The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum

Plant root systems are essential for many physiological processes, including water and nutrient absorption. MADS-box transcription factor (TF) genes have been characterized as the important regulators of root development in plants; however, the underlying mechanism is largely unknown, including chrysanthemum. Here, it was found that the overexpression of CmANR1, a chrysanthemum MADS-box TF gene, promoted both adventitious root (AR) and lateral root (LR) development in chrysanthemum. Whole transcriptome sequencing analysis revealed a series of differentially expressed unigenes (DEGs) in the roots of CmANR1-transgenic chrysanthemum plants compared to wild-type plants. Functional annotation of these DEGs by alignment with Gene Ontology (GO) terms and biochemical pathway Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that CmANR1 TF exhibited DNA binding and catalytic activity, as well as participated in phytohormone signal transduction. Both chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and gel electrophoresis mobility shift assays (EMSA) indicated the direct binding of CmPIN2 to the recognition site CArG-box motif by CmANR1. Finally, a firefly luciferase imaging assay demonstrated the transcriptional activation of CmPIN2 by CmANR1 in vivo. Overall, our results provide novel insights into the mechanisms of MADS-box TF CmANR1 modulation of both AR and LR development, which occurs by directly regulating auxin transport gene CmPIN2 in chrysanthemum.