- Email: [email protected]
Superior storage stability in low lipoxygenase maize varieties
ARTICLE IN PRESS
Journal of Stored Products Research 43 (2007) 530–534 www.elsevier.com/locate/jspr
Superior storage stability in low lipoxygenase maize varieties Jinku Lia, Ying Zhanga, Zengliang Yua, Yujuan Wanga, Ye Yangb, Zheng Liuc, Jiayue Jianga, Mei Songa, Yuejin Wua, a
Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, China b The College of Chemistry and Material Science, Anhui Normal University, Wuhu 241000, China c The College of Anhui Science and Technology, FengYang 233100, China Accepted 25 September 2006
Abstract The present study was carried out to investigate the effect of absence of lipoxygenase isoenzymes (LOXs) on deteriorative changes in seeds of maize (Zea mays L.) using accelerated ageing. Three varieties of maize seeds, nongda high-oil 115 and zhengdan 94-2 with LOX1, 2 and ZAF1 lacking LOX-1, 2, were stored at 4271 1C and 84% r.h. for a period of 15 d. Standard germination, electrical conductivity, as well as malondialdehyde tests were used to follow changes during accelerated aging tests. The results indicated that germination of all tested seeds decreased with advanced ageing while electrical conductivity of the seed-soaking solution increased. Percentage germination was correlated with increased accumulation of malondialdehyde content. For the variety lacking LOX-1,2, there was a slight change in germination during the 15-d accelerated aging experiment, but for varieties with LOX-1, 2, a decline in germinability was observed, suggesting that LOX-1, 2, may be a definitive factor which influences seed lifespan. r 2007 Elsevier Ltd. All rights reserved. Keywords: Lipoxygenase; Germination; Electrical conductivity; Malondialdehyde; Accelerated ageing; Seed deterioration
1. Introduction Maize (Zea mays L.) is the most important cereal in the world after wheat and rice and is a staple crop in China. Unfortunately, a large proportion of the annual yield is lost during storage, and these losses have been partly attributed to grain deterioration resulting in loss of seed viability. It is well documented that seed viability is affected by preharvest climatic conditions, seed type, seed structure, seed health, growth temperature and relative humidity, and seed water content (Abba and Lovato, 1999), and that lipid peroxidation is an important factor governing deteriorative rate (Wilson and McDonald, 1986). Considerable research has occurred giving a better understanding of grain deterioration. Several comprehensive reviews have identified lipid peroxidation, enzyme inactivation or protein degradation, disruption of cellular membranes, and daCorresponding author. Tel.: +86 551 5593172; fax: +86 551 5591310.
E-mail address: [email protected] (Y. Wu). 0022-474X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2006.09.005
mage to genetic (nucleic acids) integrity as major causes of seed deterioration (Walters, 1998; McDonald, 1999). While no specific pattern has emerged, the most frequently cited cause of seed deterioration is lipid peroxidation (McDonald, 1999). Similarly, some researchers have suggested that lipid degradation is responsible for deteriorative changes during storage (Aibara et al., 1986; Takano, 1993; Zhang et al., 2007). Lipoxygenase isoenzymes (LOXs) are a class of nonhaem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids with cis, cis-1, 4pentadiene structures, such as linoleic and linolenic acids, to form conjugated diene hydroperoxides. LOX activity is widespread in the plant kingdom (Siedow, 1991), occurring in vegetative tissues, and also accumulating in various seeds (Loiseau et al., 2001). In maize, two LOXs, namely, LOX-1 and LOX-2 (collectively LOX-1, 2), have been identified from dry and germinating embryos (Poca et al., 1990; Jensen et al., 1997) whereas Fauconnier et al. (1995) reported that there was no lipoxygenase activity detected in
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
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the extracts of maize that they tested. In our laboratory, a rapid method of detecting crop LOXs described by Wu et al. (2001) is used to screen for varieties lacking lipoxygenase. We obtained two inbred lines, aisinuo and zhenuo, both lacking LOX-1, 2, from 109 varieties of the inbred lines. In 2005, we procured hybrids, ZAF1, without LOX1, 2, by crossbreeding the two inbred lines — aisinuo (male) and zhenuo (female). Several functions have been proposed for lipoxygenases. For example, lipoxygenases are supposed to be active via their involvement in the biosynthesis of several growth regulators, such as abscissic acid, traumatin and jasmonates (Mueller, 1997; Sheng et al., 2000). However, there have been few reports on the effect of seed lipoxygenase on grain deterioration. One reason may be that it is not easy to screen for mutants without lipoxygenase. Suzuki et al. (1996) reported that peroxidation products of unsaturated fatty acids are lower in mutants devoid of LOX-3. Nonetheless, the authors did not establish whether mutant seeds aged faster than wild types. Therefore, the role of lipoxygenases in grain remains enigmatic. Thus, the objectives of our present study were: (i) to give a description of the role of seed lipoxygenases in maize grain during prolonged storage; (ii) to elucidate the mechanism of grain deterioration during accelerated ageing; and (iii) to provide useful information on extending seed natural life to maize breeders.
