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Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period
Available online at www.sciencedirect.com Agricultural Sciences in China OCICNCC
2006, 5 ( 6 ) : 425-430
June 2006
/
Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period LIU Ping, GUO Wen-shan, PU Han-chun, FENG Chao-nian, ZHU Xin-kai and PENG Yong-xin Jiangsu Provincial Key Laboratory of Crop Genetics and PhysiologyNheat Research Instiiuie. Yangzhou University, Yangzhou 225009, P .R. China
Abstract On the basis of the phytotron, the effects of high temperature (daily average temperature 25, 30, 35 and 40°C,respectively) on antioxidant enzymes and lipid peroxidation in flag leaves of wheat at 50% relative air moisture during grain fastest filling stage [ 19-21 days after anthesis (DAA)] were studied. The wheat cultivars tested were Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. Compared with 25"C, the higher the temperature was, the higher was the MDA content in flag leaves, while lower were the SOD, POD, and CAT activities. SOD and CAT activities in Yangmai 12 appeared to be more sensitive to high temperature than that in Yangmai 9. But POD activity in Yangmai 12 was less sensitive to high temperature. MDA content in Yangmai 12 was higher than that in Yangmai 9. The 1000-grain weight declined with increase in temperature.
Key words: wheat, high temperature, grain filling period, antioxidant enzymes, lipid peroxidation, flag leaves
INTRODUCTION Wheat grain yield and quality were affected by the environment. Temperature was a major component in environmental variation and had a marked effect on grain filling for wheat. Short period of heat shock, being more than 30°C for a few days, had happenned frequently during grain filling period in many wheat-growing areas in China and several parts of the world, and had become an important factor limiting wheat yield and quality. High temperature hastened the decline in photosynthesis and leaf area, decreased shoot and grain mass as well as weight and sugar content of kernels (Shah and Paulsen 2003; Plaut et al. 2004). The endosperm cell proliferation rate was increased during a short period of high temperature, but the proliferation duration was significantly shortened, which resulted in
a decrease of the maximum endosperm cell number and a decline in the grain weight (Feng et al. 2000). High temperature stress during grain filling stage rapidly improved the MDA and Pro content in the flag leaf of the winter wheat (Zheng et al. 2001). High temperature increased flour protein percentage, but significantly decreased dough strength, measured as mixing time and resistance breakdown (Stone et al. 1997). The effects of high temperature on antioxidant enzymes and lipid peroxidation in flag leaves of wheat during grain filling period were extensively documented (Wang et al. 2004; Guo et al. 1998; Peng et al. 1995), while little was known about the effects of different high temperatures on wheat during grain filling period for different end uses. In this study, effects of four levels of high temperatures on antioxidant enzymes and lipid peroxidation in flag leaves in wheat of weak-gluten and mediumgluten during grain filling period were investigated us-
This paper is translated from its Chinese version in Scienria Agriculrura Sinica. LIU Ping, Ph D; Correspondence GUO Wen-shan. Tel: +86-5 14-7979300.E-mail: guows@yzu,edu.cn
(92006. CAAS. All r i g k mswwd. Published by E W r Ltd.
LIU Ping er al
426
ing a phytotron. Such information would be useful for understanding the mechanism of heat damage and alleviating plant senescence under high temperature stress in wheat for different end uses.
MATERIALS AND METHODS Experimental design The experiment was carried out in the agricultural experiment field of Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology in Yangzhou from 2003 to 2004. Four temperature levels, i.e., daily average temperature of 25,30,35 and 40°C with a range o f f OS'C, were set in a phytotron, the relative air moisture was set at 50% with a range o f f 1%, and the photosynthetic photo flux density was 800 pmol m-* s-I. The wheat cultivars tested were Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. The testing plants were raised in a pot of diameter 0.3 m, height 0.27 m, filled with 10 kg loamy soil. Twelve seeds of winter wheat were sown in each pot on 28 October 2003, and six plants were kept in each pot on 30 November. The aboveground morphology of the plants appeared to be very similar to field-grown plants. The wheat plants during grain fastest filling period (19-21 DAA) were treated at 50% relative air moisture from 8:OO am to 6:OO pm. The soil moisture was controlled at 65-75% of the largest water holding in the field. The date on which 50% of spikes in a pot flowered was recorded as the date of anthesis and the date on which grains attained maximum dry weight was recorded as the date of maturity. Spikes were hand-harvested at maturity. Each treatment had four replicated pots. After high temperature treatment, the pots were transferred to the normal environment. The temperatures referred to in this paper were air temperatures within the greenhouse.
Measurements and methods Two varieties had the same date of anthesis. The spikes that flowered in the same days were marked. Samplings were carried out on Oth, 1 st, 2nd and 10th day after treatment and maturity, respectively. Twelve plants of each treatment were sampled, which was frozen
immediately for enzyme analysis. Superoxide dismutase (SOD) activity was measured spectrophotometrically on the basis of inhibition in the photochemical reduction of nitroblue tetrazolium (NBT). The 1-mL reaction mixture contained 50 mM phosphate buffer (pH 7.8), 58 mM nitroblue tetrazolium, 2. 4 mM riboflavin and 9.9 mM methionine and 0.025% (v:v) Triton X-100. One unit of SOD is defined as the amount of enzyme that inhibited the nitroblue tetrazolium reduction by 50% (Shanghai Institute of Plant Physiology 1999). Peroxidase (POD) activity was determined at 470 nm for 3 min in a reaction mixture containing 50 mM sodium phosphate buffer (pH 6.9), 3.2 mM guaiacol and 0.4 mM H,O, (Shanghai Institute of Plant Physiology 1999). Catalase (CAT) activity was measured by following the consumption of H,O, at 240 nm. The 3-mL reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0) and 10 mM H,O, (Shanghai Institute of Plant Physiology 1999). For the measurement of lipid peroxidation, the thiobarbituic acid ( T B A ) test w a s a d o p t e d w h i c h d e t e r m i n e d malondialdehyde (MDA) as an end product of lipid peroxidation. For this, leaf tissues (500 mg) were homogenized in 3-mL 0. I % trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 2 500 x g for 10 min and the supernatant was assayed for MDA concentration (Shanghai Institute of Plant Physiology 1999).
RESULTS Effects of high temperature on antioxidant enzymes in flag leaves of wheat during grain filling period Effects of high temperature on SOD activity in flag leaves of wheat during grain filling period The experimental results showed that the SOD activity declined after high temperature treatment in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten (Table 1). SOD activity in flag leaves declined with the increase in temperature. But the extent of decrease varied with temperature. SOD activity in 40°C treatment declined rapidly. Compared with the value at 25"C, a decrease in SOD activity in flag leaves at 30, 35 and
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Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period
40°C was 43.32, 71.16 and 85.67%, respectively, in Yangmai 9 at maturity; while it was 51.13, 81.23 and 91.95%, respectively, in Yangmai 12 at maturity. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 o n SOD activity. These results indicated that the transient high temperature above 30°C markedly restrained SOD activity in flag leaves, moreover resistance to high temperature varied with cultivars. Effects of high temperature on POD activity in flag leaves of wheat during grain filling period As shown in Table 2, POD activity in flag leaves of wheat declined transiently on the first day after treatment, then increased transiently on the second day after treatment, thereafter decreased till maturity in Yangmai 9. POD activity in flag leaves of wheat declined transiently on the first day after treatment, then increased until the tenth day after treatment, thereafter decreased till maturity in Yangmai 12. It was an indication that the wheat was self-regulated to compensate for the stress, which was "heat shock answer". The higher the temperature was, the lower the POD activity in flag leaves would be
421
in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. But the extent of decrease depended on different temperature. POD activity in Yangmai 9 at maturity in flag leaves at 30, 35 and 40°C was, respectively, 84.16, 53.09 and 35.55% of that at 25"C, while in Yangmai 12 at maturity, it was 98.50, 95.60 and 41.40% of that at 2 5 ° C respectively. At temperatures above 30°C. there was a significant change in grain metabolism. Active oxygen in flag leaves enhanced, which resulted in the senescence sharply. Resistance to POD activity varied with high temperature and cultivars, and Yangmai 12 was more sensitive than was Yangmai 9 on POD activity. Effects of high temperature on CAT activity in flag leaves of wheat during grain filling period As shown in Table 3, CAT activity in flag leaves of plants treated at 25, 30 and 35°C declined first, then increased, decreased in succession till maturity; while that at 40°C declined continuously after high temperature treatment in Yangmai 9. CAT activity in flag leaves of wheat declined transiently on the first day after treatment, then increased transiently on the second day after treatment,
Table 1 Effects of temperature on SOD activity in flag leaves" (Unit g I FW h ') Yangmai 9
Yangmai 12
~
Treated temperature ("C)
Days after treatment (d) I
0
__.
25 30 35 40
825.70 b 868.25 a 840.48 ah 774.14 c
808.25 a 822.19 a 796.80 a 717.90 b
2 745.34 a 704.25 a 650.57 b 601.92 c
lo
Maturity
425.44 a 387.18 b 354.34 c 332.15 c
171.24 a 97.06 b 49.38 c 24.54 d
Days after treatment (d)
0 1001.10 a 918.35 b 83.5.54 c 806.15 c
2
I 860.90 a 871.98 a 797.22 b 770.70 b
766.93 780.16 727.61 713.72
Maturity
10
a a
b b
516.19 a 469.51 b 405.95 c 388.81 c
147.38 a 72.02 b 27.66 c 11.87 d
"The maturity dates of plants treated at 25. 30. 35.40"C were 47,46,43 and 41 days after anthesis, respectively. Values followed by a common letter within a column are not significantly different at the 0.05 probability level. The same as below.
