Leonor Alegre

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.

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.

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Abstract. Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase.

Plant aging increases oxidative stress in chloroplasts

Abstract. Aging has received considerable attention in biomedicine, but little is known about the regulatory mechanisms responsible for the aging not associated with senescence in plants. This study provides new insights into the relationship between oxidative stress and plant aging, and points out chloroplasts as one of the target organelles of age-associated oxidative stress in plants. We simultaneously analyzed lipid oxidation, photosynthesis, chlorophyll content, de-epoxidation state of the xanthophyll cycle, and levels of chloroplastic antioxidant defenses such as β-carotene and α-tocopherol in leaves of the same age in 1-, 3- and 7-year-old Cistus clusii Dunal plants growing under Mediterranean field conditions. Enhanced formation of malondialdehyde in leaves (2.7-fold) and chloroplasts (2.8-fold), decreased photosynthetic activity (25%), and lower chlorophyll (ca. 20%) and chloroplastic antioxidant defense levels (ca. 25%–85%) were observed in 7-year-old plants, when compared with 1- and 3-year-old plants. The differences observed, which were associated with plant aging, were only noticeable in mature non-senescing plants (7-year-old plants). No differences were observed between pre-reproductive (1-year-old plants) and young plants (3-year-old plants). This study shows that from a certain age, oxidative stress increases progressively in chloroplasts as plants age, whereas photosynthesis is reduced. The results indicate that the oxidative stress associated with the aging in plants accumulates progressively in chloroplasts, and that the contribution of oxidative stress to aging increases as plants age.

Drought-Induced Changes in the Redox State of α-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 (α-tocopherol [α-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 α-T, a shift of the redox state of α-T toward its reduced state, and increased Asc levels in chloroplasts under stress. Lemon balm showed the highest increases in α-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 α-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.

α-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.

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 α‐tocopherol and β‐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.

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 (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 μmol 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 (∼60 pmol g−1 DW, ∼1 μmol g−1 DW, and ∼20 μmol g−1 DW) was observed.

Redox regulation of water stress responses in field-grown plants. Role of hydrogen peroxide and ascorbate

Abiotic stresses, such as drought, can increase the production of reactive oxygen species (ROS) in plants. An increase in ROS levels can provoke a partial or severe oxidation of cellular components inducing redox status changes, so continuous control of ROS and therefore of their metabolism is decisive under stress conditions. The present work focuses on the contribution of one pro-oxidant, hydrogen peroxide (H(2)O(2)) and one antioxidant, ascorbate (AA) and its redox status, in the control of plant responses to drought-oxidative stress in resistant plants growing in field conditions. After a general introduction to the concept of drought and oxidative stress and its relationship, we describe the role of H(2)O(2) in drought stress responses, emphasizing the importance of studies in H(2)O(2) subcellular localization, needed for a better understanding of its role in plant responses to stress. Although more studies are needed in the study of changes of redox status in plants subjected to stress, the AA pools and its redox status can be indicative of its involvement as a part of cellular mechanisms by which the plant respond to drought-induced oxidative stress. The mechanism of resistance and/or tolerance to drought-oxidative stress is complex, especially when studies are carried out in plants growing in field conditions, where an interaction of stresses occurs. This study sheds light on the mechanisms of plant responses to water-oxidative stress in plants growing in the field.

Interplay Between Abscisic Acid and Jasmonic Acid and its Role in Water-oxidative Stress in Wild-type, ABA-deficient, JA-deficient, and Ascorbate-deficient Arabidopsis Plants

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 Fv/Fm 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.