Simultaneous knockout of VITAMIN C DEFECTIVE 2 and 3 exacerbates ascorbate deficiency and light stress sensitivity.

Light-dependent activation of ascorbate biosynthesis is essential for accumulating ascorbate to mitigate photooxidative stress. The VTC2 and VTC3 genes play key roles in this process: VTC2 encodes GDP-L-galactose phosphorylase (GGP), the rate-limiting enzyme in ascorbate biosynthesis, and VTC3 encodes a putative protein kinase/phosphatase with an unknown function. Here, we investigated their functional and genetic relationship. In vtc3 mutants, VTC2 transcription and GGP activity were slightly enhanced, suggesting that VTC3 is not required for VTC2 expression. Additionally, the vtc3 mutation had negligible effects on the transcriptome and the activity of enzymes involved in ascorbate redox cycle regulation, narrowing down the possible roles of VTC3 in ascorbate biosynthesis. Under low-light conditions, ascorbate levels were lower in vtc2 than in vtc3 mutants, but vtc2 retained the ability to increase ascorbate levels under high-light stress, unlike vtc3. The simultaneous knockout of VTC2 and VTC3 further reduced ascorbate levels compared to single mutants and severely impaired light stress-induced ascorbate accumulation, resulting in impaired non-photochemical quenching and enhanced photooxidative damage. These findings highlight the additive effects of VTC2 and VTC3 on ascorbate biosynthesis and stress tolerance. The vtc2 vtc3 double mutants provide a valuable model for further elucidating the physiological roles of ascorbate in plants.