Daniela Carbonera

In vitro culture and genetic engineering of Populus spp.: synergy for forest tree improvement

Populus species and hybrids are intensively cultivated as sources of woody biomass for the forest products industry and for reforestation of lowlands in temperate regions of the world. However, the long generation time of trees, the presence of seasonal dormancy and the prolonged period required for evaluation of mature traits are strong limitations for classical breeding and selection. The development of methods for in vitro culture and genetic engineering has increased the possibility of producing poplar genotypes improved in insect pest resistance, herbicide tolerance, growth rate and wood quality, or reduction in undesirable traits. Poplar has become a model system in forest tree biotechnology due to several useful features: small genome size, short rotation cycle, rapid growth rate and ease of vegetative propagation. The combination of molecular techniques and classical breeding will help create forest trees with positive effects on the environment. However, risks associated with the biotechnological applications (concerning the impact on biodiversity, long-term adaptation, transgene inheritance and stability) should be carefully evaluated and field tests performed with transgenic poplar.

Seed priming: state of the art and new perspectives

Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called ‘pre-germinative metabolism’. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical–chemical and biological treatments currently available. Optimised priming protocols can be designed using the ‘hydrotime concept’ analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market.

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., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

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. The prolonged exposure to PEG6000 caused an increase in DNA oxidative damage; after 24h of treatment with the osmotic agent, the estimated amount of 8-oxo-dG was 1.25-fold higher compared to the value detected in seeds imbibed with water. Three days after imbibition, consistent cell damage and reactive oxygen species (ROS) production were also detected in radicles emerging from the PEG-treated seeds. All of the tested genes were known to be up-regulated during seed imbibition, with the highest transcript levels accumulating at approximately 8-12h of rehydration. Exposure to PEG6000 caused a delayed up-regulation of MtTdp1α and MtTdp1β genes, with transcript peaks occurring at 12-24h, when the highest levels of DNA damage were also recorded. For the top1, MtTFIIS and MtTFIIS-like genes, different expression profiles were observed in response to PEG6000. The possible roles of these genes in the repair response activated during seed imbibition are discussed.

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.), composed of two members, hereby named MtTdp1α and MtTdp1β. The expression profiles of MtTdp1α and MtTdp1β genes were evaluated in plantlets grown in vitro using copper and polyethylene glycol (PEG 6000) as stress agents. In situ detection of reactive oxygen species (ROS) was carried out by histochemical staining, while the level of oxidative DNA damage, quantified in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG), increased up to 7.4- and 6.7-fold in response to copper and PEG 6000 treatments, respectively. Quantitative real-time polymerase chain reaction revealed that both Tdp1 genes were significantly up-regulated in response to copper and PEG. The Tdp1 genes were also significantly up-regulated during seed rehydration, an aspect of seed physiology in which DNA repair is a key component. Thus, the Tdp1 genes might be used as novel tools for improving stress tolerance in crops. The expression patterns of the barrel medic top1α and top1β genes, encoding distinct isoforms of DNA topoisomerase I, were also analyzed and discussed to acquire additional information on their specific functions, closely related to that of the Tdp1 gene in animal cells.

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. To date, it is not clear why plants have retained both the OGG1 and FPG functions. In the present work, we have investigated the possible roles played in planta by MtOGG1 and MtFPG genes from Medicago truncatula Gaertn. (barrel medic). Bioinformatic investigation revealed the presence of putative mitochondrial and nuclear localization signals in the MtOGG1 and MtFPG amino acid sequences, respectively, thus suggesting for different subcellular fates. The expression profiles of both genes were evaluated by Quantitative Real-Time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2), 0.05, 0.1 and 0.2 mM) and polyethylene glycol (PEG6000, 50, 100 and 150 g L(-1)). The MtOGG1 and MtFPG genes were up-regulated in response to stress agents, at different levels, depending on treatment and tissue. As for copper, MtOGG1 showed significant up-regulation (up to 1.2- and 1.7-fold) only in roots while the MtFPG mRNA significantly increased (up to 1.3- and 2.8-fold, respectively) in roots and aerial parts. In response to PEG, the MtOGG1 expression was significantly enhanced in aerial parts (up to 1.3-fold) while the MtFPG showed significant (1.2-fold) up-regulation in roots. The expression profiles of MtOGG1 and MtFPG genes were also evaluated during seed imbibition, a physiological process which is characterized by Reactive Oxygen Species (ROS) accumulation and requires active DNA repair.

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. This technique is now emerging as a useful tool in assessing the potential of higher plants as stable sensors in ecosystems and source of information on the genotoxic impact of dangerous pollutants. Another interesting application of Single Cell Gel Electrophoresis deals with Mutation Breeding or the combined use of irradiation and in vitro culture technique to enhance genetic variability in elite plant genotypes. SCGE, in combination with in situ detection of Reactive Oxygen Species (ROS) induced by γ-rays and expression analysis of both DNA repair and antioxidant genes, can be used to gather information on the radiosensitivity level of the target plant genotypes.

Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis.

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. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ-irradiation and salinity stress. Altogether, the synergistic exposure to γ-rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival.