Transcriptional Regulation of Vitamin E Biosynthesis during Germination of Dwarf Fan Palm Seeds.

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.

Enhanced tocopherol levels during early germination events in Chamaerops humilis var. humilis seeds.

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.

Redox proteomics and physiological responses in Cistus albidus shrubs subjected to long-term summer drought followed by recovery.

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.

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1.

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.

Plant aging and excess light enhance flavan-3-ol content in Cistus clusii.

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.

How relevant are flavonoids as antioxidants in plants?

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 C) 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.

Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub, Cistus albidus L., to summer drought.

This study evaluated the possible role of hydrogen peroxide (H(2)O(2)) in the acclimation of a Mediterranean shrub, Cistus albidus L., to summer drought growing under Mediterranean field conditions. For this purpose, changes in H(2)O(2) concentrations and localization throughout a year were analysed. H(2)O(2) 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 H(2)O(2) concentrations increased 11-fold, reaching values of 10 micromol 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 H(2)O(2), but both peaked at the same time in conditions of prolonged stress. C. albidus plants tolerated high concentrations of H(2)O(2) because of its localization in the apoplast of mesophyll cells, xylem vessels, and in differentiating sclerenchyma cells. The increase in ABA, and consequently of H(2)O(2), 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, H(2)O(2), and AA to their basal levels (approximately 60 pmol g(-1) DW, approximately 1 micromol g(-1) DW, and approximately 20 micromol g(-1) DW) was observed.

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.

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea 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 quinone (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.

Enhanced alpha-tocopherol quinone levels and xanthophyll cycle de-epoxidation in rosemary plants exposed to water deficit during a Mediterranean winter.

Photosynthesis operates in a constantly shifting balance between efficient capture of solar energy and its rapid dissipation when captured in excess. In an attempt to better understand the role of alpha-tocopherol in plant photoprotection, we examined the changes in alpha-tocopherol quinone (alpha-TQ), in parallel with those of other low-molecular-weight antioxidants, in rosemary plants exposed to water deficit during a Mediterranean winter. Relative leaf water content (RWC) decreased from about 85% to approximately 65% in drought, but plants did not show symptoms of oxidative damage, as indicated by constant Fv/Fm ratios and malondialdehyde (MDA) levels. alpha-TQ was present at concentrations of 20 mmol per 100 mol of chlorophyll, and represented less than 1% of total tocopherol content in non-stressed leaves. Although alpha-tocopherol levels were not significantly altered, alpha-TQ reached up to 36 mmol per 100 mol of chlorophyll under stress (under both high light and after exposure to increasing water deficit at lower light intensities). Furthermore, both alpha-TQ and xanthophyll cycle de-epoxidation were strongly negatively correlated with the relative efficiency of photosystem II photochemistry (phiPSII) at midday. The biological significance of alpha-tocopherol and alpha-TQ in the network of photo- and antioxidative protection mechanisms evolved by plants to withstand stress is discussed.

Drought-induced changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions.

Mediterranean plants have evolved a complex antioxidant defense system to cope with summer drought. Flavonoids, and particularly flavanols and flavonols, are potent in vitro antioxidants, but their in vivo significance within the complex network of antioxidant defenses remains unclear, especially in plant responses to stress. To gain insight into the role of flavonoids in the antioxidant defense system of Cistus clusii Dunal, we evaluated drought-induced changes in flavonoids in leaves and compared the response of these compounds with that of other low molecular weight antioxidants (ascorbic acid, tocopherols and carotenoids). Among the antioxidant flavonoids analyzed, epigallocatechin gallate was present in the greatest concentrations (up to about 5 micromol dm(-2)). Other flavanols, such as epicatechin and epicatechin gallate, were found at concentrations below 0.25 and 0.03 micromol dm(-2), respectively. Neither of the antioxidant flavonols analyzed, quercetin and kaempferol, were detected in C. clusii leaves. Epigallocatechin gallate, ascorbic acid and alpha-tocopherol concentrations increased to a similar extent (up to 2.8-, 2.6- and 3.3-fold, respectively) in response to drought, but the kinetics of the drought-induced increases differed. Epigallocatechin gallate, epicatechin and epicatechin gallate concentrations increased progressively during drought, reaching maximum values after 30 days of stress. Ascorbic acid concentrations increased twofold after 15 days of drought, and maximum values were attained after 50 days of drought. In contrast, alpha-tocopherol concentrations remained constant during the first 30 days of drought, but increased sharply by 3.3-fold after 50 days of drought. The maximum efficiency of photosystem II photochemistry and the extent of lipid peroxidation remained constant throughout the drought period, whereas the redox state of ascorbic acid and alpha-tocopherol shifted toward their reduced forms in response to drought, indicating that the concerted action of low molecular weight antioxidants may help prevent oxidative damage in plants.

