Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamia pinnata) seeds during storage

Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamia pinnata) seeds during storage

South African Journal of Botany 112 (2017) 383–390 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.e...

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South African Journal of Botany 112 (2017) 383–390

Contents lists available at ScienceDirect

South African Journal of Botany journal homepage:

Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamia pinnata) seeds during storage B. Sahu a, A.K. Sahu a, V. Thomas b, S.C. Naithani a,⁎ a b

School of Life Sciences, Pt. Ravishankar Shukla University, Raipur 492 010, Chhattisgarh, India Department of Botany, St. Thomas College, Bhilai 490 006, Chhattisgarh, India

a r t i c l e

i n f o

Article history: Received 6 April 2017 Received in revised form 19 June 2017 Accepted 30 June 2017 Available online xxxx Edited by MG Kulkarni Keywords: Reactive oxygen species Lipid peroxidation Carbonylated protein Antioxidant enzyme Pongamia pinnata

1. Introduction Seed longevity, an important trait from ecological and agricultural perspectives, has been studied in considerable detail (Rajjou et al., 2008; Nonogaki et al., 2010). Seeds after harvest, undoubtedly deteriorate gradually and lose quality during extended storage (Rajjou et al., 2008). The seed storage behaviour has been categorized as orthodox and recalcitrant on the basis of desiccation tolerance and sensitivity, respectively. Orthodox seeds can be stored for longer periods if their moisture contents are reduced to 1–5% (Ellis et al., 1991a), whereas the recalcitrant seeds are killed when dehydrated below relatively high moisture content (30–50%) (Varghese and Naithani, 2008). Several seeds like Carica papaya, Coffea arabica, Elaeis guineensis (Ellis et al., 1991a,b) once categorized as recalcitrant have now been reclassified as intermediate in storage behaviour. During ageing, loss of seed vigour and viability precedes the loss in germinability (Eksi and Demir, 2011). Membrane deterioration associated enhanced solute leakage, a measure of seed vigour (Eksi and Demir, 2011), has been reported in several recalcitrant seeds during ageing (Pukacka and Ratajczak, 2007; Varghese and Naithani, 2008). ⁎ Corresponding author. E-mail address: [email protected] (S.C. Naithani). 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

Similarly, the ageing related loss of viability was reported in seeds using TTC (triphenyl tetrazolium chloride), a quick and precise test (ISTA, 2003). In dry and viable seeds, leakage of reactive oxygen species (ROS) from electron transport chain of mitochondria during seed desiccation is inevitably enhanced that in turn promotes oxidative damage of nucleic acids, proteins and lipids. Active metabolism in the hydrated pockets, reported in restricted cellular areas of dry seeds, is one of the potential sources of ROS formation (Leubner-Metzger, 2005). Additionally, non-enzymatic ROS production in anhydrate sites of dry seeds also contributes to ageing associated cellular damage (Job et al., 2005). Excessive accumulation of ROS (superoxide radical and H2O2) in orthodox, recalcitrant and intermediate seeds (Bailly, 2004; Pukacka and Ratajczak, 2007; Varghese and Naithani, 2008; Sahu et al., 2017) has been discussed as a potential cause of viability loss. ROS induced oxidative damage of proteins and lipids (Balesevic-Tubic et al., 2007; Oracz et al., 2007; Varghese and Naithani, 2008; Parkhey et al., 2012) leads to severe cellular damage that eventually results in loss of viability (Halliwell and Gutteridge, 2007). Membrane damage and generation of toxic by-products are common features of lipid peroxidation (Parkhey et al., 2012). Many proteins are specific as they are regulatory and associated with particular stages of seed development (Tunnacliffe et al., 2010; Sahu et al., 2017), dormancy (Oracz et al., 2007), germination (Nonogaki et al., 2010; Tunnacliffe et al., 2010) and longevity/ ageing (Rajjou et al., 2008). Carbonyls are produced by direct oxidative attack on Lys, Arg, Pro or Thr residues of proteins (Job et al., 2005). Accumulation of carbonyl derivatives, a diagnostic marker of oxidative stress, occurs due to ROS induced protein oxidation (Job et al., 2005). Protection against ROS is offered by a battery of antioxidases, such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) (Pukacka and Ratajczak, 2007; Sahu et al., 2017). SOD, present in all aerobic organisms, catalyses the formation of H2O2 from superoxide radical. The CAT and APX protect the cell from potentially toxic H2O2 (Sahu et al., 2017). CAT is regulatory in the removal of the H2O2 produced under various stress conditions and also during β-oxidation of the fatty acids in germinating oily seeds (Bailly, 2004). APX regulates the intracellular H2O2 present in all cell compartments and unlike CAT, participates in cell detoxification due to its high affinity for H2O2 (Kranner and Birtic, 2005). The role of APX becomes decisive in the context of metabolizing excess amount of H2O2 in the desiccating seeds (Bailly, 2004; Sahu et al., 2017). Impairment of ROS scavenging enzymes is the key determinants in the loss of viability and vigour in several


