Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

Manef Abderrabba Abha Agnihotri Andrew Allan Jean-Michel Ane Emilia L. Apostolova Klaus Appenroth Birgit Arnholdt-Schmitt Ricardo Aroca Isabel Arrillaga Folkard Asch Bayram Atasagun Keiichi Baba Shashi Babbar Frantisek Baluska Balazs Barna D. Bartels Carlos Bartoli Brigitta Basa Chris Beadle Ludger Beerhues Andres Belver Oliver Berkowitz Cincia Bertea Christine Beveridge Melike Bor Marco Borghetti Rubén Bottini Fred Bourland David Burritt Jim Burton Catalina Cabot Edward Calabrese David Chagne Subbaiah Chalivendra W.C. Chang Chiew Foan Chin Jen-Ping Chung Michael Cohen M.G.C. Costa Evelyne Costes Ashton Cowan Bert Cregg Katrien Curvers Riccardo d’Andria J. Davies Peter Davies Geert-Jan De Klerk Shantaraj Deepak C.A. Delatorre Carole Deleu Lie Deng Ming-Hua Deng Renu Deswal Georg Dittie Sidzabda Djibril Dayamba Judit Dobránszki Robert Donaldson Maria Drincovich Uwe Druege Qiaohong Duan Stephen Duke Jacques Dumais Paula Duque Rivka Elbaum J. Fettke Matthew Fidelibus David Fleisher Eloise Foo Steffi Forman Vasileios Fotopoulos Christine Foyer Philipp Franken Stephen Fry Masayuki Fujita Amy Fulcher Jean-Philippe Galaud Xiang Gao Neera Garg Norberto Gariglio Christoph Geilfus Mateja Germ Jan M. Geuns Bernard Glick Eliezer Goldschmidt Albensio Gomes Silveira H. Gong J.H. Graham William Gray K. Groten Matthias Gube Guang-Qin Guo I. Guzman Edward Gwozdz Amjad Hameed H. Hartmann Yrjo Helariutta

Background: Population structure and dynamics in natural ecosystems can be affected by seed viability and dormancy. However, how the endogenous contents of phytohormones and vitamin E in seeds relative to the environment affect viability and dormancy is not yet fully understood.Aims: We studied seed viability and germination capacity in two populations of the Mediterranean shrub, Cistus albidus, exposed to contrasting environmental conditions.Methods: We measured seed viability and germination capacity and endogenous contents of abscisic acid (ABA), gibberellins and vitamin E in seeds collected from two populations, in two environmentally contrasting environments in north-eastern Spain.Results: Plants growing in the natural site produced seeds with lower germination percentage but similar viability, indicating higher seed dormancy. Enhanced seed dormancy was paralleled with higher contents of ABA and lower contents of gibberellins (GAs). Contents of tocopherols and tocotrienols were higher in seeds of the natural population. Tocopherol contents in seeds correlated positively with the ratio living:total aerial biomass.Conclusions: Two Mediterranean populations of C. albidus growing in two sites with contrasting environmental conditions showed marked differences in seed dormancy and germination, which may be explained, at least in part, by differences in seed hormonal contents.

Changes in glutathione and ascorbate content, hydrogen peroxide and abscisic acid were studied in developing leaves in wild-type (Col-0) and glutathione-deficient mutant pad2-1 lines of Arabidopsis thaliana over a time period of 9 days of drought followed by re-watering. Glutathione deficient mutant (pad2-1) presents mechanisms of acclimation to water stress through the reduction of plant biomass and increase in endogenous concentrations of ascorbate and glutathione. These acclimation responses to stress appeared along with the first symptoms of stress and we suggest here that they are regulated by ABA and H2O2. Normally, wild type plants under conditions of stress require an initial response phase in which a decrease in antioxidants is observed before reaching acclimation through the increase in levels of antioxidants. pad2-1 is more sensitive to stress and reacts to it; however, it did not suffer more oxidative stress than Col-0 plants, even though pad2-1 had higher levels of endogenous H2O2 relative to wild-type. In both water stressed Col-0 and pad2-1 plants increases in ABA were observed, however, more sharply in wild-type stressed plants. Low levels of glutathione together with high levels of H2O2 may regulate endogenous ABA concentrations and could be related to the slow growth rates which were observed during the experiment. The results highlighted the double function of glutathione as an antioxidant and signal molecule and also, the different response patterns of wild-type and pad2-1 when faced with drought stress. The results bring new insights to the responses of pad2-1 under conditions of water stress.

