Ozone Treatment as an Approach to Induce Specialized Compounds in Melissa officinalis Plants.

Plants are constantly subjected to environmental changes that deeply affect their metabolism, leading to the inhibition or synthesis of "specialized" compounds, small organic molecules that play a fundamental role in adaptative responses. In this work, Melissa officinalis L. (an aromatic plant broadly cultivated due to the large amounts of secondary metabolites) plants were exposed to realistic ozone (O3) dosages (80 ppb, 5 h day-1) for 35 consecutive days with the aim to evaluate its potential use as elicitor of specialized metabolite production. Ozone induced stomatal dysfunction throughout the whole experiment, associated with a low photosynthetic performance, a decrease in the potential energy conversion activity of PSII, and an alteration in the total chlorophyll content (-35, -36, -10, and -17% as average compared to the controls, respectively). The production of hydrogen peroxide at 7 days from the beginning of exposure (+47%) resulted in lipid peroxidation and visible injuries. This result suggests metabolic disturbance within the cell and a concomitant alteration in cell homeostasis, probably due to a limited activation of antioxidative mechanisms. Moderate accumulated doses of O3 triggered the accumulation of hydroxycinnamic acids and the up-regulation of the genes encoding enzymes involved in rosmarinic acid, phenylpropanoid, and flavonoid biosynthesis. While high accumulated doses of O3 significantly enhanced the content of hydroxybenzoic acid and flavanone glycosides. Our study shows that the application of O3 at the investigated concentration for a limited period (such as two/three weeks) may become a useful tool to stimulate bioactive compounds production in M. officinalis.

Exogenous application of melatonin protects bean and tobacco plants against ozone damage by improving antioxidant enzyme activities, enhancing photosynthetic performance, and preventing membrane damage.

Ozone (O3) pollution is harmful to plants and ecosystems. Several chemicals have been evaluated to protect plants against O3 deleterious effects. However, they are not adequately efficient and/or the environmental safety of their application is questioned. Hence, new chemicals that provide sufficient protection while being safer for environmental application are needed. This study investigates the response of two O3-sensitive plant species (Phaseolus vulgaris L. cv. Pinto and Nicotiana tabacum L. cv. Bel-W3) leaf-sprayed with deionized water (W, control), ethylenediurea (EDU, 1 mM) or melatonin at lower (1 mM) or higher (3 mM) concentrations (Mel_L and Mel_H, respectively), and then exposed to a square wave of 200 ppb O3, lasting 1 day (5 h day-1) for bean and 2 days (8 h day-1) for tobacco. In both species, the photosynthetic activity of O3-exposed plants was about halved. O3-induced membrane damage was also confirmed by increased malondialdehyde (MDA) byproducts compared to control (W). In EDU- and Mel-treated bean plants, the photosynthetic performance was not influenced by O3, leading to reduction of the incidence and severity of O3 visible injury. In bean plants, Mel_L mitigated the detrimental effect of O3 by boosting antioxidant enzyme activities or osmoprotectants (e.g. abscisic acid, proline, and glutathione transferase). In Mel_L-sprayed tobacco plants, O3 negatively influenced the photosynthetic activity. Conversely, Mel_H ameliorated the O3-induced oxidative stress by preserving the photosynthetic performance, preventing membrane damage, and reducing the visible injuries extent. Although EDU performed better, melatonin protected plants against O3 phytotoxicity, suggesting its potential application as a bio-safer and eco-friendlier phytoprotectant against O3. It is worth noting that the content of melatonin in EDU-treated plants remained unchanged, indicating that the protectant mode of action of EDU is not Mel-related.

Genome Resources of Verticillium dahliae VdGL16: The Causal Agent of Vascular Wilt on the Invasive Species Ailanthus altissima.

Verticillium species are known as plant pathogens responsible for wilt diseases in a large variety of dicotyledon plants and crops in many parts of the world. Here we present the draft genome sequence of Verticillium dahliae Kleb. (strain VdGL16) isolated in Italy from the invasive alien species Ailanthus altissima (Mill.; commonly known as tree-of-heaven) showing Verticillium wilt symptoms. The comparison between the newly sequenced genome with those publicly available revealed candidate genes putatively involved in pathogenicity. The genome represents a new useful source for future research on Verticillium genetics and biology as well as research on novel approaches in the control of A. altissima.

