Linking tocopherols with cellular signaling in plants

Abstract

Hofius D, Hajirezaei M, Geiger M, Tschiersch H, Melzer M, Sonnewald U. 2004. Plant Physiology 135: 1256–1268. Access Plant Physiology at http://www.plantphysiol.org/ Tocopherols and tocotrienols, which differ in the degree of saturation of their hydrophobic prenyl chain, are lipophilic antioxidants that collectively constitute vitamin E, an essential dietary compound for humans and animals. Much has been revealed about the role of tocopherols in biological systems since Evans & Bishop (1922) discovered that they are indispensable dietary factors for reproduction in female rats. Although the antioxidant properties of tocopherols were not discovered until more than 40 years later (Epstein et al., 1966), dietary deficiencies of these antioxidants in humans and animals have been associated with numerous degenerative diseases and it has been shown that tocopherols may control membrane fluidity and contribute to cellular signaling in animals (Azzi et al., 1998; Brigelius-Flohéet al., 2002). Most studies on the function of tocopherols in plants have focused on their photoprotective and antioxidant roles, and it has recently been proposed that they could play a role in cellular signaling (Munné-Bosch & Alegre, 2002; Munné-Bosch & Falk, 2004). Now, Hofius et al. (2004) have shown that tocopherols may affect source–sink transitions and may alter gene expression in plants, and these authors provide the first steps towards understanding the role of tocopherols in plant cell signaling. By using a RNAi-mediated silencing approach towards sxd1, which encodes for tocopherol cyclase, Hofius et al. obtained transgenic potato plants deficient in tocopherols that show a photoassimilate export-deficient phenotype. These plants are characterized by callose occlusion of plasmodesmata in source leaves, and as a result, they show excess sugar accumulation and lower transcription of the photosynthesis-related rbcS and cab genes, which suggests a carbohydrate-mediated feedback inhibition on photosynthetic capacity. In addition, source leaves show enhanced accumulation of starch and anthocyanins, which may also be the result of excess sugar accumulation (Tsukaya et al., 1991; Mita et al., 1995). Furthermore, transgenic lines show enhanced accumulation of the transcripts of the defense-related proteinase inhibitor II (pin2), and of proline (p5cs) and jasmonic acid (aoc) biosynthesis, thus indicating that tocopherols may directly or indirectly affect gene expression significantly. A similar phenotype has been observed in the sxd1 mutant of maize, which is also deficient in tocopherols (Porfirova et al., 2002). This mutant shows a photoassimilate-deficient export phenotype with excess sugar accumulation but no alterations in photosynthesis-related gene expression in source leaves, which suggests that communication between the chloroplast and the nucleus is impaired (Provencher et al., 2001). In contrast to maize and potato plants, the photoassimilate export-deficient phenotype has not been observed in the vte1 mutant of Arabidopsis. This mutant was discovered during a screen for altered tocopherol content, and it has been shown that VTE1 and SXD1 represent single-copy orthologs, both encoding an enzyme with tocopherol cyclase activity (Porfirova et al., 2002). Despite the molecular and biochemical similarities of VTE1 and SXD1 in vte1 and sxd1 mutant leaves, the photoassimilate export-deficient phenotype is absent in vte1 Arabidopsis (Sattler et al., 2003). However, tocopherol deficiency leads to a slightly reduced growth and enhanced susceptibility to photooxidative stress in the vte1 Arabidopsis mutant (Porfirova et al., 2002). The absence of the photoassimilate export-deficient phenotype in Arabidopsis could be due to the fact that phloem loading in this species is primarily apoplastic, whereas it is symplastic in maize and potato (Van Bel, 1993). Tocopherol deficiency is therefore less apparent in Arabidopsis because it possesses few plasmodesmata between the sieve element–companion cell complex and the surrounding cells. Additionally, the effect of tocopherol deficiency on the photoassimilate export-deficient phenotype may be strongly modulated by growth conditions. Whereas experiments in maize and potato plants used individuals grown under a light intensity of 500 µmol m−2 s−1 or higher in growth chambers or glasshouses with supplemental light, the photoassimilate export-deficient phenotype in Arabidopsis has been tested only in plants grown in a growth chamber at low light (120 µmol m−2 s−1) and under a shorter photoperiod. Another factor to be considered is the plant development stage. Maize, potato