Our previous investigation revealed that GDP-Man pyrophosphorylase (VTC1), a vital ascorbic acid (AsA) biosynthesis enzyme, could be degraded through interaction with the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) in the darkness, demonstrating the posttranscriptional regulation of light signal in AsA production. Here, we further report that a point mutation in D27E of VTC1 disables the interaction with CSN5B, resulting in enhancement of AsA biosynthesis and seedling growth in Arabidopsis thaliana. To identify the interaction sites with CSN5B, we first predicted the key amino acids in VTC1 via bioinformatics analysis. And then we biochemically and genetically demonstrated that the 27th Asp was the amino acid that influenced the interaction of VTC1 with CSN5B in plants. Moreover, transgenic lines overexpressing the site-specific mutagenesis from D27 (Asp) into E27 (Glu) in VTC1 showed enhanced AsA accumulation and reduced H2O2 content in Arabidopsis seedlings, compared with the lines overexpressing the mutation from D27 into N27 (Asn) in VTC1. In addition, this regulation of VTC1 D27E mutation promoted seedling growth. Together, our data reveal that the 27th amino acid of VTC1 confers a key regulation in the interaction with CSN5B and AsA biosynthesis, as well as in Arabidopsis seedling growth.
Keywords: AsA biosynthesis; CSN5B; Point mutation; Protein stability; VTC1.