Involvement of hydrogen peroxide, calcium, and ethylene in the induction of the alternative pathway in chilling-stressed Arabidopsis callus

The roles of ethylene, hydrogen peroxide (H2O2), and calcium in inducing the capacity of the alternative respiratory pathway (AP) under chilling temperature in Arabidopsis thaliana calli were investigated. Exposure of wild-type (WT) calli, but not the calli of ethylene-insensitive mutants, etr1-3 and ein2-1, to chilling led to a marked increase of the AP capacity and triggered a rapid ethylene emission and H2O2 generation. Increasing ethylene emission by applying 1-aminocyclopropane-1-carboxylic (an ethylene precursor) markedly enhanced the AP capacity in WT calli, but not in etr1-3 and ein2-1 calli, whereas suppressing ethylene emission by applying aminooxyacetic acid (an ethylene biosynthesis inhibitor) abolished the chilling-induced AP capacity in WT calli. Furthermore, exogenous H2O2 treatment increased the AP capacity in WT calli, but not in etr1-3 and ein2-1 calli, while both catalase (H2O2 scavenger) and diphenylene iodonium (DPI, an inhibitor of NADPH oxidase) completely inhibited the chilling-induced H2O2 generation and largely inhibited the chilling-induced AP capacity. Interestingly, the chilling-induced AP capacity was completely inhibited by DPI and EGTA (calcium chelator). Further investigation demonstrated that H2O2 and calcium induced ethylene emission under chilling stress. Ethylene modulated the chilling-induced increase of pyruvate content and the expression of alternative oxidase genes (AOX1a and AOX1c). Taken together, these results indicate that H2O2-, calcium-and ethylene-dependent pathways are required for chilling-induced increase in AP capacity. However, only ethylene is indispensable for the activation of the AP capacity.