Marta Pintó-Marijuan

Photo-oxidative stress markers as a measure of abiotic stress-induced leaf senescence: advantages and limitations

Inside chloroplasts, several abiotic stresses (including drought, high light, salinity, or extreme temperatures) induce a reduction in CO2 assimilation rates with a consequent increase in reactive oxygen species (ROS) production, ultimately leading to leaf senescence and yield loss. Photo-oxidation processes should therefore be mitigated to prevent leaf senescence, and plants have evolved several mechanisms to either prevent the formation of ROS or eliminate them. Technology evolution during the past decade has brought faster and more precise methodologies to quantify ROS production effects and damage, and the capacities of plants to withstand oxidative stress. Nevertheless, it is very difficult to disentangle photo-oxidative processes that bring leaf defence and acclimation, from those leading to leaf senescence (and consequently death). It is important to avoid the mistake of discussing results on leaf extracts as being equivalent to chloroplast extracts without taking into account that other organelles, such as peroxisomes, mitochondria, or the apoplast also significantly contribute to the overall ROS production within the cell. Another important aspect is that studies on abiotic stress-induced leaf senescence in crops do not always include a time-course evolution of studied processes, which limits our knowledge about what photo-oxidative stress processes are required to irreversibly induce the senescence programme. This review will summarize the current technologies used to evaluate the extent of photo-oxidative stress in plants, and discuss their advantages and limitations in characterizing abiotic stress-induced leaf senescence in crops.

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

Ecophysiology of invasive plants: osmotic adjustment and antioxidants

Current research into plant invasiveness often attempts to predict the effect of invasions under future climate change, but most studies only focus on ecological aspects. Understanding ecophysiological responses by characterizing physiological markers such as osmotic adjustment or antioxidant protection indicators will help us to project future invasiveness patterns. In this opinion article, we highlight how the information from physiological measurements can be incorporated into effective management strategies. Furthermore, we propose how combining research strategies of physiologists and ecologists could speed up our understanding of the advantageous mechanisms adopted by invasive species. We suggest that a combined approach would also be of considerable benefit for the development of effective governmental biodiversity conservation policies.

Leaf flavonoid content in Quercus ilex L. resprouts and its seasonal variation

Here, we provide the first report on flavonoid content in holm oak (Quercus ilex L.) leaves, analyzed by HPLC–MS/MS. Flavanols and flavonols were the predominant groups, although proanthocyanidins and many soluble tannins had a relevant presence in all leaf samples. Seasonal variation of flavonoids was determined in extracts from Q. ilex leaves during resprouting after a forest fire in two Mediterranean forests. Similar seasonal trends were observed over 2 years during the two main stress seasons (winter and summer). The most abundant flavonoid was the flavanol epicatechin, which showed similar values during the two seasons. Hexosides of the flavonols, quercetin, kaempferol and rhamnetin showed considerably higher content in winter, especially at the lowest temperatures. These variations in both forests are discussed on the basis of the chlorophyll fluorescence results obtained. Anthocyanins were found practically absent in mature leaves. Nutrient or water availability differences between sites or seasons were not related to changes in leaf flavonol-hexoside content.

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase

As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast, this paper presents results on stress induced by continuous chlTGZ over-expression in transplastomic tobacco leaves. Thylakoid remodelling induced by chlTGZ over-expression in young leaves of tobacco chloroplasts has already been reported (Ioannidis et al. in Biochem Biophys Acta 1787:1215–1222, 2009). In the present work, we determined the induced alterations in the photosynthetic apparatus, in the chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways, regarding ageing and functionality of the tobacco transformed plants. The results revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. The decrease in pigment levels in the transformed leaves was accompanied by an increase in H2O2 and lipid peroxidation. The rise in H2O2 correlated with a decrease in catalase activity, whereas there was an increase in peroxidase activity. In addition, chlTGZ over-expression lead to a drop in reduced glutathione, while Fe-superoxide dismutase activity was higher in transformed than in wild-type leaves. Together with the induced oxidative stress, the over-expressed chlTGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections. Consequently, the electron transport between photosystems decrease in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced in vitro. These results are discussed in line with chlTGZ involvement in chloroplast functionality.

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non-germinated (NG) seeds treated (+GA3 ) or not treated (control) with gibberellins (GA). With GA3 treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA3 treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA3 treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1-aminocyclopropane-1-carboylic acid (ACC) decreased after imbibition. In addition, α-tocopherol and α-tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).

