Aboveground whole-plant live H2S imaging method sheds new light on the relationships between H2S, NO, and H2O2

Hydrogen peroxide (H 2 O 2 ), nitric oxide (NO), and hydrogen sulfide (H 2 S) regulate processes such as growth, development, and stress responses via post-translational modifications that alter the structure, localization, and function of multiple cellular proteins. H 2 O 2 , NO, and H 2 S are also thought to regulate the levels of each other in cells via multiple pathways. Although several methods were developed for their imaging at the cell and tissue levels, whole-plant live imaging methods were only developed for NO and H 2 O 2 . Here, we report on the development of a method for whole-plant live imaging of H 2 S that complements the H 2 O 2 and NO methods we previously developed and can be used side-by-side with them. Using H 2 S donor and scavenger, the l -CYSTEINE DESULFHYDRASE 1 ( des1 ) mutant, as well as treatment of plants with heat stress (HS), or flg22, we confirmed the specificity and biological relevance of the method developed. Using side-by-side imaging of H 2 O 2 , NO and H 2 S, in wild type and different mutants, following HS, we further reveal that H 2 O 2 accumulation could be uncoupled from H 2 S and NO accumulation, but that H 2 S and NO accumulation are mostly tied to each other, suggesting a close interplay between them. We also show that H 2 S accumulation following HS requires NO accumulation, and that mutants deficient in ASCORBATE PEROXIDASE 1 do not accumulate NO or H 2 S following HS. Our findings shed new light on the intricate relationships between H 2 O 2 , NO, and H 2 S in plants, and pave the way for future studies of these three key signalling molecules.