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seeds (Kibinza et al., 2006; Varghese and Naithani, 2008; Sahu et al., 2017). Desiccation of non-orthodox seeds below critical moisture content leads to excessive promotion in the levels of ROS due to impairment of antioxidative enzymes (Sahu et al., 2017). Karanj (Pongamia pinnata L.) an arboreal legume commonly known as Indian Beech tree, a member of subfamily Papilionaceae (Scott et al., 2008), is indigenous to the Indian subcontinent and South-East Asia (Sangwan et al., 2010). It has also been introduced to several countries as Australia, New Zealand and the United States of America (Scott et al., 2008) for controlling soil erosion, binding sand dunes because of its dense network of lateral roots (Sangwan et al., 2010). It is an excellent multipurpose tree and immensely used for its medicinal properties. Seeds yield a non-edible 40% Pongam or Karanga oil which is a potential source for biodiesel (Sangwan et al., 2010). The present work describes the seed storage behaviour and seed longevity of karanj during storage at natural ambience to offer standardized protocol for ex-situ storage in the germplasm banks. Attempt was also made to unravel the potential causes of seed ageing by monitoring the levels of ROS and its detoxifying enzymes to explain the mechanism of seed ageing as well as intermediate storage behaviour. 2. Material and methods 2.1. Seed collection, storage and determination of physical attributes Freshly mature pods of karanj (Pongamia pinnata) were collected manually from the avenue trees in and around Raipur, Chhattisgarh, India in the month of April. The pods were brought to the laboratory immediately (within 2 h) and seeds were extracted by slight hammering of the pods or by pressing the knife along the suture. Freshly harvested, healthy and uniform sized seeds were stored (for natural ageing) in well aerated baskets at ambient temperature (27–30 °C and RH 45%) and were taken out from storage at different days of storage (i.e. 0, 90, 135, 225, 315 and 405 days) for various analyses. Average seed weight was determined by weighing hundred seeds individually (DFSC/IPGRI, 1999). The initial moisture content (IMC) of seeds was determined by Varghese and Naithani (2008). The seed size, length, breadth and other physical attributes were measured for hundred individual seeds and repeated five times independently. All the analysis were performed on the seeds sample harvested from the storage on 0 (same day), 90, 135, 225, 315 and 405 days. 2.2. Water content Five independent replicates of ten seeds each were weighed before and after oven drying at 96 °C for 60 h for estimation of water content (WC) (ISTA, 1993). WC was calculated on dry mass (DM) basis and expressed as gH2O g−1 DM. 2.3. Germination and germination index Seeds were surface sterilized (in 0.1% mercuric chloride) for 15 min, thoroughly washed four times with distilled water and placed on filter paper towels saturated with distilled water in a Petri dish. Petri dishes were kept in dark (karanj seeds are negative photoblastic, unpublished) at 28–30 °C and germination was scored every 24 h as the radicle emerges to 5 mm in length. Germination was expressed in percentage. To evaluate the speed of germination, the germination index (GI) of the seeds was estimated as described elsewhere (Sahu et al., 2017). 2.4. Electrolytic leakage The electrolytic leakage of seeds was measured by determining the leachate conductivity (Sahu et al., 2017). Ten seeds were soaked in 30 ml of distilled water and leachates were collected after 24 h of imbibition. The conductivity of leachates was determined using Digital