Among the tested varieties, the oil content of ‘‘nongda high-oil 115’’ was 8.8%, while those of zhengdan 94-2 and ZAF1 were below 5%. All tested seeds were hybrid F1.
2. Materials and methods
2.4. Electrical conductivity evaluations
Seeds of maize (fresh following harvest) with and without lipoxygenase were obtained, respectively, from the local market and Professor Liu Zhen, a maize breeder at the Academy of Science and Technology Anhui.
Membrane permeability of test seed was measured by electrical conductivity (EC) according to Cordova-Tellez and Burris (2002). Three replications of 50-seed samples were rinsed with distilled water and allowed to air dry. The seeds then were weighed on an analytical balance (70.01 g), and soaked in disposable plastic cups containing 100 ml deionized water for 24 h at 25 1C. Results were recorded using a direct reading conductivity meter and were expressed in ms cm 1 g 1.
2.1. Assessment of LOXs The lipoxygenase status of the maize seeds was determined by the rapid method of Wu et al. (2001) (Chinese patent, ZL00112539.7) (Table 1). This method utilizes lipoxygenases’ coupled oxidation to an oxidation reduction indicator to develop a colour reaction which determines LOXs, status. Crude enzyme was extracted according to Fauconnier et al. (1995). Table 1 Lipoxygenase isoenzymes in the maize varieties tested Varieties
LOX1
LOX2
LOX3
Nongda high-oil 115 Zhengdan 94-2 ZAF1 Aisinuo (ZAF1 # parent) Zhenuo (ZAF1 ~ parent)
+ +
+ +
+ + + + +
+, present;
, absent.
2.2. Accelerated-ageing tests Accelerated-aging (AA) tests were conducted on 100 g of seeds in three replicates which were surface sterilized by 2% sodium hypochlorite solution. Seeds were packed into small nylon gauze packets and placed into an artificial climate chamber at 4271 1C and 84% r.h. for 15 d. Seeds were sampled at 3,6,9,12 and 15 d intervals.
2.3. Standard germination tests Standard germination was determined according to Rules for Agricultural Seed Testing-Germination Test (GB/T 3543.4-1995) (China State Bureau of Technical Supervision, 1995). Seeds (150) were divided into three replicates of 50 and randomly placed in a sterilized Petri dish containing three layers of moist filter papers. Petri dishes were incubated at 25 1C for 7 d. Seedlings with a normal root of 5 mm were counted as germinated. Results were expressed in percentages.
2.5. Malondialdehyde determination Malondialdehyde (MDA), a decomposition product from the peroxidation of polyunsaturated fatty acids, was assayed as thiobarbituric acid-reactive-substances according to Heath and Packer (1968). Naked seeds (0.6 g) were ground in 3 ml of 5% trichloroacetic acid, and then centrifuged at 8000g for 15 min (Goel and Sheoran, 2003). The resulting supernatant was used for MDA determination. Maximum absorbance of the TBA–MDA complex was measured at 532 nm and values were corrected by subtracting absorbance at 600 nm. Concentration of MDA was calculated by using its molar extinction coefficient. The value of the molar extinction coefficient was 155,000.
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
532
80
3.1. Germination percentage
70
In AA, seeds deteriorated under controlled high temperature and relative humidity for short durations. Highvigour seed lots will withstand these extreme stress conditions and deteriorate at a slower rate than low-vigour seed lots. With nongda high-oil 115 in which LOX-1, 2 were present, initial germinability was 100%, but after 3d AA, germination decreased gradually and reduced to 10% after 15 d of storage. Similarly, in the case of zhengdan 94-2 in which both LOX-1, 2 were present, the percentage of germination was 95% initially, but decreased slowly to 26%. Initial germinability in ZAF1 in which LOX-1, 2 were absent was 100% with a slight decrease from 100% to 96% observed during the 15-d storage period. From this test, we found that germination decreased significantly with lipoxygenase during AA of the tested seeds whereas there was only a slight reduction in seeds lacking LOX-1, 2 (Fig. 1).
