Potassium starvation-induced oxidative stress and antioxidant defense responses in Brassica juncea

Abstract

In this study, changes in growth, chlorophyll pigments, proline, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents, and antioxidative enzyme activities were investigated in the seedlings of four different cultivars (cvs) of mustard [Brassica juncea (L.) Czern. & Coss.], i.e. Varuna, RH-30, Rohini, and Vaibhave under potassium (K) nutrition-deficient conditions. K deficiency induced a significant decrease in concentrations of photosynthetic pigments in all four cvs, however, this decrease was higher in cvs. Varuna and RH-30. During K deficiency, proline concentration increased in all mustard cvs, but a maximum increase in this parameter was shown by cvs. Varuna and RH-30. The activity of the key proline metabolizing enzyme γ-glutamyl kinase increased more in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. The proline oxidase activity showed greater increase in cvs. Vaibhave and Rohini compared to cvs. Varuna and RH-30. K deficiency increased the concentrations of H₂O₂ and the activities of anti-oxidative defense system enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) in the seedlings of all mustard cvs, but higher activities of these enzymes were observed in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. A significant lipid peroxidation in terms of MDA contents was also observed in the K-deficient seedlings of all four mustard cvs. This study suggests that K-starvation-induced oxidative stress through the high generation of reactive oxygen species (ROS). All mustard cvs counteracted to some extent the effects of ROS by activation of antioxidant machinery. Overall, the results indicate that of all four mustard cvs, Varuna and RH-30 were tolerant to K deficiency.

Keywords:

• Brassica juncea
• K deficiency
• chlorophyll pigments
• proline contents
• H₂O₂
• lipid peroxidation
• antioxidant enzymes

Introduction

Plants are known to develop a wide range of adaptive mechanisms under various environmental stress conditions to ensure their survival and maintain growth and productivity (Ahmad & Sharma 2010; Hamdia & Shaddad 2010; Anjum et al. 2011; Shahbaz et al. 2012). Mineral nutrients are essential for the growth and development of plants, and are also an important factor in plant-environment interactions. Nutrients affect the plants' response to environment, providing resistance against the changing/adverse environment (Marschner 1995; Akram et al. 2009; Akram & Ashraf 2011). Potassium (K), an important macronutrient plays a vital role in metabolic processes, growth and adaptations to environmental stresses. K is known to be involved in activation of different enzymes and stabilization of protein synthesis of over 60 different enzyme systems in plants (Suelter 1970). K plays an active role in different physiological processes like photosynthesis, phloem solute transport of photo-assimilates into sink organs, activation of enzymes, and maintenance of cation:anion balance (Marschner 1995; Mengel & Kirkby 2001; Akram et al. 2008; Akram & Ashraf 2011).

Plant movements such as stomatal and leaf movements, and other plant tropisms are driven by K⁺ to make the surrounding guard cells either turgid or flaccid (Maathuis & Sanders 1996; Philippar et al. 1999; Franks & Farquhar 2007). K accumulation generates osmotic pressure within cells that initiates cellular and leaf expansion (Maathuis & Sanders 1996; Elumalai et al. 2002). K deficiency also known as potash deficiency is of great agricultural importance because it leads to reduced chlorophyll content and hence decreased photosynthetic activity. The rate of adenosine tri-phosphate (ATP) production is also known to be reduced under K deficiency, slowing down-regulation of the processes dependent on ATP (Hartt 1969; Pier & Berkowitz 1987; Zhao et al. 2001).

Reduced K supply is known to reduce the translocation of nitrates, phosphates, calcium, magnesium, and amino acids. The process of photosynthetic CO₂ fixation and utilization of photo-assimilates is strongly dependent on K supply (Cakmak 1994, 2005). K deficiency severely reduces the photosynthetic CO₂ fixation. It also leads to impairment in partitioning and utilization of photosynthates. Such perturbed photosynthetic activity results in accumulation of photosynthetically produced electrons that stimulate the production of reactive oxygen species (ROS) (Cakmak 2005; Ashraf & Akram 2009). ROS disrupt the normal functioning of the cell and leads to oxidative stress which in turn leads to membrane disruption, protein modifications, inhibition of enzyme activity, damage to nucleic acids, etc. (Shahbaz et al. 2008; Ashraf 2009; Perveen et al. 2010, 2011, 2012; Akram et al. 2012).

