistimulation induces asymmetric NO accumulation. Soybean roots were loaded with DAF-2DA and gravistimulated by orientating horizontally. Fluorescence intensity of dissected root tips was observed at the indicated times by confocal fluorescence microscopy. Effects of pretreatment with 20 0.01). He et al. (2004) have recently reported a similar biphasic response of Arabidopsis roots to NO. Treatment with the SNP analog sodium ferrocyanide that does not release NO had no effect (data not shown). These results suggest that the low concentration of NO in the upper side could promote root elongation, whereas the high concentration of NO in the lower side would suppress elongation, thus effecting gravitropic bending. To demonstrate that NO alone can modulate gravitropic responses, we applied NO via agar blocks containing SNP at a high concentration (5 axis: CK, Control; G, gravistimulated. Data analyzed by one-way ANOVA followed by Tukey’s test. Different symbols indicate significant differences between treatments ( 0.05). Open in a separate window Figure 5. NO and auxin stimulate cGMP accumulation in soybean roots. a, Roots were treated with SNP (?, control; , 50 nm; ?, 100 nm; ?, 500 nm). b, Roots were treated with 5 mutants defective in NO synthesis have shorter origins, a defect restored by exogenous NO (Guo et al., 2003). Our data are consistent with this and with those of He at al. (2004), suggesting that NO can exert positive or negative effects on root growth depending on its concentration and relationships with additional signaling molecules, much like auxin itself (Fu and Harberd, 2003). Our data also display clearly that auxin stimulates NO generation in soybean origins. Although the effects of auxin on NO generation were not reported for Arabidopsis origins (Guo et al., 2003), auxin offers previously been shown to stimulate NO production in cucumber (cv Williams 82) were soaked in distilled water for 6 h, placed between damp paper towels held between plastic bedding mounted vertically in trays, and germinated for 2 d (at this point there was one primary and some secondary origins). Root Treatments To induce gravitropism, the trays were turned so that the origins were orientated horizontally. For subsequent analyses, the root was excised in the indicated instances and then divided into two parts, Zone 1 and Zone 2, as explained by Joo et al. (2001), that were immediately frozen in liquid nitrogen. Zone 1 displayed the apical 4 mm of the root, including the root cap, meristem, and sometimes part of the elongation zone. Zone 2 (4C8 mm) displayed the rest of the elongation zone. To pretreat with numerous chemicals, attached origins (vertically orientated) were dipped in solutions of the appropriate chemicals (l-NNA, PBITU, cPTIO, NPA, ODQ, LY83583) for 12 h. For direct software to origins, agar blocks (0.8% [w/v], 5 mm 5 mm 20 mm) were used. Filter-sterilized solutions were added to cooled, molten agar in MES/KCl buffer (10 mm MES, pH 6.15, 10 mm KCl, 50 for 5 min. The protoplasts were washed three times with MES/KCl buffer supplemented with 0.45 m mannitol and 1 mm CaCl2 and stored in the dark. Protoplasts were incubated in 10 em /em m DAF-2DA in MES/KCl buffer supplemented with 0.45 m mannitol and 1 mm CaCl2 for 10 min and then washed in buffer (3 5 min).1). only in the above concentrations on soybean root growth: there were no negative effects ( 0.05). Confocal microscopy using the NO indication dye diaminofluorescein diacetate (DAF-2DA) shown that NO build up was asymmetric, with NO being detected mainly in the lower cells (Fig. 1c). Moreover, NO accumulation appeared to begin in probably the most apical region of the root (Fig. 1). Gravity-induced raises in fluorescence were not observed when 4-aminofluorescein diacetate (4-AF DA, the bad control for DAF-2DA) was used (data not demonstrated). Pretreatment with NaN3, l-NNA, cPTIO, or NPA all reduced NO fluorescence (Fig. 1c). At low concentrations (1 and 5 axis: C, Control; G, gravistimulated. Ideals are the mean se for five self-employed experiments. Data analyzed by one-way ANOVA followed by Tukey’s test. Different symbols show significant variations between treatments ( 0.05). c, Gravistimulation induces asymmetric NO build up. Soybean origins were loaded with DAF-2DA and gravistimulated by orientating horizontally. Fluorescence intensity of dissected root tips was observed in the indicated instances by confocal fluorescence microscopy. Effects of pretreatment with 20 0.01). He et al. (2004) have recently reported a similar biphasic response of Arabidopsis origins to NO. Treatment with the SNP analog sodium ferrocyanide that does not release NO experienced no effect (data not demonstrated). These results suggest that the low concentration of NO in the top part could promote root elongation, whereas the high concentration of NO in the lower part would suppress elongation, therefore effecting gravitropic bending. To demonstrate that NO only can modulate gravitropic reactions, we applied NO via agar blocks comprising SNP at a high concentration (5 axis: CK, Control; G, gravistimulated. Data analyzed by one-way ANOVA followed by Tukey’s test. Different symbols show significant variations between treatments ( 0.05). Open in a separate window Number 5. NO and auxin stimulate cGMP build up in soybean origins. a, Roots were treated with SNP (?, control; , 50 nm; ?, 100 nm; ?, 500 nm). b, Origins were treated with 5 mutants defective in NO synthesis have shorter origins, a defect restored by exogenous NO (Guo et al., 2003). Our data are consistent with this and with those of He at al. (2004), suggesting that NO can exert positive or negative effects on root growth depending on its concentration and relationships with additional signaling molecules, much like auxin itself (Fu and Harberd, 2003). Our data also display clearly that auxin stimulates NO generation in soybean origins. Although the effects of auxin on NO generation were not reported for Arabidopsis origins (Guo et al., 2003), auxin offers previously been shown to stimulate NO production in cucumber (cv Williams 82) were soaked in distilled water for 6 h, placed between damp paper towels held MEK inhibitor between plastic bedding mounted vertically in trays, and germinated for 2 d (at this point there was one primary and some secondary origins). Root Treatments To induce gravitropism, the trays were turned so that the origins were orientated horizontally. For subsequent analyses, the root was excised in the indicated instances and then MEK inhibitor divided into two parts, Zone 1 and Zone 2, as explained by Joo et al. (2001), that were immediately frozen in liquid nitrogen. Zone 1 displayed the apical 4 mm of the root, including the root cap, meristem, and sometimes part of the elongation zone. Zone 2 (4C8 mm) displayed the rest of the elongation zone. To pretreat with numerous chemicals, attached origins (vertically orientated) were dipped in solutions of the appropriate chemicals (l-NNA, PBITU, cPTIO, NPA, ODQ, LY83583) for 12 h. For direct software to origins, agar blocks (0.8% [w/v], 5 mm 5 mm 20 mm) were used. Filter-sterilized solutions were added to MEK inhibitor cooled, molten agar in MES/KCl buffer (10 mm MES, pH 6.15, 10 mm KCl, 50 for MEK inhibitor 5 min. The protoplasts were washed three times with MES/KCl buffer supplemented with 0.45 m mannitol and 1 mm CaCl2 and stored in the dark. Protoplasts were incubated in 10 em /em m DAF-2DA in MES/KCl buffer supplemented with 0.45 m mannitol and 1 mm CaCl2 for 10 min and then washed in buffer (3 5 min) prior to incubation in buffer plus or minus 5 em /em m IAA. Fluorescence intensity was measured by circulation cytometry using a FACScan (Becton-Dickinson, Franklin Lakes, NJ) with excitation and emission settings of 488 and 530 nm, respectively. Counting of cells halted at 30,000. Gating (gate collection at 75.
(2001), that were immediately frozen in liquid nitrogen
