Antioxidant systems from Pepper (Capsicum annuum L.): involvement in the response to temperature changes in ripe fruits

Abstract

Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes.

Figure 1

Ascorbate (ASC) and dehydroascorbate (DHA) contents in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). Column values with distinct letters are significantly different.

Figure 2

Activity and expression levels of the ascorbate-glutathione cycle enzymes in the cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Activity of ascorbate peroxidase (APX) and glutathione reductase (GR) determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Analysis of the mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the APX and GR amplification products (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 3

l-Galactono-γ-lactone dehydrogenase (GalLDH) activity and expression level in the cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Activity determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Analysis of the mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the GalLDH amplification product (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 4

Catalase (CAT) activity and expression level in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Activity determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Analysis of mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the CAT amplification product (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 5

Activity, protein content and expression levels of superoxide dismutase (SOD) in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Total activity determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Isoenzymatic pattern of SOD in the two cultivars where four isozymes were detected. Samples (100 μg protein/lane) were loaded and polyacrylamide gel electrophoresis was carried out under native conditions. Isozymes were characterized on the basis of their susceptibility to the inhibitors KCN and H₂O₂; (C) Western blotting of samples from the two pepper cultivars using specific antibodies against Mn-SOD, Fe-SOD and CuZn-SOD (see Experimental Section). Prior to transferring proteins (15 μg/lane) to PDVF membranes, polypeptides were separated in 12% polyacrylamide gels under denaturing conditions; (D) Analysis of the mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the Mn-SOD, Fe-SOD and CuZn-SOD amplification products (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 6

Activity, protein content and expression levels of the glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Total activity determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Western blotting of samples from the two pepper cultivars using specific antibodies against G6PDH and 6PGDH (see Experimental Section below). Previous to transferring proteins (15 μg/lane) to PDVF membranes, polypeptides were separated in 12% polyacrylamide gels under denaturing conditions; (C) Analysis of the mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the G6PDH and 6PGDH amplification products (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 7

NADP-isocitrate dehydrogenase (NADP-ICDH) and malic enzyme (ME) in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). (A) Total activity determined spectrophotometrically. Column values with distinct letters are significantly different; (B) Western blotting of samples from the two pepper cultivars using specific antibodies against NADP-ICDH (see Experimental Section below). Previous to transferring proteins (15 μg/lane) to PDVF membranes, polypeptides were separated by electrophoresis in 12% polyacrylamide gels under denaturing conditions; (C) Analysis of the mRNA expression. Semiquantitative reverse transcription-PCR was performed on total RNA isolated from fruits of the two cultivars. A representative agarose electrophoresis gel of the amplification products visualized by ethidium bromide staining under UV light is shown. T/C indicates the relative level of the NADP-ICDH amplification product (T) over the Actin (C, internal control) after normalization to the control samples and expresses the change in folds with respect to the corresponding controls (Actin from Harvest 1 in each cultivar). Values are means of at least three semiquantitative RT-PCR assays made in different fruits from each cultivar.

Figure 8

Lipid peroxidation and protein oxidation in cultivars Vergasa and Biela of pepper (Capsicum annuum L.) fruits harvested at two temperature conditions (Harvest 1 and 2). Lipid peroxidation was measured as μM MDA per mg protein, and protein oxidation as nmol carbonyl groups per mg protein. Column values with distinct letters are significantly different.

Figure 9

Experimental design for the analysis of ROS-related parameters in two cultivars of pepper (Capsicum annuum L.) fruits. (A) Types of pepper fruits used in this work belong to the cultivars Vergasa and Biela. Pictures of fruits correspond to the final stage in which fruits were analyzed: red for Vergasa and yellow for Biela; (B) Sampling of fruits for the analysis of enzymes and metabolites and gene expression. For enzymes and metabolite assays two strips per fruit were homogenized and analyzed. For RNA extraction the central part of the fruits was used.

Figure 10

Temperature data corresponding to Harvests 1 and 2 of the present study. The data were provided by the Meteorological Station of La Mojonera (Almería, Spain) belonging to the Junta de Andalucía, Spain (UTM Coordinates X: 526472.0, Y: 4071536.0; latitude: 36°47′19″ N, longitude: 02°42′11″ W, altitude: 142.0 m).