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 ␥-tocopherol content. This enlarged pool of ␥-tocopherol was almost entirely converted to ␣-tocopherol by crossing HPT1 overexpressing plants with lines constitutively overexpressing ␥-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 ␥-tocopherol methyltransferase genes.Tocopherols, collectively known as vitamin E, are a class of lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms. Tocopherols are essential components of the human diet because they perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radicals and protecting polyunsaturated fatty acids from lipid peroxidation (Fukuzawa and Gebicky, 1983;Neely et al., 1988; Fryer, 1993; Bramley et al., 2000). Because of these and other activities, dietary tocopherols are thought to play an important role in improving immune function and in limiting the incidence and progression of several degenerative human diseases including certain types of cancer, cataracts, neurological disorders, and cardiovascular disease (Brigelius-Flohe and Traber, 1999; Bramley et al., 2000;Pryor, 2000).In plants, indirect evidence suggests that tocopherols perform antioxidant and radical quenching functions similar to those in animals (Fryer, 1992) and that tocopherols may have additional roles related to photosynthesis (Munne-Bosch and Alegre, 2002). Plants alter their tocopherol levels during development (Molina-Torres and Martinez, 1991;Tramontano et al., 1992) and in response to a variety of stresses, including high-light, low-temperature, drought, and salt stress (Gossett et al., 1994;Streb et al., 1998; Leipner et al., 1999; Havaux et al., 2000;Munne-Bosch and Alegre, 2000). In addition, during leaf senescence, a process accompanied by chlorophyll degradation and oxid...

Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632-642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18-and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2-to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of ␤-, ␥-, and ␦-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit ␣-tocopherol synthesis. Overexpression of ␥-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of ␥-and ␦-tocopherols to ␣-and ␤-tocopherols, respectively, indicating that ␥-tocopherol methyltransferase activity limits ␣-tocopherol synthesis in stressed leaves.Tocopherols are a group of lipid soluble antioxidants collectively known as vitamin E that are essential components of animal diets. Dietary vitamin E is required for maintaining proper muscular, immune, and neural function and may be involved in reducing the risk of cancer, cardiovascular disease, and cataracts in humans (Pryor, 2000;Brigelius-Flohe et al., 2002). In plants, tocopherols are believed to protect chloroplast membranes from photooxidation and help to provide an optimal environment for the photosynthetic machinery (Fryer, 1992;Munne-Bosch and Alegre, 2002a). Many of the proposed tocopherol functions in animals and plants are related to their antioxidant properties, the most prominent of which is protection of polyunsaturated fatty acids from lipid peroxidation by quenching and scavenging various reactive oxygen species (ROS) including singlet oxygen, superoxide radicals, and alkyl peroxy radicals (Fukuzawa and Gebicky, 1983;Munne-Bosch and Alegre, 2002a).Tocopherols are only synthesized by photosynthetic organisms and consist of a polar chromanol ring and a 15-carbon lipophilic prenyl chain derived from homogentisic acid (HGA) and phytyl diphosphate (PDP; Fig. 1). In plants, HGA is formed from p-hydroxyphenyl pyruvate (HPP) by the c...

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.Tocopherols are a group of amphipathic compounds synthesized only by photosynthetic organisms. The best characterized and probably most important function of tocopherols is to act as recyclable chain reaction terminators of polyunsaturated fatty acid free radicals generated by lipid oxidation. Tocopherols have a well-documented role in mammals both as an essential nutrient (vitamin E) and general antioxidant (Fryer, 1993; Liebler, 1998; BrigeliusFlohe and Traber, 1999). A similar though less welldocumented antioxidant role is also proposed for tocopherols in photosynthetic organisms (Fryer, 1992; Niyogi, 1999).From a biosynthetic perspective, tocopherols are members of a large, multifunctional family of lipidsoluble compounds called prenylquinones that also include tocotrienols, plastoquinones, and phylloquinones (vitamin K 1 ). Structural features shared by all prenylquinones include hydrophobic prenyl tails of various lengths attached to aromatic head groups that can reversibly change their redox states. Tocopherols contain a chromanol head-group and lipophillic tail derived from the 20-carbon alcohol phytol, whereas plastoquinones contain a quinone head group and isoprenoid tails of 40, 45, or 50 carbons. Such structural features are essential for the diverse biochemical and physiological roles fulfilled by various prenylquinones.The committed step in the synthesis of all prenylquinones is the condensation of various aromatic precursors and prenyl-diphosphate (DP) substrates in reactions catalyzed by a small family of related polyprenyltransferases (Lopez et al., 1996). Most aromatic and prenyl-DP substrates are utilized by more than one polyprenyltransferase (Fig. 1). For example, the aromatic compound homogentisic acid (HGA) is used for condensation with phytyl DP (PDP), geranylgeranyl DP (G...

