Are there Specific In Vivo Roles for α- and γ-Tocopherol in Plants?

Abstract

Tocopherols belong to the Vitamin E family of amphiphilic antioxidants, together with the subfamily of tocotrienols. They are exclusively synthesized by photosynthetic organisms and consist of a polar chromanol head group and a lipophilic prenyl tail.

The Vitamin E pool in dicots is commonly dominated by α-tocopherol in leaves and by γ-tocopherol in seeds. This observation rises the question, whether α-tocopherol and γ-tocopherol are functionally equivalent in protection against various kinds of oxidative stress in planta: superoxide and singlet oxygen evolution are high during oxygenic photosynthesis in leaves, while polyunsaturated fatty acid oxidation is the main target for tocopherols in seeds.

We found that transgenic tobacco plants with a substitution of γ- for α-tocopherol in leaves are more tolerant than the wild type towards sorbitol and methyl viologen mediated oxidative stress, which increase lipid peroxidation in the chloroplast stroma. This suggests that γ-tocopherol is more potent than α-tocopherol in protecting against lipid peroxidation in both, seeds and leaves, although its natural abundance is in seeds only. If so, why has α-tocopherol accumulation in leaves been favoured during the evolution of land plants and does the abundance of γ-tocopherol in leaves conceal a disadvantage for plant fitness?

Tocopherols and tocotrienols are amphiphilic antioxidants that are commonly addressed as tocochromanols and belong to the Vitamin E family. Structurally, tocochromanols consist of a polar chromanol head, a derivative of the shikimate pathway, and a prenyl tail originating from plastidic isoprenoid biosynthesis. While tocopherols and tocotrienols can be recognized by the saturation of the prenyl moiety, α-, β, γ- and δ-tocochromanols are distinguished by the methylation pattern of the chromanol head.

Tocopherols are integrated in the cellular antioxidant network, which protects plant cells from oxidative damage that is predominantly caused by ROS (reactive oxygen species). ROS are produced in abiotic and biotic stress either (1) nonenzymatically by molecular oxygen in uncontrolled spontaneous oxidation reactions of cellular compounds or (2) enzymatically controlled after the activation of defense or stress related oxygenases and oxidases.¹ Although the soluble antioxidants ascorbic acid (Vitamin C) and glutathione span all subcellular compartments,² the capacity for ROS detoxification needs to be highest in chloroplasts, where the basic, nonenzymatic rate of ROS production is constantly high during oxygenic photosynthesis and even elevated during high light exposure. Within chloroplasts, the interplay of the soluble antioxidants ascorbate and glutathione with the amphiphilic, membrane-bound antioxidant tocopherol permits the removal of reactive species from both, stroma and thylakoid membranes.³ Several studies have shown that a deficiency in tocopherol in combination with other antioxidant deficiencies, e.g., Vitamin C,⁴ glutathione⁴ or the xanthophyll cycle^5,6 increase the light sensitivity of Arabidopsis, while single mutants did not show pronounced sensitivity towards light,^4–6 emphasizing the cooperativity of the individual components of the antioxidant network in ameliorating oxidative stress.

In planta, tocopherols can exert their antioxidant function in two different ways (reviewed in ref. 7). First, they can serve as electron donors in redox reactions with ROS-generated lipid peroxyl radicals, generating tocopheryl radicals (Toc^•), which can be regenerated by the ascorbate pool (Fig. 1A). Second, reactive singlet oxygen (¹O₂), which evolves after excitation of molecular oxygen by triplett chlorophyll (³Chl) in the PSII reaction centers can be retransformed to ground state triplet oxygen by intersystem spin crossing with tocopherol (Fig. 1B). During excess illumination of chloroplasts, i.e., high light stress, singlet oxygen evolution in the PSII reaction centers and superoxide generation by the Mehler reaction at PSI occur at high rates, representing the major source of ROS. In desiccated seeds, lipid peroxyl radicals, which originate from the reaction of molecular oxygen with polyunsaturated fatty acids (PUFAs), are the dominant ROS species.

Figure 1.