Mustard [Brassica juncea (L.) Czern & Coss.] is one of the potential oil-seed crops (Khan et al. 2002; Akmal et al. 2011). However, its growth and seed-oil-content significantly depends on the K concentration in the growth medium of the crop as K uptake begins when rapid plant growth starts and continues until grain formation (Kuo & Chen 1980). Thus, it is necessary to understand how the amount and availability of K could alter crop growth and associated physio-biochemical parameters. Published work on the effect of K deprivation on mustard plants is not much. Keeping this in view, the aim of this study was to investigate the K deficiency-induced modulation in growth, photosynthetic pigments, proline, and activities of anti-oxidant enzymes in the seedlings of four elite mustard cvs.

Materials and methods

Seeds of four B. juncea cvs (Varuna, RH-30, Rohini, and Vaibhave) were surface-sterilized with 2% (w/v) sodium hypochlorite (NaOCl) for 10 min, then washed with distilled water and kept in distilled water for 24 h. The seeds were planted in plastic pots (diameter=30 cm) which contained peat, perlite, and sand (1:1:1, v/v/v). Thinning was done by maintaining one plant per pot after germination. The seedlings were allowed to grow for three weeks under constant temperature 25±4°C for 12–13 h of photoperiod.

After 25 days, plants were transferred to plastic trays containing basic nutrient solution. The composition of the nutrient solution was as follows (mg L⁻¹): 270 N, 31 P, 234 K, 200 Ca, 64 S, 48 Mg, 2.8 Fe, 0.5 Mn, 0.5 B, 0.02 Cu, 0.05 Zn, and 0.01 Mo. The pH of the nutrient solution was monitored daily and maintained at 6.5 with 0.1 mM KOH.

Seven days after transplantation, plastic trays containing seedlings were divided into two groups. Group 1 plants received complete nutrient solution and served as control, while plants in group 2 received the nutrient solution which was deficient in K. The nutrient solution was replaced every alternate day. Symptoms of K deficiency were seen after 27 days of transplantation. After 35 days, the plants were harvested for the analyses. The experiment was laid out in a randomized block design. Each replicate comprised five plants and the treatment was replicated four times (20 plants per treatment).

Determination of chlorophyll content

Hiscox and Israelstam (1979) method was employed for the determination of chlorophyll content. Fresh leaves (100 mg) were put in a test tube containing 10 mL of dimethyl sulphoxide (DMSO). The test tubes were incubated at 65°C for 40 min. Absorbance was read at 480, 510, 645, and 663 nm spectrophotometrically (Beckman 640 D, USA). For the control, DMSO was used.

Proline determination

The method of Bates et al. (1973) was used for the quantification of proline content. Fresh leaves (300 mg) were homogenized in 10 mL 3% aqueous sulfosalicylic acid. After this, the material was subjected to centrifugation for 15 min at 10,000 rpm. The supernatant (2 mL) was mixed with 2 mL of glacial acetic acid and acid ninhydrin and incubated the mixture at 100°C for 1 h. Termination of the reaction took place in an ice bath. Toluene (4 mL) was mixed to the extract and vortexed for 20 s. The chromophore containing toluene was aspirated from the aqueous phase, and absorbance read at 520 nm using toluene as a blank.

Determination of γ-glutamyl kinase activity [ATP: l-glutamate 5-phosphotransferases (EC 2.7.2.11)]

The method of Hayzer and Leisinger (1980) was used for the determination of activity of γ-GK. Fresh leaves (1.0 g) were homogenized in Tris-HCl buffer (50 mM; pH 8.5). The homogenate was centrifuged for 30 min at 40,000 rpm. To the supernatant, ATP (10 mM) was added to prepare the reaction buffer. From this reaction buffer, 0.1 mL was taken and incubated at 37°C for 30 min. Then, 2 mL of the stop buffer were added. Absorbance was read at 535 nm spectrophotometrically (Beckman 640 D, USA). The enzyme activity was expressed in mg⁻¹ protein.

