Flower meristem maintenance by TILLERS ABSENT 1 is essential for ovule development in rice.

Plant development depends on the activity of pluripotent stem cells in meristems, such as the shoot apical meristem and the flower meristem. In Arabidopsis thaliana, WUSCHEL (WUS) is essential for stem cell homeostasis in meristems and integument differentiation in ovule development. In rice (Oryza sativa), the WUS ortholog TILLERS ABSENT 1 (TAB1) promotes stem cell fate in axillary meristem development, but its function is unrelated to shoot apical meristem maintenance in vegetative development. In this study, we examined the role of TAB1 in flower development. The ovule, which originates directly from the flower meristem, failed to differentiate in tab1 mutants, suggesting that TAB1 is required for ovule formation. Expression of a stem cell marker was completely absent in the flower meristem at the ovule initiation stage, indicating that TAB1 is essential for stem cell maintenance in the 'final' flower meristem. The ovule defect in tab1 was partially rescued by floral organ number 2 mutation, which causes overproliferation of stem cells. Collectively, it is likely that TAB1 promotes ovule formation by maintaining stem cells at a later stage of flower development.

CURLED LATER1 encoding the largest subunit of the Elongator complex has a unique role in leaf development and meristem function in rice.

The Elongator complex, which is conserved in eukaryotes, has multiple roles in diverse organisms. In Arabidopsis thaliana, Elongator is shown to be involved in development, hormone action and environmental responses. However, except for Arabidopsis, our knowledge of its function is poor in plants. In this study, we initially carried out a genetic analysis to characterize a rice mutant with narrow and curled leaves, termed curled later1 (cur1). The cur1 mutant displayed a heteroblastic change, whereby the mutant leaf phenotype appeared specifically at a later adult phase of vegetative development. The shoot apical meristem (SAM) was small and the leaf initiation rate was low, suggesting that the activity of the SAM seemed to be partially reduced in cur1. We then revealed that CUR1 encodes a yeast ELP1-like protein, the largest subunit of Elongator. Furthermore, disruption of OsELP3 encoding the catalytic subunit of Elongator resulted in phenotypes similar to those of cur1, including the timing of the appearance of mutant phenotypes. Thus, Elongator activity seems to be specifically required for leaf development at the late vegetative phase. Transcriptome analysis showed that genes involved in protein quality control were highly upregulated in the cur1 shoot apex at the later vegetative phase, suggesting the restoration of impaired proteins probably produced by partial defects in translational control due to the loss of function of Elongator. The differences in the mutant phenotype and gene expression profile between CUR1 and its Arabidopsis ortholog suggest that Elongator has evolved to play a unique role in rice development.

Antagonistic action of TILLERS ABSENT1 and FLORAL ORGAN NUMBER2 regulates stem cell maintenance during axillary meristem development in rice.

Shoot branches are formed from the axillary meristem and their formation is a key process in plant development. Although our understanding of the mechanisms underlying stem cell maintenance in the shoot apical meristem (SAM) is progressing, our knowledge of these mechanisms during the process of axillary meristem development is insufficient. To elucidate the genetic mechanisms underlying axillary meristem development in rice (Oryza sativa), we undertook a molecular genetic analysis focusing on TILLERS ABSENT1 (TAB1) and FLORAL ORGAN NUMBER2 (FON2), respective orthologs of the WUSCHEL and CLAVATA3 genes involved in SAM maintenance in Arabidopsis (Arabidopsis thaliana). We revealed that stem cells were established at an early stage of axillary meristem development in the wild-type, but were not maintained in tab1. By contrast, the stem cell region and TAB1 expression domain were expanded in fon2, and FON2 overexpression inhibited axillary meristem formation. These results indicate that TAB1 is required to maintain stem cells during axillary meristem development, whereas FON2 negatively regulates stem cell fate by restricting TAB1 expression. Thus, the genetic pathway regulating SAM maintenance in Arabidopsis seems to have been recruited to play a specific role within a narrow developmental window - namely, axillary meristem establishment - in rice.

DWARF WITH SLENDER LEAF1 Encoding a Histone Deacetylase Plays Diverse Roles in Rice Development.

