Malondialdehyde Assays in Higher Plants.

Malondialdehyde is a three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation widely used as a marker of the extent of lipid peroxidation in plants. There are several methodological approaches to quantify malondialdehyde contents in higher plants, ranging from the simplest, cheapest, and quickest spectrophotometric approaches to the more complex ones using tandem mass spectrometry. This chapter summarizes the advantages and limitations of approaches followed and provides brief protocols with some tips to facilitate the selection of the best method for each experimental condition and application.

Hormonal response to recurrent seasonal stress in coastal and mountain scabiouses growing in their natural habitat: linking ABA and jasmonates with photoprotection.

Plant species distribution across ecosystems is influenced by multiple environmental factors, and recurrent seasonal stress events can act as natural selection agents for specific plant traits and limit species distribution. For that, studies aiming at understanding how environmental constraints affect adaptive mechanisms of taxonomically closely related species are of great interest. We chose two Scabiosa species inhabiting contrasting environments: the coastal scabious S. atropurpurea, typically coping with hot-dry summers in a Mediterranean climate, and the mountain scabious S. columbaria facing cold winters in an oceanic climate. A set of functional traits was examined to assess plant performance in these congeneric species from contrasting natural habitats. Both S. atropurpurea and S. columbaria appeared to be perfectly adapted to their environment in terms of adjustments in stomatal closure, CO2 assimilation rate and water use efficiency over the seasons. However, an unexpected dry period during winter followed by the typical Mediterranean hot-dry summer forced S. atropurpurea plants to deploy a set of photoprotective responses during summer. Aside from reductions in leaf water content and Fv/Fm, photoprotective molecules (carotenoids, α-tocopherol and anthocyanins) per unit of chlorophyll increased, mostly as a consequence of a severe chlorophyll loss. The profiling of stress-related hormones (ABA, salicylic acid and jasmonates) revealed associations between ABA and the bioactive jasmonoyl-isoleucine with the underlying photoprotective response to recurrent seasonal stress in S. atropurpurea. We conclude that jasmonates may be used together with ABA as a functional trait that may, at least in part, help understand plant responses to recurrent seasonal stress in the current frame of global climate change.

English plantain deploys stress tolerance mechanisms at various organization levels across an altitudinal gradient in the Pyrenees.

High-mountain plants must withstand high solar irradiation and low temperatures during winter. Furthermore, climate change is increasing drought events, which pose an additional threat to plants. Here, we studied the stress tolerance mechanisms at various levels of biological organization in English plantain (Plantago lanceolata L.), focusing on photoprotective and antioxidant responses. The response of populations from three different altitudes in the Eastern Pyrenees (1030, 1380, and 1660 m. a.s.l.) was compared during both autumn and winter. Results showed that plants not only suffered from photoinhibition due to very low temperatures at the highest elevation during winter, but also from mild drought stress at the lowest altitude during autumn. Individuals growing at the highest elevation showed reductions in the maximum photochemical efficiency of PSII (Fv /Fm ratio), which might be caused by the lack of an increased induction of tolerance mechanisms at the highest elevation compared to the intermediate one. Although most leaves died at the highest elevation, plants could withstand stress at the organism level by generating new leaves once the stress ceased. Drought at the lowest elevation during autumn caused mild stress with small decreases in the Fv /Fm ratio, along with an increase in abscisic acid and jasmonic acid content. This study underlines the great capacity of English plantain to adapt to high elevation by activating not only photo- and antioxidant protection mechanisms and adjustments in stress-related phytohormones, but also by fully regenerating its aboveground biomass through renewed growth once the stress has ceased.

Linking Leaf Water Potential, Photosynthesis and Chlorophyll Loss With Mechanisms of Photo- and Antioxidant Protection in Juvenile Olive Trees Subjected to Severe Drought.

The identification of drought-tolerant olive tree genotypes has become an urgent requirement to develop sustainable agriculture in dry lands. However, physiological markers linking drought tolerance with mechanistic effects operating at the cellular level are still lacking, in particular under severe stress, despite the urgent need to develop these tools in the current frame of global change. In this context, 1-year-old olive plants growing in the greenhouse and with a high intra-specific variability (using various genotypes obtained either from cuttings or seeds) were evaluated for drought tolerance under severe stress. Growth, plant water status, net photosynthesis rates, chlorophyll contents and the extent of photo- and antioxidant defenses (including the de-epoxidation state of the xanthophyll cycle, and the contents of carotenoids and vitamin E) were evaluated under well-watered conditions and severe stress (by withholding water for 60 days). Plants were able to continue photosynthesizing under severe stress, even at very low leaf water potential of -4 to -6 MPa. This ability was achieved, at least in part, by the activation of photo- and antioxidant mechanisms, including not only increased xanthophyll cycle de-epoxidation, but also enhanced α-tocopherol contents. "Zarrazi" (obtained from seeds) and "Chemlali" (obtained from cuttings) showed better performance under severe water stress compared to the other genotypes, which was associated to their ability to trigger a higher antioxidant protection. It is concluded that (i) drought tolerance among the various genotypes tested is associated with antioxidant protection in olive trees, (ii) the extent of xanthophyll cycle de-epoxidation is strongly inversely related to photosynthetic rates, and (iii) vitamin E accumulation is sharply induced upon severe chlorophyll degradation.

