Tocochromanol functions in plants: antioxidation and beyond.

Tocopherols and tocotrienols, collectively known as tocochromanols, are lipid-soluble molecules that belong to the group of vitamin E compounds and are essential in the human diet. Not surprisingly, most of what is known about the biological functions of tocochromanols comes from studies of mammalian systems, yet they have been shown to be synthesized only by photosynthetic organisms. The last decade has seen a radical change in the appreciation of the biological role of tocochromanols in plants thanks to a detailed characterization of mutant and transgenic plants, including several Arabidopsis thaliana mutants, the sucrose export defective1 (sxd1) maize mutant, and some transgenic potato and tobacco lines altered in tocochromanol biosynthesis. Recent findings indicate that tocopherols may play important roles in plants beyond their antioxidant function in photosynthetic membranes. Plants deficient in tocopherols show alterations in germination and export of photoassimilates, and growth, leaf senescence, and plant responses to abiotic stresses, thus suggesting that tocopherols may influence a number of physiological processes in plants. Thus, in this review not only the antioxidant function of tocochromanols in plants, but also these new emerging possible roles will be considered. Particular attention will be paid to specific roles attributed to different tocopherol homologues (particularly alpha- and gamma-tocopherol) and the possible functions of tocotrienols, which in contrast to tocopherols are only present in a range of unrelated plant groups and are almost exclusively found in seeds and fruits.

Characterization of transplastomic tobacco plants with a plastid localized barley 4-hydroxyphenylpyruvate dioxygenase.

The 4-hydroxyphenylpyruvate dioxygenase (HPD) is the only enzyme of the biosynthetic pathway of tocopherols and tocotrienols localized outside of plastids. In order to investigate whether a plastid localized HPD could give rise to an increase in tocopherol levels of tobacco leaves and seeds, in the present study the HPD enzyme was transferred to the plastid by introducing a cDNA specific for the barley hpd gene into the plastome of tobacco. The expression of the hpd gene expression cassette was demonstrated by accumulation of the hpd gene-specific transcript and by a higher resistance of the transgenic seedlings towards the HPD specific inhibitor sulcotrione. The alpha-tocopherol content was increased in leaves of the transplastomic plants, whereas the transplastomic seeds contained a significantly increased gamma-tocochromanol level. With respect to enhanced accumulation of the tocopherol content in plants tissue, overexpression of the hpd gene in plastids did not prove to be advantageous in comparison to transgenic plants expressing high HPD level in the cytoplasm. It is hypothesized that homogentisate synthesized in plastids will have to pass the envelope membrane in order to be accessible to the following enzymes of the tocopherol biosynthetic pathway.

Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves.

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of gamma-tocopherol and gamma-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.

The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis.

The disruption of the Synechocystis open reading frame Deltaslr0090 encoding a gene with high homology to plant genes encoding 4-hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4-hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.).

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.

Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana.

Tocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in alpha-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in alpha-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging.

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

Tocopherols (alpha-, beta-, gamma- and delta-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 gamma-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.

Analysis of gene expression in amyloplasts of potato tubers.

Gene expression in amyloplasts derived from potato tubers was analyzed at the levels of transcription, mRNA accumulation and formation of polysomes. Compared with chloroplasts, overall transcriptional activity is considerably reduced in amyloplasts. Nevertheless, several transcripts are synthesized in amyloplasts during growth of tubers. Among the transcribed amyloplast genes are the ribosomal operon and the psbA gene. Primer extension analysis provided evidence that in amyloplasts the plastid encoded RNA polymerase (PEP) is the principal RNA polymerase involved in the transcription of the rrn operon. Analysis of plastid steady state transcripts showed that there are only small differences in the levels of specific transcripts between amyloplasts and chloroplasts. With respect to the low transcription rate of the accumulating RNA-species in amyloplasts, a high stability of these transcripts is obvious. Though amyloplasts possess polysomes, specific mRNAs associated with such polysomes could not be detected. This suggests that translation could be impaired in amyloplasts, which, in turn, implies that these organelles are not suitable targets for the expression of transgenes introduced into the plastid genome by plastid transformation.

Alpha-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.

A plant locus essential for phylloquinone (vitamin K1) biosynthesis originated from a fusion of four eubacterial genes.

Phylloquinone is a compound present in all photosynthetic plants serving as cofactor for Photosystem I-mediated electron transport. Newly identified seedling-lethal Arabidopsis thaliana mutants impaired in the biosynthesis of phylloquinone possess reduced Photosystem I activity. The affected gene, called PHYLLO, consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of phylloquinone in photosynthetic cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes reside as polycistronic units in eubacterial chromosomes and in plastomes of red algae strongly suggests that PHYLLO derived from a plastid operon during endosymbiosis. The principle architecture of the fused PHYLLO locus is conserved in the nuclear genomes of plants, green algae, and the diatom alga Thalassiosira pseudonana. The latter arose from secondary endosymbiosis of a red algae and a eukaryotic host indicating selective driving forces for maintenance and/or independent generation of the composite gene cluster within the nuclear genomes. Besides, individual menF genes, encoding active isochorismate synthases (ICS), have been established followed by splitting of the essential 3' region of the menF module of PHYLLO only in genomes of higher plants. This resulted in inactivation of the ICS activity encoded by PHYLLO and enabled a metabolic branch from the phylloquinone biosynthetic route to independently regulate the synthesis of salicylic acid required for plant defense. Therefore, gene fusion, duplication, and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants.

A novel nucleus-targeted protein is expressed in barley leaves during senescence and pathogen infection.

The barley (Hordeum vulgare) cDNA HvS40 represents a gene with enhanced mRNA level during leaf senescence. Biolistic transformation of onion (Allium cepa) epidermal cell layers with a glucuronidase fusion protein construct provided evidence that the 15.4-kD protein encoded by HvS40 is localized to the nucleus. Expression of the gene is induced by jasmonate and salicylic acid; both are known to act as signaling compounds during senescence and defense toward pathogens. Transcript levels of HvS40 were observed to be particularly high in leaf sectors that undergo necrosis and chlorosis after infection with Pyrenophora teres. This pathogen-related expression is, in contrast, clearly reduced in transgenic barley plants expressing a stilbene synthase from grape (Vitis vinifera), whereas the mRNA level of a gene encoding the pathogen-related protein HvPR1 is unaffected. In situ hybridization with HvS40 antisense RNA revealed that during leaf senescence, the HvS40 transcript predominantly accumulates in the mesophyll. Taken together, the findings suggest a connection between the nuclear protein encoded by HvS40 and the degeneration of chloroplasts occurring during senescence and during infection of barley wild-type plants with P. teres.