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Long terminal repeats (LTR) retrotransposons have a major role in determining genome size, structure and function, thanks to their ability to transpose. We performed a meta-analysis of LTR-retrotransposon expression in roots of sunflower plantlets treated with different plant hormones, chemicals and NaCl. By using Illumina cDNA libraries, available from public repositories, we measured the number of reads matching the retrotranscriptase domains isolated from a whole genome library of retrotransposons. LTR-retrotransposons resulted in general barely expressed, except for 4 elements, all belonging to the AleII lineage, which showed high transcription levels in roots of both control and treated plants. The expression of retrotransposons in treated plants was slightly higher than in the control. Transcribed elements belonged to specific chromosomal loci and were not abundant in the genome. A few elements resulted differentially expressed depending on the treatment. Results suggest that, although most retrotransposons are not expressed, the transcription of such elements is related to their abundance, to their position in the chromosome and to their lineage.

Simple Summary Milk fatty acid composition and gene expression in the mammary gland were evaluated in dairy ewes supplemented with linseed. The linseed supplementation improves the health and nutrition quality properties of dairy milk, besides affecting the expression of gene networks related to energy balance, RNA binding, circadian rhythm, and nutrient metabolism. This molecular knowledge on the physiology of the mammary gland might provide the basis for more detailed functional studies for future research. Abstract Several dietary strategies were adopted to reduce saturated fatty acids and increase beneficial fatty acids (FA) for human health. Few studies are available about the pathways/genes involved in these processes. Illumina RNA-sequencing was used to investigate changes in the ovine mammary gland transcriptome following supplemental feeding with 20% extruded linseed. Comisana ewes in mid-lactation were fed a control diet for 28 days (control period) followed by supplementation with 20% DM of linseed panel for 28 days (treatment period). Milk production was decreased by 30.46% with linseed supplementation. Moreover, a significant reduction in fat, protein and lactose secretion was also observed. Several unsaturated FAs were increased while short and medium chain saturated FAs were decreased by linseed treatment. Around four thousand (1795 up- and 2133 down-regulated) genes were significantly differentially regulated by linseed supplementation. The main pathways affected by linseed supplementation were those involved in the energy balance of the mammary gland. Principally, the mammary gland of fed linseed sheep showed a reduced abundance of transcripts related to the synthesis of lipids and carbohydrates and oxidative phosphorylation. Our study suggests that the observed decrease in milk saturated FA was correlated to down-regulation of genes in the lipid synthesis and lipid metabolism pathways.

Background Long Terminal Repeat retrotransposons (LTR-REs) are repetitive DNA sequences that constitute a large part of the genome. The improvement of sequencing technologies and sequence assembling strategies has achieved genome sequences with much greater reliability than those of the past, especially in relation to repetitive DNA sequences. Results In this study, we analysed the genome of Ficus carica L., obtained using third generation sequencing technologies and recently released, to characterise the complete complement of full-length LTR-REs to study their dynamics during fig genome evolution. A total of 1867 full-length elements were identified. Those belonging to the Gypsy superfamily were the most abundant; among these, the Chromovirus/Tekay lineage was the most represented. For the Copia superfamily, Ale was the most abundant lineage. Measuring the estimated insertion time of each element showed that, on average, Ivana and Chromovirus/Tekay were the youngest lineages of Copia and Gypsy superfamilies, respectively. Most elements were inactive in transcription, both constitutively and in leaves of plants exposed to an abiotic stress, except for some elements, mostly belonging to the Copia/Ale lineage. A relationship between the inactivity of an element and inactivity of genes lying in close proximity to it was established. Conclusions The data reported in this study provide one of the first sets of information on the genomic dynamics related to LTR-REs in a plant species with highly reliable genome sequence. Fig LTR-REs are highly heterogeneous in abundance and estimated insertion time, and only a few elements are transcriptionally active. In general, the data suggested a direct relationship between estimated insertion time and abundance of an element and an inverse relationship between insertion time (or abundance) and transcription, at least for Copia LTR-REs.

