Estimating heat tolerance of plants by ion leakage: a new method based on gradual heating.

Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.

Environmental plasticity of Pinot noir grapevine leaves: A trans-European study of morphological and biochemical changes along a 1,500-km latitudinal climatic gradient.

A 2-year study explored metabolic and phenotypic plasticity of sun-acclimated Vitis vinifera cv. Pinot noir leaves collected from 12 locations across a 36.69-49.98°N latitudinal gradient. Leaf morphological and biochemical parameters were analysed in the context of meteorological parameters and the latitudinal gradient. We found that leaf fresh weight and area were negatively correlated with both global and ultraviolet (UV) radiation, cumulated global radiation being a stronger correlator. Cumulative UV radiation (sumUVR) was the strongest correlator with most leaf metabolites and pigments. Leaf UV-absorbing pigments, total antioxidant capacities, and phenolic compounds increased with increasing sumUVR, whereas total carotenoids and xanthophylls decreased. Despite of this reallocation of metabolic resources from carotenoids to phenolics, an increase in xanthophyll-cycle pigments (the sum of the amounts of three xanthophylls: violaxanthin, antheraxanthin, and zeaxanthin) with increasing sumUVR indicates active, dynamic protection for the photosynthetic apparatus. In addition, increased amounts of flavonoids (quercetin glycosides) and constitutive ß-carotene and α-tocopherol pools provide antioxidant protection against reactive oxygen species. However, rather than a continuum of plant acclimation responses, principal component analysis indicates clusters of metabolic states across the explored 1,500-km-long latitudinal gradient. This study emphasizes the physiological component of plant responses to latitudinal gradients and reveals the physiological plasticity that may act to complement genetic adaptations.

Enhanced thermal stability of the thylakoid membranes from spruce. A comparison with selected angiosperms.

Recently, we have found that thermal stability of photosystem II (PSII) photochemistry in spruce needles is higher than in other plants (barley, beech) cultivated under the same temperatures. In this work, temperature dependences of various characteristics of PSII organization were studied in order to obtain complex information on the thermal stability of PSII function and organization in spruce. Temperature dependency of circular dichroism spectra revealed by about 6 °C higher thermal stability of macrodomain organization in spruce thylakoid membranes in comparison with Arabidopsis and barley ones; however, thermal disintegration of light-harvesting complex of PSII did not significantly differ among the species studied. These results thus indicate that thermal stability of PSII macro-organization in spruce thylakoid membranes is enhanced to a similar extent as thermal stability of PSII photochemistry. Clear-native polyacrylamide gel electrophoresis of preheated thylakoids demonstrated that among the separated pigment-protein complexes, only PSII supercomplexes (SCs) revealed considerably higher thermal stability in spruce thylakoids as compared to Arabidopsis and barley ones. Hence we suggest that higher thermal stability of PSII macro-organization of spruce is influenced by the maintenance of PSII SCs in the thylakoid membrane. In addition, we discuss possible effects of different PSII organizations and lipid compositions on the thermal stability of spruce thylakoid membranes.

Comprehensive Modulation of Secondary Metabolites in Terpenoid-Accumulating Mentha spicata L. via UV Radiation.

In plants, secondary metabolites change in response to environmental conditions. These changes co-regulate resilience to stressful environmental conditions, plant growth and development, and interactions between plants and the wider ecosystem, while also affecting soil carbon storage and atmospheric and climatic conditions. The objective of this study was to determine the association between UV exposure and the contents of key metabolites, including amino acids, phenolics, flavonoids, terpenoids, carotenoids, tocopherols, and phytosterols. Mentha spicata plantlets were grown in tissue culture boxes for 30 days and then exposed to a low dose of broadband UV-B (291-315 nm; 2.8 kJm-2 biologically effective UV) enriched light for eight days. Metabolite contents were quantified either immediately after the final UV exposure, or after seven days of recovery under photosynthetically active radiation. It was found that UV promoted the production of flavonoids (1.8-fold) ahead of phenolic acids (unchanged). Furthermore, the majority of monoterpenes and sesquiterpenes, constituents of valuable mint essential oil, were significantly increased through UV treatment (up to 90-fold for α-linalool). In contrast, the contents of carotenoids and tocopherols did not increase following UV exposure. A comparison between plants sampled immediately after UV exposure and after seven days of recovery showed that there was an overall increase in the content of carotenoids, mono- and sesquiterpenes, phenolics, and amino acids following recovery, while the contents of sterols and tocopherols decreased. These UV-induced changes in metabolite profile may have important consequences for agriculture, ecology, and even the global climate, and they also provide an exciting opportunity to enhance crop value, facilitating the development of improved products with higher levels of essential oils and added benefits of enhanced flavour, colour, and bioactive content.

Single and interactive effects of variables associated with climate change on wheat metabolome.

