Abstract
Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in oxidoreduction. The deficiency of this compound causes pleiotropic defects in Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT) to vkor mutant lines ultimately demonstrates that the phenotype changes are due to this gene. However, whether AtVKOR functions in Arabidopsis through its protein oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the size of the transgenic plants with a single conservative cysteine mutation (VKORC109A, VKORC116A, VKORC195A, and VKORC198A) were smaller, and two double-cysteine mutations (VKORC109AC116A and VKORC195AC198A) showed significantly stunted growth, similar with the vkor mutant line. However, mutations of two non-conservative cysteines (VKORC46A and VKORC230A) displayed little obvious changes in the phenotypes of Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII) in double-cysteine mutation plants decreased significantly to the level similar to that of the vkor mutant line both under normal growth light and high light. A significantly decreased amount of D1 protein and increased accumulation of reactive oxygen species were observed in two double-cysteine mutations under high light. All of the results above indicated that the conservative cysteines in transmembrane domains were the functional sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis thaliana.
Highlights
In chloroplasts, disulfide bond formation, a covalent bond between two cysteines, is crucial for the maturation and function of proteins (Jocelyn, 1967; Buchanan and Luan, 2005; Onda, 2013)
While the AtVKORWT transgenic plants can completely recover the phenotype defects in the vkor line, transgenic plants with a single conservative cysteine mutation (AtVKORC109A, AtVKORC116A, AtVKORC195A, AtVKORC198A) did only partly recover the phenotype defects in the vkor mutant lines (Figure 1A)
The necessity of the conservative cysteines of the AtVKOR domain is inferred from the fact that single or double cysteine mutations lead AtVKOR to losing its function of promoting disulfide bond formation in E. coli (Feng et al, 2011)
Summary
Disulfide bond formation, a covalent bond between two cysteines, is crucial for the maturation and function of proteins (Jocelyn, 1967; Buchanan and Luan, 2005; Onda, 2013). Functional analyses reveal that these two pairs of conservative cysteines are indispensable for the oxidoreductase activities of AtVKOR in the process of catalyzing disulfide bond formation in Escherichia coli (Feng et al, 2011; Karamoko et al, 2011). In M. tuberculosis, two pairs of conservative cysteines of MtbVKOR are found to play critical roles in the formation of disulfide bonds (Wang et al, 2011)
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