Direct Reading Conductivity Meter (Elico) and results were expressed as milli-Siemen (mS) seed−1. 2.5. Viability Seed viability was estimated by immersing the seeds in distilled water at 27–30 °C in dark for 18 h, as per details described in the germination test. After imbibition, the papery brown seed cover was removed carefully using forceps. The de-coated seeds were immersed in 1% solution of 2, 3, 5-triphenyl tetrazolium chloride (Sigma, USA) and incubated in dark at 27–30 °C for 12 h. The red-colored formazan formed after incubation was extracted from weighed amount of seeds with 5 ml of ethanol. The absorbance of the ethanolic solution obtained after centrifugation at 5000 rpm for 10 min was read at 520 nm (ISTA, 1996). Seed viability was expressed as A520 g−1 FW. The experiment was performed with ten seeds in five independent replicates. 2.6. ROS determination Embryonic axes were homogenized into powder using liquid nitrogen and used for ROS determination. The superoxide radical was estimated by the method of Sangeetha et al. (1990). Weighed amount (100 mg) of liquid nitrogen powder of axes was thoroughly mixed with 2 ml sodium phosphate buffer (0.2 M, pH 7.2) containing diethyle dithiocarbamate (10−3 M). The mixture was centrifuged for 15 min at 10,000 rpm and the superoxide radical in the supernatant was measured at 540 nm by its capacity to reduce nitro blue tetrazolium (2.5 × 10−4 M). The amounts of superoxide radical were expressed as −1 −1 g FW. •O− 2 min The H2O2 was estimated following Schopfer et al. (2001). Liquid nitrogen powder of axes (100 mg) was dissolved in 3 ml of KHPO4 (20 mM, pH 6.0) containing 5 μM Scopoletin (Sigma, USA) and 3 μg ml−1 horseradish peroxidase (Sigma, USA) in dark at 25 °C on a shaker for 1, 2, 3 and 4 h. The reaction mixture was centrifuged for 10 min at 10,000 rpm and the decline in fluorescence (Excitation: 346 nm, Emission: 455 nm) of scopoletin in supernatant was detected using spectrofluorometer (Shimadzu, Japan). H2O2 was expressed as nM H2O2 h−1 mg−1 FW. 2.7. Lipid peroxidation The level of lipid peroxidation was measured by following Heath and Packer (1968). Liquid nitrogen powder of axes weighing 50 mg was homogenized with 0.5% (w/v) 2-thiobarbituric acid prepared in 20% (w/v) trichloroacetic acid, incubated for 30 min in a pre-heated water bath (100 °C) and transferred at 0 °C for 30 min. Thereafter, the homogenate was centrifuged at 10,000 rpm for 15 min and the volume of clear supernatant made up to 3 ml with distilled water. The amount of malondialdehyde-thiobarbituric acid complex was measured at 540 nm and corrected for the non-specific absorbance by subtracting the value obtained at 600 nm. Interfering absorbance was removed by recording absorbance at 440 nm to eliminate the interference by sucrose. The amount of MDA was calculated from the extinction coefficient of 150 mM−1 cm− 1 (Einset and Clark, 1958) and expressed as nM MDA g−1 FW. 2.8. Carbonylated protein Carbonyl protein was estimated following Levine et al. (1994). Axes powder (50 mg) was homogenized in sodium phosphate buffer (10 mM, pH 7.2) containing 1 mM EDTA, 2 mM DTT, 0.2% (v/v) Triton X-100, 1 mM PMSF. Homogenate was centrifuged at 10,000 rpm for 20 min and protein concentration was determined in the supernatant following Bradford (1976). Protein extract, containing 50 mg protein, was incubated with 0.03% (v/v) Triton X-100 and 1% (w/v) streptomycin sulphate for 20 min to remove the nucleic acids and centrifuged at 10,000 rpm for 10 min. The supernatant was mixed with 300 μl of

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10 mM DNPH prepared in 2 M HCl and the blank was incubated in 2 M HCl (without DNPH) and incubated at 27–30 °C for 1 h. The oxidized proteins were precipitated with 10% (w/v) trichloroacetic acid and the pellets were washed three times with 500 μl of ethanol:ethyl acetate (1:1). The pellets were finally dissolved in 6 M guanidine hydrochloride prepared in potassium phosphate buffer (20 mM, pH 2.3) and the absorption was recorded at 370 nm. Carbonyl content was calculated using a molar absorption coefficient 22,000 M−1 cm−1 and expressed as mM mg−1 protein.


2.12. Statistical analysis All the experiments were performed twice with five independent replicates. Correlations between studied parameters were checked using Pearson's coefficient test. The data obtained were subjected to one way ANOVA and the mean differences were compared by Duncan's multiple range test using SPSS (Ver. 16.0). 3. Results 3.1. Physical characteristics

2.9. Protein extraction and purification The axes powder (100 mg) was homogenized in Zivy's buffer (30 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2 and 10 mM ascorbic acid) of pH 8.5 containing 1 mM DTT and 1 mM PMSF at 4 °C (Zivy et al., 1983). The mixture was centrifuged at 14000 g for 20 min and the supernatants were used as a source of enzyme for all biochemical and isoenzyme analyses of SOD, CAT and APX. Proteins were quantified in the extract following Bradford (1976).

Collected pods of karanj were D shaped, 5.83 ± 0.43 cm long, 2.36 ± 0.35 cm broad and 0.85 ± 0.21 cm thick, pointed at both ends, single seeded and light brown when ripe. On an average, each fruit weighed 3.713 ± 0.20 g with MC and WC of 15.3 ± 0.28% and 0.18 gH2O g− 1 DM, respectively. Seeds were kidney shaped and weighed 1.456 ± 0.22 g/seed. Seeds were compressed but slightly swollen, reddish brown, fairly hard, 2.59 ± 0.39 cm long, 2.23 ± 0.30 cm broad and 0.42 ± 0.08 cm thick with 14.7 ± 0.44% MC or 0.22 gH2O g−1 DM WC. 3.2. Percent germination and germination index

