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The present study aimed to characterize the genetic structure of Artemisia eriantha Ten. and the diversity of the rhizosphere microbiota. Plant leaves and rhizosphere soils were sampled from three areas of Central Italy, namely Monte Corvo, Monte Portella (both from the Gran Sasso massif), and Monte Focalone (Majella massif). The plant samples were subjected to genetic structure analysis by amplified fragment length polymorphism (AFLP) markers. The microbiota from the rhizosphere soils was investigated by 16S rRNA gene metabarcoding. The within and among population variability was typical of outbreeding species. The AFLP polymorphisms revealed a marked closeness among plant populations collected in Monte Focalone and Monte Corvo, despite the geographical proximity of the latter with Monte Portella, a result confirmed by cluster, STRUCTURE, and discriminant analyses. 16S rRNA gene metabarcoding showed higher values of diversity for Monte Corvo (H, 5.7; Chao1, 445) and Monte Focalone (H′, 5.57; Chao1, 446) than Monte Portella (H′, 5.3; Chao1, 275). At the phylum level, the communities were mainly represented by Proteobacteria, Actinobacteria, and Acidobacteria (>10%). At the genus level, the Monte Focalone and Monte Corvo microbiotas were closer than Monte Portella, thus confirming the results from the plant communities. The findings provided evidence for the first time of an association between the Artemisia eriantha plant and microbiota communities. The relevance of the results in terms of biodiversity and the conservation strategies of plant and microbiota communities in the Central Apennines are discussed.

In temperate zones, fruit trees regulate their annual growth cycle to seasonal environmental changes. During the cold season, growth is limited by both environmental and genetic factors. After the exposure to low temperature and fulfillment of chilling requirements, mild temperatures promote the growth and flowering. However, an insufficient chilling exposure may lead to nonuniform blooming, with a negative impact on fruit set. To gain insights into flower development in the fruit tree buds, peach is an interesting model, the flower and vegetative bud being distinct organs. To understand how flower bud development is regulated, we integrated cytological observations and epigenetic and chromatin genome-wide data with transcriptional changes to identify the main regulatory factors involved in flower development during chilling accumulation. We demonstrated that growth cessation does not occur in peach flower buds during chilling accumulation, but that there are changes in transcript abundance of key genes of hormone metabolism and flower bud development, distribution of histone modifications (H3K4me3 and H3K27me3) and DNA methylation. Altogether, our findings indicate that during the cold season the flower bud is in a nondormant state and that the chilling experience allows flower differentiation to be completed.

“Aglione della Valdichiana” (Allium ampeloprasum var. holmense) is a landrace of Great Headed Garlic (GHG), an historical and healthy vegetable that has been recently rediscovered and registered in the National and Regional catalog of local varieties. It is grown in small fields in a region between Tuscany and Umbria, Italy, and due to its high market value, it is at risk of commercial frauds. The morphological traits and molecular profile of 9 local populations were evaluated together with 36 accessions of leek, garlic, wild relatives and other GHGs of different geographic origins and used as controls. The 39 morphological descriptors were able to distinguish the 9 accessions from leek and garlic, but unable to distinguish among them. The 549 AFLP (Amplified Fragment Length Polymorphism) amplicons obtained from nine primer combinations were able to clearly distinguish Aglione accessions from all the others, both by UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering and by STRUCTURE analysis, and this is due to several private AFLP bands, most from M+AGC/E+CTG primer combination. Despite its vegetative propagation, the within accession variability of Aglione is higher than expected and most likely due to somatic mutations. The genetic relationships are discussed, including the closer relationship found between Aglione and garlic vs. Aglione and leek.

Aquatic habitats are vulnerable to the invasion of alien species, so early warning protocols are necessary for eradication. The presence in Italy of two alien duckweeds in freshwaters has been documented: Lemna minuta, that showed high invasivity, and L. valdiviana, still confined to south Lazio. These two species may be mistaken for each other and for the domestic L. minor and L. gibba due to morphological variation. Here, we assess the applicability of DNA barcoding as a complement to morphological analysis for monitoring the spread of alien Lemna. We chose two chloroplast genome sequences for their ability to discriminate all Lemna species: the 5’ intron of the trnK gene and the matK gene. Among 48 samples of Lemna collected at 20 sites in Central Italy, 20 were identified as L. minor, 19 as L. minuta, five as L. trisulca and four as L. gibba. L. minuta was present at most sampling sites; in particular, at six locations of Lake Trasimeno, eight L. minuta samples were found. We demonstrate that DNA sequence analyses with cost-effective barcoding techniques can effectively support expert efforts in species determination for an early alert system of invasive Lemna species.

