Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula.

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

Proteomics of developing pea seeds reveals a complex antioxidant network underlying the response to sulfur deficiency and water stress.

Pea is a legume crop producing protein-rich seeds and is increasingly in demand for human consumption and animal feed. The aim of this study was to explore the proteome of developing pea seeds at three key stages covering embryogenesis, the transition to seed-filling, and the beginning of storage-protein synthesis, and to investigate how the proteome was influenced by S deficiency and water stress, applied either separately or combined. Of the 3184 proteins quantified by shotgun proteomics, 2473 accumulated at particular stages, thus providing insights into the proteome dynamics at these stages. Differential analyses in response to the stresses and inference of a protein network using the whole proteomics dataset identified a cluster of antioxidant proteins (including a glutathione S-transferase, a methionine sulfoxide reductase, and a thioredoxin) possibly involved in maintaining redox homeostasis during early seed development and preventing cellular damage under stress conditions. Integration of the proteomics data with previously obtained transcriptomics data at the transition to seed-filling revealed the transcriptional events associated with the accumulation of the stress-regulated antioxidant proteins. This transcriptional defense response involves genes of sulfate homeostasis and assimilation, thus providing candidates for targeted studies aimed at dissecting the signaling cascade linking S metabolism to antioxidant processes in developing seeds.

High-quality genome sequence of white lupin provides insight into soil exploration and seed quality.

White lupin (Lupinus albus L.) is an annual crop cultivated for its protein-rich seeds. It is adapted to poor soils due to the production of cluster roots, which are made of dozens of determinate lateral roots that drastically improve soil exploration and nutrient acquisition (mostly phosphate). Using long-read sequencing technologies, we provide a high-quality genome sequence of a cultivated accession of white lupin (2n = 50, 451 Mb), as well as de novo assemblies of a landrace and a wild relative. We describe a modern accession displaying increased soil exploration capacity through early establishment of lateral and cluster roots. We also show how seed quality may have been impacted by domestication in term of protein profiles and alkaloid content. The availability of a high-quality genome assembly together with companion genomic and transcriptomic resources will enable the development of modern breeding strategies to increase and stabilize white lupin yield.

Water stress combined with sulfur deficiency in pea affects yield components but mitigates the effect of deficiency on seed globulin composition.

Water stress and sulfur (S) deficiency are two constraints increasingly faced by crops due to climate change and low-input agricultural practices. To investigate their interaction in the grain legume pea (Pisum sativum), sulfate was depleted at the mid-vegetative stage and a moderate 9-d water stress period was imposed during the early reproductive phase. The combination of the stresses impeded reproductive processes in a synergistic manner, reducing seed weight and seed number, and inducing seed abortion, which highlighted the paramount importance of sulfur for maintaining seed yield components under water stress. On the other hand, the moderate water stress mitigated the negative effect of sulfur deficiency on the accumulation of S-rich globulins (11S) in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the ratio of S-poor (7S) to 11S globulins. Transcriptome analysis of developing seeds at the end of the combined stress period indicated that similar biological processes were regulated in response to sulfur deficiency and to the combined stress, but that the extent of the transcriptional regulation was greater under sulfur deficiency. Seeds from plants subjected to the combined stresses showed a specific up-regulation of a set of transcription factor and SUMO ligase genes, indicating the establishment of unique regulatory processes when sulfur deficiency is combined with water stress.

Genome-wide association studies with proteomics data reveal genes important for synthesis, transport and packaging of globulins in legume seeds.

Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.

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.

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.