Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco

Abstract

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% 6 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% 6 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 (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°C, nor did superoxide and H 2 O 2 scavengers prevent BA suppression at 38°C.

Heat Reduces Nitric Oxide Production Required for Auxin-Mediated Gene Expression and Fate Determination in Tree Tobacco Guard Cell Protoplasts

“…Furthermore, indole-3-acetic acid acts in a fluctuating background of signaling molecules that are being metabolized, sequestered, and transported and for which there are tissue-specific receptors, signaling pathways, and modulators as well as redundant signaling systems. As one example, acting through a mitogen-activated protein kinase cascade, H 2 O 2 can prevent auxinresponsive gene expression in Arabidopsis and tobacco mesophyll protoplasts (Kovtun et al, 1998(Kovtun et al, , 2000, although our earlier studies provided no evidence for the involvement of H 2 O 2 in suppressed auxin signaling during sustained heat stress in cultured GCPs (Dong et al, 2007).…”

“…Thus, both heat and L-NMMA suppress auxin-responsive transgene expression in reporter-transformed GCPs ( Fig. 6; Table I; Dong et al, 2007), suggesting that many of the inhibitory phenomena observed under both treatments are likely due to failed NO-dependent auxin signaling.…”

“…Our previous studies indicated that 12 to 16 h of culture at 38°C were required before NAA-mediated BA activation was suppressed in all GCPs examined (Dong et al, 2007). While L-NMMA would be expected to act almost immediately, it could be argued that in heat experiments, long-lived transcripts produced early in the 38°C culture period could be responsible for the mGFP5-ER accumulation in 35S controls, masking inhibitory effects of heat on generalized transcription and translation processes.…”

“…In climate change studies, little attention has been given to plants like tree tobacco that have evolved to survive sustained bouts of high heat. While it is often assumed that increases in mean global temperatures will have little effect on these hardy plants, our previous studies with cultured tree tobacco guard cells (Roberts et al, 1995;Taylor et al, 1998;Gushwa et al, 2003;Dong et al, 2007) suggest that heat can disrupt growth hormone signaling in ways that drastically alter cell fate.…”

“…They do not regenerate cell walls (Taylor et al, 1998), reenter the cell cycle (Gushwa et al, 2003), dedifferentiate (Taylor et al, 1998), or divide (Roberts et al, 1995). Culturing tree tobacco GCPs for 16 h or more at 38°C suppresses activation of the auxin-responsive BA transgene promoter (Aspuria et al, 2002;Dong et al, 2007); BA activation is not suppressed when GCPs are cultured similarly at 32°C. We hypothesize that sustained heat interferes with the auxin signaling required for cell expansion, cell wall regeneration, cell cycle reentry, and cellular dedifferentiation processes that are prerequisites for cell cycle progression.…”