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.