Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid

Abstract

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.

Figure 1

Oxidative damage and survival in response to heating in Arabidopsis seedlings is affected by light and heat pretreatment. Graphs showing levels of TBARS (a and c) and survival (b and d). Arabidopsis plants were treated at different temperatures and conditions. In a and b, plants were treated 1 h at 40°C either with (white bars) or without (light-gray bars) a 1-h pretreatment a 30°C and compared with plants treated throughout at 20°C (dark-gray bars). TBARS/survival data is shown for 1, 2, and 3 d after end of 40° treatment. In c and d, plants were treated for 40°C for 1 h and allowed to recover either in the dark or in the light, and TBARS/survival was compared with control plants treated at 20°C throughout, 3 d after the end of the 40°C treatment. Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.

Figure 2

Effect of calcium channel blockers and calmodulin inhibitors on oxidative damage and survival in response to heating in Arabidopsis seedlings. Plants were pretreated in calcium channel blockers (TFP, verapimil, lanthanum chloride, or nifedipine) or calmodulin inhibitors (TFP or W7) before a 35°C treatment for 1 h. Graphs indicate TBARS levels (a) and survival (b) 3 d after the end of the 35°C treatment. Samples treated at 35°C (gray bars) were compared with controls kept at 20°C throughout (black bars). TFP was dissolved in ethanol and nifedipine in DMSO, whereas all other inhibitors were dissolved in water in Murashige and Skoog medium, and appropriate controls are presented. Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.

Figure 3

Effect of calcium, SA, ACC, and ABA on oxidative damage and survival in response to heating in Arabidopsis seedlings. Plants were pretreated with calcium, SA, ACC, or ABA before a 40°C treatment for 1 h. Graphs indicate TBARS levels (a) and survival (b) 3 d after the end of the 40°C treatment. Samples treated at 40°C (gray bars) were compared with controls kept at 20°C throughout (black bars). ABA was dissolved in ethanol, all other compounds were dissolved in water in Murashige and Skoog medium, and the appropriate control is presented. Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.

Figure 4

Effect of the etr-1 and abi-1 mutations on oxidative damage and survival in response to heating in Arabidopsis seedlings. Graphs showing levels of TBARS (a and c) and survival (b and d). Plants were given a 37°C treatment for 1 h. Graphs indicate TBARS levels (a and c) and survival (b and) 3 d after the end of the 37°C treatment. Samples treated at 37°C (gray bars) were compared with controls kept at 20°C throughout (black bars). The ethylene-insensitive mutant, etr-1, was compared with its background ecotype, Columbia (a and c), and the ABA-insensitive mutant, abi-1, was compared with its background ecotype, Landsberg erecta (b and d). Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.

Figure 5

Effect of nahG expression on oxidative damage and survival in response to heating in Arabidopsis seedlings. Plants were given a 37°C treatment for 1 h. Graphs indicate TBARS levels (a) and survival (b) 3 d after the end of the 37°C treatment. Samples treated at 37°C (gray bars) were compared with controls kept at 20°C throughout (black bars). The transgenic Arabidopsis line nahG was compared with its background ecotype. Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.

Figure 6

Cytosolic calcium responses of Arabidopsis seedlings in response to heating and recovery from heating. Photon counts emitted by individual plants heated either straight to 40°C for 1 h from 20°C (a) or to 40°C for 1 h after 30°C for 1 h (b). Time = 0 represents start time for heating (40°C [a] or 30°C [b]). Return to 20°C after heating occurred at 3,600 s (a) and 7,200 s (b). Traces are representative of the two different types of responses observed. The amount of reconstituted aequorin in each seedling was then measured and used to calibrate these measurements, and averages are presented (c) as total area under the calibrated peak, which is proportional to the [Ca²⁺]_cyt response in each case. Error bars represent se of mean over the 20 seedlings in each replicate.

Figure 7

Effect of calcium, calcium channel blockers, and calmodulin inhibitors during recovery to heating. In a and b, plants were treated with calcium channel blockers (TFP, verapamil, lanthanum chloride, or nifedipine) or calmodulin inhibitors (TFP or W7) 15 min before return to 20°C after a 1-h 35°C treatment. Graphs indicate TBARS levels (a) and survival (b) 3 d after the end of the 35°C treatment. Plants were left exposed to the antagonists for 6 h after the end of heating. Samples treated at 35°C (gray bars) were compared with controls kept at 20°C throughout (black bars). TFP was dissolved in ethanol and nifedipine in DMSO, whereas all other inhibitors were dissolved in water in Murashige and Skoog medium, and appropriate controls are presented. In c and d, plants were pretreated with calcium chloride 15 min before return to 20°C after a 40°C treatment. Graphs indicate TBARS levels (c) and survival (d) 3 d after the end of the 40°C treatment. Samples treated at 40°C (gray bars) were compared with controls kept at 20° throughout (black bars). Data shown are averages of five replicates. Error bars represent se of mean for these five replicates.