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Seed Molecular Physiology Laboratory

Lab. Fisiologia Molecolare del Seme

Referent: Prof. Alma Balestrazzi

Co-workers: Andrea Pagano (Post-Doc), Shraddha Shridhar Gaonkar (Post-Doc), Nicolò Bedotto (PhD student), Ridwan Diaguna (PhD student).

Research lines:

1. Pre-germinative metabolism: impact of DDR (DNA damage response) mechanisms on seed quality

Goal: characterisation of the molecular processes (DNA repair, antioxidant response) that determine seed vigor

The pre-germinative metabolism still represents one of the most intriguing aspects of seed biology. DNA repair is activated during early germination (imbibition phase). In this temporal window, the embryo cells must remove DNA damage accumulated along the seed life cycle under sub-optimal conditions (maturation on the mother plant, long-term storage), as well as during imbibition, to preserve genome integrity. Effective DNA repair allows cell cycle progression, consequent cell proliferation and successful germination. Antioxidant mechanisms (enzymes, metabolites) play a relevant role as reactive oxygen species (ROS) scavengers, preventing genotoxic damage.

The experimental system hereby investigated is based on seeds of the model legume Medicago truncatula L. subjected to germination in presence/absence of genotoxic agents and abiotic stress. The impact of these treatments on the pre-germinative metabolism, in terms of seed ability to effectively repair DNA damage is correlated to germination performance and, thus, to seed vigor. Samples collected at different timepoints during imbibition and at the radicle protrusion stage undergo molecular anlyses (QRT-PCR-based gene expression; genotoxicity tests as Comet Assay and Diffusion Assay, 8-OxodG-E.L.I.S.A.; ROS quantifiction assays). The molecular characterization includes high-throughput tools (RNA-Seq, proteomics, metabolomics, ATAC-Seq). 

This experimental approach has been extended to agronomically relevant species such as Medicago sativa L., Pisum sativum L., and Solanum melongena L.
 

2. Seed priming: basic and applied research for the development of molecular hallmarks

Goal: identification of genes, proteins, metabolites useful for assays for the early diagnosis of seed quality and for monitoring the seed response to priming treatments.

The study of DNA repair in the context of pre-germinative metabolism is expanded to experimental systems (model species and species of agronomic/commercial interest) consisting of seeds exposed to pre-sown treatments (seed priming). Typically, seed priming consists of two phases: i) controlled imbibition applied to delay water entry and expand the temporal window in which repair processes (DDR, antioxidant response) are active, ii) desiccation (dry-back) that brings the seed to its original moisture content. Primed seeds are then immediately used or stored. The seed industry currently relies on seed priming protocols developed on empirical bases, with poor reproducibility since their efficacy is strongly genotype- and seed lot-dependent. 

DDR and antioxidant gene expression profiles are investigated along controlled imbibition (in presence of water-hydropriming or specific priming agents such as PEG-osmopriming or phytohormones-hormopriming) and subsequently during dry-back, using Real-Time PCR. Comet Assay and DNA Diffusion Assay, and ROS quantification assays are also used to characterize the levels of genotoxic damage and antioxidant reesponse. The molecular characterization also includes high-throughput tools (RNA-Seq, proteomics, metabolomics, ATAC-Seq). Results of this investigation will allow to identify hallmarks of plant genome stability useful as indicators for the early diagnosis of seed quality, as well as to speed up the design of optimised seed priming protocols.
 

3. Novel models for the characterization of the pre-germinative metabolism: role of neglected legume species (orphan legumes)

Goal: Development of sustainable seed priming approaches for the valorization of neglected legume species

This research line focuses on the study of neglected legume species, currently cultivated by local populations within the most climate-vulnerable populations of the Mediterranean Area.

These species have been excluded from the intensive breeding programmes applied to the so-called “maajor crops” and, consequently, they have maintained key traits conferring enhanced resilience to abiotic/biotic stresses combined with a high, unique nutritional value. The severe impact of extreme climate events has evidenced the limited agronomic performance of the major crops, driving the attention of the scientific community towards orphan legumes. The characterisation of the molecular mechanisms underlying enhanced germination performance under abiotic stress (drought, salinuty, heat waves) is currently in progress. In this study, the target neglected legume species include Lathyrus sativus L. (grass pea), Pisum sativum var arvense (forage pea), Trigonella foenum-graecum L. (fenugreek). Also in this case, molecular investigations rely on Real-Time PCR, Comet Assay and DNA Diffusion Assay, ROS quantification assays, as well as  high-throughput tools (RNA-Seq, proteomics, metabolomics, ATAC-Seq). 

More details concerning the research activity at the Seed Molecular Physiology Laboratory can be found at the following links:

https://www.benefit-med.eu/
https://www.prosper-horizon.eu/ 
https://www.forecast-project.eu/ 
https://www.heatddr.eu/


National and international collaborations:

  • Università degli Studi di Pavia: Laboratorio EPR (Dip. Chimica, Prof. Daniele Dondi), Hydrogen Lab (Dip. Chimica, Prof. Chiara Milanese), Banca del Germoplasma Vegetale (Dipartimento di Scienze della Terra e dell’Ambiente, Prof. Andrea Mondoni).
  • Université Paris-Saclay (Institute of Plant Sciences Paris-Saclay - Equipe Chromosome Dynamics, Francia; Dr. Cécile Raynaud, Prof. Moussa Benhamed)
  • University of Sfax, Tunisia (Prof. Amine Elleuch, Prof. Radhouane Gdoura, Prof. Moez Hanin)
  • MORE-CoLAB, Portugal (Dr. Susana Araujo)
  • Agricultural University of Athens, Greece (Dr. Eleni Tani)
  • Benaki Phytopathological Institute, Greece (Dr. Dimosthenis Chachalis)
  • Institut Jean-Pierre Bourgin -INRAE Centre IdF Versailles Saclay, France (Dr. Massimiliano Corso)
  • Institut Jean-Pierre Bourgin - INRAE, AgroParisTech, Université Paris-Saclay, France (Prof. Loic Rajjou)
  • Aahrus University, Denmark (Prof. Fiona Hay)
  • Panora S.p.A., Italy
  • Innova-Tech Agro, Italy
  • SA.PI.SE. Sardo-Piemontese Sementi Coop Agricola, Italy
  • CREA Forests and Wood, Italy
  • CNR-IRET, Italy