Progress in the dissection and manipulation of plant vitamin E biosynthesis

Plants contain many unique biosynthetic pathways producing a diverse array of natural products that are important for plant function, agriculture, and human nutrition. The tocochromanols define one such class of compounds, comprised of four tocopherols and four tocotrienols that are collectively termed vitamin E. Tocochromanols are synthesized only by plants and other oxygenic, photosynthetic organisms, and the eight individual compounds vary widely in their vitamin E activities. Vitamin E was recognized as an essential component in mammalian diets in the 1920s and the tocochromanol biosynthetic pathway elucidated from radiotracer studies in the mid 1980s. However, it is only recently that genetic and genomics-based approaches in model photosynthetic organisms have allowed the genes and proteins for tocochromanol synthesis to be isolated, setting the stage for targeted manipulation of tocochromanol levels and types in various crops. This article reviews advancements in our molecular and genetic understanding of the tocochromanol biosynthetic pathway in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803 and highlights ongoing efforts to use this knowledge to manipulate the levels of this essential nutrient in food crops.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

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.     

Tocopherol functions in photosynthetic organisms

During the past decade, the genes required for tocopherol (vitamin E) synthesis in plants and cyanobacteria have been identified. A series of mutants in which specific pathway steps are disrupted have been generated, providing new insights into tocopherol functions in photosynthetic organisms. Tocopherols are essential for controlling non-enzymatic lipid peroxidation during seed dormancy and seedling germination. Their absence results in elevated levels of malondialdehyde and phytoprostanes, and in inappropriate activation of plant defense responses. Surprisingly, tocopherol deficiency in mature leaves has limited consequences under most abiotic stresses, including high intensity light stress. The cell wall development of phloem transfer cells under cold conditions is, however, severely impaired in mature leaves of tocopherol-deficient mutants, indicating that tocopherols are required for proper adaptation of phloem loading at low temperatures.     

Microbiological production of tocopherols: current state and prospects

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.     

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.     

Progress in the dissection and manipulation of vitamin E synthesis

Tocochromanols are a group of four tocopherols and four tocotrienols that collectively constitute vitamin E, an essential nutrient in the human diet. Tocochromanols are only synthesized in oxygenic photosynthetic organisms. Although they have similar antioxidant activities in vitro, the individual tocochromanols vary widely in their in vivo vitamin E activities. During the past several years, mutant and transgenic approaches in model organisms, particularly in Arabidopsis thaliana and Synechocystis sp. PCC6803, have allowed the full complement of core pathway genes to be isolated and studied. Pathway engineering with these genes has provided significant insights into the molecular genetic and biochemical control of tocochromanol biosynthesis in plants. The resulting knowledge base is driving efforts to manipulate the levels of this essential nutrient in staple crops for populations in the developed and developing world.     

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.     

Inactivation of genes,encoding tocopherol biosynthetic pathway enzymes,results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and γ-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.     

Functional diversity of tocochromanols in plants

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. They accumulate in photooxidative organisms, e.g. in some algae and in plants, where they localize to thylakoid membranes and plastoglobules of chloroplasts. Tocochromanols contain a polar chromanol head group with a long isoprenoid side chain. Depending on the nature of the isoprenoid chain, tocopherols (containing a phytyl chain) or tocotrienols (geranylgeranyl chain) can be distinguished in plants. The tocochromanol biosynthetic pathway has been studied in Arabidopsis and Synechocystis in recent years, and the respective mutants and genes were isolated. Mutant characterization revealed that tocopherol protects lipids in photosynthetic membranes and in seeds against oxidative stress. In addition to its antioxidant characteristics, tocopherol was shown be involved in non-antioxidant functions such as primary carbohydrate metabolism. A considerable proportion of tocopherol is synthesized from free phytol suggesting that excess amounts of phytol released from chlorophyll breakdown during stress or senescence might be deposited in the form of tocopherol in chloroplasts.     

