Abstract
Hydrogen sulfide (H2S) nowadays is regarded as a novel gaseous signaling molecule in plants during environmental stress response. The spermidine (Spd), an important plant growth regulator, plays critical roles in regulating stress tolerance in plant species. Objectives of this study were to examine whether Spd could act as a H2S signaling activator to regulate stress-responsive transcription factors (TFs) and antioxidant defense and to further explore the possible crosstalk among Spd-induced hydrogen peroxide (H2O2), nitric oxide (NO), and H2S signaling in leaves of white clover. Results showed that dehydration or exogenous application of Spd could significantly increase L/D-cysteine desulfhydrate (L/DCD) activities and then cause a quick accumulation of H2S, followed by significant improvement of antioxidant enzyme (SOD, CAT, GPOX, APX, GR, DHAR, and MDHAR) activities and transcript levels of TFs (bZIP37, bZIP107, DREB2, DREB4, and WRKY108715) and genes encoding antioxidant enzymes. The inhibitors (AOA and NH2OH) of L/DCD activities could effectively attenuate dehydration- or Spd-induced these effects in leaves. Additionally, dehydration-induced increase in H2S accumulation, L/DCD and antioxidant enzyme activities, and genes expression were remarkably abolished by the application of Spd biosynthesis inhibitor (DCHA), whereas exogenous Spd significantly alleviate the inhibitory effect of DCHA on these parameters under dehydration condition. This indicated that H2S which was derived from L/DCD pathway might act as a downstream signaling of Spd, and Spd-activated H2S signaling was involved in dehydration-regulated TFs and antioxidant defense associated with dehydration tolerance. Further analyses found that Spd-induced H2O2 production could not be attenuated by scavengers of NO and H2S, but the scavenger of H2O2 effectively inhibited Spd-induced increases in NO and H2S. The Spd-induced H2S signaling was also substantially inhibited by the application of the NO scavenger. The result implied that Spd-induced H2O2 could be an upstream signal molecule of NO and H2S, and the Spd-induced H2S might act as the downstream signaling of NO in leaves. These obtained results provide the basic evidence to understand Spd-regulated signal transduction in white clover.
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