Polyethylene microplastics alter root functionality and affect strawberry plant physiology and fruit quality traits.

Strawberry, a globally popular crop whose fruit are known for their taste and health benefits, were used to evaluate the effects of polyethylene microplastics (PE-MPs) on plant physiology and fruit quality. Plants were grown in 2-L pots with natural soil mixed with PE-MPs at two concentrations (0.2% and 0.02%; w/w) and sizes (⌀ 35 and 125 µm). Plant physiological responses, root histochemical and anatomical analyses as well as fruit biometric and quality features were conducted. Plants subjected to ⌀ 35 µm/0.2% PE-MPs exhibited the most severe effects in terms of CO2 assimilation due to stomatal limitations, along with the highest level of oxidative stress in roots. Though no differences were observed in plant biomass, the impact on fruit quality traits was severe in ⌀ 35 µm/0.2% MPs treatment resulting in a drop in fruit weight (-42%), soluble solid (-10%) and anthocyanin contents (-25%). The smallest sized PE-MPs, adsorbed on the root surface, impaired plant water status by damaging the radical apparatus, which finally resulted in alteration of plant physiology and fruit quality. Further research is required to determine if these alterations also occur with other MPs and to understand more deeply the MPs influence on fruit physio-chemistry.

Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects.

This work was aimed to provide further information about toxicology of TiO2 nanoparticles (NPs) on Vicia narbonensis L., considering different endpoints. After exposure to TiO2 nanoparticle suspension (mixture of rutile and anatase, size <100 nm) at four different concentrations (0.2, 1.0, 2.0 and 4.0 ‰), the seeds of V. narbonensis were let to germinate in controlled environmental conditions. After 72 h, the extent of the success of the whole process (seed germination plus root elongation) was recorded as the vigour index, an indicator of possible phytotoxicity. After the characterisation of the hydric state of different materials, oxidative stress and enzymatic and nonenzymatic antioxidant responses were considered as indicators of possible cytotoxicity and to assess if damage induced by TiO2 NPs was oxidative stress-dependent. Cytohistochemical detection of in situ DNA fragmentation as genotoxicity endpoint was monitored by TUNEL reaction. The treatments with TiO2 NPs in our system induced phytotoxic effects, ROS production and DNA fragmentation. The nonenzymatic and enzymatic antioxidant responses were gradually and differentially activated and were able to maintain the oxidative damage to levels not significantly different from the control. On the other hand, the results of DNA fragmentation suggested that the mechanisms of DNA repair were not effective enough to eliminate early genotoxicity effects.

The intergenic spacer region of the rDNA in Haplopappus gracilis (Nutt.) Gray.

In this paper, we provide further information on the genome organisation of Haplopappus gracilis, one of the six angiosperms showing the lowest chromosome number, i.e. 2n = 4, by determining the nucleotide sequence of the intergenic spacer region of the ribosomal RNA genes and its cytological localization on metaphase chromosomes. DNA sequence analysis reveals the occurring of a product of 4,382 bp in length, characterised by the presence of four blocks of different repeated sequences. Our analysis also evidenced putative promoter regions with three transcription initiation sites for polymerase I, as previously reported in Artemisia absinthium, belonging to the same Asteraceae family. A fluorescent in situ hybridization with the intergenic spacer probe indicates the presence of rDNA genes only in the satellited chromosomes of H. gracilis; besides, differences in the signal intensity between homologous chromosomes were frequently observed, thus suggesting for these chromosome sites the presence of a variable number of rDNA gene copies, even if a divergent chromatin organisation in corresponding regions cannot be ruled out.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia.

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion.

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.

Cytological and molecular characterization of Vicia esdraelonensis Warb. & Eig: a rare taxon.

Vicia esdraelonensis, a rare taxon belonging to section Hypechusa of subgenus Vicia, was recovered and analyzed by cytological, karyological, and molecular methods, with the aim of both characterizing this species and furthering our knowledge of the phylogeny of subgenus Vicia. Automated karyotype analysis, nuclear DNA content, and chromatin organization were determined by the Feulgen reaction, as well as chromosome banding after double staining with 4',6-diamidino-2-phenylindole (DAPI) and chromomycin A3. The chromosome number and the nuclear DNA content were in agreement with the values of the species of section Hypechusa. The GC- and AT-rich preferential sites were determined by chromomycin A3 and DAPI staining. Karyomorphological parameters indicated that V. esdraelonensis is in an intermediate position in the spatial representation of the species of section Hypechusa on the basis of symmetry indices, as well as in the dendrogram of linkage distance constructed on 37 chromosome parameters. Molecular data based on internal transcribed spacer sequences show that V. esdraelonensis can doubtlessly be included in section Hypechusa and document its closeness to V. noeana. A cladistic analysis combining the molecular data set with karyological characters is also reported. Karyological, cytological, and molecular data allow characterization of the V. esdraelonensis genome and provide information about the phylogenetic position of this species within the Hyrcanicae series of section Hypechusa.

The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis.

• Transformed aubergine plants constitutively expressing the Dm-AMP1 antimicrobial defensin (from Dahlia merckii) were generated and characterized. • Transgenic plants were selected on kanamycin and screened by polymerase chain reaction analysis. The expression of Dm-AMP1 in plant tissues and its release in root exudates were detected by Western blot analyses. Dm-AMP1 localization was performed by immunohistochemical experiments. • Dm-AMP1 expression ranged from 0.2% to 0.48% of total soluble proteins in primary transformants and from 0.16% to 0.66% in F2 plants. Transformed clones showed resistance to the pathogenic fungus Botrytis cinerea, whose development on leaves was reduced by 36-100%, with respect to controls. The protein was released in root exudates of the transformed plants and was active in reducing the growth of the co-cultured pathogenic fungus Verticillium albo-atrum, whereas it did not interfere with recognition responses and symbiosis establishment by the arbuscular mycorrhizal fungus Glomus mosseae. • Dm-AMP1 transformants may represent a useful model to study the interactions between genetically modified plants and pathogenic fungi or beneficial nontarget microorganisms.

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae).

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.