Apomixis is a rather widespread phenomenon in plants. It is defined as the asexual formation of a seed from the maternal tissues of the ovule, avoiding the processes of meiosis and fertilization. Some species are facultative apomicts and form seeds by means of sexual and apomictic pathways to different extents. This is the case of Poa pratensis, the Kentucky bluegrass, which reproduces by aposporous pseudogamous facultative apomixis. This grass is one of the most studied apomictic systems, however some aspects, such as the male meiotic behavior, have not been so far investigated. In this study the process of microsporogenesis in genotypes of P. pratensis with a different mode of reproduction was investigated. The analysis revealed an almost regular meiosis in the sexual plants whereas apomictic genotypes exhibited different levels of meiotic irregularities, mainly due to cell fusion and irregular segregation in I and II division. Our data did not reveal evident connections between the extent and types of abnormalities and the components of apomixis, apomeiosis and parthenogenesis. The meiotic behavior of the examined plants was discussed in the light of their origin.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

The involvement of DNA methylation in the response to cold stress of two different yeast species (Naganishia antarctica, psychrophilic, and Naganishia albida, psychrotolerant), exhibiting different temperature aptitudes, has been studied. Consecutive incubations at respective optimum temperatures, at 4 &deg, C (cold stress) and at optimum temperatures again, were performed. After Methylation Sensitive Amplified Polymorphism (MSAP) fingerprints a total of 550 and 423 clear and reproducible fragments were amplified from N. antarctica and N. albida strains, respectively. The two Naganishia strains showed a different response in terms of level of DNA methylation during cold stress and recovery from cold stress. The percentage of total methylated fragments in psychrophilic N. antarctica did not show any significant change. On the contrary, the methylation of psychrotolerant N. albida exhibited a nonsignificant increase during the incubation at 4 &deg, C and continued during the recovery step, showing a significant difference if compared with control condition, resembling an uncontrolled response to cold stress. A total of 12 polymorphic fragments were selected, cloned, and sequenced. Four fragments were associated to genes encoding for elongation factor G and for chitin synthase export chaperon. To the best of our knowledge, this is the first study on DNA methylation in the response to cold stress carried out by comparing a psychrophilic and a psychrotolerant yeast species.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

Globe artichoke represents one of the main horticultural species of the Mediterranean basin, and &lsquo, Spinoso sardo&rsquo, is the most widespread and economically relevant varietal type in Sardinia, Italy. In the last decades, in vitro culture of meristematic apices has increased the frequency of aberrant plants in open-field production. These off-type phenotypes showed highly pinnate-parted leaves and late inflorescence budding, and emerged from some branches of the true-to-type &lsquo, plants. This phenomenon cannot be foreseen and is reversible through generations, suggesting the occurrence of epigenetic alterations. Here, we report an exploratory study on DNA methylation patterns in off-type/true-to-type globe artichoke plants, using a modified EpiRADseq technology, which allowed the identification of 2,897 differentially methylated loci (DML): 1,998 in CG, 458 in CHH, and 441 in CHG methylation contexts of which 720, 88, and 152, respectively, were in coding regions. Most of them appeared involved in primary metabolic processes, mostly linked to photosynthesis, regulation of flower development, and regulation of reproductive processes, coherently with the observed phenotype. Differences in the methylation status of some candidate genes were integrated with transcriptional analysis to test whether these two regulation levels might interplay in the emergence and spread of the &lsquo, non-conventional phenotype.

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

DNA methylation is an epigenetic mechanism by which a methyl group is added to a cytosine or an adenine. When located in a gene/regulatory sequence it may repress or de-repress genes, depending on the context and species. Eragrostis curvula is an apomictic grass in which facultative genotypes increases the frequency of sexual pistils triggered by epigenetic mechanisms. The aim of the present study was to look for correlations between the reproductive mode and specific methylated genes or genomic regions. To do so, plants with contrasting reproductive modes were investigated through MCSeEd (Methylation Context Sensitive Enzyme ddRad) showing higher levels of DNA methylation in apomictic genotypes. Moreover, an increased proportion of differentially methylated positions over the regulatory regions were observed, suggesting its possible role in regulation of gene expression. Interestingly, the methylation pathway was also found to be self-regulated since two of the main genes (ROS1 and ROS4), involved in de-methylation, were found differentially methylated between genotypes with different reproductive behavior. Moreover, this work allowed us to detect several genes regulated by methylation that were previously found as differentially expressed in the comparisons between apomictic and sexual genotypes, linking DNA methylation to differences in reproductive mode.

