*4.2. Post-Translational Regulation*

Further studies support that light regulation of ascorbate occurs post-translationally via the three so far identified mechanisms: (i) through modulating the stability of GDP-man pyrophosphorylase (GMP/VTC1) in light/dark; (ii) through feed-back regulation of GGP (VTC2); and (iii) through a putative kinase::protein phosphatase, VTC3 (Figure 2). Constitutive photomorphogenic9-signalosome subunit 5B (CSN5B) protein has been identified as a component in light/dark regulation of ascorbate [81]. CSN5B interacts with GDP-man pyrophosphorylase (VTC1), and promotes its degradation under dark through the 26S proteasome pathway, resulting in lower ascorbate content. As mentioned above, GGP has been determined as the main regulatory point in the ascorbate biosynthetic pathway, which undergoes rapid feedback control in conditions where ascorbate levels increase [82]. An unusual open reading frame (uORF), starting with an uncommon start codon, ACG instead of AUG, has been found in the 5 -untranslated region (UTR) of GGP, which, under a high concentration of ascorbate, gets translated into a 60–65 residue peptide and further inhibits the translation of *VTC2* [82]. Interestingly, this uORF exists in a variety of plant species [82]. Although it has been hypothesized that this post-translational control might be a causative mechanism in light regulation of ascorbate, further experiments are required to validate this hypothesis. GGP has also been revealed as an important rate-limiting enzyme in *Chlamydomonas*, however, it lacks the feedback regulation mechanism existing in land plants [66].

A deeper investigation of the ascorbate deficient mutant *vtc3* led to the identification of its novel causative loci, encoding VTC3 protein, suggested as a putative kinase/phosphatase for light regulation of ascorbate [83]. Although the protein structure of the VTC3 has been proposed, harboring a kinase and a phosphatase domain at its N-terminal and C-terminal site, respectively, validation of its molecular mechanism has so far remained elusive. Given that the *vtc3* mutants were unable to accumulate ascorbate under continuous light, and localized to the chloroplast, a dual signaling function of the protein in light regulation of ascorbate was suggested by the authors. However, this remains to be experimentally validated [83].
