Elisa Pellegrini

Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone.

Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d−1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator.

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.

Salt-tolerant rootstock increases yield of pepper under salinity through maintenance of photosynthetic performance and sinks strength.

The performance of a salt-tolerant pepper (Capsicum annuum L.) accession (A25) utilized as a rootstock was assessed in two experiments. In a first field experiment under natural salinity conditions, we observed a larger amount of marketable fruit (+75%) and lower Blossom-end Root incidence (-31%) in commercial pepper cultivar Adige (A) grafted onto A25 (A/A25) when compared with ungrafted plants. In order to understand this behavior a second greenhouse experiment was conducted to determine growth, mineral partitioning, gas exchange and chlorophyll a fluorescence parameters, antioxidant systems and proline content in A and A/A25 plants under salinity conditions (80 mM NaCl for 14 days). Salt stress induced significantly stunted growth of A plants (-40.6% of leaf dry weight) compared to the control conditions, while no alterations were observed in A/A25 at the end of the experiment. Accumulation of Na(+) and Cl(-) in leaves and roots was similar in either grafted or ungrafted plants. Despite the activation of protective mechanisms (increment of superoxide dismutase, catalase, ascorbate peroxidase activity and non-photochemical quenching), A plants showed severely reduced photosynthetic CO2 assimilation (-45.6% of AN390) and substantial buildup of malondialdehyde (MDA) by-product, suggesting the inability to counteract salt-triggered damage. In contrast, A/A25 plants, which had a constitutive enhanced root apparatus, were able to maintain the shoot and root growth under salinity conditions by supporting the maintained photosynthetic performance. No increases in catalase and ascorbate peroxidase activities were observed in response to salinity, and MDA levels increased only slightly; indicating that alleviation of oxidative stress did not occur in A/A25 plants. In these plants the increased proline levels could protect enzymatic stability from salt-triggered damage, preserving the photosynthetic performance. The results could indicate that salt stress was vanished by the lack of negative effects on photosynthesis that support the maintained plant growth and increased marketable yield of the grafted plants.

How sensitive is Melissa officinalis to realistic ozone concentrations

Lemon balm (Melissa officinalis, L.; Lamiaceae) was exposed to realistic ozone (O3) dosages (80 ppb for 5 h), because high background levels of O3 are considered to be as harmful as episodic O3 regimes. Temporal alterations of different ecophysiological, biochemical and structural parameters were investigated in order to test if this species can be considered as an O3-bioindicator regarding changes in background concentrations. At the end of ozone exposure, the plants did not exhibit any visible foliar symptoms, as only at microscopic level a small number of dead cells were found. Photosynthetic processes, however, were significantly affected. During and after the treatment, ozone induced a reduction in CO2 fixation capacity (up to 52% after 12 h from the beginning of the treatment) due to mesophyllic limitations. Intercellular CO2 concentration significantly increased in comparison to controls (+90% at the end of the post-fumigation period). Furthermore impairment of carboxylation efficiency (-71% at the end of the post-fumigation period compared to controls in filtered air) and membrane damage in terms of integrity (as demonstrated by a significant rise in solute leakage) were observed. A regulatory adjustment of photosynthetic processes was highlighted during the post-fumigation period by the higher values of qNP and (1-q(P)) and therefore suggests a tendency to reduce the light energy used in photochemistry at the expense of the capacity to dissipate the excess as excitation energy. In addition, the chlorophyll a/b ratio and the de-epoxidation index increased, showing a rearrangement of the pigment composition of the photosynthetic apparatus and a marked activation of photoprotective mechanisms.

Functional Leaf Traits and Diurnal Dynamics of Photosynthetic Parameters Predict the Behavior of Grapevine Varieties Towards Ozone.

