A novel calmodulin-interacting Domain of Unknown Function 506 protein represses root hair elongation in Arabidopsis

Domain of Unknown Function 506 proteins are ubiquitous in plants. The phosphorus (P) stress-inducible REPRESSOR OF EXCESSIVE ROOT HAIR GROWTH1 (AtRXR1) gene encodes the first characterized DUF506. AtRXR1 inhibits root hair elongation by interacting with RabD2c GTPase. However, functions of other P-responsive DUF506 genes are still missing. Here, we selected two additional P-inducible DUF506 genes for further investigation. The expression of both genes was induced by auxin. Under P-stress, At3g07350 gene expressed ubiquitously in seedlings, whereas At1g62420 (AtRXR3) expression was strongest in roots. AtRXR3 overexpressors and knockouts had shorter and longer root hairs, respectively. A functional AtRXR3-green fluorescent protein fusion localized to root epidermal cells. Chromatin immunoprecipitation and quantitative reverse-transcriptase-polymerase chain reaction revealed that AtRXR3 was transcriptionally activated by RSL4. Bimolecular fluorescence complementation and calmodulin (CaM)-binding assays showed that AtRXR3 interacted with CaM in the presence of Ca²⁺ . Moreover, cytosolic Ca²⁺ ([Ca²⁺ ]_cyt ) oscillations in root hairs of rxr3 mutants exhibited elevated frequencies and dampened amplitudes compared to those of wild type. Thus, AtRXR3 is another DUF506 protein that attenuates P-limitation-induced root hair growth through mechanisms that involve RSL4 and interaction with CaM to modulate tip-focused [Ca²⁺ ]_cyt oscillations.