Heme Oxygenase-1 Delays Gibberellin-Induced Programmed Cell Death of Rice Aleurone Layers Subjected to Drought Stress by Interacting with Nitric Oxide

Abstract

Cereal aleurone layers undergo a gibberellin (GA)-regulated process of programmed cell death (PCD) following germination. Heme oxygenase-1 (HO-1) is known as a rate-liming enzyme in the degradation of heme to biliverdin IXα, carbon monoxide (CO), and free iron ions (Fe(2+)). It is a critical component in plant development and adaptation to environment stresses. Our previous studies confirmed that HO-1 inducer hematin (Ht) promotes the germination of rice seeds in drought (20% polyethylene glycol-6000, PEG) conditions, but the corresponding effects of HO-1 on the alleviation of germination-triggered PCD in GA-treated rice aleurone layers remain unknown. The present study has determined that GA co-treated with PEG results in lower HO-1 transcript levels and HO activity, which in turn results in the development of vacuoles in aleurone cells, followed by PCD. The pharmacology approach illustrated that up- or down-regulated HO-1 gene expression and HO activity delayed or accelerated GA-induced PCD. Furthermore, the application of the HO-1 inducer Ht and nitric oxide (NO) donor sodium nitroprusside (SNP) not only activated HO-1 gene expression, HO activity, and endogenous NO content, but also blocked GA-induced rapid vacuolation and accelerated aleurone layers PCD under drought stress. However, both HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) and NO scavenger 2-(4-carboxyphenyl0-4, 4,5,5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) reserved the effects of Ht and SNP on rice aleurone layer PCD under drought stress by down-regulating endogenous HO-1 and NO, respectively. The inducible effects of Ht and SNP on HO-1 gene expression, HO activity, and NO content were blocked by cPTIO. Together, these results clearly suggest that HO-1 is involved in the alleviation of GA-induced PCD of drought-triggered rice aleurone layers by associating with NO.

Keywords: Oryza sativa; aleurone layers; drought stress; gibberellin; heme oxygenase-1; nitric oxide; programmed cell death.

FIGURE 1

Hematin (Ht), sodium nitroprusside (SNP), zinc protoporphyrin IX (ZnPPIX), and 2-(4-carboxyphenyl0-4, 4, 5, 5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) up- or down-regulated HO-1 gene expression (A) and HO activity (B) in rice aleurone layers subjected to drought stress. Rice aleurone layers were treated with or without 20% (polyethylene glycol-6000) PEG, 1 μM Ht, 200 μM SNP, 10 μM ZnPPIX, 200 μM cPTIO individually or in combination for 24 h. A sample with distilled water was used as control (C). Measurement of HO-1 transcript levels in the rice aleurone layers was conducted by real-time fluorescence quantitative PCR assay (A), HO activities were also detected (B). Mean values were calculated from at least three independent experiments, bars with different letters indicate statistically significant differences at the 0.05 level, using Duncan’s multiple test.

FIGURE 2

Ht, SNP, ZnPPIX, and cPTIO raised or reduced endogenous NO content in rice aleurone layers under drought stress. Rice aleurone layers were treated with or without 20% PEG (P), 1 μM Ht (H), 200 μM SNP (S), 10 μM ZnPPIX (Zn), and 200 μM cPTIO (cP) individually or in combination for 24 h. A sample with distilled water was used as control (C). After various treatments, the aleurone layers were, respectively, stained with DAF-2DA, and then thoroughly washed to removal excess dye and immediately observed under a LSCM. Images of the distribution of NO in fluorescently labeled aleurone cells were captured (A–I). The relative DAF-2DA fluorescence intensity in the corresponding aleurone layers was also established (J). Scale bar, 100 μm. Mean values were calculated from at least three independent experiments, bars with different letters indicate statistically significant differences at the 0.05 level, according to Duncan’s multiple test.

FIGURE 3

Ht, SNP, ZnPPIX, and cPTIO up- or down-regulated the expression of HO-1 and the activity of HO in GA-induced rice aleurone layers subjected to drought stress. Aleurone layers treated with or without 20% PEG, 50 μM GA, 1 μM Ht, 200 μM SNP, 10 μM ZnPPIX, and 200 μM cPTIO alone or in combination for 24 h. A sample with distilled water treatment was used as control (C). The transcript levels of HO-1 analyzed by real-time fluorescence quantitative PCR at 24 h. The expression levels of the HO-1 gene are presented as values relative to that of the control (A). Meanwhile, the corresponding HO activity (B) was determined after different treatments for 24 h. Mean values were calculated from at least three independent experiments, bars with different letters indicate significant differences at the 0.05 level, according to Duncan’s multiple test.

FIGURE 4

Increase or decrease in HO-1 gene expression and HO activity related to the acceleration or delay of GA-triggered PCD in rice aleurone layers under drought stress. Aleurone layers were incubated in FDA (green, live cells) and FM-4-64 (red, dead cells) prior to image capture. Aleurone layers treated with or without 20% PEG (P), 50 μM GA (G), 1 μM Ht (H), 200 μM SNP (S), 10 μM ZnPPIX (Zn), and 200 μM cPTIO (cP) alone or in combination for 12, 24, and 48 h, respectively. A sample with distilled water treatment was used as control (C). Images of cells showing fluorescently labeled aleurone layers were captured (A–X). Cell survival rate was also quantified (Y) at 12, 24, and 48 h. Scale bar, 100 μm. Mean values were calculated from at least three independent experiments, bars with different letters indicated significant differences at the 0.05 level, according to Duncan’s multiple test.

FIGURE 5

The vacuolated process of aleurone cells treated with distilled water. (A–I) Vacuoles of aleurone cells treated with distilled water at 0, 2, 4, 5, 6, 7, 8, 9, and 10 days. The scale is 20 μm.

FIGURE 6

Gibberellin (GA) co-treated with PEG accelerated the vacuolated process of aleurone cells. (A–E) Vacuoles in aleurone cells treated with 50 μM GA at 1/2, 1, 2, 3, and 4 days. (F–H) Vacuoles in aleurone cells treated with 20% PEG for 1/2, 1, and 2 days. (I–K) Vacuoles in aleurone cells treated with 20% PEG + 50 μM GA for 1/2, 1, and 2 days. The scale is 20 μm.

FIGURE 7

Ht and SNP delayed vacuolation in aleurone cells treated with PEG plus GA. (A–D) Vacuoles of aleurone cells treated with 20% PEG + 50 μM GA + 1 μM Ht at 1, 2, 3, and 4 days. (E–H) Vacuoles in aleurone cells treated with 20% PEG + 50 μM GA + 200 μM SNP for 1, 2, 3, and 4 days. The scale is 20 μm.