The production of seeds without sex is considered the holy grail of plant biology. The transfer of apomixis to various crop species has the potential to transform plant breeding, since it will allow new varieties to retain valuable traits thorough asexual reproduction. Therefore, a greater molecular understanding of apomixis is fundamental. In a previous work we identified a gene, namely APOSTART, that seemed to be involved in this asexual mode of reproduction, which is very common in Poa pratensis L., and here we present a detailed work aimed at clarifying its role in apomixis. In situ hybridization showed that PpAPOSTART is expressed in reproductive tissues from pre-meiosis to embryo development. Interestingly, it is expressed early in few nucellar cells of apomictic individuals possibly switching from a somatic to a reproductive cell as in aposporic apomixis. Moreover, out of 13 APOSTART members, we identified one, APOSTART_6, as specifically expressed in flower tissue. APOSTART_6 also exhibited delayed expression in apomictic genotypes when compared with sexual types. Most importantly, the SCAR (Sequence Characterized Amplified Region) derived from the APOSTART_6 sequence completely co-segregated with apomixis.

In the last 50 years, intensive farming systems have been boosted by modern agricultural techniques and newly bred cultivars. The massive use of few and related cultivars has dramatically reduced the apple genetic diversity of local varieties, confined to marginal areas. In Central Italy a limited spread of intensive fruit orchards has made it possible to preserve much of the local genetic diversity, but at the same time the coexistence of both modern and ancient varieties has generated some confusion. The characterization and clarification of possible synonyms, homonyms, and/or labeling errors in old local genetic resources is an issue in the conservation and management of living collections. 175 accessions provided by 10 apple collections, mainly local varieties, some of unknown origin, and well-known modern and ancient varieties, were studied by using 19 SSRs, analyzed by STRUCTURE, Ward’s clustering and parentage analysis. We were able to identify 25 duplicates, 9 synonyms, and 9 homonyms. As many as 37 unknown accession were assigned to well known local or commercial varieties. Polyploids made up 20%. Some markers were found to be significantly correlated with morphological traits and the loci associated with the fruit over color were related to QTLs for resistance to biotic stresses, aroma compounds, stiffness, and acidity. In conclusion the gene pool of Central Italy seems to be rather consistent and highly differentiated compared with other European studies (FST = 0.147). The importance of safeguarding this diversity and the impact on the management of the germplasm living collection is discussed.

Requiring water and minerals to grow and to develop its organs, Maize (Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance.

The effects on bacterial, yeast and fungal communities present within an agricultural soil treated with a pig slurry-derived digestate were studied using a multidisciplinary (biochemical and 454 pyrosequencing platform) approach. Biochemical analyses showed a significant increase of CO2 emissions from soil 5 days after the amendment with digestate, whereas soil microbial biomass (C-biomass) increased significantly only after 12 and 30 days. PLFAs analysis revealed a significant increase in Gram-negative bacteria 90 days after the amendment. Results from 454 pyrosequencing revealed the presence of OTUs attributed to bacteria, yeasts and filamentous fungi. Proteobacteria, Bacteroidetes and Firmicutes exhibited a significant predominance in the first 5 days, whereas Ascomycota became predominant 90 days after the amendment. Overall, both bacterial and yeast + fungal richness exhibited a decreasing trend from 0 to 90 days after the amendment. Canonical analysis of principal coordinates showed that the cumulative effect of amendment and incubation time explained approximately 45% and 36% of the total variance observed in the bacterial and yeast + fungal communities, respectively. The correlation among some bacterial and fungal OTUs suggested the probable existence of specific biological interactions among different phyla. The results reported represent a picture of the changes of soil microbial diversity in relation with some agronomic practices, such as organic amendments.

Requiring water and minerals to grow and to develop its organs, Maize (Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance.