Table 2 Effects of temperature on POD activity in flag leaves (Unit g-l FW min-I) Yangmai 9 Days after treatment (d)
Treated temperature ("C) 0
I
25
66.38 a
58.19 a
30 35 40
62.31 a 54.43 b 50.29 b
49.62 b 44.01 c 35.42 d
In
Maturity
n
a
65.29 a
ah
65.01 a 55.02 b 45.57 c
12.94 a 10.89 a 6.87 b 4.60 b
84.29 a 71.72 b 67.54 b
2
70.71 66.57 62.69 53.03
Yangmai 12 Days after treatment (d)
b c
70.68 b
I
58.02 54.16 48.40 45.05
1
a a b h
73.43 a 64.92 b 61.27 b 62.99 b
Maturity
I I,
81.63 72.18 69.15 70.01
a
b b
b
10.00 a 9.85 a 9.56 a 4.14 b
Table 3 Effects of temperature on CAT activity in flag leaves (Unit g-I FW min-I) Treated temperature ("C)
Yangmai 9
Yangmai I ?
Days after treatment (d)
Day5 after treatment (d) I 2
0 25 30 35
0.486 a 0.41 1 b 0.358 c
0.312 ab 0.336 a 0.284 bc
0.196 a 0.197 a 0.184 a
0.238 a 0.211 a 0.191 a
40
0.300 d
0.266 c
0.161 b
0.118h
0.079 a 0.052 b 0.040 b 0.027 b
0.610 a 0.564 b 0.463 c 0.450~
0.364 a 0.318 a 0.257 b 0.169~
0.421 a 0.350 b 0.349 b 0.227~
in 0.365 a 0.253 b 0.230 b 0.163 c
Maturity 0.174 0.055 0.038 0.017
a b b b
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decreased in succession till maturity in Yangmai 12. The higher the temperature was, the lower was the CAT activity in flag leaves in both cultivars. Resistance to CAT activity varied with high temperature, and with cultivars. Compared to the value at 25"C, the decrease in CAT activity in flag leaves at 30, 35 and 40°C was 33.17,49.46 and 65.76%, respectively in Yangmai 9 at maturity, while in Yangmai 12 at maturity, it was 68.39, 78.20 and 90.04%,respectively. Yangmai 12 was more sensitive to high temperature than Yangmai 9 on CAT activity.
Effects of high temperature on lipid peroxidation in flag leaves of wheat during grain filling period The experimental results showed that MDA content in flag leaves of wheat reached minimum on the first day after treatment, then increased at maturity in Yangmai 9 and Yangmai 12 (Table 4). It might be due to some protective mechanism in the endosperm, which may protect the enzyme at elevated temperatures. The trend that the higher the temperature, the higher the MDA content in flag leaves would be in both cultivars. But the extent of enhancement depended on different
temperature. High temperature hastened the lipid peroxidation and senescence of the plant. Compared with the value at 25"C, the enhancement of MDA content in flag leaves at 30, 35 and 40°C was 4.88, 29.55 and 36.09%, respectively, in Yangmai 9 at maturity; while in Yangmai 12 at maturity, it was 9.25,33.12 and 42.10%, respectively. It followed that MDA content had different sensitivity to different high temperature stresses. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 on MDA content. These results indicated that resistance to MDA activity varied with high temperature and resistance to high temperature varied with cultivars.
Effects of high temperatureon 1 000-grain weight during grain filling period The experimental results showed that 1 000-grain weight and grain yield per spike decreased with increase of temperature at grain fastest filling stage (19-21 DAA) in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten (Table 5). The 1 000-grain weight declined due to the decrease in SOD, POD, CAT activity and increase in MDA content.
Table 4 Effects of temperature on MDA content in flag leaves (nmol g-' FW) Treated temperature ("C) 0 25 30 35 40
244.88 b 258.93 ab 277.74 a 284.01 a
Yangmai 9 Days aftermatment (d) 1 2 171.36 c 166.12 c 200.10 b 229.68 a
192.11 c 201.02 c 235.19 b 251.85 a
10
256.12 c 264.62 c 293.91 b 328.76 a
Maturity 383.22 b 401.92 b 496.45 a 521.53 a
0
166.43 a 172.25 a 178.81 a 180.26 a
Yangmai 12 Days after treatment (d) 1 2 83.52 b 103.00 b 128.32 a 138.34 a
176.01 d 193.56 c 231.79 b 256.22 a
10
Maturity
191.98 c 236.61 b 340.35 a 360.26 a
380.38 d 415.56 c 506.35 b 540.54 a
Table 5 Effects of high temperature on 1 000-grain weight during grain filling period Yangmai 9 Treated temperature ("C) 2s _.