2.10. Quantitative analysis of antioxidant enzymes SOD activity was estimated following Marklund and Marklund (1974) with minor modification described elsewhere (Sahu et al., 2017). The kinetics of pyrogallol auto-oxidation was recorded at 420 nm in a reaction mixture containing 2.94 ml of 50 mM Tris-HCl buffer of pH 8.2, 1 mM DETAPAC (Sigma, USA) and 60 μl pyrogallol, prepared in 10 mM HCl. SOD assay was performed by adding 0.2 ml enzyme extract in 2.74 ml of the Tris-HCl buffer and the reaction was initiated by adding 60 μl of pyrogallol. Absorbance was recorded at 420 nm and the enzyme activity was expressed as units of SOD min−1 mg−1 protein. CAT activity was determined as described by Chance and Maehly (1955) by recording the loss of H2O2 absorbance at 240 nm. CAT activity was performed by taking 60 μl enzymes in 2.74 ml of KHPO4 buffer (37.5 mM, pH 6.8) and 200 μl of H2O2 (60 mM) and the absorbance was recorded at 240 nm. CAT activity was expressed as A240 min−1 mg−1 protein. APX activity was measured according to the assay developed by Nakano and Asada (1981). The reaction mixture contained 2.3 ml of KHPO4 (0.025 M, pH 7.0), 500 μl ascorbic acid (0.25 mM), 190 μl EDTA (0.1 mM), 10 μl of enzyme and 20 μl of H2O2 (0.1 M). APX activity was estimated at 290 nm and expressed as A290 min−1 mg−1 protein.

Freshly harvested seeds that were shed at 0.22 gH2O g−1 DM (14.7% MC) exhibited 100% germination (Fig. 1) up to 90 days of storage at ambient conditions (27–30 °C and RH 45%). Further storage resulted in loss of germination (Fig. 1). The critical water content (CWC) for karanj seeds was 0.11 gH2O g−1DM (CMC 9%) when the percent germination dropped from 100 to 80%. Seeds dehydrated to 0.05 gH2O g−1 DM (4.6% MC) at 405 days of storage showed absolute loss of germinability (Fig. 1). Gradual fall from 80 to 40% germination in seeds dried from 0.11 (9% MC) to 0.07 (6.2% MC) gH2O g−1 DM was evident during storage from 135 to 315 days. Like percent germination and WC, the GI was maximum (833.2) in the freshly harvested seeds having 100% germination. The GI reduced to 633.2, 356.6, 104.3, 1.1 and 0 as the seeds dehydrated to 0.15, 0.11, 0.09, 0.07 and 0.05 gH2O g− 1 DM, respectively (Fig. 1). Though 100% germination was exhibited by the seeds till 90 days of storage, the GI decreased considerably (833.25 to 633.28) during this period. The percent germination of seeds displayed

2.11. Isoenzyme analysis of antioxidant enzymes Isoenzymes were separated on Native-PAGE (10%) slab gels electrophoresis (Laemmli, 1970). In each well 100 μg protein was loaded. The SOD isoenzymes were stained by incubating the gel in 2.45 mM 2,2′-dinitrophenyl-5,5′-diphenyl-3,3′-(dimethoxy-4,4′-di phenylene) ditetrazolium chloride (NBT) solution for 20 min in dark followed by soaking in 36 mM KHPO4 (pH 7.8), 28 mM TEMED and 28 μM riboflavin for 15 min, as described by Beauchamp and Fridovich (1971). CAT isoenzymes were detected (Woodbury et al., 1971) in the gels by soaking in distilled water for 15 min and then incubating in 0.03% H2O2 for 10 min. The gel was immersed in the staining mixture comprising of 1% FeCl3 and 1% K4[Fe(CN)6] solution. APX isoenzymes were detected by the method described by Mittler and Zilinskas (1993). The gels were first immersed in 50 mM KHPO4 (pH 7.0) buffer and 4 mM ascorbate for 20 min followed by 50 mM KHPO4 (pH 7.0), 4 mM ascorbate and 4 mM H2O2 for 20 min and then rinsed with 50 mM KHPO4 (pH 7.0) buffer. The gel was then incubated in 50 mM KHPO4 buffer (pH 7.8), 28 mM TEMED and 2.45 mM NBT for detecting the isoenzymes.

Fig. 1. Changes in germination (%) and germination index in karanj seeds as a function of water content and seed storage period. Inset: correlation of water content with germination (—) (r = 0.858, y = −20x + 133.3) and GI (—) (r = 0.971, y = −180.4x + 952.8). The plotted values are mean and SD of five independent replicates.


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Fig. 2. Changes in leachate conductivity and seed viability in karanj seeds during storage. Inset: correlation of water content with leachate conductivity (—) (r = −0.819, y = 1.122x − 1.672) and seed viability (—) (r = 0.901, y = −24.09x + 173.4). The plotted values are mean and SD of five independent replicates.

