Generation and characterization of a double-knockout Arabidopsis thaliana line lacking expression of AOX1a and VTC2

In higher plants, the L-galactose pathway is the main pathway for the biosynthesis of vitamin C (ascorbate, Asc), the final step of which is connected with the functioning of the mitochondrial electron transport chain (ETC). In addition to the main cytochrome pathway, plant ETC includes an alternative pathway (AP) via alternative terminal oxidase (AOX). The engagement of AOX promotes Asc synthesis, and it is hypothesized that AOX suppression under conditions of Asc deficiency may reduce plant viability. The aim of this work was to examine the consequences of simultaneously knocking out two genes in Arabidopsis thaliana: AOX1a, the most stress-inducible AOX gene, and VTC2, encoding a key enzyme of the L-galactose pathway of Asc synthesis. Two lines of A. thaliana with T-DNA insertions in the target genes were crossed to generate hybrid lines. Seed characteristics of the first (F1) and second (F2) generations were analyzed. F1 seeds were larger than those of parent lines, possibly due to heterosis. In the F2 generation, self-pollination of F1 plants resulted in seeds with significant size variation, including a group of small seeds with degenerative morphological abnormalities. Most of small seeds failed to germinate or died at the seedling stage. PCR genotyping of these seeds revealed the absence of native AOX1a and VTC2 indicating a lethal mutation caused by simultaneous knockout of both genes. One likely genetic cause is the interaction of mutations in non-allelic genes. At the physiological level, irreversible respiratory damage may occur, possibly including the impact of a cryptic mutation in the vtc2 line. Further studies are necessary to confirm these hypotheses. In general, the results obtained indicate the vital co-functioning of the AP and the L-galactose pathway of Asc biosynthesis and may be useful for the development of genetically engineered techniques for the control of vitamin C synthesis in plants.