Determination of proline oxidase activity (PROX) [L-proline: O₂oxidoreductase (EC 1.4.3.1)]

The PROX activity was determined according to Huang and Cavalieri (1979). Fresh leaves (1 g) were homogenized in 5 mL of Tris-HCl buffer (pH 8.5). After centrifugation of the homogenate at 10,000 rpm for 10 min, the supernatant was collected and again subjected to centrifugation for 20 min at 25,000 rpm. The assay mixture contained reaction extract (0.1 mL), 50 mM Tris-HCl buffer, 5 mM MgCl₂, 0.5 mM NADP⁺, 1 mM KCN, 1 mM phenazine methosulphate (PMS), 0.06 mM 2,6-dichlorophenol indophenols (DCPIP), and distilled water. Then, 3 mL of this assay mixture were used to determine the absorbance at 600 nm monitored continuously for 5 min. The activity of PROX was expressed in units mg⁻¹ protein (1 U=mM DCPIP reduced min⁻¹ mg⁻¹ protein).

Determination of hydrogen peroxide (H₂O₂) content

The protocol described by Velikova et al. (2000) was followed to determine the H₂O₂ content. Fresh leaves (500 mg) were homogenized in 5 mL of 0.1% (w/v) trichloro-acetic acid (TCA). Centrifugation of the homogenate was performed at 12,000 rpm for 15 min. To the 5 mL supernatant, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide (KI) were added. The absorbance of this mixture was read spectrophotometrically at 390 nm. The standard calibration curve was plotted using different concentrations of H₂O₂ and the content of H₂O₂ in the samples was calculated by comparison with the standard curve.

Determination of lipid peroxidation

Malondialdehyde (MDA) equivalents were used to determine lipid peroxidation rates following the method of Hodges et al. (1999). Leaf tissue (0.5 g) was crushed in 80% ethanol and the homogenate subjected to centrifugation at 3000 rpm for 10 min at 4°C. The same solvent was used for the extraction of pellet twice. The supernatant (1 mL) was taken in a test tube and equal volumes of solution of 20% TCA, 0.01% butylated hydroxyl toluene (BHT), and 0.65% (w/v) thiobarbituric acid (TBA) were added. This mixture was heated for 25 min at 95°C and then cooled at room temperature. The absorbance was read at 440, 532, and 600 nm spectrophotometrically.

Extraction of the enzymes

Fresh leaves (10 g) were crushed with 50 volumes of 100 mM Tris-HCl (pH 7.5) containing 5 mM DTT, 10 mM MgCl₂, 1 mM EDTA, 5 mM magnesium acetate, 1.5% PVP-40, 1 mM PMSF, and 1 µg mL⁻¹ aproptinin. The homogenate was filtered using a cheesecloth and subjected to centrifugation for 15 min at 10,000 rpm. The source of enzyme was the supernatant collected after centrifugation. The extraction buffer also contained serine and cysteine proteinase inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF)+1µg mL⁻¹ aproptinin]. For the analysis of ascorbate peroxidase (APX) activity, tissues were separately homogenized in the homogenizing medium (as described above for the other enzymes) with 2 mM ascorbate. All experiments were performed at 4°C. The soluble protein content was estimated according to Bradford (1976) with bovine serum albumin as blank.

Enzyme assay

Superoxide dismutase

Activity of superoxide dismutase (SOD, EC 1.15.1.1) was appraised according to Van Rossun et al. (1997) following photoreduction of nitroblue tetrazolium (NBT). The reaction mixture contained supernatant (100 µL), 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 µM NBT, 2 µM riboflavin. To initiate the reaction, the mixture was placed in tubes under 15 W fluorescent lamps. The lamps were removed after 10 min to terminate the reaction. Non-illuminated reaction mixture acted as a blank. The absorbance was read at 560 nm spectrophotometerically. The activity of SOD was expressed as unit mg⁻¹ protein. One unit of SOD was defined as the amount of protein causing 50% decrease of the SOD-inhibitable NBT reduction.

Catalase (CAT)

Catalase (CAT, EC 1.11.1.6) activity was determined according to Luck (1974). For this, 20 mM H₂O₂ (3 mL) and 50 mM phosphate buffer (pH 7.0) were added to the enzyme extract (50 µL). The decrease in absorbance was read at 249 nm. The activity of CAT was calculated using the extinction co-efficient of 36×10³ mM⁻¹ cm⁻¹ and expressed as unit mg⁻¹ protein.