In plants, reversible histone acetylation and deacetylation play a crucial role in various biological activities, including development and the response to environmental stress. Histone deacetylation, which is generally associated with gene silencing, is catalyzed by multiple histone deacetylases (HDACs). Our understanding of HDAC function in plant development has accumulated from molecular genetic studies in Arabidopsis thaliana. By contrast, how HDACs contribute to the development of rice (Oryza sativa) is poorly understood and no rice mutants of HDAC have been reported. Here we have characterized a new rice mutant showing semi-dwarfism, which we named dwarf with slender leaf1 (dsl1). The mutant showed pleiotropic defects in both vegetative and reproductive developments; e.g. dsl1 produced short and narrow leaves, accompanied by a reduction in the number and size of vascular bundles. The semi-dwarf phenotype was due to suppression of the elongation of some culm (stem) internodes. Interestingly, despite this suppression of the upper internodes, the elongation and generation of lower internodes were slightly enhanced. Inflorescence and spikelet development were also affected by the dsl1 mutation. Some of the observed morphological defects were related to a reduction in cell numbers, in addition to reduced cell division in leaf primordia revealed by in situ hybridization analysis, suggesting the possibility that DSL1 is involved in cell division control. Gene cloning revealed that DSL1 encodes an HDAC belonging to the reduced potassium dependence3/histone deacetylase1 family. Collectively, our study shows that the HDAC DSL1 plays diverse and important roles in development in rice.

TILLERS ABSENT1, the WUSCHEL ortholog, is not involved in stem cell maintenance in the shoot apical meristem in rice.

Stem cell maintenance in the shoot apical meristem (SAM) is very important for plant development and is regulated by the WUSCHEL-CLAVATA (WUS-CLV) feedback loop in Arabidopsis (Arabidopsis thaliana). WUS promotes stem cell identity, whereas CLV negatively regulates stem cell proliferation by repressing WUS expression. We previously showed that, in rice (Oryza sativa), the WUS ortholog TILLERS ABSENT1 (TAB1, also known as OsWUS) has no function in SAM maintenance, whereas it plays a crucial role in axillary meristem development. Recently, we showed that a double mutant of FLORAL ORGAN NUMBER2 (FON2) and ABERRANT SPIKELET AND PANICLE1 (ASP1) led to a marked enlargement of the inflorescence meristem, and that the TAB1 function is not associated with massive stem cells in this meristem. In this paper, we confirmed that TAB1 is also unrelated to the enlargement of the SAM in the vegetative phase of the fon2 and fon2 asp1 mutants. In addition, misexpression of TAB1 under the promoter of FON1 led to a slight reduction of the SAM size in wild type, suggesting that TAB1 is not a positive regulator of stem cells. Taking together, TAB1 seems not to be involved in meristem maintenance, irrespective of the meristem type.

Transcriptional Corepressor ASP1 and CLV-Like Signaling Regulate Meristem Maintenance in Rice.

Stem cell homeostasis is maintained by the WUSCHEL-CLAVATA (WUS-CLV) negative feedback loop in Arabidopsis (Arabidopsis thaliana). In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2) functions in the negative regulation of stem cell proliferation, similar to Arabidopsis CLV3 In this study, through genetic enhancer analysis, we found that loss of function of ABERRANT SPIKELET AND PANICLE1 (ASP1), encoding an Arabidopsis TOPLESS (TPL)-like transcriptional corepressor, enhances the fon2 flower phenotype, which displayed an increase in floral organ number. In the fon2 asp1 double mutant, the inflorescence was severely affected, resulting in bifurcation of the main axis (rachis), a phenotype that has not previously been reported. The stem cells showed marked overproliferation in fon2 asp1, resulting in extreme enlargement and splitting of the inflorescence meristem. These results suggest that ASP1 and FON2 synergistically regulate stem cell maintenance in rice. Unexpectedly, genetic analysis indicated that TILLERS ABSENT1, the rice ortholog of WUS, is not involved in promoting stem cell proliferation in this meristem. Transcriptome analysis suggested that ASP1 and FON signaling negatively regulate a set of genes with similar functions, and they act on these genes in concert. Taken together, our results suggest that TPL-like corepressor activity plays a crucial role in meristem maintenance, and that stem cell proliferation is properly maintained via the cooperation of ASP1 and FON2.

Rice Flower Development Revisited: Regulation of Carpel Specification and Flower Meristem Determinacy.