Detoxification of RoS (A) and singlet oxygen (B) by tocopherols in chloroplasts. (A) Superoxide and downstream RoS—exemplified by OH^•—attack polyunsaturated fatty acids (PUFAs) in thylakoid lipids, generating lipid radicals (PUFA^•). Upon addition of molecular oxygen, these PUFA^• react to lipid peroxyl radicals (PUFA-OO^•). Both PUFA^• and PUFA-OO^• can propagate the radical onto other PUFAs, producing either another PUFA radical or a lipid peroxide (Lipid-OOH), which is unstable and eventually disintegrates to harmless hydroxyl fatty acids. Tocopherols (Toc) can act as chain breaker by donating a hydrogen atom to lipid radicals and the formed tocopheryl radical can be quenched by the ascorbate (AsA) pool and the action of monodeydroascorbate (MDHA) reductase. (B) When the plastoquinone pool is close to full reduction and no electron acceptor for excited P680⁺ reaction centers is available, ³P680 is formed, which can transfer its excitation energy onto molecular triplet oxygen, producing singlet oxygen (¹O₂). Tocopherols are able to quench ¹O₂ by intersystem spin crossing, dissipating the transferred energy as heat. During this process, one in 120 tocopherol molecules will be oxidized to tocopherol quinone (TQ),²⁴ which reportedly dampens electron flow through PSII.²⁵ O_2,⁻ superoxide anion; OH^•, hydroxyl radical; PUFA^•, polyunsaturated fatty acid/ lipid radical, PUFA-OO^•, polyunsaturated peroxyl fatty acid/lipid peroxyl radical; Lipid-OOH, lipid peroxyl acid; Toc, tocopherol; AsA, ascorbic acid; MDHA, monodehydroascorbic acid; P680, photosystem II reaction center; P680⁺, reaction center cation (after excitation induced charge separation); Pheo⁻, Pheophytin anion (after excitation-induced charge separation); ³P680, reaction center triplett chlorophyll; ¹O₂, singlet oxygen; ³O₂, molecular triplet oxygen; TQ, tocopherol quinone. (Adapted from refs. ²⁰ and ²⁵).

Interestingly, the Vitamin E pool composition depends on the tissue type. While γ-tocopherol is predominant in dicot seeds—usually contributing to more than 90% of the tocochromanol pool⁸—tocotrienols prevail in cereal endosperm.^9,10 In contrast, the leaf tocopherol pool consists of more than 90% α-tocopherol in most plants (e.g., in potato, tobacco, Arabidopsis, maize, barley),^11–14 with only neglectable amounts of tocotrienols or γ-tocopherol.

Several approaches have been undertaken to increase the Vitamin E content in plants,^15,16 but the generated transgenics did not perform better in the tested light stress scenarios.¹⁵ In addition, information on the stress tolerance of plants with altered tocopherol pool composition is sparse. For instance, γ-tocopherol methyl transferase (γTMT) was overexpressed in Arabidopsis and Soybean, producing transgenic seeds in which the tocopherol pool was dominated by α-instead of γ-tocopherol.^13,17,18 It was observed that germinating transgenic soybean seedlings, but not dormant seeds, contained less lipid peroxidation products than wild type, suggesting a better protection of α- than by γ-tocopherol during photomorphogenesis.¹⁸ However, an increased resilience towards stress could not be discovered when Arabidopsis γTMT deficient vte4 mutants were subjected to light, heat and cold stress.¹⁹ Likewise, membrane permeable derivatives of α- and γ-tocopherol were able to reduce photoinhibition in Chlamydomonas reinhardtii caused by isoxafluole, a herbicide targeting the committed step in plastoquinone and tocopherol biosynthesis.²⁰

