Electrochemical Detection of Total Antioxidant Capacity (TAC) in Plant Tissues from Different Origins.

Total antioxidant capacity (TAC) is a reliable indicator of antioxidant content in animal and plant samples. The different experimental approaches available allow the determination of TAC using, as a reference, diverse compounds with recognized antioxidant capacities such as Trolox, ascorbic acid, gallic acid, or melatonin. A new portable device, named BRS (BQC redox system), is now commercially available that, through an electrochemical approach, allows the determination of TAC in a simple, fast, reproducible, and robust way. In this chapter, using this portable device, a comparative analysis of the TAC is assayed in different red, citrus, and Solanaceae fruits, several Allium species, and organs of different plant species, including Arabidopsis thaliana. The obtained results demonstrate the versatility of the BRS portable device.

Comparative Analysis of Catalase Activity in Plants: Spectrophotometry and Native PAGE Approaches.

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme.

Detection of Ascorbate Peroxidase (APX) Activity in Plant Tissues: Using Non-denaturing PAGE and Spectrophotometric Assay.

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H2O2-scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H2O2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

In Silico RNAseq and Biochemical Analyses of Glucose-6-Phosphate Dehydrogenase (G6PDH) from Sweet Pepper Fruits: Involvement of Nitric Oxide (NO) in Ripening and Modulation.

Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide which has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergo during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) is a recognized signal molecule that can exert regulatory functions in diverse plant processes including fruit ripening, but the relevance of NADPH as a fingerprinting of the crop physiology including ripening has also been proposed. Glucose-6-phosphate dehydrogenase (G6PDH) is the first and rate-limiting enzyme of the oxidative phase of the pentose phosphate pathway (oxiPPP) with the capacity to generate NADPH. Thus far, the available information on G6PDH and other NADPH-generating enzymatic systems in pepper plants, and their expression during the ripening of sweet pepper fruit, is very scarce. Therefore, an analysis at the transcriptomic, molecular and functional levels of the G6PDH system has been accomplished in this work for the first time. Based on a data-mining approach to the pepper genome and fruit transcriptome (RNA-seq), four G6PDH genes were identified in pepper plants and designated CaG6PDH1 to CaG6PDH4, with all of them also being expressed in fruits. While CaG6PDH1 encodes a cytosolic isozyme, the other genes code for plastid isozymes. The time-course expression analysis of these CaG6PDH genes during different fruit ripening stages, including green immature (G), breaking point (BP), and red ripe (R), showed that they were differentially modulated. Thus, while CaG6PDH2 and CaG6PDH4 were upregulated at ripening, CaG6PDH1 was downregulated, and CaG6PDH3 was slightly affected. Exogenous treatment of fruits with NO gas triggered the downregulation of CaG6PDH2, whereas the other genes were positively regulated. In-gel analysis using non-denaturing PAGE of a 50-75% ammonium-sulfate-enriched protein fraction from pepper fruits allowed for identifying two isozymes designated CaG6PDH I and CaG6PDH II, according to their electrophoretic mobility. In order to test the potential modulation of such pepper G6PDH isozymes, in vitro analyses of green pepper fruit samples in the presence of different compounds including NO donors (S-nitrosoglutathione and nitrosocysteine), peroxynitrite (ONOO-), a hydrogen sulfide (H2S) donor (NaHS, sodium hydrosulfide), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) were assayed. While peroxynitrite and the reducing compounds provoked a partial inhibition of one or both isoenzymes, NaHS exerted 100% inhibition of the two CaG6PDHs. Taken together these data provide the first data on the modulation of CaG6PDHs at gene and activity levels which occur in pepper fruit during ripening and after NO post-harvest treatment. As a consequence, this phenomenon may influence the NADPH availability for the redox homeostasis of the fruit and balance its active nitro-oxidative metabolism throughout the ripening process.

Pepper Fruit Extracts Show Anti-Proliferative Activity against Tumor Cells Altering Their NADPH-Generating Dehydrogenase and Catalase Profiles.

