Biomedical subjects

Pascal Ratet

Publications and source records attributed to Pascal Ratet.

5 recordsLinked to original sources

PubMed2006

Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Amino Acid Sequence↗

PubMed2006

Medicago truncatula transformation using leaf explants.

Legumes have long been recalcitrant to efficient Agrobacterium tumefaciens-mediated transformation. The choice and use of model legume plants (Medicago truncatula and Lotus japonicus) for molecular studies has triggered extensive studies devoted to the development of efficient Agrobacterium-mediated transformation protocols for these two plants. In M. truncatula, transformation protocols rely on the use of highly regenerable lines obtained by recurrent in vitro culture selection. These protocols are based on Agrobacterium-mediated transformation of M. truncatula followed by somatic embryogenesis-mediated plant regeneration. We describe here the protocol developed for M. truncatula R108-1 (c3).

Agrobacterium tumefaciens↗

PubMed2005

Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula.

Legumes are second only to grasses in worldwide economic importance, and understanding their molecular genetics is vital to the breeding of important grain and forage legumes. Over the past decade, Medicago truncatula has been selected as a model plant in which to study biological processes that are unique and pertinent to legumes, and that cannot easily be studied in Arabidopsis. Here, we discuss the most common tools for introducing and analyzing genetic mutations in M. truncatula. Because transformation and regeneration are still bottlenecks in studying a legume species, large-scale insertional mutagenesis poses a major challenge in M. truncatula. We discuss the tobacco retrotransposon Tnt1 as a viable and attractive option for introducing multiple independent insertions per plant for saturation mutagenesis.

Arabidopsis↗

PubMed2004

From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis.

While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

Carboxylic Ester Hydrolases↗

PubMed2003

Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals preferential tip localization of the protein in root hairs.

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.

Gene Transfer Techniques↗