Haem oxygenase delays programmed cell death in wheat aleurone layers by modulation of hydrogen peroxide metabolism

Abstract

Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H(2)O(2)) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H(2)O(2) either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H(2)O(2) production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H(2)O(2) production.

Fig. 1.

Changes of haem oxygenase (HO) activity in wheat aleurone layers treated with 50 μM GA or ABA alone. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM ABA or GA, respectively. Layers incubated in 5 mM CaCl₂ alone were regarded as the control (Con). HO activity was determined at the indicated time after various treatments. Data are means ±SD of at least three independent measurements from different experiments. Bars with asterisks are significantly different in comparison with corresponding Con samples at P <0.05 and P <0.01 (t test).

Fig. 2.

Effects of CO aqueous solution and the HO-1 inducer haematin on the cell viability of GA-treated wheat aleurone layers. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM GA alone or in the presence of CO aqueous solution (CO) or haematin (Ht) at the indicated saturations or concentrations. Quantification of viability and death for wheat aleurone layers at 48 h of incubation (A). The data are collected from at least four aleurone layers. Relative rates of O₂ consumption were measured at 48 h of incubation using an O₂ electrode (B). The rate of oxygen consumption was measured for ten layers in 3 ml of sterile water. For comparison, the percentage of live cells and relative O₂ consumption for ABA-treated (50 μM) layers are also shown. Bars denoted by the different letters are different significantly at P <0.05 according to the t test.

Fig. 3.

HO-1 transcript, HO activity, and HO-1 protein expression in wheat aleurone layers treated with ABA alone or with GA in the absence or presence of the HO-1 inducer haematin or CO aqueous solution. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM GA or ABA alone, or GA plus 10 μM haematin (Ht) or 1.0% saturation of CO aqueous solution (CO). Wheat HO-1 transcript was analysed by semi-quantitative RT-PCR after 12 h of various treatments (A), HO activity was also determined (B). Meanwhile, HO-1 protein expression was analysed by Western blotting (C), and Coomassie Brilliant Blue-stained gels are present to show that equal amounts of proteins were loaded (D). The number below the band (A) indicates the relative abundance of the corresponding gene with respect to the loading control 18S rRNA, and the number below the band (C) illustrates the relative abundance of the corresponding HO-1 protein compared with that of the ABA-treated sample. Bars denoted by the different letters (B) are different significantly at P <0.05, according to Duncan's multiple test.

Fig. 4.

PCD in wheat aleurone layers is related to the decrease in HO activity. Layers pretreated with water (–ZnPPIX) or 100 μM ZnPPIX (+ZnPPIX) for 6 h, were further incubated in a medium containing 5 mM CaCl₂ and 50 μM ABA or GA alone, or GA plus 10 μM haematin (Ht) or 1.0% saturation of CO aqueous solution for another 18 h or 42 h. HO activity was determined at 12 h (A), or 24 h (B) after various treatments. Meanwhile, digital images of fluorescently labelled wheat aleurone cells were further obtained (C). Death for wheat aleurone layers was also quantified from at least four aleurone layers (D). Scale bar, 200 μm. Data are means ±SD of at least four independent samples. Bars denoted by the different letters are significantly different at P <0.05, according to Duncan's multiple test. ND, none detected.

Fig. 5.

Effects of DTT, BHT, AsA, and H₂O₂ on PCD, HO activity, HO-1 transcript, and HO-1 protein expression in aleurone layers. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM GA, or 50 μM ABA alone or in the presence of 5 mM DTT, 100 μM BHT, 5 mM AsA, or 1 mM H₂O₂. Digital images of fluorescently labelled wheat aleurone cells were obtained at the indicated time after various treatments (A, B). HO activity was determined after 12 h of different treatments (C). Meanwhile, HO-1 transcript was analysed by semi-quantitative RT-PCR (D). The number below the band indicates the relative abundance of the corresponding gene with respect to the loading control 18S rRNA. HO-1 protein expression was analysed by Western blotting (E), and Coomassie Brilliant Blue-stained gels are present to show that equal amounts of proteins were loaded (F). Scale bar=200 μm. Data are means ±SD of at least four independent samples. Bars denoted by the different letters are different significantly at P <0.05, according to Duncan's multiple test.

Fig. 6.

Effects of an inhibitor of CAT 3-amino-1,2,4-triazole (3-AT), a ‘suicide’ inhibitor of APX p-aminophenol (p-AP), and the scavenger of hydrogen peroxide (H₂O₂) N,N'-dimethylthiourea (DMTU) on GA-induced PCD in aleurone layers and H₂O₂ production. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM ABA or GA alone, or in the presence of 100 μM 3-AT, 1 mM p-AP, or 5 mM DMTU. Digital images of fluorescently labelled wheat aleurone cells were obtained (A). Live and dead cells were also quantified (B) at 48 h after various treatments. Meanwhile, H₂O₂ content (B) was determined. Scale bar=200 μm. Data are means ±SD of at least four independent samples. Bars denoted by the different letters are significantly different at P <0.05, according to Duncan's multiple test.

Fig. 7.

Effects of ABA, GA, haematin, and CO aqueous solution on the activities or expression of ascorbate peroxidase (APX) and catalase (CAT) in wheat aleurone layers. Layers pretreated with water (–ZnPPIX) or with 100 μM ZnPPIX (+ZnPPIX, only D) for 6 h, were further incubated in a medium containing 5 mM CaCl₂ and 50 μM ABA or GA alone, or GA plus 10 μM haematin (Ht), or 1.0% saturation of CO aqueous solution for another 48 h. Layers incubated in 5 mM CaCl₂ alone were regarded as the control (Con). APX (A) and CAT activities (B) were determined at the indicated time. The corresponding mRNA expression was analysed by semi-quantitative RT-PCR (C, D). The number below the band indicates the relative abundance of the corresponding gene with respect to the loading control 18S rRNA. Data are means ±SD of at least four independent samples.

Fig. 8.

Effects of ABA, GA, haematin, CO aqueous solution, Fe²⁺, and bilirubin (BR) on the activities of ascorbate peroxidase (APX), catalase (CAT), PCD, or hydrogen peroxide (H₂O₂) content in wheat aleurone layers. Layers were incubated in a medium containing 5 mM CaCl₂ and 50 μM ABA or GA alone, or GA plus 10 μM haematin (Ht) or 1.0% saturation of CO aqueous solution, 10 μM each of FeSO₄ (Fe²⁺) or BR for 48 h. APX (A), CAT activities (B), and H₂O₂ content (D) were determined at the indicated time. Epifluorescence images of live and dead cells at 48 h were quantified as shown in C. Data are means ±SD of at least four independent samples. Bars denoted by the different letters are significantly different at P <0.05 according to Duncan's multiple test.