Malondialdehyde (nmol /g)
3. Results
Zhengdan ZAF1
60 50 40 30 20 2
4
6
8 10 12 Time of storage (days)
14
16
Fig. 2. Accumulation of malondialdehyde in maize seed during storage at 42 1C and 84% r.h
Conductivity (µs • cm-1 • g-1)
3.2. Changes in MDA MDA contents in maize (repeated three times) during the 15 d storage period are given in Fig. 2. With varieties containing LOX-1, 2, content of MDA increased significantly during storage. Initial MDA content of nongda high-oil 115 was 33.33 nmol/g, followed by a fast increase to 73.07 nmol/g after 15 d AA. In the case of zhengdan 94-2, initial MDA content was 34.26 nmol/g which increased by 25 nmol/g at the end of AA. For ZAF1, there was a slight increase from 24.89 nmol/g to 32.41 nmol/g during the 15-d AA test. Additionally, increase in MDA correlated well with decline in germination. (The value of the correlation coefficient was 0.98).
Nongda high-oil 115
28 26 24 22 20 18 16 14 12 10 8 6 4
Nongda high-oil 115 Zhengdan ZAF1
2
4
6
8 10 12 14 Time of storage (days)
16
18
Fig. 3. The increase of electrical conductivity in maize seed during storage at 42 1C and 84% r.h.
Nongda high-oil 115 Zhengdan ZAF1
3.3. EC
100
EC of the three varieties during the 15-d AA tests is shown in Fig. 3. The initial EC of nongda high-oil 115 was 12.64 ms cm 1 g 1; after the 15-d AA tests, the value reached 26.33 ms cm 1 g 1. For zhengdan 94-2, EC varied from 10.98 to 22.45 ms cm 1 g 1. In contrast with the above two varieties, a slight increase from 5.09 to 6.00 ms cm 1 g 1 was observed in ZAF1.
Germination (%)
80
60
40
4. Discussion 20
0 2
4
6
8 10 12 14 Time of storage (days)
16
18
Fig. 1. Decline of germination in maize seed during storage at 42 1C and 84% r.h.
Seed possesses its highest vigour at the time of its physiological maturity and then undergoes deteriorative change resulting in viability loss during storage. Seed deterioration is an intractable problem for maize growers worldwide. Thus, how to retain high-vigour seeds for a long period has attracted much research interest. In traditional preservation systems, maize seeds were dried
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
to a safe seed water content level after harvest, and then stored at a low temperature to avoid seed deterioration. Although this type of storage can extend the lifespan of stored seeds, it also greatly increases storage costs. As a result, the present study was conducted to ameliorate seed deterioration by examining anti-deteriorative maize genes. Several comprehensive reviews have identified lipid peroxidation, enzyme inactivation, and disruption of cellular membranes, as major causes of seed ageing (Smith and Berjak, 1995; Walters, 1998; McDonald, 1999). Therefore, to investigate the effect of lipoxygenase on grain ageing, we studied a variety lacking LOX-1, 2, namely ZAF1 (female parent: zhenuo, male parent: aisinuo), which was subjected to an accelerated-ageing test together with two other varieties containing LOX-1, 2. The results indicated that, after the 15-d AA, there was a slight reduction in germination of ZAF1 without LOX-1, 2 whereas a significant decline was observed in the normal line with LOX-1, 2. For the normal LOX-1, 2 lines, germination of nongda high-oil 115 maize seed decreased by 90% while that of zhengdan 94-2 by 69%. The probable reason for the different declines in the two normal varieties is the amount of crude fatty acids in nongda high-oil 115 which is nearly twice that of zhengdan 94-2 and could lead to greater lipid peroxidation injury. MDA, a secondary end-product of the enzymatic degradation of polyunsaturated fatty acids, is recognized as a reliable estimator of lipid peroxidation. The highest concentration of MDA accumulated in nongda high-oil 115 seed, followed by zhengdan 94-2 and ZAF1. The reason why there was only a small accumulation in ZAF1 may be the absence of LOX-1, 2, the first key enzymes in lipid peroxidation which leads to lower production of MDA. Additionally, decreased germination correlated well with accumulation of MDA. Based on the results of germination and MDA, we can infer that seed devoid of LOX-1, 2 will exhibit extended seed survival. Berjak (2005) reported that membrane breakdown is a key factor in cell debilitation and death. For seeds, that translates into a loss of viability. Similarly, Chang and Sung (1998) also suggested that ageing of seeds leads to lipid peroxidation that subsequently causes membrane perturbation. Thus, we hypothesized that there were two possible