The microRNA miR396 directly represses GROWTH-REGULATING FACTORs (OsGRFs) and has been implicated in regulating rice yield and in nitrogen assimilation. Overexpressing the miR396 targets OsGRF4 and OsGRF6 improves rice yield via increased grain size and panicle branching, respectively. Here, we used CRISPR/Cas9 to assess the function of miR396 genes in rice. Knockout of MIR396ef (MIR396e and MIR396f), but not other isoforms, enhanced both grain size and panicle branching, resulting in increased grain yield. Importantly, under nitrogen-deficient conditions, mir396ef mutants showed an even higher relative increase in grain yield as well as elevated above-ground biomass. Furthermore, we identified OsGRF8 as a new target of miR396, in addition to the known targets OsGRF4 and OsGRF6. Disruption of the miR396-targeting site in OsGRF8 was sufficient to both enlarge grain size and elongate panicles. Our results suggest that rice-seed and panicle development are regulated by miR396ef-GRF4/6/8-GIF1/2/3 modules and that miR396ef are promising targets of genome editing for breeding environmentally friendly rice varieties that require less nitrogen fertilization.

中文翻译：

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MIR396e 和 MIR396f 的中断提高了缺氮条件下的水稻产量

microRNA miR396 直接抑制生长调节因子( OsGRFs )，并参与调节水稻产量和氮同化。过表达 miR396 靶点OsGRF4和OsGRF6分别通过增加籽粒大小和分枝来提高水稻产量。在这里，我们使用 CRISPR/Cas9 来评估 miR396 基因在水稻中的功能。敲除MIR396ef（MIR396e和MIR396f），而不是其他同种型，增强了晶粒尺寸和穗分枝，从而提高了谷物产量。重要的是，在缺氮条件下，mir396ef突变体表现出更高的谷物产量相对增加以及地上生物量的增加。此外，除了已知的目标OsGRF4和OsGRF6之外，我们还将 OsGRF8确定为miR396的新目标。OsGRF8 中 miR396 靶向位点的破坏足以增大晶粒尺寸和拉长穗。我们的研究结果表明，水稻种子和穗的发育受 miR396ef-GRF4/6/8-GIF1/2/3 模块的调控，并且 miR396ef 是基因组编辑的有希望的目标，用于培育需要较少氮肥的环保水稻品种。