30 35 40
1 000-grain weight (g) 35.04 a
Grain yield (glspike) 1.37 a
32.69 b 31.43 b 30.59 b
DISCUSSION The physiological response of wheat was dependent on the environmental conditions. High temperature was one of the major constraints in the direction of increas-
1.28 b 1.21 bc 1.16 c
Yangmai 12 I 000-grain weight (g) Grain yield (glspike) 41.37 a ! 46 a 41.04 a 39.68 b 31.65 c
1.44 a 1.36 b 1.28 c
ing wheat productivity. Heat stress reduced the grain weight through a reduction in grain growth duration and grain growth rate. The increased temperature resulted in poorly filled, shriveled grain. High temperature also hastened the senescence, curtailed grain de-
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Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period
velopment and had drastic effects on the carbon and nitrogen metabolism of the plants. At high temperature, the assimilating efficiency of flag leaf was reduced and the transportation of 14C-assimilatewas disordered. The accumulation of assimilate in grain was inhibited, which resulted in a significant decrease of grain weight (Guo et al. 1998). High temperature also increased the MDA content, the membrane permeability and active oxygen of leaves of wheat (Guo rt al. 1998). The results in this study were as follows. SOD, POD, CAT activities and MDA content had different sensitivity to different high temperature stress. Different cultivars had also different sensitivity to different high temperature stress. The higher the temperature, the faster was the senescence in both cultivars. The higher the temperature, the lower the SOD, POD, and CAT activities were, while the higher was the MDA content in flag leaves, which was in line with the viewpoints expressed by Guo et al. (1998) and Zheng and He (1 999). The effects of different high temperature during grain filling period on antioxidant enzymes and lipid peroxidation in flag leaves in wheat for different end uses have been reported less in the literature. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 on SOD, CAT activities and MDA content, Yangmai 9 appeared to be more sensitive to high temperature than Yangmai 12 on POD activity. These results indicated that resistance to antioxidant enzymes and lipid peroxidation varied with high temperature and resistance to high temperature varied with cultivars due to different susceptibility of enzyme activity in flag leaves of different cultivars under different heat shock conditions. The plant resistance to stress effect was determined by the level of antioxidant enzyme activities and the ability to rapidly increase these activities. There was an indication that wheat at 2530°C was self-regulated to compensate for the disadvantage effect when the soil moisture was controlled at 65-75% of the largest water holding in the field. It might have some protective mechanism in the endosperm, which may modulate activities of antioxidant enzyme and protect the antioxidant enzyme in response to elevated temperatures. It may have the compensative growth function after heat shock. These results suggested that there could be a significant change in grain metabolism when temperature was above 30°C. The
429
metabolism of plants was turbulence and the self-regulated ability was low. The antioxidant enzymes were feeble and the lipid peroxidation increased. The plant was premature and the filling duration was shortened. It had been well documented that the effects of high temperature during grain filling period on yield and quality were also linked to soil temperature and soil moisture. The interaction between temperature and drought stress should be needed to additional studies. The mechanisms by which temperature affects the plant of wheat and the remedy measures are unclear so far, which is worthy to be studied deeply.
CONCLUSIONS The following conclusions were supported by this study. (1) The higher the temperature, the lower were the SOD, POD, CAT activities in flag leaves and IOOOgrain weight, while higher was the MDA content in flag leaves when temperature was more than 25°C. Higher MDA content in flag leaves was closely related to the decline in activities of antioxidant enzymes. (2) The antioxidant enzymes and lipid peroxidation in flag leaves have different sensitivity in response to different high temperature stress. Different cultivars had also different sensitivity in response to different high temperature stress. SOD and CAT activities and MDA content in Yangmai 12 appeared to be more sensitive to high temperature than those in Yangmai 9. But POD activity in Yangmai 12 was less sensitive to high temperature than that in Yangmai 9.
Acknowledgements This research work was financially supported by the National Natural Science Foundation of China (3057109 1, 30 170540).
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Sinica,26,399-405. (in Chinese) Guo T C, Wang C Y, Zhu Y J, Wang H C, Li J X, Zhou J Z. 1998. Effects o f high temperature on the senescene of root and top-partial of wheat plant in the later stage. Acra Agronornica
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Sinica, 24,957-962. (in Chinese)
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and lipid peroxidation and its regulation. Acta Agriculturae Universitatis Henanensis, 29, 242-245. (in Chinese)
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Plaut Z, Butow B J, Blumenthal C S. Wrigley C W. 2004. Transport of dry matter into developing wheat kernels and
its contribution to grain yield under post-anthesis water deficit and elevated temperature. Field Crops Research, 86, 185198. Shah N H, Paulsen G M. 2003. Interaction of drought and high temperature on photosynthesis and grain-filling of wheat. Plant and Soil, 257,219-226. Shanghai Institute of Plant Physiology, CAS, Shanghai Association of Plant Physiology. 1999. A Laboratory Manual
Zheng F, He Z P. 1999. Effect of high temperature stress on transportation and distribution of 14C-assimilates in grain filling period of winter wheat. Journal of China Agricultural University, 4, 73-76. (in Chinese) Zheng F, Zang X W, Huang B R, He Z P. 2001. Effects of high temperature stress on the source and sink organ of winter wheat during fillingstage and its regulation. Acta Agriculturae Boreali-Sinica, 16.99-103. (in Chinese) (Edited by ZHANG Yi-min)
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For the Vo1.5 No.5 May 2006, entitled “Induction of Root Hair Growth in a Phosphorus-Buffered Culture Solution”, authored by LIU Guodong et al. Abstract, Line 6: Change “and ryegrass (Loliurn rigidurn L.) (var. Ruanni), and the dicotyledons” to “ryegrass (Lolium rigidurn L.) (var. Ruanui), and onion (Allium cepa L.) (var. White Lisbon) and the dicotyledons”. Page 371, Section of Plant Materials, Line 5 : Change “(var. Ruanni)” to “(var. Ruanui)”. Page 371, Section of Field Crop Plants, Line 11: Delete “(better: with NaNO, as N source)”. Page 371, Section of Model Plants, Line 3: Change “the above nutrient recipe” to “ the above nutrients”. Page 371, Section of Model Plants, Line 10: Change “1OOx 15” to “90x 15”. Page 371, Section of Depletion Curve, Line 10: Change “0.5 mL 3%” to “0.25 mL 3%”. Page 372, Left column, Lines 19 and 33: Change “an hydroponic” to “a hydroponic”. Page 374, Fig.3, Line 1: Change “tri-calcium” to “tri-calcium phosphate”. Page 375, Line 1 1 of left column and Line 1 of right column: Change “hydrocarbon” to “carbohydrates”. Page 376, Acknowledgements, Lines 3-4: Change “grant numbers” to “grant number”; Line 8: Delete “of this paper in English”. Page 376, References, right column: Change “p.Part V-94” to “p. 5-94” in Ref. Lide D R. 1998.