Pear is one of the oldest fruit tree crops and the third most important temperate fruit species. Its domestication took place independently in the Far East (China) and in the Caucasus region. While the origin of Eastern Asian cultivars is clear, that of European cultivars is still in doubt. Italy has a wealth of local varieties and genetic resources safeguarded by several public and private collections to face the erosion caused by the introduction of improved varieties in specialized orchards. The objectives of the present study were: (i) to characterize the existing germplasm through nuclear (SSR) and (ii) to clarify the genetic divergence between local and cultivated populations through chloroplast DNA (cpDNA) markers in order to provide insights into phylogenetic relationships of Pyrus spp. For this reason, 95 entries from five different germplasm collections, including nine European, Mediterranean and Eastern Asian species, were analyzed, and the intergenic accD-psaI sequences were compared to the worldwide distributed dataset encompassing a total of 298 sequences from 26 different Pyrus species. The nine nuclear SSRs were able to identify a total of 179 alleles, with a loci polymorphism P = 0.89. Most of the variation (97%) was found within groups. Five accessions from different sources were confirmed to be the same. Eight out of 20 accessions of unknown origin were identified, and six synonyms were detected. Locus NH030a was found to be monomorphic in all the cultivated accessions and in reference species interfertile with P. communis, leading to hypothesize selection pressures for adaptation to cultivation. The cpDNA sequences of the 95 accessions were represented by 14 haplotypes, six of which (derived from P. communis, P. cossonii and P. ussuriensis) are recorded here for the first time and may suggest the ancient origin of some local varieties. The network analysis of the 298 cpDNA sequences allowed two different haplogroups, Eastern and Western Eurasia, to be defined, supporting recent views of a clear division between Occidental and Oriental species. By combining the results from nuclear and uniparental markers, it was possible to better define many unknown accessions.

Changes in chromosome number have played an important role in the evolution of the genus Trifolium. Along with a few species of polyploid origin there are several cases of dysploidy as evidenced by the presence of four basic chromosome numbers (x = 8, 7, 6, 5). Trifolium subterraneum and Trifolium israeliticum are related species with chromosome complements 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out to date. With this study we performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyomorphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant remodelling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karyotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but constitute distinct entities. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum is a species with 2n = 14.

The European hazelnut (Corylus avellana L.) is widespread in Europe, where it has been cultivated for centuries. Despite progress in genetics, most of the cytogenetic aspects of this species have been overlooked. The aim of this study was to fill in this gap and obtain basic information on the chromosome structure of this species. Karyomorphological analysis confirmed the chromosome number 2n = 22 and showed that, despite their apparent uniformity, the chromosomes could be separated into three groups of different size: large (L), medium (M), and small (S). As a first step towards the physical mapping of the hazelnut chromosomes, we applied FISH to localize the position of rRNA genes (rDNA). The sites of 45S and 5S rDNA enabled us to identify two chromosome pairs belonging, respectively, to the L and S groups. The self-GISH procedure revealed that repetitive DNA is concentrated in the pericentromeric regions of the chromosomes, as with other species with rather small genomes. The analysis of 5S rDNA repeats offered additional information on the hazelnut genome by obtaining the whole sequence of the transcribed region so far unpublished. The overall results constitute a substantial advance in hazelnut cytogenetics. Further investigation of other species of Corylus could be an effective approach to understanding the phylogenesis of the genus and resolving taxonomic problems.

Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal ‘short-term stress memory’ with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. DOI: http://dx.doi.org/10.7554/eLife.13546.001, eLife digest Most plants spend their entire lives in one fixed spot and so must be able to quickly adapt to any changes in their surroundings. For example, high levels of salt in the soil – which can be toxic to cells – triggers stress responses in plants that help them to mitigate any damage. Once the stress has passed, plants are able to retain a memory of it, which allows them to respond more quickly if they face the same stress in future. Furthermore, plants may pass on this ‘stress memory’ to their offspring. It is thought that stress memory is programmed by chemical modifications to DNA known as epigenetic marks. These marks do not alter the genetic information that is encoded by the DNA itself, but they can change the activity of particular genes. Environmental stress leads to changes in the epigenetic marks found on many plant genes, which can be directly passed on from the parent plant to its offspring. However, it was not clear whether the epigenetic marks that programme stress memory can be passed on in this way. Wibowo, Becker et al. investigated how a model plant called Arabidopsis thaliana is able to remember periods of salt stress. The experiments show that high levels of salt can trigger changes in the patterns of epigenetic marks associated with particular regions of DNA. This memory is reinforced by repetitive exposure to similar salt stress and can be passed onto offspring, primarily through the maternal line. However, this stress memory is not fixed in future generations as the epigenetic marks can be reset to their original patterns if plants find themselves growing and reproducing under non-stress conditions. In sum, the findings of Wibowo, Becker et al. show that epigenetic marks allow plants to inherit stress memory on a temporary basis while the stress is present, but to gradually lose the memory if the stress does not return. Future studies will focus on finding out if stress memory in crop plants works in the same way. DOI: http://dx.doi.org/10.7554/eLife.13546.002

