Effect of high-temperature stress on the activity of key enzymes in the AsA-GSH cycle in ‘Yali’ pears

Abstract

Through treating fruits at higher temperatures, choosing natural fruits on different exposures on a tree canopy and collecting exclusively both sunburn and normal fruits, the metabolic patterns were studied by comparing the activity of key enzymes in the ascorbate-glutathione (AsA-GSH) cycle including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR). The results indicated that the activity of three enzymes increased under around less than 35°C acclimating conditions, with no significant changes in membrane electrolyte leakage. The activity of APX, MDAR, and GR rose during the initial stage when fruits were treated at 45°C but decreased significantly as treating time extended. A time response existed in APX, MDAR, and GR to high temperature. For example, APX activity reached the maximum when fruits were treated for 1 h, however, MDAR and GR showed the peak when fruits were treated for 3 h and 5 h, respectively, implying the possible acting sequence relationship of three enzymes in the AsA-GSH cycle. In view of the whole cycle, APX served as the first enzyme directly scavenging active oxygen species, followed by a series of chain reactions to regenerate ascorbic acid (AsA), and GR served as the last post in the cycle. Because various ecological conditions existed in a tree canopy, there was a significant difference in the enzymatic activity among fruits bearing on either exterior or interior canopy. A significantly higher activity of APX and MDAR was found with exterior fruits, compared with interior ones, which may be regarded as a frequent acclimation to high temperature and excessive sunlight.

Keywords

‘Yali’ pear / high-temperature stress / AsA-GSH cycle / antioxidant enzyme / membrane lipid superoxidation

Introduction

Pear fruit sunburn is a common disorder caused by higher fruit surface temperatures and brings out, more or less, a loss in worldwide pear production annually (Han and Jin, 2001; Chen et al., 2006). Many studies indicate that, under high-temperature stress conditions, a great amount of active oxygen species (AOS) could be generated in plant cells, which can trigger membrane lipid superoxidation in some cases, further destroying the membrane system (Guo et al., 2003; Yu and Lin, 2006; Ke and Yang, 2007; Zhang and Huang, 2007). Due to electrolyte leakage and cell contents oxidization, fruit peel frequently shows typical symptoms like brown or dark color. It has been proved that the AsA-GSH cycle is an important antioxidant system in plants, in which APX, MDAR, and GR are key enzymes that represent the initial, middle, and final posts in the whole cycle (Li et al., 2010). Therefore, the study on changing patterns of relevant enzymes in the AsA-GSH cycle under high temperature and excessive sunlight conditions is helpful to understand how these enzymes response to environmental stress. In recent years, a lot of researches have been conducted on AOS and its function on horticultural crops (Li et al., 2010), proving that at high-temperature stress, AOS contents could pile up quickly, which may result in a damage to the function of organelles, and simultaneously, many antioxidant enzymes, such as SOD, POD, APX, MDAR, GR, etc., are inactivated or even damaged (Ding et al., 2008; Zhang et al., 2008; Jin et al., 2009).

At present, the study on antioxidant stress with ‘Yali’ pears focuses mainly on postharvest fruits in storage (Lin et al., 2006; Yan et al., 2008; Zhang et al., 2009), and little has been conducted with growing fruits, and no report has been found yet on key enzymes in the AsA-GSH cycle at high-temperature stress especially with ‘Yali’ pears. The present paper aims at revealing the dynamic changes of APX, MDAR, and GR activity under natural or artificial high-temperature conditions, in order to better understand the antioxidant mechanism of ‘Yali’ fruits.

Materials and methods

Materials

The experiment was carried out from September, 2008, to April, 2010, at the Teaching and Demonstrating Garden, Agricultural University of Hebei and the Key Laboratory of Pomology, Ministry of Agriculture, the People’s Republic of China. Twelve year-old trees (Pyrus bretschneideri Rehd. cv:‘Yali’) on Duli rootstocks (Pyrus betulaefolia Bge.) were chosen as sample plants. Those bearing trees were strong in tree vigor with a conventional management, and all of the tested trees possessed a good light reception, uniform and moderate growth, appropriate fruit load, and even fruit distribution.

