Abiotic stresses such as limited nitrogen availability can be challenging during the early stages of stand establishment. Finding physiologically/metabolically N-efficient genotypes is critical in legumes where nitrate uptake and assimilation occur before nodulation. We took advantage of barrel medic (Medicago truncatula) natural diversity (192 accessions) to identify markers associated with seedling performance with or without nitrate supply. We determined traits associated with organ elongation and seed biomass during seedling heterotrophic growth and calculated indexes of plasticity to nitrate supply. These indexes allowed us to group accessions into four nitrate response physiotypes. A genome-wide association study (GWAS) identified loci associated with seedling performance and plasticity to nitrate. In particular, we found one chromosomal region associated with seedling elongation that contained 16 genes encoding glutathione S-transferases (GSTs). This suggests the regulation of seedling elongation in absence of nitrate could be related to sulfur metabolism and/or control of oxidative stress. A new phylogenetic classification of GST in M. truncatula showed that all GST genes associated with the locus belong to class Tau, a class known to be involved in abiotic stress responses and development. Then, the monitoring of GST activity revealed a high positive relationship between hypocotyl and radicle length and GST activity during heterotrophic growth in the absence of nitrate, the correlation being strongest for hypocotyl elongation, known to be only driven by cell elongation. Our findings suggest an interaction between GSTs and nitrate to control cell elongation during seedling heterotrophic growth. They also suggest that nitrogen/sulfur metabolism appears to be a key player in the fitness of genotypes in response to nitrate availability.

Seed size is often considered to be an important trait for seed quality, i.e., vigour and germination performance. It is believed that seed size reflects the quantity of reserve material and thus the C and N sources available for post-germinative processes. However, mechanisms linking seed size and quality are poorly documented. In particular, specific metabolic changes when seed size varies are not well-known. To gain insight into this aspect, we examined seed size and composition across different accessions of barrel medic (Medicago truncatula Gaertn.) from the genetic core collection. We conducted multi-elemental analyses and isotope measurements, as well as exact mass GC–MS metabolomics. There was a systematic increase in N content (+0.17% N mg−1) and a decrease in H content (–0.14% H mg−1) with seed size, reflecting lower lipid and higher S-poor protein quantity. There was also a decrease in 2H natural abundance (δ2H), due to the lower prevalence of 2H-enriched lipid hydrogen atoms that underwent isotopic exchange with water during seed development. Metabolomics showed that seed size correlates with free amino acid and hexoses content, and anticorrelates with amino acid degradation products, disaccharides, malic acid and free fatty acids. All accessions followed the same trend, with insignificant differences in metabolic properties between them. Our results show that there is no general, proportional increase in metabolite pools with seed size. Seed size appears to be determined by metabolic balance (between sugar and amino acid degradation vs. utilisation for storage), which is in turn likely determined by phloem source metabolite delivery during seed development.

In 1907 the French palaeobotanist Octave Lignier published a study of Jurassic fossil wood that was to become a seminal palaeoxylological work. Although the names he established were often used or recombined and his contribution is still often quoted today, the original material was never re-studied, except for one of the fifteen originally published samples. This material was only recently been rediscovered, after it was thought to be lost since the 1980s. Here we make use of this opportunity to re-evaluate stratigraphical attributions and ages of Lignier's original samples, to review their nomenclatural status and discuss their taxonomical assignment. Three new combinations are introduced: (Agathoxylon tranchantii (Lignier) comb. nov., Baieroxylon divesence (Lignier ex Seward) comb. nov., Brachyoxylon blevillense (Lignier) comb. nov.), and a new species (Agathoxylon crasseradiatum sp. nov.) is published based on Lignier's material. Baieroxylon divesence is anatomically closely related to modern ginkgo and hence an interesting milestone is the little documented history of Ginkgoales wood.

Seedling pre-emergence is a critical phase of development for successful crop establishment because of its susceptibility to environmental conditions. In a context of reduced use of inorganic fertilizers, the genetic bases of the response of seedlings to nitrate supply received little attention. This issue is important even in legumes where nitrate absorption starts early after germination, before nodule development. Natural variation of traits characterizing seedling growth in the absence or presence of nitrate was investigated in a core collection of 192 accessions ofMedicago truncatula. Plasticity indexes to the absence of nitrate were calculated. The genetic determinism of the traits was dissected by genome-wide association study (GWAS). The absence of nitrate affected seed biomass mobilization and root/shoot length ratio. However, the large range of genetic variability revealed different seedling performances within natural diversity. A principal component analysis (PCA) carried out with plasticity indexes highlighted four physiotypes of accessions differing in relationships between seedling elongation and seed biomass partitioning traits in response to the absence of nitrate. Finally, GWAS revealed 45 associations with single or combined traits corresponding to coordinates of accessions on PCA, as well as two clusters of genes encoding sugar transporters and glutathione transferases surrounding loci associated with seedling elongation traits.

In Medicago truncatula, nitrate, acting as a signal perceived by MtNPF6.8, inhibits primary root growth through a reduction of root cell elongation. We evaluated here whether reactive oxygen species (ROS) could mediate the nitrate signal since ROS produced and converted (O2•−→ H2O2 → •OH) in the root tip have been reported to control cell elongation. We found that nitrate reduces the content in ROS of the primary root tip in three wild type genotypes (including R108) sensitive to nitrate, but not in the npf6.8 mutants (in the R108 genetic background), insensitive to nitrate. The decrease in ROS content observed in R108 in response to nitrate is orchestrated by cell wall peroxidases (PODs) that eliminate H2O2 and impair its conversion in •OH, the species responsible for cell wall loosening and cell elongation. These results demonstrate that ROS and PODs are downstream mediators of the nitrate signal. We further identified a NADPH oxidase (MtRBOHF), as another mediator in the nitrate signaling pathway, the primary root growth of rbohF mutants being insensitive to nitrate. We finally performed a coupled transcriptomic and proteomic analysis with R108 and npf6.8 grown in absence or presence of nitrate to uncover novel aspects of legume primary root tip response to nitrate. We found that the sensitivity of the primary root is strongly linked to the functionality of MtNPF6.8 and many nitrate responsive genes encompass genes involved in ROS homeostasis and cell wall organization, or encode transcription factors.

Hypocotyl elongation in the dark is a crucial process to ensure seedling emergence. It relies both on the cell number and cell length. The contribution of these two factors to the maximal hypocotyl length and the impact of environmental conditions on this contribution are not known. This is surprising considering the agronomic and economical importance of seedling emergence in crop establishment. Using 14 genotypes from a nested core collection representing Medicago truncatula (barrel medic) natural variation, we investigated how epidermal cell number and cell length contribute to hypocotyl length under optimal, low temperature (8°C) and water deficit (−0.50 MPa) conditions. Both cell number and length vary according to genotypes and contribute to maximal hypocotyl length differences between genotypes. This contribution, however, depends on growth conditions. Cell number is the major contributor under optimal conditions (60%) whereas cell length becomes the major determinant under stress. Maximal hypocotyl length is correlated with hypocotyl elongation rate under both stresses but not under optimal condition, revealing contrasted genotypes for cell elongation capacity under stress. To identify the genetic regulators determining cell number and cell length, quantitative trait loci (QTLs) were detected using a recombinant inbred lines population exhibiting segregation in maximal hypocotyl length. Two QTLs controlling cell number and three QTLs controlling cell length at low temperature were detected. One QTL for cell number and two for cell length were found to be associated with hypocotyl length under low temperature. This study provides new information to improve seedling emergence under abiotic stress.