Tocopherol and phylloquinone biosynthesis in chloroplasts requires the phytol kinase VITAMIN E PATHWAY GENE5 (VTE5) and the farnesol kinase (FOLK)

Abstract

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK.

Figure 1.

Arabidopsis vte5-2, folk-2, and vte5-2 folk-2 mutant plants are deficient in tocochromanols and phylloquinone. A) Tocochromanols were extracted from leaves of 4-wk-old plants and measured by HPLC. B) Tocochromanols extracted from seeds were measured by HPLC. C) Germination rate of vte5-2, folk-2, and vte5-2 folk-2 seeds. Germination rates were determined for fresh, dry seeds (stored for 1 mo at 4 °C). Seeds were placed on wet filter papers and after 1 d of stratification, the germination rate was determined every day. D) Germination rate of artificially aged seeds (40 °C, 100% humidity, 72 h). E) Phylloquinone contents in vte5-2, folk-2, and vte5-2 folk-2 leaves. Phylloquinone was extracted from leaves of 4-wk-old Arabidopsis plants and measured by fluorescence HPLC after postcolumn reduction. Data represent means ± Sd of 3 different plants. Letters within each tocochromanol class indicate significant differences; 1-way ANOVA with post hoc Tukey test, P < 0.05.

Figure 2.

VTE5 and FOLK display kinase activities with different isoprenoid alcohols. A) Supplementation of phytol, geranylgeraniol, or farnesol to S. cerevisiae cells expressing VTE5 or FOLK. The cDNAs of VTE5, FOLK, or GFP (control) were expressed in S. cerevisiae and the cells cultivated in the presence of 5 mM phytol, geranylgeraniol, or farnesol. Phosphorylation products (phytyl-P, geranylgeranyl-P, and farnesyl-P) were analyzed by LC-MS/MS and calculated as fmol per OD₆₀₀. B) Isoprenoid alcohol kinase assays with VTE5 and FOLK. Isoprenoid alcohol kinase assays were performed with microsomal fractions of VTE5 and FOLK proteins expressed in S. cerevisiae. The assays included 5 mM of phytol, geranylgeraniol, or farnesol with an equimolar mix of NTPs. Phytyl-P, geranylgeranyl-P, and farnesyl-P were analyzed by LC-MS/MS. Data represent the means ± Sd of 3 replicates. Significant differences to control: *P < 0.05; **P < 0.01, Student t-test. OD₆₀₀, optical density at 600 nm.

Figure 3.

Complementation of tocopherol deficiency in the vte5-2 mutant with FOLK. The FOLK cDNA under the control of the 35S promoter was introduced into the vte5-2 mutant background. Tocochromanols were measured A) in leaves of plants grown on soil, B) grown on a nitrogen-deficient medium, or C) in seeds. Data represent means ± Sd of 3 to 10 replicates of different plants. Letters indicate significant differences for each tocochromonal class; 1-way ANOVA with post hoc Tukey test, P < 0.05; n.d., not detectable.

Figure 4.

Isoprenoid alcohol phosphate contents in leaves and seeds of the Arabidopsis vte5-2, folk-2, and vte5-2 folk-2 mutants. A) Isoprenoid alcohol monophosphates (-P) and diphosphates (-PP) in leaves of 4-wk-old Arabidopsis plants grown on MS medium. B) Isoprenoid alcohol monophosphates and diphosphates in seeds. C) Leaf isoprenoid alcohol phosphate contents after supplementation with phytol, geranylgeraniol, or farnesol. Three-week-old plants were incubated in liquid culture supplemented with 5 mM phytol, geranylgeraniol, or farnesol for 24 h. Phytyl-P, phytyl-PP, geranylgeranyl-P, geranylgeranyl-PP, farnesyl-P, and farnesyl-PP were measured by LC-MS/MS. Data represent means ± Sd of 3 replicates of different plants. Letters indicate significant differences for each compound class; 1-way ANOVA with post hoc Tukey test, P < 0.05.

Figure 5.

