Agrochimica, Vol. LIII - N.

September-October 2009

Salt-induced biochemical changes in germinating seeds of three rice cultivars differing in salt tolerance
Plant Stress Physiology and Biochemistry Lab, Nuclear Institute for Agriculture and Biology, P.O. Box 128, Jhang Road, Faisalabad, Pakistan. E.mail: myashrafsp@yahoo.com; 2 Dept. of Botany, University of Agriculture, Faisalabad, Pakistan, Email: ashrafbot@yahoo.com; 3 Cholistan Institute of Desert Studies, Islamia University, Bahawalpur, Pakistan, Email: arshadbwp@yahoo.com
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M.Y. ASHRAF 1, M. ASHRAF 2*, N.H. NAVEED 2, N.A. AKRAM 2, M. ARSHAD 3

Received 16 September 08 - Received in revision form 24 February 2009 - Accepted 01 April 2009

Keywords: -amylase and protease activities, protein and amino acids; reducing and non-reducing sugars, rice seeds, salt stress.

Introduction. Plants are generally more sensitive to salinity during germination and early growth stages than at later growth stages (Van Hoorn, 1991; Siddiqi and Ashraf, 2008). The first physiological disorder which takes place during germination under saline conditions is reduced imbibition of water by the seed. This happens due to low solute potential of saline growth medium which causes a series of metabolic changes in plants, including changes in enzyme activities (Ashraf et al. 2002), imbalance in nitrogen metabolism (Lorenzo et al. 2001), and in the levels of plant growth regulators (Khan et al. 2004), and in general, reduction in hydrolysis and utilization of reserve food materials (Bewley and Black, 1994). Rice is very sensitive to salinity during germination and at flowering stages (Zeng and Shannon, 2000). In Pakistan, it is cultivated on about 1x106 hectares of salt affected lands as a result of which considerable reduction in rice production has been recorded (Government of Pakistan, 2007). To overcome this problem, it is crucial to devise some potential strategies. Studies of the physiological and biochemical changes in salt tolerant and sensitive varieties and identification of physiological markers may help in developing salt tolerant and high yielding rice varieties/ lines through appropriate breeding programs. Different physiological and biochemical changes are brought about by the enzyme action because enzymes are the organic compounds which act as catalysts in different chemical/biochemical reactions of biological systems (Holliday et al. 2007). Starch and proteins are reserve food materials in seeds, so studies on the breakdown of starch
*

Corresponding author: ashrafbot@yahoo.com

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and proteins and mobilization of their by-products may provide a useful information to differentiate salt tolerant and sensitive cultivars at an early stage. Most of the nitrogenous compounds are in the form of storage proteins in seeds, which undergo marked changes under salt stress (Ashraf et al. 2003). Protease becomes very active during germination, and it plays a vital role in mobilizing stored protein in seed as free amino acids, which are utilized to synthesize proteins and enzymes required for the growing embryo (Ashraf et al. 1998). For example, in cotton, total soluble protein increased in the germinating seeds of all cultivars under salt stress (Cusido et al. 1987), although a lack of salt stress effect was found on cotyledonary soluble protein content in the germinating achenes of sunflower (Ashraf et al. 2003). The three rice cultivars chosen for the present study have shown different degree of salt tolerance at the vegetative and adult stages (Ashraf et al. 1998; Ali et al. 2006), i.e., IRRI-6 being salt tolerant, NIAB-IRRI9 moderately salt tolerant, and 38/88 salt sensitive. The objective of the present study was to assess under saline conditions whether these rice cultivars which differ in their response to salt stress at the adult stage also differ at the initial growth stages (germination and seedling), in terms of -amylase and protease activities and mobilization of sugars and amino acids to the growing parts.
Materials and Methods. The seed of three rice cultivars i.e., IRRI-6 (salt tolerant), 38/88 (salt sensitive), and NIAB IRRI-9 (moderately salt tolerant) (Ashraf et al. 1998; Ali et al. 2006) was obtained from the Mutation Breeding Division of NIAB, Faisalabad, Pakistan. All seed samples were surface sterilized in 0.1% HgCl2 for 5 min, washed thoroughly in sterile distilled water and transferred on to filter papers in 14 cm Petri dishes. Ten cm3 of saline solutions (0, 50, or 100 mmol NaCl L-1 in Hoaglands nutrient solution) were added to each Petri dish. Five replications of each treatment were kept in a completely randomized design. The seeds were allowed to germinate in dark at 28 2C in growth chambers. Fresh samples were collected at every 24 h interval after sowing for the estimation of fresh weight and for the assay of -amylase, protease, soluble sugars, soluble protein and total amino acids. Water uptake. The gradual daily increase in weight of seeds was recorded regularly up to 120 h after sowing. The difference between the weight of dry seeds with that of germinating ones indicates the water uptake by the germinating seeds. Alpha amylase. The activity of -amylase was determined according to Chrispeel and Varner (1967). The pre-chilled leaf material (1 g) was homogenized in a mortar, extracted with cold 1% NaCl, 5 mL phosphate buffer (pH 7.2), 2 mL alkaline starch

