The spikelet is a unique inflorescence structure in grass. The molecular mechanisms behind the development and evolution of the spikelet are far from clear. In this study, a dominant rice mutant, lateral florets 1 (lf1), was characterized. In the lf1 spikelet, lateral floral meristems were promoted unexpectedly and could generally blossom into relatively normal florets. LF1 encoded a class III homeodomain-leucine zipper (HD-ZIP III) protein, and the site of mutation in lf1 was located in a putative miRNA165/166 target sequence. Ectopic expression of both LF1 and the meristem maintenance gene OSH1 was detected in the axil of the sterile lemma primordia of the lf1 spikelet. Furthermore, the promoter of OSH1 could be bound directly by LF1 protein. Collectively, these results indicate that the mutation of LF1 induces ectopic expression of OSH1, which results in the initiation of lateral meristems to generate lateral florets in the axil of the sterile lemma. This study thus offers strong evidence in support of the "three-florets spikelet" hypothesis in rice.lateral floret | three-florets spikelet | evolution | yield | rice F lower development is a key process in the reproduction of angiosperms. Under suitable conditions, flowering signals are transmitted to shoot apical meristems (SAMs), which are transformed first into inflorescence meristems (IMs). Floral meristems (FMs) are then initiated on the top and/or lateral domains of the IMs and subsequently transformed into the four whorls of floral organs. The spikelet is a unique unit of inflorescence architecture in grasses and consists of a pair of glumes and a fixed or variable number of florets. Some grassspecific genes are involved in regulating spikelet development. For example, FRIZZY PANICLE (FZP) functions in regulating spikelet meristem (SM) identity in rice. In the fzp mutant, axillary meristems (AMs) are formed instead of FMs, and these then develop into higher-order branches (1). Three genes that encode members of the AP2/ERF superfamily, SUPERNUMERARY BRACT (SNB), INDETERMINATE SPIKELET 1 (OsIDS1), and MULTI-FLORETS SPIKELET 1 (MFS1), are involved in regulating spikelet determinacy in rice (2, 3). In these mutants, the transition from SM to FM is delayed, and extra organs or florets are produced. However, our knowledge about the details of spikelet development in rice remains limited.In most members of Oryzeae, the spikelet is composed of one pair of rudimentary glumes, one pair of sterile lemmas, and one terminal fertile floret, which consists of one pair of hulls (lemma and palea) and inner floral organs (4). Regarding the origin of the sterile lemmas, the "three-florets spikelet" hypothesis proposes that the putative ancestor of the rice spikelet contained two lateral florets in addition to a terminal fertile floret. Subsequently, the lemmas of the two lateral florets degenerated into sterile lemmas, and the inner floral organs and palea degenerated markedly and disappeared during evolution (5). In recent years, several reports have supported this hypot...