Figure 1.
Comparative effects of HO-1 inducer hematin, CO-saturated aqueous solution, and light on the restoration of chlorophyll content in etiolated wheat seedling leaves.
14-day-old wheat seedlings were kept at 25°C in continuous darkness for 5 days. After that, some were transferred into light, while others were still left in darkness without (D→D) or with different contentions of hematin (1.0, 10, and 100 μM, D→D+H1.0, 10, 100), and different saturations of CO aqueous solution (0.1, 1.0, 10, and 50%, D→D+CO0.1, 1.0, 10, 50%). L→L+ZnPPIX, D→D+ZnPPIX, and D→L+ZnPPIX D→H10+Hb stand for combination with HO-1 specific inhibitor ZnPPIX (100 μM), and CO/NO scavenger hemoglobin (Hb, 0.1 gL−1), for additional 3 days. L→L stands for wheat seedlings were grown in normal light cycle. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 2.
Time course of chlorophyll accumulation following incubation in HO-1 inducer hematin in wheat seedling leaves.
Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark for 5 days. Afterwards, seedlings were cultured in the Hoagland solution with or without 10 μM HO-1 inducer hematin (H) treatment, in the dark (D) for another 5 days. L→L stands for wheat seedlings were grown in normal light cycle. Chlorophyll was extracted and quantified at various times. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 3.
Time course of HO-1 gene expression in wheat seedling leaves during transition from dark to light for additional hours.
A, HO-1 mRNA expression was analyzed by quantitative real-time RT-PCR as described in Materials and Methods. B, CO release under different treatments at 12 h. Values were the mean ± SE of three independent experiments. Bars denoted by the different letter differed significantly at P<0.05 according to Duncan's multiple range tests.
Table 1.
Time course of changes in HO activities (U mg protein−1) during transition from dark to light.
Figure 4.
Effects of NO donor SNP, hematin and CO-saturated aqueous solution on HO activities in wheat seedling leaves after dark pretreatment.
Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark (D) for 5 days. Afterwards, seedlings were cultured in the Hoagland solution without (D→D) or with SNP (S, 100 μM), HO-1 inducer hematin (H, 10 μM), 1.0% CO-saturated aqueous solution (CO) in completely darkness for another 3 days. Values were the mean ± SE for at least three independent experiments. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 5.
HO-1 inducer hematin, exogenous CO and light treatments induce HO-1 gene expression in wheat seedling leaves after dark pretreatment.
Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark (D) for 5 days. Afterwards, seedlings were cultured in the Hoagland solution without (D→D) or with HO-1 inducer hematin (H, 10 μM), and 1.0% CO-saturated aqueous solution (CO) for another 12 h. HO-1 mRNA expression was analyzed by quantitative real-time RT-PCR as described in Materials and Methods. Three independent experiments were performed, bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 6.
SNP, hematin, CO-saturated aqueous solution, CO scavenger hemoglobin, mammalian NOS-like inhibitor L-NAME, and the NO specific scavenger cPTIO differentially influence the chlorophyll content in etiolated wheat seedling leaves after dark pretreatment.
Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark (D) for 5 days. Afterwards, seedlings were cultured in the Hoagland solution without or with 100 μΜ SNP (S), 10 μM HO-1 inducer hematin (H), 1.0% CO aqueous solution (CO), 0.1 g L−1 Hb, 200 μM L-NAME, 100 μM cPTIO, or the above combination treatments, in the light (L) or dark (D) for another 3 days. Values were the mean ± SE for at least three independent experiments. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 7.
Time course of endogenous nitric oxide (NO) generation induced by SNP, hematin and CO aqueous solution in wheat seedling leaves.
Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark (D) for 5 days (25°C). Afterwards, seedlings were cultured in the Hoagland solution without or with 100 μΜ SNP (S), 10 μM HO-1 inducer hematin (H), and 1.0% CO aqueous solution (CO), in the light (L) or dark (D) for another 3 days. NO contents were detected by using Greiss reagent. Values were the mean ± SE for at least three independent experiments. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests.
Figure 8.
NO accumulation in etiolated distilled water was taken as control.
The distribution of nitric oxide (NO) in wheat seedling leaves induced by SNP, hematin, and CO aqueous solution in wheat seedling leaves. Before starting the experiments, 14-day-old wheat seedlings cultured in the Hoagland solution were kept in the light (L, 300 μmmol m−2s−1) or dark (D) for 5 days. Afterwards, seedlings were cultured in the Hoagland solution without or with 100 μΜ SNP (S), 10 μM HO-1 inducer hematin (H), and 1.0% CO aqueous solution (CO), in the light (L) or dark (D) for another 2 days. A, The NO distribution was detected by fluorescence probe DAF-2 DA and negative probe AF 4-DA 2 days after different treatments under fluorescence microscopy (TCS-SP2 confocal laser scanning microscope; Leica Lasertechnik GmbH). B, Mean relative DAF-2 DA and AF 4-DA fluorescence densities corresponding to samples without cPTIO treatment was given. Values were the mean ± SE for at least three independent experiments. Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple range tests. Bars = 20 μm.
Figure 9.
Effects of treatment with SNP, hematin, CO-saturated aqueous solution, ZnPPIX, and cPTIO on Pfr contents (3 d) and expression profiles of PHYA expression (2 d).
14-day-old wheat seedlings were grown for 5 days at 25°C in continuous darkness (D) or light (L) at 300 μmmol m−2s−1 before either being transferred into light or left in continuous darkness without or with 100 μM SNP, 10 μM hematin, 1.0% CO aqueous solution, 100 μM HO-1 specific inhibitor ZnPPIX, and 100 μM cPTIO, or their combination treatments, for another 3 days. PHYA mRNA expression was analyzed by quantitative real-time RT-PCR as described in Materials and Methods. Values were the mean ± SE for at least three independent experiments (n = 20). Bars denoted by the same letter did not significantly differ at P<0.05 according to Duncan's multiple tests.