In contrast to the previous studies, we aimed at inducing desiccation stress in leaves by growing tobacco on up to 300 mM NaCl and sorbitol, mimicking the situation in desiccated seeds.¹² Both stresses cause oxidative stress by hyperosmolarity (reviewed in ref. 21), although different subcellular compartments are involved. Excess salt endangers protein integrity and needs to be sequestered in the vacuole, concomitantly disturbing pH and ion homeostasis across the tonoplast and the plasma membrane.²² In contrast to sodium chloride, sorbitol more specifically causes desiccation. In consequence, the reduced water availability in chloroplasts favors the generation of reactive oxygen species (ROS) in the stroma. Transgenics silenced for homogentisate phytyl transferase (HPT) and γTMT were employed to compare the effect of tocopherol deficiency in HPT:RNAi versus the substitution of γ- for α-tocopherol in γTMT:RNAi plants with wild type. Both transgenics exhibited an elevated susceptibility towards salinity, while an exchange of γ- for α-tocopherol in γTMT:RNAi tobacco rendered the plants more tolerant towards oxidative stress in chloroplasts caused by sorbitol, which was supported by an increased tolerance of γTMT transgenics towards methyl viologen,¹² suggesting that γ-tocopherol is superior over α-tocopherol in protecting plant tissue against oxidative stress in chloroplast.

Are there indications, whether the physicochemical properties of α-tocopherol predestine it as (1) antioxidant in thylakoids, where singlet oxygen and superoxide are produced at high rates, and why (2) γ-tocopherol is favoured in seeds, where lipid peroxidation is the main target for tocopherols?

$$\text{α}-\text{Toc}\stackrel{-1{\text{ e}}^{-}}{\to }\text{α}-{\text{Toc}}^{•\text{+}}\stackrel{-1{\text{ H}}^{+}}{\to }\text{α}-{\text{Toc}}^{•}\stackrel{-1{\text{ e}}^{-}}{\to }\text{α}-{\text{Toc}}^{\text{+}}$$

Recent in vitro studies on the reaction kinetics of tocopherol vitamers have revealed that the α-tocopheryl radical cation (αToc^•+), an intermediate in αToc^• formation from αToc (see equation), is less stable than the γ-tocopheryl radical cation (γToc^•+) in organic solvents.¹⁶ This demonstrates a higher reactivity for αToc^•+, which might be crucial for its effectiveness in thylakoids.

In contrast, the stability of α-tocopheryl phenoxonium cations (αToc⁺), the tocopheryl radical oxidation product, is 10⁴ times greater than the γToc⁺ cation.^23,24 In vivo, tocopheryl phenoxonium cations might, if at all, play a role as intermediates in tocopherol quinone (TQ) formation, a negative modulator of PSII activity.^25,26 The different stability of α- and γ-tocopherol oxidation products could thus potentially influence the response of the photosynthetic apparatus to light stress in the presence of α- and γ-tocopherol, but direct experimental evidence is lacking to date.

However, there is evidence that known signaling pathways can be modulated by tocopherol deficiency. A distinct transcriptional response in tocopherol deficient Arabidopsis vte2 seedlings was observed that addressed target genes of both, oxidative and biotic stress responses.²⁷ Concomitantly, the content of 12-OPDA, a lipid peroxidation product and precursor in jasmonate biosynthesis increased in the vte2 mutants, which can be attributed to nonenzymatic peroxidation. Interestingly, the transcriptional response did not involve many MeJA regulated genes,²⁷ indicating that the involved signaling differed from jasmonate signaling.

In conclusion, tocopherol pool composition and tocopherol abundance seem to not just affect plant stress responses but also cellular signaling, underlining the importance of tocopherols beyond their mere antioxidant activity in plants.

Abbreviations

³Chl

triplett chlorophyll

HPT

homogentisate phytyl transferase

γTMT

γ-tocopherol methyl transferase

NaCl

sodium chloride

MeJA

methyl jasmonate

1O₂

singlet oxygen

12-OPDA

12-oxo-phytodienoic acid

PUFA

poly unsaturated fatty acid

PS

photosystem

ROS

reactive oxygen species

Toc•

tocopheryl radical

Toc•+

tocopheryl radical cation

Toc+

tocopheryl phenoxonium cation

TQ

tocopheryl quinone

vte2

vitamin E deficient

Addendum to: Abbasi AR, Hajirezaei M, Hofius D, Sonnewald U, Voll LM. Specific Roles of α- and γ-Tocopherol in Abiotic Stress Responses of Transgenic Tobacco. Plant Physiol. 2007;143:1720–1738. doi: 10.1104/pp.106.094771.