Transposable element activity can be harmful to the host’s genome integrity, but it can also provide selective advantages. One strategy to cope with transposons is epigenetic control through DNA base modifications. We report the non-canonic DNA modification dynamics of fig (Ficus carica L.) by exploiting high-quality genome reference and related N4-methylcytosine (4mC) and N6-methyladenine (6mA) data. Overall, 1.49% of transposon nucleotides showed either 4mC or 6mA modifications: the 4mC/6mA ratio was similar in Class I and Class II transposons, with a prevalence of 4mC, which is comparable to coding genes. Different percentages of 4mC or 6mA were observed among LTR-retrotransposon lineages and sub-lineages. Furthermore, both the Copia and Gypsy retroelements showed higher modification rates in the LTR and coding regions compared with their neighbour regions. Finally, the unconventional methylation of retrotransposons is unrelated to the number of close genes, suggesting that the 4mC and 6mA frequency in LTR-retrotransposons should not be related to transcriptional repression in the adjacency of the element. In conclusion, this study highlighted unconventional DNA modification patterns in fig transposable elements. Further investigations will focus on functional implications, in regards to how modified retroelements affect the expression of neighbouring genes, and whether these epigenetic markers can spread from repeats to genes, shaping the plant phenotype.

Transposable elements (TEs) contribute to genomic innovations, as well as genome instability, across a wide variety of species. Popular designations such as 'selfish DNA' and 'junk DNA,' common in the 1980s, may be either inaccurate or misleading, while a more enlightened view of the TE-host relationship covers a range from parasitism to mutualism. Both plant and animal hosts have evolved epigenetic mechanisms to reduce the impact of TEs, both by directly silencing them and by reducing their ability to transpose in the genome. However, TEs have also been co-opted by both plant and animal genomes to perform a variety of physiological functions, ranging from TE-derived proteins acting directly in normal biological functions to innovations in transcription factor activity and also influencing gene expression. Their presence, in fact, can affect a range of features at genome, phenotype, and population levels. The impact TEs have had on evolution is multifaceted, and many aspects still remain unexplored. In this review, the epigenetic control of TEs is contextualized according to the evolution of complex living systems.

Transposable elements (TEs) are DNA sequences that can change their position within genomes. TEs are present in most organisms and can be an important genomic component. Their activities are manifold: restructuring of genome size, chromosomal rearrangements, induction of gene mutations, and alteration of gene activity by insertion near or within promoters, intronic regions, or enhancer. There are several examples of mutations and other genetic variations determined by the activity of TEs, associated with the evolution of prokaryotic and eukaryotic organisms and the domestication of plants. Generally, TE mobilization occurs when the organism is subjected to stress, which can include both biotic and abiotic stresses, polyploidy conditions, and interspecific hybridizations, very common events in plants. TEs are widely distributed among organisms. TEs also play essential roles in evolution, but most of them are either dormant or inactive. This is mainly determined by epigenetic silencing mechanisms, regulatory systems, and control systems that aim to limit its proliferation. Furthermore, the host has recruited many genes originated from TEs as transcriptional regulators, especially in defense against pathogens and invasive genetic elements; this phenomenon is called molecular domestication. Therefore, TEs are responsible for horizontal gene transfer and the movement of genetic material between organisms, even phylogenetically distant, with a consequent remixing of their gene pools.

Much has been said about sunflower (Helianthus annuus L.) retrotransposons, representing the majority of the sunflower’s repetitive component. By contrast, class II transposons remained poorly described within this species, as they present low sequence conservation and are mostly lacking coding domains, making the identification and characterization of these transposable elements difficult. The transposable element Tetu1, is a non-autonomous CACTA-like element that has been detected in the coding region of a CYCLOIDEA (CYC) gene of a sunflower mutant, tubular ray flower (turf). Based on our knowledge of Tetu1, the publicly available genome of sunflower was fully scanned. A combination of bioinformatics analyses led to the discovery of 707 putative CACTA sequences: 84 elements with complete ends and 623 truncated elements. A detailed characterization of the identified elements allowed further classification into three subgroups of 347 elements on the base of their terminal repeat sequences. Only 39 encode a protein similar to known transposases (TPase), with 10 TPase sequences showing signals of activation. Finally, an analysis of the proximity of CACTA transposons to sunflower genes showed that the majority of CACTA elements are close to the nearest gene, whereas a relevant fraction resides within gene-encoding sequences, likely interfering with sunflower genome functionality and organization.

