Class III Peroxidases (POD) in Pepper (Capsicum annuum L.): Genome-Wide Identification and Regulation during Nitric Oxide (NO)-Influenced Fruit Ripening

Abstract

The class III peroxidases (PODs) catalyze the oxidation of several substrates coupled to the reduction of H₂O₂ to water, and play important roles in diverse plant processes. The POD family members have been well-studied in several plant species, but little information is available on sweet pepper fruit physiology. Based on the existing pepper genome, a total of 75 CaPOD genes have been identified, but only 10 genes were found in the fruit transcriptome (RNA-Seq). The time-course expression analysis of these genes showed that two were upregulated during fruit ripening, seven were downregulated, and one gene was unaffected. Furthermore, nitric oxide (NO) treatment triggered the upregulation of two CaPOD genes whereas the others were unaffected. Non-denaturing PAGE and in-gel activity staining allowed identifying four CaPOD isozymes (CaPOD I-CaPOD IV) which were differentially modulated during ripening and by NO. In vitro analyses of green fruit samples with peroxynitrite, NO donors, and reducing agents triggered about 100% inhibition of CaPOD IV. These data support the modulation of POD at gene and activity levels, which is in agreement with the nitro-oxidative metabolism of pepper fruit during ripening, and suggest that POD IV is a target for nitration and reducing events that lead to its inhibition.

Keywords: fruit ripening; nitration; nitric oxide; pepper; peroxidase.

Figure 1

Representative model of the experimental design used in this study with the phenotype of sweet pepper (C. annuum L.) fruits at different stages and treatments: immature green, breaking point 1 (BP1), breaking point 2 without nitric oxide (NO) treatment (BP2 − NO), breaking point 2 with NO treatment (BP2 + NO), and ripe red. Pepper fruits were subjected to a NO-enriched atmosphere (5 ppm) in a methacrylate box for one hour and then were stored at room temperature (RT) for 3 days. Reproduced from [35] with permission from Oxford University Press Journal and Copyright Clearance Center (2019).

Figure 2

Synteny analysis of CaPOD genes. Pepper chromosomes (Chr. 1–12) are represented in different colors. Grey curved lines within the scheme refer to the co-localization of genetic loci in the pepper genome. Collinear relationships between CaPOD genes are drawn in orange. Tandem duplication for CaPOD genes is indicated with blue lines. Red asterisks indicate those CaPOD genes identified in the transcriptome of sweet pepper fruit.

Figure 3

Heatmap of cis-regulatory elements corresponding to the 1500 bp upstream region from the transcription start point of CaPOD genes. The distribution of these elements in the promoter sequence is also shown. The cis-regulatory elements were grouped according to their functional implications as hormone-responsive elements and abiotic/biotic stress-responsive elements. CaPOD genes identified in the sweet pepper fruit transcriptome are highlighted in red. Motifs were identified from the PlantCARE database.

Figure 4

Phylogenetic relationships between pepper and Arabidopsis POD genes. Clusters (I–IV) are displayed in different colors. Clusters were divided into different subgroups (a–d) according to their evolutionary distance. Species abbreviations: At (Arabidopsis thaliana), Ca (Capsicum annuum). Those CaPODs identified in the transcriptome of sweet pepper fruit are highlighted in red.

Figure 5

Identification and position of consensus amino acid motifs for pepper CaPODs. (a) Distribution of conserved motifs. The distribution of conserved motifs numbered 1–10 of the 75 pepper PODs is represented by boxes of different colors. (b) Amino acids sequence of the motifs. Ten amino acid motifs with various were identified and are represented with different sizes. The height of each amino acid symbol is proportional to the degree of conservation in the consensus sequences depicted in the ten motifs. Sequence logos of conserved motifs were created by MEME.

Figure 6

Time-course expression analysis of CaPOD genes (RNA-Seq) from pepper fruits. Differences in temporal expression patterns between different ripening stages of sweet pepper fruit and the effect of NO treatment on genes encoding different POD isoforms are shown. Samples corresponding to immature green (G), breaking point 1 (BP1), breaking point 2 with and without NO treatment (BP2 + NO and BP2 − NO, respectively), and red (R) were used. Diamonds indicate statistically significant changes in expression levels (p < 0.05) in comparison to immature green fruits (G). Green line: BP2 fruits treated with NO. Red line: untreated fruits.

Figure 7

Isozyme peroxidase (POD) activity of sweet pepper fruits at different ripening stages: immature green (G), breaking point 1 (BP1), breaking point 2 with and without NO treatment (BP2 + NO and BP2 − NO, respectively), and red ripe (R). (a) In-gel isozyme profile of POD activity). (b) Total relative quantification of POD isoenzyme activity. Protein samples (28 µg per lane) were separated by non-denaturing polyacrylamide gel electrophoresis (PAGE; 8% acrylamide), and the activity was detected by the 3,3-diaminobenzidine method. POD isozymes were labeled I–IV, according to their increasing electrophoretic mobility.

Figure 8

Effect of nitration, S-nitrosation, and reducing agents on the green pepper fruit peroxidase (POD) isozymes analyzed in non-denaturing gels. (a) In-gel isozyme profile of POD activity in 8% acrylamide gels. (b) Densitometric analysis of POD isozymes and their relative quantification (%) was made by the ImageJ program. SIN-1 is a peroxynitrite donor and a nitrating compound. GSNO (S-nitrosoglutathione) and CysNO (nitrosocysteine) are NO donors and nitrosating agents. L-Cys: cysteine. GSH: reduced glutathione. NaHS: sodium hydrosulfide as H₂S donor. H₂O₂: hydrogen peroxide. All treatments were done by pre-incubating the green pepper samples (28 µg protein per lane) with these compounds (5 mM) at 25 °C for 1 h, except with SIN-1, which was pre-incubated at 37 °C for 1 h. The number assigned to each peak indicates the percentage of either isozyme activity inhibition (red) or activation (blue) in relation to the control samples (green fruit crude extracts) after the quantification made with the help of the ImageJ program.