The Effects of Elevated Tropospheric Ozone on Carbon Fixation and Stable Isotopic Signatures of Durum Wheat Cultivars with Different Biomass and Yield Stability.

Tropospheric ozone (O3) enrichment caused by human activities can reduce important crop yields with huge economic loss and affect the global carbon cycle and climate change in the coming decades. In this study, two Italian cultivars of durum wheat (Claudio and Mongibello) were exposed to O3 (80 ppb, 5 h day-1 for 70 consecutive days), with the aim to investigate the changes in yield and biomass, ecophysiological traits, and stable carbon and nitrogen isotope values in plants, and to compare the stable isotope responses under environmental stressors. Both cultivars showed a relative O3 tolerance in terms of photosynthetic performance, but in cultivar Mongibello, O3 was detrimental to the grain yield and plant biomass. The δ13C values in the leaves of plants identified that the impact of O3 on CO2 fixation by RuBisCO was dominant. The δ15N value showed significant differences between treatments in both cultivars at seven days from the beginning of the exposure, which could be considered an early indicator of ozone pollution. Under increasingly frequent extreme climates globally, the relationships among stable isotope data, ecophysiological traits, and agronomic parameters could help breed future cultivars.

Antioxidants and Phytohormones Act in Coordination to Regulate Sage Response to Long Term Ozone Exposure.

Antioxidants and phytohormones are hallmarks of abiotic stress responses in plants. Although it is known that they can offer cell protection or accelerate programmed cell death (PCD) depending on the level of stress, the involvement of these metabolites in stress acclimation is still not fully elucidated. Here, we showed the role of antioxidants and phytohormones in Salvia officinalis tolerance to long-term ozone (O3) exposure (120 ppb for 36 days, 5 h day-1). Salicylic acid (SA) content was increased under O3 throughout the whole experiment (+150%, as average compared with control), being required to maintain the cellular redox state and potentiate defense responses. This accumulation was induced before the production of ethylene (ET), suggesting that ET was controlled by SA during O3 exposure to modulate the magnitude of chlorosis formation and the cell redox balance (by regulating ascorbate and glutathione levels). The synthesis and/or regeneration of these antioxidants did not protect membranes from lipid peroxidation, as demonstrated by the accumulation of malondialdehyde (+23% as average). However, these processes of lipid oxidation did not include the synthesis of the membrane breakdown products, as confirmed by the unchanged values of jasmonic acid, thus indicating that this compound was not involved in the regulation of PCD strategies.

Season-long exposure of bilberry plants to realistic and future ozone pollution improves the nutraceutical quality of fruits.

Ozone (O3) is a phytotoxic air pollutant capable of limiting plant yield and growth, and altering the quality of edible plant products. This study aimed to investigate the effects of long-term O3 exposure at realistic and future concentrations (applied during fruit development) not only on morphological, physiological, and biochemical plant/leaf traits of Vaccinium myrtillus but also on its fruit yield and quality. Three-year-old saplings were grown from May to July under three levels of O3 concentration [1.0, 1.5 and 2.0 times the ambient air concentrations, denoted as AA, 1.5_AA and 2.0_AA], using a new-generation O3 Free Air Controlled Exposure system. Ozone induced oxidative pressure and membrane denaturation as confirmed by the accumulation of anion superoxide, hydrogen peroxide (•O2-: +39 and + 29%; H2O2: +55 and + 59% in 1.5_AA and 2.0_AA, respectively, compared with AA), and malondialdehyde by-product (1.4- and 2.5-fold higher than AA, in 1.5_AA and 2.0_AA, respectively). The observed oxidative burst likely affected several cellular structures interested by photosynthetic processes (e.g., decrease of the maximum rate of carboxylation: -30%). This constraint likely induced a decline in plant vitality and a different partitioning of biomass allocation between above and below organs. An accelerated maturation of bilberries due to O3 was reported, suggesting that plants grown under harsher environmental conditions suffered from metabolic changes associated with early ripening. Increasing O3 concentrations might be responsible for an alteration of the ratio between oxidation and reduction processes mechanisms that was followed by a loss of integrity of membranes, so limiting the availability of energy/resources, triggering enzymatic oxidation of phenols to red/purple pigments, and promoting fruit maturation. To the best of our knowledge, this is the first research showing that long-term O3 exposure during bilberry fruit development influenced not only several plant/leaf traits, but also fruit nutraceutical quality at the time of harvest.

