H(2) enhances arabidopsis salt tolerance by manipulating ZAT10/12-mediated antioxidant defence and controlling sodium exclusion

Abstract

Background: The metabolism of hydrogen gas (H(2)) in bacteria and algae has been extensively studied for the interesting of developing H(2)-based fuel. Recently, H(2) is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H(2) in plants as well as its signalling cascade remain unknown.

Methodology/principal findings: In this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H(2) in enhancing Arabidopsis salt tolerance. An increase of endogenous H(2) release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H(2)-saturated liquid medium, mimicking the induction of endogenous H(2) release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H(2) pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS) overproduction and lipid peroxidation. Additionally, H(2) pretreatment maintained ion homeostasis by regulating the antiporters and H(+) pump responsible for Na(+) exclusion (in particular) and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H(2) signalling.

Conclusions: Overall, our findings indicate that H(2) acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.

Figure 1. H₂ alleviates salt stress-induced Arabidopsis seedlings growth inhibition.

Effects of H₂-saturated aqueous solution pretreatments with the indicated saturations for 24 hr on endogenous H₂ production (A), fresh weight and primary root growth (B) in 5-day-old seedlings grown in the liquid MS medium with or without NaCl treatment for the indicated times (A) or 120 hr (B). Effects of H₂ pretreatment on morphology (C), chlorophyll content and fresh weight (D) in Arabidopsis plants. 25-day-old seedlings were pretreated with or without 50% H₂-saturated aqueous solution for 24 hr and then exposed to the liquid MS medium in the presence or absence of 150 mM NaCl for another 8 days. Sample without chemicals was the control (Con). Bar  = 2 cm (C). Statistical analysis was performed using SPSS 16.0 software. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at P<0.05 according to Duncan’s multiple range test.

Figure 2. H₂ protects Arabidopsis seedlings against salt stress-induced lipid peroxidation and ROS homeostasis.

Seedlings were pre-incubated in 50% H₂-saturated MS liquid medium for 24 hr, and then exposed to the MS liquid medium in the presence or absence of 150 mM NaCl. Sample without chemicals was the control (Con). Levels of lipid peroxidation (thiobarbituric acid reactive substance, TBARS) were measured at the indicated times (A). To detect O₂ ⁻ and H₂O₂, seedlings were stained with NBT (B) and DAB (C) 120 hr after various treatments, respectively. Bar  = 2 mm. (D) Transcript levels of zinc finger protein10 (ZAT10; At1g27730), zinc finger protein12 (ZAT12; At5g59820), cytosolic ascorbate peroxidase1 (cAPX1, At1g07890) and Fe superoxide dismutase1 (FSD1, Ag4g25100) after 120 hr of indicated treatments were analyzed by real-time RT-PCR. Expression levels were presented as values relative to corresponding untreated control samples (Con), after normalization to actin2/7 (At3g18780) levels. Statistical analysis was performed using SPSS 16.0 software. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at P<0.05 according to Duncan’s multiple range test.

Figure 3. Modulation of APX by H₂ and phenotypes of cAPX1 knockout plants.

(A) Changes of APX activity in Arabidopsis seedlings. (B) Up panel, stromal APX (sAPX) and cytoplasmic APX (cAPX) gene expression in Arabidopsis seedlings. Bottom panel, Coomassie Brilliant Blue-stained gels that showing equal amounts of proteins were loaded. The numbers above/below the band indicate the relative abundance of the corresponding sAPX/cAPX protein compared with that of the control sample. (C) Determination of total APX activity in 5-day-old wild-type and capx1 mutant seedlings. (D–F) Changes of fresh weight, chlorophyll content, and primary root growth of capx1 mutant seedlings. Corresponding samples without chemicals were regarded as a control (Con, 100%). 5-day-old wild-type and capx1 mutant seedlings were pretreated with or without 50% H₂-saturated aqueous solution for 24 hr, followed by the exposure to the liquid MS medium in the presence or absence of 150 mM NaCl for another 120 hr, and then phenotypic indicators were determined, respectively. The dashed lines denoted the inhibition rate of wild-type grown under NaCl stress, taking corresponding wild-type samples without chemicals as a 100%. Statistical analysis was performed using SPSS 16.0 software. Data are means ± SE from three independent experiments. Bars denoted by the different letters were different significantly at P<0.05 according to Duncan’s multiple range test (A, D-F). Additionally, the asterisk above the bar indicates significantly different in comparison with the wild-type at P<0.05 according to t test (C).

Figure 4. H₂ regulates ion homeostasis and phenotypes of sos1 knockout plants.

(A) Changes of Na/K ratio in Arabidopsis seedlings. (B) Relative gene expression of salt overly sensitive1 (SOS1; At2g01980), Arabidopsis H⁺-ATPase3 (AHA3; At5g57350) in Arabidopsis seedling roots. (C) Up panel, plasma membrane (PM) H⁺-ATPase protein level in Arabidopsis seedling roots. Bottom panel, Coomassie Brilliant Blue-stained gels that showing equal amounts of proteins were loaded. The number above the band indicates the relative abundance of the corresponding H⁺-ATPase_protein compared with that of the control sample. (D-F) Changes of fresh weight, chlorophyll content, and primary root growth of sos1 mutant seedlings. Corresponding samples without chemicals were regarded as a control (Con, 100%). 5-day-old wild-type and sos1 mutant seedlings were pretreated with or without 50% H₂-saturated aqueous solution for 24 hr, followed by the exposure to the liquid MS medium in the presence or absence of 150 mM NaCl for another 120 hr, and then phenotypic indicators were determined, respectively. The dashed lines denoted the inhibition rate of wild-type grown under NaCl, taking corresponding wild-type samples without chemicals as a 100%. Statistical analysis was performed using SPSS 16.0 software. Data are means ± SE from three independent experiments. The asterisk above the bar indicates significantly different in comparison with NaCl-treated alone sample at P<0.05 according to t test (A). Additionally, bars denoted by the different letters were different significantly at P<0.05 according to Duncan’s multiple range test (B, D–F).

Figure 5. Regulation of transcripts responsible for Na compartmentation by H₂.

(A, B) Relative gene expression of Arabidopsis V-type proton ATPase proteolipid subunit c4 (AVAP4; At1g75630), sodium hydrogen exchanger2 (NHX2; At3g05030), sodium hydrogen exchanger5 (NHX5; At1g54370), Arabidopsis vacuolar membrane proton pump1 (AVP1; At1g15690), sodium hydrogen exchanger1 (NHX1; At5g27150), and sodium hydrogen exchanger3 (NHX3; At5g55470) in Arabidopsis seedling roots or leaves, respectively. Seedlings were pre-incubated in 50% H₂-saturated MS liquid medium for 24 hr, and then exposed to the MS liquid medium in the presence or absence of 150 mM NaCl for anther 120 hr. Sample without chemicals was the control (Con). Plot key illustrated each bar shown in A and B. Statistical analysis was performed using SPSS 16.0 software. Data are means ± SE from three independent experiments. Differences among treatments were analyzed by one-way ANOVA, taking P<0.05 level as significant according to Duncan’s multiple range test.