MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.