*4.1. Transcriptional Regulation*

Despite observing alterations in gene expression patterns of the ascorbate biosynthetic pathway under light stress in multiple studies, the upstream signal transduction pathway controlling this phenomenon is largely unknown. Studies were performed to decipher light-regulated *cis-*elements in rice [74] and subsequently in Arabidopsis [75]. The conserved sequences (the GT1 box and the TGACG motif) in the promoter regions of the *L-galactose-1-phosphate phosphatase (GPP*) and *GLDH* genes were found to be responsible for light induction of these genes in rice [74]. Further efforts have been made to find such consensus elements in Arabidopsis, however, authors identified a different, but critical region for light regulation of *VTC2*, in –40 to –70 bp of its promoter [75]. Information on whether such a casual promoter region exist upstream of other genes of the pathway is not yet available.

Ascorbic acid mannose pathway regulator 1 (AMR1) has been identified as a negative regulator of multiple genes encoding early and late enzymes of the Man/L-Gal pathway, including GMP, GME, GGP, GPP, GDH, and GLDH, with the highest effect on GME and GGP [76]. The expression of AMR1 has been validated to be decreased by light and to be accompanied by an increase in ascorbate levels [76]. In contrast to AMR1, the ethylene response factor98 (*At*ERF98) has been identified as a positive regulator of D-Man/L-Gal pathway, by directly binding to the promoter of *VTC1*; encoding GMP; and also enhancing the expression of multiple genes of the pathway including, *VTC1*, *VTC2*, *GDH,* and *GLDH* [77]. Although the essential role of ERF98 has been revealed under the salt stress [77], no investigations have been done to verify its role under the light stress.

Recently, implementing genome wide association study (GWAS) on 302 tomato accessions identified a basic helix–loop–helix (bHLH) transcription factor, *Sl*bHLH59, which positively regulates ascorbate content in tomato fruits [78]. The most similar protein to *Sl*bHLH59 in Arabidopsis appears to be unfertilized embryo sac 12 (UNE12), regulating fertilization [78]. Further investigations are, however, needed to clarify whether this protein has links to the accumulation of ascorbate in Arabidopsis. The schematic representation of the regulatory factors is depicted in Figure 2.

**Figure 2.** Overview of light regulation on ascorbate biosynthesis. Ascorbate biosynthesis is regulated transcriptionally by the ascorbic acid mannose pathway regulator 1 (AMR1), ethylene response factor 98 (ERF98), and *Solanum lycopersicum* basic helix–loop–helix (bHLH) transcription factor 59 (*sl*bHLH59; tomato-specific). AMR1 has negative effects on *GMP*, *GME*, *GGP*, *GPP*, *GDH*, and *GLDH* in Arabidopsis. *AMR1* expression decreases rapidly under light; thereafter, ascorbate levels increase. *At*ERF98 is the positive regulator of the ascorbate pathway by directly binding to the promoter of *VTC1*, and up-regulating the expression of *VTC1*, *VTC2, GDH*, and *GLDH.* The light-specific functionality of ERF98 has yet to be investigated. *sl*bHLH59 activates the genes of the pathway in tomato fruits and increases ascorbate levels under the light. The close homolog of this transcription factor (TF) in Arabidopsis is unfertilized embryo sac 12 (UNE12), however, its role for ascorbate biosynthesis has yet to be investigated. Ascorbate undergoes post-translational regulation via constitutive photomorphogenic9-signalosome subunit 5B (CSN5B), VTC3, and the feedback regulation of *VTC2.* CSN5B binds to VTC1 and promotes its degradation under the dark. VTC3 is a putative kinase/phosphatase for light regulation of ascorbate. Feedback regulation of ascorbate is controlled by an unusual open reading frame (uORF), located upstream of the *VTC2* gene. uORF functionality under the light needs further investigations. Ascorbate is also controlled via photosynthetic and mitochondria electron transport chains, designated as photosynthetic electron transport chain (pETC) and mitochondrial electron transport chain (mETC), respectively. This relationship is bidirectional.

Other components linked to ascorbate regulation have been proposed in multiple studies that are beyond the scope of this review because their roles in acclimation responses have not been fully characterized. Readers are referred to other comprehensive reviews covering the general regulatory components of ascorbate biosynthesis [67,79,80].