Cancer is considered one of the main causes of human death worldwide, being characterized by an alteration of the oxidative metabolism. Many natural compounds from plant origin with anti-tumor attributes have been described. Among them, capsaicin, which is the molecule responsible for the pungency in hot pepper fruits, has been reported to show antioxidant, anti-inflammatory, and analgesic activities, as well as anti-proliferative properties against cancer. Thus, in this work, the potential anti-proliferative activity of pepper (Capsicum annuum L.) fruits from diverse varieties with different capsaicin contents (California < Piquillo < Padrón < Alegría riojana) against several tumor cell lines (lung, melanoma, hepatoma, colon, breast, pancreas, and prostate) has been investigated. The results showed that the capsaicin content in pepper fruits did not correspond with their anti-proliferative activity against tumor cell lines. By contrast, the greatest activity was promoted by the pepper tissues which contained the lowest capsaicin amount. This indicates that other compounds different from capsaicin have this anti-tumor potentiality in pepper fruits. Based on this, green fruits from the Alegría riojana variety, which has negligible capsaicin levels, was used to study the effect on the oxidative and redox metabolism of tumor cell lines from liver (Hep-G2) and pancreas (MIA PaCa-2). Different parameters from both lines treated with crude pepper fruit extracts were determined including protein nitration and protein S-nitrosation (two post-translational modifications (PTMs) promoted by nitric oxide), the antioxidant capacity, as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), among others. In addition, the activity of the NADPH-generating enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and NADP-isocitrate dehydrogenase (NADP-ICDH) was followed. Our data revealed that the treatment of both cell lines with pepper fruit extracts altered their antioxidant capacity, enhanced their catalase activity, and considerably reduced the activity of the NADPH-generating enzymes. As a consequence, less H2O2 and NADPH seem to be available to cells, thus avoiding cell proliferation and possibly triggering cell death in both cell lines.

NADP-Dependent Malic Enzyme Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO).

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50-75% ammonium-sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

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

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.

Soybean (Glycine max L.) Lipoxygenase 1 (LOX 1) Is Modulated by Nitric Oxide and Hydrogen Sulfide: An In Vitro Approach.

Hydrogen sulfide (H2S) and nitric oxide (NO) are two relevant signal molecules that can affect protein function throughout post-translational modifications (PTMs) such as persulfidation, S-nitrosation, metal-nitrosylation, and nitration. Lipoxygenases (LOXs) are a group of non-heme iron enzymes involved in a wide range of plant physiological functions including seed germination, plant growth and development, and fruit ripening and senescence. Likewise, LOXs are also involved in the mechanisms of response to diverse environmental stresses. Using purified soybean (Glycine max L.) lipoxygenase type 1 (LOX 1) and nitrosocysteine (CysNO) and sodium hydrosulfide (NaHS) as NO and H2S donors, respectively, the present study reveals that both compounds negatively affect LOX activity, suggesting that S-nitrosation and persulfidation are involved. Mass spectrometric analysis of nitrated soybean LOX 1 using a peroxynitrite (ONOO-) donor enabled us to identify that, among the thirty-five tyrosine residues present in this enzyme, only Y214 was exclusively nitrated by ONOO-. The nitration of Y214 seems to affect its interaction with W500, a residue involved in the substrate binding site. The analysis of the structure 3PZW demonstrates the existence of several tunnels that directly communicate the surface of the protein with different internal cysteines, thus making feasible their potential persulfidation, especially C429 and C127. On the other hand, the CysNO molecule, which is hydrophilic and bulkier than H2S, can somehow be accommodated throughout the tunnel until it reaches C127, thus facilitating its nitrosation. Overall, a large number of potential persulfidation targets and the ease by which H2S can reach them through the diffuse tunneling network could be behind their efficient inhibition.

Advances in Nitric Oxide Signalling and Metabolism in Plants.

More than 15,000 scientific articles published since the late 1950s related to RNS action or detection in various plant materials are listed in the Web of Science database [...].

H2S-Generating Cytosolic L-Cysteine Desulfhydrase and Mitochondrial D-Cysteine Desulfhydrase from Sweet Pepper (Capsicum annuum L.) Are Regulated During Fruit Ripening and by Nitric Oxide.

Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5'-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO-), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO-, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species.

Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes Through Fluorescent Protein Containing a Type 1 Peroxisomal Targeting Signal (PTS1).

Plant peroxisomes have an active nitro-oxidative metabolism. However, the assay of reactive oxygen and nitrogen species (ROS/RNS) could be a challenge since the purification of peroxisomes is technically a high time-consuming approach that needs to be optimized for each tissue/organ (root, leaf, fruit) and plant species. Arabidopsis thaliana, as a model plant for biochemical and molecular studies, has become a useful tool to study the basic metabolism, including also that of ROS/RNS. The combination of specific fluorescent probes with Arabidopsis plants expressing a fluorescent protein containing a type 1 peroxisomal targeting signal (PTS1) is a powerful tool to address the profile of ROS/RNS in peroxisomes by confocal laser scanning microscope (CLSM). This chapter provides a detailed description to detect the content and distribution of ROS and RNS in Arabidopsis peroxisomes, together with a critical analysis of their potentialities and limitations, since these approaches require appropriate controls to corroborate the obtained data.

