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.

Keywords: Sunflower; calcium; glutathione reductase; peroxidase; protein kinase C; reactive oxygen species; seed development; superoxide dismutase.

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.

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.

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.

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.

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