A probable crosstalk between Ca⁺², reactive oxygen species accumulation and scavenging mechanisms and modulation of protein kinase C activity during seed development in sunflower

Abstract

Seed development in sunflower involves a gradual dehydration and accumulation of oil bodies in the cells of developing cotyledons during transition from 30 to 40 DAA stage. Reactive oxygen species (ROS) content decreased with seed maturation. NO content and NO contributed by putative nitric oxide synthase, however, did not change markedly. Superoxide dismutase (SOD) activity exhibited a peak at 30 DAA stage, indicating its scavenging role at the mid-stage of seed development. H₂O₂ produced as a result of SOD action is subsequently scavenged primarily by elevation of GR activity. Significant temporal differences were evident in GR and POD activity during seed development. Protein kinase C (PKC) activity also showed modulation during early stages of embryo and seed development. Use of PKC-specific fluorescent probe, Fim-1, and PKC inhibitors (staurosporine and bisindoylmaleamide) provided evidence for increase in PKC activity at 40 DAA stage with an increase in protein concentration (50 to 200 µg). Endogenous calcium content also increased with seed maturation. Tissue homogenates from 40 DAA stage showed enhanced fluorescence due to Fim-1-PKC binding in presence of calcium ions and its lowering due to calcium chelating agent (BAPTA). Western blot analysis revealed an increase in the intensity of 2 bands representing PKC with the advancement of seed maturation and their further upregulation by calcium. Present findings, thus, provide new information on the biochemical regulation of seed development in sunflower, with evidence for a possible correlation between calcium, ROS, their scavenging enzymes and “conventional” PKC activity.

Introduction

A developing seed consists of heterogeneous and highly organized tissue systems of maternal and filial origin. Embryo development is followed by differentiation of filial tissue from the meristem into specialized storage organs. Thus, seed development is a sequential and gradual process of cell division, cell expansion, and accumulation of storage products. The process of seed filling in sunflower involves an accumulation of oil bodies containing triacylglycerols in the developing cotyledons, which correlates well with the phase of dehydration. Upon maturation, sunflower seeds exhibit orthodox storage response, whereby they are able to tolerate dehydration for a significant storage period. The genetically programmed process of seed development exhibits a clear correlation with several other metabolic events as well. The transitional stage of seed development (30 DAA onwards) is of special interest since it involves significant reprogramming of metabolic events.¹ The complex spatial and temporal interplay of the regulation of gene expression and biochemical and morphogenic events during seed development is regulated by a variety of external and internal stimuli. These stimuli are perceived and further propagated in the cells by various signal transduction mechanisms, among which protein phosphorylation is a major component.

Protein kinase C (PKC) constitutes a group of enzymes which are a component of various signal transduction processes in living systems. These enzymes phosphorylate serine and threonine residues on a number of protein. Many signal transduction processes triggered by an increased production of intracellular Ca⁺² and diacylglycerol (DAG) involve PKC.² Twelve different isoforms of PKC have been classified into 3 different subclasses (conventional, novel, and atypical) according to their sequence homology and activation requirements. Conserved C3 and C4 domains bearing the catalytic sites are present across all the isoforms, whereas C1 domain is present in conventional and novel isoforms. It bears the site responsible for the interaction of PKC with activators like DAG and phorbol esters.^3-5 C1 domain in the conventional subclass is responsible for binding of Ca⁺² and phospholipids and calcium-dependent activation of theses isozymes.⁶ α, βI, βII, and γ isoforms come under “conventional” PKCs category. These isoforms are diacylglycerol-sensitive and Ca⁺² responsive.⁷ “Novel” PKCs include δ, ε, θ, and η. They are Ca⁺² -independent.⁸ “Atypical” PKCs are not DAG-sensitive but are activated by other lipids (ceramides) or proteins. They include λ and ζ forms. They seem to be regulated mainly through their intracellular localization which is modulated by their interaction with regulatory proteins, nuclear localization signals and nuclear export signals in their regulatory domain.⁹ Phosphorylation of PKC isoforms regulates their maturation, signaling, and downregulation. Translocation of inactive PKC from the cytosol toward a cellular membrane implies its activation.¹⁰ Translocation to the cellular membrane also results in the interaction of PKC with various cytoskeletal and membrane proteins.¹¹

Different PKC isoforms are commonly co-expressed in the same cell performing different cellular functions. Activation of various PKC isoforms depends on hormones and growth factors. These stimulators, when bound to their respective receptors, activate the members of phospholipase C family, which generate DAG.¹² DAG is often co-produced with inositol-1,4,5-trisphosphate [(Ins(1,4,5)P₃)], triggering the release of calcium ions into the cytosol from the intracellular stores, thereby elevating cytosolic calcium levels. High cytosolic concentration of calcium can directly activate phospholipase C, leading to PKC activation in the absence of a receptor. PKC activity can be modulated by several pharmacological agents either directly or indirectly through a signaling cascade. Most PKC isozymes are ubiquitously expressed in all tissues at all times of development.