Transient Waterlogging Events Impair Shoot and Root Physiology and Reduce Grain Yield of Durum Wheat Cultivars.

Durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn) is a staple crop of the Mediterranean countries, where more frequent waterlogging events are predicted due to climate change. However, few investigations have been conducted on the physiological and agronomic responses of this crop to waterlogging. The present study provides a comprehensive evaluation of the effects of two waterlogging durations (i.e., 14 and 35 days) on two durum wheat cultivars (i.e., Svevo and Emilio Lepido). An integrated analysis of an array of physiological, biochemical, biometric, and yield parameters was performed at the end of the waterlogging events, during recovery, and at physiological maturity. Results established that effects on durum wheat varied depending on waterlogging duration. This stress imposed at tillering impaired photosynthetic activity of leaves and determined oxidative injury of the roots. The physiological damages could not be fully recovered, subsequently slowing down tiller formation and crop growth, and depressing the final grain yield. Furthermore, differences in waterlogging tolerance between cultivars were discovered. Our results demonstrate that in durum wheat, the energy maintenance, the cytosolic ion homeostasis, and the ROS control and detoxification can be useful physiological and biochemical parameters to consider for the waterlogging tolerance of genotypes, with regard to sustaining biomass production and grain yield.

Stress markers and physiochemical responses of the Mediterranean shrub Phillyrea angustifolia under current and future drought and ozone scenarios.

Mediterranean plants are particularly threatened by the exacerbation of prolonged periods of summer drought and increasing concentrations of ground-level ozone (O3). The aims of the present study were to (i) test if selected markers (i.e., reactive oxygen species, ROS; malondialdehyde, MDA; photosynthetic pigments) are able to discriminate the oxidative pressure due to single and combined stress conditions, and (ii) elucidate the physiochemical adjustments adopted by Phillyrea angustifolia (evergreen woody species representative of the maquis, also known as narrow-leaved mock privet) to perceive and counter to drought and/or O3. Plants were grown from May to October under the combination of two levels of water irrigation [i.e., well-watered (WW) and water-stressed (WS)] and three levels of O3 [i.e., 1.0, 1.5 and 2.0 times the ambient air concentrations, i.e. AA (current O3 scenario), 1.5 × AA and 2.0 × AA (future O3 scenarios), respectively], using a new-generation O3 Free Air Controlled Exposure (FACE) system. Overall, this species appeared relatively sensitive to drought (e.g., net CO2 assimilation rate and stomatal conductance significantly decreased, as well as total chlorophyll and carotenoid contents), and tolerant to O3 (e.g., as confirmed by the absence of visible foliar injury, the unchanged values of total carotenoids, and the detrimental effects on stomatal conductance, total chlorophylls and terpene emission only under elevated O3 concentrations). The combination of both stressors led to harsher oxidative stress. Only when evaluated together (i.e., combining the information provided by the analysis of each stress marker), ROS, MDA and photosynthetic pigments, were suitable stress markers to discriminate the differential oxidative stress induced by drought and increasing O3 concentrations applied singly or in combination: (i) all these stress markers were affected under drought per se; (ii) hydrogen peroxide (H2O2) and MDA increased under O3per se, following the gradient of O3 concentrations (H2O2: about 2- and 4-fold higher; MDA: +22 and + 91%; in 1.5 × AA_WW and 2.0 × AA_WW, respectively); (iii) joining together the ROS it was possible to report harsher effects under 2.0 × AA_WS and 1.5 × AA_WS (both anion superoxide and H2O2 increased) than under 2.0 × AA_WW (only H2O2 increased); and (iv) MDA showed harsher effects under 2.0 × AA_WS than under 1.5 × AA_WS (increased by 49 and 18%, respectively). Plants activated physiological and biochemical adjustments in order to partially avoid (e.g., stomatal closure) and tolerate (e.g., increased terpene emission) the effects of drought when combined with increasing O3 concentrations, suggesting that the water use strategy (isohydric) and the sclerophyllous habit can further increase the plant tolerance to environmental constraints in the Mediterranean area.

