LpMPK6 regulates leaf color in perennial ryegrass by inhibiting LpMYBR1‐modulated transcription of <i>LpNRT1.5</i>

Leaf color is an important trait for the quality and ornamental value of turfgrass. As an essential component of chlorophyll molecules and related pigments, nitrogen is pivotal for leaf coloration. However, the mechanisms underlying nitrogen-mediated leaf color regulation in perennial turfgrass species remain unclear. In this study, we demonstrate that mitogen-activated protein kinase 6 (LpMPK6) regulates leaf color in perennial ryegrass by phosphorylating LpMYBR1. LpMPK6 overexpression (OE) plants showed leaf chlorosis under soil conditions without nutrient supplementation or in low-nitrogen hydroponic cultivation, accompanied by reduced chlorophyll and nitrogen content. Conversely, LpMPK6 RNAi lines maintained less chlorotic leaves and higher levels of chlorophyll and nitrogen under nitrogen-deficient conditions. Yeast two-hybrid, pull-down, co-immunoprecipitation, and luciferase complementation imaging indicated that LpMPK6 interacted with transcription factor LpMYBR1. Phenotypic analysis revealed that LpMYBR1 functionally antagonized LpMPK6, with more chlorotic leaves and lower levels of chlorophyll and nitrogen in LpMYBR1 RNAi lines, whereas LpMYBR1 OE plants retained more green leaves under nitrogen-deficient conditions. DNA affinity purification, yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays demonstrated that LpMYBR1 bound to the promoter of LpNRT1.5 and activated its transcription. Furthermore, LpNRT1.5 regulated leaf color by mediating NO₃ ^- root-to-shoot transport. Notably, LpMPK6 negatively regulated this transport process by phosphorylating LpMYBR1 and suppressing its transactivation of LpNRT1.5. Taken together, our results revealed a mechanism whereby LpMPK6 suppresses LpMYBR1-mediated transcriptional activation of LpNRT1.5 via phosphorylation, thereby regulating NO₃ ^- transport and leaf coloration in perennial ryegrass. These findings provide insights and offer candidate genes for turfgrass quality improvement.