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solution (0.1%), and 2 mL 0.01 N HCl. The material was centrifuged at 10,000 x g and the supernatant was used to determine the enzyme activity. The enzyme activity was expressed as mg of starch hydrolyzed g-1 fresh weight h-1. Reducing and non-reducing sugars. Reducing sugars in the plant samples were extracted in 80% alcohol (1:10, w:v) following the method of Nelson (1944). One mL of the extract was added to 5 mL of o-toluidine and heated at 97C for 15 min. It was cooled in ice cold water and read at 630 nm with a spectrophotometer (Hitachi-220 Japan). Total sugars were determined from the same alcoholic extracts after their digestion with 0.5 N HCl. The non-reducing sugars were calculated according to the method of Loomis and Shull (1937). Ethanol extract (0.1 mL) tube, added of 0.1 mL of 5.4 N KOH, heated at 97C for 10 min and cooled in ice cold water. Then 3 mL of anthrone were added and heated at 97C for 5 min. It was again cooled in ice cold water, incubated at room temperature for 20 min and read at 620 nm. Protease activity. Protease activity was determined following the method of Ainouz et al. (1972). Five germinating seeds were homogenized in a mortar, extracted with cold 1% NaCl in 0.2 M phosphate buffer (pH 7.0) and centrifuged at 12000 x g for 30 min. One ml of the supernatant was incubated at 50C with 5 cm3 of 1% casein in 0.2 M phosphate buffer (pH 6.0). The reaction was terminated after 60 min with 1 cm3 of 40% TCA (trichloroacetic acid). The proteolytic activity was measured at 570 nm in TCA soluble fraction after reaction with Folin phenol reagent (Lowry et al. 1951). Total soluble proteins. Five germinating seeds were ground in 0.1 mol NaCl dm-3 solution (Ainouz, 1970) and filtered through a nylon cloth. The filtrate was precipitated with an equal volume of 10% TCA and centrifuged at 10,000 x g for 5 min. The pellet was re-suspended in 0.1 mol NaOH dm-3 and soluble protein was then estimated following Lowry et al. (1951). Total amino acids. Fresh leaf material (0.1 g) was extracted in 10 mL citrate buffer (20 g citric acid monohydrate dissolved in 200 mL of 1N NaOH and diluted to 500 mL with distilled water, pH 5), incubated at room temperature for 10 min and centrifuged it at 15000 x g at 15C for 10 min. Then, to one mL of the supernatant, 1 mL of the acid ninhydrin solution (0.8 g SnCl2 dissolved in 500 mL citrate buffer mixed with 0.2 g ninhydrin in 200 mL methyl cellosolve) was added and heated for 20 min. After cooling it, 5 mL of diluent solvent (n-propanol and distilled water in 1:1 ratio) were added, mixed and incubated at room temperature for 15 min. The optical density of each sample was measured at 570 nm using a spectrophotometer (IRMECO U2020). Concentration of total amino acids was calculated following Moore and Stein (1948). Statistical Analysis. A two way analysis of variance was performed for all attributes using the MSTAT Computer Program (MSTAT Development Team, 1989). Two factors were cultivars and salt treatments. The Duncans New Multiple Range test at the 5% level of probability was used to test the differences among mean values following Steel and Torrie (1986).