In the tripartite combination ozone-poplar-Chrysomela populi, the pollutant alters the plant-insect interaction via primary metabolites of foliage.

Ozone (O3)-induced metabolic changes in leaves are relevant and may have several ecological significances. Here, variations in foliar chemistry of two poplar clones (Populus deltoides × maximowiczii, Eridano, and P. × euramericana, I-214) under a chronic O3 treatment (80 ppb, 5 h d-1 for 10 consecutive days) were investigated. The aim was to elucidate if leaf age and/or O3-sensitivity (considering Eridano and I-214 as O3-sensitive and O3-resistant, respectively) can affect suitability of poplar foliage for Chrysomela populi L. (Coleoptera Chrysomelidae), in terms of palatability. Comparing controls, only low amino acid (AA) contents were reported in Eridano [about 3- and 4-fold in mature and young leaves (ML and YL, respectively)], and all the investigated primary metabolites [i.e. water soluble carbohydrates (WSC), proteins (Prot) and AA] were higher in YL than in ML of I-214 (+23, +54 and + 20%, respectively). Ozone increased WSC only in YL of Eridano (+24%, i.e. highest values among samples; O3 effects are always reported comparing O3-treated plants with the related controls). A concomitant decrease of Prot was observed in both ML and YL of Eridano, while only in YL of I-214 (-41, -45 and -51%, respectively). In addition, O3 decreased AA in YL of Eridano and in ML of I-214 (-40 and -14%, respectively). Comparing plants maintained under charcoal-filtered air, total ascorbate (Asc) was lower in Eridano in both ML and YL (around -22%), and abscisic acid (ABA) was similar between clones; furthermore, higher levels of Asc were reported in YL than in ML of Eridano (+19%). Ozone increased Asc and ABA (about 2- and 3-fold, respectively) in both ML and YL of Eridano, as well as ABA in YL of I-214 (about 2-fold). Comparing leaves maintained under charcoal-filtered air, the choice feeding test showed that the 2nd instar larvae preferred YL, and the quantity of YL consumed was 9 and 4-fold higher than ML in Eridano and I-214, respectively. Comparing leaves exposed to O3-treatment, a significant feeding preference for YL disks was also observed, regardless of the clone. The no-choice feeding test showed that larval growth was slightly higher on untreated YL than on untreated ML (+19 and + 10% in Eridano and I-214, respectively). The body mass of larvae fed with O3-treated YL was also significantly higher than that of larvae fed with untreated YL (3- and 2-fold in Eridano and I-214). This study highlights that realistic O3 concentrations can significantly impact the host/insect interactions, a phenomenon dependent on leaf age and O3-sensitivity of the host.

The Biosynthesis of Phenolic Compounds is an Integrated Defence Mechanism to Prevent Ozone Injury in Salvia officinalis.

Specialized metabolites constitute a major antioxidant system involved in plant defence against environmental constraints, such as tropospheric ozone (O3). The objective of this experiment was to give a thorough description of the effects of an O3 pulse (120 ppb, 5 h) on the phenylpropanoid metabolism of sage, at both biochemical and molecular levels. Variable O3-induced changes were observed over time among the detected phenylpropanoid compounds (mostly identified as phenolic acids and flavonoids), likely because of their extraordinary functional diversity. Furthermore, decreases in the phenylalanine ammonia-lyase (PAL), phenol oxidase (PPO), and rosmarinic acid synthase (RAS) activities were reported during the first hours of treatment, probably due to an O3-induced oxidative damage to proteins. Both PAL and PPO activities were also suppressed at 24 h from the beginning of exposure, whereas enhanced RAS activity occurred at the end of treatment and at the recovery time, suggesting that specific branches of the phenolic pathways were activated. The increased RAS activity was accompanied by the up-regulation of the transcript levels of genes like RAS, tyrosine aminotransferase, and cinnamic acid 4-hydroxylase. In conclusion, sage faced the O3 pulse by regulating the activation of the phenolic biosynthetic route as an integrated defence mechanism.