Loss of vigor and viability accompanying the storage of desiccation tolerant orthodox seeds is believed to be correlated with the modulation of ROS accumulation in the tissue. Recalcitrant seeds have earlier been reported to exhibit a disturbance in the antioxidative mechanisms, thereby leading to oxidative damage and its subsequent lethal impact.¹³ Thus, an upregulation of antioxidant genes is supposed to be one of the major mechanisms operating during the maturation of orthodox seeds, such as sunflower. A documentary evidence for these assumptions is, however, scanty except for the observations on changes in catalase activity and its isoform pattern and expression, in developing sunflower seeds during desiccation phase.¹⁴

Present investigations aim at understanding the possible correlation among the various phases of seed development, generation, and enzymatic scavenging of ROS during these phases, activation of protein kinase C, and its modulation by calcium. In this context, qualitative and quantitative analysis of major ROS scavenging enzymes, namely superoxide dismutase (SOD), peroxidase (POD), and glutathione reductase (GR), has been undertaken at 3 stages of sunflower seed development (20, 30, and 40 d after anthesis). Modulation of the activity of these enzymes with seed development has been examined for a possible correlation with calcium-stimulated protein kinase C (PKC) activity. The results provide new information on the phasing of the activity of specific ROS scavenging enzymes, PKC activity in relation with seed development, and their possible crosstalk with calcium ions.

Results and Discussion

Developing sunflower seeds exhibit optimal fresh weight at 30 DAA stage and a rapid increase in dry weight with the advancement of development. Thus, mature seeds at 40 DAA stage have negligible water content (1.7%). Young seeds at 20 DAA stage exhibit 63% water. Sharp depletion of water (desiccation) is evident during the transition phase from 30 to 40 DAA stage (Fig. 1A, B). Seed development in sunflower at 20, 30, and 40 d after anthesis (DAA) exhibits noteworthy changes in the degree of vacuolation, cell size, and size and degree of abundance of oil bodies in the developing cotyledons (Fig. 1C). Thus, cotyledon cells at 20 DAA stage have been observed to be longer, vacuolated, and exhibited a sparse distribution of large oil bodies. With seed maturation, cell size is relatively reduced and cells get filled with clusters of small oil bodies. Earlier investigations have revealed that oil body biogenesis in developing oilseeds starts within a few days after anthesis whereby oleosins and some other minor proteins are expressed on the oil body membrane.¹⁵ Not much information is available on lipid accumulation and fatty acid desaturation during seed development in sunflower.¹⁶ Recent findings from the author’s laboratory have revealed a rapid accumulation of proteins and lipids up to 30 DAA stage during the process of seed filling. Oil body biogenesis is evident at a stage as early as 10 DAA. It coincides with slower expression of the 16 kDa oleosin isoform than that of 17.5 and 20 kDa isoforms.¹⁷

Figure 1. Changes in fresh weight and dry weight (A), dry weight per gram fresh weight and water content (B) accompanying seed development. Each value represents the mean and standard errors from 12 seeds. Triple asterisks (***) represents one way ANOVA to be significant P < 0.001. (C) Microscopic examination of 7 µm thick sections of developing cotyledons. Magnification –400 X.

