Liver transcriptome analysis in gilthead sea bream upon exposure to low temperature.

BACKGROUND: Water temperature greatly influences the physiology and behaviour of teleost fish as other aquatic organisms. While fish are able to cope with seasonal temperature variations, thermal excursions outside their normal thermal range might exceed their ability to respond leading to severe diseases and death.Profound differences exist in thermal tolerance across fish species living in the same geographical areas, promoting for investigating the molecular mechanisms involved in susceptibility and resistance to low and high temperatures toward a better understanding of adaptation to environmental challenges. The gilthead sea bream, Sparus aurata, is particularly sensitive to cold and the prolonged exposure to low temperatures may lead to the "winter disease", a metabolic disorder that significantly affects the aquaculture productions along the Northern Mediterranean coasts during winter-spring season. While sea bream susceptibility to low temperatures has been extensively investigated, the cascade of molecular events under such stressful condition is not fully elucidated. RESULTS: In the present study two groups of wild sea bream were exposed for 21 days to two temperature regimes: 16 ± 0.3°C (control group) and 6.8 ± 0.3°C (cold-exposed group) and DNA microarray analysis of liver transcriptome was carried out at different time points during cold exposure.A large set of genes was found to be differentially expressed upon cold-exposure with increasingly relevant effects being observed after three weeks at low temperature. All major known responses to cold (i.e. anti-oxidant response, increased mitochondrial function, membrane compositional changes) were found to be conserved in the gilthead sea bream, while, evidence for a key role of unfolded protein response (UPR) to endoplasmic reticulum (ER) stress, during short- and long-term exposure to cold is reported here for the first time. CONCLUSIONS: Transcriptome data suggest a scenario where oxidative stress, altered lipid metabolism, ATP depletion and protein denaturation converge to induce ER stress. The resulting UPR activation further promotes conditions for cell damage, and the inability to resolve ER stress leads to severe liver dysfunction and potentially to death.

Uptake and translocation of pharmaceutically active compounds by olive tree (Olea europaea L.) irrigated with treated municipal wastewater.

Introduction: The use of treated municipal wastewater (TWW) represents a relevant opportunity for irrigation of agricultural crops in semi-arid regions to counter the increasing water scarcity. Pharmaceutically active compounds (PhACs) are often detected in treated wastewater, posing a risk to humans and the environment. PhACs can accumulate in soils and translocate into different plant tissues, reaching, in some cases, edible organs and entering the food chain. Methods: This study evaluated the uptake and translocation processes of 10 PhACs by olive trees irrigated with TWW, investigating their accumulation in different plant organs. The experiment was conducted in southern Italy, in 2-year-old plants irrigated with three different types of water: freshwater (FW), TWW spiked with 10 PhACs at a concentration of 200 µg L-1 (1× TWW), and at a triple dose (3× TWW), from July to October 2021. The concentration of PhACs in soil and plant organs was assessed, collecting samples of root, stem, shoot, leaf, fruit, and kernel at 0 (T0), 50 (T1), and 107 (T2) days of irrigation. PhACs extraction from soil and plant organs was carried out using the QuEChERS method, and their concentrations were determined by high-resolution mass spectrometry coupled with liquid chromatography. Results: Results of uptake factors (UF) showed a different behavior between compounds according to their physicochemical properties, highlighting PhACs accumulation and translocation in different plant organs (also edible part) in 1× TWW and 3× TWW compared to FW. Two PhACs, carbamazepine and fluconazole, showed interactions with the soil-plant system, translocating also in the aerial part of the plant, with a translocation factor (TF) greater than 1, which indicates high root-to-leaf translocation. Discussion: Findings highlight that only few PhACs among the selected compounds can be uptaken by woody plants and accumulated in edible parts at low concentration. No effects of PhACs exposure on plant growth have been detected. Despite the attention to be paid to the few compounds that translocate into edible organs, these results are promising for adapting wastewater irrigation in crops. Increasing knowledge about PhACs behavior in woody plants can be important for developing optimized wastewater irrigation and soil management strategies to reduce PhACs accumulation and translocation in plants.

Carbon isotope discrimination and water use efficiency in interspecific Prunus hybrids subjected to drought stress.

