Hélène Zuber

A gene expression atlas of the model legume Medicago truncatula

Legumes played central roles in the development of agriculture and civilization, and today account for approximately one-third of the worlds primary crop production. Unfortunately, most cultivated legumes are poor model systems for genomic research. Therefore, Medicago truncatula, which has a relatively small diploid genome, has been adopted as a model species for legume genomics. To enhance its value as a model, we have generated a gene expression atlas that provides a global view of gene expression in all major organ systems of this species, with special emphasis on nodule and seed development. The atlas reveals massive differences in gene expression between organs that are accompanied by changes in the expression of key regulatory genes, such as transcription factor genes, which presumably orchestrate genetic reprogramming during development and differentiation. Interestingly, many legume-specific genes are preferentially expressed in nitrogen-fixing nodules, indicating that evolution endowed them with special roles in this unique and important organ. Comparative transcriptome analysis of Medicago versus Arabidopsis revealed significant divergence in developmental expression profiles of orthologous genes, which indicates that phylogenetic analysis alone is insufficient to predict the function of orthologs in different species. The data presented here represent an unparalleled resource for legume functional genomics, which will accelerate discoveries in legume biology.

A Combined Proteome and Transcriptome Analysis of Developing Medicago truncatula Seeds Evidence for Metabolic Specialization of Maternal and Filial Tissues

A comparative study of proteome and transcriptome changes during Medicago truncatula (cultivar Jemalong) seed development has been carried out. Transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. These data, combined with the analysis of transcript and protein distribution in the isolated seed coat, endosperm, and embryo, demonstrated the major contribution made to the embryo by the surrounding tissues. First, a remarkable compartmentalization of enzymes involved in methionine biosynthesis between the seed tissues was revealed that may regulate the availability of sulfur-containing amino acids for embryo protein synthesis during seed filling. This intertissue compartmentalization, which was also apparent for enzymes of sulfur assimilation, is relevant to strategies for modifying the nutritional value of legume seeds. Second, decreasing levels during seed filling of seed coat and endosperm metabolic enzymes, including essential steps in Met metabolism, are indicative of a metabolic shift from a highly active to a quiescent state as the embryo assimilates nutrients. Third, a concomitant persistence of several proteases in seed coat and endosperm highlighted the importance of proteolysis in these tissues as a supplementary source of amino acids for protein synthesis in the embryo. Finally, the data revealed the sites of expression within the seed of a large number of transporters implied in nutrient import and intraseed translocations. Several of these, including a sulfate transporter, were preferentially expressed in seeds compared with other plant organs. These findings provide new directions for genetic improvement of grain legumes.

Exploring the nuclear proteome of Medicago truncatula at the switch towards seed filling

Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatin-modifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.

The Seed Composition of Arabidopsis Mutants for the Group 3 Sulfate Transporters Indicates a Role in Sulfate Translocation within Developing Seeds

Sulfate is required for the synthesis of sulfur-containing amino acids and numerous other compounds essential for the plant life cycle. The delivery of sulfate to seeds and its translocation between seed tissues is likely to require specific transporters. In Arabidopsis (Arabidopsis thaliana), the group 3 plasmalemma-predicted sulfate transporters (SULTR3) comprise five genes, all expressed in developing seeds, especially in the tissues surrounding the embryo. Here, we show that sulfur supply to seeds is unaffected by T-DNA insertions in the SULTR3 genes. However, remarkably, an increased accumulation of sulfate was found in mature seeds of four mutants out of five. In these mutant seeds, the ratio of sulfur in sulfate form versus total sulfur was significantly increased, accompanied by a reduction in free cysteine content, which varied depending on the gene inactivated. These results demonstrate a reduced capacity of the mutant seeds to metabolize sulfate and suggest that these transporters may be involved in sulfate translocation between seed compartments. This was further supported by sulfate measurements of the envelopes separated from the embryo of the sultr3;2 mutant seeds, which showed differences in sulfate partitioning compared with the wild type. A dissection of the seed proteome of the sultr3 mutants revealed protein changes characteristic of a sulfur-stress response, supporting a role for these transporters in providing sulfate to the embryo. The mutants were affected in 12S globulin accumulation, demonstrating the importance of intraseed sulfate transport for the synthesis and maturation of embryo proteins. Metabolic adjustments were also revealed, some of which could release sulfur from glucosinolates.

Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization

BackgroundSulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole.ResultsIn the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes.ConclusionsThis study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.

Legume adaptation to sulfur deficiency revealed by comparing nutrient allocation and seed traits in Medicago truncatula

Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

Metabolic Specialization of Maternal and Filial Tissues

The seed consists of three principal components of maternal (seed coats) or zygotic (embryo and endosperm) origin with distinct functions, but that interplay throughout their development to ensure the accumulation of storage compounds for successful germination and early seedling growth. The reserves stored in mature seeds represent major human and livestock food sources. Therefore, much research and breeding efforts are concentrated on optimizing seed quality and yield. The principal filial storage organ differs between species. For example, it is the endosperm for cereal grains accumulating high amount of starch, and the embryo for protein-rich legume seeds. These organs are surrounded by tissues of maternal and/or zygotic origin, depending on the species, which represent a protective barrier and play a role in furnishing the filial organ with nutrients and oxygen. Seed tissues and cell types have been individually studied by the omics approaches with a view to dissecting the molecular processes underlying reserve accumulation. The most comprehensive analyses have been performed at the transcriptome and/or proteome levels in various species, including Medicago, soybean, Arabidopsis, sugar beet, barley, wheat, maize, rice, and tomato. Here, we report the division of metabolic activities between seed tissues, based on the identification and ontological classification of gene products differentially accumulated between seed tissues. The work allowed metabolic networks to be proposed in specific tissue-types and regulatory factors to be identified, two fundamental tasks in systems biology, with an ultimate goal to undertake a computational reconstruction of tissue-specific metabolic models.