Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance
Sunday, 2017/10/08 | 05:05:09
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Jung H, Chung PJ, Park SH, Reveche Redillas MCF, Kim YS, Suh JW, Kim JK. Plant Biotechnology Journal; Mar. 27, 2017; DOI: 10.1111/pbi.12716 SummaryThe 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 (ROXOsERF48) or whole-body (OXOsERF48) 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, ROXOsERF48 plants showed a more vigorous root growth than OXOsERF48 and NT plants. In addition, the ROXOsERF48 plants exhibited higher grain yield than OXOsERF48 and NT plants under field-drought conditions. We constructed a putative OsERF48 regulatory network by cross-referencing ROXOsERF48 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.
See: http://onlinelibrary.wiley.com/doi/10.1111/pbi.12716/full
Figure 1: OsERF48 is a nuclear protein with transcriptional activation. (a) Relative expression of OsERF48 in response to abiotic stresses. Two-week-old seedlings were exposed to air-drying (drought), 400 mm NaCl (salt), 100 μm abscisic acid (ABA) at 28 °C and at 4 °C (low temperature) for the indicated time points. OsUbi1 expression was used as an internal control. Values are the means ± SD (standard deviation) of three independent experiments. (b) Subcellular localization of OsERF48 in rice protoplasts. Protoplasts were transiently co-transformed with OsERF48-GFP and the nuclear localization control OsNF-YA7-mCherry. Fluorescence was observed using a confocal microscope. (c–d) Transactivation activity of OsERF48 using a yeast system. (c) Schematic structure of OsERF48 full length and deletion mutants. (d) Transformed yeast cells harbouring the indicated constructs on SD/-Trp and SD/-Trp/AbA200/X-α-gal. NC, negative control (pGBKT7); PC, positive control (pGBKT7-53 + pGADT7-RecT); AP2/ERF, AP2/ERF domain, CMI, conserved motif of group I, GBD, GAL4 DNA-binding domain. |
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