Main conclusion The interaction between enzymatic and non-enzymatic antioxidants, endogenous levels of ABA and ABA-GE, the rapid recuperation of photosynthetic proteins under re-watering as well the high level of antioxidant proteins in previously drought-stressed plants under re-watering conditions, will contribute to drought resistance in plants subjected to a long-term drought stress under Mediterranean field conditions. This work provides an overview of the mechanisms of Cistus albidus acclimation to long-term summer drought followed by re-watering in Mediterranean field conditions. To better understand the molecular mechanisms of drought resistance in these plants, a proteomic study using 2-DE and MALDI-TOF/TOF MS/MS was performed on leaves from these shrubs. The analysis identified 57 differentially expressed proteins in water-stressed plants when contrasted to well watered. Water-stressed plants showed an increase, both qualitatively and quantitatively, in HSPs, and downregulation of photosynthesis and carbon metabolism enzymes. Under drought conditions, there was considerable upregulation of enzymes related to redox homeostasis, DHA reductase, Glyoxalase, SOD and isoflavone reductase. However, upregulation of catalase was not observed until after re-watering was carried out. Drought treatment caused an enhancement in antioxidant defense responses that can be modulated by ABA, and its catabolites, ABA-GE, as well as JA. Furthermore, quantification of protein carbonylation was shown to be a useful marker of the relationship between water and oxidative stress, and showed that there was only moderate oxidative stress in C. albidus plants subjected to water stress. After re-watering plants recovered although the levels of ABA-GE and antioxidant enzymes still remain higher than in well-watered plants. We expect that our results will provide new data on summer acclimation to drought stress in Mediterranean shrubs.

Most angiosperms accumulate vitamin E in the form of tocopherols in seeds, exerting a protective antioxidant role. However, several palm trees principally accumulate tocotrienols, rather than tocopherols, in seeds, as it occurs in other monocots. To unravel the protective role of either tocopherols or tocotrienols against lipid peroxidation during seed germination in Chamaerops humilis var. humilis; seed viability, natural and induced germination capacity, seed water content, malondialdehyde levels (as an indicator of the extent of lipid peroxidation) and vitamin E levels (including both tocopherols and tocotrienols) were examined at various germination phases in a simulated, natural seed bank. At the very early stages of germination (operculum removal), malondialdehyde levels increased 2.8-fold, to decrease later up to 74%, thus indicating a transient lipid peroxidation at early stages of germination. Tocopherol levels were absent in quiescent seeds and did not increase during operculum removal, but increased later presumably dampening malondialdehyde accumulation. Thereafter, tocopherols continued increasing, while lipid peroxidation levels decreased. By contrast, tocotrienols levels remained constant or even decreased as germination progressed, showing no correlation with lipid peroxidation levels. We hypothesize that despite their high tocotrienol content, seeds synthesize tocopherols during germination to protect lipids from peroxidation events.

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.

