Effects of Exogenous Gibberellic Acid₃ on Iron and Manganese Plaque Amounts and Iron and Manganese Uptake in Rice

<div><p>Gibberellins (GA) regulate various components of plant development. Iron and Mn plaque result from oxiding and hydroxiding Fe and Mn, respectively, on the roots of aquatic plant species such as rice (<i>Oryza sativa</i> L.). In this study, we found that exogenous gibberellic acid₃ (GA₃) spray decreased Fe plaque, but increased Mn plaque, with applications of Kimura B nutrient solution. Similar effects from GA₃, leading to reduced Fe plaque and increased Mn plaque, were also found by scanning electron microscopy and energy dispersive X-ray spectrometric microanalysis. Reduced Fe plaque was observed after applying GA₃ to the groups containing added Fe²⁺ (17 and 42 mg•L^-1) and an increasing trend was detected in Mn plaques of the Mn²⁺ (34 and 84 mg•L^-1) added treatments. In contrast, an inhibitor of GA₃, uniconazole, reversed the effects of GA₃. The uptake of Fe or Mn in rice plants was enhanced after GA₃ application and Fe or Mn plaque production. Strong synergetic effects of GA₃ application on Fe plaque production were detected. However, no synergetic effects on Mn plaque production were detected.</p></div

Scanning electron micrographs and energy-dispersive X-ray analysis of rice root surfaces after GA₃ treatment.

<p>Four-leaf rice seedlings were grown in KB solution (1.2 mg·L^-1 Fe²⁺ and 0.5 mg·L^-1 Mn²⁺, pH 5.0) and harvested at the 60 h after spraying with 0.18 mM GA₃ (G-M) or distilled water (A-F). Rice root morphology was assessed by scanning electron microscopy (SEM) with (G, H) or without (A, B) GA₃ spraying. The images of any section on root surfaces marked in pink color after GA₃ (I) or control (C) treatments and the energy-dispersive X-ray analysis (EDX) dissection of ion-distribution in pink region of root surfaces which was shown with white dots (D-F, J-M). These regions exhibited different nutrient compositions: C (D, J), O (E, K), Fe (F, L) and Mn (M). The micrographs are representative of the general morphology. Bar = 50 nm.</p

Time course of Fe and Mn plaques after exogenous GA₃ treatment.

<p>Four-leaf rice seedlings grown in KB (1.2 mg·L^-1 Fe²⁺ and 0.5 mg·L^-1 Mn²⁺, pH 5.0) were treated with 0.18 mM GA₃ foliar spray for 96 h. Plants without GA₃ treatments were the control. The Fe (A) and Mn (B) plaques were extracted using the DCB method and detected by ICP-OES. Data are the means ± SE of at least three independent experiments (n = 15) with similar results. The control and exogenous GA₃ treatments were compared using a <i>t</i>-test.</p

An inhibitor of glycolate oxidase influences the GA₃-induced plaque changing.

<p>Four-leaf rice seedlings grown in KB (pH 5.0) were treated by foliar spray of exogenous GA₃ (0.18mM), NaHSO₃ (100mg·L^-1) and GA₃ with NaHSO₃ (0.18mM, 100mg·L^-1) respectively. Then roots were harvested to determine Fe and Mn plaque content after 60 h. The Fe (B) and Mn (C) plaques were extracted using the dithionite-citrate-carbonate (DCB) method and detected by ICP-OES. Data are the means ± SE of at least three independent experiments (n = 15) with similar results. Within each set of experiments, bars with different letters are significantly different <i>P</i><0.05 according to Duncan’s multiple range tests. (A) Images were taken by digital camera after 60 h of the different treatments. Bar = 1cm.</p

Dose response of Fe and Mn plaques to exogenous GA₃ or S3307 treatment.