Ascorbate peroxidase

The method of Nakano and Asada (1981) was followed for the determination of APX activity. The assay mixture (3 mL) contained: enzyme extract (0.1 mL), 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H₂O₂, and 50 mM potassium phosphate buffer (pH 7.0). The decrease in absorbance was read at 265 nm. APX activity was assayed by measuring the oxidation of ascorbate at 290 nm. APX activity was expressed as unit mg⁻¹ protein.

Glutathione reductase (GR)

The activity of GR (EC 1.6.4.2) was determined according to Carlberg and Mannervik (1985). The assay mixture (1 mL) contained: potassium phosphate buffer (0.75 µL) (pH 7.0), 2 mM EDTA, 2 mM NADPH (75 µL), and 75 µL (20 mM) oxidized glutathione (GSSG). Addition of enzyme extract (0.1 mL) to the assay mixture initiated the reaction. The decrease in absorbance was read at 340 nm for 2 min. The activity of GR was calculated using the extinction co-efficient of NADPH of 6.2 mM⁻¹ cm⁻¹ and expressed as unit mg⁻¹ protein.

Statistical analysis

One-way analysis of variance (ANOVA) was used for statistical analysis followed by Duncan's Multiple Range Test (DMRT). The values obtained were the mean±S.E. for five samples in each group. P values at 0.05 were considered as significant.

Results

Biomass yield

Analyses of variance and mean data for shoot and root lengths (Table 1; Figure 1A, B) show that a maximum decrease in shoot length (39.9%) was found to be in cv. Vaibhave and minimum (30%) in cv. Varuna under K-deficient conditions as compared to control conditions. The decrease in root length was observed to be 53.3% in cv. Varuna and 63.6% in cv. Vaibhave. A decrease in shoot dry weight (DW) was also observed which was 51.1% in cv. Varuna, 54.4% in cv. RH-30, 59.3% in cv. Rohini and 63.4% in cv. Vaibhave under K-deficient conditions as compared to control conditions. The decrease in root DW was highest in cv. Vaibhave (66%) and lowest (57.1%) in cv. Varuna under K-deficient conditions (Table 1; Figure 1C, D).

Table 1. Mean squares from analyses of variance of data for shoot and root lengths, shoot and root dry weights (DW), photosynthetic pigments, proline contents, activities of antioxidant enzymes and levels of non-enzymatic antioxidants of four mustard [B. juncea (L.) Czern & Coss.] cultivars grown under control, or potassium (K)-deficient conditions.

Source of variation	df	Shoot length	Root length	Shoot DW	Root DW
Cultivars (cvs)	3	23.07**	8.745**	2.98**	0.523**
Potassium (K	1	2115.0**	900.4**	283.6**	64.35**
Cvs×K	3	1.333*	0.505ns	0.183ns	0.004ns
		Chlorophyll a	Chlorophyll b	Total chlorophyll	Carotenoids
Cvs	3	0.028**	0.0066*	0.10*	0.006ns
K	1	1.717**	0.531**	4.699**	0.673**
Cvs×K	3	0.0014ns	0.0004ns	0.01ns	0.00005ns
		Proline	γ-GK	Proline oxidase	H2O2
Cvs	3	548.2**	0.98*	0.0063*	0.0757ns
K	1	49959.3**	16.335**	0.109**	8.108**
Cvs×K	3	425.9**	0.295ns	0.0018ns	0.047ns
		MDA	SOD	CAT	APX
Cvs	3	2.673**	305.5**	1901.4**	0.75ns
K	1	47.88**	8336.5**	22143.4**	50.46**
Cvs×K	3	1.013*	146.7**	184.4*	0.03ns
		GR
Cvs	3	1.605**
K	1	33.13**
Cvs×K	3	0.405ns

ns, non-significant; * and **, significant at 0.01 and 0.001 levels, respectively.

Figure 1. Potassium (K) deficiency-induced changes in shoot and root length (A,B), shoot and root DWs (C,D), Chlorophyll a, b (E,F), total chlorophyll (G), and carotenoids (H) in different cultivars of mustard.