and Arabidopsis plants are all annuals, in which flowering plays a key role in source–sink transition and the progression of leaf senescence. Because tocopherol deficiency blocks photoassimilate export in source leaves, the effects of this deficiency might be more apparent as the plants age and the flowering transition is initiated. Compelling evidence indicates that the phenotypes described to date for tocopherol deficiency, characterized by reduced growth, photoassimilate export deficiency and higher susceptibility to photooxidation (i) are species-specific, (ii) depend on the extent of tocopherol deficiency, (iii) depend on growth conditions (light intensity, photoperiod, etc.) and plant developmental stage, and (iv) are more evident as growth conditions become more stressful (Porfirova et al., 2002; Hofius et al., 2004; Munné-Bosch & Falk, 2004). Although direct evidence to explain the mechanism(s) of action of tocopherols in regulating signal transduction and gene expression in plants has not been provided so far, tocopherols are part of an intricate signaling network controlled by reactive oxygen species (ROS), antioxidants and phytohormones, and are therefore good candidates to influence plant cell signaling. Tocopherols, ascorbate and glutathione are interdependent in the control of ROS levels (Asada, 1999; Kanwischer et al., 2005), and ROS are crucial modulators of gene expression in chloroplasts (Apel & Hirt, 2004) and the nucleus (Vandenabeele et al., 2003). Furthermore, ROS and hormones interact in the regulation of signal transduction and gene expression in plants (Pastori & Foyer, 2002). Although several tocopherol-induced changes in gene expression observed in transgenic potato plants may be indirect and may result from excess sugar accumulation (Gibson, 2005), tocopherols may be linked to the plant cell signaling network and may give rise to the observed phenotype through other mechanisms (Fig. 1). Proposed model showing the interactions between α-tocopherol, ROS and phytohormones in cellular signaling. OH•, 1O2, tocopherol, jasmonic acid and salicylic acid may directly or indirectly regulate gene expression. JA, jasmonic acid; PUFA, polyunsaturated fatty acid; SA, salicylic acid. Although studies on animal models suggest direct nonantioxidant roles for tocopherols in the regulation of gene expression (Azzi et al., 1998; Brigelius-Flohéet al., 2002), data available in plants tend to favor a direct role of tocopherols in regulating the cell redox homeostasis rather than directly regulating gene expression. Enhanced callose synthesis, and the consequent occlusion of specific plasmodesmata, is the most significant characteristic of the photoassimilate export-deficient phenotype in tocopherol-deficient plants. Callose formation is often attributed to ROS-derived signalling and it is strongly correlated with oxidative damage to membrane lipids (Yamamoto et al., 2001), indicating a mechanistic links between ROS, lipid peroxidation and callose synthesis. It has therefore been proposed that tocopherol deficiency indirectly affects callose synthesis by increasing the extent of lipid peroxidation in the chloroplast membranes (Hofius et al., 2004). By controlling ROS levels and the extent of lipid peroxidation, and therefore the hydroperoxide content in chloroplasts, tocopherols may indirectly regulate the amounts of jasmonic acid in leaves and may affect jasmonic acid-dependent gene expression. In addition to enhanced callose synthesis, tocopherol-deficient plants show increased accumulation of jasmonic acid-responsive pin2 transcripts and up-regulation of the jasmonic acid biosynthetic gene aoc, which supports this contention. Jasmonic acid also regulates the expression of anthocyanin biosynthetic genes (Creelman & Mullet, 1997), which further supports the relationship between tocopherol deficiency and anthocyanin accumulation in the source leaves of plants with the photoassimilate export-deficient phenotype. Other hormones involved in stress and ROS signaling, such as salicylic acid, are also strongly correlated with tocopherol levels in plants (Munné-Bosch & Peñuelas, 2003a) and may regulate callose synthesis (Ostergaard et al., 2002). The expression of the genes encoding for tyrosine aminotransferase (tat) and p-hydroxyphenylpyruvate dioxygenase (hpd), which catalyze the transamination from tyrosine to p-hydroxyphenylpyruvate and its conversion to homogentisate in tocopherol biosynthesis, respectively, is regulated by jasmonic acid (Falk et al., 2002; Sandorf & Holländer-Czytko, 2002). Jasmonic acid is synthesized as a result of lipid peroxidation in chloroplasts, its synthesis takes place in chloroplasts, cytoplasm and