Fig. 3. Effect of dehydration on the ROS level (superoxide radical and H2O2) in karanj seeds during storage. Inset: correlation of water content with superoxide radical (—) (r = − 0.883, y = 170.8x + 49.00) and H2O2 (—) (r = − 0.949, y = 13.29x + 8.98). The plotted values are mean and SD of five independent replicates.

a negative correlation (r = − 0.97) with the seed storage period. On the contrary, a positive correlation (r = 0.86) was obtained between germination and seed WC.

3.5. Reactive oxygen species

Sharp increase from 0.30 to 0.83 mS seed− 1 was observed in the leachate conductivity when the fresh seeds dehydrated from 0.22 to 0.11 gH2O g− 1 DM for 135 days (Fig. 2). Thereafter, the seed leachate conductivity was promoted to 1.90, 4.40 and 5.60 mS seed− 1 as the seeds desiccated to 0.09, 0.07 and 0.05 gH2O g− 1 DM, respectively. A negative correlation (r = − 0.82) was established between leachate conductivity and WC of the seeds, but a positive correlation (r = 0.96) was obtained between leachate conductivity and seed storage period.

The accumulation of superoxide radical showed a low profile in the fresh seeds but increased rapidly with the dehydration of seeds during storage (Fig. 3). Almost 3.7-fold promotion in the levels of superoxide radical was registered in the seeds desiccated from 0.22 to 0.05 gH2O g−1 DM. Superoxide radical showed a negative correlation (r = −0.88) with the seed WC, but exhibited a positive correlation (r = 0.99) with seed storage period. Like superoxide, the amount of H2O2 also increased with seed dehydration during storage (Fig. 3). Initially the fresh seeds registered 21.3 μM H2O2 that increased gradually to 39.3, 45.3, 64.5, 71.3 and 91.3 nM H2O2 h−1 mg− 1 FW as the seeds dehydrated to 0.15, 0.11, 0.09, 0.07 and 0.05 gH2O g− 1 DM, respectively. The accumulation of H2O2 showed positive correlation (r = 0.99) with seed storage period whereas negative correlation (r = −0.95) with seed WC.

3.4. Viability

3.6. Lipid peroxidation and carbonylated protein

Declining trend in TTC reduction was discernible with increasing periods of seed storage and/or decreasing WC (Table 1). Initially, fresh seeds (0.22 gH2O g− 1 DM) showed the highest viability (139.9 A520 g−1 FW), which declined to 34.7 A520 g−1 FW when dehydrated to 0.05 gH2O g−1 DM (Fig. 2). The seed viability observed positive correlation (r = 0.90) with seed WC and negative correlation (r = −0.98) with seed storage periods.

The patterns of lipid peroxidation and carbonylated protein, estimated in the embryonic axes were similar to those of superoxide and H2O2 (Fig. 4). The levels of lipid peroxidized product estimated in the axes increased from 6.94 nM MDA g−1 FW to 91.86 nM MDA g−1 FW as the fresh seeds desiccated to 0.05 gH2O g−1 DM within 405 days of storage. The lipid peroxidation exhibited positive correlation (r = 0.96) with storage period whereas negative correlation (r =

3.3. Electrolyte leakage

Table 1 Seed viability in relation to germination and water content in natural drying karanj seeds. Water content (gH2O g−1 DM)







Germination (%) Pictures of TZ test







Viability (A520 g−1 FW)







Dry mass, DM; tetrazoleum, TZ; fresh weight, FW

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The APX activity detected in the axes was higher (24.9 A290 min−1 mg−1 protein) in the 100% viable seeds but reduced sharply as the seeds dehydrated during storage and showed a net 6.5fold loss in the axes of non-viable seed (3.8 A290 min−1 mg−1 protein) (Fig. 5). A positive correlation (r = 0.97) was recorded between APX activity and WC, while a negative correlation (r = −0.99) was discernible between APX activity and storage period of seeds. Total 4 isoenzymes of APX were recorded in the axes of 100% viable seeds. The intensity of all the four isoenzymes declined gradually with the loss of germination (Fig. 6C). 4. Discussion

Fig. 4. Levels of lipid and protein oxidation in karanj seeds during storage. Inset: correlation of water content with lipid peroxidation (—) (r = −0.843, y = 16.23x − 19.75) and protein oxidation (—) (r = −0.957, y = 4.072x + 1.348). The plotted values are mean and SD of five independent replicates.