In C3 plants, carbon isotope composition (δ13C) is influenced by isotopic effects during diffusion from the atmosphere to the chloroplasts and carboxylation reactions. This work aimed to demonstrate if δ13C of leaf soluble carbohydrates (δ13Cleaves) and of dry matter from new-growth shoots (δ13Cshoots) of Prunus plants subjected to a period of water deficit was related to water use efficiency (WUE). For this purpose, three interspecific Prunus hybrids rootstocks (6-5, 7-7 and G × N) were gradually subjected to drought and then rewatered. Soil water content (SWC) decreased from 26.1 to 9.4% after 70 days of water shortage, when plants reached values of predawn leaf water potential (LWP) ranging from -3.12 to -4.00 MPa. Gas exchange, particularly net photosynthetic and transpiration rates, differed among the three hybrids, leading to different values of WUE. After 70 days of drought, a significant δ13C increase of 5.86, 4.28 and 4.99‰ was observed in 6-5, 7-7 and G × N, respectively. Significant correlations between δ13C and other parameters (substomatal CO2/atmospheric CO2 ratio, stomatal conductance and stem water potential) were found in all hybrids. The rewatering phase caused a recovery of the physiological status of the plants. The isotope composition of δ13Cshoots was correlated with the average WUE measured during the whole experiment. δ13Cleaves and δ13Cshoots were positively related (r = 0.87; p < 0.001). The isotopic signature was a reliable screening tool to identify Prunus genotypes tolerant to drought stress. The results suggest the possibility of using δ13C as an integrated indicator of level of drought stress in plants subjected to prolonged stress conditions.

Soil management type differentially modulates the metabolomic profile of olive xylem sap.

In conventional olive growing, frequent soil tillage strongly reduces the complexity and diversity of the agro-ecosystem. Here, a metabolomic analysis was carried out on the xylem sap (XS) of olive plants (Olea europaea L.) from a grove located in Southern Italy (Basilicata region). The orchard has been divided in two plots that have been managed for 18 years with two different systems: a) 'sustainable management' (Smng), with no-tillage, fertigation and internal C-inputs (spontaneous weeds and pruning residues), and b) an adjacent rainfed 'conventional management' (Cmng), that included soil tillage and mineral fertilization. The XS was extracted from olive shoots in two sampling times (ST1: May; ST2: October) using a Sholander pressure chamber, and its metabolome analyzed by ultra-high performance liquid chromatography (UHPLC) coupled to a hybrid quadrupole-time-of-flight mass spectrometer (QTOF-MS). The discriminating compounds were 94 at ST1 and 119 at ST2, and 35 of them were in common between the two sampling times. The majority of the discriminating metabolites (73 on 94 at ST1, and 109 on 119 at ST2) were found at higher concentration in the XS of Smng plants, compared to that of Cmng ones. Most of the discriminating metabolites found in XS (about 80%, both at ST1 and ST2) were involved in plant secondary metabolism, mainly for plant chemical defense, growth regulation and signal transduction. The most prevailing class of compounds included terpenoids, phytohormones, alkaloids, sterols/steroids, retinols/retinoids, tocopherols and carotenoids. For the first time, we have demonstrated that the XS of a tree crop significantly responds to a shift of soil management. Generally, the plants of the Smng plot showed an up-regulated secondary metabolism. The results of our study encourage the use of a set of sustainable agricultural practices in a productive orchard, in order to enhance plant physiological status, increase yield quantity/quality, safeguard the environment and ameliorate human health.

Evaluation of the possible persistence of potential human pathogenic bacteria in olive orchards irrigated with treated urban wastewater.

