Data_Sheet_1_Downregulation of miR156-Targeted PvSPL6 in Switchgrass Delays Flowering and Increases Biomass Yield.docx

MiR156/SQUAMOSA PROMOTER BINDING-LIKEs (SPLs) module is the key regulatory hub of juvenile-to-adult phase transition as a critical flowering regulator. In this study, a miR156-targeted PvSPL6 was identified and characterized in switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. Overexpression of PvSPL6 in switchgrass promoted flowering and reduced internode length, internode number, and plant height, whereas downregulation of PvSPL6 delayed flowering and increased internode length, internode number, and plant height. Protein subcellular localization analysis revealed that PvSPL6 localizes to both the plasma membrane and nucleus. We produced transgenic switchgrass plants that overexpressed a PvSPL6-GFP fusion gene, and callus were induced from inflorescences of selected PvSPL6-GFPOE transgenic lines. We found that the PvSPL6-GFP fusion protein accumulated mainly in the nucleus in callus and was present in both the plasma membrane and nucleus in regenerating callus. However, during subsequent development, the signal of the PvSPL6-GFP fusion protein was detected only in the nucleus in the roots and leaves of plantlets. In addition, PvSPL6 protein was rapidly transported from the nucleus to the plasma membrane after exogenous GA3 application, and returned from the plasma membrane to nucleus after treated with the GA3 inhibitor (paclobutrazol). Taken together, our results demonstrate that PvSPL6 is not only an important target that can be used to develop improved cultivars of forage and biofuel crops that show delayed flowering and high biomass yields, but also has the potential to regulate plant regeneration in response to GA3.

MiR156/SQUAMOSA 促进子结合类 (SPLs) 模块是幼年期至成年期转变的关键调控枢纽，作为重要的开花调控因子。在本研究中，我们鉴定并表征了在牧草（ Panicum virgatum L.）中的一种miR156靶基因PvSPL6，牧草是一种兼具饲料和生物燃料用途的作物。在牧草中过表达PvSPL6促进了开花，并减少了节间长度、节间数量和植株高度；而PvSPL6的下调则延缓了开花，并增加了节间长度、节间数量和植株高度。蛋白质亚细胞定位分析显示，PvSPL6定位于质膜和细胞核。我们构建了过表达PvSPL6-GFP融合基因的转基因牧草植株，并从选定的PvSPL6-GFPOE转基因系的穗部诱导出愈伤组织。我们发现，PvSPL6-GFP融合蛋白主要在愈伤组织的细胞核中积累，并在再生的愈伤组织中存在于质膜和细胞核。然而，在随后的发育过程中，PvSPL6-GFP融合蛋白的信号仅在植株的根和叶的细胞核中被检测到。此外，在施加外源GA3后，PvSPL6蛋白迅速从细胞核转运至质膜，而在用GA3抑制剂（苯并噻二唑）处理后则从质膜返回细胞核。综合以上结果，我们得出结论，PvSPL6不仅是一个重要的靶标，可用于培育开花延迟、生物量产量高的饲料和生物燃料作物改良品种，而且还具有调节植物再生以响应GA3的潜力。