− 0.84) with seed WC. The accumulation of carbonylated protein increased 4-fold (5.4 to 24.5 mM mg−1 protein) as the seeds dehydrated from 0.22 to 0.05 gH2O g−1 DM. Amounts of carbonylated protein were positively correlated with storage period of seeds (r = 0.98), whereas negatively correlated with seed WC (r = −0.96). 3.7. Antioxidative enzymes: quantitative and isoenzymes The patterns of SOD, CAT and APX activities detected in the axes of karanj seeds were similar during storage. The activities of all the enzymes were comparatively higher in the 100% viable seeds of karanj but declined gradually as the seeds dehydrated during storage (Fig. 5). For example, the axes of the fresh seeds observed 106.3 Unit SOD min−1 mg−1 protein that declined gradually to 102.2, 90.2, 45.8, 26.1 and 18.5 Unit SOD min−1 mg−1 protein in the seeds dehydrated to 0.15, 0.11, 0.09, 0.07 and 0.05 gH20 g−1 DM. A negative correlation (r = −0.97) existed between SOD activity and storage period whereas a positive correlation (r = 0.88) was discernible between SOD activity and WC in dehydrating seeds. Total 2 bands (isoenzyme) of SOD were noticed in the axes of 100% viable karanj seeds (Fig. 6A). The isoenzyme 1 was present in the axes of viable seeds (80 to 100%) showing WC of 0.22, 0.15 and 0.11 gH2O g−1 DM, whereas it disappeared once seeds desiccated to or below 0.09 gH2O g−1 DM. The isoenzyme 2 was constantly present throughout the analysis although with decreasing intensity. The axes of fresh seeds observed substantially higher activities of CAT (0.55 A240 min−1 mg−1 protein) that declined by 4.5-fold in the non-viable seeds with WC 0.05 gH2O g−1 DM (Fig. 5). The correlation (r = −0.99) between CAT activity and seed storage period was negative whereas it was positive (r = 0.96) when calculated with seed WC. Two isoenzymes of CAT were recorded in the axes of dehydrating seeds during storage (Fig. 6B). Isoenzyme-2 appeared only at first two stages of dehydrating seeds (i.e. at WC of 0.22 and 0.15 gH2O g− 1 DM), and disappeared in the later stages of drying. The isoenzyme-1 was present with remarkably high intensity in the axes of seeds showing 100% viability (0.22 and 0.15 gH2O g−1 DM), but the intensity decreased gradually with loss in viability.

The kidney shaped fresh karanj seeds (length: 2.59 cm, breadth: 2.23 cm) extracted from light brown fruits, were shed with 14.7% (0.22 gH2O g−1 DM) moisture content and 100% germination. Storage of fresh mature seeds in ambient conditions, permitting slow drying, leads to gradual loss of germination and GI with absolute loss of viability at 405 days of storage (Fig. 1). For example, slow drying of seeds for 135 days at ambient conditions during storage resulted in 80% survival at WC of 0.11 gH2O g−1 DM, whereas dehydration to 0.07 gH2O g−1 DM for 315 days reduced the seed germination to 40% (Fig. 1). The large number of tree seeds viz., Pometia pinnata, Hopea odorata, Aesculus indica, Mangifera indica and Madhuca indica (Côme and Corbineau, 1989; Uniyal and Nautiyal, 1996; Varghese et al., 2002) that are desiccation-sensitive showed loss of viability with prolonged drying during storage. The critical moisture content (CMC) or critical water content (CWC) estimated for karanj seeds were 9% or 0.11 gH2O g−1 DM, respectively, confirming its intermediate storage behaviour. The CMC (9%) of karanj seeds was similar to several intermediate seeds viz., Carica papaya, Coffea arabica, Elaeis guinensis and Azadirachta indica (Ellis et al., 1991a,b; Varghese and Naithani, 2002) showing CMC in the range of 9–13%. The loss of germinability in the karanj seeds was further supported by recording GI. Although, dehydration of fresh mature seeds from 0.22 to 0.15 gH2O g−1 DM caused no change in germination percentage, the GI decreased substantially from 833 to 633. Our results clearly revealed that loss of GI precedes the decrease in percent germination as also reported in several other desiccation-sensitive seeds viz., Azadirachta indica (Varghese, 2000), Madhuca indica (Varghese et al., 2002). Further storage reduced the GI to 356, 104 and 1 with decreased in WC to 0.11, 0.09 and 0.07 gH2O g−1 DM, respectively. The percent germination and GI tests, both are widely used in the field and laboratory conditions for the assessment of salt stress, drought stress and priming effect (Sadeghi et al., 2011). The non-orthodox seeds are desiccation sensitive (Sahu et al., 2017) because they are like germinating propagules (Berjak et al., 2007), perhaps due to short or abbreviated maturation drying phase. In these seeds, germination commences immediately after shedding due to relatively high WC that is enough to support the germination related events (Walters, 2015). Thus, these seeds or propagules become more intolerant to desiccation during storage (Berjak et al., 2007). Prolonged drying (natural drying) of karanj seeds to 0.07 and 0.05 gH2O g−1 DM resulted in 40 and 0% germination, respectively. Removal of bound water at 0.15 gH2O g−1 DM probably leads to deleterious sub-cellular changes and desiccation-induced accumulation of oxidants that causes oxidative stress (Leprince et al., 1993) consequently leading to loss in viability (Varghese and Naithani, 2002; Sahu et al., 2017). The drying induced loss of germinability, vigour and viability of karanj seed was further established by observing increased solute leakage and decline in TTC reduction. Increasing pattern of electrolyte leakage was evident as the fresh mature karanj seeds deteriorate due to dehydration during storage (Fig. 2). Drying of seeds to 0.11 gH2O g−1 DM for 135 days resulted in 2-fold promotion in solute leakage (0.83 mS seed− 1) with nearly 20% loss of germination. Further, 18fold increase in the electrolyte leakage obtained at 405 days of storage in the non-viable seeds than the fresh mature seeds indicated severe


