MULTI-FLORET SPIKELET1, Which Encodes an AP2/ERF Protein,Determines Spikelet Meristem Fate and Sterile Lemma Identity in Rice

The spikelet is a unique inflorescence structure of grass. The molecular mechanism that controls the development of the spikelet remains unclear. In this study, we identified a rice (Oryza sativa) spikelet mutant, multi-floret spikelet1 (mfs1), that showed delayed transformation of spikelet meristems to floral meristems, which resulted in an extra hull-like organ and an elongated rachilla. In addition, the sterile lemma was homeotically converted to the rudimentary glume and the body of the palea was degenerated in mfs1. These results suggest that the MULTI-FLORET SPIKELET1 (MFS1) gene plays an important role in the regulation of spikelet meristem determinacy and floral organ identity. MFS1 belongs to an unknown function clade in the APETALA2/ethylene-responsive factor (AP2/ERF) family. The MFS1-green fluorescent protein fusion protein is localized in the nucleus. MFS1 messenger RNA is expressed in various tissues, especially in the spikelet and floral meristems. Furthermore, our findings suggest that MFS1 positively regulates the expression of LONG STERILE LEMMA and the INDETERMINATE SPIKELET1 (IDS1)-like genes SUPERNUMERARY BRACT and OsIDS1.In the reproductive phase of angiosperms, the shoot meristem is transformed into an inflorescence meristem, which then produces a floral meristem from which floral organs begin to develop, according to the mechanism known as the ABCDE model (Coen and Meyerowitz, 1991; Coen and Nugent, 1994; Dreni et al., 2007; Ohmori et al., 2009). An inflorescence can be classified as determinate or indeterminate based on whether its apical meristem is transformed into a terminal floral meristem. In an indeterminate inflorescence, the lateral meristem is permanently differentiated from the apical meristem, which is not converted into the terminal floral meristem, as occurs during the development of the inflorescences of Arabidopsis (Arabidopsis thaliana) and snapdragon (Antirrhinum majus). In contrast, in a determinate inflorescence, the apical meristem is transformed into the terminal floral meristem after the production of a fixed number of lateral meristems, as occurs during the development of the inflorescences of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum; Bradley et al., 1997; Ratcliffe et al., 1999; Sussex and Kerk, 2001; Chuck et al., 2008).In general, inflorescences in grasses consist of branches and spikelets (Coen and Nugent, 1994; Itoh et al., 2005; Kobayashi et al., 2010). In these organisms, the branch meristem is determinate. It produces several lateral spikelet meristems, followed by the final production of a terminal spikelet meristem. The spikelet, the specific unit of the grass inflorescence, comprises a pair of bracts and one to 40 florets; it shows determinacy or indeterminacy depending on the species (Clifford, 1987; Malcomber et al., 2006). In species with a determinate spikelet, such as rice (Oryza sativa), after the production of fixed lateral floral meristems, the spikelet meristems are converted into terminal floral meristems, resulting in termination of the spikelet meristem fate. In contrast, in species with an indeterminate spikelet, such as wheat (Triticum aestivum), the spikelet meristem fate is maintained continuously and produces a variable number of lateral floral meristems.In Arabidopsis, the gene TERMINAL FLOWER1 (TFL1) was shown to maintain indeterminacy in the fate of the inflorescence. In the tfl1 mutant, the inflorescence meristems were converted into floral meristems earlier than in the wild type, but the ectopic expression of TFL1 resulted in the transformation of floral meristems at a later stage of development to secondary inflorescence meristems (Bradley et al., 1997; Ratcliffe et al., 1999; Mimida et al., 2001). In rice, overexpression of either of the TFL1-like genes, RICE CENTRORADIALIS1 (RCN1) or RCN2, delayed the transition of branch meristems to spikelet meristems and finally resulted in the production of a greater number of branches and