Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light-Induced Ascorbate Accumulation.

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called ∆dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆dhar Arabidopsis plants as well as a quadruple mutant line combining ∆dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ∼30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with ∆dhar, there was near-complete inhibition of high-light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in ∆dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis.

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.

Controlling the Nucleation and Growth of Salt from Bodily Fluid for Enhanced Biosensing Applications.

Surface-enhanced Raman spectroscopy (SERS) represents a transformative tool in medical diagnostics, particularly for the early detection of key biomarkers such as small extracellular vesicles (sEVs). Its unparalleled sensitivity and compatibility with intricate biological samples make it an ideal candidate for revolutionizing noninvasive diagnostic methods. However, a significant challenge that mars its efficacy is the throughput limitation, primarily anchored in the prerequisite of hotspot and sEV colocalization within a minuscule range. This paper delves deep into this issue, introducing a never-attempted-before approach which harnesses the principles of crystallization-nucleation and growth. By synergistically coupling lasers with plasmonic resonances, we navigate the challenges associated with the analyte droplet drying method and the notorious coffee ring effect. Our method, rooted in a profound understanding of crystallization's materials science, exhibits the potential to significantly increase the areal density of accessible plasmonic hotspots and efficiently guide exosomes to defined regions. In doing so, we not only overcome the throughput challenge but also promise a paradigm shift in the arena of minimally invasive biosensing, ushering in advanced diagnostic capabilities for life-threatening diseases.

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.