Experimental design

Comparison of enzymatic activity at different temperatures

In August, comparatively uniform bagged fruits were picked up and treated at 25, 35, and 45°C with a shade cover of black cloth above the fruits. An electro-heating standing-temperature incubator was used to maintain the designed temperatures for 1, 3, 5, and 7 h. To precisely evaluate the effect of different temperatures, pretreatment was made at a given temperature until fruit surface temperature reached the designed value, and then, time counting was started. During treatment, a multiple-point temperature sensor (Model: TH–74 manufactured by Shanghai Qiujing Instrument and Meter Plant, China) was used to monitor the fruit surface temperature. After heating treatment finished, fruits were peeled off with a sharp knife immediately (thickness: about 1-1.5 mm), and then, the peel was put into a -72°C refrigerator for assay. The experiment was done by a completely randomized design with three replicates.

Comparison of enzymatic activity between exterior and interior fruits on a canopy

Three sample trees were used for this trail. Three exposed and three shaded normal fruits with south-west exposure of a tree canopy from each sample tree were labeled earlier and harvested at 14: 00 in a sunny day of August. The peel on the exposed side of fruits was removed immediately in a laboratory, frozen in liquid nitrogen, and finally put into a -72°C refrigerator for assay.

Comparison of enzymatic activity between normal and browning fruits

Also, in August, five normal or browning fruits were specially picked from the field and brought back to the laboratory. The peeling on the exposed side of fruits was removed immediately, frozen in liquid nitrogen, and then put into a -72°C refrigerator for assay. The experiment was done by a completely randomized design with five replicates.

Assay for physiologic or biochemical indexes

The assay of relative electric conductivity was applied according to Zhang et al. (2002) and that of APX, MDAR, and GR activities was based on a method by Song (2005).

Data were statistically analyzed with the SPSS software to examine the significance among different treatments.

Results

Effect of high-temperature stress on membrane integrity and the activity of key enzymes in the AsA-GSH cycle

Effect of high temperatures on membrane integrity

As shown in Fig. 1, there was a significant difference (P<0.05) in electrolyte leakage among different treatments. There was no significant change in electric conductivity when fruit treated at either 25°C or 35°C. However, when fruits were heated and maintained at 45°C, a significant difference (P<0.05) in conductivity occurred as treating time extended although no substantial change appeared at the first hour. Compared with the control (25°C), the electrolyte leakage at 45°C for 3, 5, and 7 h increased by 32.9%, 26.62%, and 54.6%, respectively.

Comparison of APX activity at different temperatures

The APX activity in fruits varied with different temperatures (Fig. 2). No significant change in enzymatic activity was observed with fruits treated at 25°C during treatment. There was no significant change within 3 h when fruit temperature remained at 35°C, and thereafter, a remarkable rise in enzymatic activity took place with a significantly higher activity (P<0.05) exhibiting during treatment than that in the control. However, the enzymatic activity of fruits treated at 45°C showed a fluctuant change with the maximal value appearing at 1 h, significantly higher (P<0.05) than the control, and subsequently declined sharply and reached the minimum at 3 h, significantly lower (P<0.05) than at both 35°C and 25°C. Moreover, the increment of enzyme activity at 5 h was significantly higher (P<0.05) than the control, but there was no significance compared to fruits treated at 35°C. The enzymatic activity with treatment at 45°C for 7 h decreased to below the control level. Those results suggested that high-temperature stress at 45°C could induce the enzymatic activity to rise rapidly within 1 h, but 35°C high-temperature acclimation could induce enzymatic activity in fruits to increase for a longer time.

Comparison of MDAR activity at different temperatures

At different temperatures, the changes of MDAR activity in fruits were shown in Fig. 3. There was no significant change in enzymatic activity during treatment with fruits kept at 25°C. When fruits were subject to 35°C high temperature, the enzymatic activity exhibited a rising trend. Within 3 h, there was no significant difference in enzymatic activity compared to the treatment at 25°C, but thereafter, the activity increased quickly, significantly higher than the control (P<0.05). Under 45°C high-temperature stress, the enzymatic activity increased significantly before 3 h and then decreased to below the control level quickly.