Hyperspectral Reflectance of Light-Adapted Leaves Can Predict Both Dark- and Light-Adapted Chl Fluorescence Parameters, and the Effects of Chronic Ozone Exposure on Date Palm (Phoenix dactylifera).

High-throughput and large-scale measurements of chlorophyll a fluorescence (ChlF) are of great interest to investigate the photosynthetic performance of plants in the field. Here, we tested the capability to rapidly, precisely, and simultaneously estimate the number of pulse-amplitude-modulation ChlF parameters commonly calculated from both dark- and light-adapted leaves (an operation which usually takes tens of minutes) from the reflectance of hyperspectral data collected on light-adapted leaves of date palm seedlings chronically exposed in a FACE facility to three ozone (O3) concentrations (ambient air, AA; target 1.5 × AA O3, named as moderate O3, MO; target 2 × AA O3, named as elevated O3, EO) for 75 consecutive days. Leaf spectral measurements were paired with reference measurements of ChlF, and predictive spectral models were constructed using partial least squares regression. Most of the ChlF parameters were well predicted by spectroscopic models (average model goodness-of-fit for validation, R2: 0.53-0.82). Furthermore, comparing the full-range spectral profiles (i.e., 400-2400 nm), it was possible to distinguish with high accuracy (81% of success) plants exposed to the different O3 concentrations, especially those exposed to EO from those exposed to MO and AA. This was possible even in the absence of visible foliar injury and using a moderately O3-susceptible species like the date palm. The latter view is confirmed by the few variations of the ChlF parameters, that occurred only under EO. The results of the current study could be applied in several scientific fields, such as precision agriculture and plant phenotyping.

Red versus green leaves: transcriptomic comparison of foliar senescence between two Prunus cerasifera genotypes.

The final stage of leaf ontogenesis is represented by senescence, a highly regulated process driven by a sequential cellular breakdown involving, as the first step, chloroplast dismantling with consequent reduction of photosynthetic efficiency. Different processes, such as pigment accumulation, could protect the vulnerable photosynthetic apparatus of senescent leaves. Although several studies have produced transcriptomic data on foliar senescence, just few works have attempted to explain differences in red and green leaves throughout ontogenesis. In this work, a transcriptomic approach was used on green and red leaves of Prunus cerasifera to unveil molecular differences from leaf maturity to senescence. Our analysis revealed a higher gene regulation in red leaves compared to green ones, during leaf transition. Most of the observed DEGs were shared and involved in transcription factor activities, senescing processes and cell wall remodelling. Significant differences were detected in cellular functions: genes related to photosystem I and II were highly down-regulated in the green genotype, whereas transcripts involved in flavonoid biosynthesis, such as UDP glucose-flavonoid-3-O-glucosyltransferase (UFGT) were exclusively up-regulated in red leaves. In addition, cellular functions involved in stress response (glutathione-S-transferase, Pathogen-Related) and sugar metabolism, such as three threalose-6-phosphate synthases, were activated in senescent red leaves. In conclusion, data suggests that P. cerasifera red genotypes can regulate a set of genes and molecular mechanisms that cope with senescence, promoting more advantages during leaf ontogenesis than compared to the green ones.

Early Detection of Sage (Salvia officinalis L.) Responses to Ozone Using Reflectance Spectroscopy.

Advancements in techniques to rapidly and non-destructively detect the impact of tropospheric ozone (O3) on crops are required. This study demonstrates the capability of full-range (350-2500 nm) reflectance spectroscopy to characterize responses of asymptomatic sage leaves under an acute O3 exposure (200 ppb for 5 h). Using partial least squares regression, spectral models were developed for the estimation of several traits related to photosynthesis, the oxidative pressure induced by O3, and the antioxidant mechanisms adopted by plants to cope with the pollutant. Physiological traits were well predicted by spectroscopic models (average model goodness-of-fit for validation (R2): 0.65-0.90), whereas lower prediction performances were found for biochemical traits (R2: 0.42-0.71). Furthermore, even in the absence of visible symptoms, comparing the full-range spectral profiles, it was possible to distinguish with accuracy plants exposed to charcoal-filtered air from those exposed to O3. An O3 effect on sage spectra was detectable from 1 to 5 h from the beginning of the exposure, but ozonated plants quickly recovered after the fumigation. This O3-tolerance was confirmed by trends of vegetation indices and leaf traits derived from spectra, further highlighting the capability of reflectance spectroscopy to early detect the responses of crops to O3.