effects of the presence of LOX-1, 2 leading to loss of membrane integrity during ageing: (i) the LOX-1, 2 will react with the polyunsaturated fatty acids of phospholipid, a main component of the membrane, leading to a decline in membrane flexibility; (ii) during lipid peroxidation, free radicals are formed which initiate additional attack on membranes. Bewley and Black (1994) reported that values of EC measured in seed soaking solutions vary with leaching and are directly related to cellular membrane integrity. The results of EC demonstrated that the EC of ZAF1 changed only slightly from 5.09 to 6 ms cm 1 g 1 while a much greater increase was observed in the LOX-1, 2 containing line. The EC test provides strong experimental support for
533
what we supposed — the line devoid of LOX-1, 2 greatly resists membrane injury during ageing. In conclusion, seed varieties lacking LOX-1, 2 can retain higher vigour during AA than lines containing LOX-1, 2 and the absence of LOX-1, 2 may be very important in minimizing deterioration in maize during storage. As an inherited genetic factor, LOX-1, 2 is widespread in maize seeds, and mutants without LOX-1, 2 can be screened easily by the rapid method of Wu et al. (2001).Thus, breeding maize varieties without LOX-1, 2 could basically delay the deterioration during maize storage. Acknowledgement The present study was supported by the Knowledge Innovative Program of the Chinese Academy of Sciences, Grant no.: KSCX-SW-324. References Abba, E.J., Lovato, A., 1999. Effect of seed storage temperature and relative humidity on maize (Zea mays L.) seed viability and vigor. Seed Science and Technology 27, 101–114. Aibara, S., Ismail, I.A., Yamashita, H., Ohta, H., Sekiyama, F., Morita, Y., 1986. Changes in rice bran lipids and fatty acids during storage. Agricultural Biological Chemistry 50, 665–673. Berjak, P., 2005. Protector of the seeds: seminal reflections from Southern Africa. Science 307, 47–49. Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York. Chang, S.M., Sung, J.M., 1998. Deteriorative changes in primed sweet corn seeds during storage. Seed Science and Technology 26, 613–626. China State Bureau of Technical Supervision, 1995. Rules for Agricultural Seed Testing-Germination test. Standards Press of China, Beijing. Cordova-Tellez, L., Burris, J.S., 2002. Embryo drying rates during the acquisition of desiccation tolerance in maize seed. Crop Science 42, 1989–1995. Fauconnier, M.L., Vanzeveren, E., Marlier, M., Lognay, G., Wathelet, J.P., Severin, M., 1995. Assessment of lipoxygenase activity in seed extracts from 35 plant species. Grasas y Aceites 46, 6–10. Goel, A., Sheoran, I.S., 2003. Lipid peroxidation and peroxide-scavenging enzymes in cotton seeds under natural ageing. Biologia Plantarum 46, 429–434. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 12, 189–198. Jensen, A.B., Poca, E., Rigaud, M., Freyssinet, G., Pages, M., 1997. Molecular characterization of L2 lipoxygenase from maize embryos. Plant Molecular Biology 33, 605–614. Loiseau, J., Vu, B.L., Macherel, M.H., Deunff, Y.L., 2001. Seed lipoxygenases: occurrence and functions. Seed Science Research 11, 199–211. McDonald, M.B., 1999. Seed deterioration: physiology, repair and assessment. Seed Science and Technology 27, 177–237. Mueller, M.J., 1997. Enzymes involved in jasmonic acid biosynthesis. Physiologia Plantarum 100, 653–663. Poca, E., Rabinovitch-Chable, H., Cook-Moreau, J., Pages, M., Rigaud, M., 1990. Lipoxygenases from Zea mays L.: purification and physicochemical characteristics. Biochimica et Biophysica Acta 1045, 107–114. Sheng, J., Luo, Y., Wainwright, H., 2000. Studies on lipoxygenase and the formation of ethylene in tomato. Journal of Horticultural Science and Biotechnology 75, 69–71.
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Siedow, J.N., 1991. Plant lipoxygenase: structure and function. Annual Review of Plant Molecular Biology 42, 145–188. Smith, M.T., Berjak, P., 1995. Deteriorative changes associated with the loss of viability of stored desiccation tolerant and desiccation sensitive seeds. In: Kigel, J., Galili, G. (Eds.), Seed Development and Germination. Marcel Dekker Inc., New York, pp. 701–746. Suzuki, Y., Yasui, T., Matsukura, U., Terao, J., 1996. Oxidative stability of bran lipids from rice variety [Oryza sativa (L.)] lacking lipoxygenase-3 in seeds. Journal of Agricultural and Food Chemistry 44, 3479–3483. Takano, K., 1993. Advances in cereal chemistry and technology in Japan. Cereal Foods World 38, 695–698.