Isoprenoid alcohols, phytenal, and fatty acid isoprenoid alcohol esters in leaves and seeds of Arabidopsis vte5-2, folk-2, and vte5-2 folk-2 plants. A) Isoprenoid alcohols were extracted from leaves or seeds and analyzed by GC-MS after silylation. Only phytol and geranylgeraniol were detected in leaves and only phytol in the seeds, while farnesol in leaves and geranylgeraniol and farnesol in seeds were below the detection limit. Phytenal was extracted from leaves, derivatized with methoxylhydroxylamine, and analyzed by LC-MS/MS. B) FAPEs, geranylgeranyl esters, and farnesyl esters were determined by direct infusion Q-TOF MS/MS in leaves or seeds of soil-grown plants. The composition of isoprenoid alcohol esters is provided in Supplementary Fig. S4. Data represent means ± Sd of 3 to 4 replicates. Letters indicate significant differences for each compound class; 1-way ANOVA with post hoc Tukey test, P < 0.05.

Figure 6.

Tocochromanol and phylloquinone/menaquinone-4 contents in vte5-2, folk-2, and vte5-2 folk-2 plants after homogentisate or DHNA supplementation. A) Two-week-old Arabidopsis plants were grown on a control medium (− homogentisate) or medium supplemented with 5 mM homogentisate (+ homogentisate) for 9 d. Tocopherols and PC-8 were extracted from leaves and measured by HPLC. B) Tocotrienol contents in plants grown on control or on homogentisate-supplemented medium. C) Two-week-old plants were transferred to solid medium containing 2.5 mM DHNA (+ DHNA) or without DHNA (− DHNA) and grown for another 9 d. Phylloquinone and menaquinone-4 contents were measured by HPLC with postcolumn reduction. Data represent means ± Sd of 3 to 4 replicas of different plants. A, B) Letters indicate significant differences for each compound class; 1-way ANOVA with post hoc Tukey test, P < 0.05. Asterisks indicate differences between treatments; Student t-test: *P < 0.05; **P < 0.01. C) Significant differences to Col-0; **P < 0.01, Student t-test. Significant differences between treatments; ^##P < 0.01, Student t-test. n.d., not detectable.

Figure 7.

Subcellular localization of VTE5 and FOLK. Leaves of N. benthamiana plants were infiltrated with Agrobacterium tumefaciens cells carrying the constructs pLH9000-VTE5-eGFP (top) or pLH9000-FOLK-eGFP (bottom panels). After 4 d, leaves were analyzed by CLSM. Colocalization of the GFP fluorescence and chlorophyll autofluorescence is indicated with white arrows. Red arrows indicate chloroplasts in untransformed cells, which lack green fluorescence. The absence of green fluorescence from these chloroplasts indicates that there was no overflow of chlorophyll fluorescence to the green channel. Bars = 10 µm.

Figure 8.

Geranylgeranyl-PP from the MEP pathway and phytyl-PP contribute to isoprenoid synthesis in the chloroplast. Geranylgeranyl-PP is derived from the isoprenoid de novo synthesis pathway (MEP pathway) and is employed for the synthesis of carotenoids, ABA, and geranylgeranyl-chlorophyll. GG-chlorophyll is converted into chlorophyll by GGR. Hydrolysis of GG-chlorophyll and chlorophyll gives rise to the release of geranylgeraniol and phytol, respectively. Geranylgeraniol and phytol are phosphorylated by VTE5 or FOLK. Geranylgeranyl-PP and phytyl-PP are derived from a second phosphorylation by VTE6. The percent numbers indicate the relative contribution of geranylgeranyl-PP or phytyl-PP to the different isoprenoids. During homogentisate (HGA) supplementation, or in the presence or absence of DHNA, tocotrienols and menaquinone-4 are produced in vte5-2 folk-2 plants (dashed arrows). Phylloquinone is produced not only from phytyl-PP derived from the kinase reactions but also from geranylgeranyl-PP from the MEP pathway, which is potentially converted into phytyl-PP by a side reaction of GGR (dashed arrow).