Evidence of Drought Stress Memory in the Facultative CAM, Aptenia cordifolia: Possible Role of Phytohormones

Although plant responses to drought stress have been studied in detail in several plant species, including CAM plants, the occurrence of stress memory and possible mechanisms for its regulation are still very poorly understood. In an attempt to better understand the occurrence and possible mechanisms of regulation of stress memory in plants, we measured the concentrations of phytohormones in Aptenia cordifolia exposed to reiterated drought, together with various stress indicators, including leaf water contents, photosynthesis and mechanisms of photo- and antioxidant protection. Results showed that plants exposed to drought stress responded differently if previously challenged with a first drought. Gibberellin levels decreased upon exposure to the first drought and remained lower in double-stressed plants compared with those exposed to stress for the first time. In contrast, abscisic acid levels were higher in double- than single-stressed plants. This occurred in parallel with alterations in hydroperoxide levels, but not with malondialdehyde levels, thus suggesting an increased oxidation state that did not result in oxidative damage in double-stressed plants. It is concluded that (i) drought stress memory occurs in double-stressed A. cordifolia plants, (ii) both gibberellins and abscisic acid may play a role in plant response to repeated periods of drought, and (iii) changes in abscisic acid levels in double-stressed plants may have a positive effect by modulating changes in the cellular redox state with a role in signalling, rather than cause oxidative damage to the cell.

Response of transglutaminase activity and bound putrescine to changes in light intensity under natural or controlled conditions in Quercus ilex leaves

In order to further study a previously observed relationship between polyamine (PA) content and changes in irradiation, we examined the level of free and bound PAs, the activity of transglutaminase (TGase, EC 2.3.2.13) and chlorophyll fluorescence in holm oak (Quercus ilex L.) leaves in response to different levels of light intensity and amount. A diurnal trend of free and bound putrescine (F-Put and B-Put, respectively) and TGase activity was observed in plants under natural conditions in the forest, with the highest value corresponding to the maximum light intensity and amount of light received by the leaves. In another set of experiments, potted Q. ilex plants in experimental fields were subjected to a range of periods of natural photosynthetic photon flux density (PPFD) by covering or not covering the whole trees. Under a natural photoperiod (uncovered leaves), B-Put content and TGase activity paralleled the diurnal PPFD pattern, reaching a maximum at the highest PPFD; prior to this maximum, free PAs showed a significant rise. Plants that were in darkness until midday and suddenly exposed to high light intensity showed enhanced TGase activity, resulting in the maximum accumulation of B-Put. The involvement of the accumulation of B-Put reflected in the changes of the B-Put/bound spermidine ratio during the photoprotective responses to high light stress in forest plants is discussed in relation to the chlorophyll fluorescence parameters observed.

Sex-related differences in lipid peroxidation and photoprotection in Pistacia lentiscus

Sex-related differences in the response of dioecious plants to abiotic stress have been poorly studied to date. This work explored to what extent sex may affect plant stress responses in Pistacia lentiscus L. (Anacardiaceae), a tree well adapted to Mediterranean climatic conditions. It was hypothesized that a greater reproductive effort in females may increase oxidative stress in leaves, particularly when plants are exposed to abiotic stress. Measurements of oxidative stress markers throughout the year revealed increased lipid peroxidation in females, but only during the winter. Enhanced lipid peroxidation in females was associated with reduced photoprotection, as indicated by reduced tocopherol levels and nonphotochemical quenching (NPQ) of chlorophyll fluorescence. Enhanced lipid peroxidation in females was also observed at predawn, which was associated with increased lipoxygenase activity and reduced cytokinin levels. An analysis of the differences between reproductive (R) and nonreproductive (NR) shoots showed an enhanced photoprotective capacity in R shoots compared to NR shoots in females. This capacity was characterized by an increased NPQ and a better antioxidant protection (increased carotenoid and tocopherol levels per unit of chlorophyll) in R compared to NR shoots. It is concluded that (i) females exhibit higher lipid peroxidation in leaves than males, but only during the winter (when sex-related differences in reproductive effort are the highest), (ii) this is associated with a lower photoprotective capacity at midday, as well as enhanced lipoxygenase activity and reduced cytokinin levels at predawn, and (iii) photoprotection capacity is higher in R relative to NR shoots in females.

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

AbstractMain conclusionThe 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.