02006.CMS. All ri~hts reserved. Publishedby Elsevier LM.
2006, 5 ( 6 ) : 425-430
June 2006
/
Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period LIU Ping, GUO Wen-shan, PU Han-chun, FENG Chao-nian, ZHU Xin-kai and PENG Yong-xin Jiangsu Provincial Key Laboratory of Crop Genetics and PhysiologyNheat Research Instiiuie. Yangzhou University, Yangzhou 225009, P .R. China
Abstract On the basis of the phytotron, the effects of high temperature (daily average temperature 25, 30, 35 and 40°C,respectively) on antioxidant enzymes and lipid peroxidation in flag leaves of wheat at 50% relative air moisture during grain fastest filling stage [ 19-21 days after anthesis (DAA)] were studied. The wheat cultivars tested were Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. Compared with 25"C, the higher the temperature was, the higher was the MDA content in flag leaves, while lower were the SOD, POD, and CAT activities. SOD and CAT activities in Yangmai 12 appeared to be more sensitive to high temperature than that in Yangmai 9. But POD activity in Yangmai 12 was less sensitive to high temperature. MDA content in Yangmai 12 was higher than that in Yangmai 9. The 1000-grain weight declined with increase in temperature.
Key words: wheat, high temperature, grain filling period, antioxidant enzymes, lipid peroxidation, flag leaves
INTRODUCTION Wheat grain yield and quality were affected by the environment. Temperature was a major component in environmental variation and had a marked effect on grain filling for wheat. Short period of heat shock, being more than 30°C for a few days, had happenned frequently during grain filling period in many wheat-growing areas in China and several parts of the world, and had become an important factor limiting wheat yield and quality. High temperature hastened the decline in photosynthesis and leaf area, decreased shoot and grain mass as well as weight and sugar content of kernels (Shah and Paulsen 2003; Plaut et al. 2004). The endosperm cell proliferation rate was increased during a short period of high temperature, but the proliferation duration was significantly shortened, which resulted in
a decrease of the maximum endosperm cell number and a decline in the grain weight (Feng et al. 2000). High temperature stress during grain filling stage rapidly improved the MDA and Pro content in the flag leaf of the winter wheat (Zheng et al. 2001). High temperature increased flour protein percentage, but significantly decreased dough strength, measured as mixing time and resistance breakdown (Stone et al. 1997). The effects of high temperature on antioxidant enzymes and lipid peroxidation in flag leaves of wheat during grain filling period were extensively documented (Wang et al. 2004; Guo et al. 1998; Peng et al. 1995), while little was known about the effects of different high temperatures on wheat during grain filling period for different end uses. In this study, effects of four levels of high temperatures on antioxidant enzymes and lipid peroxidation in flag leaves in wheat of weak-gluten and mediumgluten during grain filling period were investigated us-
This paper is translated from its Chinese version in Scienria Agriculrura Sinica. LIU Ping, Ph D; Correspondence GUO Wen-shan. Tel: +86-5 14-7979300.E-mail: guows@yzu,edu.cn
(92006. CAAS. All r i g k mswwd. Published by E W r Ltd.
LIU Ping er al
426
ing a phytotron. Such information would be useful for understanding the mechanism of heat damage and alleviating plant senescence under high temperature stress in wheat for different end uses.
