**6. AsA in plants: coping with oxygen and beyond**

Excess oxygen conditions (hyperoxia) increase the risk of ROS overproduction. Hyperoxic conditions typically occur in the lungs of animals [68], but also in plant green tissues, where oxygen is produced at high rates in the photosynthetic process [69]. Photosynthetic organisms have developed, in addition to SOD, CAT and other antioxidant enzymes, also a family of AsA-specific peroxidases present in multiple isoforms in most cell compartments, possibly by duplication of the original chloroplastic forms [70]. Interestingly, accurate studies have ascertained that AsA peroxidases are not simply scavenging enzymes, but their catalytic activity is calibrated so that hydrogen peroxide can still act as a molecular signal [71, 72].

Plants produce AsA using a complex biosynthetic (Smirnoff-Wheeler) pathway that evolved in photosynthetic organisms [73]. In difference to AsA synthesizing animals, the last step in the pathway is catalyzed by a mitochondrial enzyme (l-galactono-1,4-lactone dehydrogenase). Notably, all plants must synthesize their own AsA in order to survive, because they cannot take it from other organisms.

### **Figure 3.**

*The central role of ascorbate (AsA) in plant cell signaling. The last step in AsA biosynthesis is catalyzed by the mitochondrial enzyme l-Galactono-1,4-lactone dehydrogenase (l-GalLDH). Reactive Oxygen Species (ROS) are formed in mitochondria, chloroplasts and as a consequence of different stress conditions. ROS (red dots) can be reduced by AsA in uncatalyzed reactions, and hydrogen peroxide can be removed in the reaction catalyzed by AsA peroxidases (AsA px) located in different cell compartments and organelles. AsA is also specifically used by 2-oxoglutarate-dependent dioxygenases (2-ODDs), including enzymes involved in the synthesis and/or catabolism of plant hormones (ABA: abscisic acid; GA: gibberellin; strigo: stigolactones). AsA oxidation produces dehydroascorbic acid (DHA, in the most stable dimeric form). In the nucleus, AsA cooperates in the regulation of the activity of Ten-Eleven-Translocation (TET) methylcytosine dioxygenases, a class of enzymes involved in the epigenetic control of gene expression.*

The only case described of a plant unable to synthesize AsA is the *Arabidopsis vtc2*/*vtc5* double mutant, which is not viable if not supplemented with AsA [74]. Plant mutants with relatively low AsA content (about 30–50% of the wild type) generally show increased sensitivity to environmental stresses [75, 76]. The expression of pathogenesis-related proteins and, in general, of defense-related genes is also induced by AsA deficiency [77, 78].

In plants, 2-ODDs are the second most represented class of enzymes [19, 79]. A large number of them are involved in the synthesis and/or the catabolism of plant hormones and growth regulators, including auxins [80], gibberellins [81], ethylene [82], abscisic acid [83], strigolactones [84]. Cysteine oxidases in plants perform an oxygen-sensing mechanism similar to the animal HIF-mediated pathway [85–87]. A novel dioxygenase using directly AsA (and not 2-oxoglutarate) for an unusual modification of cytosine has been recently observed in the green algae *Chlamydomonas reinhardtii* [88]. All this information from plants confirms that AsA functions not only as an antioxidant, but also as a regulator of many signaling routes through the action of different dioxygenases. As compared to animals, plants developed even more specialized antioxidants (e.g. AsA peroxidases), but their action is in equilibrium with ROS production, which is required for the activation of crucial signaling pathways [89].

A schematic representation of AsA centrality in different aspects of plant cell signaling and regulation is given in **Figure 3**.
