H(2)O(2), plasma membrane H(+)-ATPase (PM H(+)-ATPase) and salicylic acid (SA) play important roles in sensing external stimulation and activating defense responses in plants. However, it remains uncertain whether they are involved and interrelated in response to heat acclimation. Experiments were performed by pharmacological methods, and the relationship and the connection between endogenous H(2)O(2), free SA and PM H(+)-ATPase were investigated in pea plants (Pisum sativum L.) during heat acclimation. The results showed that an accumulation peaks of H(2)O(2), free SA and PM H(+)-ATPase, were detected during heat acclimation at 37 degrees C for 2 h and H(2)O(2) burst appeared before SA accumulation that followed by increase of PM H(+)-ATPase activity (Fig.1). Pretreatments with either scavengers of active oxygen species (dimethyl sulfoxide and ascorbic acid) or antioxidant (reduced glutathione) inhibited the increases in both H(2)O(2) and free SA contents as a part of heat acclimation (Fig.2). Additionally, changes in activity of plasma membrane NADPH oxidase paralleled with H(2)O(2) level during heat acclimation (Figs.1 and 3), implicating that H(2)O(2) might be generated by plasma membrane NADPH oxidase. Moreover, pretreatments with either diphenylene iodonium (DPI), a suicide substrate inhibitor of plasma membrane NADPH oxidase, or dimethylthiourea (DMTU), a quencher of H(2)O(2), could block the increase in free SA content and activity of plasma membrane NADPH oxidase as a part of heat acclimation (Fig.4). According to the assay described above, it is suggested that both H(2)O(2) and PM H(+)-ATPase participate in SA signaling that leads to the development of thermotolerance in pea plant, and H(2)O(2) functions upstream and PM H(+)-ATPase functions downstream of the SA signal. Also, the regulation mechanism of PM H(+)-ATPase activity was investigated, which showed that during heat acclimation, increase of PM H(+)-ATPase activity was independent of PM H(+)-ATPase amount and the enzyme activity may be modulated at post-translational level that may involve in reversible protein phosphorylation (Fig.5).