**2. Ascorbate Biosynthesis and Subcellular Distribution**

Ascorbate is present in Arabidopsis leaves as one of the most abundant primary metabolites [18]. In plants, ascorbate is generally synthesized through one dominant pathway, so-called the D-mannose/L-galactose (Smirnoff–Wheeler) pathway [18]. Three other pathways, so-called the *Myo*-inositol [34], L-gulose [35], and L-galacturonate [36], have been also suggested as alternative routes for ascorbate biosynthesis [37]. However, strong evidence on the existence of all these alternative pathways has not yet been reported. Moreover, it appears that even if the alternative pathways exist, their roles in ascorbate biosynthesis should be minor at least in Arabidopsis, where ascorbate loss in the *vtc2vtc5* double mutants appears not to be compensated by the other pathways [38].

Therefore, here, we only demonstrate the details of the major pathway (Figure 1). In this pathway, D-glucose-6-phosphate is converted to ascorbate in nine enzymatic reactions, as depicted in Figure 1, with the last step catalized by L-galactono-1,4-lactone dehydrogenase (GLDH), located in the inner membrane of the mitochondria in which L-galactono-1, 4-lactone, the direct precursor of ascorbate, is converted to ascorbate.

**Figure 1.** D-Mannose/L-Galactose pathway of ascorbate biosynthesis in plants. The genes of the pathway are highlighted in purple and written in italics. The enzymes are highlighted in green. Phosphoglucose isomerase (PGI), phosphomannose isomerase (PMI), and phosphomannomutase (PMM) are responsible for the conversion of D-glucose-6-P to D-mannose-1-P, the direct precursor of GDP-D-mannose pyrophosphorylase (GMP), the first committed enzyme of the pathway encoded by *VTC1*. GDP-mannose-3 -5 -epimerase (GME), GDP-L-galactose transferase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GLDH) are the next enzymes of the pathway. GGP is the key enzyme of the pathway encoded by *VTC2* and *VTC5* paralogs. This enzyme undergoes feedback regulation by ascorbate pool size. GLDH is located in the intermembrane of mitochondria and is connected to the mitochondria respiratory chain.

Ascorbate specific immunogold labelling and quantitative transmission electron microscopy showed that ascorbate was found in most cellular organelles, including cytosol, nuclei, peroxisomes, vacuoles, mitochondria, and chloroplasts, but not in cell walls and intercellular spaces. Moreover, it has been shown that, despite showing a strong increase in chloroplasts (104%) under high light conditions (700 μmol m-2s−1), vacuoles even demonstrated a stronger ascorbate specific labeling (395%) than chloroplasts. This highlights the relevance of vacuoles in ascorbate metabolism in response to high light acclimation, which deserves further investigations [23].

Given that ascorbate distributes across all the cellular compartments, despite exclusive production in mitochondria [39], the involvement of ascorbate transporters is necessary for its function. The identification of ascorbate transporters has long been considered as a difficult task [40] but eventually, a phosphate transporter 4 family protein (*At*PHT4;4) was identified as an ascorbate transporter located at the chloroplast envelope membrane [41]. However, transporters localized to other membranes remain unknown and information concerning subcellular ascorbate concentration is rare and normally confined to single environmental conditions.

#### **3. Role of Ascorbate in Light Acclimation**

Under the light acclimation process, the chloroplast undergoes coordinated metabolic adjustments with extra-chloroplastic metabolism in order to maintain the overall fitness of plants and avoid damage [42]. Several metabolites produced in the plastids and motochondria are subsequently transmitted to the nucleus and modulate nuclear gene expression. This phenomenon is termed as retrograde signaling and known as a critical component of plant acclimation responses. Several light-shift experiments have been conducted to unravel the early and late metabolic responses to different light intensities. Changes in light intensity rapidly manipulate the electron pressure generated in the photosynthetic electron transport chain (pETC); therefore, the ROS signals generated from pETC are considered as important retrograde signals for short- and long-term acclimation [42]. Ascorbate and glutathione are known as redox signals, playing roles on longer time scales [42]. It has been revealed that total ascorbate levels increased after an hour in plants exposed to high light (800 μmol photons m−<sup>2</sup> s−1). This increase is even delayed by 3 h in plants transferred to high light following acclimation to low light intensity (8 μmol photons m−<sup>2</sup> s<sup>−</sup>1) [43].

