The loss of RBOHD function modulates root adaptive responses to combined hypoxia and salinity stress in Arabidopsis

Salinity stress in nature is often accompanied by soil waterlogging. The constraints imposed by this additional stress have a profound effect on Na⁺ and Cl⁻ transport from roots to shoots, thus affecting homeostasis of some essential ions such as K⁺ or Ca²⁺ and influencing plant growth. The underlying mechanisms, however, remain largely unknown. In this study, we used a range of electrophysiological (ion flux measuring MIFE) and imaging (fluorescence dyes) techniques to investigate the role of Respiratory Burst Oxidase Homolog protein D (RBOHD) in Arabidopsis root responses to combined salinity and hypoxia stress. We found that combined stress causes more damage to plants than salinity stress alone, and the rbohD mutant is more sensitive to both treatments compared with wild type (WT). Mild hypoxia stress (root exposure to N₂-bullbed solution for 48 h) reduced detrimental impact of salinity on the magnitude of NaCl-induced K⁺ loss from the root in wild type; this effect, however, was not observed in rbohD mutant. In salt-treated plants, onset of hypoxia led to increased uptake of Na⁺ and Cl⁻ in plants lacking functional RBOHD protein but not in a wild type. The rbohD mutant lacked ability for stress-induced H₂O₂ production and accumulated more Ca²⁺ and Na⁺ than WT under both salinity and combined stress. These results suggested RBOHD plays an important role in the regulation of downstream Ca²⁺ signal and H₂O₂ production, thus affecting plant ionic homeostasis, and that the lack of functional RBOHD proteins compromises plant ability to minimise Na⁺ accumulation under salinity and combined stress.