In the present investigations, maximum ROS accumulation is evident in young seeds at 20 DAA stage, coinciding with high metabolic activity (Fig. 2A). Total ROS content gradually decreases with seed maturation. Thus, a reduction of 31.33% in total ROS content is evident in the tissue homogenate when comparing seeds at 20 DAA stage with those at 40 DAA stage. In contrast with ROS, total NO content and NO contributed by putative nitric oxide synthase does not change markedly with the change in the stage of development (Fig. 2B), thereby indicating its non-involvement as a regulatory molecule during seed development. Superoxide dismutase (SOD) activity exhibits a peak at 30 DAA stage without any noteworthy change in the activity of the 3 isoforms detected zymographically (Fig. 2C). These observations indicate the scavenging role of SOD at the mid-stage of seed development more than any other enzyme [glutathione reductase (GR) and peroxidase (POD)]. H₂O₂ thus produced as a result of the action of SOD on superoxide ions, is subsequently scavenged primarily by GR activity. Although both peroxidase (POD) and glutathione reductase (GR) play the role of scavenging H₂O₂ produced in the tissue system, significant differences are evident in their activity during seed development. Thus, POD activity is high in the tissue system in young (20 DAA) developing seeds (Fig. 2D). This is responsible for scavenging of H₂O₂ generated in the metabolically active cells of developing seeds at 20 DAA stage. Subsequent SOD activity (optimal at 30 DAA stage) leads to another phase of H₂O₂ production later. But it may be noted that the scavenging of this later produced H₂O₂ is not being undertaken by POD, its activity being very low in the later phase of seed development. H₂O₂ produced in the tissue system at the later stages of seed development was effectively scavenged by GR activity. GR activity is enhanced by 36 and 43% at 30 and 40 DAA stages (% of GR activity in 20 DAA seeds), respectively (Fig. 2E). It is, thus, evident that the 3 ROS scavenging enzymes (SOD, POD, and GR) are contributing in the reduction of ROS produced in developing seeds through a temporal distribution of their maximal activities at various stages of seed development. Thus, POD seems to be responsible for scavenging H₂O₂ in the young (20 DAA) seeds whereas GR is responsible for scavenging H₂O₂ produced in the developing seeds from mid to maturing stages (30 and 40 DAA), as a consequence of conversion of superoxide ions into H₂O₂ by the peak SOD activity at 30 DAA stage. Except for an isolated report on changes in catalase activity during seed dessication,¹⁴ current data provide first detailed report on the modulation of ROS and its scavenging enzymes during different stages of seed development in sunflower. It further indicates an active mechanism operative in orthodox seeds to prevent damage to developing seeds by ROS.

Figure 2. Estimation of reactive oxygen species (ROS) content, quantitative, and zymographic analysis of ROS scavenging enzymes (POD, SOD, and GR) and NO accumulation in relation with seed development in sunflower. Data represent mean values and standard errors from 3 replicates. Double asterisks (**) represents one way ANOVA to be significant at P < 0.01 and triple asterisks (***) represents one way ANOVA to be significant P < 0.001.

A probable crosstalk between ROS and PKC seems evident from earlier investigations on animal systems, with calcium ions regulating both PKC activity and ROS homeostasis through various scavenging mechanisms. Different cellular and subcellular components of cells witness a complex network involving a crosstalk between calcium and ROS signaling pathways.¹⁸ These interactions can be either stimulatory or inhibitory. It involves the regulation of calcium-dependency of mechanisms engaged in ROS generation and their annihilation. Calcium regulates mitochondrial ROS generation by increasing metabolic activities in the cells, particularly by activating the enzymes involved in TCA cycle, ATP synthase, and adenine nucleotide translocase, thereby increasing cell metabolism. Cytosolic ROS generation is regulated by calcium-dependent, direct or indirect, regulation of ROS generating enzymes, like plasma membrane-associated NADPH oxidase complex, etc.¹⁹ Calcium is also likely to be involved in the regulation of different ROS scavenging enzymes (catalase, POD), thereby regulating ROS scavenging mechanisms in the cell.^19-21

Protein kinase C activity exhibits development-dependent modulation during seed maturation in sunflower. This has been observed both during early stages of embryo development and during the development of seeds by the use of PKC specific fluorescent probe Fim-1, a membrane permeant derivative of bisindoylmaleamide, as well as PKC inhibitors, namely staurosporine and bisindoylmaleamide. Fim-1-diacetate selectively inhibits PKC by binding to its catalytic domain by competing with ATP, which exhibits consistent binding with the catalytic domain of the enzyme. Thus, use of this fluorescent derivative does not disturb the activity of PKC associated with its regulatory domain. Selectivity of PKC toward Fim-1 is 16 times more as compared with PKA. Staining pattern produced by Fim-1 has been shown to be very similar to that of an anti-PKC antibody.²²

At the globular stage of embryo development, fluorescence due to PKC is more prominent in the peripheral cells than in the deep-seated ones (Fig. 3A). Interestingly, even at this stage, which is marked with no polar axis differentiation, fluorescence due to PKC in the parietal cells is more in one specific region than the rest of the globular embryo (see Fig. 3A insets). Probably, this non-uniformity in the abundance of PKC in the peripheral cells of the globular embryos is an indication of the sign of initiation of biochemical polarity prior to cellular differentiation. At the heart-shaped stage of embryo development, PKC distribution in the deep-seated cell is more profuse than at the globular stage. Peripheral cells and the cells of suspensor exhibit greater fluorescence due to PKC than the deep-seated cells (Fig. 3B). At the cotyledonary stage of embryo development, the relative abundance of fluorescence due to PKC is less than at the heart-shaped stage (Fig. 3C).

