Overexpression of <i>Os<scp>ERF</scp>48</i> causes regulation of <i>Os<scp>CML</scp>16</i>, a calmodulin‐like protein gene that enhances root growth and drought tolerance

Abstract

The AP2/ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible OsERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root-specific (ROX^O^s^ERF⁴⁸ ) or whole-body (OX^O^s^ERF⁴⁸ ) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic (NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROX^O^s^ERF⁴⁸ plants showed a more vigorous root growth than OX^O^s^ERF⁴⁸ and NT plants. In addition, the ROX^O^s^ERF⁴⁸ plants exhibited higher grain yield than OX^O^s^ERF⁴⁸ and NT plants under field-drought conditions. We constructed a putative OsERF48 regulatory network by cross-referencing ROX^O^s^ERF⁴⁸ root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in OsERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation-qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.