Note: Different letters (a–f) indicate least significant difference between and within means. Means±S.E.; n=3; Cvs, cultivars; K, K-deficiency.

Chlorophyll and carotenoid content

The results for chlorophyll and carotenoid contents in the four mustard cvs under K⁺ deficiency (Table 1; Figure 1E–H) show a minimum reduction in chlorophyll a (35.3%) in cv. Varuna and maximum (41%) in cv. Vaibhave as compared to control conditions. The decrease in chlorophyll b in cvs. Varuna, RH-30, and Rohini was 44% and that in cv. Vaibhave 46%. Of all cultivars, cvs. Varuna and RH-30 showed a minimum decrease (38%) in total chlorophyll, while cvs. Rohini and Vaibhave showed a maximum decrease (42%) in total chlorophyll. The carotenoid content was 55.7% in cv. Varuna, 57.8% in cv. RH-30, 61.1% in cv. Rohini, and 62.9% in cv. Vaibhave (Table 1; Figure 1E–H).

Proline content

Generally, there was a substantial increase in proline content in all four mustard cvs due to K deficiency as compared to control conditions. For example, proline content was found to increase 3 times in cv. Varuna, 2.6 times in cv. RH-30, 2.4 times in both cvs. Rohini and Vaibhave as compared to control under K deficiency (Table 1; Figure 2A).

Figure 2. Potassium (K) deficiency-induced changes in proline (A), γ-glutamyl kinase (B), proline oxidase (C), H2O2 (D), MDA (E), and activities of SOD, APX, CAT, and GR enzymes (F–I) in different cultivars of mustard.

Note: Different letters (a–f) indicate least significant difference between and within means. Means±S.E.; n=3; Cvs, cultivars; K, K-deficiency.

Proline metabolizing enzymes

γ- Glutamyl kinase

Of all four mustard cultivars, cv. Varuna showed 38.5% increase in γ- glutamyl kinase, whereas cv. Vaibhave showed 27.9% increase in this enzyme under K deficiency as compared to control conditions (Table 1; Figure 2B).

Proline oxidase

A maximum increase in proline oxidase was observed in cv. Vaibhave (35.4%) and minimum in cv. Varuna (26.3%) under K deficiency as compared to control conditions (Table 1; Figure 2C).

H₂O₂ content and lipid peroxidation

K deficiency enhanced the accumulation of H₂O₂ in all mustard varieties as compared to control conditions, however, the minimum being in cv. Varuna (62%) and maximum in the remaining three cvs (Table 1; Figure 2D).

A significant increase in MDA content was observed due to K deficiency in all mustard cvs as compared to control conditions. The K deficiency induced was 35.1% in cv. Varuna, 39.3% in cv. RH-30, 46.5% in cv. Rohini, and 47.4% in cv. Vaibhave compared with control (Table 1; Figure 2E).

Antioxidant enzymes

The activity of SOD enzyme increased in all mustard cvs due to K deficiency as compared to control conditions (Table 1; Figure 2F). A maximum increase in SOD activity was observed in cv. Varuna (40.8%) and minimum (30%) in cvs. Rohini and Vaibhave.

K deficiency increased the CAT activity in all mustard cvs as compared to control conditions. Cv. Varuna showed a increase of 32.6% in CAT activity, cv. RH-30 31.2%, cv. Rohini and Vaibhave showed 28.6% compared with control under K-deficient regimes (Table 1; Figure 2G).

The APX activity increased in all mustard varieties with K deficiency as compared to control conditions, the highest increase being in cv. Varuna (42.3%) and the minimum (39.1%) in cvs. Rohini and Vaibhave. Of all mustard cultivars, cv. RH-30 showed 40% increase in APX activity under K deficiency as compared to control (Table 1; Figure 2H).

A significant increase (36.5% and 33.3%) in GR activity was observed in cvs. Varuna and RH-30, respectively, due to K deficiency, however, a minimum increase (28.1%) was observed in cv. Vaibhave compared with control (Table 1; Figure 2I).