Walters, C., 1998. Understanding the mechanisms and kinetics of seed ageing. Seed Science Research 8, 223–244. Wilson, D.O., McDonald, M.B., 1986. The lipid peroxidation model of seed deterioration. Seed Science and Technology 14, 259–268. Wu, Y.J., Wu, J.D., Zhang, Y., 2001. A new rapid crop lipoxygenase isoenzymes detective technique. China Patent Bureau, Invention Patent Communique´ 17, 21–53. Zhang, Y., Yu, Z., Lu, Y., Wang, Y., She, D., Song, M., Wu, Y., 2007. Effect of the absence of lipoxygenase isoenzymes on the storage characteristics of rice grains. Journal of Stored Products Research 43, 87–91.
Journal of Stored Products Research 43 (2007) 530–534 www.elsevier.com/locate/jspr
Superior storage stability in low lipoxygenase maize varieties Jinku Lia, Ying Zhanga, Zengliang Yua, Yujuan Wanga, Ye Yangb, Zheng Liuc, Jiayue Jianga, Mei Songa, Yuejin Wua, a
Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, China b The College of Chemistry and Material Science, Anhui Normal University, Wuhu 241000, China c The College of Anhui Science and Technology, FengYang 233100, China Accepted 25 September 2006
Abstract The present study was carried out to investigate the effect of absence of lipoxygenase isoenzymes (LOXs) on deteriorative changes in seeds of maize (Zea mays L.) using accelerated ageing. Three varieties of maize seeds, nongda high-oil 115 and zhengdan 94-2 with LOX1, 2 and ZAF1 lacking LOX-1, 2, were stored at 4271 1C and 84% r.h. for a period of 15 d. Standard germination, electrical conductivity, as well as malondialdehyde tests were used to follow changes during accelerated aging tests. The results indicated that germination of all tested seeds decreased with advanced ageing while electrical conductivity of the seed-soaking solution increased. Percentage germination was correlated with increased accumulation of malondialdehyde content. For the variety lacking LOX-1,2, there was a slight change in germination during the 15-d accelerated aging experiment, but for varieties with LOX-1, 2, a decline in germinability was observed, suggesting that LOX-1, 2, may be a definitive factor which influences seed lifespan. r 2007 Elsevier Ltd. All rights reserved. Keywords: Lipoxygenase; Germination; Electrical conductivity; Malondialdehyde; Accelerated ageing; Seed deterioration
1. Introduction Maize (Zea mays L.) is the most important cereal in the world after wheat and rice and is a staple crop in China. Unfortunately, a large proportion of the annual yield is lost during storage, and these losses have been partly attributed to grain deterioration resulting in loss of seed viability. It is well documented that seed viability is affected by preharvest climatic conditions, seed type, seed structure, seed health, growth temperature and relative humidity, and seed water content (Abba and Lovato, 1999), and that lipid peroxidation is an important factor governing deteriorative rate (Wilson and McDonald, 1986). Considerable research has occurred giving a better understanding of grain deterioration. Several comprehensive reviews have identified lipid peroxidation, enzyme inactivation or protein degradation, disruption of cellular membranes, and daCorresponding author. Tel.: +86 551 5593172; fax: +86 551 5591310.
E-mail address: [email protected] (Y. Wu). 0022-474X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2006.09.005
mage to genetic (nucleic acids) integrity as major causes of seed deterioration (Walters, 1998; McDonald, 1999). While no specific pattern has emerged, the most frequently cited cause of seed deterioration is lipid peroxidation (McDonald, 1999). Similarly, some researchers have suggested that lipid degradation is responsible for deteriorative changes during storage (Aibara et al., 1986; Takano, 1993; Zhang et al., 2007). Lipoxygenase isoenzymes (LOXs) are a class of nonhaem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids with cis, cis-1, 4pentadiene structures, such as linoleic and linolenic acids, to form conjugated diene hydroperoxides. LOX activity is widespread in the plant kingdom (Siedow, 1991), occurring in vegetative tissues, and also accumulating in various seeds (Loiseau et al., 2001). In maize, two LOXs, namely, LOX-1 and LOX-2 (collectively LOX-1, 2), have been identified from dry and germinating embryos (Poca et al., 1990; Jensen et al., 1997) whereas Fauconnier et al. (1995) reported that there was no lipoxygenase activity detected in
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
531
the extracts of maize that they tested. In our laboratory, a rapid method of detecting crop LOXs described by Wu et al. (2001) is used to screen for varieties lacking lipoxygenase. We obtained two inbred lines, aisinuo and zhenuo, both lacking LOX-1, 2, from 109 varieties of the inbred lines. In 2005, we procured hybrids, ZAF1, without LOX1, 2, by crossbreeding the two inbred lines — aisinuo (male) and zhenuo (female). Several functions have been proposed for lipoxygenases. For example, lipoxygenases are supposed to be active via their involvement in the biosynthesis of several growth regulators, such as abscissic acid, traumatin and jasmonates (Mueller, 1997; Sheng et al., 2000). However, there have been few reports on the effect of seed lipoxygenase on grain deterioration. One reason may be that it is not easy to screen for mutants without lipoxygenase. Suzuki et al. (1996) reported that peroxidation products of unsaturated fatty acids are lower in mutants devoid of LOX-3. Nonetheless, the authors did not establish whether mutant seeds aged faster than wild types. Therefore, the role of lipoxygenases in grain remains enigmatic. Thus, the objectives of our present study were: (i) to give a description of the role of seed lipoxygenases in maize grain during prolonged storage; (ii) to elucidate the mechanism of grain deterioration during accelerated ageing; and (iii) to provide useful information on extending seed natural life to maize breeders.
