Heat suppresses activation of an auxin-responsive promoter in cultured guard cell protoplasts of tree tobacco.

Cultured guard cell protoplasts (GCP) of tree tobacco (Nicotiana glauca) comprise a novel system for investigating the cell signaling mechanisms that lead to acquired thermotolerance and thermoinhibition. At 32 degrees C in a medium containing an auxin (1-naphthaleneacetic acid [NAA]) and a cytokinin (6-benzylaminopurine), GCP expand, regenerate cell walls, dedifferentiate, and divide. At 38 degrees C, GCP acquire thermotolerance within 24 h, but their expansion is limited and they neither regenerate walls nor reenter the cell cycle. These putative indicators of auxin insensitivity led us to hypothesize that heat suppresses induction of auxin-regulated genes in GCP. Protoplasts were transformed with BA-mgfp5-ER, in which the BA auxin-responsive promoter regulates transcription of mgfp5-ER encoding thermostable green fluorescent protein (GFP) or with a similar 35S-cauliflower mosaic virus constitutive promoter construct. Heat suppressed NAA-mediated activation of BA. After 21 h at 32 degrees C in media with NAA, 49.0% +/- 3.9% of BA-mgfp5-ER transformants strongly expressed GFP; expression percentages were similar to those of 35S-mgfp5-ER transformants at 32 degrees C or 38 degrees C. After 21 h at 38 degrees C in media with NAA, 7.9% +/- 1.6% of BA-mgfp5-ER transformants weakly expressed GFP, similar to GCP cultured at 32 degrees C in media lacking NAA. Expression at 38 degrees C was not increased by incubating for 48 h or increasing NAA concentrations 20-fold. After 9 to 12 h at 38 degrees C, BA was no longer activated when cells were transferred to 32 degrees C. Heat-stressed cells accumulate reactive oxygen species, and hydrogen peroxide (H(2)O(2)) suppresses auxin-responsive promoter activation in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. H(2)O(2) did not suppress BA activation at 32 degrees C, nor did superoxide and H(2)O(2) scavengers prevent BA suppression at 38 degrees C.