Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT‐independent pathway for tocotrienol synthesis has also been shown to occur by de‐regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co‐expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi‐functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2–1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA‐expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA‐induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT‐catalyzed tocotrienol synthesis.     

Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

转基因水稻中过表达OsHGGT基因提高种子三烯生育酚含量的研究

旨在提高稻米中三烯生育酚的含量,将来源于日本晴尿黑酸牻牛儿基牛儿基牻转移酶(homogentisic acid gerany-lgeranyl transferase,HGGT)基因导入粳稻品种武育粳3号过量表达。经PCR和RT-PCR分析证明外源基因已导入水稻中并能够在水稻胚乳中表达。HPLC测定结果表明,过表达HGGT后,转基因水稻种子糠层及胚乳中γ-三烯生育酚和总三烯生育酚的含量分别是未转化对照的1.52和1.67倍,且三烯生育酚的积累并未导致总生育酚含量的降低,最终糠层及胚乳中总三烯生育酚与总生育酚的比值分别提高到0.82和1.82,极显著高于(P<0.01)未转化对照(分别为0.54和1.27)。     

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.     

Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis

Tocopherols are essential components of the human diet and are synthesized exclusively by photosynthetic organisms. These lipophilic antioxidants consist of a chromanol ring and a 15-carbon tail derived from homogentisate (HGA) and phytyl diphosphate, respectively. Condensation of HGA and phytyl diphosphate, the committed step in tocopherol biosynthesis, is catalyzed by HGA phytyltransferase (HPT). To investigate whether HPT activity is limiting for tocopherol synthesis in plants, the gene encoding Arabidopsis HPT, HPT1, was constitutively overexpressed in Arabidopsis. In leaves, HPT1 overexpression resulted in a 10-fold increase in HPT specific activity and a 4.4-fold increase in total tocopherol content relative to wild type. In seeds, HPT1 overexpression resulted in a 4-fold increase in HPT specific activity and a total seed tocopherol content that was 40% higher than wild type, primarily because of an increase in gamma-tocopherol content. This enlarged pool of gamma-tocopherol was almost entirely converted to alpha-tocopherol by crossing HPT1 overexpressing plants with lines constitutively overexpressing gamma-tocopherol methyltransferase. Seed of the resulting double overexpressing lines had a 12-fold increase in vitamin E activity relative to wild type. These results indicate that HPT activity is limiting in various Arabidopsis tissues and that total tocopherol levels and vitamin E activity can be elevated in leaves and seeds by combined overexpression of the HPT1 and gamma-tocopherol methyltransferase genes.     

Accumulation of tocopherols and tocotrienols during seed development of grape (Vitis vinifera L. cv. Albert Lavallée)

Tocopherols and tocotrienols are present in mature seeds. Yet, little is known about the physiological role and the metabolism of these compounds during seed development. Here we present data on tocopherol and tocotrienol accumulation during seed development in Vitis vinifera L. cv. Albert Lavallée (Royal). This species was chosen for its ability to synthesize both tocopherols and tocotrienols. It is shown here for the first time that during seed development there are significant differences in localization and accumulation kinetics of tocopherols and tocotrienols. Tocopherols are found homogeneously dispersed throughout all tissues of the seed, in concentrations ranging from 20 to 100 microg tocopherol per g dry weight. Tocopherol levels decrease gradually during seed development. In contrast, tocotrienols are only found in the endosperm of the seeds, accumulating in a sigmoid fashion during the maturation period of seed development. Tocotrienol levels were found to be (54+/-7.4) microg/g dry seed in 90-day-old seeds of V. vinifera L. Furthermore, tocotrienol biosynthesis is demonstrated in these seeds during tocotrienol accumulation and in an endosperm fraction isolated at 75 days after flowering.     

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (??-, ??-, ??- and ??-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.     

Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana

Vitamin E is considered a major antioxidant in biomembranes, but little evidence exists for this function in plants under photooxidative stress. Leaf discs of two vitamin E mutants, a tocopherol cyclase mutant (vte1) and a homogentisate phytyl transferase mutant (vte2), were exposed to high light stress at low temperature, which resulted in bleaching and lipid photodestruction. However, this was not observed in whole plants exposed to long-term high light stress, unless the stress conditions were extreme (very low temperature and very high light), suggesting compensatory mechanisms for vitamin E deficiency under physiological conditions. We identified two such mechanisms: nonphotochemical energy dissipation (NPQ) in photosystem II (PSII) and synthesis of zeaxanthin. Inhibition of NPQ in the double mutant vte1 npq4 led to a marked photoinhibition of PSII, suggesting protection of PSII by tocopherols. vte1 plants accumulated more zeaxanthin in high light than the wild type, and inhibiting zeaxanthin synthesis in the vte1 npq1 double mutant resulted in PSII photoinhibition accompanied by extensive oxidation of lipids and pigments. The single mutants npq1, npq4, vte2, and vte1 showed little sensitivity to the stress treatments. We conclude that, in cooperation with the xanthophyll cycle, vitamin E fulfills at least two different functions in chloroplasts at the two major sites of singlet oxygen production: preserving PSII from photoinactivation and protecting membrane lipids from photooxidation.     

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.     