Antioxidative responses of three oak species under ozone and water stress conditions.

Plants are frequently exposed to adverse environmental conditions such as drought and ozone (O3). Under these conditions, plants can survive due to their ability to adjust their metabolism. The aim of the present study was to compare the detoxification mechanisms of three oak species showing different O3 sensitivity and water use strategy. Two-year-old seedlings of Quercus ilex, Q. pubescens and Q. robur were grown under the combination of three levels of O3 (1.0, 1.2 and 1.4 times the ambient O3 concentration) and three levels of water availability (on average 100, 80 and 42% of field capacity i.e. well-watered, moderate drought and severe drought, respectively) in an O3 Free Air Controlled Exposure facility. Ozone and drought induced the accumulation of reactive oxygen species (ROS) and this phenomenon was species-specific. Sometimes, ROS accumulation was not associated with membrane injury suggesting that several antioxidative defence mechanisms inhibited or alleviated the oxidative damage. Both O3 and drought increased total carotenoids that were able to prevent the peroxidation action by free radicals in Q. ilex, as confirmed by unchanged malondialdehyde by-product values. The concomitant decrease of total flavonoids may be related to the consumption of these compounds by the cell to inhibit the accumulation of hydrogen peroxide. Unchanged total phenols confirmed that Q. ilex has a superior ability to counteract oxidative conditions. Similar responses were found in Q. pubescens, although the negative impact of both factors was less efficiently faced than in the sympatric Q. ilex. In Q. robur, high O3 concentrations and severe drought induced a partial rearrangement of the phenylpropanoid pathways. These antioxidative mechanisms were not able to protect the cell structure (as confirmed by ROS accumulation) suggesting that Q. robur showed a lower degree of tolerance than the other two species.

Can nutrient fertilization mitigate the effects of ozone exposure on an ozone-sensitive poplar clone?

We tested the independent and interactive effects of nitrogen (N; 0 and 80 kg ha-1), phosphorus (P; 0, 40 and 80 kg ha-1), and ozone (O3) application/exposure [ambient concentration (AA), 1.5 × AA and 2.0 × AA] for five consecutive months on biochemical traits of the O3-sensitive Oxford poplar clone. Plants exposed to O3 showed visible injury and an alteration of membrane integrity, as confirmed by the malondialdehyde by-product accumulation (+3 and +17% under 1.5 × AA and 2.0 × AA conditions, in comparison to AA). This was probably due to O3-induced oxidative damage, as documented by the production of superoxide anion radical (O2-, +27 and +63%, respectively). Ozone per se, independently from the concentrations, induced multiple signals (e.g., alteration of cellular redox state, increase of abscisic acid/indole-3-acetic acid ratio and reduction of proline content) that might be part of premature leaf senescence processes. By contrast, nutrient fertilization (both N and P) reduced reactive oxygen species accumulation (as confirmed by the decreased O2- and hydrogen peroxide content), resulting in enhanced membrane stability. This was probably due to the simultaneous involvement of antioxidant compounds (e.g., carotenoids, ascorbate and glutathione) and osmoprotectants (e.g., proline) that regulate the detoxification processes of coping with oxidative stress by reducing the O3 sensitivity of Oxford clone. These mitigation effects were effective only under AA and 1.5 × AA conditions. Nitrogen and P supply activated a free radical scavenging system that was not able to delay leaf senescence and mitigate the adverse effects of a general peroxidation due to the highest O3 concentrations.

When "thirsty" means "less able to activate the signalling wave trigged by a pulse of ozone": A case of study in two Mediterranean deciduous oak species with different drought sensitivity.