peroxisomes, and it regulates gene expression in the nucleus (Creelman & Mullet, 1997). α-Tocopherol could therefore modulate its own synthesis by regulating lipid peroxidation in chloroplasts and jasmonic acid contents within the cell and therefore modulate the expression of tat and hpd. Other hormones that may be involved in the regulation of tocopherol biosynthesis include abscisic acid. An abscisic acid-specific motif has been identified in the promoter region of the hpd gene, which indicates that tocopherol biosynthesis may be stimulated by this compound (J. Falk, University of Kiel, pers. comm.). Further research is needed for a better understanding of the interactions between tocopherols and phytohormones in plants. Tocopherol levels change significantly during plant growth and development and in response to stress as a result of the altered expression of pathway-related genes, degradation and recycling. By quenching and scavenging ROS and reducing the extent of lipid peroxidation in membranes, tocopherols participate in regulating the extent of oxidative stress, either associated with leaf senescence or stress tolerance. The response of tocopherols during leaf senescence is characterized by two phases (Chrost et al., 1999; Munné-Bosch & Peñuelas, 2003b). In the first phase, there is an increase in tocopherol synthesis, which is followed by a second phase of net tocopherol loss. It has been suggested that initial enhanced tocopherol levels contribute to the high photoprotection required during the first phase of leaf senescence, in which protein degradation occurs slowly and protection of photosynthetic membranes is required to optimize nutrient remobilization from chloroplasts (Munné-Bosch & Peñuelas, 2003b). However, increases in tocopherols at this phase may have additional roles related to cellular signaling. On the basis of the results obtained by Hofius et al. (2004), it may be hypothesized that tocopherols may affect leaf senescence by modulating source–sink transitions. Enhanced tocopherol levels may limit lipid peroxidation and callose synthesis, thus avoiding the occlusion of plasmodesmata and allowing photoassimilate export from senescing leaves (source) to young growing leaves or reproductive organs (sinks). When the senescence-induced nutrient remobilization has been accomplished, tocopherol degradation exceeds its synthesis and levels decrease (phase II). Consequently, callose synthesis increases and the occlusion of plasmodesmata prevents further export of photoassimilates. A number of environmental stress factors have been associated with an increase in photosynthesis-derived ROS, and the levels of tocopherols and other antioxidants have been related to stress tolerance. Whereas stress-tolerant plants usually display increased tocopherol levels, the most sensitive ones show net tocopherol loss under stress, which leads to oxidative damage and cell destruction (reviewed in Munné-Bosch & Alegre, 2002). However, the function of tocopherols might be more complex, especially considering their role in cellular signaling. In plant responses to stress, increased tocopherol levels may not only increase photoprotection but also favor photoassimilate export from sources to sinks. In addition, a decrease in tocopherols may not necessarily lead to irreversible injuries, but to the activation of genes responsible for proline and anthocyanin synthesis, as observed in tocopherol-deficient plants (Hofius et al., 2004). Proline and anthocyanins effectively protect plants from a number of environmental stress factors (e.g. drought, salt stress, high light) and may serve a protective role by compensating for tocopherol deficiency. The recent findings on tocopherol-deficient plants indicating a role for these compounds in redox signaling open a new field for tocopherol research in plant biology. The elucidation of signal transduction pathways and gene expression modulated by tocopherols in plants will probably bring some of the most exciting discoveries in the near future and will provide us with a better understanding of the role of these compounds in plant development and stress tolerance. Further research is needed to understand better the senescence and stress responses in tocopherol-deficient plants, the link between tocopherol deficiency and changes in photoassimilate transport, and the function of different tocopherol forms in plants. I thank Kozi Asada, Jon Falk, Leonor Alegre and other colleagues for their helpful discussions during the preparation of the manuscript. Three anonymous reviewers are also gratefully acknowledged for their comments. This work was partly supported by the Spanish Government (grant no. BOS2003-01032).