A comparative study on functional leaf treats and the diurnal dynamics of photosynthetic processes was conducted on 2-year-old potted plants of two grape (Vitis vinifera L.) varieties (Aleatico, ALE, and Trebbiano giallo, TRE), exposed under controlled conditions to realistic concentrations of the pollutant gas ozone (80 ppb for 5 h day-1, 8:00–13:00 h, + 40 ppb for 5 h day-1, 13:00–18:00 h). At constitutive levels, the morphological functional traits of TRE improved leaf resistance to gas exchange, suggesting that TRE is characterized by a potential high degree of tolerance to ozone. At the end of the treatment, both varieties showed typical visible injuries on fully expanded leaves and a marked alteration in the diurnal pattern of photosynthetic activity. This was mainly due to a decreased stomatal conductance (-27 and -29% in ALE and TRE, in terms of daily values in comparison to controls) and to a reduced mesophyllic functioning (+33 and +16% of the intercellular carbon dioxide concentration). Although the genotypic variability of grape regulates the response to oxidative stress, similar detoxification processes were activated, such as an increased content of total carotenoids (+64 and +30%, in ALE and TRE), enhanced efficiency of thermal energy dissipation within photosystem II (+32 and +20%) closely correlated with the increased de-epoxidation index (+26 and +22%) and variations in content of some osmolytes. In summary, we can conclude that: the daily photosynthetic performance of grapevine leaves was affected by a realistic exposure to ozone. In addition, the gas exchange and chlorophyll a fluorescence measurements revealed a different quali-quantitative response in the two varieties. The genotypic variability of V. vinifera and the functional leaf traits would seem to regulate the acclimatory response to oxidative stress and the degree of tolerance to ozone. Similar photoprotective mechanisms were activated in the two varieties, though to a different extent.

Ozone tolerance in lichens: a possible explanation from biochemical to physiological level using Flavoparmelia caperata as test organism.

Lichens are among the best biomonitors of airborne pollutants, but surprisingly they reveal high tolerance to ozone (O3). It was recently suggested that this might be due to the high levels of natural defences against oxidative stress, related to their poikilohydric life strategy. The objective of this work is to give a thorough description of the biochemical and physiological mechanisms that are at the basis of the O3-tolerance of lichens. Chlorophyll a fluorescence (ChlaF) emission, histochemical ROS localization in the lichen thallus, and biochemical markers [enzymes and antioxidants involved in the ascorbate/glutathione (AsA/GSH) cycle; hydrogen peroxide (H2O2) and superoxide anion (O2(-))] were used to characterize the response of the epiphytic lichen Flavoparmelia caperata (L.) Hale exposed to O3 (250 ppb, 5 hd(-1), 2 weeks) at different watering regimes and air relative humidity (RH) in a fumigation chamber. After two-week exposure ChlaF was affected by the watering regime but not by O3. The watering regime influenced also the superoxide dismutase activity and the production of ROS. By contrast O3 strongly influenced the AsA/GSH biochemical pathway, decreasing the reduced ascorbate (AsA) content and increasing the enzymatic activity of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) independently from the watering regime and the relative humidity applied. This study highlights that F. caperata can face the O3-induced oxidative stress thanks to high levels of constitutive enzymatic and non-enzymatic defences against ROS formed naturally during the dehydration-rehydration cycles to which lichens are frequently exposed.

How do background ozone concentrations affect the biosynthesis of rosmarinic acid in Melissa officinalis

Lemon balm (Melissa officinalis; Lamiaceae) plants were exposed to background ozone (O3) dosages (80ppb for 5h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Immediately at the end of fumigation the plants appeared visually symptomless, but necrotic lesions were observed later. The biosynthesis of rosmarinic acid (RA) comprises eight enzymes, among them phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and rosmarinic acid synthase (RAS). The transcript levels of these genes have been investigated by quantitative RT-PCR. There was a quick up-regulation of all genes at 3h of O3 exposure, but at 24h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease of RA concentration in lemon balm leaves. The specific activity of PAL increased at 12h FBE to 163% in comparison to control levels. This work provides insight into the effect of O3 stress on the formation of the main phenolic ingredient of the pharmaceutically important plant M. officinalis.

