1). test). Treatment with both a NOS and NR inhibitor did reduce both NO content MLN2480 (BIIB-024) and gravicurvature more than 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 NO being detected predominantly in the lower cells (Fig. 1c). Moreover, NO accumulation MLN2480 (BIIB-024) appeared to begin in the most apical region of the root (Fig. 1). Gravity-induced increases in fluorescence were not observed when MLN2480 (BIIB-024) 4-aminofluorescein diacetate (4-AF DA, the negative control for DAF-2DA) was used (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 Rabbit Polyclonal to ARG2 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 roots, 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 MLN2480 (BIIB-024) effects on root growth depending on its concentration and interactions with other signaling molecules, similar to auxin itself (Fu and Harberd, 2003). Our data also show clearly that auxin stimulates NO generation in soybean roots. Although the effects of auxin on NO generation were not reported for Arabidopsis roots (Guo et al., 2003), auxin has previously been shown to stimulate NO production in cucumber (cv Williams 82) were soaked in distilled water for 6 h, placed between wet paper towels held between plastic sheets mounted vertically in trays, and germinated for 2 d (at this point there was one primary and some secondary roots). Root Treatments To induce gravitropism, the trays were turned so that MLN2480 (BIIB-024) the roots were orientated horizontally. For subsequent analyses, the root was excised at the indicated times and then divided into two parts, Zone 1 and Zone 2, as described by Joo et al. (2001), that were immediately frozen in liquid nitrogen. Zone 1 represented the apical 4 mm of the root, including the root cap, meristem, and sometimes part of the elongation zone. Zone 2 (4C8 mm) represented the rest of the elongation zone. To pretreat with various chemicals, attached roots (vertically orientated) were dipped in solutions of the appropriate chemicals (l-NNA, PBITU, cPTIO, NPA, ODQ, LY83583) for 12.