One of the key challenges linked with future food and nutritional security is to evaluate the interactive effect of climate variables on plants' growth, fitness, and yield parameters. These interactions may lead to unique shifts in the morphological, physiological, gene expression, or metabolite accumulation patterns, leading to an adaptation response that is specific to future climate scenarios. To understand such changes, we exposed spring wheat to 7 regimes (3 single and 4 combined climate treatments) composed of elevated temperature, the enhanced concentration of CO2, and progressive drought stress corresponding to the predicted climate of the year 2100. The physiological and metabolic responses were then compared with the current climate represented by the year 2020. We found that the elevated CO2 (eC) mitigated some of the effects of elevated temperature (eT) on physiological performance and metabolism. The metabolite profiling of leaves revealed 44 key metabolites, including saccharides, amino acids, and phenolics, accumulating contrastingly under individual regimes. These metabolites belong to the central metabolic pathways that are essential for cellular energy, production of biosynthetic pathways precursors, and oxidative balance. The interaction of eC alleviated the negative effect of eT possibly by maintaining the rate of carbon fixation and accumulation of key metabolites and intermediates linked with the Krebs cycle and synthesis of phenolics. Our study for the first time revealed the influence of a specific climate factor on the accumulation of metabolic compounds in wheat. The current work could assist in the understanding and development of climate resilient wheat by utilizing the identified metabolites as breeding targets for food and nutritional security.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs-indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase-suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that-in line with the Dynamic Exchange Model-the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Temperature alters susceptibility of Picea abies seedlings to airborne pollutants: The case of CdO nanoparticles.

Although plants are often exposed to atmospheric nanoparticles (NPs), the mechanism of NP deposition and their effects on physiology and metabolism, and particularly in combination with other stressors, are not yet understood. Exploring interactions between stressors is particularly important for understanding plant responses in urban environments where elevated temperatures can be associated with air pollution. Accordingly, 3-year-old spruce seedlings were exposed for 2 weeks to aerial cadmium oxide (CdO) NPs of environmentally relevant size (8-62 nm) and concentration (2 × 105 cm-3). While half the seedlings were initially acclimated to high temperature (35 °C) and vapour pressure deficit (VPD; 2.81 kPa), the second half of the plants were left under non-stressed conditions (20 °C, 0.58 kPa). Atomic absorption spectrometry was used to determine Cd content in needles, while gas and liquid chromatography was used to determine changes in primary and secondary metabolites. Photosynthesis-related processes were explored with gas-exchange and chlorophyll fluorescence systems. Our work supports the hypothesis that atmospheric CdO NPs penetrate into leaves but high temperature and VPD reduce such penetration due to stomatal closure. The hypothesis that atmospheric CdO NPs influences physiological and metabolic processes in plants was also confirmed. This impact strengthens with increasing time of exposure. Finally, we found evidence that plants acclimated to stress conditions have different sensitivity to CdO NPs compared to plants not so acclimated. These findings have important consequences for understanding impacts of global warming on plants and indicates that although the effects of elevated temperatures can be deleterious, this may limit other forms of plant stress associated with air pollution.

Long-term fertilization determines different metabolomic profiles and responses in saplings of three rainforest tree species with different adult canopy position.

BACKGROUND: Tropical rainforests are frequently limited by soil nutrient availability. However, the response of the metabolic phenotypic plasticity of trees to an increase of soil nutrient availabilities is poorly understood. We expected that increases in the ability of a nutrient that limits some plant processes should be detected by corresponding changes in plant metabolome profile related to such processes. METHODOLOGY/PRINCIPAL FINDINGS: We studied the foliar metabolome of saplings of three abundant tree species in a 15 year field NPK fertilization experiment in a Panamanian rainforest. The largest differences were among species and explained 75% of overall metabolome variation. The saplings of the large canopy species, Tetragastris panamensis, had the lowest concentrations of all identified amino acids and the highest concentrations of most identified secondary compounds. The saplings of the "mid canopy" species, Alseis blackiana, had the highest concentrations of amino acids coming from the biosynthesis pathways of glycerate-3P, oxaloacetate and α-ketoglutarate, and the saplings of the low canopy species, Heisteria concinna, had the highest concentrations of amino acids coming from the pyruvate synthesis pathways. CONCLUSIONS/SIGNIFICANCE: The changes in metabolome provided strong evidence that different nutrients limit different species in different ways. With increasing P availability, the two canopy species shifted their metabolome towards larger investment in protection mechanisms, whereas with increasing N availability, the sub-canopy species increased its primary metabolism. The results highlighted the proportional distinct use of different nutrients by different species and the resulting different metabolome profiles in this high diversity community are consistent with the ecological niche theory.

Wet effluent diffusion denuder: The tool for determination of monoterpenes in forest.

