Heterotrimeric G-protein subunits regulate plant architecture, pod development, seed size, and symbiotic nodulation in Medicago truncatula.

Heterotrimeric G proteins are crucial transducers of signaling from receptors, participating in growth and development, as well as in responses to biotic and abiotic stimuli. However, little is known about their roles in regulating various yield-related traits in legumes. In this study, we systematically analyzed the functions of two G-protein-encoding genes, MtGα1 and MtGβ1, along with Regulator of G-protein Signaling1 (MtRGS1), in Medicago truncatula. All three genes were ubiquitously expressed in roots, stems, leaves, nodules, flowers, and pods. We generated the knockout mutants Mtgα1, Mtgβ1, and Mtrgs1 using CRISPR/Cas9 and assessed their growth and development. MtGα1 knockout resulted in slightly shorter plants with smaller pods and shorter spines, but larger seeds, without affecting overall biomass or other traits. MtGβ1 knockout led to dwarfism, weak root development, a severe drop in biomass production, smaller legume pods with shorter spines, and smaller seeds. However, the Mtrgs1 mutants were largely similar to wild-type plants, with few significant defects in growth and development. We also investigated the symbiotic nodulation-related phenotypes of these mutants, discovering that Mtgβ1 mutants produce lighter nodules, whereas Mtgα1 and Mtrgs1 mutants have normal nodulation phenotypes similar to those of wild-type plants. These observations suggest that MtGβ1 positively regulates nodulation, although the detailed mechanisms by which G proteins regulate symbiotic nitrogen fixation in M. truncatula remain to be explored. This work provides potentially valuable genetic resources for further functional analysis and elucidation of the molecular mechanisms of G proteins in this model legume. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00210-x.