Conservation and developmental roles of MtSMAX1 in Medicago truncatula

Karrikin (KAR) signaling plays crucial roles in plant development, regulating key traits such as photomorphogenesis, root hair development, and arbuscular mycorrhizal symbiosis. SUPPRESSOR OF MAX2 1 (SMAX1), a key negative regulator of the KAR pathway, has been functionally studied in several plant species. However, the SMAX1 protein remains poorly understood in Medicago truncatula, an important model legume, which limits the comprehensive understanding and application of the KAR pathway in legumes. In this study, we identified the single SMAX1 ortholog MtSMAX1 in M. truncatula. Sequence alignment and structural modeling analyses revealed that MtSMAX1 is highly conserved with SMAX1 homologs from other plant species. Yeast two-hybrid assays demonstrated that MtSMAX1 interacts with two KAR signaling receptors KARRIKIN INSENSITIVE 2 (MtKAI2a/b). In addition, both MtKAI2a/b interact with the F-box protein MORE AXILLARY GROWTH 2 (MtMAX2), suggesting a conserved KAR signaling pathway in M. truncatula. To further investigate the biological functions of MtSMAX1, we generated two distinct CRISPR-edited mutant lines. The loss-of-function mutant Mtsmax1, which carries a premature termination mutation, displays defective phenotypes including reduced seed size, dwarfism, and delayed flowering. In contrast, the Mtsmax1^ΔQ210 mutant, harboring a single glutamine deletion at position 210, exhibits specific defects only in seed development. These phenotypic differences indicate a previously unreported role of MtSMAX1 in regulating legume seed development, with the conserved Q210 residue potentially involved in this process. Furthermore, MtSMAX1 overexpression lines exhibit phenotypes opposite to those of the mutants, further validating the biological functions of MtSMAX1. Gene expression analysis of key developmental marker genes revealed altered expression levels in MtSMAX1 mutants, which are tightly consistent with the corresponding phenotypic variations. These results suggest that MtSMAX1 likely functions as an important transcriptional regulator to modulate the expression of downstream developmental genes. Collectively, our findings establish MtSMAX1 as a pivotal regulator of multiple developmental processes in legumes and provide a foundation for elucidating the broader biological functions and regulatory mechanisms of the KAR signaling pathway.