Figure 3. Localization and quantitative detection of PKC during embryo (A, B, and C) and seed (D and E) development. Free calcium concentration in the water soluble tissue homogenates of the developing seed was estimated by generating a standard curve (F) and estimation of [Ca⁺²] were undertaken by incubating CaCl₂ (for standard curve) and tissue homogenates (G) with 5 μM of Oregon green BAPTA-AM-1, followed by measurement of emission upon excitation at 488 nm (em. 520 nm). Histograms represent mean values and standard errors from 3 replicates. Triple asterisks (***) represents one way ANOVA to be significant P < 0.001.

Biochemical analysis of PKC activity has been undertaken by observing an increase in fluorescence in the tissue homogenates of developing seeds at 40 DAA stage with an increase in protein concentration from 50 to 200 µg (Fig. 3D). This observation provides the first indication of PKC activity in the tissue homogenates of the developing seeds and it was further substantiated by the use of PKC inhibitors (staurosporine and bisindoylmaleamide) which leads to a significant inhibition of enzyme activity, as is evident from reduced fluorescence due to PKC binding with Fim-1 (Fig. 3E). Water soluble tissue homogenates (10,00 g supernatants) of developing seeds at 20, 30, and 40 DAA stages, when monitored for free calcium concentration against a standard curve (Fig. 3F) generated using Oregon green as calcium specific probe, revealed relatively lower endogenous calcium content at 20 DAA stage, it being higher at 30 and 40 DAA stages (Fig. 3F and G). Tissue homogenates from cotyledons at 40 DAA stage also revealed enhanced fluorescence due to Fim-1-PKC binding in the presence of externally provided increasing concentrations of calcium ions (Fig. 4A andD). Lowering down of calcium-induced PKC activity in the presence of calcium chelating agents (BAPTA) further confirmed the role of calcium levels in modulating PKC activity (Fig. 4BandE). It is further evident from Figure 4C that PKC activity in the tissue homogenates of developing seeds increased with the stage of development from 20 to 40 DAA, apparently coinciding with increasing availability of free calcium in the tissue at the later stage of seed development (Fig. 3B). Furthermore, positive modulation of PKC activity is evident in all the 3 stages of seed development with the co-addition of 10 mM Ca⁺², more so in maturing seeds at 40 DAA seeds. The impact of exogenous calcium ions is further confirmed by western blot analysis of the protein derived from the tissue homogenate of cotyledons at 40 DAA stage and its reversal by the co-incubation by BAPTA (Fig. 4F). Western blot analysis revealed an increase in the intensity of 2 bands representing PKC detected using immunochemical techniques by employing anti-PKC polyclonal antibody.

Figure 4. (A) Spectrofluorometric monitoring of calcium modulation of putative protein kinase C activity in 10 000 g supernatant obtained from mature sunflower seeds at 40 DAA stage (ex: 480 nm, em: 520 nm). (B) Effect of BAPTA (a calcium chelating agent) on calcium-modulated increase in putative protein kinase C activity in 10 000 g supernatant obtained from developing seeds (40 DAA). (C) Differential activity of putative protein kinase C at the 3 developmental stage (i.e., 20, 30, and 40 d after anthesis) in the presence and absence of 10 mM Ca⁺². Tissue homogenates each representing 100 µg of protein, were incubated with 2.5 µM Fim-1 diacetate followed by spectrofluorometric monitoring of fluorescence emission. (D) Relative change in putative PKC activity in tissue homogenates (10 000 g supernatant) from seeds at 40 DAA stage (% of control i.e., protein + probe) in the presence of different concentrations of calcium. (E) Relative change in putative PKC activity (10 000 g supernatant) from seeds at 40 DAA stage (% of control, i.e., protein + probe) in the presence of calcium, without and with BAPTA. Data represent mean values and standard errors from 3 replicates. Double asterisks (**) represents one way ANOVA to be significant at P < 0.01 and triple asterisks (***) represents one way ANOVA to be significant P < 0.001. (F) Immunochemical detection of protein kinase C at the 3 stages of sunflower seed development i.e., 20, 30, and 40 DAA by western blot analysis.