There is a lack of knowledge about the possibility that plants facing abiotic stressors, such as drought, have an altered perception of a pulse of O3 and incur in alterations of their signalling network. This poses some concerns as to whether defensive strategy to cope episodic O3 peaks in healthy plants may fail under stress. In this study, a set of saplings of two Mediterranean deciduous species, Quercus cerris and Q. pubescens, was subjected to water withholding (20% of daily evapotranspiration for 15 days) while another set was kept well-watered. Saplings were then subjected to a pulse of O3 (200 nl l-1 for 5 h) or maintained in filtered air. Q. pubescens had a more severe decline of photosynthesis and leaf PDΨw (about -65% and 5-fold lower than in well-watered ones) and events of cell death were observed under drought when compared to Q. cerris, which is supportive for a higher sensitivity to drought exhibited by this species. When O3 was applied after drought, patterns of signalling compounds were altered in both species. Only in Q. pubescens, the typical O3-induced accumulation of apoplastic reactive oxygen species, which is the first necessary step for the activation of signalling cascade, was completely lost. In Q. cerris the most frequent changes encompassed the weakening of peaks of key signalling molecules (ethylene and salicylic acid), whereas in Q. pubescens both delayed (salicylic and jasmonic acid) or weakened (ethylene and salicylic acid) peaks were observed. This is translated to a higher ability of Q. cerris to maintain a prompt activation of defensive reaction to counteract oxidative damage due to the pollutant. Our results reveal the complexity of the signalling network in plants facing multiple stresses and highlight the need to further investigate possible alteration of defensive mechanism of tree species to predict their behavior.

Cross-talk between physiological and biochemical adjustments by Punica granatum cv. Dente di cavallo mitigates the effects of salinity and ozone stress.

Plants are exposed to a broad range of environmental stresses, such as salinity and ozone (O3), and survive due to their ability to adjust their metabolism. The aim of this study was to evaluate the physiological and biochemical adjustments adopted by pomegranate (Punica granatum L. cv. Dente di cavallo) under realistic field conditions. One-year-old saplings were exposed to O3 [two levels denoted as ambient (AO) and elevated (EO) O3 concentrations] and salinity [S (salt, 50 mM NaCl)] for three consecutive months. No salt (NS) plants received distilled water. Despite the accumulation of Na+ and Cl- in the aboveground biomass, no evidence of visible injury due to salt (e.g. tip yellow-brown lesions) was found. The maintenance of leaf water status (i.e. unchanged values of electrolytic leakage and relative water content), the significant increase of abscisic acid, proline and starch content (+98, +65 and +59% compared to AO_NS) and stomatal closure (-24%) are suggested to act as adaptive mechanisms against salt stress in AO_S plants. By contrast, EO_NS plants were unable to protect cells against the negative impact of O3, as confirmed by the reduction of the CO2 assimilation rate (-21%), accumulation of reactive oxygen species (+10 and +225% of superoxide anion and hydrogen peroxide) and malondialdehyde by-product (about 2-fold higher than AO_NS). Plants tried to preserve themselves from further oxidative damage by adopting some biochemical adjustments [i.e. increase in proline content (+41%) and induction of catalase activity (8-fold higher than in AO_NS)]. The interaction of the two stressors induced responses considerably different to those observed when each stressor was applied independently. An analysis of the antioxidant pool revealed that the biochemical adjustments adopted by P. granatum under EO_S conditions (e.g. reduction of total ascorbate; increased activities of superoxide dismutase and catalase) were not sufficient to ameliorate the O3-induced oxidative stress.

Signalling molecules responsive to ozone-induced oxidative stress in Salvia officinalis.