Mesophyll conductance: the leaf corridors for photosynthesis.

Besides stomata, the photosynthetic CO2 pathway also involves the transport of CO2 from the sub-stomatal air spaces inside to the carboxylation sites in the chloroplast stroma, where Rubisco is located. This pathway is far to be a simple and direct way, formed by series of consecutive barriers that the CO2 should cross to be finally assimilated in photosynthesis, known as the mesophyll conductance (gm). Therefore, the gm reflects the pathway through different air, water and biophysical barriers within the leaf tissues and cell structures. Currently, it is known that gm can impose the same level of limitation (or even higher depending of the conditions) to photosynthesis than the wider known stomata or biochemistry. In this mini-review, we are focused on each of the gm determinants to summarize the current knowledge on the mechanisms driving gm from anatomical to metabolic and biochemical perspectives. Special attention deserve the latest studies demonstrating the importance of the molecular mechanisms driving anatomical traits as cell wall and the chloroplast surface exposed to the mesophyll airspaces (Sc/S) that significantly constrain gm. However, even considering these recent discoveries, still is poorly understood the mechanisms about signaling pathways linking the environment a/biotic stressors with gm responses. Thus, considering the main role of gm as a major driver of the CO2 availability at the carboxylation sites, future studies into these aspects will help us to understand photosynthesis responses in a global change framework.

Global gene flow releases invasive plants from environmental constraints on genetic diversity.

When plants establish outside their native range, their ability to adapt to the new environment is influenced by both demography and dispersal. However, the relative importance of these two factors is poorly understood. To quantify the influence of demography and dispersal on patterns of genetic diversity underlying adaptation, we used data from a globally distributed demographic research network comprising 35 native and 18 nonnative populations of Plantago lanceolata Species-specific simulation experiments showed that dispersal would dilute demographic influences on genetic diversity at local scales. Populations in the native European range had strong spatial genetic structure associated with geographic distance and precipitation seasonality. In contrast, nonnative populations had weaker spatial genetic structure that was not associated with environmental gradients but with higher within-population genetic diversity. Our findings show that dispersal caused by repeated, long-distance, human-mediated introductions has allowed invasive plant populations to overcome environmental constraints on genetic diversity, even without strong demographic changes. The impact of invasive plants may, therefore, increase with repeated introductions, highlighting the need to constrain future introductions of species even if they already exist in an area.

Oxidative Stress: A Master Regulator of Plant Trade-Offs?

Trade-offs between growth, reproduction, and defence have been documented. Oxidative stress is one of the physiological mechanisms that underlie trade-offs at the cellular and organ levels. The diversity of plant life forms and the complexity of scaling up limit our knowledge of oxidative stress as a universal mediator of life-history trade-offs at the organism level. Joint efforts by plant physiologists and ecologists will undoubtedly provide novel insights into this topic in the near future.

Seasonal, Sex- and Plant Size-Related Effects on Photoinhibition and Photoprotection in the Dioecious Mediterranean Dwarf Palm, Chamaerops humilis.

In Mediterranean-type ecosystems plants are exposed to several adverse environmental conditions throughout the year, ranging from drought stress during the warm and dry summers to chilling stress due to the typical drop in temperatures during winters. Here we evaluated the ecophysiological response, in terms of photoinhibition and photoprotection, of the dioecious Mediterranean palm, Chamaerops humilis to seasonal variations in environmental conditions. Furthermore, we considered as well the influence of plant size, maturity, and sexual dimorphism. Results showed evidence of winter photoinhibition, with a marked decrease of the F v /F m ratio below 0.7 between January and March, which was coincident with the lowest temperatures. During this period, the de-epoxidation state of the xanthophyll cycle and zeaxanthin levels increased, which might serve as a photoprotection mechanism, owing the full recovery from winter photoinhibition during spring. Furthermore, mature plants showed lower chlorophyll levels and higher ß-carotene levels per unit of chlorophyll than juvenile plants, and females displayed lower leaf water contents and higher photoinhibition than males during summer, probably due to increased reproductive effort of females. However, neither low temperatures during winter nor reproductive events in females during the summer led to irreversible damage to the photosynthetic apparatus. We conclude that (i) the Mediterranean dwarf palm, C. humilis, suffers from photoinhibition during winter, but this is transient and does not lead to irreversible damage, and (ii) females from this plant species are more sensitive than males to photoinhibition during reproductive events.