The dynamics of long-terminal-repeat retrotransposons in two poplar species (Populus deltoides and P. nigra) and in an interspecific hybrid, recently synthesized, were investigated by analyzing the genomic abundance and transcription levels of a collection of 828 full-length retroelements identified in the genome sequence of P. trichocarpa, all occurring also in the genomes of P. deltoides and P. nigra. Overall, genomic abundance and transcription levels of many retrotransposons in the hybrid resulted higher or lower than expected by calculating the mean of the parental values. A bioinformatics procedure was established to ascertain the occurrence of the same retrotransposon loci in the three genotypes. The results indicated that retrotransposon abundance variations between the hybrid and the mean value of the parents were due to i) co-segregation of retrotransposon high- or low-abundant haplotypes; ii) new retroelement insertions; iii) retrotransposon loss. Concerning retrotransposon expression, this was generally low, with only 14/828 elements over- or under-expressed in the hybrid than expected by calculating the mean of the parents. It is concluded that interspecific hybridisation between the two poplar species determine quantitative variation and differential expression of some retrotransposons, with possible consequences for the genetic differentiation of the hybrid.

Seagrasses as Posidonia oceanica reproduce mostly by vegetative propagation, which can reduce genetic variability within populations. Since, in clonally propagated species, insurgence of genetic variability can be determined by the activity of transposable elements, we have estimated the activity of such repeat elements by measuring their expression level in the leaves of plants from a Mediterranean site, for which Illumina complementary DNA (cDNA) sequence reads (produced from RNAs isolated by leaves of plants from deep and shallow meadows) were publicly available. Firstly, we produced a collection of retrotransposon-related sequences and then mapped Illumina cDNA reads onto these sequences. With this approach, it was evident that Posidonia retrotransposons are, in general, barely expressed, only nine elements resulted transcribed at levels comparable with those of reference genes encoding tubulins and actins. Differences in transcript abundance were observed according to the superfamily and the lineage to which the retrotransposons belonged. Only small differences were observed between retrotransposon expression levels in leaves of shallow and deep Posidonia meadow stands, whereas one TAR/Tork element resulted differentially expressed in deep plants exposed to heat. It can be concluded that, in P. oceanica, the contribution of retrotransposon activity to genetic variability is reduced, although the nine specific active elements could actually produce new structural variations.

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.

An overall picture comparing the repetitive components of the genomes of three Quercus species was obtained by genome skimming with Illumina sequence reads. Read sets of Q. lobata, Q. robur, and Q. suber species were subjected to hybrid clustering in order to assemble a repeatome of the Quercus genus and to annotate it. The repeatome was composed of 8573 clusters. The abundance of repeated sequences in the three species was assessed by mapping Illumina reads of each species onto the repeatome. The repetitive portion of the genome was similar among the three species. The most abundant repetitive sequences were long terminal repeat-retrotransposons. Copia elements were overrepresented when compared with Gypsy ones. The most abundant retrotransposon lineages were SIRE for the Copia superfamily and Ogre/TAT for the Gypsy superfamily. Some of the clusters belonging to these lineages showed different transpositional time profiles among the three species. Ribosomal DNAs accounted for 3.64–6.75% of the repetitive component. Satellite DNAs were much more abundant in Q. lobata (8.68% of the genome) than in the two other species. However, different satellite DNAs showed large variations in their abundances. Overall, the composition of the repetitive portion of the genome showed some differences among oak species, suggesting a possible role of repeats for Quercus species differentiation. In the cases of Q. lobata and Q. robur, both of which belong to the Quercus section of the Quercus genus, such differences may be related to the different geographical origins of the species.