The interplay between jasmonic acid (JA) and abscisic acid (ABA) in plant responses to water stress and in water-stress-enhanced oxidative stress was investigated in Arabidopsis thaliana plants subjected to water stress by water deprivation. For this purpose a drought assay was conducted using Arabidopsis mutants impaired in ABA (aba2), JA (aos), and ascorbate (vtc1) biosynthesis. Our results show an interaction between ABA and JA during their biosynthesis. Moreover, the coordinated action of ABA and JA protected wild-type, aba2, and aos plants from the effects of stress. However, this effect was not observed in the vtc1 mutant, which showed a distinct decrease in the F (v)/F (m) ratio, concomitant with a marked fall in relative water content (RWC), despite high endogenous concentrations of JA and ABA. This finding indicates the relevance of ascorbate metabolism in plant acclimation to stress. Despite the interaction between the two phytohormones, drought-associated stomatal closure is regulated mainly by ABA and weakly by JA, whereas JA plays a role in the formation of antioxidants regulating ascorbate and glutathione metabolism. A time course analysis revealed the relevance of plant age and stress duration in the responses of the mutants compared to wild-type plants. Here we discuss the relationship between ABA, JA, ascorbate, and glutathione in plants under water stress.

Physiological studies on aging in perennials are mainly focused either on the primary metabolism or the hormonal regulation of the process. However, to our knowledge, the involvement of the secondary metabolism in this process has not yet been explored. Cistus clusii, a Mediterranean sclerophyllous evergreen bush, shows considerable amounts of flavan-3-ols in leaves. In the present study, we aimed at determining the impact of environmental conditions and plant aging in the flavan-3-ol content in C. clusii plants grown in field conditions, which included summer drought and recovery periods. Six-year-old plants suffered more from photo-oxidative stress, especially during excess light periods, and showed lower maximum photosynthetic rates than 1-year-old plants. C. clusii leaves accumulated (−)-epigallocatechin gallate in early summer, in a strong positive correlation with both the photon flux density and the photoperiod, but not with the plant water status. Moreover, C. clusii plants accumulated proanthocyanidins (polymeric flavan-3-ols) in leaves during summer. Older plants showed higher levels of proanthocyanidins and (−)-epicatechin, but only during late spring and summer. From the result of the present study, we conclude that excess light enhances flavan-3-ol content in C. clusii, a process enhanced as plants age due to increased excess light stress.

Here we examined the contribution of methyl jasmonate (MeJA) to the acclimation of a Mediterranean shrub, Cistus albidus L., to water stress under natural climatic conditions. For this purpose, changes in MeJA, hydrogen peroxide (H2O2), ascorbate (AA) and the maximum efficiency of PSII photochemistry (Fv/Fm ratio) and lipid peroxidation were monitored in young leaves of two sets of plants: well-watered plants and plants exposed to water stress and then re-watered. We also estimated plant water relations and plant growth. MeJA accumulation in water-stressed plants showed a maximum increase after 11 weeks of water stress and a second increase after the re-watering, which was performed in week 17 of the experiment. Like MeJA, H2O2 variations showed a biphasic time course, reaching the first peak under mild water stress and the second peak during plant recovery. H2O2 accumulation was not associated with oxidative damage, and in addition to showing intact cell ultratructure, water-stressed plants showed lower lipid peroxidation than well-watered plants. Furthermore, the Fv/Fm ratio remained above 0.75 throughout the experiment in both sets of plants. AA concentrations began to increase at the beginning of water stress, before the increase in MeJA. AA reached a steady maximum, which was maintained during water stress, and returned to initial values when plants were re-watered. On the basis of these results, we conclude that MeJA, H2O2 and AA are involved in the mechanisms of plant resistance to water stress as follows: MeJA arrests the growth of young leaves and AA prevents oxidative damage.

