Prof. Alma Balestrazzi, Prof. Daniela Carbonera, Prof. Anca Macovei
1) Role of DNA repair in the plant response to abiotic stress. Aim of the research is the molecular characterisazion of genes involved in the response to genotoxic stress (DNA Damage Response-DDR), as useful tools to detect the level of abiotic stress tolerance in crops. The research activity at the Plant Biotechnology Laboratory has led to the isolation and molecular characterisation of novel genes involved in DNA repair processes in planta. This investigation, carried out in the model legume Medicago truncatula, has disclosed for the first time in plants the presence of the small gene family Tdp1 (Tyrosyl-DNA phosphodiesterase) and the TFIIS-like gene. MtTdp1a, MtTdp1b, and MtTFIIS-like genes are up-regulated in response to oxidative/genotoxic stress caused by exposure to heavy metals and osmotic stress, thus confirming their key role in the DDR activated under adverse environmental conditions. These novel gene functions have been investigated using gene silecing approaches, RNA-Seq analysis and genotoxicity tests (Comet Assay-Single Cell Gel Electrophoresis, DNA diffusion).
2) Molecular profile of seed quality. Aim of the research is the identification of molecular indicators of seed quality (vigor). DNA repair pathways are activated during the early phase of seed germination (imbibition), when the so-called ‘pre-germinative metabolism’ is triggered. A working system has been established, using imbibed seeds from model plants (Legumes, Medicago truncatula; Solanacee, Petunia hybrida) in order to validate the role of novel DNA repair genes during the pre-germinative metabolism. A translational research project has been subsequently started focusing on commercially relevant horticultural species and cereals, in collaboration with Seed Companies (ATLAS srl, APSOVSEMENTI srl, Bejo BV, Hoopmann Group), Breeders (NIRP International), and with the Research Institutes CREA-SCV (S. Angelo Lodigiano, LO) and CREA-FSO (Sanremo, IM).
3) DNA repair mechanisms induced by ionizing radiations (IR) in plant cells: basic and applied (in vitro breeding) aspects. DNA repair pathways and the antioxidant response are investigated in plant cells characterised by natural radio-tolerance (Petunia hybrida, Medicago truncatula) irradiated with gamma-rays LDR (Low Dose Rate) and HDR (High Dose Rate) in order to identify the key molecular players of LD(Low Dose)/LDR response in planta. The plant response to IR-induced genotoxic damage results in the activation of different DNA repair mechanisms, some of them are ‘error-prone’. Consequently, there are some DNA repair enzymes which can introduce changes in the original DNA sequence while removing the lesions. This mutagenic effect can be modulated, based on the physical parameters (total dose and dose rate) of irradiation treatments as well as on the qualitative/quantitative features of the DNA damage response (DDR) specific for each plant genotype/variety/cultivar. A deeper understanding of the molecular components of DDR involved in the response to LDR and HDR ionizing radiations is an essential requisite for designing improved breeding protocols of ‘elite’ varieties.
CREA-FSO, Unità di Ricerca per la Floricoltura e le Specie Ornamentali (Sanremo-IM), Dr. Annalisa Giovannini.
CREA-SCV, Unità di Ricerca per la Selezione dei Cereali e la Valorizzazione delle Varietà Vegetali (S. Angelo Lodigiano-LO), Dr. Patrizia Vaccino.
CNR-IPSP, Bari, Dr. Paola Leonetti.
Instituto de Tecnologia Quimica e Biologica (ITQB)-Università Nuova di Lisbona (Portogallo). Dr. Susana Araujo, Dr. Pedro Fevereiro.
Institute of Experimental Botany, Praga. Czech Academy of Science. Dr. Karel Angelis.
Institute of Plant Genetics, Poznan. Polish Academy of Science. Dr. Jorge Paiva.
ICGEB, New Delhi (India). Dr. Narendra Tuteja.
National Technical University, Atene (Grecia). Dr. Alex Georgakilas.
Plant Molecular Biology
Prof. Rino Cella
1) “Molecular farming”: production by tobacco transplastomic plants of recombinant enzymes useful for the digestion of lignocellulosic biomass, and their use for the production of second-generation biofuels
This research&development program aims at developing a sustainable process for the production of second-generation biofuels. Particularly, using the “molecular farming” notion, we have obtained tobacco plants that accumulate five enzymes that are necessary for the degradation of lignocellulosic biomass. To this end, we have transformed the plastid genome (plastome) since this procedure permits to accumulate a high amount of the recombinant enzyme of interest within the chloroplast. With respect to biosafety, since in tobacco the plastome is maternally inherited, there is no transgene dispersal by pollen. We are currently investigating the best conditions that increase and accelerate biogas production by anerobic biodigestion.
2) Molecular-genetic analysis of the mechanism of translesion synthesis and base removal in Arabidopsis
The reactive oxigen species (ROS), which form during cell metabolism, can generate genotoxic modification that might interfere with DNA replication. The formation of oxidized bases among which 7,8-diidro-oxo-guanine (8-oxo-G) is harmful for the cell, and in some occasions can stop replicative DNA polymerases. Nonetheless, some specialized polymerases are able to overcome the block by a process called translesion (TLS) synthesis. However, the latter might be mutagenic because of abnormal base pairing. Base removal can occur by a process called Base Excision Repair (BER). The Arabidopsis genome contain all the genes encoding BER glycosylases namely uracil-DNA-glycosylase (UDG), oxoguanin glycosylase (OGG1) and MUTYH glycosylase, while the gene encoding DNA polymerase beta (PolB) is missing. Thus in Arabidopsis, the only X-family polymerase is polymerase lamda (PolL). This poses the problem of the mechanism of BER. Besides polymerase eta (PolH, Y-family) and zeta (PolZ, B-family), PolL carry out TLS. In fact, human PolL is more efficient than PolH in the presence of PCNA e RPA. Arabidopsis has two genes encoding PCNA but we have shown that only PCNA2 interacts with PolL during TLS. We are currently investigating how AtPolL interacts with OGG1 and MUTYH during the oxidative stress.
Prof. Erik Nielsen
In the course of 2015, the basic research carried out in Plant Biochemistry Laboratory of DBB concerned mainly:
Quantification of goitrogenic substances (belonging to the family of C-glucosyl flavones) and of phytin in 150 pure lines of millet, research carried out in the frame of a project funded by the Cariplo (new pearl millet) aimed at improving the nutritional quality of this cereal.
The applied research in 2015 mainly concerned:
Analysis and characterization of triglycerides and xanthophylls produced and accumulated in different stressful environmental conditions by a previously isolated strain of the microalga Scenedesmus obliquus and by microcysts of the microalgae Haematococcus pluvialis received from an Argentine research group with which exists an ongoing collaboration.
Verification of the presence and the levels of compounds with nutraceutical / cosmoceutical activitiy in waste parts of horticultural plants used to prepare products of fourth gamma, or in waste fruits (products of first gamma).
Characterization of lignocellulolitic activities of some species of fungi that degrade poplar wood (in collaboration with the Department of Land Ecology, UNIPV)