Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues.

Tocopherols and tocotrienols are vitamin E compounds, differing only in the saturation state of the isoprenoid side chain. Tocopherol biosynthesis, physiology and distribution have been studied in detail. Tocopherols have been found in many different plant species, and plant tissues. In contrast, comparatively little is known about the physiology and distribution of tocotrienols. These compounds appear to be considerably less widespread in the plant kingdom. In this study 80 different plant species were analysed for the presence of tocotrienols. Twenty-four species were found to contain significant amounts of tocotrienols. No taxonomic relation was apparent among the 16 dicotyledonous species that were found to contain tocotrienol. Monocotyledonous species (eight species) belonged either to the Poaceae (six species) or the Aracaceae (two species). A more detailed analysis of tocotrienol accumulation revealed the presence of tocotrienols in several non-photosynthetic tissues and organs, i.e. seeds, fruits and in latex, in concentrations up to 2000 ppm. No tocotrienols could be detected in mature photosynthetic tissues. However, we found the transient accumulation of low levels of tocotrienols in the young coleoptiles of plant species whose seeds contained tocotrienols. No measurable tocotrienol biosynthesis was apparent in coleoptiles, or in chloroplasts isolated from such coleoptiles. In line with these results, we found that tocotrienol accumulation in coleoptiles was not associated with chloroplasts. Based on our data, we conclude that tocotrienols may be transiently present in photosynthetically active tissues, however, it remains to be proven whether the tocotrienols are biosynthesised in such tissues, or imported from elsewhere in the plant.

Tissue-specific expression and developmental regulation of cytochrome b561 genes in Arabidopsis thaliana and Raphanus sativus.

Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes-Artb561-1 and Artb561-2-using relative-quantitative RT-PCR and in situ hybridization (ISH) techniques. The genes show overlapping and distinct tissue- and organ-specific expression patterns. Transcripts of both genes are found in leaf epidermal cells, and expression seems to correlate with leaf maturation and cessation of cell elongation. Both genes are also expressed in the epidermal cell layer of stems and roots in the L1 layer of the shoot apex, in the vascular system of leaves, stems and roots, and in the root pericycle. In addition, Artb561-1 is expressed in the root cap, whereas Artb561-2 mRNA is found in the epidermis of lateral roots, in the root meristem, and in unfertilized ovules. These observations provide important information for the elucidation of the physiological function of cyts b561 in plants.

Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular level.

Plant stress responses are a key factor in steering the development of cells, tissues, and organs. However, the stress-induced signal transduction cascades that control localized growth and cell size/differentiation are not well understood. It is reported here that oxidative stress, exerted by paraquat or alloxan, induced localized cell proliferation in intact seedlings, in isolated root segments, and at the single cell level. Analysis of the stress-induced mitotic activity revealed that oxidative stress enhances auxin-dependent growth cycle reactivation. Based on the similarities between responses at plant, tissue, or single cell level, it is hypothesized that a common mechanism of reactive oxygen species enhanced auxin-responsiveness underlies the stress-induced re-orientation of growth, and that stress-induced effects on the protoplast growth cycle are directly relevant in terms of understanding whole plant behaviour.

Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures.

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.

Non-photochemical quenching kinetics during the dark to light transition in relation to the formation of antheraxanthin and zeaxanthin.

Nonlinear regression analysis (NLR) is applied to quantify the dynamic response of non-photochemical fluorescence quenching (NPQ) of Trifolium repens cv. Regal upon dark to light transition. Commonly, only steady-state levels of NPQ are evaluated, ignoring transient kinetics. Experimental NPQ kinetics are fitted best with a sum of two functions: a sigmoidal Hill function plus a transient logarithmic normal function. It is shown that not only steady-state level of NPQ, but also the speed at which steady state is reached, increased with light intensity. The question is raised which biological processes cause the induction of the components of NPQ kinetics. The NPQ kinetics are found to resemble the kinetics of antheraxanthin and zeaxanthin formation during a dark to light transition. Furthermore, both molecules are known to induce NPQ. The hypothesis is put forward that a transient phase of NPQ (0-2 min after transition) is dependent upon concentrations of antheraxanthin, whereas the saturating phase corresponds with the production of zeaxanthin. A mathematical model, based on the presented hypothesis, predicts the effect of increasing light intensity on concentrations of antheraxanthin and zeaxanthin which correspond with experimental results. Implications of the hypothesis are discussed as well as the role of NLR in evaluating chlorophyll a fluorescence kinetics.

Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism.

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.