Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance

Abstract

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.

Figure 1.

Thermotolerance phenotype of mutants showing heat sensitivity as 7-d-old seedlings. Seedlings grown on agar plates in the light for 7 d were heated to 38°C for 90 min, cooled to room temperature for 120 min, then heated to 45°C for 180 min (acquired thermotolerance, gray bars) or heated to 45°C for 60 min (basal thermotolerance, white bars). Percentage of survival of plants relative to the wild-type control on the same plate was determined 5 d after heat stress. A, ABA biosynthesis mutants; B, ABA-insensitive mutants; C, SA signaling transgenic line and mutant; D, ethylene signaling mutants; E, NADPH oxidase mutants; F, antioxidant mutants; G, uvh6. Each experiment was performed on a minimum of five separate plates, each with at least 20 plants of each line, including the wild-type and hot1 controls. Further controls were performed using unheated plants and plants given only the 38°C pretreatment, and all of these plants survived (data not shown). Data for the hot1 controls in each section of the figure were derived from the same plates as the mutants in that section; the exact percentage of survival varied from plate to plate due to minor differences in temperature within the incubator. B does not show the hot1 control, as the abi1 and abi2 mutants are in the Ler background, while hot1 is in Col. Mutants in C and D were tested on the same plate, so the hot1 controls are identical. Error bars represent the sd from the average value over all experiments. WT, Wild type.

Figure 2.

Heat-induced oxidative damage in mutants with decreased thermotolerance. Plants were heat treated as described in Figure 1, and after 2 d of recovery, seedlings were harvested and stored in liquid nitrogen until the assay was performed. The TBARS level determined from the mutants relative to the wild-type control on each plate was determined. Values are graphed as percentage of greater than the wild type (WT; i.e. a mutant with 2 times the TBARS level seen in the wild type is recorded as a value of 100% greater than the wild type). A, ABA biosynthesis mutants; B, ABA-insensitive mutants; C, SA signaling mutant/transgenic line; D, ethylene signaling mutants; E, NADPH oxidase mutants; F, antioxidant mutants; G, uvh6. Experimental replication and controls were as described in Figure 1. Error bars represent the sd over all experiments.

Figure 3.

High light intensity during recovery increases heat sensitivity. Seven-day-old plants were heated on plates to 45°C for 30 min without pretreatment and allowed to recover under normal or high light conditions (100 or 250 μmol m⁻² s⁻¹) for 5 d. Averages of the survival of plants relative to the wild type (WT) control on each plate are shown. Repetition and controls were as described in Figure 1, with unheated samples being allowed to recover under either high or low light conditions. Survival of unheated control plants under both high and low light conditions was 100% (data not shown). Error bars represent the sd over all experiments.

Figure 4.

Heat stress phenotypes of uvh6 plants at different life stages. A, Germination of seeds after heating to 45°C for 220 min and scoring 1 week after heat treatment. B, Hypocotyl elongation of 2.5-d-old seedlings and 2.5 d recovery following a 38°C pretreatment, 120 min at room temperature, and 180 min heat stress at 45°C. C, Root elongation 5 d after heat stress for plants heated to 45°C for 180 min after pretreatment; plants were heated 4 d after germination. D, Photograph of 7-d plants heated and allowed to recover for 5 d. Plants were unheated (22), heated to 38°C for 90 min only (38), heated to 45°C for 180 min after pretreatment at 38°C (38–45), or heated directly to 45°C for 60 min (45). E, Photograph of leaves removed from 25-d plants and heated to 45°C for 180 min in a water bath and left to recover for 2 d. Error bars in A, B, and C represent the sd over five replicate experiments, each containing at least 10 plants of each line.

Figure 5.

HSP accumulation in mutant plants. Seedlings were treated at 38°C for 90 min and cooled to room temperature for 30 min prior to harvesting tissue for protein extraction. HSP levels were determined by SDS-PAGE followed by western blotting with antibodies against Hsp101 and class I and class II sHsps.

Figure 6.

Comparison of basal and acquired thermotolerance in selected mutants. The survival of 7-d plants (A) or root growth of 4-d plants (B) of each mutant was determined after progressively longer heat treatments. For basal thermotolerance, heat treatments were from 30 to 120 min at 45°C at 5-min intervals. For acquired thermotolerance, heat treatments were from 90 to 250 min at 10-min intervals following pretreatment. In each case, the time of heat treatment required to reduce survival or root growth to 50% of the unheated controls was determined. The data are presented as the additional time at 45°C required for 50% survival of that mutant as compared to hot1 (the most severe mutant). Error was ±10 min for acquired thermotolerance and ±5 min for basal thermotolerance.

Figure 7.

Framework for heat stress responses and thermotolerance in Arabidopsis. The different signaling components and protective factors postulated to be involved in thermotolerance are shown grouped into pathways where there is evidence that there are interactions between a specific signaling component and a particular end response. Question marks indicate some of the places in the schema where components remain unidentified. The areas highlighted in dark gray represent pathways shown to be critical for basal thermotolerance, those in white are critical for acquired thermotolerance, and areas shaded in light gray are critical for both. The pathways indicated by black arrows are critical for basal and acquired thermotolerance at all stages in development; white arrows indicate pathways critical only in 4-d plants and older after exposure to light. HSFs, heat shock transcription factors.