Leaf Gas Film 1 promotes glycerol ester accumulation and formation of a tight root barrier to radial O2 loss in rice.

Rice (Oryza sativa L.) and many other wetland plants form an apoplastic barrier in the outer parts of the roots to restrict radial O2 loss to the rhizosphere during soil flooding. This barrier facilitates longitudinal internal O2 diffusion via gas-filled tissues from shoot to root apices, enabling root growth in anoxic soils. We tested the hypothesis that Leaf Gas Film 1 (LGF1), which influences leaf hydrophobicity in rice, plays a crucial role in tight outer apoplastic barriers formation in rice roots. We examined the roots of a rice mutant (dripping wet leaf 7, drp7) lacking functional LGF1, its wild type, and an LGF1 overexpression line for their capacity to develop outer apoplastic barriers that restrict radial O2 loss. We quantified the chemical composition of the outer part of the root and measured radial O2 diffusion from intact roots. The drp7 mutant exhibited a weak barrier to radial O2 loss compared to the wild type. However, introducing functional LGF1 into the mutant fully restored tight barrier function. The formation of a tight barrier to radial O2 loss was associated with increased glycerol ester levels in exodermal cells, rather than differences in total root suberization or lignification. These results demonstrate that, in addition to its role in leaf hydrophobicity regulation, LGF1 plays an important role in controlling the function of the outer apoplastic barriers in roots. Our study suggests that increased deposition of glycerol esters in the suberized root exodermis establishes a tight barrier to radial O2 loss in rice roots.

Signalling responses in the bark and foliage of canker-susceptible and -resistant cypress clones inoculated with Seiridium cardinale.

The necrotrophic fungus Seiridium cardinale is the main responsible for Cypress Canker Disease (CCD), a pandemic affecting many Cupressaceae worldwide. The present study aims to elucidate the signalling of the early responses in the bark and foliage of CCD-susceptible and -resistant C. sempervirens clones to S. cardinale inoculation (SI and RI, respectively). In the bark of SI, a peaking production of ethylene (Et) and jasmonic acid (JA) occurred at 3 and 4 days post inoculation (dpi), respectively, suggesting an attempted plant response to the pathogen. A response that, however, was ineffective, as confirmed by the severe accumulation of malondialdehyde by-products at 13 dpi (i.e., lipid peroxidation). Differently, Et emission peaked in RI bark at 3 and 13 dpi, whereas abscisic acid (ABA) accumulated at 1, 4 and 13 dpi, resulting in a lower MDA accumulation (and unchanged levels of antioxidant capacity). In the foliage of SI, Et was produced at 1 and 9 dpi, whereas JA and salicylic acid (SA) accumulated at 1 and 3 dpi. Conversely, an increase of ABA and SA occurred at 1 dpi in the RI foliage. This outcome indicates that some of the observed metabolic alterations, mainly occurring as local defence mechanisms, might be able to gradually shift to a systemic resistance, although an accumulation of MDA was observed in both SI and RI foliage (but with an increased antioxidant capacity reported only in the resistant clone). We believe that the results reported here will be useful for the selection of clones able to limit the spread and damage of CCD.

Exogenous melatonin application helps late-sown durum wheat to cope with waterlogging under Mediterranean environmental conditions.