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

To cite this article: Aina R, Asero R, Ghiani A, Marconi G, Albertini E, Citterio S. Exposure to cadmium-contaminated soils increases allergenicity of Poa annua L. pollen. Allergy 2010; 65: 1313–1321. Abstract Background: Pollution is considered as one main cause for the increase of allergic diseases. Air pollutants may cause and worsen airway diseases and are probably able to make pollen allergens more aggressive. Previous studies looked at traffic-related air pollution, but no data about the effects of polluted soils on pollen allergens are available. We aimed to assess the effects of plant exposure to cadmium-contaminated soil on allergenicity of the annual blue grass, Poa annua L, pollen. Methods: Poa plants were grown in soil contaminated or not contaminated (control) with cadmium. At flowering, mature pollen was analyzed by microscopy, to calculate the percentage of pollen grains releasing cytoplasmic granules, and by proteomic techniques to analyze allergen proteins. Allergens were identified by sera from grass pollen–allergic patients and by mass spectrometry. Results: Pollen from Cd-exposed plants released a higher amount of allergenic proteins than control plants. Moreover, Cd-exposed pollen released allergens-containing cytoplasmic grains much more promptly than control pollen. Group 1 and 5 allergens, the major grass pollen allergens, were detected both in control and Cd-exposed extracts. These were the only allergens reacting with patient’s sera in control pollen, whereas additional proteins strengthening the signal in the gel region reacting with patient’s sera were present in Cd-exposed pollen. These included a pectinesterase, a lipase, a nuclease, and a secretory peroxydase. Moreover, a PR3 class I chitinase-like protein was also immunodetected in exposed plants. Conclusion: Pollen content of plants grown in Cd-contaminated soils is more easily released in the environment and also shows an increased propensity to bind specific IgE.

The seed in the mature and dry state is metabolically inactive (quiescent) and is thus able to withstand extreme environmental conditions, such as drought and cold. Germination commences when the dry seed, shed from its parent plant, takes up water (imbibition) and ends when the root emerges through the seed coat. During seedling establishment, the reserves stored in the seed are metabolized, whereas the subsequent vegetative and reproductive growth is supported by photosynthesis. Here, we describe the functional characterization of the PH-START protein AtAPO1 (Arabidopsis thaliana APOSTART1), the putative homologue of PpAPO1 (Poa pratensis APOSTART1) in Arabidopsis thaliana. By using translational fusion of the AtAPO1 promoter to the uiaD gene and in situ hybridization analyses, we show that AtAPO1 is expressed in mature embryo sacs and developing embryos. The functional analysis of two at-apostart mutant alleles suggests that AtAPO1 is involved in the control of seed germination.

Poa pratensis L. is a forage and turf grass species well adapted to a wide range of mesic to moist habitats. Due to its genome complexity little is known regarding evolution, genome composition and intraspecific phylogenetic relationships of this species. In the present study we investigated the morphological and genetic diversity of 33 P. pratensis accessions from 23 different countries using both nuclear and chloroplast molecular markers as well as flow cytometry of somatic tissues. This with the aim of shedding light on the genetic diversity and phylogenetic relationships of the collection that includes both cultivated and wild materials. Morphological characterization showed that the most relevant traits able to distinguish cultivated from wild forms were spring growth habit and leaf colour. The genome size analysis revealed high variability both within and between accessions in both wild and cultivated materials. The sequence analysis of the trnL-F chloroplast region revealed a low polymorphism level that could be the result of the complex mode of reproduction of this species. In addition, a strong reduction of chloroplast SSR variability was detected in cultivated materials, where only two alleles were conserved out of the four present in wild accessions. Contrarily, at nuclear level, high variability exist in the collection where the analysis of 11 SSR loci allowed the detection of a total of 91 different alleles. A Bayesian analysis performed on nuclear SSR data revealed that studied materials belong to two main clusters. While wild materials are equally represented in both clusters, the domesticated forms are mostly belonging to cluster P2 which is characterized by lower genetic diversity compared to the cluster P1. In the Neighbour Joining tree no clear distinction was found between accessions with the exception of those from China and Mongolia that were clearly separated from all the others.

Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.

