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

Abstract

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.

Keywords: S-nitrosation; Fruit ripening; hydrogen peroxide; melatonin; nitration; nitric oxide; pepper fruit; peroxynitrite; persulfidation; post-translational modifications.

Fig. 1.

Some representative families of bioactive compounds with antioxidant capacity present in climacteric and non-climacteric fleshy fruits.

Fig. 2.

Enzymes directly involved in ascorbate metabolism in plants. The mitochondrial enzyme l-galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) catalyzes the conversion of l-galactono-1,4 lactone to l-ascorbate, which can be oxidized either by ascorbate peroxidase (APX; EC 1.11.1.11) to decompose H₂O₂, or by ascorbate oxidase (AO; EC 1.10.3.3) using O₂. In both cases, l-ascorbate is converted into monodehydroascorbate (MDA) which is then converted into dehydroascorbate (DHA) in a non-enzymatic manner. l-Ascorbate is recycled from MDA and DHA by monodehydroascorbate reductase (MDAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1), respectively, two enzymes of the glutathione–ascorbate pathway.

Fig. 3.

Ascorbate peroxidase (APX) activity in pepper fruits. (A) Total APX activity and (B) APX isozymes separated by non-denaturing PAGE in sweet pepper fruits at different stages of ripening correspond to immature green (G), breaking point (BP1), and red ripe (R). Reproduced from González-Gordo et al. (2019, 2022b).

Fig. 4.

Post-translational modifications (PTMs) that affect ascorbate peroxidase (APX). (A) Identification of the PTMs mediated by NO (nitration and S-nitrosation), H₂S (persulfidation), and GSH (S-glutathionylation) that positively or negatively affect the activity of APX (see Table 2 for details). (B) Model of the plastidial/mitochondrial APX3 isozyme from pepper fruits where the residue Cys32, susceptible to undergo thiol-based oxidative post-translational modifications (oxiPTMs), and Tyr235 which undergoes tyrosine nitration thus triggering its inhibition, are highlighted. Another characteristic is the loop (in orange color) formed by residues 188–203 which is located in the vicinity of the propionate side chain of the heme group.

Fig. 5.

Simple model explaining the effect of the exogenous application of NO, H₂S, and melatonin on ascorbate peroxidase (APX) and H₂O₂ in fruits. NO and H₂S can mediate a direct action on APX activity through post-translational modifications (PTMs) such as tyrosine nitration, S-nitration, or persulfidation, modulating the H₂O₂ content or affecting expression of the APX gene. Melatonin can also interact with H₂O₂ to generate several hydroxymelatonin derivatives. TFs, transcription factors.