Pomegranate (Punica granatum L.) Fruits: Characterization of the Main Enzymatic Antioxidants (Peroxisomal Catalase and SOD Isozymes) and the NADPH-Regenerating System

Abstract

Pomegranate (Punica granatum L.) is a common edible fruit. Its juice can be used as a source of antioxidative compounds, primarily polyphenols and vitamin C, in addition to other vitamins and minerals. Nevertheless, little is still known about how the enzymatic machinery, mainly that related to oxidative metabolism, is influenced by the genotype and the environmental and climate conditions where pomegranate plants grow. In this work, seeds and juices from two pomegranate varieties (Valenciana and Mollar) grown in two different Spanish locations were assayed. Both varieties showed clear differences in their respective polypeptide profiles. The analysis of the isoenzymatic superoxide dismutase (SOD) activity pattern displayed one Mn-SOD and five CuZn-SODs (I–V) whose abundances depended on the variety. Furthermore, by immunoblot assays, at least one additional Fe-SOD with a subunit size of about 23 kDa was also detected in both varieties. Besides this, the presence of the H2O2-scavenging peroxisomal catalase in seeds and juice indicates that an active metabolism of reactive oxygen species (ROS) takes place in this fruit, but the two pomegranate varieties showed opposite activity profiles. The activities of the main NADPH-regenerating enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphlogluconate dehydrogenase (6PGDH), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADP-dependent malic enzyme (NADP-ME), were studied in the same plant materials, and they behaved differently depending on the genotype. Finally, our data demonstrate the presence of two specific enzymes of the peroxisomal glyoxylate cycle, malate synthase (MS) and isocitrate lyase (ICL). These enzymes participate in oilseeds by channeling the lipid catabolism to the carbohydrate synthesis for further use in seed germination and early seedling growth. The results obtained in this work indicate that a similar mechanism to that reported in oilseeds may also operate in pomegranate.

Antioxidant Profile of Pepper (Capsicum annuum L.) Fruits Containing Diverse Levels of Capsaicinoids

“…For the identification of the different SOD isozymes, before the staining procedure, pre-incubation of gels was carried out in the presence of specific inhibitors, either 5 mM KCN or 5 mM H 2 O 2 . Copper- and zinc-containing SODs (CuZn-SODs) are inhibited by both KCN and H 2 O 2 ; iron-containing SODs (Fe-SODs) are inactivated by H 2 O 2 ; and Mn-SODs are resistant to both inhibitors [ 58 , 59 ].…”

“…For the identification of the different SOD isozymes, before the staining procedure, pre-incubation of gels was carried out in the presence of specific inhibitors, either 5 mM KCN or 5 mM H 2 O 2 . Copper-and zinc-containing SODs (CuZn-SODs) are inhibited by both KCN and H 2 O 2 ; iron-containing SODs (Fe-SODs) are inactivated by H 2 O 2 ; and Mn-SODs are resistant to both inhibitors [58,59]. NADP-dependent dehydrogenase (NADP-DHs) activities were determined by recording the NADPH formation at 340 nm and 25 • C. The assay was performed in a reaction medium containing 50 mM HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)], pH 7.6, 2 mM MgCl 2 and 0.8 mM NADP.…”

Superoxide Radical Metabolism in Sweet Pepper (Capsicum annuum L.) Fruits Is Regulated by Ripening and by a NO-Enriched Environment

“…•− into O 2 and H 2 O 2 and are associated with stress, development, and senescence conditions in plants (Houmani et al, 2016;del Río et al, 2018). Moreover, the type and number of SOD isozymes can differ between different plant species and their corresponding organs (Asada et al, 1973;Bridges and Salin, 1981;Almansa et al, 1989;Corpas et al, 1991Corpas et al, , 2006Pinilla et al, 2019). In preliminary studies, the presence of four SOD and seven O 2…”

Antioxidant Profile of Pepper (<em>Capsicum annuum</em> L.) Fruits Containing Diverse Levels of Capsaicinoids

“…For the identification of the different SOD isozymes, before staining pre-incubation of gels in the presence of specific inhibitors, either 5 mM KCN or 5 mM H2O2, was carried out. Copper,zinc-containing SODs (CuZn-SODs) are inhibited by both KCN and H2O2; iron-containing SODs (Fe-SODs) are inactivated by H2O2; and Mn-SODs are resistant to both inhibitors [59,60].…”