Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula

• Published in: The Plant cell 2012-09, Vol.24 (9), p.3838-3852
• Main Authors: Ariel, Federico, Brault-Hernandez, Marianne, Laffont, Carole, Huault, Emeline, Brault, Mathias, Plet, Julie, Moison, Michael, Blanchet, Sandrine, Ichanté, Jean Laurent, Chabaud, Mireille, Carrere, Sébastien, Crespi, Martin, Chan, Raquel L., Frugier, Florian
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Consensus Sequence; Cytokinins; Cytokinins - pharmacology; Datasets; Gene Expression Profiling; Gene expression regulation; Gene Expression Regulation, Plant; Genes; Legumes; Life Sciences; Medicago truncatula - drug effects; Medicago truncatula - genetics; Medicago truncatula - microbiology; Medicago truncatula - physiology; MicroRNAs - genetics; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Nitrogen Fixation; Nodulation; Nodules; Oligonucleotide Array Sequence Analysis; Phylogeny; Plant cells; Plant Growth Regulators - pharmacology; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Root Nodulation - genetics; Plant roots; Plants; Promoter Regions, Genetic - genetics; Root Nodules, Plant - drug effects; Root Nodules, Plant - genetics; Root Nodules, Plant - microbiology; Root Nodules, Plant - physiology; Seedlings - drug effects; Seedlings - genetics; Seedlings - microbiology; Seedlings - physiology; Sequence Alignment; Signal Transduction; Sinorhizobium meliloti - physiology; Symbiosis; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptome; Vegetal Biology
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.112.103267

Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogénesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulationrelated Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.