Arbuscular mycorrhizal fungi (AMF) play a fundamental role in plant growth and nutrition in natural and agricultural ecosystems. Despite the importance of such symbionts, the different developmental changes occurring during the AMF life cycle have not been fully elucidated at the molecular level. Here, the RNA-seq approach was used to investigate Rhizoglomus irregulare specific and common transcripts at two different time points of mycorrhizal establishment in Helianthus annuus in vivo. Four days after inoculation, transcripts related to cellular remodeling (actin and tubulin), cellular signaling (calmodulin, serine/threonine protein kinase, 14-3-3 protein, and calcium transporting ATPase), lipid metabolism (fatty acid desaturation, steroid hormone, and glycerophospholipid biosynthesis), and biosynthetic processes were detected. In addition to such transcripts, 16 days after inoculation, expressed genes linked to binding and catalytic activities; ion (K+, Ca2+, Fe2+, Zn2+, Mn2+, Pi, ammonia), sugar, and lipid transport; and those involved in vacuolar polyphosphate accumulation were found. Knowledge of transcriptomic changes required for symbiosis establishment and performance is of great importance to understand the functional role of AMF symbionts in food crop nutrition and health, and in plant diversity in natural ecosystems.

Due to DNA heterozygosity and repeat content, assembly of non-model plant genomes is challenging. Herein, we report a high-quality genome reference of one of the oldest known domesticated species, fig (Ficus carica L.), using Pacific Biosciences single-molecule, real-time sequencing. The fig genome is ~333 Mbp in size, of which 80% has been anchored to 13 chromosomes. Genome-wide analysis of N6 -methyladenine and N4 -methylcytosine revealed high methylation levels in both genes and transposable elements, and a prevalence of methylated over non-methylated genes. Furthermore, the characterization of N6 -methyladenine sites led to the identification of ANHGA, a species-specific motif, which is prevalent for both genes and transposable elements. Finally, exploiting the contiguity of the 13 pseudomolecules, we identified 13 putative centromeric regions. The high-quality reference genome and the characterization of methylation profiles, provides an important resource for both fig breeding and for fundamental research into the relationship between epigenetic changes and phenotype, using fig as a model species.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-5260-2) contains supplementary material, which is available to authorized users.

Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.

The availability of transcriptomic data sequence is a key step for functional genomics studies. Recently, a repertoire of predicted genes of a Japanese cultivar of fig (Ficus carica L.) was released. Because of the great phenotypic variability that can be found in this species, we decided to study another fig genotype, the Italian cv. Dottato, in order to perform comparative studies between the two cultivars and extend the pan genome of this species. We isolated, sequenced and assembled fig genomic DNA from young fruits of cv. Dottato. Then, putative gene sequences were predicted and annotated. Finally, a comparison was performed between cvs. Dottato and Horaishi predicted transcriptomes. Our data provide a resource (available at the Sequence Read Archive database under SRP109082) to be used for functional genomics of fig, in order to fill the gap of knowledge still existing in this species concerning plant development, defense and adaptation to the environment.

The identification of genes regulating milk secretion by mammary glands is a key-step for exploiting changes in milk composition induced by different diet regimens. Linseed supplementation is a reliable feeding strategy to enhance polyunsaturated fatty acid content in milk fat from sheep, increasing milk quality and nutraceutical value. To investigate the molecular bases of diet induced differences in milk composition, we collected milk from dairy ewes at 90day of lactation and after 3weeks of diet supplementation with extruded linseed. The milk of dairy ewes contains milk somatic cells mostly derived by mammary glands. After isolating milk somatic cells by centrifugation, RNAs were purified from these cells, and Illumina RNA sequencing was performed to analyze RNA synthesis. Our data provide a resource (available at Gene Expression Omnibus database under GSE89163) to be employed for comparative analyses of gene expression in milk somatic cells in different breeds and different diets, with the long-term aim of developing strategies to improve sheep milk quality.