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Fig. 5. Effect of dehydration during storage on the activities of antioxidant enzyme (SOD, CAT and APX) in karanj seeds. Inset: correlation of water content with SOD (—) (r = 0.883, y = −20.33x + 136.0), CAT (—) (r = 0.956, y = − 0.088x + 0.626) and APX (·····) (r = 0.970, y = −4.051x + 28.96). The plotted values are mean and SD of five independent replicates.

impairment of selectivity of plasma membrane. Our study corroborates the findings of others (Berjak et al., 2007; Pukacka and Ratajczak, 2007; Varghese and Naithani, 2008; Sahu et al., 2017) and postulates that the magnitude of electrolyte leakage is directly proportional to membrane damage that leads to proportionate loss of seed germinability, vigour and viability (Berjak et al., 2007; Pukacka and Ratajczak, 2007; Varghese and Naithani, 2008; Eksi and Demir, 2011). The positive correlation of leachate conductivity with storage period (r = 0.96) and negative correlation with seed WC (r = −0.82), further validated our conclusions. Similarly, strong association of seed viability with the percent germination was revealed from TTC test performed on dehydrating karanj seeds during storage. The fresh seeds showed intense red coloration (139.9 A520 g−1 FW), due to TTC reduction, that gradually declined (34.7 A520 g−1 FW) as the seeds exhibited loss of viability (Table 1). Gradual reduction in the development of red colored formazan, as a

result of TTC reduction was discernible with increasing desiccation during storage (Fig. 2). Superoxide radical and H2O2 levels that were stimulated in response to desiccation showed negative correlation with germination percentage (Fig. 3). Accumulation of superoxide radical and H2O2 during prolonged storage was positively associated with loss of seed germinability and viability in orthodox (Kibinza et al., 2006; Cheng and Song, 2008) and recalcitrant/intermediate seeds (Varghese and Naithani, 2002, 2008; Sahu et al., 2017). The ROS-triggered oxidative stress is responsible for molecular and structural damage that adversely affects seed storability and survival (Berjak et al., 2007; Pukacka and Ratajczak, 2007). In dehydrating non-orthodox seeds, excessive accumulation of ROS, an attribute of low viable dry seeds, is primarily due to failure of the antioxidant system and excessive leakage of ROS from ETC of mitochondria (Varghese and Naithani, 2008; Sahu et al., 2017). Alteration in the respiratory pathway and energy metabolism has been reported as being a key reason for loss of germinability during seed ageing (Kibinza et al., 2006; Oracz et al., 2007). It is suggested that the disturbance in the ROS metabolic pathway (perhaps failure of antioxidative enzyme) generated surplus or damaging levels of ROS in desiccation-sensitive karanj seed when dehydrated below CWC, consequently resulting in a cascade of deteriorative primary and secondary reactions involving oxidation/peroxidation of lipids, proteins and DNA. ROS induced oxidative damage to proteins and lipids are one of the modifications leading to severe cellular damage resulting in loss of viability (Halliwell and Gutteridge, 2007). ROS mediated lipid peroxidation is a widely described mechanism of cellular injury in dry seeds (Bailly, 2004; Parkhey et al., 2012). Membrane damage and generation of toxic by-products are common features of lipid peroxidation (Parkhey et al., 2012). The karanj seeds of high viability observed very low levels of peroxidized lipids, which gradually accumulated by 3-fold as the seeds desiccated from 0.22 to 0.11 gH2O g−1 DM. Further dehydration from 0.11 to 0.05 gH2O g− 1 DM noticed 4-fold accumulations of peroxidized lipids (Fig. 4). Lipid peroxidation is the most frequently suggested cause of loss of viability in orthodox seeds of Helianthus annuus, Pisum sativum (Balesevic-Tubic et al., 2007) and recalcitrant seeds of Azadirachta indica, Shorea robusta (Varghese and Naithani, 2002; Parkhey et al., 2012). A close relationship between increasing ROS level with simultaneous increase in efflux of solutes in imbibing seeds is considered causal factor for membrane perturbations that leads to loss of viability in desiccation sensitive seeds (Varghese and Naithani, 2008). During seed ageing, oxidative modification of proteins resulted in their structural and functional loss thus targeting these defective

Fig. 6. Isoenzyme expression of SOD (6A), CAT (6B) and APX (6C) in karanj seeds during storage. Hundred micrograms of proteins were loaded in each well and similar results were obtained in at least five independent experiments.

