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 mays L.). 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 to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. 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.

Comparative Transcriptome Profiling of Maize Coleoptilar Nodes during Shoot-Borne Root Initiation

“…It has been demonstrated that LBD proteins can bind specifically to the 59-GCGGCG-39 LBD consensus sequence motif in the promoter of target genes (Husbands et al, 2007). It has been demonstrated that RTCS can bind to the LBD promoter element of arf34 that is located 277 bp upstream of the ATG start codon (Majer et al, 2012). Among the differentially expressed genes identified in this study, 100 of 828 contained at least one LBD motif in their 1-kb promoter.…”

“…The maize LBD genes rtcs-neighbour (rtcn; GRMZM2G092483_T02; Taramino et al, 2007) and lbd34 (GRMZM2G075499_T01; Majer et al, 2012) are preferentially expressed in wild-type versus rtcs coleoptilar nodes at all three developmental stages dissected in this study. rtcn is physically located next to the rtcs gene in the maize genome (Taramino et al, 2007).…”

“…The rtcs gene (GRMZM2G092542) encodes a member of the Lateral Organ Boundaries (LOB) domain (LBD) transcription factor family (Taramino et al, 2007) and is localized in the nucleus (Majer et al, 2012). The conserved LBD is plant specific and contains a C block that is predicted to contain a secondary structure that resembles a DNA-binding zinc finger (Majer and Hochholdinger, 2011).…”

“…It has been demonstrated that LBD proteins recognize a specific 59-GCGGCG-39 DNA consensus sequence termed the LBD motif (Husbands et al, 2007). The RTCS protein can bind to an LBD motif of the arf34 gene, while the ARF34 protein can bind to an LBD motif in the rtcs promoter (Majer et al, 2012). Moreover, LBD proteins contain a C-terminal GAS block that forms a coiled-coil structure, which is predicted to be a Leu zipper motif required for proteinprotein interactions (Majer and Hochholdinger, 2011).…”

“…Moreover, LBD proteins contain a C-terminal GAS block that forms a coiled-coil structure, which is predicted to be a Leu zipper motif required for proteinprotein interactions (Majer and Hochholdinger, 2011). It has also been demonstrated by yeast two-hybrid experiments that the RTCS protein can homointeract with RTCS and heterointeract with its closely related homolog RTCS-like (Majer et al, 2012).…”