The MtSNF4b subunit of the sucrose non-fermenting-related kinase complex connects after-ripening and constitutive defense responses in seeds of Medicago truncatula

Dormant seeds are capable of remaining alive in the hydrated state for extended periods of time without losing vigor, until environmental cues or after-ripening result in the release of dormancy. Here, we investigated the possible role of the regulatory subunit of the sucrose non-fermenting-related kinase complex, MtSNF4b, in dormancy of Medicago truncatula seeds. Expression of MtSNF4b and its involvement in a high-molecular-weight complex are found in dormant seeds, whereas imbibition of fully after-ripened, non-dormant seeds leads to dissociation of the complex. MtSNF4b is capable of complementing the yeast Δsnf4 mutant and of interacting with the MtSnRK1 α-subunit in a double hybrid system. Transcriptome analyses on freshly harvested and after-ripened RNAi Mtsnf4b and wild-type embryos implicate MtSNF4b in the defense response in hydrated dormant embryonic tissues, affecting the expression of genes encoding enzymes of flavonoid and phenylpropanoid metabolism, WRKY transcription factors and pathogenesis-related proteins. Silencing MtSNF4b also increased the speed of after-ripening during dry storage, an effect that appears to be related to a change in base water potential. No significant difference in ABA content or sensitivity was detected between mutant and wild-type seeds. Pharmacological studies using hexoses and sugar analogs revealed that mannose restored germination behavior and expression of the genes PAL, CHR and IFR in RNAi Mtsnf4b seeds towards that of the wild-type, suggesting that MtSNF4b might act upstream of sugar-sensing pathways. Overall, the results suggest that MtSNF4b participates in regulation of a constitutively activated defense response in hydrated, dormant seeds

Characterization of dormancy behaviour in seeds of the model legume Medicago truncatula

Seeds of Medicago truncatula, a genomic model species for legumes, exhibit physiological and physical dormancy. Here, the factors influencing the germination behaviour of freshly harvested and stored seeds were investigated using several genotypes. Hardseededness is promoted when mature seeds are equilibrated at relative humidities (RH) below 75%. The release of physical dormancy during imbibition was dependent on the initial water content/RH that the seeds were dried to: the drier the seeds, the longer the imbibition time needed to break physical dormancy. The kinetics of physical dormancy release was slower than that of physiological dormancy, making it possible to uncouple both phenomena. Freshly harvested embryos without seed coverings germinated at the same speed as afterripened seeds. The depth of dormancy varied between different M. truncatula genotypes, from more to less dormant: DZA315.16>A17 (Jemalong)>R108>DZA45.5. This difference was eliminated by removing the endosperm. Collectively, these observations indicate that the endosperm is likely the main factor in the reduced germination of freshly harvested seeds. White light decreased germination speed of dormant seeds whereas it had no effect on non-dormant seeds. Recently harvested seeds were most dormant at temperatures above 17°C, whereas afterripened seeds germinated over a wider range of temperature. Fluridone could efficiently break dormancy, reinforcing the role of abscisic acid (ABA) synthesis. However, dormancy was not affected by gibberellic acid (100 μM GA3) or nitrate. The particular dormancy features unravelled here for M. truncatula, combined with the available genomic resources, make it a new, useful model for genetic and molecular studies which can complement those developed for Arabidopsis

Identification of a molecular dialogue between developing seeds of Medicago truncatula and seedborne xanthomonads.

Plant pathogenic bacteria disseminate and survive mainly in association with seeds. This study addresses whether seeds are passive carriers or engage a molecular dialogue with pathogens during their development. We developed two pathosystems using Medicago truncatula with Xanthomonas alfalfae subsp. alfalfae (Xaa), the natural Medicago sp. pathogen and Xanthomonas campestris pv. campestris (Xcc), a Brassicaceae pathogen. Three days after flower inoculation, the transcriptome of Xcc-infected pods showed activation of an innate immune response that was strongly limited in Xcc mutated in the type three secretion system, demonstrating an incompatible interaction of Xcc with the reproductive structures. In contrast, the presence of Xaa did not result in an activation of defence genes. Transcriptome profiling during development of infected seeds exhibited time-dependent and differential responses to Xcc and Xaa. Gene network analysis revealed that the transcriptome of Xcc-infected seeds was mainly affected during seed filling whereas that of Xaa-infected seeds responded during late maturation. The Xcc-infected seed transcriptome exhibited an activation of defence response and a repression of targeted seed maturation pathways. Fifty-one percent of putative ABSCISIC ACID INSENSITIVE3 targets were deregulated by Xcc, including oleosin, cupin, legumin and chlorophyll degradation genes. At maturity, these seeds displayed decreased weight and increased chlorophyll content. In contrast, these traits were not affected by Xaa infection. These findings demonstrate the existence of a complex molecular dialogue between xanthomonads and developing seeds and provides insights into a previously unexplored trade-off between seed development and pathogen defence