<p>Four-leaf rice seedlings grown in KB (1.2 mg·L^-1 Fe²⁺ and 0.5 mg·L^-1 Mn²⁺, pH 5.0) were harvested at a concentration gradient of GA₃ (0.03, 0.06, 0.12, 0.18, and 0.24 mM) or its inhibitor, S3307 (0.04, 0.08, 0.12, 0.16, and 0.20 mM) spraying treatments after 60 h, and compared with distilled-water-treated samples (0 mM). The Fe (A) and Mn (B) plaques were extracted using the dithionite-citrate-carbonate (DCB) method and detected by ICP-OES. Data are the means ± SE of at least three independent experiments (n = 15) with similar results. * and ** significantly different between the distilled water (0 mM) and the exogenous GA₃ or S3307 treatment at the <i>P</i> < 0.05 or 0.01 level according to a <i>t</i>-test.</p

Impact of pre-treatment with DMTU or DPI on SA-induced ARF in mung bean hypocotyl cuttings.

<p>The primary roots were removed from seedlings of 5-day-old germinated mung beans, incubated in DMTU or DPI for 4 h, moved into 0.4 mM SA for 24 h, washed three times and cultivated in distilled water for another five days. The number of adventitious roots was quantified and is expressed as the mean from three independent experiments with 30 explants for each treatment. The different letters above the bars indicate significant differences among the treatments (P<0.05), according to the LSD test. </p

Time course of H₂O₂ accumulation (a) and O^2– production (b) in the hypocotyls of mung beans treated with water or SA.

<p>Explants were incubated with SA or water for 24 h, and the H₂O₂ levels were monitored at the indicated time points. The mean values shown are the averages of three different experiments. The error bars represent the SE (n=5). The asterisks indicate that the mean values are significantly different compared with the control values (P<0.05). FW, fresh weight.</p

Interaction between SA in combination with H₂O₂ on ARF in mung bean hypocotyls.

<p>Hypocotyls were treated with different test solutions for 24 h, and then the cuttings were transferred to distilled water and continuously grown for 5 days at 25±2°C, with a 14-h photoperiod (PAR of 200 µmol m⁻² s⁻¹). The distilled water was replaced every day, and the number of adventitious roots of more than 1 mm long was recorded. The number of roots was determined after 5 d of treatment. The values represent the means of 30 explants, and the error bars represent the SE (P<0.05). H₂O₂:10 mM H₂O₂.</p

Histochemical and cytochemical detection of H₂O₂ accumulation induced by SA in mung bean hypocotyl cuttings.

<div><p>a Mung bean hypocotyl cuttings were incubated in SA for 24 h, and H₂O₂ levels were monitored at the indicated time points. All experiments were repeated at least three times with similar results. Bar=1 cm.</p> <p>b Mung bean hypocotyl cuttings were incubated in SA for 12 h. Hypocotyls treated with distilled water under the same conditions served as controls. All experiments were repeated at least three times with similar results. Abbreviations: CW, cell wall; IS, intercellular space. Bar=1 µm.</p></div

SA-induced ARF increases in mung bean hypocotyl cuttings.

<p>(a) Hypocotyls were incubated with 0, 0.1, 0.2, 0.4, 0.6 and 0.8 mM SA for 24 h and washed three times, and then the cuttings were transferred into distilled water. The cuttings were continuously grown for 5 days in this distilled water at 25±2°C, with a 14-h photoperiod (PAR of 200 µmol m⁻² s⁻¹). The distilled water was replaced every day. The root numbers were determined at 5 d after treatment. In addition, adventitious roots of more than 1 mm long were quantified. The values represent the means of 30 explants, and the different letters above the bars indicate significant differences among the treatments at a P<0.05 level, according to the LSD test. (b) Time course of adventitious root formation induced by application of SA or water in mung bean hypocotyls. Hypocotyls were treated with 0.4 mM SA (the optimal concentration) or CK (water) for 24 h and were then transferred into distilled water and continuously grown for 7 days at 25±2°C, with a 14-h photoperiod (PAR of 200 µmol m⁻² s⁻¹). The root numbers were determined in 24-h intervals. The distilled water was replaced every day, and the number of adventitious roots of more than 1 mm long was recorded. The values represent the means of 30 explants, and the error bars represent the SE (P<0.05) .</p