Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways

Abstract

Hydrogen sulfide (H2S) has been recently found to act as a potent priming agent. This study explored the hypothesis that hydroponic pretreatment of strawberry (Fragaria × ananassa cv. Camarosa) roots with a H2S donor, sodium hydrosulfide (NaHS; 100 μM for 48 h), could induce long-lasting priming effects and tolerance to subsequent exposure to 100mM NaCI or 10% (w/v) PEG-6000 for 7 d. Hydrogen sulfide pretreatment of roots resulted in increased leaf chlorophyll fluorescence, stomatal conductance and leaf relative water content as well as lower lipid peroxidation levels in comparison with plants directly subjected to salt and non-ionic osmotic stress, thus suggesting a systemic mitigating effect of H2S pretreatment to cellular damage derived from abiotic stress factors. In addition, root pretreatment with NaHS resulted in the minimization of oxidative and nitrosative stress in strawberry plants, manifested via lower levels of synthesis of NO and H(2)O(2) in leaves and the maintenance of high ascorbate and glutathione redox states, following subsequent salt and non-ionic osmotic stresses. Quantitative real-time RT-PCR gene expression analysis of key antioxidant (cAPX, CAT, MnSOD, GR), ascorbate and glutathione biosynthesis (GCS, GDH, GS), transcription factor (DREB), and salt overly sensitive (SOS) pathway (SOS2-like, SOS3-like, SOS4) genes suggests that H2S plays a pivotal role in the coordinated regulation of multiple transcriptional pathways. The ameliorative effects of H2S were more pronounced in strawberry plants subjected to both stress conditions immediately after NaHS root pretreatment, rather than in plants subjected to stress conditions 3 d after root pretreatment. Overall, H2S-pretreated plants managed to overcome the deleterious effects of salt and non-ionic osmotic stress by controlling oxidative and nitrosative cellular damage through increased performance of antioxidant mechanisms and the coordinated regulation of the SOS pathway, thus proposing a novel role for H2S in plant priming, and in particular in a fruit crop such as strawberry.

Fig. 1.

Phenotypic effects of H₂S donor NaHS (100 μΜ) on strawberry plants exposed to 100mM NaCl or 10% (w/v) PEG-6000, for 7 d, with respective controls. (A) Control, pretreated with H₂O, no acclimation, not stressed. (B) H₂S, pretreated with H₂S, no acclimation, not stressed. (C) NaCl, pretreated with H₂O, no acclimation, 100mM NaCl stressed. (D) H₂S₍₀₎→NaCl, pretreated with H₂S, no acclimation, 100mM NaCl stressed). (E) H₂S₍₃₎→NaCl, pretreated with H₂S, 3 d acclimation, 100mM NaCl stressed. (F) PEG, pretreated with H₂O, no acclimation, 10% (w/v) PEG-6000 stressed. (G) H₂S₍₀₎→PEG, pretreated with H₂S, no acclimation, 10% (w/v) PEG-6000 stressed. (H) H₂S₍₃₎→PEG, pretreated with H₂S, 3 d acclimation, 10% (w/v) PEG-6000 stressed. Red arrows indicate wilted, necrotic leaves.

Fig. 2.

Effect of 100 μΜ NaHS root pretreatment on leaf H₂S content at the initiation (day 0, white bars) and 7 d (grey bars) after stress imposition. Treatment acronyms are as described in the legend to Fig. 1. Data are means ± SE of three replications. Bars with different letters are significantly different (P < 0.05). FW, freshweight.

Fig. 3.

Effect of 100 μΜ NaHS on chlorophyll fluorescence (A) and stomatal conductance (B) in leaves of strawberry plants after 7 d of exposure to 100mM NaCl or 10% (w/v) PEG-6000. Treatment acronyms are as described in the legend to Fig. 1. Data are means ± SE of three replications. Bars with different letters are significantly different (P < 0.05).

Fig. 4.

Lipid peroxidation, measured as leaf malondialdehyde (MDA) content, as affected by H₂S donor NaHS (100 μΜ), at the initiation of stress imposition (day 0, white bars) and 7 d (grey bars) after treatment with 100mM NaCl or 10% (w/v) PEG-6000. Treatment acronyms are as described in the legend to Fig. 1. Data are means ± SE of three replications. Bars with different letters are significantly different (P < 0.05). FW, freshweight.

Fig. 5.

Effect of 100 μΜ NaHS on H₂O₂ (A) and NO (B) leaf content in strawberry plants at the initiation of stress imposition (Day 0; white columns) and 7 d (grey bars) after treatment with 100mM NaCl or 10% (w/v) PEG-6000. Treatment acronyms are as described in the legend to Fig. 1. Data are means ± SE of three replications. Bars with different letters are significantly different (P < 0.05). FW, freshweight.

Fig. 6.

Effects of 100 μΜ NaHS on ascorbate and glutathione pool and redox state at the initiation of stress imposition (day 0, white bars) and 7 d after stress treatments (grey bars): (A) reduced ascorbate, ASC, (B) oxidized ascorbate, DHA, (C) ascorbate redox state, (D) reduced glutathione, GSH, (E) oxidized glutathione, GSSG, and (F) glutathione redox state. Treatment acronyms are as described in the legend to Fig. 1. Data are means ± SE of three replications. Bars with different letters are significantly different (P < 0.05). FW, freshweight.

Fig. 7.

Heat map showing temporal expression pattern in selected genes associated with enzymatic antioxidants, RNS biosynthesis, redox homeostasis, and SOS pathway in leaves of strawberry plants under non-stress and NaCl/PEG stress conditions. Following root pretreatment with 100 μΜ NaHS 3 d before stress imposition or until application of the stress factor, plants were grown with or without 100mM NaCl or 10% (w/v) PEG-6000 for 7 d as described schematically in Supplementary Fig. S1. Tissues were sampled immediately after H₂S pretreatment (day 0) and 7 d after pretreatment. Relative mRNA abundance was evaluated by quantitative real-time RT-PCR using three biological repeats. Upregulation is indicated in green; downregulation is indicated in red; diagonal dotted lines represent statistically significant differences compared with control samples (P < 0.05). A scale of colour intensity is presented as a legend. Actual relative expression data, obtained from three independent replicates, are shown in Supplementary Table S2.