Jane Larkindale

Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.

To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.

Plants can acclimate rapidly to environmental conditions, including high temperatures. To identify molecular events important for acquired thermotolerance, we compared viability and transcript profiles of Arabidopsis thaliana treated to severe heat stress (45 degrees C) without acclimation or following two different acclimation treatments. Notably, a gradual increase to 45 degrees C (22 degrees C to 45 degrees C over 6 h) led to higher survival and to more and higher-fold transcript changes than a step-wise acclimation (90 min at 38 degrees C plus 120 min at 22 degrees C before 45 degrees C). There were significant differences in the total spectrum of transcript changes in the two treatments, but core components of heat acclimation were apparent in the overlap between treatments, emphasizing the importance of performing transcriptome analysis in the context of physiological response. In addition to documenting increases in transcripts of specific genes involved in processes predicted to be required for thermotolerance (i.e. protection of proteins and of translation, limiting oxidative stress), we also found decreases in transcripts (i.e. for programmed cell death, basic metabolism, and biotic stress responses), which are likely equally important for acclimation. Similar protective effects may also be achieved differently, such as prevention of proline accumulation, which is toxic at elevated temperatures and which was reduced by both acclimation treatments but was associated with transcript changes predicted to either reduce proline synthesis or increase degradation in the two acclimation treatments. Finally, phenotypic analysis of T-DNA insertion mutants of genes identified in this analysis defined eight new genes involved in heat acclimation, including cytosolic ascorbate peroxidase and the transcription factors HsfA7a (heat shock transcription factor A7a) and NF-X1.

The dehydration-responsive element binding protein (DREB)/C-repeat binding factor (CBF) family are the classical transcriptional regulators involved in plant responses to drought, salt and cold stress. Recently it was demonstrated that DREB2A is induced by heat stress (hs) and is a regulator of the hs response of Arabidopsis. Here we provide molecular insights into the regulation and function of hs transcription factor HsfA3. Among the 21 members of the Arabidopsis Hsf family, HsfA3 is the only Hsf that is transcriptionally induced during hs by DREB2A, and HsfA3 in turn regulates the expression of Hsp-encoding genes. This transcription factor cascade was reconstructed in transient GUS reporter assays in mesophyll protoplasts by showing that DREB2A could activate the HsfA3 promoter, whereas HsfA3 in turn was shown to be a potent activator on the promoters of Hsp genes. Direct binding to the corresponding promoters was demonstrated by electrophoretic mobility shift assays, and the involvement of HsfA3 in the hs response in vivo was shown directly by observation of reduced thermotolerance in HsfA3 mutant lines. Altogether these data demonstrate that HsfA3 is transcriptionally controlled by DREB2A and important for the establishment of thermotolerance.