Tropospheric ozone (O3) is the most important gaseous pollutant and induces a mass of negative impacts on vegetation at functional and genic levels. The aim of the present study was to investigate the role of reactive oxygen species and signalling molecules in sage plants exposed to O3 (200 ppb, 5 h). Ozone exposure induced only a transient oxidative burst, as confirmed by the rapid peak of anion superoxide during the first hours of exposure (+16% compared to controls). The spontaneous reaction of O3 with membrane fatty acids stimulates peroxidative processes, as demonstrated by the rise of thiobarbituric acid reactive substances concentration starting after 1 h of exposure (+25%). The formation of lipid-based signalling molecules (e.g. jasmonic acid) may be regarded as a sort of O3-perception. The concomitant accumulation of salicylic acid suggests that sage responds early to O3 by inducing cellular antioxidants mechanisms in order to minimize O3-oxidative burst. The transient increase of abscisic acid (+25% at the end of the treatment) twinned with the maximal ethylene emission (about two-fold higher than controls) could be interpreted as a first attempt by plants to regulate the signalling responses induced by O3. In order to investigate the involvement of transcription factors in managing oxidative protection, BLASTX analysis against the Salvia miltiorrhiza sequence genome was carried out using Arabidopsis thaliana WRKY sequences as queries. Six gene sequences were identified for sage WRKYs and their relative gene expression analyses were characterized. WRKY4, WRKY5, WRKY11 and WRKY46 were up-regulated by O3 at 2 and 5 h of exposure and they showed similarity with AtWRKY48, AtWRKY22 and AtWRKY53 in A. thaliana. These results suggest that WRKYs could play a pivotal role in the signalling mechanisms during the responses of plants to O3.

How Quercus ilex L. saplings face combined salt and ozone stress: a transcriptome analysis.

BACKGROUND: Similar to other urban trees, holm oaks (Quercus ilex L.) provide a physiological, ecological and social service in the urban environment, since they remove atmospheric pollution. However, the urban environment has several abiotic factors that negatively influence plant life, which are further exacerbated due to climate change, especially in the Mediterranean area. Among these abiotic factors, increased uptake of Na + and Cl - usually occurs in trees in the urban ecosystem; moreover, an excess of the tropospheric ozone concentration in Mediterranean cities further affects plant growth and survival. Here, we produced and annotated a de novo leaf transcriptome of Q. ilex as well as transcripts over- or under-expressed after a single episode of O3 (80 nl l-1, 5 h), a salt treatment (150 mM for 15 days) or a combination of these treatments, mimicking a situation that plants commonly face, especially in urban environments. RESULTS: Salinity dramatically changed the profile of expressed transcripts, while the short O3 pulse had less effect on the transcript profile. However, the short O3 pulse had a very strong effect in inducing over- or under-expression of some genes in plants coping with soil salinity. Many differentially regulated genes were related to stress sensing and signalling, cell wall remodelling, ROS sensing and scavenging, photosynthesis and to sugar and lipid metabolism. Most differentially expressed transcripts revealed here are in accordance with a previous report on Q. ilex at the physiological and biochemical levels, even though the expression profiles were overall more striking than those found at the biochemical and physiological levels. CONCLUSIONS: We produced for the first time a reference transcriptome for Q. ilex, and performed gene expression analysis for this species when subjected to salt, ozone and a combination of the two. The comparison of gene expression between the combined salt + ozone treatment and salt or ozone alone showed that even though many differentially expressed genes overlap all treatments, combined stress triggered a unique response in terms of gene expression modification. The obtained results represent a useful tool for studies aiming to investigate the effects of environmental stresses in urban-adapted tree species.

Multiple Consequences Induced by Epidermally-Located Anthocyanins in Young, Mature and Senescent Leaves of Prunus.

Anthocyanic morphs are generally less efficient in terms of carbon gain, but, in turn, are more photoprotected than anthocyanin-less ones. To date, mature leaves of different morphs or leaves at different developmental stages within the same species have generally been compared, whereas there is a lack of knowledge regarding different stages of development of red vs. green leaves. Leaves (1-, 7-, and 13-week-old) of red- (RLP) and green-leafed (GLP) Prunus in terms of photosynthetic rate, carbon metabolism and photoprotective mechanisms were compared to test whether anthocyanin-equipped leaves perform better than anthocyanin-less leaves and whether photoprotection is the primary role of epidermally-located anthocyanins, using for the first time a recently-developed parameter of chlorophyll fluorescence (qPd). GLP leaves had a higher photosynthetic rate in 1- and 7-week-old leaves, but RLP leaves performed better at an early stage of senescence and had a longer leaf lifespan. Anthocyanins contributed to leaf photoprotection throughout the leaf development, but were tightly coordinated with carotenoids. Besides photoprotecting, we propose that epidermal anthocyanins may be principally synthetized to maintain an efficient carbon-sink strength in young and senescent leaves, thus extending the RLP leaf lifespan.