Comparison of GR activity at different temperatures

There was no significant change in GR activity during treatment at 25°C. There was also no significant difference in GR activity within 1 h with fruits treated at 35°C as compared to those at 25°C, but thereafter, the activity increased quickly and significantly higher than the control (P<0.05). Fruits treated at 45°C showed a higher activity than the control (P<0.05) during initial stage as time prolonged, achieving the maximum at 5 h, and then declined to below the control level (Fig. 4).

Comparison of APX and MDAR activity between exterior and interior fruits

Under natural conditions, there was generally a great difference in sunlight reception and fruit surface temperatures among fruits on different exposures of a tree canopy, which resulted surely in a corresponding variation in fruit antioxidant ability. As shown in Fig. 5, APX and MDAR activity differed significantly between exposed and shaded fruits on a tree canopy, with a very significantly higher activity in exposed fruits than in shaded ones (P<0.01), increasing by 120% and 25%, respectively. This phenomenon may be explained as a result of frequent acclimation of exposed fruits to higher temperatures and excessive sunlight, and vice versa.

Comparison of key enzymes activity in the AsA-GSH cycle between normal and browning fruits

There was a significant difference (P<0.05) in APX, MDAR, and GR activity between normal and browning fruits (Fig. 6). The activity of three enzymes was consistently lower in browning fruits than in normal ones, decreasing by 16%, 18%, and 21%, respectively, and achieving significant (P<0.05) or very significant level (P<0.01). Under both high temperature and excessive sunlight stress, fruits showed browning symptoms, resulting from an abnormal accumulation in AOS amount that caused damage to membrane. Meanwhile, the antioxidant enzymes may be also severely affected at extremely higher temperatures. The browning fruits showed a very significant (P<0.01) electrolyte leakage (Fig. 7).

Discussion

High-temperature stress cannot only raise the AOS accumulating level in cells but also positively induce plants to establish a defensive system to avoid or lessen the damage caused by free radicals. The present experiment indicated that acclimation of fruits at 35°C could induce APX, MDAR, and GR activity to increase to some extent, enhance the membrane integrity of fruit surface cells, and decrease the electrolyte leakage. After the fruits encountered high-temperature stress at 45°C, APX, GR, and MDAR activity increased initially, but the time for peak activity occurrence varied with individual enzymes, with APX at 1 h, 33.9% higher than the control, and with MDAR and GR at 3 and 5 h, respectively, 10% and 19.4% higher than the control. The results suggested that there was a given sequence in the time response of APX, MDAR, and GR to high-temperature stress, which was related closely to their posts in the cycle (Li et al., 2010). In this experiment, although APX, MDAR, and GR activity showed a certain degree of increase at 25°C, the level of membrane lipid superoxidation in high-temperature treated fruits was significantly higher than that of the control after 3 h, showing that during initial stage of high-temperature treatment, the activity increase for each of the antioxidant enzymes was merely a kind of heat-shock and self-defensive response. However, as treating time extended, successive AOS accumulation in fruits inhabited the enzymatic activity and reduced plant defensive ability for itself on one hand and even actively attacked the membrane system on the other, incurring a membrane lipid superoxidation and, finally, resulting in fruit injury.

Zhang et al. (2004a) reported that there was a great difference in light reception and fruit surface temperatures on different exposures of a tree canopy, which would result in a corresponding change in fruit antioxidant ability (Zhang et al., 2004b). In the present experiment, it was found that there absolutely existed a great difference in antioxidant enzymes activity between exposed and shaded fruits, with higher APX and MDAR activity in exterior fruits than in interior ones, implying that a frequent acclimation of exposed fruits to high temperature, and excessive sunlight may be responsible for inducing a higher activity of antioxidant enzymes.

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