Functions of NO and H2S Signal Molecules Against Plant Abiotic Stress.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two recognized signal molecules in higher plants involved in a wide range of physiological processes and the mechanisms of response against adverse environmental conditions. These molecules can interact to provide an adequate response to palliate the negative impact exerted by stressful conditions, particularly by regulating key components of the metabolism of reactive oxygen species (ROS) to avoid their overproduction and further oxidative damage which, finally, affects cellular functioning. NO and H2S can exert the regulation over the function of susceptible proteins by posttranslational modifications (PTMs) including nitration, S-nitrosation, and persulfidation but also through the regulation of gene expression by the induction of specific transcription factors which modulate the expression of genes encoding proteins related to stress resistance. This chapter encompasses a wide perspective of the signaling and functional relationships between NO and H2S to modulate the overproduction of reactive oxygen species, particularly under abiotic stress conditions.

Analysis of Plant L-Cysteine Desulfhydrase (LCD) Isozymes by Non-denaturing Polyacrylamide Gel Electrophoresis.

Hydrogen sulfide (H2S) is a signaling molecule that achieves different regulatory functions in animal and plant cells. The cytosolic enzyme L-cysteine desulfhydrase (LCD; EC 4.4.1.28) catalyzes the conversion of cysteine (L-Cys) to pyruvate and ammonium with the concomitant generation of H2S, this enzyme being considered one of the main sources of H2S in higher plants. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a specific assay for LCD activity, the present protocol allows identifying diverse LCD isozymes present in different organs (roots, shoots, leaves, and fruits) and plant species including pea, garlic, Arabidopsis, and pepper.

Fruit Physiology through Signaling Processes: Latest Advances and Future Challenges.

Fruits are unique to flowering plants and confer a selective advantage to these species by facilitating seed maturation and dispersal [...].

Small Heat Shock Protein (sHSP) Gene Family from Sweet Pepper (Capsicum annuum L.) Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO).

Small heat shock proteins (sHSPs) are usually upregulated in plants under diverse environmental stresses. These proteins have been suggested to function as molecular chaperones to safeguard other proteins from stress-induced damage. The ripening of pepper (Capsicum annuum L.) fruit involves important phenotypic, physiological, and biochemical changes, which have associated endogenous physiological nitro-oxidative stress, but they can also be significantly affected by environmental conditions, such as temperature. Based on the available pepper genome, a total of 41 sHSP genes were identified in this work, and their distributions in the 12 pepper chromosomes were determined. Among these genes, only 19 sHSP genes were found in the transcriptome (RNA-Seq) of sweet pepper fruits reported previously. This study aims to analyze how these 19 sHSP genes present in the transcriptome of sweet pepper fruits are modulated during ripening and after treatment of fruits with nitric oxide (NO) gas. The time-course expression analysis of these genes during fruit ripening showed that 6 genes were upregulated; another 7 genes were downregulated, whereas 6 genes were not significantly affected. Furthermore, NO treatment triggered the upregulation of 7 sHSP genes and the downregulation of 3 sHSP genes, whereas 9 genes were unchanged. These data indicate the diversification of sHSP genes in pepper plants and, considering that sHSPs are important in stress tolerance, the observed changes in sHSP expression support that pepper fruit ripening has an associated process of physiological nitro-oxidative stress, such as it was previously proposed.

Lipoxygenase (LOX) in Sweet and Hot Pepper (Capsicum annuum L.) Fruits during Ripening and under an Enriched Nitric Oxide (NO) Gas Atmosphere.

Lipoxygenases (LOXs) catalyze the insertion of molecular oxygen into polyunsaturated fatty acids (PUFA) such as linoleic and linolenic acids, being the first step in the biosynthesis of a large group of biologically active fatty acid (FA)-derived metabolites collectively named oxylipins. LOXs are involved in multiple functions such as the biosynthesis of jasmonic acid (JA) and volatile molecules related to the aroma and flavor production of plant tissues, among others. Using sweet pepper (Capsicum annuum L.) plants as a model, LOX activity was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE) and specific in-gel activity staining. Thus, we identified a total of seven LOX isozymes (I to VII) distributed among the main plant organs (roots, stems, leaves, and fruits). Furthermore, we studied the FA profile and the LOX isozyme pattern in pepper fruits including a sweet variety (Melchor) and three autochthonous Spanish varieties that have different pungency levels (Piquillo, Padrón, and Alegría riojana). It was observed that the number of LOX isozymes increased as the capsaicin content increased in the fruits. On the other hand, a total of eight CaLOX genes were identified in sweet pepper fruits, and their expression was differentially regulated during ripening and by the treatment with nitric oxide (NO) gas. Finally, a deeper analysis of the LOX IV isoenzyme activity in the presence of nitrosocysteine (CysNO, a NO donor) suggests a regulatory mechanism via S-nitrosation. In summary, our data indicate that the different LOX isozymes are differentially regulated by the capsaicin content, fruit ripening, and NO.