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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco^1,[OA]

Willamette University, Department of Biology, Salem, Oregon 97301 (M.A.D., J.L.B., K.C., M.Q.D., K.M., G.T.); and Japan Atomic Energy Agency, 1233 Watanuki, Takasaki 370–1292, Japan (Y.O.)

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°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°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°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°C or 38°C. After 21 h at 38°C in media with NAA, 7.9% ± 1.6% of BA-mgfp5-ER transformants weakly expressed GFP, similar to GCP cultured at 32°C in media lacking NAA. Expression at 38°C was not increased by incubating for 48 h or increasing NAA concentrations 20-fold. After 9 to 12 h at 38°C, BA was no longer activated when cells were transferred to 32°C. Heat-stressed cells accumulate reactive oxygen species, and hydrogen peroxide (H₂O₂) suppresses auxin-responsive promoter activation in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. H₂O₂ did not suppress BA activation at 32°C, nor did superoxide and H₂O₂ scavengers prevent BA suppression at 38°C.

² Present address: Department of Energy Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI 48824.

³ Present address: University of Kentucky College of Medicine, Box 40, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536–0298.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gary Tallman (gtallman{at}willamette.edu).

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.104646

^* Corresponding author; e-mail gtallman{at}willamette.edu.

Received June 26, 2007; accepted August 13, 2007; published August 17, 2007.