This study evaluated the possible role of hydrogen peroxide (H2O2) in the acclimation of a Mediterranean shrub, Cistus albidus L., to summer drought growing under Mediterranean field conditions. For this purpose, changes in H2O2 concentrations and localization throughout a year were analysed. H2O2 changes in response to environmental conditions in parallel with changes in abscisic acid (ABA) and oxidative stress markers, together with lignin accumulation, xylem and sclerenchyma differentiation, and leaf area were also investigated. During the summer drought, leaf H2O2 concentrations increased 11-fold, reaching values of 10 mu mol g(-1) dry weight (DW). This increase occurred mainly in mesophyll cell walls, xylem vessels, and sclerenchyma cells in the differentiation stage. An increase in ABA levels preceded that of H2O2, but both peaked at the same time in conditions of prolonged stress. C. albidus plants tolerated high concentrations of H2O2 because of its localization in the apoplast of mesophyll cells, xylem vessels, and in differentiating sclerenchyma cells. The increase in ABA, and consequently of H2O2, in plants subjected to drought stress might induce a 3.5-fold increase in ascorbic acid (AA), which maintained and even decreased its oxidative status, thus protecting plants from oxidative damage. After recovery from drought following late-summer and autumn rainfall, a decrease in ABA, H2O2, and AA to their basal levels (similar to 60 pmol g(-1) DW, similar to 1 mu mol g(-1) DW, and similar to 20 mu mol g(-1) DW) was observed.

Flavonoids are a large family of plant secondary metabolites, principally recognized for their health-promoting properties in human diets. Most flavonoids outperform well-known antioxidants, such as ascorbate (vitamin Q and alpha-tocopherol (vitamin E), in in vitro antioxidant assays because of their strong capacity to donate electrons or hydrogen atoms. However, experimental evidence for an antioxidant function in plants is limited to a few individual flavonoids under very specific experimental and developmental conditions. As we discuss here, although flavonoids have been demonstrated to accumulate with oxidative stress during abiotic and biotic environmental assaults, a convincing spatio-temporal correlation with the flavonoid oxidation products is not yet available. Thereby, the widely accepted antioxidant function of flavonoids in plants is still a matter of debate.

BACKGROUND: Carnosic acid and carnosol, the two major phenolic diterpenes present in rosemary and sage extracts, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, these compounds have been identified as being present in some species of the family Lamiaceae, but there is still little information about their distribution within the plant kingdom. The aim of the present study was to gain a better understanding of the occurrence of these compounds within leaf extracts of the genus Salvia and also to examine to what extent leaf senescence may influence their accumulation in relation to that of alpha-tocopherol (vitamin E).RESULTS: Methanolic leaf extracts of 60 species of the genus Salvia were tested for the presence of carnosic acid and carnosol. These two diterpenes were detected in 48 and 27 species respectively. In contrast, alpha-tocopherol was present in all species examined. Leaf senescence in Salvia officinalis resulted in alpha-tocopherol increases by up to 5.5-fold, while carnosic acid and carnosol increased by up to IS and 290% respectively. Isorosmanol, an oxidation product of carnosic acid, increased by up to 2.1-fold in senescing leaves.CONCLUSION: It was found that, while alpha-tocopherol is ubiquitous in the genus Salvia, phenolic diterpenes are present in several but not all species. It was also shown that leaf senescence may increase the yield of both phenolic diterpenes and alpha-tocopherol in sage extracts. (c) 2008 Society of Chemical Industry

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.

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC qui:none (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo. (c) 2006 Elsevier Ltd. All rights reserved.

Abscisic acid (ABA) accumulation has been analyzed in irrigated and water-stressed wild-type and the vtc-1 mutant of Arabidopsis thaliana, which shows an ascorbate deficiency in leaves of approximately 60%. The amounts of ABA increased progressively up to 2.3-fold in water-stressed wild-type plants, whereas levels were kept at low levels in the irrigated plants. In contrast, initial increases followed by a sharp decrease of abscisic acid levels were observed in water-stressed vtc-1 mutants. Furthermore, the levels of this phytohormone increased up to fivefold in irrigated mutants. This differential accumulation of ABA in the mutant strongly correlated with the ascorbate redox state, but not with ascorbate levels. Changes in ABA levels in leaves paralleled those of chloroplasts. Immunolocalization studies showed a differential ABA accumulation in chloroplasts of vtc-1 mutants, which displayed the highest ABA labeling in irrigated plants. Our results indicate an altered pattern of ABA accumulation in the vtc-1 mutant compared to the wild type, under both irrigated conditions and water-stress conditions, which is strongly dependent on the ascorbate redox state.