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Results. Salt stress significantly affected water uptake in the germinating seeds of all rice cultivars (Table 1). There was a gradual increase in water uptake from 24 h to 72 h and then a sharp increase after 72 h up to 96 h reaching a maximum value at 120 h (Figure 1A, B, C) in the germinating seeds of all rice cultivars. Water uptake by the germinating seeds was high under control conditions in all rice cultivars, but it gradually decreased with increase in salinity of the growth medium. Under saline conditions, the germinating seeds of cv. IRRI-6 maintained the highest water contents among the three cultivars (Figure 1 A). Salt stress significantly affected germination percentage in all rice cultivars (Table 2). A maximum germination percentage was recorded under control conditions, followed by that under 50 and 100 mmol NaCl L-1 (Table 3). A maximum value of germination percentage was recorded in cv. IRRI-6 followed by cvs. NIAB-IRRI-9 and 38/88 (Table 3). All cultivars responded in a similar way under control conditions in terms of mean germination percentage. As the salt concentration increased, germination percentage significantly decreased in all rice cultivars. However, cv. IRRI-6 performed better than the other two cultivars at 50 and 100 mmol L-1 NaCl.
Table 1. Mean squares from analysis of variance of data for different parameters of germinating seeds of three rice cultivars grown under saline conditions. Parameters Time Variety Variety Salinity Salinity Salinity Salinity Error x x x interval X Time Variety Time x Variety Time df 4 2 8 2 8 4 16 90 Water 80.22 ** 5.09 ** 1.95 * 47.68 ** 5.47 ** 2.86 ** 1.49 ** 0.22 uptake 156.60 ** 21.78 *** 0.18 * 175.78 ** 1.70 * 0.84 * 0.14 NS 0.16 -amylase ** ** * ** * NS Protease 5.24 0.35 0.04 2.91 0.43 0.007 0.004 NS 0.008 Protein 1.899 ** 0.020 * 0.013 * 1.617 ** 0.079 * 0.003 NS 0.011 NS 0.007 ** * ** * Amino 4.63 0.65 7.03 0.08 0.009 NS 0.02 * 0.01 * 0.001 acids Reducing 5.03 ** 0.5 * 0.007 * 3.42 ** 0.48 * 0.001 NS 0.003 NS 0.004 sugars Non262.91 ** 1.05 * 1.16 * 29.53 ** 9.44** 0.07 NS 0.09 NS 0.24 reducing sugars
*, **, *** NS

significant at 0.05, 0.01, and 0.001 levels, respectively. non-significant

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Fig. 1 Time course of water uptake (A, B, C), and -amylase activity (D, E, F) in germinating seeds of three rice cultivars under saline conditions.

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Table 2. Mean squares from analysis of variance of data for different parameters of germinating seeds of three rice cultivars grown under saline conditions. Parameters df Shoot length Root length Fresh weight Germination (%)
* ** NS

Variety 2 0.80 * 1.51 ** 2.49 ** 27.40 **

Salinity 2 29.35 ** 0.77 * 31.42 ** 1421.02 **

Variety x Salinity 4 0.14 NS 0.28 * 0.30 NS 78.26 *

Error 18 0.17 0.23 0.34 21.05

significant at 0.05, and 0.01 levels, respectively. non-significant

Table 3. Effect of salinity (NaCl) on germination and seedling growth of three rice cultivars. Rice Variety/Treatment IRRI-6 t1 (0 mmol L-1 NaCl) t2 (50 mmol L-1 NaCl) t3 (100 mmol L-1 NaCl) 38/88 t1 t2 t3 NIAB-IR-9 t1 t2 t3 Germination (%) 85.57 a 65.67 b 57.73 c 70.00 a 63.43 b 49.10 c 82.33 a 61.20 b 56.63 c Shoot length (cm) 7.26 a 5.48 b 3.79 c 7.02 a 4.60 b 3.26 c 6.86 a 4.67 b 3.54 c Root length (cm) 4.26 a 3.86 b 3.56 b 4.23 a 4.05 a 3.03 b 4.16 a 4.07 b 3.25 c Fresh weight of 100 seedlings (g) 9.86 a 7.40 b 6.84 c 9.58 a 6.12 b 5.59 c 9.27 a 6.14 b 6.05 b

The mean values sharing same letter in the same column do not differ significantly at p 0.05.