Three methods, i.e., the cylindrical wet effluent diffusion denuder (CWEDD)-GCMS, Tenax tubes-GCMS and Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS) were compared for the determination of monoterpenes in forest. While the on-line technique (PTR-TOF-MS) allows only for the measurement of sum of monoterpenes, both the off-line preconcentration techniques (CWEDD and Tenax tubes) are suitable for the determination of concentrations of individual monoterpenes due to subsequent analysis of samples by GCMS. The CWEDD-GCMS is the only method that allows sampling of individual monoterpenes with short time intervals of 2-5min. Monoterpenes are absorbed into a liquid (n-heptane), flowing down on the inner wall of the CWEDD, and then the collected liquid with monoterpenes is immediately taken away for the GCMS analysis, which minimizes time when collected monoterpenes are exposed to oxidants presented in the air during sampling. The limits of detection of CWEDD-GCMS are in the range 1-7pptv for individual monoterpenes.

WITHDRAWN: Wet effluent diffusion denuder: The tool for determination of monoterpenes in forest.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, 10.1016/j.talanta.2016.03.032. The duplicate article has therefore been withdrawn.The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Changes of primary and secondary metabolites in barley plants exposed to CdO nanoparticles.

The environmental fate of airborne nanoparticles and their toxicity to plants is not yet fully understood. Pot-grown barley plants with second leaves developed were therefore exposed to CdO nanoparticles (CdONPs) of ecologically relevant size (7-60 nm) and concentration (2.03 ± 0.45 × 105 particles cm-3) in air for 3 weeks. An experiment was designed to test the effects of different treatments when only leaves (T1); leaves and soil substrate (T2); and leaves, soil, and water supply were exposed to nanoparticles (T3). A fourth, control group of plants was left without treatment (T0). Although CdONPs were directly absorbed by leaves from the air, a part of leaf-allocated Cd was also transported from roots by transpiration flow. Chromatographic assays revealed that CdONPs had a significant effect on total content of primary metabolites (amino acids and saccharides) but no significant effect on total content of secondary metabolites (phenolic compounds, Krebs cycle acids, and fatty acids). In addition, the compositions of individual metabolite classes were affected by CdONP treatment. For example, tryptophan and phenylalanine were the most affected amino acids in both analysed organs, while ferulic acid and isovitexin constituted the polyphenols most affected in leaves. Even though CdONP treatment had no effect on total fatty acids content, there were significant changes in the composition of saturated and unsaturated fatty acids in both the roots and leaves of treated plants. Although the results indicate the most pronounced effect in T3 plants as compared to T1 and T2 plants, even just leaf exposure to CdONPs has the potential to induce changes in plant metabolism.

Morphological, biochemical and physiological traits of upper and lower canopy leaves of European beech tend to converge with increasing altitude.

The present work has explored for the first time acclimation of upper versus lower canopy leaves along an altitudinal gradient. We tested the hypothesis that restrictive climatic conditions associated with high altitudes reduce within-canopy variations of leaf traits. The investigated beech (Fagus sylvatica L.) forest is located on the southern slope of the Hrubý Jeseník Mountains (Czech Republic). All measurements were taken on leaves from upper and lower parts of the canopy of mature trees (>85 years old) growing at low (400 m above sea level, a.s.l.), middle (720 m a.s.l.) and high (1100 m a.s.l.) altitudes. Compared with trees at higher altitudes, those growing at low altitudes had lower stomatal conductance, slightly lower CO(2) assimilation rate (A(max)) and leaf mass per area (LMA), and higher photochemical reflectance index, water-use efficiency and Rubisco content. Given similar stand densities at all altitudes, the different growth conditions result in a more open canopy and higher penetration of light into lower canopy with increasing altitude. Even though strong vertical gradients in light intensity occurred across the canopy at all altitudes, lower canopy leaves at high altitudes tended to acquire the same morphological, biochemical and physiological traits as did upper leaves. While elevation had no significant effect on nitrogen (N) and carbon (C) contents per unit leaf area, LMA, or total content of chlorophylls and epidermal flavonoids in upper leaves, these increased significantly in lower leaves at higher altitudes. The increases in N content of lower leaves were coupled with similar changes in A(max). Moreover, a high N content coincided with high Rubisco concentrations in lower but not in upper canopy leaves. Our results show that the limiting role of light in lower parts of the canopy is reduced at high altitudes. A great capacity of trees to adjust the entire canopy is thus demonstrated.

Opposite metabolic responses of shoots and roots to drought.

Shoots and roots are autotrophic and heterotrophic organs of plants with different physiological functions. Do they have different metabolomes? Do their metabolisms respond differently to environmental changes such as drought? We used metabolomics and elemental analyses to answer these questions. First, we show that shoots and roots have different metabolomes and nutrient and elemental stoichiometries. Second, we show that the shoot metabolome is much more variable among species and seasons than is the root metabolome. Third, we show that the metabolic response of shoots to drought contrasts with that of roots; shoots decrease their growth metabolism (lower concentrations of sugars, amino acids, nucleosides, N, P, and K), and roots increase it in a mirrored response. Shoots are metabolically deactivated during drought to reduce the consumption of water and nutrients, whereas roots are metabolically activated to enhance the uptake of water and nutrients, together buffering the effects of drought, at least at the short term.