Functional modeling identifies paralogous solanesyl-diphosphate synthases that assemble the side chain of plastoquinone-9 in plastids

Abstract

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.

Keywords: Arabidopsis; Photosystem II; Quinones; Terpenoids; Vitamin E.

FIGURE 1.

Metabolic connections among the biosynthetic pathways of plastoquinone-9, plastochromanol-8, and tocopherols in plants. Knock-out mutants corresponding to HPPD, HST, and MSBQ/MPBQ methyltransferase are devoid of plastoquinone-9 and are seedling-lethal. DMPBQ, 2,3-dimethyl-5-phytyl-1,4-benzoquinone; HPPD, 4-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyl transferase; HST, homogentisate solanesyl transferase; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol; MSBQ, 2-methyl-6-solanesyl-1,4-benzoquinol; NDC1, NAD(P)H quinone oxidoreductase C1; SAH, s-adenosyl-homocysteine; SAM, s-adenosyl-methionine.

FIGURE 2.

Co-expression network of the Arabidopsis solanesyl-diphosphate synthase family. The top 100 co-expressors of AtSPS1, AtSPS2, and AtSPS3 were mined from the ATTED-II database and then aggregated using GeneVenn (29). Subcellular localization data supported by direct experimental evidence and/or manual curation were compiled from the TAIR and SUBA-2 databases. The “other” category regroups proteins that are located in the endoplasmic reticulum, the plasma membrane, the vacuole, or the plasmodesma or that are secreted outside of the cells.

FIGURE 3.

Subcellular localization of AtSPS1. A–C, transient expression of AtSPS1-GFP in tobacco leaves. D–F, leaf tissue of Arabidopsis AtSPS1-GFP transgenics. G–I, leaf tissue of wild type Arabidopsis. A, D, and G, green pseudocolor channel; B, E, and H, red pseudocolor channel; C, F, and I, overlay of green and red pseudocolor channels. Transgenics and wild type Arabidopsis plants were imaged with the same settings.

FIGURE 4.

Phylogenetic relationships of plant trans-long chain prenyl-diphosphate synthases to their closest relatives in prokaryotes and eukaryotes. The closest homologs of AtSPS1 and AtSPS2 were mined from fully sequenced prokaryotic and eukaryotic genomes using BLASTp searches. Protein sequences were then processed using the following algorithm suite at Phylogeny.fr (30): MUSCLE (multiple alignment), Gblocks (curation of misalignments and divergent regions), PhyML (maximum likelihood reconstruction), and TreeDyn (tree visualization). E. coli octaprenyl-diphosphate synthase (IspB; YP_491372.1) served as an outgroup to root the tree. Branches of the strictly eukaryotic clade are shown in orange (metazoans and fungi) and red (plants); members of this clade are orthologous to yeast COQ1 that is targeted to the mitochondrion to make the side chain of ubiquinone. Light green branches mark the subfamily of cyanobacterial and plastid-encoded solanesyl-diphosphate synthases, and the dark green branches mark their nuclear encoded orthologs in plants; green arrowheads point to those that are paralogous. Bootstrap values out of 100 replications are given next to each branching. ATSPS1 (ABF58968.1), ATSPS2 (NP_173148.2), ATSPS3 (AAM13005.1), B. distachyon (1, XP_003567824.1; 2, XP_003576903.1; 3, XP_003563402.1; 4, XP_003559297.1), Caenorhabditis elegans SPS (NP_491588.1), C. reinhardtii (1, XP_001693430.1; 2, XP_001691069.1), C. merolae (1, NP_849058.1 plastid encoded; 2, BAM80356.1), Cucumis sativus (1, XP_004137246.1; 2, XP_004134571.1), Danio rerio (NP_001017656.1), D. melanogaster (NP_733425.1), Gallus gallus DPS (XP_418592.3), Glycine max (1, XP_003543174.1; 2, XP_003546747.1; 3, XP_003525839.1), Homo sapiens DPS (AAD28559.1), Hordeum vulgare (1, BAK00672.1; 2, BAK05302.1), Neurospora crassa (XP_959949.1), N. sp. PCC 7120 (NP_484140.1), Oryza sativa (SPS1, NP_001058362.1; SPS2, Q75HZ9.2, 3, TIGR contig TC495672), P. patens (1, XP_001775803.1; 2, XP_001762387.1, 3, XP_001753309.1), Prochlorococcus marinus (YP_001014501.1), S. cerevisiae HPS (EGA88084.1), S. moellendorffii (1, XP_002985427.1; 2, XP_002979762.1; 3, XP_002977539.1), Solanum lycopersicum (SPS, XP_004244308.1; DPS, NP_001234089.1), S. sp. PCC 6803 (NP_439899.1), Trichodesmium erythraeum (YP_720124.1), Vitis vinifera (1, XP_002285665.1; 2, XP_002268229.2), Zea mays (1, ACN25661.1, 2, NP_001149100.1, 3, ACG33955.1). DPS, decaprenyl-diphosphate synthase; HPS, hexaprenyl-diphosphate synthase; SPS, solanesyl-diphosphate synthase.

