SQUAMOSA Promoter Binding Protein-Likes (SPLs) proteins are plant-specific transcription factors that play many crucial roles in plant growth and development. However, there is little information about SPL family in the model legume Medicago truncatula. In this study, a total of 23 MtSPL genes were identified in M. truncatula genome, in which 17 of the MtSPLs contained the putative MtmiR156 binding site at the coding or 3′ UTR regions. Tissue-specific expression pattern analysis showed that most MtmiR156-targeted MtSPLs were highly expressed in seed and pod. The observation of MtmiR156B-overexpressing plants reveals that MtmiR156/MtSPL modules are not only involved in the development of leaves and branches, but also in the seed pod development, especially the formation of spine on pod. The spines on pods are developed in many plant species, which allow pods to adhere to the animals, and then be transported on the outside. This study sheds light on the new function of SPL family in seed dispersal by controlling the formation of spiky pod, and provides insights on understanding evolutionary divergence of the members of SPL gene family among plant species.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Class I KNOTTED-like homeobox (KNOXI) genes are parts of the regulatory network that control the evolutionary diversification of leaf morphology. Their specific spatiotemporal expression patterns in developing leaves correlate with the degrees of leaf complexity between simple-leafed and compound-leafed species. However, KNOXI genes are not involved in compound leaf formation in several legume species. Here, we identify a pathway for dual repression of MtKNOXI function in Medicago truncatula. PINNATE-LIKE PENTAFOLIATA1 (PINNA1) represses the expression of MtKNOXI, while PINNA1 interacts with MtKNOXI and sequesters it to the cytoplasm. Further investigations reveal that UNUSUAL FLORAL ORGANS (MtUFO) is the direct target of MtKNOXI, and mediates the transition from trifoliate to pinnate-like pentafoliate leaves. These data suggest a new layer of regulation for morphological diversity in compound-leafed species, in which the conserved regulators of floral development, MtUFO, and leaf development, MtKNOXI, are involved in variation of pinnate-like compound leaves in M. truncatula.

Abiotic stress, such as drought, osmotic, and salinity stresses, seriously affects plant growth and crop production. Studying stress-resistant genes that enhance plant stress tolerance is an efficient way to facilitate the breeding of crop species with high stress tolerance. In this study, we reported that the core circadian clock component, the LATE ELONGATED HYPOCOTYL (LHY) orthologue MtLHY, plays a positive role in salt stress response in Medicago truncatula. The expression of MtLHY was induced by salt stress, and loss-of-function mutants of MtLHY were shown to be hypersensitive to salt treatment. However, overexpression of MtLHY improved salt stress tolerance through a higher accumulation of flavonoids. Consistently, exogenous flavonol application improved the salt stress tolerance in M. truncatula. Additionally, MtLHY was identified as a transcriptional activator of the flavonol synthase gene, MtFLS. Our findings revealed that MtLHY confers plant salt stress tolerance, at least by modulating the flavonoid biosynthesis pathway, which provides insight into salt stress tolerance that links the circadian clock with flavonoid biosynthesis.

Nitrogen is an indispensable nutrient for plant growth and crop production, but it is not directly accessible to plants without the help of nitrogen-fixing bacteria. Legume plants can form root nodules in symbiosis with rhizobia. NODULE INCEPTION (NIN), a founding member of the NIN-like protein (NLP) family, is essential for nodulation in legume species. However, the knowledge of functional characteristics of the NLP family members in peanuts is limited. In this study, a genome-wide analysis of the NLP genes was carried out. A total of 16 NLP genes were identified in the peanut genome, including 2 AhNIN and 14 AhNLP, which were unevenly distributed on nine chromosomes of the peanut genome. Furthermore, transcriptomic profiles and expression pattern analysis showed that both AhNINa and AhNINb genes were specifically expressed in root nodules. Subcellar localization and transcriptional activity analysis revealed that both AhNINa and AhNINb encode transcriptional activators. In addition, the roots that down-regulated the expression of AhNINa and AhNINb genes failed to form nodules. These findings provide significant insights into the molecular functions of AhNINa and AhNINb genes in regulating peanut nodule development.