Although Ficus carica L. (fig) is one of the most resistant fruit tree species to salinity, no comprehensive studies are currently available on its molecular responses to salinity. Here we report a transcriptome analysis of F. carica cv. Dottato exposed to 100 mM sodium chloride for 7 weeks, where RNA-seq analysis was performed on leaf samples at 24 and 48 days after the beginning of salinization; a genome-derived fig transcriptome was used as a reference. At day 24, 224 transcripts were significantly up-regulated and 585 were down-regulated, while at day 48, 409 genes were activated and 285 genes were repressed. Relatively small transcriptome changes were observed after 24 days of salt treatment, showing that fig plants initially tolerate salt stress. However, after an early down-regulation of some cell functions, major transcriptome changes were observed after 48 days of salinity. Seven weeks of 100 mM NaCl dramatically changed the repertoire of expressed genes, leading to activation or reactivation of many cell functions. We also identified salt-regulated genes, some of which had not been previously reported to be involved in plant salinity responses. These genes could be potential targets for the selection of favourable genotypes, through breeding or biotechnology, to improve salt tolerance in fig or other crops.

The relationship between variation of the repetitive component of the genome and domestication in plant species is not fully understood. In previous work, variations in the abundance and proximity to genes of long terminal repeats (LTR)-retrotransposons of sunflower (Helianthus annuus L.) were investigated by Illumina DNA sequencingtocompare cultivars and wild accessions. In this study, we annotated and characterized 22 specific retrotransposon families whose abundance varies between domesticated and wild genotypes. These families mostly belonged to the Chromovirus lineage of the Gypsy superfamily and were distributed overall chromosomes. They were also analyzed in respect to their proximity to genes. Genes close to retrotransposon were classified according to biochemical pathways, and differences between domesticated and wild genotypes are shown. These data suggest that structural variations related to retrotransposons might have occurred to produce phenotypic variation between wild and domesticated genotypes, possibly by affecting the expression of genes that lie close to inserted or deleted retrotransposons and belong to specific biochemical pathways as those involved in plant stress responses.

Arbuscular mycorrhizal (AM) fungi are essential elements of soil fertility, plant nutrition and productivity, facilitating soil mineral nutrient uptake. Helianthus annuus is a non-model, widely cultivated species. Here we used an RNA-seq approach for evaluating gene expression variation at early and late stages of mycorrhizal establishment in sunflower roots colonized by the arbuscular fungus Rhizoglomus irregulare. mRNA was isolated from roots of plantlets at 4 and 16 days after inoculation with the fungus. cDNA libraries were built and sequenced with Illumina technology. Differential expression analysis was performed between control and inoculated plants. Overall 726 differentially expressed genes (DEGs) between inoculated and control plants were retrieved. The number of up-regulated DEGs greatly exceeded the number of down-regulated DEGs and this difference increased in later stages of colonization. Several DEGs were specifically involved in known mycorrhizal processes, such as membrane transport, cell wall shaping, and other. We also found previously unidentified mycorrhizal-induced transcripts. The most important DEGs were carefully described in order to hypothesize their roles in AM symbiosis. Our data add a valuable contribution for deciphering biological processes related to beneficial fungi and plant symbiosis, adding an Asteraceae, non-model species for future comparative functional genomics studies.

Retrotransposon expression during arbuscular mycorrhizal (AM) fungal colonisation of sunflower roots (Helianthus annuus) was analysed using Illumina RNA-Seq, in order to verify whether mycorrhizal symbiosis can activate retrotransposable elements. Illumina cDNA libraries were produced from RNAs isolated from the roots of sunflower plants at 4 and 16 days after inoculation with the AM fungus Rhizoglomus irregulare and from their respective control plants. Illumina reads were mapped to a library of reverse transcriptase-encoding sequences, putatively belonging to long terminal repeat retrotransposons of Gypsy and Copia superfamilies. Forty-six different reverse transcriptase sequences were transcribed, although at a low rate, in mycorrhizal or control roots and only four were significantly over-expressed at day 16, compared with control roots. Almost all expressed or over-expressed sequences belonged to low-copy elements, mostly, of the Copia superfamily. A meta-analysis, using publicly available Illumina cDNA libraries obtained from sunflower plants treated with different hormones and chemicals, mimicking stimuli produced by abiotic and biotic stresses, was also conducted. Such analyses indicated that the four reverse transcriptase sequences over-expressed in mycorrhizal roots were explicitly induced only by AM symbiosis, showing the specificity of AM stimuli compared to that of other fungal/plant interactions.

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.