Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress

Abstract

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from a-tocopherol to g-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.Vitamin E encompasses a class of lipid antioxidants consisting of four forms each of tocopherol and tocotrienol. Because of its high economical value and importance for human nutrition, much effort has been invested to elucidate the tocopherol biosynthetic pathway in plants and Cyanobacteria and to identify limiting steps by overexpression of candidate genes in transgenic plants. The hydroquinone ring of tocopherol is derived from the shikimate pathway of aromatic amino acid synthesis. Homogentisate, the precursor for the synthesis of tocopherol, tocotrienol, and plastoquinone, is synthesized by p-hydroxyphenylpyruvate dioxygenase (HPPD; Fig. 1; Norris et al., 1998) (Collakova et al., 2003a). Final methylation by g-tocopherol methyltransferase (g-TMT, VTE4) results in the production of a-tocopherol. a-Tocopherol is the predominant form in leaves, whereas g-tocopherol is most abundant in seeds of Arabidopsis (Shintani and DellaPenna, 1998). Side reactions of VTE1, VTE3, and VTE4 result in the production of minor tocopherol forms in Arabidopsis seeds or leaves (d-tocopherol, b-tocopherol; Fig. 1).Under oxidative stress, the amounts of different antioxidants strongly increase in plants, and it is believed that this results in an increased capacity to scavenge reactive oxygen species (Noctor and Foyer, 1998;Collakova et al., 2003b). The accumulation of antioxidants under stress is at least in part mediated by induction of gene expression. For example, HPPD and HPT1, two steps of tocopherol synthesis, were shown to be induced under light stress (Coll...

Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes

“…Furthermore, plastoquinone and phylloquinone are critical for photosynthetic function as electron acceptors and donors, whereas tocopherol is an important protector against oxidative damage to the thylakoid membrane (Havaux et al, 2005;Kanwischer et al, 2005). The presence of proteins somehow involved with lipid metabolism and/or breakdown is also consistent with the proposition that PGs form a lipid/fatty acid storage space (Fig.…”

The Influence of Diluted Seawater and Ripening Stage on the Content of Antioxidants in Fruits of Different Tomato Genotypes

“…This marked synthesis upon ripening in Gimar gf could be explained with the maintenance of residual chloroplasts (16) where tocopherols are synthesized and accumulate (34). Increases in R-tocopherol contents were also reported in leaves of rosemary, sage, and lemon balm subjected to water deficit (33) whereas a decrease was observed in wheat roots exposed to a high concentration of copper (35).…”

Tocopherol Cyclase (VTE1) Localization and Vitamin E Accumulation in Chloroplast Plastoglobule Lipoprotein Particles

“…The final destination of tocopherol is most likely the thylakoid membrane, where it has been shown to protect the photosystem II and to prevent the oxidative degradation of fatty acids by scavenging reactive oxygen species (72,73). Under high light stress conditions, levels of tocopherols increase (72), and VTE1 is up-regulated (45). The presence of VTE1 and tocopherols (this paper) together with the reported increase in size and number of plastoglobules (33)(34)(35) provides a molecular explanation for the role of plastoglobules under conditions resulting in oxidative stress.…”

“…I. Kubigsteltig (44)), chlorophyll a/b-binding protein (CAB; kindly provided by Dr. K. Apel, ETH Zurich, Switzerland), RuBisCO large subunit (kindly provided by Dr. S. Crafts-Brandner, United States Department of Agriculture, Phoenix, AZ), AtVTE1 (45) or AtTIC110 (46), or with affinitypurified antibodies specific for AtTOC75 (36) and AtPGL35 (this paper). Chlorophyll concentration was determined by the method of Arnon (47), and proteins were quantified following the Bradford method (48).…”