Under suitable conditions, low-quality, treated urban wastewater (TWW) is an additional water resource for irrigation in water-scarce environments but its use in agriculture requires a careful monitoring of a range of hygiene parameters, including human pathogenic bacteria (HPB). DNA-based microbiological analyses on soil, xylem sap, and leaves surface (phyllosphere) were carried out in an olive (Olea europaea L.) grove located in Southern Italy (Basilicata region). The experimental grove has been managed in two plots for 18 years. The experimental plot (WWtr) was drip irrigated daily with TWW (2800 m3 ha-1 year-1), while the control plot (RFtr) was rainfed. The results of the 16S-rRNA-based metagenomic analysis demonstrated that the phyllosphere had the lowest number of potential HPB (6), compared to soil (22) and xylem (26) compartments. Gammaproteobacteria, including potential HPB, like Pseudomonas and Acinetobacter spp., were significantly higher in WWtr soil and xylem sap, compared to RFtr. A similar trend was observed for Burkholderia spp. (Betaproteobacteria) and Mycobacterium spp. (Actinobacteria). The Firmicutes Enterococcus, Staphylococcus and Streptococcus spp. were more abundant in WWtr xylem sap. The pathogenic Clostridium perfringens was found higher on WWtr leaves (relative abundance 7.17 in WWtr and 1.33 in RFtr) and Enterococcus faecalis in WWtr xylem sap (93.22 in WWtr and 7.08 in RFtr). On the basis of the results obtained, the irrigation with TWW can be considered a realistic and safe agronomic practice in Mediterranean orchards, and an opportunity for farmers and consumers.

Development and validation of a gene expression oligo microarray for the gilthead sea bream (Sparus aurata).

BACKGROUND: Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on biological processes in farmed species and overcome such bottlenecks. Here we present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture. RESULTS: A public data base was constructed, consisting of 19,734 unique clusters (3,563 contigs and 16,171 singletons). Functional annotation was obtained for 8,021 clusters. Over 4,000 sequences were also associated with a GO entry. Two 60mer probes were designed for each gene and in-situ synthesized on glass slides using Agilent SurePrint technology. Platform reproducibility and accuracy were assessed on two early stages of sea bream development (one-day and four days old larvae). Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. A two class SAM test identified 1,050 differentially expressed genes between the two developmental stages. Functional analysis suggested that down-regulated transcripts (407) in older larvae are mostly essential/housekeeping genes, whereas tissue-specific genes are up-regulated in parallel with the formation of key organs (eye, digestive system). Cross-validation of microarray data was carried out using quantitative qRT-PCR on 11 target genes, selected to reflect the whole range of fold-change and both up-regulated and down-regulated genes. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates. Good concordance between qRT-PCR and microarray data was observed between 2- and 7-fold change, while fold-change compression in the microarray was present for differences greater than 10-fold in the qRT-PCR. CONCLUSION: A highly reliable oligo-microarray platform was developed and validated for the gilthead sea bream despite the presently limited knowledge of the species transcriptome. Because of the flexible design this array will be able to accommodate additional probes as soon as novel unique transcripts are available.

Fruit calcium accumulation coupled and uncoupled from its transpiration in kiwifruit.

Accumulation of Ca in several fleshy fruit is often supposed to depend, among others, by climatic variables driving fruit transpiration. This study tests the whole causal chain hypothesis: VPD → fruit transpiration → Ca accumulation. Also there are evidences that relationship between fruit transpiration and Ca content is not always clear, hence the hypothesis that low VPD reduces the fraction of xylemic water destined to transpiration was tested by examining the water budget of fruit. Attached fruits of Actinidia deliciosa were subjected to Low (L) and High (H) VPD. Their transpiration was measured from early after fruit-set to day 157 after full bloom (DAFB). Fruits were picked at 70, 130 and 157 DAFB for Ca and K determinations and for water budget analysis. Cumulative transpired water was ∼ 70 g and ∼ 16 g H2O f(-1) in HVPD and LVPD, respectively. Calcium accumulated linearly (R(2) = 0.71) with cumulative transpiration when VPD was high, while correlation was weaker (R(2) = 0.24) under LVPD. Under low VPD the fraction of xylem stream destined to transpiration declined to 40-50%. Results suggest that Ca accumulation is coupled to cumulative transpiration under high VPD because under that condition cumulative transpiration equals xylem stream (which carry the nutrient). At LVPD, Ca gain by fruit is uncoupled from transpiration because ∼ 60% of the xylemic water is needed to sustain fruit growth. Results will apply to most fruits (apples, tomatoes, capsicum, grapes etc.) since most suffer Ca deficiency disorders and grow in changing environments with variable VPD, also they could be supportive for the implementation of fruit quality models accounting also for mineral compositions and for a reinterpretation of certain field practices aimed at naturally improve fruit Ca content.