Research Report

Changes in Enzyme Activity during Accelerated Ageing in Soybean (Glycine max (L.) Merrill)  

Rahul Kumar Chandel1 ,  Geetanjly2 , Zeba Khan3 , Radhamani J.4
1 Department of Biotechnology, Krishna College of Science and R. Technology, Agra, Uttar Pradesh, India
2 Division of Entomology, Indian Agricultural Research Institute, New Delhi, India
3 Department of Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
4 Germplasm Conservation Division, National Bureau Of Plant Genetic Resources, New Delhi, India
Author    Correspondence author
Legume Genomics and Genetics, 2016, Vol. 7, No. 9   doi: 10.5376/lgg.2016.07.0009
Received: 30 May, 2016    Accepted: 29 Jun., 2016    Published: 24 Aug., 2016
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Rahul K.C., Geetanjly, Zeba K., and Radhamani J., 2006, Changes in Enzyme Activity during Accelerated Ageing in Soybean (Glycine max (L.) Merrill), Legume Genomics and Genetics, 7(9): 1-7 (doi: 10.5376/lgg.2016.07.0009)


The object of this work was to study the effect of accelerated ageing on enzyme activity for soybean (Glycine max (L.) Merrill). Under accelerated ageing conditions at 42°C temperature and 100% relative humidity on eight varieties of soybean, namely: PK-416, PK-472, PK-327, PS-1241, PK-262, PUSA-16, PUSA-37 and SL-525, were studied. Their seeds were examined under differential ageing conditions namely: 1st day, 3rd day, 5th day and 6th day of aging at different moisture levels of 3, 4, 6 and 7%. The best germination result was shown at 6 and 7% moisture content which can hence be considered suitable for the long term storage. The effect of aging was least on PK-262 and SL-525 that means these varieties can easily be stored for long time without getting deteriorated in their quality. Varieties PK-327 and PK-416 were maximally affected by ageing and as the ageing proceeded their viability was almost negligible. This test reflects the ability of the seed sample to withstand the stresses of high temperature and humidity and estimates the relative storability (longetivity) and vigour of seeds. For this, initial moisture of eight varieties was measured and after that moisture was brought to the level of 3, 4, 6 and 7% by keeping them in beads, sodium hydroxide solution and silica gel, respectively. The germination percentage vigour index and conductivity values were taken for one day, three days, five days and six days aged seeds of all samples respectively and enzyme activity was taken on 0, 3 and 5 days aged seeds in four varieties, PK-262, SL-525, PK-327 and PK-416. Seed deterioration leads to impairing go enzyme activity. The extent of seed deterioration can be best assessed in terms of damage to the membrane bound enzyme. A steady decrease in activities of catalase, paroxidase, superoxide dismutase, ascorbate peroxidase enzymes were observed except in lipoxygenase activity which increases consistently with ageing.

Accelerated ageing; Deterioration; Germination; Humidity; Malonaldehyde content; Peroxidase; Vigour

1 Background

Soybean (Glycine max (L.) Merrill) is one of the most important oil seed crops in the world. The major problem faced for soybean cultivation is its poor storability under ambient storage leading to poor plant stand. The loss of germination potential in soybean occurs more frequently in tropical countries as compared to temperate environment. The environmental conditions of high temperature and humidity in tropical countries such as India make it difficult to produce good quality soybean seeds or to maintain its viability, during storage (Singh and Ram, 1986; Bhatia, 1996). Seed deterioration is a natural phenomenon that occurs in all seed which leads to a gradual decline of seed viability during storage. The process is most pronounced in oil-rich seeds such soybean, because of the high susceptibility to peroxidation of polyunsaturated fatty acids present in these seeds (Priestly and Leopold, 1983). One of the most common tests to determine the physiological potential of seeds is the accelerated ageing (Delouche and Baskin, 1973) due to the possibility of a standardized methodology and reproductibility of results (Tomes et al., 1988). The efficiency of this test in estimating the storage of seed lots as well as providing a good relationship with seeding emergence in the field for various species (Spinola et al., 1998; Rodo et al., 1998). Accelerated ageing test has been recognized as a good predictor of seed storability. Therefore the mechanisms of seeds deterioration have been mostly studied by manipulating the ageing rate by exposing the seed to high temperature as normally used in the accelerated ageing test (Priestley et al., 1985; Sun and Leopold, 1995; Sung, 1996; Basra et al., 2003). According to Priestley and Leopold (1983), accelerated ageing, although reduces seed vigour, it also induces alterations other than those observed in the natural ageing. Accelerated ageing can inactivate enzymes to different extents or affect their synthesis as reported by Bailly et al. (1996) who founded a clear relation between the activity loss of scavenging enzymes, lipid peroxidation and deterioration of sunflower seeds during accelerated ageing. Basavarajappa et al. (1991) reported that in ageing maize seeds while the total protein content decreased, free amino acid concentration increased, which indicates protein degradation by proteases.