The harsh life of an urban tree: the effect of a single pulse of ozone in salt-stressed Quercus ilex saplings

Ozone (O3) and salinity are usually tested as combined factors on plant performance. However, the response to a single episode of O3 in plants already stressed by an excess of NaCl as occurs in the natural environment has never been investigated, but is important given that it is commonly experienced in Mediterranean areas. Three-year-old Quercus ilex L. (holm oak) saplings were exposed to salinity (150 mM NaCl, 15 days), and the effect on photosynthesis, hydric relations and ion partitioning was evaluated (Experiment I). In Experiment II, salt-treated saplings were exposed to 80 nl l-1 of O3 for 5 h, which is a realistic dose in a Mediterranean environment. Gas exchanges, chlorophyll fluorescence and antioxidant systems were characterized to test whether the salt-induced stomatal closure limited O3 uptake and stress or whether the pollutant represents an additional stressor for plants. Salt-dependent stomatal closure depressed the photosynthetic process (-71.6% of light-saturated rate of photosynthesis (A380)) and strongly enhanced the dissipation of energy via the xanthophyll cycle. However, salt-treated plants had higher values of net assimilation rate/stomatal conductance (A/gs) than the controls, which was attributable to a greater mesophyll conductance gm/gs and carboxylation efficiency (higher gm/maximal rate of Rubisco carboxylation (Vcmax)), thus suggesting no damage to chloroplasts. O3 did not exacerbate the effect of salinity on photosynthesis, however a general enhancement of the Halliwell-Asada cycle was necessary to counteract the O3-triggered oxidative stress. Despite the 79.4% gs reduction in salt-stressed plants, which strongly limited the O3 uptake, a single peak in the air pollutant led to an additional burden for the antioxidant system when plants had been previously subjected to salinity.

Trichoderma harzianum T-22 induces systemic resistance in tomato infected by Cucumber mosaic virus.

Understanding the induction of plant defenses against viruses using biocontrol agents is essential for developing new strategies against these pathogens, given the ineffectiveness of chemical treatments. The ability of Trichoderma harzianum, strain T-22 (T22) to control Cucumber mosaic virus (CMV) in Solanum lycopersicum var. cerasiforme plants and the changes in the physiology of tomato treated/infected with T22/CMV were examined. Plant growth-promoting effects, photosynthetic performance, reactive oxygen species scavenging enzymes, and phytohormones were investigated. T22 improved tomato growth in terms of plant height and improved photosynthesis, total chlorophyll content and plant gas exchange. In contrast, CMV induced a negative effect on dry matter accumulation and inhibited the photosynthetic capacity. The analysis of plant hormones demonstrated that treating with T22 before or simultaneously to CMV infection, led to a systemic resistance by jasmonic acid/ethylene and salicylic acid signaling pathways. Conversely, systemic resistance was abscissic acid-dependent when T22 treatment was administered after the CMV infection. In conclusion, the data reported here indicate that the T22-based strategy may be the most effective measure against CMV.

Ecophysiology of Tilia Americana under ozone fumigation

Abstract The negative effects of the pollutant gas ozone are widely studied in many plant species, but the intimate mechanisms of toxicity have not been completely defined. Generally this contaminant or its free radical by–products impair membrane functions, leading to declines in physiological processes, accelerated foliar senescence and premature leaf abscission. Trees of the genus Tilia do not show any foliar injury induced by ozone under natural conditions. In this study, we investigated the effects of this pollutant on ecophysiological and biochemical parameters of T. Americana saplings exposed to a fumigation (120 ppb for 45 consecutive days, 5 h d –1 ). At the end of treatment, even if plants did not exhibit any visible foliar injury, several parameters were significantly affected: stomatal conductance for water vapor (–15% compared to control), net photosynthesis (–39%), intercellular CO 2 concentration (+30%), as well as chlorophyll fluorescence indexes. After 45 days of fumigation neo–, viola– and anteraxanthin content significantly decreased (–25%, –34% and –63%, respectively, in comparison with controls), but no zeaxanthin induction was detected, suggesting that exposure did not activate the xanthopyll cycle. Under these circumstances, this species should be regarded as “middle tolerant/sensitive”.