Differential gene expression and chemical patterns of an intertidal crab inhabiting a polluted port and an adjacent marine protected area.

The acquisition of data to safeguard marine protected areas located close to ports is important in order to develop plans that allow effective protection from pollution as well as sustainable development of the port. The area Secche della Meloria is a Marine Protected Area (MPA-MEL) three miles from Livorno Harbour (LH), which is characterized by a long history of pollution. Here we studied the bioaccumulation and transcriptomic patterns of the marbled crab, Pachygrapsus marmoratus (Fabricius, 1787) (Crustacea; Brachyura, Grapsidae), inhabiting the two selected sites. Results showed that the two crab populations are significantly different in their chemical composition of trace elements and Polyciclic Aromatic Hydrocarbons (PAHs), and gene expression patterns (1280 DEGs). Enrichment analysis indicated that crabs at LH had the highest stress response genes, and they were associated with higher levels of bioaccumulation detected in body tissues. We are confident that the significant differential gene expression profiles observed between crabs, characterized by significant chemical differences, is associated with responses to contaminant exposure.

Himantoglossum adriaticum H. Baumann × Himantoglossum robertianum (Loisel.) P. Delforge: A New Interspecific Hybrid Assessed by Barcoding Analysis.

Most cultivated orchids, contributing to a worldwide highly profitable industry, are originated from tropic regions. Conversely, a considerable number of spontaneous orchids, belonging to the terrestrial orchids and widely diffused throughout the European continent, are not considered for trading due to their less gorgeous appearance and for technical difficulties in seed propagation. However, a breeding programme was undertaken aimed at developing a new hybrid between Himantoglossum adriaticum H. Baumann and H. robertianum (Loisel.) P. Delforge [syn. Barlia robertiana (Loisel.) Greuter] by applying techniques of anther conservation, manual pollination and in vitro asymbiotic germination of the obtained seeds. The plantlets that originated from the protocorms after seed germination were successfully acclimatised after potting in a proper medium. The parentage of the progenies of the hybridisation experiment was assessed by sequencing the Internal Transcribed Spacer assembly (ITS) and plastid barcoding markers of the parental lines and of the hybrids. The method proved to be effective in revealing the origin of the hybrids and to validate the maternal inheritance of the plastid DNA.

Effect of ammonia on the gene expression levels of the freshwater cyclopoid Eucyclops serrulatus.

Ammonia pollution is a critical issue in Europe, since more than half of the European freshwater bodies actually fail to meet EU quality standards for this chemical. In this study, the response of stress-related genes to a sublethal ammonia concentration has been investigated in the adults of the freshwater cyclopoid Eucyclops serrulatus. Two short-term exposures (12h and 24h) at 12mg/L NH4+ have been tested. Results indicate that 12mg/L NH4+ causes a significant increase in the expression of some proteins, namely CAT, HSP90 and HSP40, suggesting an activation of the protecting antioxidant system after both 12h and 24h.

Polyphenolic profiles and antioxidant and antiradical activity of Italian berries from Vaccinium myrtillus L. and Vaccinium uliginosum L. subsp. gaultherioides (Bigelow) S.B. Young.

Total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), radical scavenging activity (RSA), ferric reducing antioxidant power (FRAP), and a number of anthocyanins, phenolic acids, coumarins, flavanols, dihydrochalcones and flavonols were investigated in Tuscan bilberry (i.e. Vaccinium myrtillus) and "false bilberry" (i.e. Vaccinium uliginosum subsp. gaultherioides Bigelow). V. myrtillus berries showed much higher TSP, TMA, RSA and FRAP values than V. uliginosum subsp. gaultherioides fruits. Moreover, very different profiles of individual phenolics were observed in the two species, being V. myrtillus mainly characterised by delphinidin and cyanidin glycosides, together with chlorogenic acid, and V. uliginosum subsp. gaultherioides dominated by malvidin derivatives and flavonols. Strong differences between the two species regarded also metabolites investigated herein for the first time, such as scopoletin, which was approximately two magnitude orders higher in V. uliginosum subsp. gaultherioides than in V. myrtillus berries. Very different abundances were also highlighted for cryptochlorogenic acid and quercetin-3-rhamnoside that were about ten-fold higher in bilberry than in "false bilberry". When the anthocyanin composition pattern of Tuscan "false bilberry" was compared to those elsewhere reported for V. uliginosum fruits harvested in different world areas, some important differences were observed.

Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment.

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO2 assimilation rate at saturating light and daily assimilated CO2 were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (Φ(PSII)) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chl(tot)) exceeded 200 mmol mol⁻¹ Chl(tot) in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H2O2. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H2O2 in excess light-stressed leaves. Nonetheless, the concentration of H2O2 and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H2O2 within a sub-lethal concentration range.

The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves.

Flavonoids have recently been suggested to have the potential to serve as antioxidants other than effective UV attenuators in photoprotection. Here, we tested the hypothesis that flavonoids accumulate in response to "excess light" in the presence or in the absence of UV radiation. In a UV exclusion experiment, we grew Ligustrum vulgare plants outdoors under 30% or 100% sunlight irradiance, by cutting-off the whole UV waveband. These plants were also exposed to UV irradiance or supplied with 125 mM NaCl at the root zone. Leaves of plants under 100% sunlight irradiance suffered from excess light, which was exacerbated greatly by root zone salinity stress. Salinity stress repressed the activities of antioxidant enzymes, particularly in full sunlight, and led to severe leaf oxidative damage. Dihydroxy B-ring-substituted flavonoids, namely quercetin 3-O- and luteolin 7-O-glycosides, accumulated steeply in response to sunlight irradiance in the absence of UV radiation. UV radiation and root zone NaCl increased, to a similar degree, the concentration of these flavonoids, which have a great potential to scavenge various forms of reactive oxygen. Treatment-induced changes in leaf phenylpropanoid concentration affected antioxidant activities to a greater extent than the UV-screening capacities of leaf extracts. Early responses to an abrupt increase in sunlight irradiance included a steep increase in the concentrations of quercetin derivatives and cyanidin 3-O-glucoside, with the latter negligibly absorbing in the UV-spectral region. In contrast, effective UV attenuators, such as hydroxycinnamates and monohydroxy B-ring flavonoids, were unresponsive to the light treatments. Overall, these findings lead to the hypothesis that flavonoids may have an important antioxidant function in photoprotection. This hypothesis is further corroborated by the large distribution of quercetin and luteolin derivatives in the vacuoles of mesophyll, not only in the corresponding compartments of epidermal cells, but also in full sunlight-treated leaves in the absence of UV radiation. Future experiments aimed at evaluating the relative contribution of flavonoids within the complex antioxidant defense systems operating in the leaf are needed to help conclusively address the relevance of their antioxidant functions in photoprotection.

Ozone tolerance in Phaseolus vulgaris depends on more than one mechanism.

Two bean cultivars with different sensitivity to ozone, i.e. the O3-sensitive Cannellino and the O3-tolerant Top Crop, were exposed to acute O3-stress (165 nL L(-1)) with the aim of evaluating physiological and biochemical traits that may confer O3-tolerance. Stomatal conductance was smaller and the ability to dissipate excess energy, via regulated and unregulated nonphotochemical quenching mechanisms was greater in Top Crop than in Cannellino. These morphological and physiological-traits allowed the O3-tolerant cultivar to compensate for the light-induced declines in PhiPSII, to preserve photosystem II from excitation-energy, and likely to prevent the generation of ROS to a superior degree than the O3-sensitive cultivar. Furthermore, the potential capacities to reducing the superoxide anion and H2O2 were significantly greater in Top Crop than in Cannellino. These findings are consistent with the early accumulation of H2O2, the almost complete disruption of cell structure, and irreversible damages to the photosynthetic apparatus observed in the O3-sensitive cultivar.

Mesophyll distribution of 'antioxidant' flavonoid glycosides in Ligustrum vulgare leaves under contrasting sunlight irradiance.

