Grain weight and grain number are two important traits directly determining grain yield in rice. To date, a lot of genes related to grain weight and grain number have been identified; however, the regulatory mechanism underlying these genes remains largely unknown. In this study, we studied the biological function of OsSPL18 during grain and panicle development in rice. Knockout (KO) mutants of OsSPL18 exhibited reduced grain width and thickness, panicle length and grain number, but increased tiller number. Cytological analysis showed that OsSPL18 regulates the development of spikelet hulls by affecting cell proliferation. qRT-PCR and GUS staining analyses showed that OsSPL18 was highly expressed in developing young panicles and young spikelet hulls, in agreement with its function in regulating grain and panicle development. Transcriptional activation experiments indicated that OsSPL18 is a functional transcription factor with activation domains in both the N-terminus and C-terminus, and both activation domains are indispensable for its biological functions. Quantitative expression analysis showed that DEP1, a major grain number regulator, was significantly down-regulated in OsSPL18 KO lines. Both yeast one-hybrid and dual-luciferase (LUC) assays showed that OsSPL18 could bind to the DEP1 promoter, suggesting that OsSPL18 regulates panicle development by positively regulating the expression of DEP1. Sequence analysis showed that OsSPL18 contains the OsmiR156k complementary sequence in the third exon; 5ʹ RLM-RACE experiments indicated that OsSPL18 could be cleaved by OsmiR156k. Taken together, our results uncovered a new OsmiR156k-OsSPL18-DEP1 pathway regulating grain number in rice.

粒重和粒数是直接决定水稻籽粒产量的两个重要特征。迄今为止，已经鉴定出许多与籽粒重量和籽粒数量有关的基因。然而，这些基因的潜在调控机制仍是未知之数。在这项研究中，我们研究了OsSPL18在水稻籽粒和穗发育过程中的生物学功能。OsSPL18的敲除（KO）突变体表现出降低的籽粒宽度和厚度，穗长和籽粒数量，但增加了分till数量。细胞学分析表明，OsSPL18通过影响细胞增殖来调节小穗壳的发育。qRT-PCR和GUS染色分析表明OsSPL18在发育中的幼穗和幼穗壳中高度表达，与其在调节谷粒和穗发育中的功能一致。转录激活实验表明，OsSPL18是一种功能性转录因子，在N端和C端均具有激活域，而两个激活域对于其生物学功能都是必不可少的。定量表达分析表明，DEP1，一个重要的粮食数调节器，被显著在下调OsSPL18 KO线。酵母单杂交和双荧光素酶（LUC）分析均显示OsSPL18可以与DEP1启动子结合，这表明OsSPL18通过正向调节OsSPL18的表达来调节穗的发育。DEP1。序列分析表明，OsSPL18在第三个外显子中包含OsmiR156k互补序列。5次RLM-RACE实验表明OsSPL18可以被OsmiR156k裂解。两者合计，我们的结果发现了一个新的OsmiR156k - OsSPL18 - DEP1途径调节水稻的粒数。