**5. Role of Sugars, Photosynthesis, and Respiration in Light Regulation of Ascorbate**

Given that carbohydrates are substrates for ascorbate synthesis, attempts have been made to decipher the putative links between these metabolites in light acclimation responses. Schmitz et al. [21] examined the roles of sugar and starch metabolism in the acclimation process to high light by using Arabidopsis mutants deficient in either the triose phosphate/phosphate translocator (*tpp*) or ADP-glucose pyrophosphorylase (*AGPase*) or both of them (*adg1-1*/*tpt-2*). While soluble sugars, mainly glucose, accumulated in both wild type and mutant plants within four hours of high light exposure, the acclimation response was impaired in the mutants after only two days. The comparison of transcriptomic results with publicly available ones revealed a correlation between responses to high light and those to sugar levels following four hours of high light treatment, while the responses at 48 h were also similar to those of ROS accumulation. These results suggest that soluble sugars act as modulators in the short term, but this role is replaced by ROS in the long term. Ascorbate levels increased in all lines over time upon exposure to high light. Interestingly, the redox state of ascorbate was not affected in the triose phosphate/phosphate translocator (TPT) mutant, but reduced in all other lines, suggesting the effect of sugar localization on the redox state of ascorbate [21]. These results emphasize the signaling functions of soluble sugars in high light acclimation and, further, the involvement of ascorbate redox state in signaling pathways.

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

Although it has been identified that sugars affect the redox state of ascorbate in light acclimation, as described above, regulation of ascorbate accumulation under light appears to be independent of sugars, but dependent on the photosynthetic electron transport chain [59]. Leaf ascorbate levels and transcript levels of the ascorbate biosynthetic genes, *GMP*, *GPP*, *GDH*, and *VTC2*, were decreased, and plants were unable to accumulate ascorbate upon inhibition of photosynthetic electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and atrazine (ATZ) treatment even under continuous light [59]. In the same study, the effect of sugars on foliar ascorbate levels was examined by transferring two-week-old Arabidopsis seedlings to the media in the presence or absence of sucrose, with subsequent transferal to the dark for 48 h to reduce the internal carbon sources and total ascorbate levels. In effect, darkness led to a significant decline in leaf sugar levels by 90% in both the presence and absence of sucrose, accompanied by a reduction in ascorbate levels. External supplementation of sucrose did not restore the leaf ascorbate pool sizes. In an opposite way, the levels of sugar increased in both the presence and absence of sucrose after transferring them from the dark to the light, and again, the levels of ascorbate could not be restored to normal levels in the sucrose supplemented plants [59]. This observation is in line with the research where the absence of a correlation between carbohydrates and ascorbate levels could be observed in the ripening period of tomato fruits under irradiances that stimulate ascorbate biosynthesis [84]. Despite a remarkable increase in ascorbate levels upon ripening under light, carbohydrate levels remained unchanged. Similarly, alteration in carbohydrate levels upon flower pruning did not show effects on ascorbate levels [84]. Furthermore, these authors confirmed in separate studies that high light has positive effects on ascorbate upregulation only in green tomato fruits, determining photosynthesis as an integral part of this mechanism [84]. Therefore, it appears that photosynthesis is a key component in controlling the leaf ascorbate pool size under light, however, carbon supply through photosynthesis appeared not to be the determinant of the ascorbate levels in Arabidopsis and tomato. Further studies are required to explore the signaling mechanisms in this process. Yet, it has been proposed that the effects of sugars on ascorbate is a genotype-specific phenomenon, which varies in different plant species [73,85]. In contrast to what was observed in the above-mentioned studies, it has been revealed that sucrose feeding in tomato fruits increased the expression of key ascorbate biosynthetic genes such as *VTC1*, *VTC2*, *GDH*, and *GLDH*, as well as of recycling and turnover genes *APX*, *MDHAR*, *DHAR*, and *GR*, pointing to yet unknown signaling components in modulating the ascorbate biosynthetic and recycling gene expression patterns mediated by sugars [86]. That being said, sucrose and glucose feeding had no effects on ascorbate levels in barley and pea embryonic axes [73]. As sucrose feeding in these plant species has not been examined in light shift experiments, it is, however, difficult to draw a solid conclusion on the role of sugars on light regulation of ascorbate.

Given that GLDH, the last enzyme of the ascorbate pathway, lies in the inner membrane of the motochondria [87], being designated as part of the complex I of the respiratory electron transport chain [88], the relationship between ascorbate biosynthesis and respiration is rendered inevitable [73]. Bartoli et al. [87] observed that isolated mitochondria from potato leaves were able to synthesize ascorbate from L-GaL, and subsequently, L-GaL stimulated mitochondrial electron transport rates. Besides, it has been demonstrated that cytochrome C (cytC), located between complexes III and IV, is the electron acceptor of GLDH and treatment of intact mitochondria with potassium cyanide (KCN, an inhibitor of respiration) blocked ascorbate production [69,87]. Following that, Bartoli et al. [69] examined the effects of high light and respiration on ascorbate synthesis in Arabidopsis WTs and transgenic plants overexpressing the mitochondrial alternative oxidase [69]. It has been observed that plants under high light had a higher amount of ascorbate, GLDH, cytochrome C, and cytochrome C oxidase (CCO) activities, accompanied by an improved capacity of the AOX and CCO electron transport rates. Furthermore, *AOX*-overexpressing lines exhibited higher ascorbate levels than WT, especially at high light [69]. AOX is an enzyme in the plant mitochondria that bypasses cytC by directly accepting the electrons from the ubiquinone pool, which prevents over-reduction of the respiratory electron transport chain, and reduces the risk of ROS overproduction [69,73,89]. These studies demonstrate an

important mechanism in light regulation of ascorbate through the AOX pathway and further highlight important bidirectional interconnections between the mitochondrial electron transport chain and ascorbate biosynthesis, through both cytC and AOX respiratory pathways.