Tocotrienols in Vellozia gigantea leaves: occurrence and modulation by seasonal and plant size effects

Vitamin E occurs in all photosynthetic organisms examined to date. Tocopherols predominate in photosynthetic tissues (α-tocopherol being the major form), while either tocopherols or tocotrienols (or both) are present in seeds. Tocotrienols have not been described in photosynthetic tissues thus far. Here, we report on the presence of tocotrienols in leaves of higher plants. Both tocopherols and tocotrienols accumulated in leaves of Vellozia gigantea, an endemic plant found in the rupestrian fields of Serra do Cipó, Brazil. Increased plant size had a remarkable effect on the vitamin E composition of leaves, α-tocopherol and β-tocotrienol levels being highest in the largest individuals, but only during the dry season. Vitamin E levels positively correlated with lipid hydroxyperoxide levels, which also increased in the largest individuals during the dry season. However, the maximum efficiency of PSII photochemistry (F _v/F _m ratio) kept above 0.75 throughout the experiment, thus indicating absence of photoinhibitory damage to the photosynthetic apparatus. It is concluded that higher plants, such as V. gigantea, can accumulate tocotrienols in leaves, aside from tocopherols, and that the levels of both tocopherols and tocotrienols in the leaves of this species are strongly modulated by seasonal and plant size effects.     

Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function

Tocopherols are lipophilic antioxidants synthesized exclusively by photosynthetic organisms and collectively constitute vitamin E, an essential nutrient for both humans and animals. Tocopherol cyclase (TC) catalyzes the conversion of various phytyl quinol pathway intermediates to their corresponding tocopherols through the formation of the chromanol ring. Herein, the molecular and biochemical characterization of TCs from Arabidopsis (VTE1 [VITAMIN E 1]), Zea mays (SXD1 [Sucrose Export Deficient 1]) and Synechocystis sp. PCC6803 (slr1737) are described. Mutations in the VTE1, SXD1, or slr1737 genes resulted in both tocopherol deficiency and the accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), a TC substrate. Recombinant SXD1 and VTE1 proteins are able to convert DMPBQ to gamma-tocopherol in vitro. In addition, expression of maize SXD1 in a Synechocystis sp. PCC6803 slr1737 knockout mutant restored tocopherol synthesis, indicating that TC activity is evolutionarily conserved between plants and cyanobacteria. Sequence analysis identified a highly conserved 30-amino acid C-terminal domain in plant TCs that is absent from cyanobacterial orthologs. vte1-2 causes a truncation within this C-terminal domain, and the resulting mutant phenotype suggests that this domain is necessary for TC activity in plants. The defective export of Suc in sxd1 suggests that in addition to presumed antioxidant activities, tocopherols or tocopherol breakdown products also function as signal transduction molecules, or, alternatively, the DMPBQ that accumulates in sxd1 disrupts signaling required for efficient Suc export in maize.     

Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes

Tocopherols are lipid-soluble compounds synthesized only by photosynthetic eukaryotes and oxygenic cyanobacteria. The pathway and enzymes for tocopherol synthesis are homologous in cyanobacteria and plants except for 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ MT), which catalyzes a key methylation step in both tocopherol and plastoquinone (PQ) synthesis. Using a combined genomic, genetic, and biochemical approach, we isolated and characterized the VTE3 (vitamin E defective) locus, which encodes MPBQ/MSBQ MT in Arabidopsis. The phenotypes of vte3 mutants are consistent with the disruption of MPBQ/MSBQ MT activity to varying extents. The ethyl methanesulfonate-derived vte3-1 allele alters tocopherol composition but has little impact on PQ levels, whereas the null vte3-2 allele is deficient in PQ and alpha- and gamma-tocopherols. In vitro enzyme assays confirmed that VTE3 is the plant functional equivalent of the previously characterized MPBQ/MSBQ MT (Sll0418) from Synechocystis sp PCC6803, although the two proteins are highly divergent in primary sequence. Sll0418 orthologs are present in all fully sequenced cyanobacterial genomes, Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana but absent from vascular and nonvascular plant databases. VTE3 orthologs are present in all vascular and nonvascular plant databases and in C. reinhardtii but absent from cyanobacterial genomes. Intriguingly, the only prokaryotic genomes that contain VTE3-like sequences are those of two species of archea, suggesting that, in contrast to all other enzymes of the plant tocopherol pathway, the evolutionary origin of VTE3 may have been archeal rather than cyanobacterial. In vivo analyses of vte3 mutants and the corresponding homozygous Synechocystis sp PCC6803 sll0418::aphII mutant revealed important differences in enzyme redundancy, the regulation of tocopherol synthesis, and the integration of tocopherol and PQ biosynthesis in cyanobacteria and plants.     

