A RSL4-CPKs feedback loop integrates auxin and ethylene signals to regulate root hair growth in Arabidopsis

Root hairs are essential for nutrient and water uptake and environmental interactions. RSL4 is a key transcription factor regulating their growth, but how hormones regulate RSL4 post-translationally remains unclear. Here, we show that root hair-enriched CPK4/5/6/11 directly phosphorylate RSL4 at Ser94/95/111/131. Phosphodeficient RSL4^{S94/95/111/131A} shows reduced transcriptional activity and fails to rescue the rsl4 mutant, whereas phosphomimetic RSL4^{S94/95/111/131D} enhances activity and promotes root hair elongation. Loss of function of CPKs results in slower root hair growth compared with wild-type, and leads to shorter root hairs under normal conditions and in response to auxin and ethylene. Notably, promoters of CPK4 and CPK11 contain binding motifs for RSL4, ethylene-responsive EIN3, and auxin-responsive ARF5. RT-qPCR, LUC reporter assays, and ChIP assays confirm that these transcription factors directly regulate CPK4 and CPK11 expression. Our findings reveal that RSL4 is post-translationally activated by CPK-mediated phosphorylation, while CPK4/11 are transcriptionally induced by RSL4. Both are further modulated by ethylene and auxin signaling via EIN3 and ARFs, forming a positive feedback loop that integrates transcriptional and phosphorylation events downstream of hormonal signals to drive root hair growth. This work uncovers a hierarchical regulatory network underlying root hair development.