Among the tested varieties, the oil content of ‘‘nongda high-oil 115’’ was 8.8%, while those of zhengdan 94-2 and ZAF1 were below 5%. All tested seeds were hybrid F1.
2. Materials and methods
2.4. Electrical conductivity evaluations
Seeds of maize (fresh following harvest) with and without lipoxygenase were obtained, respectively, from the local market and Professor Liu Zhen, a maize breeder at the Academy of Science and Technology Anhui.
Membrane permeability of test seed was measured by electrical conductivity (EC) according to Cordova-Tellez and Burris (2002). Three replications of 50-seed samples were rinsed with distilled water and allowed to air dry. The seeds then were weighed on an analytical balance (70.01 g), and soaked in disposable plastic cups containing 100 ml deionized water for 24 h at 25 1C. Results were recorded using a direct reading conductivity meter and were expressed in ms cm 1 g 1.
2.1. Assessment of LOXs The lipoxygenase status of the maize seeds was determined by the rapid method of Wu et al. (2001) (Chinese patent, ZL00112539.7) (Table 1). This method utilizes lipoxygenases’ coupled oxidation to an oxidation reduction indicator to develop a colour reaction which determines LOXs, status. Crude enzyme was extracted according to Fauconnier et al. (1995). Table 1 Lipoxygenase isoenzymes in the maize varieties tested Varieties
LOX1
LOX2
LOX3
Nongda high-oil 115 Zhengdan 94-2 ZAF1 Aisinuo (ZAF1 # parent) Zhenuo (ZAF1 ~ parent)
+ +
+ +
+ + + + +
+, present;
, absent.
2.2. Accelerated-ageing tests Accelerated-aging (AA) tests were conducted on 100 g of seeds in three replicates which were surface sterilized by 2% sodium hypochlorite solution. Seeds were packed into small nylon gauze packets and placed into an artificial climate chamber at 4271 1C and 84% r.h. for 15 d. Seeds were sampled at 3,6,9,12 and 15 d intervals.
2.3. Standard germination tests Standard germination was determined according to Rules for Agricultural Seed Testing-Germination Test (GB/T 3543.4-1995) (China State Bureau of Technical Supervision, 1995). Seeds (150) were divided into three replicates of 50 and randomly placed in a sterilized Petri dish containing three layers of moist filter papers. Petri dishes were incubated at 25 1C for 7 d. Seedlings with a normal root of 5 mm were counted as germinated. Results were expressed in percentages.
2.5. Malondialdehyde determination Malondialdehyde (MDA), a decomposition product from the peroxidation of polyunsaturated fatty acids, was assayed as thiobarbituric acid-reactive-substances according to Heath and Packer (1968). Naked seeds (0.6 g) were ground in 3 ml of 5% trichloroacetic acid, and then centrifuged at 8000g for 15 min (Goel and Sheoran, 2003). The resulting supernatant was used for MDA determination. Maximum absorbance of the TBA–MDA complex was measured at 532 nm and values were corrected by subtracting absorbance at 600 nm. Concentration of MDA was calculated by using its molar extinction coefficient. The value of the molar extinction coefficient was 155,000.