FIGURE 5.

Molecular characterization of the atsps1 and atsps2 T-DNA insertion mutants. A, structure of the atsps1 locus. B, genotyping PCR of WT Arabidopsis plant and atsps1 T-DNA insertion mutant (SALK_126948). C, RT-PCR analyses of RNA abundance in wild type and atsps1 plants. D, structure of the atsps2 locus. E, genotyping PCR of WT Arabidopsis plant and atsps2 T-DNA insertion mutant (SALK_064292). Note that amplifications with both RP2 and LP2 gene-specific primers in combination with the LBb1 T-DNA-specific primer in SALK_064292 mutant are diagnostic of a tandem insertion. F, RT-PCR analyses of RNA abundance in wild type and atsps2 plants. Boxes and lines represent exons and introns, respectively. RP1, LP1, RP2, LP2, and LBb1 indicate the location of the genotyping primers; RTfwd1, RTrvs1, RTfwd2, and RTrvs2 indicate the location of the RT-PCR primers. −RT, controls for genomic DNA contamination performed without reverse transcriptase.

FIGURE 6.

Levels of prenylated quinones and tocochromanols in Arabidopsis leaves and seeds. Extracts from leaves of 4-week-old plants grown at 120 μE·m⁻²·s⁻¹ and seeds were analyzed by HPLC in spectrophotometric detection mode for plastoquinone-9 (PQ-9) and ubiquinone-9 (UQ-9) or fluorometric detection mode for plastochromanol-8 (PC-8) and tocopherols (α/γ-TC). A, plastoquinone-9 (open bars) and plastochromanol-8 (black bars) in leaves. B, α- and γ-tocopherols in leaves. C, ubiquinone-9 in leaves. D, plastochromanol-8 in seeds. E, tocopherols in seeds. Data are the means of 8–12 replicates ± S.E. for the measurements of plastoquinone-9, plastochromanol-8, and α- + γ-tocopherols in leaves, 5 replicates ± S.E. for that of ubiquinone-9 in the wild type and the atsps1 and atsps2 mutants, duplicates ± S.E. for that of ubiquinone-9 in the atsps1/atsps2 double knock-out, and 3–6 replicates ± S.E. for plastochromanol-8 and tocopherols in seeds. Differing asterisk annotations indicate that the corresponding values are significantly different as determined by Fisher's least significant difference test (p < α = 0.05) from an analysis of variance.

FIGURE 7.

The atsps1 atsps2 double homozygous mutant is devoid of plastoquinone-9 and plastochromanol-8. A, phenotype comparison between WT and atsps1 atsps2 double homozygous mutant plants. Three-week-old seedlings from the atsps1 × atsps2 F2 segregating progeny were grown at 120 μE·m⁻²·s⁻¹ on Murashige and Skoog solid medium. B, genotyping PCR of wild type Arabidopsis and atsps1 atsps2 double homozygous mutant plants. Primer pairs were the same as those described in the legend for Fig. 5. C, plastoquinone-9 (PQ-9), plastochromanol-8 (PC-8), and tocopherol (TC) content in the cotyledons of wild type and atsps1 atsps2 double homozygous mutant plants. N.D., not detected.