Salt stress significantly affected fresh weight of seedlings of all cultivars (Table 3). The highest seedling fresh weight was recorded under control conditions followed by that in 50 and 100 mmol NaCl L-1. Cultivar IRRI-6 was the highest in seedling fresh weight followed by cvs. NIAB-IRRI-9 and 38/88. Salt stress significantly affected seedling shoot and root length in all cultivars (Table 2). The highest shoot and root length was recorded under control conditions. Cultivar IRRI-6 responded better than the other two cultivars in shoot and root length under saline conditions (Table 3). However, a marked reduction in root length was observed at 50 and 100 mmol NaCl L-1. Comparison

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of the cultivars under saline conditions showed that cv. IRRI-6 had the maximum root length followed by cvs. NIAB-IRRI-9 and 38/88 (Table 3). Alpha-amylase activity in the germinating seeds of all cultivars was reduced significantly due to salt stress which caused changes in reducing and non-reducing sugars in the germinating seeds of all rice cultivars (Figures 1, 2, 3). There was a slow increase in sugar content in the germinating seeds of all rice cultivars up to 48 h; then there was a sharp increase up to 96 h and thereafter it remained steady up to 120 h. The effect of salt stress was very severe in cv. 38/88 where the reduction in -amylase activity was 54 and 40 percent at 50 and 100 mmol NaCl L-1, respectively. Highest -amylase activity was observed in the germinating seeds of cv. NIAB-IRRI-9 under control conditions. The release of both reducing and non-reducing sugars from germinating seeds followed a trend similar to that of -amylase activity in all three rice cultivars (Figure 2). The levels of sugars gradually increased in the germinating seeds of all cultivars with time, but the sugar content decreased with the increase in salt concentration of the growth medium. In the germinating seeds of all three cultivars, non-reducing sugars were higher than reducing sugars at all levels of salinity (Figure 2). Germinating seeds of cv. NIAB-IRRI-9 had the highest and those of cv. 38/88 the lowest reducing sugars of all cultivars (Figure 2, B, C, E, F). For non-reducing sugars, the germinating seeds of IRRI-6 responded better than the other two cultivars (Figure 2 D), while cv. 38/88 was the poor performer in terms of non-reducing sugars (Figure 2 E). There was a positive correlation between -amylase activity and release of sugars (Figure 1 and 2) in the germinating seeds of all rice cultivars. The proteolytic activity in the germinating seeds of all rice cultivars increased with time under all salt treatments, but it significantly decreased with increase in external salt level (Figure 3). Protease activity showed a slow increase up to 48 h in the germinating seeds; thereafter, a steep increase in the activity was recorded, and it was the highest at 120 h in all three rice cultivars. Protease activity was maximum in the germinating seeds of cv. NIAB-IRRI-9 and minimum in those of cv. 38/88 (Figure 3 B). The germinating seeds of cv. 38/88 had significantly higher level of protein than those of cvs. IRRI-6 and NIAB-IRRI-9 (Figure 3 D, E, F). Salt stress significantly reduced the protease activity thereby resulting in a decrease in protein breakdown in the germinating seeds of all cultivars. The protein contents in the germinating seeds of all cultivars progressively decreased with increase in growth period. The

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Fig. 2 Time-course of reducing (A, B, C) and non-reducing (D, E, F) sugars in germinating seeds of three rice cultivars under saline conditions.

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Fig. 3 Time-course of protease activity (A, B, C) and protein (D, E, F) in germinating seeds of three rice cultivars grown under saline conditions.

