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.

H2S in Horticultural Plants: Endogenous Detection by an Electrochemical Sensor, Emission by a Gas Detector, and Its Correlation with L-Cysteine Desulfhydrase (LCD) Activity.

H2S has acquired great attention in plant research because it has signaling functions under physiological and stress conditions. However, the direct detection of endogenous H2S and its potential emission is still a challenge in higher plants. In order to achieve a comparative analysis of the content of H2S among different plants with agronomical and nutritional interest including pepper fruits, broccoli, ginger, and different members of the genus Allium such as garlic, leek, Welsh and purple onion, the endogenous H2S and its emission was determined using an ion-selective microelectrode and a specific gas detector, respectively. The data show that endogenous H2S content range from pmol to µmol H2S · g-1 fresh weight whereas the H2S emission of fresh-cut vegetables was only detected in the different species of the genus Allium with a maximum of 9 ppm in garlic cloves. Additionally, the activity and isozymes of the L-cysteine desulfhydrase (LCD) were analyzed, which is one of the main enzymatic sources of H2S, where the different species of the genus Allium showed the highest activities. Using non-denaturing gel electrophoresis, the data indicated the presence of up to nine different LCD isozymes from one in ginger to four in onion, leek, and broccoli. In summary, the data indicate a correlation between higher LCD activity with the endogenous H2S content and its emission in the analyzed horticultural species. Furthermore, the high content of endogenous H2S in the Allium species supports the recognized benefits for human health, which are associated with its consumption.

Identification of Compounds with Potential Therapeutic Uses from Sweet Pepper (Capsicum annuum L.) Fruits and Their Modulation by Nitric Oxide (NO).

Plant species are precursors of a wide variety of secondary metabolites that, besides being useful for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities have been reported in the literature. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop. To achieve this, we applied a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analyzed metabolites depended on the ripening stage and on the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research perspectives on the pepper fruit's bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.