The role of the salvage pathway of phytol, geranylgeraniol, and farnesol phosphorylation in <em>Arabidopsis thaliana</em>

Abstract

The isoprenoid alcohol phytol can be phosphorylated in Arabidopsis to phytyl-phosphate (phytyl-P) by the phytol kinase (VTE5) and further to phytyl-diphosphate (phytyl-PP) by the phytyl phosphate kinase (VTE6). Phytyl-PP derived from phytol phosphorylation enters the biosynthesis of tocopherol and phylloquinone. Arabidopsis phytol kinase mutant plants (<em>vte5</em>) still contain reduced levels of tocopherol, while phytyl phosphate kinase mutant plants (<em>vte6</em>) are tocopherol deficient. One homolog of <em>VTE5</em> in Arabidopsis was identified and later designated <em>FOLK</em> for farnesol kinase. FOLK is able to phosphorylate farnesol, geraniol, and geranylgeraniol. In this work, the role of phytol, geranylgeraniol, and farnesol phosphorylation, for the biosynthesis of different metabolites including tocopherol (vitamin E) and phylloquinone (vitamin K1), was investigated in Arabidopsis <em>vte5-2</em>, <em>folk-2</em>, and <em>vte5-2 folk-2</em> mutant plants. <br /> Phytyl-P and geranylgeranyl-P levels are reduced, while phytol accumulates in <em>vte5-2</em> and <em>vte5-2 folk-2</em>. Geranylgeraniol accumulates in <em>folk-2</em> and <em>vte5-2 folk-2</em>. The <em>vte5-2</em> single mutant shows reduced tocopherol levels, while the <em>folk-2</em> single mutant plants have similar tocopherol levels as the WT. The phylloquinone levels are similar to the WT in <em>vte5-2</em> and in <em>folk-2</em>. The <em>vte5-2 folk-2</em> double mutant plants are tocopherol deficient and have a reduced content of phylloquinone in comparison to the WT. Therefore, the farnesol kinase (FOLK) and the phytol kinase (VTE5) are involved in tocopherol and phylloquinone biosynthesis. Enzyme assays with recombinant proteins expressed in <em>E. coli</em> revealed that VTE5 is able to phosphorylate phytol, geranylgeraniol, and farnesol, while FOLK only phosphorylated geranylgeraniol and farnesol but not phytol. Geranylgeranyl-PP is the precursor for the biosynthesis of chlorophyll and carotenoids. The contents of chlorophyll and carotenoids were not altered in the mutants indicating that VTE5 and FOLK are not required for the biosynthesis of these compounds. The sterol content was slightly reduced in the <em>vte5-2 folk-2</em> double mutant suggesting that farnesol phosphorylation by VTE5 and FOLK contributes to sterol synthesis. <br /> Arabidopsis mutant plants lacking phytyl phosphate kinase (VTE6) activity show a strong growth retardation. The decreased growth of <em>vte6-1</em> could not be rescued by supplementation with gibberellic acid. Arabidopsis <em>vte6</em> mutants were described to accumulate phytyl-P and phytol derived from chlorophyll degradation. Accumulation of phytyl-P might be detrimental for plant growth and therefore cause the severe growth phenotype. The introduction of the <em>pao1</em> mutation, affected in chlorophyll degradation, into the <em>vte6-1</em> background did not rescue the retarded growth of <em>vte6-1</em>. The <em>vte6-1 pao1</em> plants were still only viable on synthetic medium supplemented with sucrose. Nevertheless, some plants grew slightly better and developed shoots but no siliques nor seeds. <br /> In conclusion, Arabidopsis farnesol kinase FOLK and the phytol kinase VTE5 are involved in the biosynthesis of tocopherol and phylloquinone. Tocopherol and phylloquinone synthesis depends on the availability of phytyl-PP. However, the recombinant FOLK protein does not show <em>in vitro</em> phytol kinase activity. Presumably, the geranylgeraniol kinase activity of FOLK contributes to tocopherol and phylloquinone biosynthesis. This shows the important role of geranylgeraniol phosphorylation next to phytol phosphorylation for tocopherol and phylloquinone biosynthesis in Arabidopsis.