Adaptation of the Long-Lived Monocarpic Perennial Saxifraga longifolia to High Altitude.

Global change is exerting a major effect on plant communities, altering their potential capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high altitude in an endemic long-lived monocarpic, Saxifraga longifolia, by combining demographic and physiological approaches. Plants from three altitudes (570, 1100, and 2100 m above sea level [a.s.l.]) were investigated in terms of leaf water and pigment contents, and activation of stress defense mechanisms. The influence of plant size on physiological performance and mortality was also investigated. Levels of photoprotective molecules (α-tocopherol, carotenoids, and anthocyanins) increased in response to high altitude (1100 relative to 570 m a.s.l.), which was paralleled by reduced soil and leaf water contents and increased ABA levels. The more demanding effect of high altitude on photoprotection was, however, partly abolished at very high altitudes (2100 m a.s.l.) due to improved soil water contents, with the exception of α-tocopherol accumulation. α-Tocopherol levels increased progressively at increasing altitudes, which paralleled with reductions in lipid peroxidation, thus suggesting plants from the highest altitude effectively withstood high light stress. Furthermore, mortality of juveniles was highest at the intermediate population, suggesting that drought stress was the main environmental driver of mortality of juveniles in this rocky plant species. Population structure and vital rates in the high population evidenced lower recruitment and mortality in juveniles, activation of clonal growth, and absence of plant size-dependent mortality. We conclude that, despite S. longifolia has evolved complex mechanisms of adaptation to altitude at the cellular, whole-plant and population levels, drought events may drive increased mortality in the framework of global change.

Ecophysiological response to seasonal variations in water availability in the arborescent, endemic plant Vellozia gigantea.

The physiological response of plants growing in their natural habitat is strongly determined by seasonal variations in environmental conditions and the interaction of abiotic and biotic stresses. Here, leaf water and nutrient contents, changes in cellular redox state and endogenous levels of stress-related phytohormones (abscisic acid (ABA), salicylic acid and jasmonates) were examined during the rainy and dry season in Vellozia gigantea, an endemic species growing at high elevations in the rupestrian fields of the Espinhaço Range in Brazil. Enhanced stomatal closure and increased ABA levels during the dry season were associated with an efficient control of leaf water content. Moreover, reductions in 12-oxo-phytodienoic acid (OPDA) levels during the dry season were observed, while levels of other jasmonates, such as jasmonic acid and jasmonoyl-isoleucine, were not affected. Changes in ABA and OPDA levels correlated with endogenous concentrations of iron and silicon, hydrogen peroxide, and vitamin E, thus indicating complex interactions between water and nutrient contents, changes in cellular redox state and endogenous hormone concentrations. Results also suggested crosstalk between activation of mechanisms for drought stress tolerance (as mediated by ABA) and biotic stress resistance (mediated by jasmonates), in which vitamin E levels may serve as a control point. It is concluded that, aside from a tight ABA-associated regulation of stomatal closure during the dry season, crosstalk between activation of abiotic and biotic defences, and nutrient accumulation in leaves may be important modulators of plant stress responses in plants growing in their natural habitat.

Tocotrienols in Vellozia gigantea leaves: occurrence and modulation by seasonal and plant size effects.

Vitamin E occurs in all photosynthetic organisms examined to date. Tocopherols predominate in photosynthetic tissues (α-tocopherol being the major form), while either tocopherols or tocotrienols (or both) are present in seeds. Tocotrienols have not been described in photosynthetic tissues thus far. Here, we report on the presence of tocotrienols in leaves of higher plants. Both tocopherols and tocotrienols accumulated in leaves of Vellozia gigantea, an endemic plant found in the rupestrian fields of Serra do Cipó, Brazil. Increased plant size had a remarkable effect on the vitamin E composition of leaves, α-tocopherol and ß-tocotrienol levels being highest in the largest individuals, but only during the dry season. Vitamin E levels positively correlated with lipid hydroxyperoxide levels, which also increased in the largest individuals during the dry season. However, the maximum efficiency of PSII photochemistry (F v/F m ratio) kept above 0.75 throughout the experiment, thus indicating absence of photoinhibitory damage to the photosynthetic apparatus. It is concluded that higher plants, such as V. gigantea, can accumulate tocotrienols in leaves, aside from tocopherols, and that the levels of both tocopherols and tocotrienols in the leaves of this species are strongly modulated by seasonal and plant size effects.