Apoplastic synthesis of nitric oxide by plant tissues

Abstract

Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase-type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.

Figure 1.

The NO Scavenger cPTIO Promotes PCD in H. vulgare Aleurone Layers. Freshly prepared aleurone layers were treated with ABA or GA. cPTIO (200 μM) alone or with the NO donor SNP (100 μM) was added to some layers 18 h later. Data represent the mean of ∼300 cells in each of 12 aleurone layers. The data are pooled from two independent experiments, both of which showed similar results. Means that are significantly different at the level of P = 0.05 in a Student's t test are indicated by different numbers of asterisks.

Figure 2.

Synthesis of NO by H. vulgare Aleurone Layers as Measured by Mass Spectrometry. (A) Ten aleurone layers were placed in 2 mL of 20 mM CaCl₂. At the times indicated, 125 μM SNP, 125 μM NaNO₂, and 50 μM cPTIO were added. Note the increase in NO signal after SNP and NaNO₂ additions and the quenching of both signals by cPTIO. (B) Peak NO production after addition of 800 μM NaNO₂ to 10 aleurone layers in 2 mL of 20 mM CaCl₂ (control) or 2 mL of 2 mM Tris-Mes, pH 5.8.

Figure 3.

NO Production by Aleurone Layer Medium. (A) Incubation medium from aleurone layers treated with GA or ABA for the indicated times was diluted 1:10 and placed in the sample cuvette of a mass spectrometer. NaNO₂ (800 μM) was added, and the peak NO signal was determined. (B) pH of the medium in (A).

Figure 4.

Nitrite-Dependent NO Production from Aleurone Layer Media. Nitrite at 0 to 20 μM was added to medium in which GA-treated H. vulgare aleurone layers were incubated for 18 h. SNP (100 μM) was added to the same medium without added nitrite. The rate of NO production was determined using the NO-reactive fluorescent probe DAF-FM. Data are means +sd of media from six flasks of aleurone layers and are representative of two independent replicates.

Figure 5.

Reducing Equivalents Accumulate in the Incubation Medium around ABA- and GA-Treated H. vulgare Aleurone Layers. Media were assayed for their ability to reduce an iron-dipyridyl complex. Reduced ascorbate was used as a standard. Data points are means ±sd for four flasks of aleurone layers, and the data are pooled from three independent experiments.

Figure 6.

Phenolic Compounds Accumulate in the Incubation Medium around ABA- and GA-treated H. vulgare Aleurone Layers. Incubation media were assayed for the presence of total phenolics using Folin and Ciocalteu's phenol reagent (A) and for vanillin-reactive flavanols using vanillin (B). Data points in (A) are means ±sd for four (0 h) or eight flasks of aleurone layers. Data points in (B) are means ±sd of 4 (0 h), 5 (72 h), or 10 (24 and 48 h) flasks of aleurone layers.

Figure 7.

Proanthocyanidins in H. vulgare Grain Are Concentrated in the Testa. Imbibed H. vulgare grain were sectioned longitudinally ([A] and [B]), and isolated aleurone layers were sectioned transversely ([C] and [D]). The deep-red peripheral band of vanillin staining in (B) is lacking in the unstained grain (A). The same section through the aleurone layers and attached testa is shown before (C) and after (D) staining. Note that only the region immediately outside of the aleurone layer is stained with vanillin.

Figure 8.

The Rate of Nonenzymatic NO Production from Nitrite Depends on the pH and Phenolic Content of the Medium. (A) Relative NO production rates (log scale) at pH 3, 4, and 5 in the presence or absence of 340 μM catechin. Data are means ±sd of three or four determinations and are representative of two replicates. (B) The effect of catechin and ferulic acid on NO production. Data are means ±sd of four determinations and are representative of two replicates.

Figure 9.

Catechin Accelerates the Rate of Nitrite Loss from Aleurone Layer Medium. Nitrite (100 μM) was added to medium in which ABA-treated (A) or GA-treated (B) aleurone layers had been incubated for 24 h or to medium containing added catechin (340 μM). Medium nitrite concentration was determined at the times indicated. Data points are means ±sd of medium from two or three flasks of aleurone layers, and the data in (A) and (B) are representative of at least two independent replicates.