spikelets than in the wild type (Nakagawa et al., 2002; Rao et al., 2008).To date, no gene that acts to maintain the indeterminacy of the spikelet meristem has been reported. However, two classes of genes have been shown to be involved in termination of the indeterminacy of spikelet meristems. One of these is the group of terminal floral meristem identity genes. A grass-specific LEAFY HULL STERILE1 (LHS1) clade in the SEPALLATA (SEP) subfamily belongs to this class. LHS1-like genes were found to be expressed only in the terminal floral meristem in species with spikelet determinacy, which suggested that they exclusively determine the production of the terminal floral meristem, by which the spikelet meristem acquires determinacy (Cacharroón et al., 1999; Malcomber and Kellogg, 2004; Zahn et al., 2005). The other class comprises the INDETERMINATE SPIKELET1 (IDS1)-like genes, which belong to the APETALA2/ethylene-responsive factor (AP2/ERF) family. Unlike LHS1-like genes, this class of genes regulates the correct timing of the transition of the spikelet meristem to the floral meristem but does not specify the identity of the terminal floral meristem. In maize (Zea mays), loss of IDS1 function produces extra florets (Chuck et al., 1998). In addition, mutation of SISTER OF IDS1 (SID1), a paralog of IDS1 in maize, resulted in no defects in terms of spikelet development. However, the ids1+sid1 double mutant failed to generate floral organs and instead developed more bract-like structures than are found in wild-type plants (Chuck et al., 2008). The rice genome contains two IDS1-like genes, SUPERNUMERARY BRACT (SNB) and OsIDS1. Loss of activity of SNB or OsIDS1 produced extra rudimentary glumes, and snb+osids1 double mutant plants developed more rudimentary glumes than either of its parental mutants (Lee et al., 2007; Lee and An, 2012). These results revealed that the mutated IDS1-like genes prolonged the activity of the spikelet meristem.In most members of Oryzeae, the spikelet is distinct from those of other grasses, in that it comprises a pair of rudimentary glumes, a pair of sterile lemmas (empty glumes), and one floret (Schmidt and Ambrose, 1998; Ambrose et al., 2000; Kellogg, 2009; Hong et al., 2010). The rudimentary glumes are generally regarded as severely reduced bract organs, but the origin of sterile lemmas has been widely debated. Recent studies suggested that the sterile lemmas are the vestigial lemmas of two lateral florets. The gene LONG STERILE LEMMA (G1)/ELONGATED EMPTY GLUME1 (ELE1) is a member of a plant-specific gene family. In the g1/ele1 mutant, sterile lemmas were found to be homeotically transformed into lemmas (Yoshida et al., 2009; Hong et al., 2010). The OsMADS34 and EXTRA GLUME1 (EG1) genes were also shown to determine the identities of sterile lemmas. In the osmads34 and eg1 mutants, the sterile lemmas were enlarged and acquired the identities of lemmas (Li et al., 2009; Gao et al., 2010; Kobayashi et al., 2010). Additionally, the SEP-like gene LHS1/OsMADS1, which specifies the identities of both the lemma and the palea, was not expressed in sterile lemmas, and ectopic expression in sterile lemmas resulted in the transformation of sterile lemmas to lemmas (Jeon et al., 2000; Li et al., 2009; Tanaka et al., 2012). These findings suggest that the sterile lemma may be homologous to the lemma. Nevertheless, some researchers still considered that the sterile lemmas are instead vestigial bract-like structures similar to the rudimentary glumes (Schmidt and Ambrose, 1998; Kellogg, 2009; Hong et al., 2010).In this study, we isolated the rice MULTI-FLORET SPIKELET1 (MFS1) gene, which belongs to a clade of unknown function in the AP2/ERF gene family. The mutation of MFS1 was shown to delay the transformation of the spikelet meristem to the floral meristem and to result in degeneration of the sterile lemma and palea. These results suggest that MFS1 plays an important role in the regulation of spikelet determinacy and organ identity. Our findings also reveal that MFS1 positively regulates the expression of G1 and the IDS1-like genes SNB and OsIDS1.