Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Cells Overexpressing the Gene.

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5'-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, suspension cultures overexpressing the gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.

DNA Diffusion Assay Applied to Plant Cells.

DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4',6-diamidino-2-phenylindole).

Pollen Grain Preservation and Fertility in Valuable Commercial Rose Cultivars.

In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution (generally made in Europe and North America) and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen.

Systems biology and genome-wide approaches to unveil the molecular players involved in the pre-germinative metabolism: implications on seed technology traits.

The pre-germinative metabolism is among the most fascinating aspects of seed biology. The early seed germination phase, or pre-germination, is characterized by rapid water uptake (imbibition), which directs a series of dynamic biochemical events. Among those are enzyme activation, DNA damage and repair, and use of reserve storage compounds, such as lipids, carbohydrates and proteins.

Overexpression of PDH45 or SUV3 helicases in rice leads to delayed leaf senescence-associated events.

Senescence is a very complex process characterized by a highly regulated series of degenerative events which include changes in cell structure, metabolism and gene expression. In animals, one of the indicators of senescence is telomere shortening. In plants, this aspect is more puzzling because telomere shortening is not always correlated with senescence.

Physical Methods for Seed Invigoration: Advantages and Challenges in Seed Technology.

In the context of seed technology, the use of physical methods for increasing plant production offers advantages over conventional treatments based on chemical substances. The effects of physical invigoration treatments in seeds can be now addressed at multiple levels, ranging from morpho-structural aspects to changes in gene expression and protein or metabolite accumulation. Among the physical methods available, "magneto-priming" and irradiation with microwaves (MWs) or ionizing radiations (IRs) are the most promising pre-sowing seed treatments.

Exploring the molecular and chemical-physical aspects of low-dose irradiation using radio-tolerant plant cells.

Animals and plants show different levels of radio-sensitivity, with safe dose values in the 0.001-1 and 1-100 Gy range, respectively. The increased radio-tolerance observed in plant cells might represent a valuable tool to investigate the events underlying the low dose (LD) response in the highly radio-sensitive animal cells.

Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and activity.

In the present work, eleven saponins and three sapogenins purified from Medicago sativa were tested for their cytotoxicity against highly proliferating white poplar (Populus alba L.) cell suspension cultures. After preliminary screening, four saponins with different structural features in terms of aglycone moieties and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5, monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a bidesmoside of zanhic acid) and different cytotoxicity were selected and used for further investigation on their structure-activity relationship.

White poplar (Populus alba L.) suspension cultures as a model system to study apoptosis induced by alfalfa saponins.

In animal cells, the anticancer function played by plant saponins involves a complex network of molecular processes that still deserves investigation and apoptosis seems to be the outstanding pathway. An intriguing aspect of the biological activity of saponins is related to their effects on genome integrity. As demonstrated by the studies carried out in white poplar (Populus alba L.

Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants.

Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress. Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.

Synergistic exposure of rice seeds to different doses of γ-ray and salinity stress resulted in increased antioxidant enzyme activities and gene-specific modulation of TC-NER pathway.

Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ -rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ -rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life.

Single Cell Gel Electrophoresis (Comet) assay with plants: research on DNA repair and ecogenotoxicity testing.

Single Cell Gel Electrophoresis is currently used to investigate the cell response to genotoxic agents as well as to several biotic and abiotic stresses that lead to oxidative DNA damage. Different versions of Single Cell Gel Electrophoresis have been developed in order to expand the range of DNA lesions that can be detected and guidelines for their use in genetic toxicology have been provided. Applications of Single Cell Gel Electrophoresis in plants are still limited, compared to animal systems.

Plant hormone signaling and modulation of DNA repair under stressful conditions.

The role played by phytohormone signaling in the modulation of DNA repair gene and the resulting effects on plant adaptation to genotoxic stress are poorly investigated. Information has been gathered using the Arabidopsis ABA (abscisic acid) overly sensitive mutant abo4-1, defective in the DNA polymerase ε function that is required for DNA repair and recombination. Similarly, phytohormone-mediated regulation of the Ku genes, encoding the Ku heterodimer protein involved in DNA repair, cell cycle control and telomere homeostasis has been demonstrated, highlighting a scenario in which hormones might affect genome stability by modulating the frequency of homologous recombination, favoring plant adaptation to genotoxic stress.

RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula.

An intron-spliced hairpin RNA approach was used for the targeted silencing of the MtTdp1α gene encoding the αisoform of tyrosyl-DNA phosphodiesterase 1 in Medicago truncatula Gaertn. Tyrosyl-DNA phosphodiesterase 1, involved in the repair of DNA topoisomerase I-mediated DNA damage, has been poorly investigated in plants. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed different levels of transcriptional modulation (up- and down-regulation, alternative splicing, activation of alternative promoter) in genes involved in DNA damage sensing, DNA repair, and chromatin remodelling.

γ irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells.

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.

Understanding the molecular pathways associated with seed vigor.

Farmers and growers are constantly looking for high quality seeds able to ensure uniform field establishment and increased production. Seed priming is used to induce pre-germinative metabolism and then enhance germination efficiency and crop yields. It has been hypothesized that priming treatments might also improve stress tolerance in germinating seeds, leaving a sort of 'stress memory'.

New insights on the barrel medic MtOGG1 and MtFPG functions in relation to oxidative stress response in planta and during seed imbibition.

In plants, 8-oxoguanine DNA glycosylase/lyase (OGG1) and formamidopyrimidine-DNA glycosylase (FPG) play similar roles within the base excision repair (BER) pathway involved in the removal of oxidized bases, e.g. 7,8-dihydro-8-oxoguanine (8-oxo-dG) and formamidopyrimidine (FAPy) lesions.

Unraveling the response of plant cells to cytotoxic saponins: role of metallothionein and nitric oxide.

A wide range of pharmacological properties are ascribed to natural saponins, in addition to their biological activities against herbivores, plant soil-borne pathogens and pests. As for animal cells, the cytotoxicity and the chemopreventive role of saponins are mediated by a complex network of signal transduction pathways which include reactive oxygen species (ROS) and nitric oxide (NO). The involvement of other relevant components of the saponin-related signaling routes, such as the Tumor Necrosis Factor(TNF)-α, the interleukin(IL)-6 and the Nuclear Transcription Factor-kB (NF-κB), has been highlighted in animal cells.

Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity.

Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g.

Seed imbibition in Medicago truncatula Gaertn.: Expression profiles of DNA repair genes in relation to PEG-mediated stress.

The expression profiles of genes involved in DNA repair, namely MtTdp1 (tyrosyl-DNA phosphodiesterase), top1 (DNA topoisomerase I), MtTFIIS (transcription elongation factor II-S) and MtTFIIS-like, were evaluated in Medicago truncatula Gaertn. during seed imbibition carried out with the osmotic agent polyethylene glycol (PEG6000, 100g/L). The use of PEG6000 resulted in delayed water up-take by seeds, and reduced levels of oxidative DNA damage, measured in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG) were observed compared to seeds imbibed with water.

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins.

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar 'Villafranca') cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses.

The TFIIS and TFIIS-like genes from Medicago truncatula are involved in oxidative stress response.

The cDNA sequence coding for a novel putative TFIIS (transcription elongation factor II-S), hereby named MtTFIIS-like, was isolated from barrel medic (Medicago truncatula Gaertn.) by reverse transcriptase-polymerase chain reaction. The nucleotide sequence contains an open reading frame of 1074 bp, predicting a 40.

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA.

In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase).

The tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress.

The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this enzyme in the repair of topoisomerase I-DNA covalent lesions. In contrast, information in this regard is totally missing in plants. We report for the first time in plants on the Tdp1 gene family from barrel medic (Medicago truncatula Gaertn.

Soil persistence of DNA from transgenic poplar.

The presence of recombinant DNA in soil cultivated with white poplars (Populus alba L.) expressing either the bar transgene for herbicide tolerance or the StSy transgene for resveratrol production, respectively, was investigated in a greenhouse over a 20-month period. The bar trial included the transgenic lines 5P56 and 6EA22P56 and the untransformed line, while the StSy trial was established with the transgenic lines 5EAC1 and 12EAC1 and with the untransformed line.