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total amino acids increased significantly with the increase in protease activity in the germinating seeds of all rice cultivars under salt treatments (Figure 4). The amount of amino acids increased with time and was the highest at 120 h after sowing in the germinating seeds of all rice cultivars. A maximum increase in amino acids was recorded in cv. NIAB-IRRI-9 followed by cv. IRRI-6 and 38/88 (Figure 4 A, B, C). The concentration of total amino acids in the germinating seeds of all cultivars decreased significantly with increase in salinity, and cv. 38/88 was the lowest of all cultivars in seed amino acid content (Figure 4 B). Discussion. It is now well evident that in germinating seeds, regulation of a multitude of metabolic processes takes place (Bewley and Black, 1994). Increase in the activities of certain key enzymes is vital for the supply of food reserves to the growing embryo. Alpha-amylase along with -glucosidase and starch phosphorylase are the key enzymes, which hydrolyse the starch present in the endoderm of cereal grains (Sun and Henson, 1990). Alpha-amylase attacks the (1-4) linkages in either amylose or amylopectin in a random fashion to split the macromolecules into various low molecular weight (short chain) dextrins (Sun and Henson, 1990). The decrease in -amylase activity in the germinating seeds of all rice cultivars following salt stress, and its increase with time shows that although salinity reduced the activity of enzyme, it had the ability to break starch into sugars to fulfill the necessary requirement of the growing seedlings during germination. There are some reports which also show that both salinity and water stress can reduce -amylase activities in germinating seeds (Cordeiro et al. 2002). The difference observed in rice cultivars in -amylase activity is similar to what has been earlier observed in some sorghum varieties grown under saline conditions (Khan et al. 1989). Reducing and non-reducing sugars increased with time in the germinating seeds, but the increase was substantially low in those seedlings which had been growing under saline conditions. It was observed that germinating seeds with higher -amylases were successful in maintaining the higher levels of reducing and non-reducing sugars during germination (Chanda et al. 1999). The same was true in the salt tolerant rice cv. IRRI-6 (Figures 1, 2). Breakdown of stored protein in the germinating seeds by protease is a vital phenomenon so as to supply amino acids to the growing embryo for the synthesis of new proteins (Tan-Wilson et al. 1996). Proteases which catalyze limited or complete protein degradation are synthesized

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Fig. 4 Time-course of total soluble amino acids (A, B, C) in three rice cultivars grown under saline conditions.

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as precursors in germinating seeds (Moutou et al. 2004). The results of the present study show that proteolytic activity increased gradually during germination with a steep increase after 48 h, being higher in cv. IRRI-6 and lower in cv. 38/88. This is in agreement with previous findings observed in pearl millet (Ramana and Radhakrishnan, 1987), sorghum (Khan et al. 1989), and cotton (Ashraf et al. 2002). The protease activity, however, decreased with the increase in salinity. The decrease in proteolysis caused by salinity resulted in slower mobilization of reserve protein during seed germination, thus the residual protein level was higher in the germinating rice seeds with the increase in salinity. Dubey and Rani (1990) reported that NaCl reduced protease activity in the germinating seeds of rice so that protein breakdown decreased. The reserve protein in the germinating seeds of all three rice cultivars decreased gradually with time, which may have been due to time-course of protein hydrolysis. Khan et al. (1993) found that in wheat seeds germinating under different NaCl levels, total protein concentration decreased in controls on third day of germination, but remained relatively high in saline solutions. They reported slow breakdown of protein in the germinating seeds with increasing salinity levels. Breakdown of amino acids is generally linked to the production of metabolites for other biosynthetic pathways like enzymes, proteins, plant hormones etc. (Tempone et al. 2007). However, the amount of amino acids in growing plant tissues is maintained to a certain level to fulfill the requirements for the biosynthesis of proteins (Iqbal et al. 2006). The results of the present study show that the levels of amino acids increased gradually with time and decreased with the increase in external salt concentration. Similarly, Debouba et al. (2006) reported that NaCl salinity reduces or delays biosynthesis of protein necessary for plant growth. The results of the present study also indicate that the activity of protease and other associated metabolic changes were reduced or delayed in germinating rice seeds due to salinity. As a result, germination, emergence, fresh weight of seedlings, and shoot and root lengths were reduced significantly in rice cultivars in agreement with what had been already observed in rice and cotton (Chachar et al. 2008). In conclusion, in the germinating seeds of all three rice cultivars salt stress reduced shoot fresh weight, germination rate, water uptake, -amylase and protease activities, and accumulation of sugars. Cultivar IRRI-6 was the highest and cv. 38/88 the lowest in growth, -amylase and protease activities, and reducing and non-reducing sugars. Thus, all