BACKGROUND AND AIMS: Flavonoids have the potential to serve as antioxidants in addition to their function of UV screening in photoprotective mechanisms. However, flavonoids have long been reported to accumulate mostly in epidermal cells and surface organs in response to high sunlight. Therefore, how leaf flavonoids actually carry out their antioxidant functions is still a matter of debate. Here, the distribution of flavonoids with effective antioxidant properties, i.e. the orthodihydroxy B-ring-substituted quercetin and luteolin glycosides, was investigated in the mesophyll of Ligustrum vulgare leaves acclimated to contrasting sunlight irradiance. METHODS: In the first experiment, plants were grown at 20 % (shade) or 100% (sun) natural sunlight. Plants were exposed to 100 % sunlight irradiance in the presence or absence of UV wavelengths, in a second experiment. Fluorescence microspectroscopy and multispectral fluorescence microimaging were used in both cross sections and intact leaf pieces to visualize orthodihydroxy B-ring-substituted flavonoids at inter- and intracellular levels. Identification and quantification of individual hydroxycinnamates and flavonoid glycosides were performed via HPLC-DAD. KEY RESULTS: Quercetin and luteolin derivatives accumulated to a great extent in both the epidermal and mesophyll cells in response to high sunlight. Tissue fluorescence signatures and leaf flavonoid concentrations were strongly related. Monohydroxyflavone glycosides, namely luteolin 4'-O-glucoside and two apigenin 7-O-glycosides were unresponsive to changes in sunlight irradiance. Quercetin and luteolin derivatives accumulated in the vacuoles of mesophyll cells in leaves growing under 100 % natural sunlight in the absence of UV wavelengths. CONCLUSIONS: The above findings lead to the hypothesis that flavonoids play a key role in countering light-induced oxidative stress, and not only in avoiding the penetration of short solar wavelengths in the leaf.

Contrasting response mechanisms to root-zone salinity in three co-occurring Mediterranean woody evergreens: a physiological and biochemical study.

The present study investigated the extent to which physiological and biochemical traits varied because of root-zone salinity in three Mediterranean evergreens differing greatly in their strategies of salt allocation at an organismal level: the 'salt-excluders', Olea europaea L. and Phillyrea latifolia L. (both Oleaceae), and Pistacia lentiscus L., which, instead, largely uses Na+ and Cl- for osmotic adjustment. Both Oleaceae spp. underwent severe leaf dehydration and reduced net photosynthesis and whole-plant growth to a significantly greater degree than did P. lentiscus. Osmotic adjustment in Oleaceae mostly resulted from soluble carbohydrates, which, in turn, likely feedback regulated net photosynthesis. Salt stress reduced the actual efficiency of PSII photochemistry (ΦPSII) and enhanced the concentration of de-epoxided violaxanthin-cycle pigments in O. europaea and P. latifolia. Phenylpropanoid metabolism was upregulated by salt stress to a markedly greater degree in O. europaea and P. latifolia than in P. lentiscus. In contrast, species-specific variations in leaf lipid peroxidation were not observed in response to salinity stress. The results suggest that the species-specific ability to manage the allocation of potentially toxic ions out of sensitive leaf organs, other than affecting physiological responses, largely determined the extent to which leaf biochemistry, mostly aimed to counter salt-induced oxidative damage, varied in response to salinity stress.

Osmyb4 expression improves adaptive responses to drought and cold stress in transgenic apples.

Constitutive expression of the rice cold-inducible Osmyb4 gene in transgenic Arabidopsis (Arabidopsis thaliana) plants improves adaptive responses to cold and drought stress, most likely due to the constitutive activation of several stress-inducible pathways and to the accumulation of several compatible solutes (e.g., glucose, fructose, sucrose, proline, glycine betaine and some aromatic compounds). Although the Osmyb4 gene seems able to activate stress responsive pathways in different species, we previously reported that its specific effect on stress tolerance depends on the transformed species. In the present work, we report the effects of the Osmyb4 expression for improving the stress response in apple (Malus pumila Mill.) plants. Namely, we found that the ectopic expression of the Myb4 transcription factor improved physiological and biochemical adaptation to cold and drought stress and modified metabolite accumulation. Based on these results it may be of interest to use Osmyb4 as a tool for improving the productivity of woody perennials under environmental stress conditions.

Transgene expression in the vegetative tissues of apple driven by the vascular-specific rolC and CoYMV promoters.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of beta-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.