There are several reports suggesting that the polyunsaturated fatty acid present in the oil seeds are highly susceptible to non-enzymatic peroxidation in the presence of oxygen (Sung and Jeng, 1994; Trawatha et al., 1995). Thus, there is a general belief that lipid peroxidation is the basic cause of seed deterioration and the major changes related to this process are the depletion of lipid reserves resulting in production of fatty acids (Basra et al., 2003) enzyme degradation and inactivation (Hsu et al., 2003) and loss of membrane integrity (Sung, 1996). Deterioration process also affects the activity of several peroxide scavenging enzymes such as catalase, peroxidase, superoxide dismutase and acid phosphate (Baily et al., 1996; Spinola et al., 2000; Hsu et al., 2003). Estimation of enzyme was more sensitive than vigour test and other parameters in soybean during accelerated ageing.


2 Results

Steady decreases in the activities of catalase, peroxidase, superoxide dismutase and ascorbate peroxidase enzymes were observed except lipoxygenase enzyme activity which increased consistently with ageing. The increase in malonealdehyade content in the seeds has been reported by several workers (Harman and Mattick, 1976; Stewart and Bewley, 1980). The lipid peroxidation was observed in different accessions of soybean PK2-262 showed minimum activity while PK-416 showed maximum activity on 1, 3 and 5-days aged seeds. Catalase catalyzes the reduction of H2O2 to water and molecular oxygen. Its activity is a good indicator of seeds storability. Seeds with higher catalase activity during ageing perform well as compare to those with lower catalase activity. PK-262 showed the maximum activity in un-aged or control seeds while PK-416 showed lowest. Similarly, in 3-days aged seeds PK-262 showed maximum catalase activity followed by PK-327, PK-416 and SL-525. In 5-days aged seeds, there were no significant difference among SL-525, PK-416 and PK-327 while PK-262 again showed maximum enzymatic activity. The peroxidase showed sharp reduction in its activity under accelerated ageing conditions. Its activities in unaged or control seeds were almost double that of 5-days aged seeds. Decreased activity of enzymes was observed in 3 and 5-days aged seeds as compared to un-aged seeds. PK-262 showed maximum activity, followed by SL-525, PK-416 and PK-327. Superoxide dismutase catalyze the dismutation of superoxide radical (O2) to hydrogen peroxide (H2O2). It was observed that in un-aged seeds, PK-262 showed the maximum activity followed by PK-327, SL-525 and PK-416, respectively. Similar patterns were observed for 3 and 5-days aged seeds. Highest SOD activity was found in PK-262. This shows that PK-262 is most resistant to ageing treatment and thus can be potentially stored for a longer time. The germination pattern showed a steady decline with faster deterioration rate. Ascorbate peroxidase reduces hydrogen peroxide to water with the help of ascorbic acid. Seeds with higher Ascorbate peroxidase activity during ageing perform well as compared to seeds with lower ascorbate peroxidase activity. PK-262 showed the maximum activity in un-aged seeds where its activity was highest while PK- 416 activity was lowest, similarly 3-days aged PK-262 showed maximum ascorbate peroxidase activity followed by PK-327, SL-525 and PK-416. In 5-days aged seeds, there were no significant difference among SL-525, PK-416 and PK-327 while PK-262 again showed maximum enzymatic activity. The enzymatic activities are graphically represented (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). The germination percentage, vigour index and conductivity values decreased for one day, three days, five days and six days aged seeds of all samples respectively.


Based on the present investigation, it was evident that PK-262 was best stored among all cultivars. This was further proved by our observations of physiological parameters namely: germination percentage vigour, and electrical conductivity.



Figure 1 Lipoxygenase (nm/g.f.w) activity on zero day, three days and five days respectively in different accessions (SL 525, PK 262, PK 416 and PK 327) of soybean



Figure 2 Catalase (µ mol g-1 dw/min) activity on zero day, three days and five days respectively in different accessions (SL 525, PK 262, PK 416 and PK 327) of soybean



Figure 3 Peroxidase (µ mol g-1 dw/min) activity on zero day, three days and five days respectively in different accessions (SL 525, PK 262, PK 416 and PK 327) of soybean



Figure 4 Superoxide dismutase (SOD) (unit/mgdw) activity on zero day, three days and five days respectively in different accessions (SL 525, PK 262, PK 416 and PK 327) of soybean



Figure 5 Ascorbate peroxidase (APX) activity on zero day, three days and five days respectively in different accessions (SL 525, PK 262, PK 416 and PK 327) of soybean


3 Discussion

It was observed that due to ageing, seed viability and vigour decreased but electrical conductivity of seed leachate increased in all the soybean varieties tested. Decrease in germination of aged seeds might be due to either decline of alpha amylase activity and sugar content or denaturation of protein (Nautiyal et al., 1997). The electrical conductivity is related to the deterioration processes of seeds as degradation of cell membranes and leakage out of the cells (Delouche and Baskin, 1973). Thus many researchers have used electrical conductivity test to indicate seed vigour.  Germination percentage was found to be positively correlated with vigour index and negatively correlated with electrical conductivity of seed leachate. The germination percentage, vigour index and conductivity values were taken for 1, 3, 5 and 6-days aged seeds of the samples, respectively.