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.Tocopherols are a group of amphipathic compounds synthesized only by photosynthetic organisms. The best characterized and probably most important function of tocopherols is to act as recyclable chain reaction terminators of polyunsaturated fatty acid free radicals generated by lipid oxidation. Tocopherols have a well-documented role in mammals both as an essential nutrient (vitamin E) and general antioxidant (Fryer, 1993; Liebler, 1998; BrigeliusFlohe and Traber, 1999). A similar though less welldocumented antioxidant role is also proposed for tocopherols in photosynthetic organisms (Fryer, 1992; Niyogi, 1999).From a biosynthetic perspective, tocopherols are members of a large, multifunctional family of lipidsoluble compounds called prenylquinones that also include tocotrienols, plastoquinones, and phylloquinones (vitamin K 1 ). Structural features shared by all prenylquinones include hydrophobic prenyl tails of various lengths attached to aromatic head groups that can reversibly change their redox states. Tocopherols contain a chromanol head-group and lipophillic tail derived from the 20-carbon alcohol phytol, whereas plastoquinones contain a quinone head group and isoprenoid tails of 40, 45, or 50 carbons. Such structural features are essential for the diverse biochemical and physiological roles fulfilled by various prenylquinones.The committed step in the synthesis of all prenylquinones is the condensation of various aromatic precursors and prenyl-diphosphate (DP) substrates in reactions catalyzed by a small family of related polyprenyltransferases (Lopez et al., 1996). Most aromatic and prenyl-DP substrates are utilized by more than one polyprenyltransferase (Fig. 1). For example, the aromatic compound homogentisic acid (HGA) is used for condensation with phytyl DP (PDP), geranylgeranyl DP (G...

HighlightUMAMIT14, a member of the Usually Multiple Acids Move In and out Transporters 14 family of amino acid transporters, is involved in unloading amino acids from the phloem in roots in addition to a previously described role in seed loading.

5-Formyltetrahydrofolate (5-CHO-THF) is formed viaa second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 mol of CO 2 mol ؊1 ) and non-photorespiratory (3200 mol of CO 2 mol ؊1 ) conditions. The mutation had only mild visible effects at 370 mol of CO 2 mol ؊1 , reducing growth rate by ϳ20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 mol of CO 2 mol ؊1 , the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants. 5-Formyltetrahydrofolate (5-CHO-THF)1 is formed from 5,10-methenyltetrahydrofolate (5,10-CHϭTHF) by a hydrolytic reaction catalyzed by serine hydroxymethyltransferase (SHMT) in the presence of glycine (1, 2). Spontaneous chemical hydrolysis of 5,10-CHϭTHF may be a minor additional source (3). 5-CHO-THF is the most stable natural folate and the most enigmatic, for it is the only one that does not serve as a cofactor in one-carbon metabolism. Instead, 5-CHO-THF is a potent inhibitor of SHMT and most other folate-dependent enzymes in vitro (4, 5). 5-CHO-THF probably acts as a stable storage form of folate in seeds and fungal spores (5-7), but it is not clear what role, if any, it plays in metabolically active tissues (8). This question is particularly pertinent for leaves. Leaf mitochondria have very high levels of SHMT and, during photorespiration, receive a massive influx of glycine (which leads to a matching SHMT-mediated glycine 3 serine flux) (9). Conditions in leaf mitochondria therefore favor 5-CHO-THF formation (Fig. 1). Indeed, 5-CHO-THF can comprise 50% of the folate pool in leaf mitochondria (10, 11), which is far more than in mammalian mitochondria (12)(13)(14). Furthermore, 5-CHO-THF is reported to make up 14 -40% of the folate pool in leaves and o...

In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5-and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.

Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.