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

Hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) regulate processes such as growth, development, and stress responses via post-translational modifications that alter the structure, localization, and function of multiple cellular proteins. H2O2, NO, and H2S 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 H2O2. Here, we report on the development of a method for whole-plant live imaging of H2S that complements the H2O2 and NO methods we previously developed and can be used side-by-side with them. Using H2S 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 H2O2, NO and H2S, in wild type and different mutants, following HS, we further reveal that H2O2 accumulation could be uncoupled from H2S and NO accumulation, but that H2S and NO accumulation are mostly tied to each other, suggesting a close interplay between them. We also show that H2S accumulation following HS requires NO accumulation, and that mutants deficient in ASCORBATE PEROXIDASE 1 do not accumulate NO or H2S following HS. Our findings shed new light on the intricate relationships between H2O2, NO, and H2S in plants, and pave the way for future studies of these three key signalling molecules.