Tocotrienols inhibit human glutathione S-transferase P1-1

Tocopherols and tocotrienols are food ingredients that are believed to have a positive effect on health. The most studied property of both groups of compounds is their antioxidant action. Previously, we found that tocopherols and diverse tocopherol derivatives can inhibit the activity of human glutathione S-transferase P1-1 (GST P1-1). In this study we found that GST P1-1 is also inhibited, in a concentration-dependent manner, by alpha- and gamma-tocotrienol. The concentration giving 50% inhibition of GST P1-1 is 1.8 +/- 0.1 microM for alpha-tocotrienol and 0.7 +/- 0.1 microM for gamma-tocotrienol. This inhibition of GST P1-1 is noncompetitive with respect to both substrates CDNB and GSH. We also examined the 3D structure of GST P1-1 for a possible tocopherol/tocotrienol binding site. The enzyme contains a very hydrophobic pit-like structure where the phytyl tail of tocopherols and tocotrienols could fit in. Binding of tocopherol and tocotrienol to this hydrophobic region might lead to bending of the 3D structure. In this way tocopherols and tocotrienols can inhibit the activity of the enzyme; this inhibition can have far-reaching implications for humans.     

Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Tyamagondlu V. Venkatesh, and Balasulojini Karunanandaa have equally contributed.     

A review of characterization of tocotrienols from plant oils and foods

Tocotrienols, members of the vitamin E family, are natural compounds found in a number of vegetable oils, wheat germ, barley and certain types of nuts and grains. Vegetable oils provide the best sources of these vitamin E forms, particularly palm oil and rice bran oil contain higher amounts of tocotrienols. Other sources of tocotrienols include grape fruit seed oil, oats, hazelnuts, maize, olive oil, buckthorn berry, rye, flax seed oil, poppy seed oil and sunflower oil. Tocotrienols are of four types, viz. alpha (α), beta (β), gamma (γ) and delta (δ). Unlike tocopherols, tocotrienols are unsaturated and possess an isoprenoid side chain. A number of researchers have developed methods for the extraction, analysis, identification and quantification of different types of vitamin E compounds. This article constitutes an in-depth review of the chemistry and extraction of the unsaturated vitamin E derivatives, tocotrienols, from various sources using different methods. This review article lists the different techniques that are used in the characterization and purification of tocotrienols such as soxhlet and solid–liquid extractions, saponification method, chromatography (thin layer, column chromatography, gas chromatography, supercritical fluid, high performance), capillary electrochromatography and mass spectrometry. Some of the methods described were able to identify one form or type while others could analyse all the analogues of tocotrienol molecules. Hence, this article will be helpful in understanding the various methods used in the characterization of this lesser known vitamin E variant.     

Does lack of tocopherols and tocotrienols put women at increased risk of breast cancer?

Breast cancer is the leading site of new cancers in women and the second leading cause (after lung cancer) of cancer mortality in women. Observational studies that have collected data for dietary exposure to alpha-tocopherol with or without the other related tocopherols and tocotrienols have suggested that vitamin E from dietary sources may provide women with modest protection from breast cancer. However, there is no evidence that vitamin E supplements confer any protection whatever against breast cancer. Observational studies that have assessed exposure to vitamin E by plasma or adipose tissue concentrations of alpha-tocopherol have failed to provide consistent support for the idea that alpha-tocopherol provides any protection against breast cancer. In addition, evidence from studies in experimental animals suggest that alpha-tocopherol supplementation alone has little effect on mammary tumors. In contrast, studies in breast cancer cells indicate that alpha- gamma-, and delta-tocotrienol, and to a lesser extent delta-tocopherol, have potent antiproliferative and proapoptotic effects that would be expected to reduce risk of breast cancer. Many vegetable sources of alpha-tocopherol also contain other tocopherols or tocotrienols. Thus, it seems plausible that the modest protection from breast cancer associated with dietary vitamin E may be due to the effects of the other tocopherols and the tocotrienols in the diet. Additional studies will be required to determine whether this may be the case, and to identify the most active tocopherol/tocotrienol.     

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.     

Tocopherols play a crucial role in low-temperature adaptation and Phloem loading in Arabidopsis

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.     

Tocotrienols in health and disease: the other half of the natural vitamin E family

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. Structurally, natural vitamin E includes eight chemically distinct molecules: -, β-, γ- and δ-tocopherol; and -, β-, γ- and δ-tocotrienol. Symptoms caused by -tocopherol deficiency can be alleviated by tocotrienols. Thus, tocotrienols may be viewed as being members of the natural vitamin E family not only structurally but also functionally. Palm oil and rice bran oil represent two major nutritional sources of natural tocotrienol. Taken orally, tocotrienols are bioavailable to all vital organs. The tocotrienol forms of natural vitamin E possesses powerful hypocholesterolemic, anti-cancer and neuroprotective properties that are often not exhibited by tocopherols. Oral tocotrienol protects against stroke-associated brain damage in vivo. Disappointments with outcomes-based clinical studies testing the efficacy of -tocopherol need to be handled with caution and prudence recognizing the untapped opportunities offered by the other forms of natural vitamin E. Although tocotrienols represent half of the natural vitamin E family, work on tocotrienols account for roughly 1% of the total literature on vitamin E. The current state of knowledge warrants strategic investment into investigating the lesser known forms of vitamin E.     

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10–20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K_m value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.     

The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.