Metabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, Korea.
Int J Mol Sci. 2021 Mar 31;22(7):3647. doi: 10.3390/ijms22073647.
The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice () protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct , consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with , , , or alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.
细胞分裂素 (CK) 信号通路的主要成分已从受体鉴定到其下游转录因子。然而，由于信号蛋白是由多基因家族编码的，因此表征和量化每个成分或它们的组合对信号级联的贡献一直具有挑战性。在这里，我们描述了一种在水稻（）原生质体中瞬时表达基因的系统，该系统使用 CK 报告基因构建体 ，该构建体由合成的 CK 反应启动子和萤火虫荧光素酶基因组成，可作为信号输出的敏感读数，适用于重建 CK 信号分支。我们使用该系统系统地测试了 CK 信号成分的单独或组合（有无 CK 处理）的贡献。类型-B 应答调节蛋白（RR）OsRR16、OsRR17、OsRR18 和 OsRR19 均强烈激活 ，而 OsRR18 和 OsRR19 对 CK 的诱导最强。单独转染报告基因 、 、 或 仅导致相对较弱的效应，而低于类型-B OsRRs 的效应。当我们测试 OsHK03、OsHPs 和 OsRRs 的组合时，每种组合都表现出不同的 CK 信号活性。因此，该系统允许在水稻中快速高通量探索 CK 信号。