In Mediterranean countries, late-sown durum wheat (Triticum turgidum L. subsp. durum) may face waterlogging (WL) at early stages. As mitigation of waterlogging by melatonin (MT) has been poorly explored, we analyzed the effects of exogenous MT foliar application to WL-stressed durum wheat on its ecophysiological performance, growth and biomass production. Late-sown plants of a relatively tolerant cultivar (i.e., Emilio-Lepido) were subjected to two WL durations (i.e., 14 and 35 days of WL; DOW) at tillering, with or without exogenous MT application (i.e., 0 and 100 µM). Prolonged WL reduced shoot biomass (-43%), but the application of MT mitigated this detrimental effect. Waterlogging impaired photosynthesis, reducing leaf CO2 assimilation and chlorophyll content (-61 and - 57%, at 14 and 35 DOW). In control, MT increased the photosynthetic pigments (+48%), whereas it exacerbated the decrease in photosynthesis under both WL conditions (-72%, on average). Conversely, MT reduced WL-induced oxidative damage in both shoots and roots (-25% hydrogen peroxide production), facilitating osmotic adjustments and mitigating oxidative stress. The accumulation of osmotic regulators in MT + WL plants (+140 and + 42%, in shoots and roots at 35 DOW; respectively) and mineral solutes (+140 and + 104%, on average, in shoots and roots at 14 DOW) likely mitigated WL stress, limiting the impact of oxidative stress and promoting biomass accumulation. Our results highlight the potential of MT as a bioactive compound in mitigating the adverse effects of WL on late-sown durum wheat and the importance of the complex interactions between physiological responses and environmental stressors.

Containing alien plants in coastal dunes: Evidence from a soil manipulation experiment.

Biological invasion is recognised as one of the major threats to biodiversity, particularly in disturbance-prone ecosystems such as costal dunes. Many studies have associated alien plant invasion of dune ecosystem to human disturbances, but less is known about the role of soil properties in invasion after disturbance. Soil properties are crucial filters during plant succession and soil-related changes in the initial stage of species colonization might shape the final success of the invaders. We performed a manipulative experiment aimed at elucidating the effects of soil properties on plant colonization processes in highly invaded dune systems, as a proxy for plausible management actions to curb the success of exotic plant species over native ones, which was measured through species richness and abundance. In a barrier island of the Marano and Grado lagoon, Northern Adriatic Sea, we mechanically removed all the native and alien vegetation present in the back dune (also known as secondary dune), triggering a new ecological succession and further altered, for the following three months, soil properties by adding salt, nitrogen, and organic matter in a full factorial design with randomized blocks. The soil treatments reduced the overall species richness and abundance of alien plants. Further, soil treatment interactions strongly shaped community evenness and species richness. Soil salinity had a positive effect on native cover while decreasing the overall number of alien species, especially in soil with added organic matter. Our findings suggest that soil salinity, and its interplay with organic matter, might significantly reduce the initial success of alien species propagule pressure (i.e. alien plant germination), with likely implications for the trajectories of future plant communities. This study highlights that alien plant containment should be focused on early stages of succession, giving new perspective on future environmental management actions for dune restoration and conservation.

Responses of key root traits in the genus Oryza to soil flooding mimicked by stagnant, deoxygenated nutrient solution.

Excess water can induce flooding stress resulting in yield loss, even in wetland crops such as rice (Oryza). However, traits from species of wild Oryza have already been used to improve tolerance to abiotic stress in cultivated rice. This study aimed to establish root responses to sudden soil flooding among eight wild relatives of rice with different habitat preferences benchmarked against three genotypes of O. sativa. Plants were raised hydroponically, mimicking drained or flooded soils, to assess the plasticity of adventitious roots. Traits included were apparent permeance (PA) to O2 of the outer part of the roots, radial water loss, tissue porosity, apoplastic barriers in the exodermis, and root anatomical traits. These were analysed using a plasticity index and hierarchical clustering based on principal component analysis. For example, O. brachyantha, a wetland species, possessed very low tissue porosity compared with other wetland species, whereas dryland species O. latifolia and O. granulata exhibited significantly lower plasticity compared with wetland species and clustered in their own group. Most species clustered according to growing conditions based on PA, radial water loss, root porosity, and key anatomical traits, indicating strong anatomical and physiological responses to sudden soil flooding.

Restricted O2 consumption in pea roots induced by hexanoic acid is linked to depletion of Krebs cycle substrates.

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O2 consumption rate. HxA treatment resulted in root tissue extrusion of K+ , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential.

Evaluation of Fruquintinib in the Continuum of Care of Patients with Colorectal Cancer.

