Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants.

Plants accumulate high concentrations of ascorbate, commonly in their leaves, as a redox buffer. While ascorbate levels have increased during plant evolution, the mechanisms behind this phenomenon are unclear. Moreover, has the increase in ascorbate concentration been achieved without imposing any detrimental effects on the plants? In this review, we focus on potential transitions in two regulatory mechanisms related to ascorbate biosynthesis and the availability of cellular dehydroascorbate (DHA) during plant evolution. The first transition might be that the trigger for the transcriptional induction of VTC2, which encodes the rate-limiting enzyme in ascorbate biosynthesis, has shifted from oxidative stress (in green algae) to light/photosynthesis (in land plants), probably enabling the continuous accumulation of ascorbate under illumination. This could serve as a preventive system against the unpredictable occurrence of oxidative stress. The second transition might be that DHA-degrading enzymes, which protect cells from the highly reactive DHA in green algae and mosses, have been lost in ferns or flowering plants. Instead, flowering plants may have increased glutathione concentrations to reinforce the DHA reduction capacity, possibly allowing ascorbate accumulation and avoiding the toxicity of DHA. These potential transitions may have contributed to strategies for plants' safe and effective accumulation of ascorbate.

The demand for ascorbate recycling capacity rises as the ascorbate pool size increases in Arabidopsis plants.

Ascorbate recycling is required for high ascorbate accumulation. Hence, when the ascorbate pool size is small, does the demand for ascorbate recycling decrease? We herein investigate the impact of ascorbate recycling capacity on ascorbate pool size in an ascorbate-deficient background. Our findings demonstrate that a smaller ascorbate pool size lowers the need for ascorbate recycling capacity even under light stress.

Chloroplast dehydroascorbate reductase and glutathione cooperatively determine the capacity for ascorbate accumulation under photooxidative stress conditions.

Ascorbate is an indispensable redox buffer essential for plant growth and stress acclimation. Its oxidized form, dehydroascorbate (DHA), undergoes rapid degradation unless it is recycled back into ascorbate by glutathione (GSH)-dependent enzymatic or non-enzymatic reactions, with the enzymatic reactions catalyzed by dehydroascorbate reductases (DHARs). Our recent study utilizing an Arabidopsis quadruple mutant (∆dhar pad2), which lacks all three DHARs (∆dhar) and is deficient in GSH (pad2), has posited that these GSH-dependent reactions operate in a complementary manner, enabling a high accumulation of ascorbate under high-light stress. However, as Arabidopsis DHAR functions in the cytosol or chloroplasts, it remained unclear which isoform played a more significant role in cooperation with GSH-dependent non-enzymatic reactions. To further comprehend the intricate network of ascorbate recycling systems in plants, we generated mutant lines lacking cytosolic DHAR1/2 or chloroplastic DHAR3, or both, in another GSH-deficient background (cad2). A comprehensive comparison of ascorbate profiles in these mutants under conditions of photooxidative stress induced by various light intensities or methyl viologen unequivocally demonstrated that chloroplastic DHAR3, but not cytosolic isoforms, works in concert with GSH to accumulate ascorbate. Our findings further illustrate that imbalances between stress intensity and recycling capacity significantly impact ascorbate pool size and tolerance to photooxidative stress. Additionally, it was found that the absence of DHARs and GSH deficiency do not impede ascorbate biosynthesis, at least in terms of transcription or activity of biosynthetic enzymes. This study provides insights into the robustness of ascorbate recycling.

Distribution and Functions of Monodehydroascorbate Reductases in Plants: Comprehensive Reverse Genetic Analysis of Arabidopsis thaliana Enzymes.

Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes. Thus, the number and distribution of MDAR isoforms differ according to plant species. Moreover, the physiological significance of MDARs remains poorly understood. In this study, we classify plant MDARs into three classes: class I, chloroplastic/mitochondrial enzymes; class II, peroxisomal membrane-attached enzymes; and class III, cytosolic/peroxisomal enzymes. The cytosol-specific isoforms form a subclass of class III and are conserved only in Brassicaceae plants. With some exceptions, all land plants and a charophyte algae, Klebsormidium flaccidum, contain all three classes. Using reverse genetic analysis of Arabidopsis thaliana mutants lacking one or more isoforms, we provide new insight into the roles of MDARs; for example, (1) the lack of two isoforms in a specific combination results in lethality, and (2) the role of MDARs in ascorbate redox regulation in leaves can be largely compensated by other systems. Based on these findings, we discuss the distribution and function of MDAR isoforms in land plants and their cooperation with other recycling systems.