The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. The genetic constitution of the offspring is, therefore, usually identical to the maternal parent, a trait of great interest to plant breeders. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A fundamental contribution to the understanding of the genetic control of the apomictic pathway could be provided by a deep knowledge of molecular mechanisms that regulate the reproductive events. In Poa pratensis the cDNA-AFLP method of mRNA profiling allowed us to visualize a total of 2248 transcript-derived fragments and to isolate 179 sequences that differed qualitatively or quantitatively between apomictic and sexual genotypes at the time of flowering when the primary stages of apomixis occur. Three ESTs were chosen for further molecular characterization because of their cDNA-AFLP expression pattern and BLAST information retrieval. The full-lengths of the newly isolated genes were recovered by RACE and their temporal expression patterns were assessed by RT-PCR. Their putative role in cell signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis and embryogenesis in plants is reported and discussed.

Methylation-sensitive amplified polymorphism (MSAP) is a technique developed for assessing the extent and pattern of cytosine methylation and has been applied to genomes of several species (Arabidopsis, grape, maize, tomato, and pepper). The technique relies on the use of isoschizomers that differ in their sensitivity to methylation.

Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences in the rapeseed genome, as detected by MSAP analysis. © 2013 Marconi et al.

The identification of a bi-univocal correspondence between geographical origin of saffron (Crocus sativus L.) and the composition of its stigmas has recently been the subject of many research papers, which have focused on the analysis of the differences among the so called “minor components”, such as flavonoids and volatiles, in the secondary metabolic pattern of this spice. Saffron pigments (crocetin esters), on the other hand, constitute the majority of the metabolites found in its stigmas, and their spectrophotometric measurement is still used as an official method to determine the quality of the spice in terms of coloring power. To our knowledge, no attempts have been made to find a correspondence between the geographical origin of different saffron samples and their morphological traits and pigments pattern. In this paper, we have demonstrated that saffron corms of different origins, grown in the same experimental field, produce daughter corms with different dimensions and still produce stigma samples with different pigment profiles. Furthermore, daughter corm dimensions and pigment profile even more so, may be related to the origin of the sample, and therefore pigments can be used as chemotaxonomic markers. Compositional analyses results were corroborated by genetic data obtained using AFLP molecular markers.

The olive is considered one of the most important fruit crops of the Mediterranean basin where it shows a wide range of variability, with about 2,000 cultivars. Italy, with about 500 cultivars, plays a fundamental role. The ability to discriminate olive cultivars and estimate genetic variability are important factors in better management of genetic resources and in helping to understand how genetic diversity is partitioned among cultivars. The two main objectives of the present investigation were to evaluate the identity of cultivars grown in Abruzzo region, central Italy, and to study their genetic structure. We applied amplified fragment length polymorphism (AFLP) methodology on 84 genotypes belonging to the most relevant and oldest varieties cultivated in Abruzzo and on six unknown genotypes. The information content of data was evaluated using the Marker Ratio index and the Polymorphic Index Content. Moreover, STRUCTURE software was used to investigate the genetic population structure. The analysis enabled us to clearly distinguish eight cultivars within seven clusters. Additionally, one cluster was found to have various minor cultivars and showed a relatively high level of diversity. The partitioning of genetic diversity showed that the largest amount of molecular variance was within groups. Our data suggest that both sexual and clonal propagation have played an important role in the evolution of olive cultivars. In our hypothesis, some ancestral population spread in central Italy with a relevant role of seed propagation, followed by a selection of superior clones from which more traditional varieties originated. In a few cases, hybridization should be taken into consideration to explain the diffusion of recently developed cultivars.

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. © 2011 Elsevier B.V.

Here we report the first results of a study of 5S rDNA of Vitis vinifera . 5S rDNA sequences from seven genotypes were amplified by PCR, cloned, and sequenced. Three types of repeats were found. Two variants, denominated long repeat and short repeat, appeared to be the main components of the 5S rDNA of this species, since they were found in all genotypes analyzed. They differed markedly from each other in both the length and the nucleotide composition of the spacers. The third variant, classified as DEL short repeat, differs from the short repeat owing to a large deletion in the spacer region. It appears to be the most recent repeat type, since it was identified in only one genotype. The organization of the 5S rDNA repeat unit variants was investigated by amplifying the genomic DNA with primers designed on the sequence of the long and short spacers. The PCR-amplified fragments showed that the long repeat is associated with the other two repeats, indicating that in V. vinifera different repeat units coexist within the same tandem array. FISH analysis demonstrated that 5S rRNA genes are localized at a single locus. The variability of 5S rDNA repeats is discussed in relation to the putative allopolyploid origin of V. vinifera.

Background Classical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae. All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered. Results We report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits. Conclusion Previous reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.