Karine Gallardo

Proteomics of Medicago truncatula Seed Development Establishes the Time Frame of Diverse Metabolic Processes Related to Reserve Accumulation

We utilized a proteomic approach to investigate seed development in Medicago truncatula, cv Jemalong, line J5 at specific stages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twenty proteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins had previously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, and lipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study also revealed proteins presumably involved in cell division during embryogenesis (β-tubulin and annexin). Their abundance decreased before the accumulation of the major storage protein families, which itself occurs in a specific temporal order: vicilins (14 d after pollination [DAP]), legumins (16 DAP), and convicilins (18 DAP). Furthermore, the study showed an accumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved in embryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipid synthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation of proteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursor accumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulation of enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydrolase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seed development.

Genetic diversity for seed protein composition in Medicago truncatula

Fifty lines of Medicago truncatula, derived from ecotypes or cultivars of diverse geographical origin, were grown under uniform conditions, and variation in seed protein composition and quantity was investigated. One-dimensional electrophoretic profiles revealed 46 major seed polypeptides, of which 26 were polymorphic within the collection. The vicilin/convicilin (7S) and the legumin (11S) type proteins were identified by immunoblotting using antibodies raised against the homologous pea proteins. The polymorphism for the major seed protein classes allowed the clustering of the genotypes into four groups. There was no evidence of clustering according to geographical origin of the lines. However, all lines not belonging to either M. truncatula ssp. truncatula or ssp. longispina were clustered in a single group, demonstrating the value of seed protein profiles in delimiting species boundaries. The Jemalong line group was differentiated early in the dendrogram, and thus represents an ancient clade in the seed diversity of M. truncatula. Within-accession variation was investigated for one-dimensional seed profiles, with additional lines obtained from the same ecotypes. As expected for an autogamous species, within-accession variation was low. Seed protein content was highly variable among the 50 lines examined. Lines contrasting for qualitative traits and seed protein content were identified to allow for the genetic determination of these characters.

Targeting Induced Local Lesions IN Genomes (TILLING) in Medicago truncatula

TILLING is a reverse genetics strategy that combines the high density of point mutations provided by traditional chemical mutagenesis with rapid screening of DNA pools from a mutagenized population for induced mutations (McCallum et al., Nat Biotechnol 18:455-457, 2000). This high-throughput technique allows the identification of point mutations in any gene of interest.

A QTL analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigor in Medicago truncatula.

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.