Article Figures & Data
Figures
- Figure 1.
Tocopherol biosynthetic pathway. This figure represents the enzymatic reactions and intermediates that are involved in tocopherol synthesis. 1, HPPD. 2, Homogentisate phytyl transferase (HPT). 3, 2-Methyl-6-phytyl-1,4-benzoquinone (MPBQ) methyltransferase. 4, TC. 5, γ-Tocopherol methyltransferase (γ-TMT). HPP, p-Hydroxyphenylpyruvate; HGA, homogentisic acid; SAM, S-adenosyl l-Met.
- Figure 2.
HPLC analysis of tocopherols in wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Tocopherols present in Arabidopsis, maize, and Synechocystis sp. PCC6803 lipid extracts were separated by normal phase HPLC and detected using a fluorescence detector with 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. A, Arabidopsis leaf tissue: solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize leaf tissue: solid line, wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803: solid line, wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1 expressed in the Δslr1737 insertional mutant. Retention times of α-, β-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards. LU, Luminescence units.
- Figure 3.
HPLC analysis of the prenyl quinones from wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Lipids were extracted from Arabidopsis, maize, and Synechocystis sp. PCC6803, and total prenyl quinines were isolated by thin-layer chromatography (TLC) and then analyzed by normal phase HPLC (see “Materials and Methods”) A, Arabidopsis. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize. Solid line, Wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803. Solid line, Wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1cDNA expressed in the Δslr1737 mutant background. Insets, Spectra of the peak labeled DMPBQ. Phyllo, Phylloquinone; PQ, Plastoquinone.
- Figure 4.
HPLC analysis of seed tocopherols in wild-type Arabidopsis, vte1-1, and vte1-2. Total seed lipids were extracted, and the tocopherols present were separated by reverse phase HPLC and detected using a fluorescence detector; 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. Retention times of α-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards.
- Figure 5.
Alignment of the carboxy termini of TC orthologs from plants and cyanobacteria. The asterisk above the At4g32770 protein sequence denotes the position of the vte1-2 mutation. Anabaena, Anabaena sp. PCC7120 (all0245); Chlamy, Chlamydomonas reinhardtii; Medicago, M. truncatula; Nostoc, N. punctiforme (506-74); Physcomitrella, Physcomitrella patens; Synecho, Synechococcus sp. PCC7002. The P. patens, rice (Oryza sativa), and wheat (Triticum aestivum) sequences are partial sequences obtained from ESTs. Dark shading, Amino acid identity; light shading, Amino acid similarity. The threshold for amino acid consensus identity or similarity is 51%.
- Figure 6.
TC activity of proteins expressed in E. coli. E. coli cell lysates from cells overexpressing the empty pET vector or pET engineered to express TC proteins from Arabidopsis, maize, and Synechocystis sp. PCC6803 were incubated with radiolabeled 2,3-methyl-6-phytyl-1,4-benzonequinol (3 methyl 14C) for 4 h as described in “Materials and Methods.” Total lipids were extracted, separated by TLC, and radiolabeled products were detected by phosphor imager analysis. Products were identified by comigration with standards. The 14C incorporation into γ-tocopherol was quantified densitometrically and expressed as pixels per microgram of total protein.
Tables
- Table I
Pairwise comparisons of VTE1 orthologs from plants and cyanobacteria
Pair-wise comparisons were performed using ClustalW and are expressed as percentage amino acid similarity. The predicted mature plant protein sequences were used for alignments. Anabaena, Anabaena sp. PCC7120 (all0245); Nostoc, Nostoc punctiforme (506-74); Synecho, Synechococcus sp. PCC7002.
SXD1 VTE1 Medicago truncatula Barley (Hordeum vulgare) slr1737 Synecho Nostoc % VTE1 79 M. truncatula 80 84 Barley 92 80 83 slr1737 46 46 48 48 Synecho 47 49 49 48 63 Nostoc 55 55 54 55 59 67 Anabaena 55 54 54 55 61 67 85 - Table II.
Analysis of Glc, Suc, and starch in wild-type Arabidopsis and vte1-1
Glc, Suc, and starch levels were analyzed spectrophotometrically using the enzyme-coupled assays described in “Materials and Methods.” Glc and Suc are expressed as nanomoles per milligram fresh wt (n = 4).
Beginning of Photoperiod End of Photoperiod Carbohydrates Columbia vte1-1 Columbia vte1-1 Glc 2.32 ± 0.62 2.46 ± 0.58 2.36 ± 0.79 2.63 ± 0.83 Suc 0.07 ± 0.07 0.16 ± 0.16 1.56 ± 0.60 1.75 ± 0.28 Starcha 13 ± 3 13 ± 4 86 ± 14 85 ± 11 -
↵a Starch is expressed as nanomoles of Glc monomers per milligram fresh wt
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