Ethylene participates in strigolactone-triggered stomatal closure via Gα protein-activited hydrogen peroxide and hydrogen sulfide synthesis in Arabidopsis

The signaling cascade of strigolactone (SL)-regulated stomatal closure in Arabidopsis thaliana was investigated using pharmacological assays, fluorescence microscopy, spectrophotometry, gas chromatography, RT-PCR, and qRT-PCR. In wild-type plants, GR24 (a synthetic strigolactone analog)-induced stomatal closure was significantly inhibited by ethylene biosynthesis/perception inhibitors or Gα inhibitor pertussis toxin (PTX). GR24 obviously promoted closure in eto1-1 , cGα1 and wGα1 mutants, but failed to do so in mutants etr1-1 , etr1-3 , gpa1–1 and gpa1-2 . GR24 also upregulated 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) gene ACS and heterotrimeric G-protein α subunit 1 gene GPA1 transcript levels, showing that both ethylene synthesis and Gα activation were required for SL-induced stomatal closure. Ethylene biosynthesis/perception inhibitors significantly suppressed GR24-induced hydrogen peroxide (H 2 O 2 ) production, hydrogen sulfide (H 2 S) synthesis, and L-/D-cysteine desulfhydrase (L-/D-CDes) activity increase in wild-type plants. These responses occurred in eto1–1 mutant but not in etr1 mutants. ACC-induced stomatal closure, H 2 O 2 and H 2 S synthesis, and L-/D-CDes activity increase were obviously abolished in AtrbohD , AtrbohF , AtrbohD/F , Atl-cdes and Atd-cdes mutants, and exogenous H 2 O 2 or sodium hydrosulfide (NaHS) could significantly rescue the defect in etr1 mutants, showing that ethylene acted via H 2 O 2 and H 2 S synthesis in SL-induced stomatal closure. Furthermore, PTX obviously suppressed GR24-induced H 2 O 2 and H 2 S synthesis, and L-/D-CDes increase in the wild type. These responses persisted in cGα1 and wGα1 mutants but were absent in gpa1 mutants. Finally, cholera toxin (CTX)-induced stomatal closure and downstream responses required AtrbohD/F and AtL-/D-CDe s, and exogenous H 2 O 2 and H 2 S rescued GR24-induced closure in gpa1 mutants, suggesting that Gα activation acted upstream of H 2 O 2 and H 2 S synthesis in SL-induced stomatal closure. In contrast, ACC failed to rescue the defect in GR24-induced stomatal closure treated with PTX in the wild type and in gpa1 mutants. Notably, GR24 substantially increased ethylene production in both PTX-treated wild-type plants and in gpa1 mutants, indicating that ethylene functioned by activating Gα in SL-induced stomatal closure. In summary, SL induced ethylene biosynthesis, which activated Gα. Activated Gα then promoted H 2 O 2 production via AtrbohD/F and subsequent H 2 S synthesis via AtL-CDes/AtD-CDes, finally leading to stomatal closure.