The management of patients with metastatic colorectal cancer (mCRC) has the continuum of care as the treatment paradigm. To date, trifluridine/tipiracil, a biochemically modulated fluoropyrimidine, and regorafenib, a multi-kinase inhibitor, remain the main options for the majority of patients who progressed to standard doublet- or triplet-based chemotherapies, although a tailored approach could be indicated in certain circumstances. Being highly selective for vascular endothelial growth factor receptor (VEGFR)-1, -2 and -3, fruquintinib demonstrated a strong anti-tumor activity in preclinical models and received approval from China's National Medical Products Administration (NMPA) in 2018 for the treatment of patients with chemo-refractory mCRC. The approval was based on the results of the phase III FRESCO trial. Then, in order to overcome geographic differences in clinical practice, the FRESCO-2 trial was conducted in the US, Europe, Japan, and Australia. In a heavily pretreated patient population, the study met its primary endpoint, demonstrating an advantage of fruquintinib over a placebo in overall survival (OS). Here, we review the clinical development of fruquintinib and its perspectives in gastrointestinal cancers. Then, we discuss the introduction of fruquintinib in the continuum of care of CRC paying special attention to unmet needs, including the identification of cross-resistant and potentially susceptible populations, evaluation of radiological response, and identification of novel biomarkers of clinical benefit.

Novel functions of the root barrier to radial oxygen loss - radial diffusion resistance to H2 and water vapour.

The root barrier to radial O2 loss (ROL) is a trait enabling waterlogging tolerance of plants. The ROL barrier restricts O2 diffusion to the anoxic soil so that O2 is retained inside root tissues. We hypothesised that the ROL barrier can also restrict radial diffusion of other gases (H2 and water vapour) in rice roots with a barrier to ROL. We used O2 and H2 microsensors to measure ROL and permeability of rice roots, and gravimetric measurements to assess the influence of the ROL barrier on radial water loss (RWL). The ROL barrier greatly restricted radial diffusion of O2 as well as H2 . At 60 kPa pO2 , we found no radial diffusion of O2 across the barrier, and for H2 the barrier reduced radial diffusion by 73%. Similarly, RWL was reduced by 93% in roots with a ROL barrier. Our study showed that the root barrier to ROL not only completely blocks radial O2 diffusion under steep concentration gradients but is also a diffusive barrier to H2 and to water vapour. The strong correlation between ROL and RWL presents a case in which simple measurements of RWL can be used to predict ROL in screening studies with a focus on waterlogging tolerance.

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.

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.

Date palm responses to a chronic, realistic ozone exposure in a FACE experiment.

Date palms are highly economically important species in hot arid regions, which may suffer ozone (O3) pollution equivalently to heat and water stress. However, little is known about date palm sensitivity to O3. Therefore, to identify their resistance mechanisms against elevated O3, physiological parameters (leaf gas exchange, chlorophyll fluorescence and leaf pigments) and biomass growth responses to realistic O3 exposure were tested in an isoprene-emitting date palm (Phoenix dactylifera L. cv. Nabut Saif) by a Free-Air Controlled Exposure (FACE) facility with three levels of O3 (ambient [AA, 45 ppb as 24-h average], 1.5 x AA and 2 x AA). We found a reduction of photosynthesis only at 2 x AA although some foliar traits known as early indicators of O3 stress responded already at 1.5 x AA, such as increased dark respiration, reduced leaf pigment content, reduced maximum quantum yield of PSII, inactivation of the oxygen evolving complex of PSII and reduced performance index PITOT. As a result, O3 did not affect most of the growth parameters although significant declines of root biomass occurred only at 2 x AA. The major mechanism in date palm for reducing the severity of O3 impacts was a restriction of stomatal O3 uptake due to low stomatal conductance and O3-induced stomatal closure. In addition, an increased respiration in elevated O3 may indicate an enhanced capacity of catabolizing metabolites for detoxification and repair. Interestingly, date palm produced low amounts of monoterpenes, whose emission was stimulated in 2 x AA, although isoprene emission declined at both 1.5 and 2 x AA. Our results warrant more research on a biological significance of terpenoids in plant resistance against O3 stress.

Water use strategy affects avoidance of ozone stress by stomatal closure in Mediterranean trees-A modelling analysis.