Seed ageing affected seed germination, seed vigour, electrical conductivity and activities of enzymes. In support of our results, Sung (1996) reported that both natural and accelerated ageing enhanced lipid peroxidation and reduced seed germination, ageing also inhibits the activity of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. Thus, it has been observed that lipid peroxidation is not only sole factor for determining the longevity patterns of soybean seeds but also the activities of catalase, peroxidase and superoxide dismutase and ascorbate peroxidase (APX) also play an important role in seed storage.


4 Materials and Methods

The study took place at National Bureau of Plant Genetic Resources New Delhi India. The test samples comprised of 100 seeds of soybean (Glycine max (L.) Merrill) (initial moisture, 10-11%) from each line, placed on wire mesh screen suspended over a 40 ml of water inside a plastic accelerated aging box (11.0 × 11.0 × 3.5 cm). Boxes were kept at 42°C and 100% relative humidity for 72 h (Hampton and Tekrony, 1995; Marcos-Filho, 1999). After the aging period, two replicates of 10 seeds each were submitted to the standard germination test. Seeds were germinated at 27°C in rolled towel papers moistened with water equivalent to 2.5 times the substratum weight. Seedlings were counted 5 and 8 days after sowing. The degree of seed longevity was expressed as the percent seed germination, vigour of the seed and conductivity. For this, initial moisture of eight varieties were measured and after that the moisture was brought to the level of 3, 4, 6 and 7% by keeping them in beads, sodium hydroxide solution and silica gel respectively for few days till the required moisture was attained. The samples were kept in air tight desiccators so that there was no hindrance due to atmospheric humidity. After regular intervals of 16 h, the moisture content of the respective eight varieties was recorded and was further kept in the desiccators if the moisture level is yet to be reached. The silica gel, beads and the sodium hydroxide solution were regularly replaced with the fresh ones so that the required moisture level can be reached. Once the required moisture level was reached, the seeds were assessed for viability and conductivity, the remaining material was kept for enzymatic assay.


4.1 Seed germination

Germination tests were conducted by taking 20 seeds of each variety in three replicates, which were placed in towel paper and kept in germinator maintaining adequate humidity and temperature of 27 ± 2°C. Germination percentage was recorded everyday up to 7th day. Germination % was calculated on the basis of normal and abnormal seedlings.


4.2 Vigour index

Seeding vigour index was calculated by multiplying percentage of germination with total seedling length (Abdul and Anderson, 1980).


4.3 Electrical conductivity

This test is based on the leakage of solutes that occurs through the cell membrane from all seeds into deionized water. Measurement of electrical conductivity of seed soaked in water, with a conductivity meter, the amount of electrolyte leakage can be accessed. From each accession, two replicates of seeds were taken and weighed. The seeds were soaked in 25 ml of distilled water at 25 ± 2°C and electrical conductivity was measured after an interval of 27 h. The electrical conductivity was measured by using conductivity meter and expressed as µs/cm/gm fresh weight basis.


4.4 Lipoxygenase activity

This involves measuring the amount of the product of lipid per oxidation monoaldehydes (1, 3 - propondial) by calorimetric method. 0.25 g seed samples were weighed and homogenized in 2 ml of distilled water using a pestle and mortar. Four milliliters of TBA-TCA reagent (0.5% 2- thiobarbituric acid in 20% trichloro acetic acid) was added. The sample was incubated at 95°C for 30 min. The reaction was stopped by placing the reaction tubes in the ice bucket. The samples were then centrifuged at 10,000 rpm for 10 min. The red colored supernatant were collected and absorbance was read at 532 nm and the value for non specific absorption at 600 nm was read and subtracted from specific absorption.


4.5 Catalase activity

Catalase assay was based on the absorbance of H2O2 at 240 nm in UV-range. A decrease in the absorbance was recorded over a time period as described by Aebi (1984).


4.6 Peroxidase activity

Peroxidase activity is assayed as increase in optical density due to the oxidation of guaiacol to terra-guaiasol (Castillo et al., 1984). The absorbance due to the formation of tetra-guaiacol was recorded at 470 nm and enzyme activity was calculated as per extinction coefficient of its oxidation product, tetra-guaiacol. Enzyme activity is expressed as mol tetra-guaiacol formed per min per g fresh weight or per mg protein.


4.7 Superoxide dismutase activity

The assay is based on the formation of blue colored formazone by nitro-blue tetrazolium and O2 radical, which is absorbed at 560 nm and the enzyme (SOD) decreases this absorbance due to reduction in the formation of radical (Dhindsa et al., 1981). The blank without enzyme consequently gave the highest absorbance, which decreased with the increasing enzymatic activity.


4.8 Ascorbate peroxidase activity

The assay is based on the decrease in absorbance of ascorbic acid at 290 nm, due to oxidation of ascorbic acid to mono- dehydroascorbic acid and dehydroascorbic acid. Enzyme assay was done according to Chen et al. (1989), and Nakano et al. (1981).



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Legume Genomics and Genetics
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