Discussion

In the present study, K-deficient plants possessed low amount of photosynthetic pigments (chlorophyll a and b), which may have been due to K deficiency-induced enhanced generation of ROS which can frequently damage the organelle membranes (Cakmak 2005; Ashraf 2009). Long ago, it was reported that photosynthetic components mainly the enzymes involved in Calvin cycle are inhibited by K deficiency (Kaiser 1976). K deficiency is also known to induce closing of stomata (Desikan et al. 2004), which adds on to the increase in ROS generation by limiting CO₂ uptake efficiency. The reduction in the quantity of photosynthetic pigments leads to appearance of chlorosis and necrosis in K-deficient plants, which could be a consequence of enhanced generation of ROS (Cakmak 1994; Cakmak 2005). K deficiency also reduces ribulose bisphosphate carboxylase activity and increases mesophyll resistance thus subsequently lowering down the rate of photosynthesis (Cakmak & Engels 1999; Zhao et al. 2001).

Low K accumulation induced high production of ROS. H₂O₂, one of the important ROS, is known to disrupt the function of cell which in turn reduces growth and development of the plant (Cakmak 2005; Ashraf 2009; Noreen & Ashraf 2009; Noreen et al. 2010, 2012). For example, in Arabidopsis and maize plants, Shin and Schachtman (2004) observed that K deficiency enhanced H₂O₂ production in root as well as leaf tissues. Generally, enhanced K deficiency is associated with a marked increase in the activities of antioxidant enzymes and play significant role in detoxification of ROS (Cakmak 2005), and plants have developed a complex defense system to combat the oxidative stress-induced damage (Noctor & Foyer 1998; Mittler 2002; Ashraf 2009). For example, SOD regulates the cellular concentration of , H₂O₂, CAT and several classes of peroxidases play important role to scavenge H₂O₂ (Scandalios 1993; Tsai et al. 2005). In the present study, K deficiency enhanced the activities of antioxidant enzymes including SOD, APX, CAT, and glutathione reductase (GR), which might have protected mustard seedlings from oxidative damage caused by K deficiency-induced excessive ROS production. Previously, while working with mulberry plants, Tewari et al. (2004) also observed high activities of antioxidant enzymes but under Mg-deficient conditions.

During environmental stresses, proline accumulation occurs in most plants in large amounts (Akram et al. 2007; Ashraf & Foolad 2007; Kamran et al. 2009; Koyro et al. 2011; Shahbaz et al. 2011). In an earlier study with maize, salt-induced high proline concentration improved plant growth and development of maize plants (Cha-um & Kirdmanee 2009). Recently, transgenic approach to overproduce proline in plants has been adovocated to be an effective means of increasing stress tolerance in plants (Ashraf & Foolad 2007; Akram et al. 2012). Generally, high proline contents help to protect various vital enzymes, membranes and proteins in the plant cells by scavenging ROS (Banu et al. 2010), bringing about osmotic adjustment (Rhodes & Hanson 1993), and facilitating water uptake (Flowers 2004; Hassine & Lutts 2010; Hassine et al. 2010). In the present study, proline contents increased significantly in K-deficient mustard seedlings. Synthesis of proline in large amount might serve as a compatible solute that can help plant tissues to tolerate the oxidative stress caused by K deficiency.

Proline oxidase and γ-glutamyl kinase play an important role in controlling the level of proline. Proline oxidase catalyzes the conversion of proline to glutamate and γ-glutamyl kinase plays an important role in the synthesis of proline in plants (Jaleel et al. 2007). In the present study, increase in γ-glutamyl kinase and decrease in proline oxidase were observed due to K deficiency in mustard seedlings. Similarly, in an earlier study with sunflower, Manivannan et al. (2007) observed an increase in the activity of γ-glutamyl kinase and decreased activity of proline oxidase underwater deficit conditions.

In conclusion, K deficiency led to decreased growth in terms of shoot and root lengths and DWs and photosynthetic pigments in all four cvs of mustard. Also, K deficiency initiated the generation of ROS, leading to oxidative stress in the seedlings of all mustard cvs. This triggered distinct redox changes in the cellular metabolism in response to the oxidative stress, e.g. activation of antioxidant machinery like SOD, CAT, APX, and GR. The mustard cvs. Varuna and RH-30 showed more accumulation in proline and higher activities of antioxidants, which suggests their enhanced tolerance to K-deficiency stress as compared to cvs. Rohini and Vaibhave.