Hydrogen Peroxide and Abscisic Acid Mediate Salicylic Acid-Induced Freezing Tolerance in Wheat

Abstract

Salicylic acid (SA) can induce plant resistance to biotic and abiotic stresses through cross talk with other signaling molecules, whereas the interaction between hydrogen peroxide (H₂O₂) and abscisic acid (ABA) in response to SA signal is far from clear. Here, we focused on the roles and interactions of H₂O₂ and ABA in SA-induced freezing tolerance in wheat plants. Exogenous SA pretreatment significantly induced freezing tolerance of wheat via maintaining relatively higher dark-adapted maximum photosystem II quantum yield, electron transport rates, less cell membrane damage. Exogenous SA induced the accumulation of endogenous H₂O₂ and ABA. Endogenous H₂O₂ accumulation in the apoplast was triggered by both cell wall peroxidase and membrane-linked NADPH oxidase. The pharmacological study indicated that pretreatment with dimethylthiourea (H₂O₂ scavenger) completely abolished SA-induced freezing tolerance and ABA synthesis, while pretreatment with fluridone (ABA biosynthesis inhibitor) reduced H₂O₂ accumulation by inhibiting NADPH oxidase encoding genes expression and partially counteracted SA-induced freezing tolerance. These findings demonstrate that endogenous H₂O₂ and ABA signaling may form a positive feedback loop to mediate SA-induced freezing tolerance in wheat.

Keywords: endogenous signal; freezing tolerance; inhibitor; salicylic acid; wheat.

FIGURE 1

SA pretreatment improves freezing tolerance of wheat plants. (A) Image of the maximum PS II quantum yield (Fv/Fm). (B) The average value of Fv/Fm and electron transport rates (ETR). (C) Electrolyte leakage (EL) and MDA content. (D) The phenotype of wheat plants in response to water and SA (100 μM) under freezing stress. Plants treated with 0, 10, 100, and 1000 μM SA were challenged with freezing stress at –2°C for 1 day, and were denoted as F, SA10 + F, SA100 + F, SA1000 + F, respectively. CT indicates the control (no SA and no freezing stress). The color code depicted on the top of the image (A) ranges from 0 (blue) to 1.0 (red). Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 2

SA regulates H₂O₂ production in leaves of wheat plants. (A) Cytochemical localization of H₂O₂ in wheat leaves with CeCl₃ staining and transmission electron microscopy. Arrows, CeCl₃ precipitates; IS, intercellular space; CW, cell wall; Bars, 2 μm. The last fully expanded leaves were harvested at 3 h after water or 100 μM SA treatment. (B) Effects of DPI (diphenyleneiodonium, an inhibitor of NADPH oxidase) and SHAM (salicylhydroxamic acid, an inhibitor of cell wall Prx) on SA-induced H₂O₂ production. (C) Effects of DPI on SA increased the activity of cell wall Prx. (D) Effects of SHAM on SA up-regulated expression of RbohD and RbohF. (E) Staining H₂O₂ with H₂DCF-DA and detected by confocal laser scanning microscope system (CLSM). Bars, 200 μm. Plants pretreated with 100 μM DPI or 5 mM SHAM were treated with water or 100 μM SA. The last fully expanded leaves were used for the analysis of H₂O₂ content and cell wall Prx activity at 3 h, Rboh expression at 12 h after water or 100 μM SA treatment. Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 3

The relationship between SA-induced H₂O₂ and ABA. (A) Effects of DMTU (dimethylthiourea, a scavenger of H₂O₂ and OH•), DPI or SHAM on SA-induced ABA accumulation. (B) Effects of Flu (fluorine, an inhibitor of ABA synthesis) on SA-induced H₂O₂ accumulation. (C) Effects of Flu on SA up-regulated expression of RbohD and RbohF. Plants pretreated with water, 2 mM DMTU, 100 μM DPI, or 5 mM SHAM were treated with water or 100 μM SA treatment. The last fully expanded leaves were collected for the analysis of H₂O₂ content at 3 h, ABA content and Rboh expression at 12 h after water or 100 μM SA treatments. Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 4

DMTU and Flu offset the SA-induced freezing tolerance. (A) Effects of DMTU on SA-induced H₂O₂ accumulation at 3 h after water or 100 μM SA treatment. (B) Effects of Flu on SA-induced ABA accumulation 12 h after water or 100 μM SA treatment. (C) Effects of DMTU or Flu on the images of Fv/Fm in SA treated plants under freezing stress. (D) Effects of DMTU or Flu on the average value of Fv/Fm in SA treated plants under freezing stress. Plants pretreated with water, 2 mM DMTU or 1 μM Flu were treated with water or 100 μM SA. After 12 h, the plants were challenged with freezing stress at –2°C for 1 day. Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 5

DMTU and Flu reduce the SA-induced increase of antioxidative capacity in wheat under freezing stress. Plants pretreated with water, 2 mM DMTU or 1 μM Flu were treated with water or 100 μM SA. After 12 h, the plants were challenged with freezing stress at –2°C for 1 day. Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 6

DMTU and Flu down-regulate the SA-induced expression of cold-responsive genes in wheat under freezing stress. Plants pretreated with water, 2 mM DMTU or 1 μM Flu were treated with water or 100 μM SA. After 12 h, the plants were challenged with freezing stress at –2°C for 1 day. Data are the means ± SD of three replicates. Significant differences at P < 0.05 level are denoted by different lowercase letters according to Duncan’s multiple range test.

FIGURE 7

A conclusive model for the induction of freezing tolerance by exogenous SA in wheat plants. SA activates cell wall Prx to produce H₂O₂ in the apoplast, which functions as a signal molecule to induce the increase of antioxidative capacity, to trigger the expression of cold-responsive genes and ABA synthesis. The increased ABA further activates NADPH oxidase, which could induce more production H₂O₂ in the apoplast. In addition, ABA signal also plays a role in SA-induced antioxidant capacity, and expression of cold-responsive genes in wheat plants under freezing stress.