**4. Light Regulation of Ascorbate**

Despite existing cumulative evidence on the importance of ascorbate on light acclimation responses, regulatory mechanisms of the ascorbate pool size by light remained poorly understood. It appears that ascorbate pool size is highly sensitive to both the light intensity and time of the day because transcript profiles of the genes encoding the enzymes of the pathway behaved unpredictably in different light-shift experiments and vary between species. Therefore, conclusions on the correlation between the gene expression, activity of the corresponding enzymes of the pathway, and the ascorbate pool size are inconsistent between studies. That being said, the comparison of multiple light-shift experiments revealed GDP-L-galactose phosphorylase (GGP) as the key enzyme of the pathway controlling the ascorbate levels under high light [54,56–59]. The corresponding genes encoding this enzyme, the first committed step of the ascorbate biosynthetic pathway, are *VTC2* and *VTC5* paralogs, which were identified to be induced in concert upon 24 h exposure to high light, leading to a 20-fold increase in the activity of the corresponding enzyme, and an increase in ascorbate levels [56].

In a study where the authors explored the transcriptional regulation of ascorbate by RNA-seq following a step change of light intensity in Arabidopsis, *VTC2* and, to lesser extent, *VTC5* were validated as regulatory points in light accumulation of ascorbate, the expression of both genes were correlated with different light intensities, however, a minor change in GDP mannose pyrophosphorylase (GMP) could also be observed [57]. Moreover, GGP has been proposed as a key rate-limiting step for ascorbate biosynthesis not only in Arabidopsis [60], but also in other species including tobacco [61], apple [62], and kiwifruit [60,63]. Besides, the light-responsiveness of *VTC2* expression has been

observed in tomato fruits following an observation on *VTC2* reduction under a continuous shading [64]. GGP has also been validated as a highly regulated enzyme in the green algea, *Chlamydomonas reinhardtii,* where it is thought to exhibit protective function against oxidative stress [65,66]. Further, a potential regulatory role for GGP has been proposed owing to the evidence on nuclear localization of the protein; however, as yet, no mechanistic evidence proposed has been supposed as a hypothesis [58].

Besides GGP, GLDH has been also suggested as an important controlling point for light regulation of ascorbate biosynthesis at the level of the enzyme activity [67,68]. Arabidopsis plants, grown under high light after supplementation with L-galactone-1,4-lactone (L-Gal; the precursor of ascorbate), accumulated up to twofold ascorbate levels and had twice as high GLDH activities of the low-light grown plants, assumed as higher respiration rates [69]. GLDH is located in the inner membrane of the mitochondria, which carries a redox-sensitive thiol residue (Cys-340), critical for the conversion of L-Gal into ascorbate [70]. This residue has been validated to be irreversibly oxidized by H2O2, inactivating GLDH [70], and has been suggested to be responsible for regulation of GLDH activity during the early stages of heat stress produced programmed cell death [67,71]. Moreover, Arabidopsis *GLDH* overexpressing lines accumulated higher ascorbate levels and demonstrated higher chlorophyll fluorescence parameters after exposure to high light for 14 days, which led them to have lower sensitivity to light stress [72].

It should be noted that, despite observing multiple studies on light effects on the ascorbate biosynthetic pathway, so far, few reports exist concerning the effects of light on the components of ascorbate recycling and turnover [73]. One report, however, does demonstrate that the activities of dehydroascorbate reductase and monodehydroascorbate reductase are enhanced in the Arabidopsis plants, grown under high light [69].
