Root-specific overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. Oryza sativa

The AP2/ERF family is one of the plant-specific transcription factors (TFs) 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 that contains four conserved motifs, CMI-1, 2, 3 and 4. Transactivation assay in yeast revealed that the CMI-1 at the C-terminal end was essential for its transcriptional activity. When the OsERF48 was overexpressed in an either root-specific (ROXOsERF48) or whole-body (OXOsERF48) expression manner, both transgenic plants showed a longer and denser root phenotype than the nontransgenic (NT) controls. When plants were grown on a 40% PEG-infused medium, an in vitro drought condition, ROXOsERF48 plants showed a more vigorous root growth over OXOsERF48 and NT plants. In addition, the ROXOsERF48 plants exhibited higher grain yield under field-drought conditions than OXOsERF48 and NT plants. We constructed a putative regulatory network of OsERF48 by cross-referencing of RNA-seq data of ROXOsERF48 roots with a co-expression network database, revealing an involvement of 20 drought-related genes. These include genes for stress signaling, carbohydrate metabolism, cell-wall proteins, and drought-response. More importantly, OsCML16, a key gene for calcium signaling during abiotic stress, was identified to be the direct target of OsERF48 by the ChIP-qPCR and the protoplast transient assay. Thus, our results demonstrated that OsERF48 regulates OsCML16, a calmodulin-like protein gene that enhance root growth and drought tolerance. Overall design: RNA-Seq on roots of nontransgenic control (NT) and root-specific OsERF48 overexpression transgenic rice.

AP2/ERF家族是一类植物特异性转录因子（transcription factors, TFs），其成员参与调控多种植物发育过程与胁迫耐受应答。本研究针对干旱诱导型水稻ERF家族Ib亚族成员OsERF48开展了功能表征，该蛋白包含CMI-1、CMI-2、CMI-3与CMI-4四个保守基序。酵母反式激活实验结果显示，位于C端的CMI-1基序是其转录激活活性的必需核心元件。
分别通过根特异性表达（ROXOsERF48）与全株组成型表达（OXOsERF48）两种方式过表达OsERF48，两类转基因水稻的根系均较非转基因（nontransgenic, NT）对照更长且更致密。当植株培养于40%聚乙二醇（PEG）浸润的培养基（模拟体外干旱胁迫环境）中时，根特异性过表达株系ROXOsERF48的根系生长态势显著优于全株过表达株系OXOsERF48与非转基因对照。此外，在田间干旱胁迫条件下，ROXOsERF48株系的籽粒产量显著高于OXOsERF48与非转基因对照。
通过将ROXOsERF48根系的RNA测序（RNA sequencing, RNA-seq）数据与共表达网络数据库交叉比对，研究人员构建了OsERF48的潜在调控网络，共筛选得到20个干旱相关基因，涵盖胁迫信号通路、碳水化合物代谢、细胞壁蛋白及干旱响应相关基因。尤为关键的是，借助染色质免疫沉淀定量PCR（Chromatin Immunoprecipitation quantitative PCR, ChIP-qPCR）与原生质体瞬时转化实验，证实非生物胁迫下钙信号通路关键基因OsCML16是OsERF48的直接靶标基因。综上，本研究结果表明，OsERF48通过调控钙调蛋白样蛋白基因OsCML16，增强水稻根系生长与干旱耐受能力。
实验整体设计：对非转基因对照（NT）与根特异性OsERF48过表达转基因水稻的根系进行RNA测序分析。