Two direct targets of cytokinin signaling control symbiotic nodulation

ARIEL FD; BRAULT-HERNANDEZ M; LAFFONT C; HUAULT E; BRAULT M; PLET J; MOISON M; BLANCHET S; ICHANTÉ JL; CHABAUD M; CARRERE S; CRESPI M; CHAN RL; FRUGIER F

PLANT CELL

AMER SOC PLANT BIOLOGISTS

Lugar: Rockville; Año: 2012 vol. 24 p. 3838 - 3852

1040-4651

Cytokinins control many aspects of plant development and in legumes, the CRE1 (Cytokinin Response 1) receptor is crucial to set up symbiotic nodule organogenesis, allowing these crops to fix atmospheric nitrogen. A transcriptomic analysis of the short-term cytokinin response of Medicago truncatula roots revealed diverse regulatory pathways controlled by this hormone. To identify direct links between cytokinins and symbiotic nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced MtRR1 Response Regulator, and searched for related sequences in genes rapidly regulated by cytokinins. Among genes containing this so-called RR Binding Site (RRBS) were the nodulation-related Type-A RR MtRR4 and the Nodulation Signaling Pathway 2 (NSP2) TF that is critical for early stages of the symbiotic interaction. Site-directed mutagenesis revealed that RRBS cis-elements in the MtRR4 and MtNSP2 promoters are essential for expression during nodule initiation and for cytokinin induction. Furthermore, a microRNA targeting MtNSP2 (miR171h) is also rapidly induced by cytokinins and shows an expression pattern anticorrelated with its target. Other MtCRE1-dependent cytokinin primary targets were a cytokinin oxydase/deshydrogenase (MtCKX1) and a basic Helix-Loop-Helix TF (MtbHLH476), and we showed that the bHLH TF is positively involved in nodulation. Hence, NSP2 and a new bHLH TFs are direct targets of the CRE1-dependent cytokinin signaling pathway acting in symbiotic nodulation.