Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers

Abstract

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

Figure 1

PCD in barley aleurone layers is delayed by the NO donors SNP and SNAP. Digital images of fluorescently labeled barley aleurone cells are shown in A through E. Layers were incubated in FDA (green, live cells) and FM 4-64 (red, dead cells) prior to image capture. Layers were treated with 5 μm GA (A–C and E) alone (A) or with 100 μm SNP (B), 300 μm SNAP (C), 300 μm SNAP plus 300 μm cPTIO (E), or with 5 μm ABA alone (D) for the times indicated.

Figure 2

Barley aleurone layers treated with GA and NO donors remain viable longer than layers treated with GA alone. Layers were incubated in 5 μm GA alone (A and B) or in the presence of 100 μm SNP (A) or 300 μm SNAP (B). Relative rates of O₂ consumption were measured at various times of incubation using an O₂ electrode. For comparison, O₂ consumption rates for ABA-treated (5 μm) layers are shown in A and B. Data are means + sd of at least four individual layers.

Figure 3

NO donors act in a concentration-dependent manner on GA-treated layers, but do not affect O₂ consumption by ABA-treated barley aleurone layers. GA or ABA layers were incubated for 48 h with SNP (A) or SNAP (B) at the concentrations shown, and relative rates of O₂ consumption were determined using an O₂ electrode. Data are means ± sd of at least four independent layers.

Figure 4

NO donors increase the amount of α-amylase secreted by GA-treated barley aleurone layers. α-Amylase activity in the medium surrounding barley aleurone layers treated with ABA alone (5 μm; A and B) or GA (5 μm) in the absence (A and B) or presence of 300 μm SNAP (A) or 100 μm SNP (B) is shown at incubation times between zero and 56 h. Data are means ± sd for three flasks of layers.

Figure 5

The NO donors SNP and SNAP do not inhibit the accumulation of α-amylase mRNA. Barley aleurone layers were treated with GA alone, GA plus SNAP, GA plus SNP, or ABA alone, and total RNA was isolated at the times indicated. Northern blots were probed with a cDNA for α-amylase. An rRNA probe was used as a loading control. Data are from one experiment and are representative of two independent experiments.

Figure 6

The NO donor SNAP delays the GA-induced loss of CAT mRNA and protein. The amount of barley Cat2 mRNA (A) was determined by probing northern blots with a Cat2 cDNA probe. The amount of CAT protein (B) was determined by probing protein blots with an antibody against maize (Zea mays) CAT. Total RNA and protein were extracted at the times indicated from barley aleurone layers treated with GA, GA plus SNAP, or ABA. Data are from a single experiment and are representative of at least two replicates.

Figure 7

The NO donor SNAP delays the GA-induced loss of SOD mRNA and protein. The amount of barley Sod4 mRNA (A) was determined by probing northern blots with a maize Sod4 cDNA probe. The amount of SOD protein (B) was determined by probing protein blots with an antibody against maize SOD4. Total RNA and protein were extracted at the times indicated from barley aleurone layers treated with GA, GA plus SNAP, or ABA, and total protein was also extracted from layers treated with GA plus SNP at the times indicated. Data are from a single experiment and are representative of two replicates.

Figure 8

BHT delays GA-induced PCD in barley aleurone layers. Aleurone layers were incubated with GA alone or with GA plus 1 mm BHT, and were then loaded with the fluorescent probes FDA and FM 4-64 at 24, 36, and 48 h. Epifluorescence images (A) of live (green) and dead (red) cells were quantified as shown in C. α-Amylase secretion by layers from the same experiment is quantified in D. Note that cells in layers treated with ABA alone or ABA plus BHT do not die (B).

Figure 9

Fluorescent probes report the presence of NO in barley aleurone protoplasts. Epifluorescence (A–F) and differential interference contrast (G–I) images of barley aleurone protoplasts incubated with DAQ (A), 1-AQ (B), DAF-2 DA (D and G), 1-AF DA (E and H), or no probe (C, F, and I). Arrows point to damaged cells.