Present observations reporting calcium-modulated enhancement of PKC indicate the presence of “conventional” type of PKC reported from animal systems, which possesses calcium-binding site in the regulatory domain. Most PKC isozymes are ubiquitously expressed in all tissues. The discovery of calcium-dependent protein kinases further provides evidence for the critical role of calcium ions in signal transduction mechanisms.²³ These kinases are dependent on calcium but act independent of calmodulin and they have been purified from a number of plant species.²⁴ Purification of a PKC-type kinase and its characterization from maize has also been reported.²⁴ This 78 kDa protein is stimulated by phosphatidylserine and oleylacetyl glycerol in the presence of calcium ions, suggesting it to be a “conventional” serine/threonine protein kinase C (CPKC). Similar calcium and phospholipid-dependent kinase activity has earlier been demonstrated in the cytosolic fractions of zucchini, oat, Amaranthus tricolor, and rice.^25-28 Specific isoforms of “conventional” PKC (α, β, and γ) are modulated by calcium. A probable crosstalk between ROS and PKC seems likely from earlier investigations on animal systems, with calcium ions regulating both PKC activity and ROS homeostasis through various scavenging mechanisms (Fig. 5).²⁹ Low levels of oxidants in the tissue can modify cell signaling proteins. PKC is a potential candidate for redox modifications by oxidants and antioxidants since it contains susceptible and unique structural features.²⁹ Oxidation of the autoinhibitory function of the regulatory domain of PKC is known to stimulate its activity through suppression of its autoinhibitory function. Likewise, the C-terminal catalytic domain contains reactive cysteine molecules which can be the targets for various antioxidant enzymes, thereby inhibiting enzyme activity. Thus, the 2 domains of PKC respond differently.

Figure 5. A model depicting probable interaction between ROS, antioxidant enzymes, and modulation of PKC activity (modified from Gopalakrishna and Jaken, 2000).²⁹ This model is based on earlier information from literature and offers scope for future work in this direction in relation with seed development.

To sum up, the genetically programmed process of seed development exhibits a clear correlation with several metabolic events. An upregulation of antioxidant genes seems to be one of the major mechanisms operating during the maturation of orthodox seeds, such as sunflower. Loss of vigor and viability accompanying the storage of desiccation-tolerant orthodox seeds is correlated with the modulation of ROS accumulation in the cells of cotyledons. The 3 ROS scavenging enzymes (SOD, POD, and GR) contribute in the reduction of the ROS produced in the developing seeds through a temporal distribution of their maximal activities during various stages of development. Protein kinase C is a potential candidate for redox modifications by oxidants and antioxidants since it contains susceptible and unique structural features. Thus, present observations reveal the presence of “conventional” type of PKC in developing seeds which possess a calcium-binding site in the regulatory domain. ROS and PKC interaction is likely through direct action of ROS and antioxidant enzymes on the regulatory and catalytic domains of PKC, with calcium ions regulating both PKC activity and ROS homeostasis through various scavenging mechanisms.

Materials and Methods

Plant material

Sunflower (Helianthus annuus L. cv KBSH-44) seeds were procured from National Seeds Corporation (Hyderabad, India) and plants were raised in the botanical garden of Department of Botany, University of Delhi, during October to February. Developing seeds were harvested from the 2 peripheral whorls 20, 30, and 40 d after anthesis (DAA). Collection of seeds was undertaken from a number of inflorescences maturing on a specific date for each stage. After removal of hull, freshly harvested seeds were used for various analyses. For biochemical analyses, seeds were counted/weighed and stored in liquid nitrogen until further use.

Microscopic analysis of wax sections of seeds

Freshly harvested cotyledons of different developmental stages were fixed in a mixture of 0.05% glutaraldehyde and 4% paraformaldehyde prepared in phosphate buffer saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 6.5 mM Na₂HPO₄, and 1.5 mM KH₂PO₄, pH 7.3) for 1 h at 24 °C. Fixed cotyledons were subjected to dehydration at 24 °C for 1 h each in an increasing gradation of ethanol (70, 80, 90, and 100%) diluted in PBS. Cotyledons were dehydrated overnight in 100% ethanol followed by dehydration for 3 h each in 1:1 and 1:3 proportion of ethanol: xylene and finally in 100% xylene at 24 °C for 2 h. Tissues were cold infiltrated overnight with paraffin wax. Ten cold-infiltrated cotyledons of each stage were then embedded in paraffin wax and serial sections (7 µm thick) were prepared using a rotary microtome. The sections were dewaxed and then observed with light microscope.

