Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea maysL.)

Abstract

Rootless concerning crown and seminal roots(Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea maysL.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds toLBDDNA motifs. RTCS binding to anLBDmotif in the promoter of the auxin response factor (ARF)ZmArf34and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter ofRtcswas shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versusrtcscoleoptilar nodes suggested RTCS-dependent activation ofZmArf34expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.