FIGURE 8.

Quantification of the photoactive and non-photoactive pool sizes of plastoquinone-9 in Arabidopsis leaves. A, photoactive pool. The quantity of plastoquinol-9 was first determined in the leaves of plants that had been kept in the dark for at least 2 h. Plastoquinol-9 was then requantified after the plants had been exposed to high light intensity (1100 μE·m⁻²·s⁻¹) for 5 min. The size of the photoactive pool of plastoquinone-9 was calculated by subtracting the amount of plastoquinol-9 measured in the light from that measured in the dark. B, non-photoactive pools of plastoquinol-9 (PQH₂-9, not reoxidized in the dark) and plastoquinone-9 (PQ-9, not reduced in the light). Data are the means of 4–6 replicates ± S.E. Differing asterisk annotations next to each error bar indicate that the corresponding values are significantly different as determined by Fisher's least significant difference test (p < α = 0.05) from an analysis of variance.

FIGURE 9.

Phenotypes and analysis of photosystem II of wild type, atsps1, and atsps2 plants. A, 1-month-old plants grown at 110 μE·m⁻²·s⁻¹ for 16-h days. B, 1-month-old plants grown at 500 μE·m⁻²·s⁻¹ for 16-h days. C, imaging of maximum quantum efficiency (F_v/F_m) of photosystem II of wild type, atsps1, and atsps2 plants grown at moderate light intensity (120 μE·m⁻²·s⁻¹). D–F, representative FluorCam 700 MF images acquired after the plants had been exposed to high light intensity (800 μE·m⁻²·s⁻¹) for 2, 24, and 48 h, respectively. Plants were dark-adapted for 20 min prior to illumination with actinic light. G, immunodetection of photosystem II polypeptide D1 in thylakoid membranes prepared from wild type, atsps1, and atsps2 plants grown at moderate light intensity (120 μE·m⁻²·s⁻¹) or exposed to high light regime (800 μE·m⁻²·s⁻¹) for 24 h. Each of the lanes contains 20 μg of proteins.

FIGURE 10.

Analysis of photosystem I of wild type, atsps1, and atsps2 plants. A and B, representative kinetics of change in absorbance during photooxidation of P700 in the leaves of wild type, atsps1, and atsps2 plants grown at moderate light intensity (120 μE·m⁻²·s⁻¹) or exposed to high light regime (800 μE·m⁻²·s⁻¹) for 48 h. Open and filled arrowheads indicate the start and end of far-red illumination, respectively. Leaves were dark-adapted for 20 min and then preilluminated with far-red light for 2 min prior to the measurements. C, immunodetection of photosystem I subunit PsaA in thylakoid membranes prepared from wild type, atsps1, and atsps2 plants grown at moderate light intensity (120 μE·m⁻²·s⁻¹) or exposed to high light regime (800 μE·m⁻²·s⁻¹) for 48 h. Each of the lanes contained 20 μg of proteins.

FIGURE 11.

Plastoquinone-9 and tocochromanol levels of Arabidopsis leaves during acclimation to high light intensity. Three-week-old wild type, atsps1, and atsps2 plants grown at moderate light intensity (110 μE·m⁻²·s⁻¹; 16-h days) were exposed to continuous high light (600 μE·m⁻²·s⁻¹) for 48 h. Rosette leaves were sampled just before the switch to high light and then at 24 and 48 h after. A, plastoquinone-9; B, plastochromanol-8; C, total tocopherols. The inset in A shows the RT-PCR analyses of RNA abundance for AtSPS1, AtSPS2, and the actin control in the leaves of the wild type.

FIGURE 12.

Sequence alignment of AtSPS1 (ABF58968) and AtSPS2 (NP_173148) with their octaprenyl-diphosphate synthase homologue in E. coli (EcIspB; YP_491372). Note the N-terminal extensions of the plant enzymes (in green boxes) as predicted by both WoLF PSORT and TargetP 1.1 to encode plastid targeting peptides. AtSPS1 and AtSPS2 share 80% identical residues, pointing to a recent gene duplication. Identical residues are shaded in black and similar ones in gray. Dashes symbolize gaps introduced to maximize alignment.