“Seed Molecular Physiology” Lab
Referent: Prof. Alma Balestrazzi
Co-workers: Andrea Pagano (Research fellow Post-Doc), Paola Pagano (PhD student), Shraddha Shridhar Gaonkar (PhD student), Francesca Messina (PhD student).
1. Pre-germinative metabolism: impact of DDR (DNA damage response) mechanisms on seed quality.
Objective: characterization of the molecular processes (DNA repair, antioxidant response) that determine the vigor of the seed.
Pre-germinative metabolism is still one of the most fascinating aspects of seed biology. The initial phase of germination is associated with molecular events that not only allow the transition of the seed from a phase of quiescence to a phase of active proliferation but also define its quality. DNA repair mechanisms are activated during the early stage of seed germination (imbibition). In this time window, the embryo cells must remove the DNA damage that accumulates during the life cycle of the seed in sub-optimal conditions (maturation on the mother plant, storage for prolonged times) and during imbibition, to preserve the integrity of the genome. The efficient repair of DNA damage allows the progression of the cell cycle, the initiation of proliferation and the completion of the germination process. Antioxidant mechanisms (enzymes and metabolites) play an equally important role, since they remove reactive oxygen species (ROS, reactive oxygen species) and prevent genotoxic damage. An experimental reference system has been set up consisting of seeds of the leguminous species model Medicago truncatula L. subjected to germination tests in the presence / absence of genotoxic agents or abiotic stress (eg water stress). The impact of treatments at the level of pre-germination metabolism, meant as the ability of the seed to efficiently repair DNA damage, is related to the germination performance and therefore to the level of vigor of the seed. Samples taken at subsequent times during soaking and subsequently in correspondence with the protusion phase of the radicle are subjected to molecular analysis (gene expression measured by Real-Time PCR; genotoxicity test, in particular Comet Assay and DNA Diffusion Assay; quantification assays of ROS). The characterization includes high-throughput investigations (RNA-Seq, proteomics, metabolomics, ATAC-Seq). This experimental approach has been extended to species of agronomic interest (Medicago sativa L .; Pisum sativum L .; Solanum melongena L.).
2. Seed priming: basic and applied research for the development of molecular indicators.
Objective: identification of genes, proteins, metabolites that can be used in early diagnosis assays of semen quality and for monitoring the seed response to vigorization treatments.
The study of DNA repair mechanisms in the context of pre-germinative metabolism are extended to experimental systems (model species and species of agronomic-commercial interest) consisting of seeds subjected to pre-sowing or vigorization treatments ("seed priming"). A typical priming protocol consists of two phases: i) controlled imbibition of the seed to slow down the entry of water and thus extend the time window in which the repair mechanisms are active (DNA damage response-DDR, antioxidant response), ii ) dehydration (dry-back) to bring the seed back to its initial water content, store it or use it directly. The seed industry currently has seed priming protocols defined on an empirical basis, which are not very reproducible since their effectiveness strongly depends on genotype and lot of seeds. The expression profiles of genes encoding key components of the DDR network and of the antioxidant response are analyzed during the controlled imbibition treatment (in the presence of water-hydropriming or specific priming agents, e.g. PEG-osmopriming or phytohormones-ormopriming ) and subsequently during the dehydration phase (dry-back) with the support of molecular analysis techniques (Real-Time PCR; Comet Assay and DNA Diffusion Assay; ROS quantification assays). The characterization of such systems includes high-throughput investigations (RNA-Seq, proteomics, metabolomics, ATAC-Seq). The results allow to identify hallmarks of plant genome stability useful to be used for the early diagnosis of seed quality and for the design of optimized priming protocols.
National and international collaborations:
This line of research makes use of the collaboration of the TEM Laboratory (Prof. Marco Biggiogera), the EPR Laboratory (Dept. Chemistry, University of Pavia, Prof. Daniele Dondi) and the Vegetable Germplasm Bank (Department of Earth Sciences and of the Environment, University of Pavia, Prof. Andrea Mondoni).
At the international level, collaborations are currently underway with:
- Università Paris-Saclay (Institute of Plant Sciences Paris Saclay - Equipe Chromosome Dynamics, Francia;
- Dr. Cécile Raynaud, Prof. Moussa Benhamed), BLC3 (Technology and Innovation Campus, Portogallo;
- Dr. Susana Araújo), Leibniz Institute of Plant Genetics and Crop Plant Research-IPK, Genebank Department, Germania; Prof. Andreas Börner).
The basic knowledge acquired is used as a starting point for translational research activities on horticultural species of commercial interest and cereals carried out in collaboration with Italian (ISI Sementi S.p.A .; SA.PI.SE. Coop. Agricola) and foreign industries. (Hoopman Group) and with CREA Institutes (CREA-RGB). A research network has recently been set up (BENEFIT-Med Consortium: 11 Institutions from Greece, France, Portugal, Germany, Tunisia, Morocco and Algeria coordinated by UNIPV-Prof. Balestrazzi) as part of a project financed with EU funds that will work on the issues of seed priming applied to neglected legume species (orphan legumes).