Die and let live: leaf senescence contributes to plant survival under drought stress.

Leaf senescence is a highly regulated physiological process that leads to leaf death and is, as such, the last developmental stage of the leaf. Plant aging and environmental stresses may induce the process of senescence. Here we will focus on the role of leaf senescence in field-grown plants as a response to adverse climatic conditions and, more specifically, on how it contributes to plant survival under drought stress. Drought induces several responses in plants including leaf senescence, which plays a major role in the survival of several species. Drought-induced leaf senescence contributes to nutrient remobilisation during stress, thus allowing the rest of the plant (i.e. the youngest leaves, fruits or flowers) to benefit from the nutrients accumulated during the life span of the leaf. In addition, drought-induced leaf senescence, especially when accompanied by leaf abscission, avoids large losses through transpiration, thus contributing to the maintenance of a favourable water balance of the whole plant. Drought-induced leaf senescence occurs gradually and is characterised by specific macroscopic, cellular, biochemical and molecular changes. Leaf yellowing (i.e. chlorophyll degradation) and specific changes in cell ultrastructure (e.g. chromatin condensation, thylakoid swelling, plastoglobuli accumulation), metabolism (e.g.protein degradation, lipid peroxidation) and gene expression occur during leaf senescence in drought-stressed plants. Cytokinins and ABA have been shown to be involved in the regulation of drought-induced leaf senescence, although the possible role of other plant hormones should not be excluded. Reactive oxygen species, whose concentrations increase during drought-induced leaf senescence, are also known to be regulators of this process. The complex mechanisms of regulation of leaf senescence in drought-stressed plants are discussed, and attention is drawn to those aspects that still require investigation.

Drought-induced changes in the redox state of alpha-tocopherol, ascorbate, and the diterpene carnosic acid in chloroplasts of Labiatae species differing in carnosic acid contents.

To assess antioxidative protection by carnosic acid (CA) in combination with that of other low-molecular weight (M(r)) antioxidants (alpha-tocopherol [alpha-T] and ascorbate [Asc]) in chloroplasts, we measured endogenous concentrations of these antioxidants, their redox states, and other indicators of oxidative stress in chloroplasts of three Labiatae species, differing in their CA contents, exposed to drought stress in the field. Damage to the photosynthetic apparatus was observed neither in CA-containing species (rosemary [Rosmarinus officinalis]) and sage [Salvia officinalis]) nor in CA-free species (lemon balm [Melissa officinalis]) at relative leaf water contents between 86% and 58%, as indicated by constant maximum efficiency of photosystem II photochemistry ratios and malondialdehyde levels in chloroplasts. The three species showed significant increases in alpha-T, a shift of the redox state of alpha-T toward its reduced state, and increased Asc levels in chloroplasts under stress. Lemon balm showed the highest increases in alpha-T and Asc in chloroplasts under stress, which might compensate for the lack of CA. Besides, whereas in rosemary and sage, the redox state of CA was shifted toward its oxidized state and the redox state of Asc was kept constant, lemon balm displayed a shift of the redox state of Asc toward its oxidized state under stress. In vitro experiments showed that both CA and Asc protect alpha-T and photosynthetic membranes against oxidative damage. These results are consistent with the contention that CA, in combination with other low-M(r) antioxidants, helps to prevent oxidative damage in chloroplasts of water-stressed plants, and they show functional interdependence among different low-M(r) antioxidants in chloroplasts.

Enhanced photo- and antioxidative protection, and hydrogen peroxide accumulation in drought-stressed Cistus clusii and Cistus albidus plants.

Mechanisms of drought stress resistance were studied in Cistus clusii Dunal and Cistus albidus L., two native Mediterranean shrubs that can withstand severe summer drought. While water deficit, solar radiation and temperature increased from winter to summer in the field, C. clusii and C. albidus reduced leaf area, increased root mass per leaf area, and showed diurnal changes in stomatal conductance to minimize water loss. In both species, the consequent reductions in CO2 assimilation were accompanied by reduced efficiency of photosystem II photochemistry, and protection against stress was afforded by enhanced de-epoxidation of violaxanthin in the xanthophyll cycle and increases in alpha-tocopherol and beta-carotene. In addition, hydrogen peroxide (H2O2) accumulation was observed in mesophyll cell walls of both species during the first stages of drought, although no accumulation of H2O2 was observed in chloroplasts or other organelles during the study. Despite these common responses, C. albidus and C. clusii differed in the extent of photo- and antioxidative protection. In response to drought, C. clusii showed a higher de-epoxidation state of the xanthophyll cycle and higher alpha-tocopherol and beta-carotene concentrations than C. albidus. We conclude that several structural and biochemical mechanisms underlie stress resistance in C. clusii and C. albidus, and are indicative of the different degrees of stress resistance of these shrubs.

Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water-stressed Arabidopsis plants.

The effects of low ascorbic acid (Asc) on lipophilic antioxidant defences and lipid peroxidation in chloroplasts were evaluated in the vtc-1 mutant of Arabidopsis thaliana, which had an Asc deficiency in chloroplasts of ca. 60%. Although low Asc did not cause oxidative stress in optimal growth conditions, it increased malondialdehyde levels in chloroplasts by ca. 60%, and reduced alpha-tocopherol and beta-carotene by ca. 85% and 40%, respectively, in water-stressed (WS) mutants. These results are indicative of the interplay between Asc and lipophilic antioxidants in chloroplasts of WS plants in vivo, and show that Asc contributes to the protection of thylakoid membrane lipids from oxidation in stressed plants.

Effect of drought and high solar radiation on 1-aminocyclopropane-1-carboxylic acid and abscisic acid concentrations in Rosmarinus officinalis plants.

The endogenous concentrations of ACC and ABA were measured, at predawn and at maximum solar radiation, during a summer drought, and recovery after autumn rainfalls, in rosemary (Rosmarinus officinalis L.), a drought-tolerant species, growing under Mediterranean field conditions. During the summer, plants were subjected to both water deficit and high solar radiation. Plants showed severe reductions in shoot water potential to -3 MPa, which were associated with drastic stomatal closure (73%), a decrease in net photosynthesis, reaching almost zero, and a severe chlorophyll loss (74%). Despite the severity of the stress, plants recovered after the autumn rainfalls. The concentration of ACC was not enhanced by drought, and at predawn these concentrations remained constant at approximately 600 pmol ACC-1 DW throughout the experiment. Thus, ethylene did not regulate the response of rosemary to drought. However, a sharp increase in ACC levels between predawn and midday was observed. This increase was positively correlated to the intensity of the incident solar radiation. ACC levels recorded in June at midday reached 16 000 pmol g DW and in October values of 1000 pmol g-1 DW were observed. In contrast, in drought-stressed plants predawn concentrations of ABA were up to 130-fold those of recovered plants, and the levels of ABA scored at midday were double of those scored at predawn. In conclusion, although drought-stressed rosemary plants showed a relatively moderate ABA accumulation (approximately 500 pmol g-1 DW#, at predawn), it seems to be an essential factor for the regulation of the plant response to stress, thereby enabling a rapid recovery after stress release, although other mechanisms can not be excluded. As drought stress did not induce ACC accumulation, it was concluded that ethylene production was not a major factor in the drought stress resistance of rosemary plants. The increased ACC and ABA concentrations at midday were correlated with day length and light intensity and not with the water status of the plant.

An increase in water deficit has no impact on the photosynthetic capacity of field-grown Mediterranean plants.

In the Mediterranean, annual mean precipitation has continuously decreased over the last three years (by ca 36% in Barcelona), and the decrease has been dramatic during the summer (by ca 78 and 64% during July and August, respectively). The impact of increased drought on the photosynthetic capacity of Mediterranean vegetation is currently unknown. In this study, two native Mediterranean plants [rosemary (Rosmarinus officinalis L.) and lavender (Lavandula stoechas L.)] were grown outdoors and subjected to two water regimes (50 mm month-1 during the summer, or no supplementary water at all). Rosemary and lavender plants watered with 50 mm month-1 during the summer had higher relative leaf water content and water potential than non-watered plants. Changes in water status were accompanied by large decreases in parameters of gas exchange [i.e. the light-saturated rate of CO2 assimilation, the maximum velocity of ribulose-1,5-bisphosphate (RuBP) carboxylation by Rubisco and the capacity for RuBP] and of modulated chlorophyll fluorescence (i.e. the relative quantum efficiency of PSII photochemistry and the efficiency of energy capture by open PSII reaction centres) during the summer, but no differences were found in any photosynthetic parameters for leaves subjected to the two water regimes. The drought-induced decreases in the relative quantum efficiency of PSII photochemistry in rosemary and lavender plants were attributable to 'downregulation' of electron transport. Photodamage to PSII in the field appeared to be a later effect of drought in these plants. Photorespiration was not a major mechanism protecting the photosynthetic apparatus of these plants from photodamage in the field. After the autumn rainfall, photosynthetic capacity fully recovered. We conclude that rosemary and lavender are well adapted to drought, and that an increase in water deficit is unlikely to have a significant impact on the photosynthetic capacity of leaves.