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three rice cultivars, known for their differing salt tolerance at the adult stage, maintained their respective degree of salt tolerance at the initial growth stages i.e., germination and seedling stage. Attributes appraised in the rice cultivars at the initial growth stages might be used as potential indicators for breeding for salt tolerance in rice.
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Moore, S. and Stein, W.H.: Method in enzymology. Vol. III, (Eds. Colowick and N.O. Kaplan). Academic Press, Inc. NewYork. R.G.D (1948). Moutou, K.A., Panagiotaki, P. and Mamuris, Z.: Effects of salinity on digestive protease activity in the euryhaline sparid Sparus aurata L.: a preliminary study. Aquaculture Res., 35, 912-914 (2004). MSTAT DEVELOPMENT TEAM.: MSTAT users guide: A microcomputer program for the design management and analysis of agronomic research experiments. Michigan State Univ. East Lansing, USA (1989). Nelson, N.: A photometric adaptation on the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375380 (1944). Ramana, T. and Radhakrishanan, T.M.: De novo synthesis of protease during germination of pearl millet seeds. Curr. Sci., 59, 347-400 (1987). Siddiqi, E.H. and Ashraf, M.: Can leaf water relation parameters be used as selection criteria for salt tolerance in safflower (Carthamus tinctorius L.). Pak. J. Bot., 40, 221-228 (2008). Steel, R.G.D. and Torrie, J.H.: 1986. Principles and procedures of statistics. McGraw Hill Book Co., Inc. New York. Sun, Z. and Henson, C.A.: Degradation of native starch granules by barley -glucosidases. Plant Physiol., 94, 320-327 (1990). Tan-Wilson, A.L., Xiaowen, L., Ruoying, C., Xiaoquin, Q.I. and Wilson, K.A.: An acidic amino acid-specific protease from germinating soybeans. Phytochemistry, 42, 313-319 (1996). Tempone, A.G., Sartorelli, P., Mady, C. and Fernandes, F.: Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. Cell Mol. Biol., 15, 15-25 (2007). Van Hoorn, J.W.: Development of soil salinity during germination and early seedling growth and its effect on several crops. Agric. Water Manag., 20, 17-21 (1991). Zeng, L. and Shannon, M.C.: Salinity effects on seedling growth and yield components of rice. Crop Sci., 40, 996-1003 (2000).

SUMMARY. A study was conducted to assess some salt-induced physiological and biochemical changes in the germinating seeds of three rice cultivars (IRRI-6, salt tolerant; NIAB-IRRI-9, moderately salt tolerant; 38/88, relatively salt sensitive) under controlled conditions. The results from different biochemical analyses showed that increase in NaCl concentration in the growth medium reduced the activities of -amylase and protease in the germinating seeds of all three rice cultivars examined, however, the reduction in enzymatic activities was more pronounced in cv. 38/88 than that in the other cultivars. A decrease in the concentration of reducing and non-reducing sugars in the germinating seeds of all cultivars was observed, particularly during the period from 24 to 72 h. The decrease in sugar content in the germinating seeds was found to be attributable to salt-induced reduction in -amylase activity. Similarly, as a result of saltinduced reduction in protease activity, a decrease in the breakdown of reserve proteins and accumulation of amino acids were recorded in the germinating seeds of all cultivars. Under saline conditions cv. IRRI-6 followed by NIAB-IRRI-9 was the highest, whereas cv. 38/88 the lowest in germination, growth, -amylase and protease activities, reducing and non-reducing sugars. Overall, the three rice cultivars, known for their different degree of salt tolerance at the adult stage, maintained their respective salt tolerance potential at the germination stage.