Effects of Abscisic Acid and Nitric Oxide on Chilling Resistance and Activation of the Antioxidant System in Walnut Shoots In Vitro

in Journal of the American Society for Horticultural Science

The roles of abscisic acid (ABA) and nitric oxide (NO) and the relationship between NO and ABA on chilling resistance and activation of antioxidant activities in walnut (Juglans regia) shoots in vitro under chilling stress were investigated. Walnut shoots were treated with ABA, the NO donor sodium nitroprusside (SNP), ABA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), PTIO, SNP in combination with the ABA biosynthesis inhibitor fluridone (Flu), and Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels, and the antioxidant defense system were analyzed. The results showed that ABA treatment markedly alleviated the decreases in the maximal photochemical efficiency and survival and the increases in electrolyte leakage and lipid peroxidation induced by chilling stress, suggesting that application of ABA could improve the chilling tolerance. Further analyses showed that ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. Similar results were observed when exogenous SNP was applied. ABA in combination with PTIO or PTIO alone differentially abolished these protective effects of ABA. However, treatment with NO in combination with Flu or Flu alone did not affect the SNP-induced protective effect against CI or the activation of antioxidant activities under conditions of chilling stress. In addition, ABA treatment increased the NO content under chilling conditions, which was suppressed by the ABA biosynthesis inhibitor Flu or NO scavenger PTIO. Conversely, SNP application induced the same ABA rise observed in control plants in response to chilling. Taken together, these results suggested that ABA may confer chilling tolerance in walnut shoots in vitro by enhancing the antioxidant defense system, which is partially mediated by NO, preventing the overproduction of ROS to alleviate the oxidative injury induced by chilling.

Contributor Notes

We sincerely thank Dong Pei (Lab of Tree Breeding and Cultivation, State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry) for her kind help with experiment design and the equipment. This work was supported by the National Natural Science Foundation of China (Grant No. 31401817) and the Commonweal Special Foundation of State Forestry Administration of China (No. 201304712).

Corresponding author. E-mail: jinhetaojht@263.net.

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    In vitro walnut shoots of 4.5–5.0 cm height used for the investigation of effects of abscisic acid (ABA), sodium nitroprusside, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, and fluridone on the electrolyte leakage, malondialdehyde content, maximal fluorescence ratio, percentage survival, nitric oxide contents, and ABA contents in the leaves of walnut shoots in vitro after 3 d of cultivation at 25 or 4 °C.

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    Effects of different abscisic acid (ABA) concentrations on electrolyte leakage (A), malondialdehyde (MDA) content (B) and maximal fluorescence ratio (Fv/Fm) (C) and different SNP concentrations on electrolyte leakage (D), MDA content (E), and Fv/Fm (F) in the leaves of walnut shoots in vitro after 3 d of cultivation at 25 or 4 °C. Values represent the means of three replicates ± sd as shown by the vertical error bars. Different letters above the bars indicate significant differences via Duncan’s multiple-range test at P < 0.05.

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    Effects of different 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) concentrations on nitric oxide (NO) content (A) and different fluridone (Flu) concentrations on abscisic acid content (B) in the leaves of walnut shoots in vitro after 3 d of cultivation at 25 or 4 °C. Each value represents mean of three replicates ± sd shown by vertical error bar.

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    Phenotypes in leaves of walnut shoots in vitro subjected to different treatments for 3 d: (A) control, (B) chilling, (C) chilling + abscisic acid (ABA), (D) chilling + sodium nitroprusside (SNP), (E) chilling + ABA + 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), (F) chilling + PTIO, (G) chilling + SNP + fluridone (Flu), and (H) chilling + Flu.

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    Effects of abscisic acid (ABA), sodium nitroprusside (SNP), 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), and fluridone (Flu) on the electrolyte leakage (A), malondialdehyde (MDA) content (B), maximal fluorescence ratio (Fv/Fm) (C), percentage survival (D), nitric oxide (NO) contents (E), and ABA contents (F) in the leaves of walnut shoots in vitro after 3 d of cultivation at 25 or 4 °C. Values represent the means of three replicates ± sd as shown by the vertical error bars. Different letters above the bars indicate significant differences via Duncan’s multiple-range test at P < 0.05.

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    Effects of abscisic acid (ABA), sodium nitroprusside (SNP), 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), and fluridone (Flu) on H2O2 content (A), superoxide radicals (O2) production rate (B), and hydroxyl radicals (OH) content (C) in the leaves of walnut shoots in vitro after 3 d of cultivation at 25 or 4 °C. Values represent the means of three replicates ± sd as shown by the vertical error bars. Different letters above the bars indicate significant differences via Duncan’s multiple-range test at P < 0.05.

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    Effects of abscisic acid (ABA), sodium nitroprusside (SNP), 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), and fluridone (Flu) on the activities of superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and glutathione reductase (GR) (D), and the contents of ascorbic acid (AsA) (E) and reduced glutathione (GSH) (F) in the leaves of walnut shoots in vitro after cultivation at 25 or 4 °C for 3 d. Values represent the means of three replicates ± sd as shown by the vertical error bars. Different letters above the bars indicate significant differences via Duncan’s multiple-range test at P < 0.05.

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