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proteins for preferential degradation (Job et al., 2005; Oracz et al., 2007). Various proteins, especially enzymes, are specific and control the regulation of particular stage of seed development (Tunnacliffe et al., 2010; Sahu et al., 2017), dormancy (Oracz et al., 2007), germination (Nonogaki et al., 2010; Tunnacliffe et al., 2010) and longevity/ageing (Rajjou et al., 2008). The carbonylated proteins accumulated by 4.5fold (5.40 to 24.5 mM mg−1 protein) in karanj seeds dehydrated from 0.22 to 0.05 gH2O g−1 DM (Fig. 4). The levels of carbonyls, produced by direct oxidative attack on Lys, Arg, Pro or Thr residues of proteins (Job et al., 2005), rise significantly during loss of viability in seeds of Spartina alterniflora (Hammond, 2008). The ROS induced multiple cellular damage is reflected in the accumulation of carbonyl derivatives as a result of protein oxidation, a diagnostic marker of oxidative stress (Job et al., 2005; Oracz et al., 2007). Efficient scavenging system that maintains low levels of ROS is a prerequisite for seed survival and prolonged storability. Dehydrationinduced sharp loss in the levels of scavenging enzymes (SOD, CAT and APX) was closely correlated with the accumulation of ROS during storage (Figs. 3 and 5). The axes of non-desiccated 100% viable seeds of karanj were characterized by maximum antioxidant enzyme activity that gradually reduced as the seed dehydrated with concomitant loss in germinability, viability and vigour. The protective roles of SOD, CAT and APX have been extensively discussed in relation to ROS mediated damage of cellular membranes (Bailly, 2004; Kibinza et al., 2006) and oxidation of biomolecules (Job et al., 2005; Varghese and Naithani, 2008). Failure of antioxidant enzymes eventually leads to impairment in seed germination and vigour associated cellular metabolism (Cheng and Song, 2008; Varghese and Naithani, 2008). The quantitative data obtained for SOD in karanj seed was supported by isoenzyme analysis (Fig. 6A). The karanj seeds showed total two isoenzymes (band) of SOD, out of which the isoenzyme 2, marker for viable seeds, appeared with maximum intensity in the seeds showing 100% germination (at WC of 0.22 and 0.15 gH2O g−1 DM), but it absolutely disappeared in the later stages as the seeds observed loss of germination from 80 to 0% in seeds dried to 0.15, 0.11, 0.09 and 0.05 gH2O g−1 DM (Fig. 6A). More SOD bands appeared in the seeds with higher viability than in the low viable seeds indicated close relationship of protective role of SOD in maintaining 100% germinability of karanj seeds. Similarly, higher levels of CAT and APX were recorded in the axes of 100% viable seeds. The levels of CAT and APX were 0.55 A240 min−1 mg−1 protein and 24.9 A290 min−1 mg−1 protein, respectively, in the axes of 100% viable seeds, but reduced to 0.12 A240 min−1 mg−1 protein and 3.8 A290 min−1 mg−1 protein, respectively, when subjected to storage for 405 days (Fig. 5). Like SOD, the activities of CAT and APX were positively correlated (above r = 0.90) with percent germination in karanj seed and was indicative of active involvement of these antioxidant enzymes in seed longevity and germination. Life span of seed is defined by its ability to evoke antioxidant enzyme activities to detoxify the excess amounts of ROS generated (Bailly, 2004; Rajjou et al., 2008). The expression of CAT and APX isoenzymes of further substantiated the quantitative analysis in drying karanj seeds. Out of the two CAT isoenzymes, isoenzyme-2 was present only in the 100% viable seeds (like SOD isoenzymes-1). The intensity of band 1 was very high in the 100% viable seeds (at WC of 0.22 and 0.15 gH2O g−1 DM) but reduced sharply as the karanj seeds dehydrated and showed reduced seed germinability (Fig. 6B). Similarly, the intensities of four isoenzymes of APX reduced gradually as the fresh seeds dehydrated during storage (Fig. 6C). Considering the exclusive presence of SOD (band 2) and CAT (band 2) isoenzymes only in 100% viable karanj seeds suggested their paramount role in protecting the seed from dehydration induced damage and their absence was marked by initiation of loss of viability and vigour. In conclusion, our results provide strong evidence that accumulation of ROS (superoxide radical and H2O2) stimulated loss of viability and vigour in karanj seeds during prolonged storage. Dehydration, especially below CWC (0.11 gH2O g−1 DM) plays a key role in the impairment of


antioxidative enzymes (quantitative and isoenzymes) in desiccation sensitive seeds by permitting excessive build-up of damaging levels of ROS that eventually leads to massive lipid peroxidation and membrane damage (enhanced solute leakage). Our study also established explicitly the key role of antioxidative enzymes (SOD, CAT and APX) in maintaining seed quality; high viability and vigour in the karanj seeds during storage.

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