Quantification of reactive oxygen species (ROS) in tissue homogenates

500 mg of developing seeds from each of the 3 developing stages were powdered in liquid nitrogen and homogenized in 3 ml grinding medium [50 mM Tris (pH 7.5), 0.25 M sucrose] containing 1 mM PMSF.³⁰ Tissue homogenates were filtered through 4 layers of muslin cloth and centrifuged at 10 000 g for 20 min at 4 °C. ROS was estimated using de-esterified 2,7-dichlorofluorescein (DCFH) obtained from DCFH-DA by the hydrolysis in NaOH.³¹ Protein equivalent to 100 μg from each sample was incubated with 5 μM of the probe (de-esterified DCFH) for 20 min at 4 °C. Fluorescence was measured after 20 min of incubation using a spectrofluorometer (Perkin Elmer, USA) at an excitation wavelength of 485 nm. Emission was observed at 535 nm. Data have been presented as intensity of fluorescence (at the 3 stages of seed development).

Zymographic detection of peroxidase activity

Peroxidase (EC 1.11.1.7) isoforms were detected zymographically.³² Homogenates were prepared by grinding the tissue in 50 mM of sodium acetate buffer (pH 4) and filtered through 4 layers of muslin cloth. The filtrates were centrifuged at 10 000 g for 20 min at 4 °C. Protein was quantified from the unternatant.³³ Each total soluble protein (TSP) aliquot, equivalent to 100 μg protein, was mixed with reducing Laemmli sample buffer (1:1) and loaded in the stacking gel of a 12.5% flat mini vertical gel. Electrophoresis was performed at 75 V for 30 min and at 25 mA for rest of the time at 4 °C. After electrophoresis, gel was incubated for 20–30 min in 0.2 M sodium acetate buffer (pH 5.0) containing 1.3 mM benzidine (24 mg in 100 ml) and 1.3 mM H₂O₂ (4 μl in 100 ml) until brown peroxidase isoform bands appeared.

Estimation of POD activity

Spectrophotometric analysis of peroxidase activity was performed by mixing 30 μg of protein from the tissue homogenate from different seed developmental stages, with 2.4 ml of substrate solution (0.6 mM o-dianisidine and 8.8 mM H₂O₂ in 50 mM sodium phosphate buffer, pH 6.0).³² Change in absorbance was recorded at 460 nm up to 5 min against a blank containing 2.4 ml of substrate solution mixed with sodium acetate buffer (pH 6.0).

Zymographic detection of superoxide dismutase (SOD)

Superoxide dismutase (EC 1.5.1.1) isoforms were detected zymographically by grinding 500 mg of seeds to powder in liquid nitrogen and homogenizing it in 3 mL of TRIS-HCl buffer (50 mM, pH 7.0) containing 50 mM NaCl, 0.05% Tween-80 and 1mM phenylmethylsulfonyl fluoride (PMSF).^34,35 The homogenates were centrifuged at 10 000 g for 20 min and the unternatants thus obtained after centrifugation were used for estimating SOD activity. 60 μg of TSP from each homogenate was mixed with non-reducing Laemmli sample buffer (1:1) and loaded in the stacking gel of a 12.5% flat vertical gel. Electrophoresis was performed at 75 V for 0.5 h and at 25 mA for rest of the time at 4 °C. The gel was then soaked in 2.5 mM nitrobluetetrazolium (NBT) for 25 min and then incubated for 20 min in dark in 50 mM phosphate buffer (pH 7.8) containing 28 μM riboflavin and 28 mM TEMED. The gel was then transferred to distilled water and exposed to light for 10–15 min at room temperature. During illumination, gel became uniformly blue, except at positions containing SOD isoforms. Illumination was stopped when maximum contrast between achromatic zone and blue color was achieved.

Estimation of SOD activity

SOD activity was estimated spectrophotometrically by incubating a mixture of 60 μg of total soluble protein with 3 mL of substrate solution (9.9 mM l-methionine, 1.67 x10–4 M nitrobluetetrazolium and 2.4 x10–6 M riboflavin in 50 mM phosphate buffer, pH 7.8) at 27 °C.³⁴ Reaction was started by placing the tubes (containing reaction mixture) near 60 W tungsten bulbs for 10 min. Blank and controls were run in the same manner but in the absence of illumination and enzyme, respectively. Photochemical reaction was stopped by covering the tubes with black cloth after 10 min of incubation in light and absorbance was recorded immediately thereafter at 560 nm. One unit of SOD activity was defined as the amount of enzyme required for 50% inhibition of NBT reduction in the assay conditions.