Tocopherols and associated derivatives track the phytoplanktonic response to evolving pelagic redox conditions spanning Oceanic Anoxic Event 2

Tocopherols serve a critical role as antioxidants inhibiting lipid peroxidation in photosynthetic organisms, yet are seldom used in geobiological investigations. The ubiquity of tocopherols in all photosynthetic lifeforms is often cited as an impediment to any diagnostic paleoenvironmental potential, while the inability to readily analyze these compounds via conventional methods, such as gas chromatography–mass spectrometry, further diminishes the capacity to serve as useful ‘biomarkers’. Here, we analyzed an exceptionally preserved black shale sequence from the Demerara Rise that spans Oceanic Anoxic Event 2 (OAE-2) to reexamine the significance of tocopherols and associated derivatives (i.e. tocol derivatives) in ancient sediments. Tocol derivatives were analyzed via liquid chromatography–quadrupole time-of-flight–mass spectrometry and included tocopherols, a methyltrimethyltridecylchroman, and the first reported detection of tocopherol quinones and methylphytylbenzoquinones in the geologic record. Strong correlations between tocol derivatives were observed over the studied interval. Tocol derivative concentrations and ratios, which normalized tocopherols to potential derivatives, revealed absolute and relative increases in tocopherols as exclusive features of OAE-2 that can be explained by two possible mechanisms related to tocopherol production and preservation. The development of photic zone euxinia during OAE-2 likely forced an upward migration of oxygenic photoautotrophs, increasing oxidative stress that elicited heightened tocopherol biosynthesis. However, shoaling euxinic conditions may have simultaneously acted to enhance tocopherol preservation given the relatively high lability of tocopherols in the water column. Both scenarios could produce the observed stratigraphic distribution of tocol derivatives in this study, although the elevated tocopherol concentrations that define OAE-2 at the Demerara Rise are primarily attributed to enhanced tocopherol production by shoaling phytoplanktonic communities. Thus, the occurrence of tocopherols and associated derivatives in sediments and rocks of marine origin is likely indicative of shallow-water anoxia, tracking the phytoplanktonic response to the abiotic stresses associated with vertical fluctuations in pelagic redox.     

α-Tocopherol may influence cellular signaling by modulating jasmonic acid levels in plants

Most studies on the function of tocopherols in plants have focused on their photo-protective and antioxidant properties, and it has been recently suggested, though not yet demonstrated, that they may also play a role in cellular signaling. By using vte1 mutants of Arabidopsis thaliana, with an insertion in the promoter region of the gene encoding tocopherol cyclase, we demonstrate here for the first time that tocopherol deficiency may alter endogenous phytohormone levels in plants, thereby reducing plant growth and triggering anthocyanin accumulation in leaves. In plants grown under a combination of high light and low temperature conditions to induce anthocyanin accumulation, we evaluated age-dependent changes in tocopherols, indicators of photo-oxidative stress, phytohormone levels, plant growth and anthocyanin levels in wild type and vte1 mutants. These mutants showed lower tocopherol levels, reduced growth and enhanced anthocyanin accumulation compared with the wild type, while both the maximum and relative efficiencies of PSII, chlorophylls, and carotenoids were not significantly altered. Analyses of phytohormone levels revealed that reduced growth and enhanced anthocyanin accumulation in tocopherol-deficient plants were preceded by increased jasmonic acid levels. This is the first study suggesting a direct effect of tocopherols on phytohormones levels in plants and will undoubtedly help us to better understand the multiple functions tocopherols play in plants, as well as the cellular signaling mechanisms responsible for the phenotypes thus far described in tocopherol-deficient plants.     