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
532
80
3.1. Germination percentage
70
In AA, seeds deteriorated under controlled high temperature and relative humidity for short durations. Highvigour seed lots will withstand these extreme stress conditions and deteriorate at a slower rate than low-vigour seed lots. With nongda high-oil 115 in which LOX-1, 2 were present, initial germinability was 100%, but after 3d AA, germination decreased gradually and reduced to 10% after 15 d of storage. Similarly, in the case of zhengdan 94-2 in which both LOX-1, 2 were present, the percentage of germination was 95% initially, but decreased slowly to 26%. Initial germinability in ZAF1 in which LOX-1, 2 were absent was 100% with a slight decrease from 100% to 96% observed during the 15-d storage period. From this test, we found that germination decreased significantly with lipoxygenase during AA of the tested seeds whereas there was only a slight reduction in seeds lacking LOX-1, 2 (Fig. 1).
Malondialdehyde (nmol /g)
3. Results
Zhengdan ZAF1
60 50 40 30 20 2
4
6
8 10 12 Time of storage (days)
14
16
Fig. 2. Accumulation of malondialdehyde in maize seed during storage at 42 1C and 84% r.h
Conductivity (µs • cm-1 • g-1)
3.2. Changes in MDA MDA contents in maize (repeated three times) during the 15 d storage period are given in Fig. 2. With varieties containing LOX-1, 2, content of MDA increased significantly during storage. Initial MDA content of nongda high-oil 115 was 33.33 nmol/g, followed by a fast increase to 73.07 nmol/g after 15 d AA. In the case of zhengdan 94-2, initial MDA content was 34.26 nmol/g which increased by 25 nmol/g at the end of AA. For ZAF1, there was a slight increase from 24.89 nmol/g to 32.41 nmol/g during the 15-d AA test. Additionally, increase in MDA correlated well with decline in germination. (The value of the correlation coefficient was 0.98).
Nongda high-oil 115
28 26 24 22 20 18 16 14 12 10 8 6 4
Nongda high-oil 115 Zhengdan ZAF1
2
4
6
8 10 12 14 Time of storage (days)
16
18
Fig. 3. The increase of electrical conductivity in maize seed during storage at 42 1C and 84% r.h.
Nongda high-oil 115 Zhengdan ZAF1
3.3. EC
100
EC of the three varieties during the 15-d AA tests is shown in Fig. 3. The initial EC of nongda high-oil 115 was 12.64 ms cm 1 g 1; after the 15-d AA tests, the value reached 26.33 ms cm 1 g 1. For zhengdan 94-2, EC varied from 10.98 to 22.45 ms cm 1 g 1. In contrast with the above two varieties, a slight increase from 5.09 to 6.00 ms cm 1 g 1 was observed in ZAF1.
Germination (%)
80
60
40
4. Discussion 20
0 2
4
6
8 10 12 14 Time of storage (days)
16
18
Fig. 1. Decline of germination in maize seed during storage at 42 1C and 84% r.h.
Seed possesses its highest vigour at the time of its physiological maturity and then undergoes deteriorative change resulting in viability loss during storage. Seed deterioration is an intractable problem for maize growers worldwide. Thus, how to retain high-vigour seeds for a long period has attracted much research interest. In traditional preservation systems, maize seeds were dried
ARTICLE IN PRESS J. Li et al. / Journal of Stored Products Research 43 (2007) 530–534
to a safe seed water content level after harvest, and then stored at a low temperature to avoid seed deterioration. Although this type of storage can extend the lifespan of stored seeds, it also greatly increases storage costs. As a result, the present study was conducted to ameliorate seed deterioration by examining anti-deteriorative maize genes. Several comprehensive reviews have identified lipid peroxidation, enzyme inactivation, and disruption of cellular membranes, as major causes of seed ageing (Smith and Berjak, 1995; Walters, 1998; McDonald, 1999). Therefore, to investigate the effect of lipoxygenase on grain ageing, we studied a variety lacking LOX-1, 2, namely ZAF1 (female parent: zhenuo, male parent: aisinuo), which was subjected to an accelerated-ageing test together with two other varieties containing LOX-1, 2. The results indicated that, after the 15-d AA, there was a slight reduction in germination of ZAF1 without LOX-1, 2 whereas a significant decline was observed in the normal line with LOX-1, 2. For the normal LOX-1, 2 lines, germination of nongda high-oil 115 maize seed decreased by 90% while that of zhengdan 94-2 by 69%. The probable reason for the different declines in the two normal varieties is the amount of crude fatty acids in nongda high-oil 115 which is nearly twice that of zhengdan 94-2 and could lead to greater lipid peroxidation injury. MDA, a secondary end-product of the enzymatic degradation of polyunsaturated fatty acids, is recognized as a reliable estimator of lipid