MATERIALS AND METHODS Experimental design The experiment was carried out in the agricultural experiment field of Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology in Yangzhou from 2003 to 2004. Four temperature levels, i.e., daily average temperature of 25,30,35 and 40°C with a range o f f OS'C, were set in a phytotron, the relative air moisture was set at 50% with a range o f f 1%, and the photosynthetic photo flux density was 800 pmol m-* s-I. The wheat cultivars tested were Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. The testing plants were raised in a pot of diameter 0.3 m, height 0.27 m, filled with 10 kg loamy soil. Twelve seeds of winter wheat were sown in each pot on 28 October 2003, and six plants were kept in each pot on 30 November. The aboveground morphology of the plants appeared to be very similar to field-grown plants. The wheat plants during grain fastest filling period (19-21 DAA) were treated at 50% relative air moisture from 8:OO am to 6:OO pm. The soil moisture was controlled at 65-75% of the largest water holding in the field. The date on which 50% of spikes in a pot flowered was recorded as the date of anthesis and the date on which grains attained maximum dry weight was recorded as the date of maturity. Spikes were hand-harvested at maturity. Each treatment had four replicated pots. After high temperature treatment, the pots were transferred to the normal environment. The temperatures referred to in this paper were air temperatures within the greenhouse.
Measurements and methods Two varieties had the same date of anthesis. The spikes that flowered in the same days were marked. Samplings were carried out on Oth, 1 st, 2nd and 10th day after treatment and maturity, respectively. Twelve plants of each treatment were sampled, which was frozen
immediately for enzyme analysis. Superoxide dismutase (SOD) activity was measured spectrophotometrically on the basis of inhibition in the photochemical reduction of nitroblue tetrazolium (NBT). The 1-mL reaction mixture contained 50 mM phosphate buffer (pH 7.8), 58 mM nitroblue tetrazolium, 2. 4 mM riboflavin and 9.9 mM methionine and 0.025% (v:v) Triton X-100. One unit of SOD is defined as the amount of enzyme that inhibited the nitroblue tetrazolium reduction by 50% (Shanghai Institute of Plant Physiology 1999). Peroxidase (POD) activity was determined at 470 nm for 3 min in a reaction mixture containing 50 mM sodium phosphate buffer (pH 6.9), 3.2 mM guaiacol and 0.4 mM H,O, (Shanghai Institute of Plant Physiology 1999). Catalase (CAT) activity was measured by following the consumption of H,O, at 240 nm. The 3-mL reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0) and 10 mM H,O, (Shanghai Institute of Plant Physiology 1999). For the measurement of lipid peroxidation, the thiobarbituic acid ( T B A ) test w a s a d o p t e d w h i c h d e t e r m i n e d malondialdehyde (MDA) as an end product of lipid peroxidation. For this, leaf tissues (500 mg) were homogenized in 3-mL 0. I % trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 2 500 x g for 10 min and the supernatant was assayed for MDA concentration (Shanghai Institute of Plant Physiology 1999).
RESULTS Effects of high temperature on antioxidant enzymes in flag leaves of wheat during grain filling period Effects of high temperature on SOD activity in flag leaves of wheat during grain filling period The experimental results showed that the SOD activity declined after high temperature treatment in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten (Table 1). SOD activity in flag leaves declined with the increase in temperature. But the extent of decrease varied with temperature. SOD activity in 40°C treatment declined rapidly. Compared with the value at 25"C, a decrease in SOD activity in flag leaves at 30, 35 and
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Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period
40°C was 43.32, 71.16 and 85.67%, respectively, in Yangmai 9 at maturity; while it was 51.13, 81.23 and 91.95%, respectively, in Yangmai 12 at maturity. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 o n SOD activity. These results indicated that the transient high temperature above 30°C markedly restrained SOD activity in flag leaves, moreover resistance to high temperature varied with cultivars. Effects of high temperature on POD activity in flag leaves of wheat during grain filling period As shown in Table 2, POD activity in flag leaves of wheat declined transiently on the first day after treatment, then increased transiently on the second day after treatment, thereafter decreased till maturity in Yangmai 9. POD activity in flag leaves of wheat declined transiently on the first day after treatment, then increased until the tenth day after treatment, thereafter decreased till maturity in Yangmai 12. It was an indication that the wheat was self-regulated to compensate for the stress, which was "heat shock answer". The higher the temperature was, the lower the POD activity in flag leaves would be
421
in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. But the extent of decrease depended on different temperature. POD activity in Yangmai 9 at maturity in flag leaves at 30, 35 and 40°C was, respectively, 84.16, 53.09 and 35.55% of that at 25"C, while in Yangmai 12 at maturity, it was 98.50, 95.60 and 41.40% of that at 2 5 ° C respectively. At temperatures above 30°C. there was a significant change in grain metabolism. Active oxygen in flag leaves enhanced, which resulted in the senescence sharply. Resistance to POD activity varied with high temperature and cultivars, and Yangmai 12 was more sensitive than was Yangmai 9 on POD activity. Effects of high temperature on CAT activity in flag leaves of wheat during grain filling period As shown in Table 3, CAT activity in flag leaves of plants treated at 25, 30 and 35°C declined first, then increased, decreased in succession till maturity; while that at 40°C declined continuously after high temperature treatment in Yangmai 9. CAT activity in flag leaves of wheat declined transiently on the first day after treatment, then increased transiently on the second day after treatment,
Table 1 Effects of temperature on SOD activity in flag leaves" (Unit g I FW h ') Yangmai 9
Yangmai 12
~
Treated temperature ("C)