The role of homogentisate phytyltransferase and other tocopherol pathway enzymes in the regulation of tocopherol synthesis during abiotic stress

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 beta-, gamma-, and delta-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit alpha-tocopherol synthesis. Overexpression of gamma-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of gamma- and delta-tocopherols to alpha- and beta-tocopherols, respectively, indicating that gamma-tocopherol methyltransferase activity limits alpha-tocopherol synthesis in stressed leaves.     

Current opinions on the functions of tocopherol based on the genetic manipulation of tocopherol biosynthesis in plants

As a member of an important group of lipid soluble antioxidants,tocopherols play a paramount role In the daily diet of humans and animals.Recently,genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp.PCC 6803.At the basis of these foundations,genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol.In addition,genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems.The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants.The roles of tocopherol in the network of reactive oxygen species,antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed.     

Methyl jasmonate differentially affects tocopherol content and tyrosine amino transferase activity in cultured cells of Amaranthus caudatus and Chenopodium quinoa

Tocopherols are lipid-soluble compounds synthesised exclusively by photosynthetic organisms. In this study, in vitro callus cultures were established from two plants that are naturally rich in tocopherols, Amaranthus caudatus and Chenopodium quinoa, in order to examine whether callus cultures were able to produce these compounds at levels comparable to those observed in planta . In both species, cotyledon explants produced the best callus induction and, once established, callus cultures were grown under two different hormonal treatments to check for effects of growth and to induce chloroplast differentiation in the cells. A rapid differentiation of chloroplasts occurred only in C. quinoa cell aggregates grown in the presence of benzyladenine, leading to the production of a homogeneous green callus. In both species, only α-tocopherol was produced by callus cultures, although levels were much lower than in planta, and the production was not influenced by the hormonal conditions. Interestingly, cell cultures of the two species responded in different ways to methyl jasmonate (MJ). In A. caudatus cultures, treatment with 100 μ m MJ increased the production of α-tocopherol up to fivefold, and the inductive effect was influenced by the hormonal composition of the medium. This increase in α-tocopherol was associated with a proportional increase in tyrosine aminotransferase (TAT) activity, one of the key enzymes involved in tocopherol biosynthesis. By contrast, in C. quinoa cultures, elicitation with MJ did not have any effect, neither on tocopherol production, nor on TAT activity. These results are discussed in relation to chloroplast differentiation and the interplay between jasmonates and phytohormones.     

Expression of tobacco tocopherol cyclase in rice regulates antioxidative defense and drought tolerance

Tocopherols (α-, β-, γ-, and δ-tocopherol) represent a group of lipophilic antioxidants that are synthesized only by photosynthetic organisms. It is widely believed that the main functions of tocopherols are protection of pigments and proteins of photosystem and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species. In the present study, we report on the cloning and characterization of NtTC, which is a tocopherol cyclase (TC) ortholog isolated from tobacco. To enhance tocopherol contents, we generated independent transgenic rice events in expressing NtTC or NtTC along with Perilla γ-tocopherol methyltransferase genes. The transgenic TC line significantly increased α-, total tocopherol, total glutathione, and total antioxidant status activity levels compared with the wild type. Furthermore, TC rice plants showed higher tolerance to drought than wild-type rice plants. On the basis of these studies, we concluded that overexpression of NtTC could increase the tolerance to drought stress and that the increase in tocopherol affects cellular signaling and antioxidant defense of plants in response to drought.