Both ozone (O3 ) and drought can limit carbon fixation by forest trees. To cope with drought stress, plants have isohydric or anisohydric water use strategies. Ozone enters plant tissues through stomata. Therefore, stomatal closure can be interpreted as avoidance to O3 stress. Here, we applied an optimization model of stomata involving water, CO2 , and O3 flux to test whether isohydric and anisohydric strategies may affect avoidance of O3 stress by stomatal closure in four Mediterranean tree species during drought. The data suggest that stomatal closure represents a response to avoid damage to the photosynthetic mechanisms under elevated O3 depending on plant water use strategy. Under high-O3 and well-watered conditions, isohydric species limited O3 fluxes by stomatal closure, whereas anisohydric species activated a tolerance response and did not actively close stomata. Under both O3 and drought stress, however, anisohydric species enhanced the capacity of avoidance by closing stomata to cope with the severe oxidative stress. In the late growing season, regardless of the water use strategy, the efficiency of O3 stress avoidance decreased with leaf ageing. As a result, carbon assimilation rate was decreased by O3 while stomata did not close enough to limit transpirational water losses.

Somatostatin analogs in pregnant patients with neuroendocrine tumor.

Somatostatine analogs (SSAs) are currently indicated in the treatment of acromegaly and neuroendocrine tumors (NETs). Actually, pregnancy in patients with acromegaly and NETs does not represent an exceptional event because reproductive behavior has changed in the last decades and patients with NETs show more frequently long-term survival. The safety profile of SSAs during pregnancy is still controversial. Concerning acromegaly, based on case reports and series, SSAs administration during pregnancy seems to be relatively well tolerated. Concerning patients with NETs, up to date only one patient with NET receiving SSA during pregnancy has been reported in literature. We report two cases of gastroenteropancreatic-NET patients receiving SSA lanreotide for the entire course of their pregnancy, with favorable outcomes for both mothers and babies. Our experience supports the possibility to continue safely SSA lanreotide during pregnancy in patients with NET.

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.

Immune Checkpoint Inhibitors in the Treatment of Renal Cancer: Current State and Future Perspective.

Systemic treatment of renal cancer (RCC) has undergone remarkable changes over the past 20 years with the introduction of immunotherapeutic agents targeting programmed cell death (PD-1)/programmed death-ligand 1 (PD-L1) axis, as a single-agent or combined with anti-CTLA-4 monoclonal antibodies (MoAbs) or a multi-target vascular endothelial growth factor-(VEGF) tyrosine kinase inhibitor (TKI). In this paper, we review the main evidence on the use of Immune Checkpoint Inhibitors (ICIs) for RCC treatment from the first demonstration of activity of a nivolumab single agent in a phase I trial to the novel combination strategies (anti-PD-1 plus anti-CTLA4 or anti-PD-1 plus TKI). In addition, we discuss the use of anti-PD-1/PD-L1 agents in patients with non-clear cells and rare histological subtype RCC. Then, we critically examine the current findings in biomarkers that have been proposed to be prognostic or predictive to the response of immunotherapy including immune gene expression signature, B7-H1 expression, PBRM1 loss of function, PD-L1 expression, frame shift indel count, mutations in bromodomain-containing genes in patients with MiT family translocation RCC (tRCC), high expression of the T-effector gene signature, and a high myeloid inflammation gene expression pattern. To date, a single biomarker as a predictor of response has not been established. Since the dynamic behavior of the immune response and the different impact of ICI treatment on patients with specific RCC subtypes, the integration of multiple biomarkers and further validation in clinical trials are needed.

Drought stress modulates secondary metabolites in Brassica oleracea L. convar. acephala (DC) Alef, var. sabellica L.