Estimation of nitric oxide content and putative nitric oxide synthase (NOS) activity

Five hundred mg of tissue from each stage was ground to fine powder in liquid nitrogen and homogenized in 1.5 ml extraction buffer (50 mM TRIS-HCl, 250 mM sucrose, pH 7.5). The pooled homogenate was filtered through 4 layers of muslin cloth pre-soaked in the above-stated buffer and centrifuged at 10 000 g for 20 min at 4 °C. Protein concentration was determined by Bradford assay.³³ Total NO content was estimated by adding samples to the reaction buffer containing 1 mM l-arginine, 1µM β-NADPH, and 2.5 µM DAF-2DA. The reaction mixture was incubated at 25 °C for 30 min.^36,37 In order to determine NO content due to putative NOS activity, samples were preincubated for 30 min with 10 mM aminoguanidine in the reaction buffer. This was followed by the addition of 1 mM l-arginine, 1 µM β-NADPH, and 2.5 µM DAF-2DA.³⁸ The reaction mixture was incubated at 25 °C for 30 min. Reaction mixtures without protein served as control for their respective samples. Fluorescence was estimated using a spectrofluorometer (Perkin Elmer, USA) at the required excitation and emission wavelengths of 495 and 515 nm, respectively.³⁹ Data was presented as arbitrary fluorescence units.

Zymographic detection of glutathione reductase (GR) activity

Glutathione reductase (EC 1.8.1.7) was detected according to Foyer et al. (1991).⁴⁰ Homogenates were prepared by grinding 500 mg tissue in 1 ml of extraction buffer [5 mM ascorbate and 1 mM EDTA in 0.1 M sodium phosphate buffer (pH 7)] and filtered through 4 layers of muslin cloth. The filtrates were centrifuged at 10 000 g for 20 min at 4 °C. Protein was quantified from the unternatent according to Bradford (1976). Aliquot of each TSP, equivalent to 75 μg, was mixed with reducing Laemmli sample buffer (1:1) and loaded in the stacking gel of a 15% flat mini gel. Vertical electrophoresis was performed at 75 V for 0.5 h and at 25 mA for rest of the time at 4 °C. Gel was then incubated for 10 min in 0.25 M TRIS-HCl buffer (pH 8.4), 4 mM GSSG (oxidized glutathione), 1.5 mM NADPH, and 2 mM DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] until yellow GR bands appear.

Estimation of GR activity

Spectrophotometric analysis of glutathione reductase (GR) activity was performed according to Sairam et al. (2002).⁴¹ Enzyme assay was performed by mixing 30 μg of protein from the tissue homogenate with 2.8 ml of reaction mixture [1 ml 0.2 M potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA, 0.5 ml 3 mM DTNB in 0.01 M phosphate buffer (pH 7.5), 0.1 ml 2 mM NADPH]. Reaction was initiated by adding 0.1 ml of 2 mM GSSG (oxidized glutathione) just before taking the observation. Absorbance change was recorded at 412 nm up to 5 min against a blank containing 2.9 ml of substrate solution mixed with 0.1 ml 0.2 M potassium phosphate buffer (pH 7.5).

In vivo localization of protein kinase C (PKC) activity in developing embryos

Putative protein kinase C was localized using Fim-1-diacetate.⁴² Different embryo developmental stages were dissected out and 5 embryos from each developmental stage were incubated with the 2 µM Fim-1-diacetate in dark for 30 min followed by rinsing in distilled water for 10 min. Observations were taken by confocal laser scanning microscopy (ex. 480 nm; em. 520 nm).

Estimation of putative protein kinase C activity (PKC)

Seeds at different stages of development were ground to fine powder in liquid nitrogen and were extracted in buffer (25 mM Tris-sucrose buffer containing 1 mM phenylmethanesulfonyl fluoride) for 30 min at 4 °C. Protein suspensions were then subjected to centrifugation at 10 000 g for 20 min at 4 °C. Protein concentration was determined according to Bradford (1976).³³ Tissue homogenates, each containing 100 μg of protein, were incubated with 2.5 μM Fim-1-diacetate in the extraction buffer for 30 min at 25 °C. Reaction mixtures without protein served as control. Fluorescence was estimated using a spectrofluorometer (Perkin Elmer, USA) at the required wavelengths (ex. 480nm; em. 520 nm). Presence of putative PKC in the tissue homogenates was further confirmed by the use of PKC specific inhibitors, namely staurosporine and bisindolymaleamide (100 nM each). Calcium modulation of PKC activity was examined spectrofluorometrically using 0.1, 10, and 50 mM of CaCl₂. Similar range of calcium has been previously used to monitor modulation of thiol protease activity.⁴³ Calcium modulation of PKC was further ascertained by using BAPTA (2.5 and 5 mM), a calcium chelating agent, in the tissue homogenates.