peroxidation. The highest concentration of MDA accumulated in nongda high-oil 115 seed, followed by zhengdan 94-2 and ZAF1. The reason why there was only a small accumulation in ZAF1 may be the absence of LOX-1, 2, the first key enzymes in lipid peroxidation which leads to lower production of MDA. Additionally, decreased germination correlated well with accumulation of MDA. Based on the results of germination and MDA, we can infer that seed devoid of LOX-1, 2 will exhibit extended seed survival. Berjak (2005) reported that membrane breakdown is a key factor in cell debilitation and death. For seeds, that translates into a loss of viability. Similarly, Chang and Sung (1998) also suggested that ageing of seeds leads to lipid peroxidation that subsequently causes membrane perturbation. Thus, we hypothesized that there were two possible effects of the presence of LOX-1, 2 leading to loss of membrane integrity during ageing: (i) the LOX-1, 2 will react with the polyunsaturated fatty acids of phospholipid, a main component of the membrane, leading to a decline in membrane flexibility; (ii) during lipid peroxidation, free radicals are formed which initiate additional attack on membranes. Bewley and Black (1994) reported that values of EC measured in seed soaking solutions vary with leaching and are directly related to cellular membrane integrity. The results of EC demonstrated that the EC of ZAF1 changed only slightly from 5.09 to 6 ms cm 1 g 1 while a much greater increase was observed in the LOX-1, 2 containing line. The EC test provides strong experimental support for
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what we supposed — the line devoid of LOX-1, 2 greatly resists membrane injury during ageing. In conclusion, seed varieties lacking LOX-1, 2 can retain higher vigour during AA than lines containing LOX-1, 2 and the absence of LOX-1, 2 may be very important in minimizing deterioration in maize during storage. As an inherited genetic factor, LOX-1, 2 is widespread in maize seeds, and mutants without LOX-1, 2 can be screened easily by the rapid method of Wu et al. (2001).Thus, breeding maize varieties without LOX-1, 2 could basically delay the deterioration during maize storage. Acknowledgement The present study was supported by the Knowledge Innovative Program of the Chinese Academy of Sciences, Grant no.: KSCX-SW-324. References Abba, E.J., Lovato, A., 1999. Effect of seed storage temperature and relative humidity on maize (Zea mays L.) seed viability and vigor. Seed Science and Technology 27, 101–114. Aibara, S., Ismail, I.A., Yamashita, H., Ohta, H., Sekiyama, F., Morita, Y., 1986. Changes in rice bran lipids and fatty acids during storage. Agricultural Biological Chemistry 50, 665–673. Berjak, P., 2005. Protector of the seeds: seminal reflections from Southern Africa. Science 307, 47–49. Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York. Chang, S.M., Sung, J.M., 1998. Deteriorative changes in primed sweet corn seeds during storage. Seed Science and Technology 26, 613–626. China State Bureau of Technical Supervision, 1995. Rules for Agricultural Seed Testing-Germination test. Standards Press of China, Beijing. Cordova-Tellez, L., Burris, J.S., 2002. Embryo drying rates during the acquisition of desiccation tolerance in maize seed. Crop Science 42, 1989–1995. Fauconnier, M.L., Vanzeveren, E., Marlier, M., Lognay, G., Wathelet, J.P., Severin, M., 1995. Assessment of lipoxygenase activity in seed extracts from 35 plant species. Grasas y Aceites 46, 6–10. Goel, A., Sheoran, I.S., 2003. Lipid peroxidation and peroxide-scavenging enzymes in cotton seeds under natural ageing. Biologia Plantarum 46, 429–434. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 12, 189–198. Jensen, A.B., Poca, E., Rigaud, M., Freyssinet, G., Pages, M., 1997. Molecular characterization of L2 lipoxygenase from maize embryos. Plant Molecular Biology 33, 605–614. Loiseau, J., Vu, B.L., Macherel, M.H., Deunff, Y.L., 2001. Seed lipoxygenases: occurrence and functions. Seed Science Research 11, 199–211. McDonald, M.B., 1999. Seed deterioration: physiology, repair and assessment. Seed Science and Technology 27, 177–237. Mueller, M.J., 1997. Enzymes involved in jasmonic acid biosynthesis. Physiologia Plantarum 100, 653–663. Poca, E., Rabinovitch-Chable, H., Cook-Moreau, J., Pages, M., Rigaud, M., 1990. Lipoxygenases from Zea mays L.: purification and physicochemical characteristics. Biochimica et Biophysica Acta 1045, 107–114. Sheng, J., Luo, Y., Wainwright, H., 2000. Studies on lipoxygenase and the formation of ethylene in tomato. Journal of Horticultural Science and Biotechnology 75, 69–71.
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