BACKGROUND: Consumer preference today is for the consumption of functional food and the reduction of chemical preservatives. Moreover, the antimicrobial properties and health-promoting qualities of plant secondary metabolites are well known. Due to forecasted climate changes and increasing human population, agricultural practices for saving water have become a concern. In the present study, the physiological responses of curly kale Brassica oleracea L. convar. Acephala (DC) var. sabellica to drought stress and the impact of water limitation on the concentration of selected secondary metabolites were investigated under laboratory-controlled conditions. RESULTS: Results indicated that drought stress increased the content of trans-2-hexenal, phytol and δ-tocopherol, and decreased chlorophyll content. Moreover, drought stress increased antioxidant capacity and the expression of AOP2, a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates, and of three genes - TGG1, TGGE and PEN2 - encoding for myrosinases, the enzymes involved in glucosinolate breakdown. CONCLUSION: The present study shows that water limitation during the growing phase might be exploited as a sustainable practice for producing curly kale with a high concentration of nutritionally important health-promoting bioactive metabolites. © 2019 Society of Chemical Industry.

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.

Effects of nitrogen and phosphorus imbalance on photosynthetic traits of poplar Oxford clone under ozone pollution.

Ozone (O3) pollution and the availability of nitrogen (N) and phosphorus (P) in the soil both affect plant photosynthesis and chlorophyll (Chl) content, but the interaction of O3 and nutrition is unclear. We postulated that the nutritional condition changes plant photosynthetic responses to O3. An O3-sensitive poplar clone (Oxford) was subject to two N levels (N0, 0 kg N ha- 1; N80, 80 kg N ha- 1), two P levels (P0, 0 kg P ha- 1; P80, 80 kg P ha- 1) and three levels of O3 exposure (ambient concentration, AA; 1.5 × AA; 2.0 × AA) over a growing season in an O3 free air controlled exposure (FACE) facility. The daily change of leaf gas exchange and dark respiration (Rd) were investigated at mid-summer (August). Chl a fluorescence was measured three times in July, August and September. At the end of the growing season, Chl content was measured. It was found that Chl content, the maximum quantum yield (Fv/Fm), Chl a fluorescence performance index (PI) and gas exchange were negatively affected by elevated O3. Phosphorus may mitigate the O3-induced reduction of the ratio of photosynthesis to stomatal conductance, while it exacerbated the O3-induced loss of Fv/Fm. Nitrogen alleviated negative effects of O3 on Fv/Fm and PI in July. Ozone-induced loss of net photosynthetic rate was mitigated by N in medium O3 exposure (1.5 × AA). However, such a mitigation effect was not observed in the higher O3 level (2.0 × AA). Nitrogen addition exacerbated O3-induced increase of Rd suggesting an increased respiratory carbon loss in the presence of O3 and N. This may result in a further reduction of the net carbon gain for poplars exposed to O3.

Variations in physiological and biochemical traits of oak seedlings grown under drought and ozone stress.

Despite the huge biodiversity characterizing the Mediterranean environment, environmental constraints, such as high sunlight and high temperatures alongside with dry periods, make plant survival hard. In addition, high irradiance leads to increasing ozone (O3 ) concentrations in ambient air. In this era of global warming, it is necessary to understand the mechanisms that allow native species to tolerate these environmental constraints and how such mechanisms interact. Three Mediterranean oak species (Quercus ilex, Quercus pubescens and Quercus cerris) with different features (drought tolerant, evergreen or deciduous species) were selected to assess their biometrical, physiological and biochemical responses under drought and/or O3 stress (80-100 nl l(-1) of O3 for 5 h day(-1) for 77 consecutive days). Leaf visible injury appeared only under drought stress (alone or combined with O3 ) in all three species. Drought × O3 induced strong reductions in leaf dry weight in Q. pubescens and Q. cerris (-70 and -75%, respectively). Alterations in physiological (i.e. decrease in maximum carboxylation rate) and biochemical parameters (i.e. increase in proline content and build-up of malondialdehyde by-products) occurred in all the three species, although drought represented the major determinant. Quercus ilex and Q. pubescens, which co-occur in dry environments, were more tolerant to drought and drought × O3 . Quercus ilex was the species in which oxidative stress occurred only when drought was applied with O3 . High plasticity at a biochemical level (i.e. proline content) and evergreen habitus are likely on the basis of the higher tolerance of Q. ilex.