One important function of ascorbate is as a cofactor in the xanthophyll cycle, in which the excess excitation energy is dissipated as heat from excited chlorophylls to xanthophyll carotenoids, a photoprotection mechanism termed as non-photochemical fluorescence quenching (NPQ) [44]. In this cycle, the violaxanthin de-epoxidase (VDE) enzyme, localized in the thylakoid lumen, uses ascorbate as a cofactor to reduce the epoxide group of the substrate violaxanthin and converts it to antheraxanthin and zeaxanthin [45]. In a light-shift experiment (from 160 μmol photons m−<sup>2</sup> s−<sup>1</sup> to 1800 μmol photons m−<sup>2</sup> s−1), roles of ascorbate in light acclimation were investigated using the Arabidopsis mutant deficient in VDE enzyme (*npq1)*, ascorbate deficient mutant (*vtc2)*, along with *vtc2npq1* double knockouts [46]. It has been revealed that the *vtc2* mutants, having 10%–30% of the wild type (WT) ascorbate levels, lost their acclimation capacity after long-term exposure to high light (up to five days at 1800 μmol photons m−<sup>2</sup> s<sup>−</sup>1). In contrast to the *npq1* single mutants, deficient in zeaxanthin, which were slightly more sensitive to high light than the WTs, *vtc2* and *vtc2npq1* double mutants showed an increased degree of bleached leaves, lipid peroxidation, and photoinhibition (increased degree of damage to (Photosystem II) PSII, measured by Fv/Fm). These data confirmed the importance of ascorbate in light acclimation responses and also showed that ascorbate has even more important roles than other photoprotective metabolites such as xanthophylls in acclimation to high light stress. Further, loss of PSII efficiency was not observed after short-term high light exposure (up to 2 h) in *vtc2* mutants, however, the conversion rate of violaxanthin to zeaxanthin was reduced owing to the dependency of VDE to ascorbate [47]. These data further corroborated the importance of ascorbate on long-term acclimation to high light rather than short-term.

*Plants* **2020**, *9*, 101

In a subsequent study in which they investigated the thylakoid-associated proteome of Arabidopsis WT and *vtc2* after transition to high light (1000 μmol photons m−<sup>2</sup> s<sup>−</sup>1), differential protein accumulation could be observed in a number of stress-associated proteins between WT and *vtc2* including Fe-superoxide dismutase (Fe-SOD), Cu, Zn-SOD, HSP70s (cpHSP70-1 and 2), PsbS protein, and a chloroplast-localized glyoxalate I [48]. SODs are metalloenzymes, which have been long known as stable markers for abiotic stress tolerance against ROS [49]. Also, it has been shown that HSP70-2 in *Chlamydomonas reinhardtii* chloroplasts has photoprotective roles for PSII reaction centers during photoinhibition and PSII repair [48,50]. Apart from the xanthophyll zeaxanthin, PsbS is known as another component of NPQ [51]. PsbS-dependent quenching site has been recently deciphered to be in Light-harvesting complex II (LHCII), and in the PSII core, most likely in the core antenna complexes CP43 and/or CP47 [52]. In the study of Giacomelli and coworkers, PsbS protein was up-regulated more than twofold upon transition to high light, however, it remained unchanged in the *vtc2*, which is in line with the observation that *vtc2* mutants have reduced levels of non-photochemical quenching [47]. This study shows that ascorbate has a significant impact on chloroplast proteome linking to oxidative stress and quenching, however, it cannot be entirely ruled out that these changes are the consequences of a direct or indirect effect of ascorbate deficiencies in the *vtc2* mutants. Moreover, the ascorbate deficient mutants, *vtc1*, *vtc2*, and *vtc3*, were found to accumulate visibly and quantitatively less anthocyanin compared with the wild types during the high light treatment in several studies [48,53,54]. *vtc1* and *vtc2* mutants were also unable to induce the expression of anthocyanin biosynthesis enzymes, and the corresponding transcription factors of the pathway, PAP1, GL3, and EGL3 under high light acclimation [54], whereas the transcripts related to anthocyanin biosynthesis and regulation are known to be up-regulated rapidly by high light in Arabidopsis WT plants [54,55]. Further, given the fact that both ascorbate and anthocyanin have been shown to accumulate in a similar time-scale (days) and in similar ranges of light intensities after high light exposure, and that the *vtc* mutants had defects in the accumulation of anthocyanin, the existing interconnection between them has been proposed in the study of Page and coworkers [54]. The authors observed a tight correlation of ascorbate and anthocyanin levels across six different Arabidopsis ecotypes under normal and high light conditions, which adds further proof to the relationship between them [54]. More investigation should be done to explore the co-regulatory mechanism of ascorbate levels with anthocyanin under high light acclimation.
