The knowledge on post-transcriptional regulation mechanisms implicated in seed development (SD) is still limited, particularly in one of the most consumed grain legumes, Phaseolus vulgaris L. We explore for the first time the miRNA expression dynamics in P. vulgaris developing seeds. Seventy-two known and 39 new miRNAs were found expressed in P. vulgaris developing seeds. Most of the miRNAs identified were more abundant at 10 and 40 days after anthesis, suggesting that late embryogenesis/early filling and desiccation were SD stages in which miRNA action is more pronounced. Degradome analysis and target prediction identified targets for 77 expressed miRNAs. While several known miRNAs were predicted to target HD-ZIP, ARF, SPL, and NF-Y transcription factors families, most of the predicted targets for new miRNAs encode for functional proteins. MiRNAs-targets expression profiles evidenced that these miRNAs could tune distinct seed developmental stages. MiRNAs more accumulated at early SD stages were implicated in regulating the end of embryogenesis, postponing the seed maturation program, storage compound synthesis and allocation. MiRNAs more accumulated at late SD stages could be implicated in seed quiescence, desiccation tolerance, and longevity with still uncovered roles in germination. The miRNAs herein described represent novel P. vulgaris resources with potential application in future biotechnological approaches to modulate the expression of genes implicated in legume seed traits with impact in horticultural production systems.

Trehalose, a non-reducing disaccharide with multiple functions, among which source of energy and carbon, stress protectant, and signaling molecule, has been mainly studied in relation to plant development and response to stress. The trehalose pathway is conserved among different organisms and is composed of three enzymes: trehalose-6-phosphate synthase (TPS), which converts uridine diphosphate (UDP)-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P), trehalose-6-phosphatase (TPP), which dephosphorylates T6P to produce trehalose, and trehalase (TRE), responsible for trehalose catabolism. In plants, the trehalose pathway has been mostly studied in resurrection plants and the model plant Arabidopsis thaliana, where 11 AtTPS, 10 AtTPP, and 1 AtTRE genes are present. Here, we aim to investigate the involvement of the trehalose pathway in the early stages of seed germination (specifically, seed imbibition) using the model legume Medicago truncatula as a working system. Since not all the genes belonging to the trehalose pathway had been identified in M. truncatula, we first conducted an in silico analysis using the orthologous gene sequences from A. thaliana. Nine MtTPSs, eight MtTPPs, and a single MtTRE gene were hereby identified. Subsequently, the expression profiles of all the genes (together with the sucrose master-regulator SnRK1) were investigated during seed imbibition with water or stress agents (polyethylene glycol and sodium chloride). The reported data show a temporal distribution and preferential expression of specific TPS and TPP isoforms during seed imbibition with water. Moreover, it was possible to distinguish a small set of genes (e.g., MtTPS1, MtTPS7, MtTPS10, MtTPPA, MtTPPI, MtTRE) having a potential impact as precocious hallmarks of the seed response to stress. When the trehalose levels were measured by high-performance liquid chromatography, a significant decrease was observed during seed imbibition, suggesting that trehalose may act as an energy source rather than osmoprotectant. This is the first report investigating the expression profiles of genes belonging to the trehalose