Early events in signalling high-temperature stress in tobacco BY2 cells involve alterations in membrane fluidity and enhanced hydrogen peroxide production

Alterations in membrane fluidity are among the early events in plants that detect changes in ambient temperature. However, signal transduction downstream of the membrane-associated processes is still not well understood. We have focused here on the role of hydrogen peroxide (H2O2) in high-temperature signalling in relation to changes in membrane fluidity in cells of tobacco (Nicotiana tabacum L.) cv. Bright Yellow 2 (BY2). As final indicators of the heat-signalling cascade, we have monitored the synthesis of small heat-shock proteins (sHSPs). Elevation of temperature between 32 and 38 degrees C resulted in a fast, transient stimulation of H2O2 production in the tobacco cells. A similar H2O2 burst could be induced at lower temperatures (28-32 degrees C) by membrane fluidization using benzyl alcohol (BA). Diphenylene iodonium (DPI), a NADPH oxidase inhibitor, prevented both the heat- and BA-triggered H2O2 rise. The synthesis of sHSPs (14.5 and 16 kDa) was shifted to lower temperatures by BA application and was suppressed by DPI treatment in the same way. The results indicate that H2O2 is an early component of the heat-signalling pathway, which responds rapidly to changes in membrane fluidity and is required for the activation of sHSP synthesis.