Days after treatment (d) I
0
__.
25 30 35 40
825.70 b 868.25 a 840.48 ah 774.14 c
808.25 a 822.19 a 796.80 a 717.90 b
2 745.34 a 704.25 a 650.57 b 601.92 c
lo
Maturity
425.44 a 387.18 b 354.34 c 332.15 c
171.24 a 97.06 b 49.38 c 24.54 d
Days after treatment (d)
0 1001.10 a 918.35 b 83.5.54 c 806.15 c
2
I 860.90 a 871.98 a 797.22 b 770.70 b
766.93 780.16 727.61 713.72
Maturity
10
a a
b b
516.19 a 469.51 b 405.95 c 388.81 c
147.38 a 72.02 b 27.66 c 11.87 d
"The maturity dates of plants treated at 25. 30. 35.40"C were 47,46,43 and 41 days after anthesis, respectively. Values followed by a common letter within a column are not significantly different at the 0.05 probability level. The same as below.
Table 2 Effects of temperature on POD activity in flag leaves (Unit g-l FW min-I) Yangmai 9 Days after treatment (d)
Treated temperature ("C) 0
I
25
66.38 a
58.19 a
30 35 40
62.31 a 54.43 b 50.29 b
49.62 b 44.01 c 35.42 d
In
Maturity
n
a
65.29 a
ah
65.01 a 55.02 b 45.57 c
12.94 a 10.89 a 6.87 b 4.60 b
84.29 a 71.72 b 67.54 b
2
70.71 66.57 62.69 53.03
Yangmai 12 Days after treatment (d)
b c
70.68 b
I
58.02 54.16 48.40 45.05
1
a a b h
73.43 a 64.92 b 61.27 b 62.99 b
Maturity
I I,
81.63 72.18 69.15 70.01
a
b b
b
10.00 a 9.85 a 9.56 a 4.14 b
Table 3 Effects of temperature on CAT activity in flag leaves (Unit g-I FW min-I) Treated temperature ("C)
Yangmai 9
Yangmai I ?
Days after treatment (d)
Day5 after treatment (d) I 2
0 25 30 35
0.486 a 0.41 1 b 0.358 c
0.312 ab 0.336 a 0.284 bc
0.196 a 0.197 a 0.184 a
0.238 a 0.211 a 0.191 a
40
0.300 d
0.266 c
0.161 b
0.118h
0.079 a 0.052 b 0.040 b 0.027 b
0.610 a 0.564 b 0.463 c 0.450~
0.364 a 0.318 a 0.257 b 0.169~
0.421 a 0.350 b 0.349 b 0.227~
in 0.365 a 0.253 b 0.230 b 0.163 c
Maturity 0.174 0.055 0.038 0.017
a b b b
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decreased in succession till maturity in Yangmai 12. The higher the temperature was, the lower was the CAT activity in flag leaves in both cultivars. Resistance to CAT activity varied with high temperature, and with cultivars. Compared to the value at 25"C, the decrease in CAT activity in flag leaves at 30, 35 and 40°C was 33.17,49.46 and 65.76%, respectively in Yangmai 9 at maturity, while in Yangmai 12 at maturity, it was 68.39, 78.20 and 90.04%,respectively. Yangmai 12 was more sensitive to high temperature than Yangmai 9 on CAT activity.
Effects of high temperature on lipid peroxidation in flag leaves of wheat during grain filling period The experimental results showed that MDA content in flag leaves of wheat reached minimum on the first day after treatment, then increased at maturity in Yangmai 9 and Yangmai 12 (Table 4). It might be due to some protective mechanism in the endosperm, which may protect the enzyme at elevated temperatures. The trend that the higher the temperature, the higher the MDA content in flag leaves would be in both cultivars. But the extent of enhancement depended on different
temperature. High temperature hastened the lipid peroxidation and senescence of the plant. Compared with the value at 25"C, the enhancement of MDA content in flag leaves at 30, 35 and 40°C was 4.88, 29.55 and 36.09%, respectively, in Yangmai 9 at maturity; while in Yangmai 12 at maturity, it was 9.25,33.12 and 42.10%, respectively. It followed that MDA content had different sensitivity to different high temperature stresses. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 on MDA content. These results indicated that resistance to MDA activity varied with high temperature and resistance to high temperature varied with cultivars.
Effects of high temperatureon 1 000-grain weight during grain filling period The experimental results showed that 1 000-grain weight and grain yield per spike decreased with increase of temperature at grain fastest filling stage (19-21 DAA) in Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten (Table 5). The 1 000-grain weight declined due to the decrease in SOD, POD, CAT activity and increase in MDA content.