Estimation of free cytosolic calcium concentration

Seeds were ground to powder in liquid nitrogen and powder was dispersed in the extraction buffer (50 mM TRIS-HCl, 20 mM sucrose, pH 7.2). The homogenates were centrifuged at 10 000 g for 20 min at 4 °C and the unternatants were used for the spectrofluorometric analysis of free cytosolic calcium concentrations, using Oregon Green BAPTA-AM-1 (Invitrogen Bioservices Pvt. Ltd, USA), a fluorescence probe for detection of calcium. Equal volumes (5 μl) of supernatants were mixed with 5 μM of Oregon Green in 600 µl of extraction buffer (50 mM TRIS-HCl, 20 mM sucrose, pH 7.2) and fluorescence was estimated (ex. 488 nm; em. 520 nm). Reaction mixtures without tissue homogenates served as controls for their respective samples.

Cytosolic free calcium concentration was estimated from the calibration curve obtained using standard CaCl₂ solutions (0.2, 0.4, 0.6, 0.8, and 2 mM). Free cytosolic calcium concentration (nM) in the tissue homogenates was calculated using the following equation:

[Ca⁺²]_free = K_d(F – F_min)/(F_max – F)

The dissociation constant (K_d) of calcium for Oregon Green BAPTA-AM-1 is 170 nM. F represents fluorescence due to calcium ions in the samples. F_max is the fluorescence for calcium-saturated probe obtained from standard calibration at the highest concentration of calcium (2 mM) and F_min is the fluorescence of the lowest concentration of calcium used for standard calibrations.

Western blot analysis of protein kinase C (PKC)

Subsequent to resolution of polypeptides on a 12.5% vertical SDS-PAGE, the gel was washed in transfer buffer [20% glycine, 5% Tris (hydroxymethyl)aminomethane, and 10% methanol, 4 °C] for at least 15 min. PVDF membrane was washed in methanol (100%) for 10 s followed by deionized water (5 min) and transfer buffer (10–15 min) sequentially. Filter paper (GE Healthcare, USA), cut to the size of PVDF membrane, was also pre-soaked in transfer buffer. The transfer sandwich was prepared by packing together filter papers (4 pcs), activated PVDF membrane, gel and filter paper (4 pcs). The sandwich was inserted in the wet transfer unit. The current was gradually enhanced from 80 to 120 and 150 mA after 30 min interval each and protein transfer was accomplished by running the system for 2 hours. Subsequently, the blot was rinsed with PBS buffer [8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0,24 g KH₂PO₄; final volume = 1L) pH 6.8] and then incubated in blocking buffer (5% BSA, 0.1% Tween 20 in PBS) for 2 hours at 25 °C. Blocking prevents non-specific binding of primary and secondary antibodies to membrane. The membrane was then incubated overnight at 4 °C in an orbital shaker with anti-PKC antibody (1:500) in blocking buffer. Thereafter, the membrane was washed thrice in wash buffer (0.1% tween 20 in PBS, pH 6.8) for 5 min each and incubated in secondary antibody (anti-rabbit IgG conjugated to alkaline phosphatase, antibody) (1:3000 in wash buffer) for 1 h at room temperature on an orbital shaker. Finally, the membrane was washed in wash buffer 3 times for 5 min each and developed using freshly prepared BCIP/NBT (1 Sigma Fast tablet dissolved in 10 mL MilliQ water) for 10–30 min. BCIP/NBT (5-Bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium) is used as a precipitating substrate for detection of alkaline phosphatase activity. Once the desirable color intensity was obtained, the membrane was placed in MilliQ water to stop color development. Membrane dried between tissue paper could be stored. Primary antibody was procured from Abcam (USA) and secondary antibodies were obtained from Sigma Aldrich (USA).

Statistical analysis

Effects of various treatments were analyzed by SPSS 16.0 statistical program (SPSS Inc, Chicago, IL, USA) using one way ANOVA. All experiments were performed at least thrice.