In seeds, desiccation tolerance (DT) and the ability to survive the dry state for prolonged periods of time (longevity) are two essential traits for seed quality that are consecutively acquired during maturation. Using transcriptomic and metabolomic profiling together with a conditional-dependent network of global transcription interactions, we dissected the maturation events from the end of seed filling to final maturation drying during the last 3 weeks of seed development in Medicago truncatula. The network revealed distinct coexpression modules related to the acquisition of DT, longevity, and pod abscission. The acquisition of DT and dormancy module was associated with abiotic stress response genes, including late embryogenesis abundant (LEA) genes. The longevity module was enriched in genes involved in RNA processing and translation. Concomitantly, LEA polypeptides accumulated, displaying an 18-d delayed accumulation compared with transcripts. During maturation, gulose and stachyose levels increased and correlated with longevity. A seed-specific network identified known and putative transcriptional regulators of DT, including ABSCISIC ACID-INSENSITIVE3 (MtABI3), MtABI4, MtABI5, and APETALA2/ ETHYLENE RESPONSE ELEMENT BINDING PROTEIN (AtAP2/EREBP) transcription factor as major hubs. These transcriptional activators were highly connected to LEA genes. Longevity genes were highly connected to two MtAP2/EREBP and two basic leucine zipper transcription factors. A heat shock factor was found at the transition of DT and longevity modules, connecting to both gene sets. Gain-and loss-of-function approaches of MtABI3 confirmed 80% of its predicted targets, thereby experimentally validating the network. This study captures the coordinated regulation of seed maturation and identifies distinct regulatory networks underlying the preparation for the dry and quiescent states.

Seed longevity, the maintenance of viability during storage, is a crucial factor for preservation of genetic resources and ensuring proper seedling establishment and high crop yield. We used a systems biology approach to identify key genes regulating the acquisition of longevity during seed maturation of Medicago truncatula. Using 104 transcriptomes from seed developmental time courses obtained in five growth environments, we generated a robust, stable coexpression network (MatNet), thereby capturing the conserved backbone of maturation. Using a trait-based gene significance measure, a coexpression module related to the acquisition of longevity was inferred from MatNet. Comparative analysis of the maturation processes in M. truncatula and Arabidopsis thaliana seeds and mining Arabidopsis interaction databases revealed conserved connectivity for 87% of longevity module nodes between both species. Arabidopsis mutant screening for longevity and maturation phenotypes demonstrated high predictive power of the longevity cross-species network. Overrepresentation analysis of the network nodes indicated biological functions related to defense, light, and auxin. Characterization of defense-related wrky3 and nf-x1-like1 (nfxl1) transcription factor mutants demonstrated that these genes regulate some of the network nodes and exhibit impaired acquisition of longevity during maturation. These data suggest that seed longevity evolved by co-opting existing genetic pathways regulating the activation of defense against pathogens.

The preservation of our genetic resources and production of high-quality seeds depends on their ability to remain viable and vigorous during storage. In a quantitative trait locus analysis on seed longevity in Medicago truncatula, we identified the bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5). Characterization of Mt-abi5 insertion mutant seeds revealed that both the acquisition of longevity and dormancy were severely impaired. Using transcriptomes of developing Mt-abi5 seeds, we created a gene coexpression network and revealed ABI5 as a regulator of gene modules with functions related to raffinose family oligosaccharide (RFO) metabolism, late embryogenesis abundant (LEA) proteins, and photosynthesis-associated nuclear genes (PhANGs). Lower RFO contents in Mt-abi5 seeds were linked to the regulation of SEED IMBIBITION PROTEIN1 Proteomic analysis confirmed that a set of LEA polypeptides was reduced in mature Mt-abi5 seeds, whereas the absence of repression of PhANG in mature Mt-abi5 seeds was accompanied by chlorophyll and carotenoid retention. This resulted in a stress response in Mt-abi5 seeds, evident from an increase in α-tocopherol and upregulation of genes related to programmed cell death and protein folding. Characterization of abi5 mutants in a second legume species, pea (Pisum sativum), confirmed a role for ABI5 in the regulation of longevity, seed degreening, and RFO accumulation, identifying ABI5 as a prominent regulator of late seed maturation in legumes.

Seed longevity, defined as the ability to remain alive during storage, is an important agronomic factor. Poor longevity negatively impacts seedling establishment and consequently crop yield. This is particularly problematic for soybean as seeds have a short lifespan. While the economic importance of soybean has fueled a large number of transcriptome studies during embryogenesis and seed filling, the mechanisms regulating seed longevity during late maturation remain poorly understood. Here, a detailed physiological and molecular characterization of late seed maturation was performed in soybean to obtain a comprehensive overview of the regulatory genes that are potentially involved in longevity. Longevity appeared at physiological maturity at the end of seed filling before maturation drying and progressively doubled until the seeds reached the dry state. The increase in longevity was associated with the expression of genes encoding protective chaperones such as heat shock proteins and the repression of nuclear and chloroplast genes involved in a range of chloroplast activities, including photosynthesis. An increase in the raffinose family oligosaccharides (RFO)/sucrose ratio together with changes in RFO metabolism genes was also associated with longevity. A gene co-expression network analysis revealed 27 transcription factors whose expression profiles were highly correlated with longevity. Eight of them were previously identified in the longevity network of Medicago truncatula, including homologues of ERF110, HSF6AB, NFXL1 and members of the DREB2 family. The network also contained several transcription factors associated with auxin and developmental cell fate during flowering, organ growth and differentiation. A transcriptional transition occurred concomitant with seed chlorophyll loss and detachment from the mother plant, suggesting the activation of a post-abscission program. This transition was enriched with AP2/EREBP and WRKY transcription factors and genes associated with growth, germination and post-transcriptional processes, suggesting that this program prepares the seed for the dry quiescent state and germination.