Table 4 Effects of temperature on MDA content in flag leaves (nmol g-' FW) Treated temperature ("C) 0 25 30 35 40
244.88 b 258.93 ab 277.74 a 284.01 a
Yangmai 9 Days aftermatment (d) 1 2 171.36 c 166.12 c 200.10 b 229.68 a
192.11 c 201.02 c 235.19 b 251.85 a
10
256.12 c 264.62 c 293.91 b 328.76 a
Maturity 383.22 b 401.92 b 496.45 a 521.53 a
0
166.43 a 172.25 a 178.81 a 180.26 a
Yangmai 12 Days after treatment (d) 1 2 83.52 b 103.00 b 128.32 a 138.34 a
176.01 d 193.56 c 231.79 b 256.22 a
10
Maturity
191.98 c 236.61 b 340.35 a 360.26 a
380.38 d 415.56 c 506.35 b 540.54 a
Table 5 Effects of high temperature on 1 000-grain weight during grain filling period Yangmai 9 Treated temperature ("C) 2s _.
30 35 40
1 000-grain weight (g) 35.04 a
Grain yield (glspike) 1.37 a
32.69 b 31.43 b 30.59 b
DISCUSSION The physiological response of wheat was dependent on the environmental conditions. High temperature was one of the major constraints in the direction of increas-
1.28 b 1.21 bc 1.16 c
Yangmai 12 I 000-grain weight (g) Grain yield (glspike) 41.37 a ! 46 a 41.04 a 39.68 b 31.65 c
1.44 a 1.36 b 1.28 c
ing wheat productivity. Heat stress reduced the grain weight through a reduction in grain growth duration and grain growth rate. The increased temperature resulted in poorly filled, shriveled grain. High temperature also hastened the senescence, curtailed grain de-
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Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period
velopment and had drastic effects on the carbon and nitrogen metabolism of the plants. At high temperature, the assimilating efficiency of flag leaf was reduced and the transportation of 14C-assimilatewas disordered. The accumulation of assimilate in grain was inhibited, which resulted in a significant decrease of grain weight (Guo et al. 1998). High temperature also increased the MDA content, the membrane permeability and active oxygen of leaves of wheat (Guo rt al. 1998). The results in this study were as follows. SOD, POD, CAT activities and MDA content had different sensitivity to different high temperature stress. Different cultivars had also different sensitivity to different high temperature stress. The higher the temperature, the faster was the senescence in both cultivars. The higher the temperature, the lower the SOD, POD, and CAT activities were, while the higher was the MDA content in flag leaves, which was in line with the viewpoints expressed by Guo et al. (1998) and Zheng and He (1 999). The effects of different high temperature during grain filling period on antioxidant enzymes and lipid peroxidation in flag leaves in wheat for different end uses have been reported less in the literature. Yangmai 12 appeared to be more sensitive to high temperature than Yangmai 9 on SOD, CAT activities and MDA content, Yangmai 9 appeared to be more sensitive to high temperature than Yangmai 12 on POD activity. These results indicated that resistance to antioxidant enzymes and lipid peroxidation varied with high temperature and resistance to high temperature varied with cultivars due to different susceptibility of enzyme activity in flag leaves of different cultivars under different heat shock conditions. The plant resistance to stress effect was determined by the level of antioxidant enzyme activities and the ability to rapidly increase these activities. There was an indication that wheat at 2530°C was self-regulated to compensate for the disadvantage effect when the soil moisture was controlled at 65-75% of the largest water holding in the field. It might have some protective mechanism in the endosperm, which may modulate activities of antioxidant enzyme and protect the antioxidant enzyme in response to elevated temperatures. It may have the compensative growth function after heat shock. These results suggested that there could be a significant change in grain metabolism when temperature was above 30°C. The
429
metabolism of plants was turbulence and the self-regulated ability was low. The antioxidant enzymes were feeble and the lipid peroxidation increased. The plant was premature and the filling duration was shortened. It had been well documented that the effects of high temperature during grain filling period on yield and quality were also linked to soil temperature and soil moisture. The interaction between temperature and drought stress should be needed to additional studies. The mechanisms by which temperature affects the plant of wheat and the remedy measures are unclear so far, which is worthy to be studied deeply.
CONCLUSIONS The following conclusions were supported by this study. (1) The higher the temperature, the lower were the SOD, POD, CAT activities in flag leaves and IOOOgrain weight, while higher was the MDA content in flag leaves when temperature was more than 25°C. Higher MDA content in flag leaves was closely related to the decline in activities of antioxidant enzymes. (2) The antioxidant enzymes and lipid peroxidation in flag leaves have different sensitivity in response to different high temperature stress. Different cultivars had also different sensitivity in response to different high temperature stress. SOD and CAT activities and MDA content in Yangmai 12 appeared to be more sensitive to high temperature than those in Yangmai 9. But POD activity in Yangmai 12 was less sensitive to high temperature than that in Yangmai 9.
Acknowledgements This research work was financially supported by the National Natural Science Foundation of China (3057109 1, 30 170540).
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