The ABC model in flower development represents a milestone of plant developmental studies and is essentially conserved across a wide range of angiosperm species. Despite this overall conservation, individual genes in the ABC model are not necessarily conserved and sometimes play a species-specific role, depending on the plant. We previously reported that carpels are specified by the YABBY gene DROOPING LEAF (DL) in rice (Oryza sativa), which bears flowers that are distinct from those of eudicots. In contrast, another group reported that carpels are specified by two class C genes, OsMADS3 and OsMADS58. Here, we have addressed this controversial issue by phenotypic characterization of floral homeotic gene mutants. Analysis of a complete loss-of-function mutant of OsMADS3 and OsMADS58 revealed that carpel-like organs expressing DL were formed in the absence of the two class C genes. Furthermore, no known flower organs including carpels were specified in a double mutant of DL and SUPERWOMAN1 (a class B gene), which expresses only class C genes in whorls 3 and 4. These results suggest that, in contrast to Arabidopsis, class C genes are not a key regulator for carpel specification in rice. Instead, they seem to be involved in the elaboration of carpel morphology rather than its specification. Our phenotypic analysis also revealed that, similar to its Arabidopsis ortholog CRABS CLAW, DL plays an important function in regulating flower meristem determinacy in addition to carpel specification.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

Two-Color In Situ Hybridization: A Technique for Simultaneous Detection of Transcripts from Different Loci.

Over the years, in situ hybridization has been used for visualizing spatial gene expression patterns. Direct comparison of the expression patterns of different genes is useful for studying various biological activities in many situations. If we can distinguish signals derived from different probes in the same hybridization reaction, the localization of multiple gene transcripts can be detected simultaneously. In this chapter, we describe a two-color in situ hybridization procedure, which enables us to compare the expression patterns of two genes in a single tissue section. First, we explain how to prepare RNA probes and tissue samples. Then, we explain how to perform an in situ experiment, including pre-hybridization, hybridization, post-hybridization, and detection steps. A stepwise detection procedure is used to obtain the two color signals.

WUSCHEL-RELATED HOMEOBOX4 acts as a key regulator in early leaf development in rice.

Rice (Oryza sativa) has long and narrow leaves with parallel veins, similar to other grasses. Relative to Arabidopsis thaliana which has oval-shaped leaves, our understanding of the mechanism of leaf development is insufficient in grasses. In this study, we show that OsWOX4, a member of the WUSCHEL-RELATED HOMEOBOX gene family, plays important roles in early leaf development in rice. Inducible downregulation of OsWOX4 resulted in severe defects in leaf development, such as an arrest of vascular differentiation, a partial defect in the early cell proliferation required for midrib formation, and a failure to maintain cellular activity in general parenchyma cells. In situ analysis showed that knockdown of OsWOX4 reduced the expression of two LONELY GUY genes, which function in the synthesis of active cytokinin, in developing vascular bundles. Consistent with this, cytokinin levels were downregulated by OsWOX4 knockdown. Transcriptome analysis further showed that OsWOX4 regulates multiple genes, including those responsible for cell cycle progression and hormone action, consistent with the effects of OsWOX4 downregulation on leaf phenotypes. Collectively, these results suggest that OsWOX4 acts as a key regulator at an early stage of leaf development. Our previous work revealed that OsWOX4 is involved in the maintenance of shoot apical meristem in rice, whereas AtWOX4 is specifically associated with the maintenance of vascular stem cells in Arabidopsis. Thus, the function of the two orthologous genes seems to be diversified between rice and Arabidopsis.

Characterization of a half-pipe-like leaf1 mutant that exhibits a curled leaf phenotype.

Leaf forms are diverse in angiosperms, and different types of cells are differentiated depending on the species. Rice leaves are composed of a leaf blade, a leaf sheath and the junction region between them. Cells with characteristic features, such as bulliform cells and sclerenchyma cells, are differentiated in the leaf blade, together with standard epidermal and mesophyll cells. To understand the genetic mechanism underlying leaf morphogenesis in rice, we focused on a mutant, half-pipe-like leaf1 (hal1), whose leaves are adaxially curled. Histological observation revealed that the bulliform cells, which are responsible for leaf rolling under dry conditions, were small in size and abnormal in shape in a semidominant hal1-d mutant. Bulliform cell files were often ambiguous in semi-transparent hal1-d leaves cleared by the TOMEI method, suggesting that the bulliform cells were undeveloped. Therefore, a reduction in the growth of the bulliform cells seemed to be a major cause of leaf curling in the hal1-d mutant. The hal1-d mutation also affected the size of the leaf blade and the spikelet.

Three TOB1-related YABBY genes are required to maintain proper function of the spikelet and branch meristems in rice.

YABBY genes play important roles in the development of lateral organs such as leaves and floral organs in Angiosperms. However, the function of YABBY genes is poorly understood in monocots. We focused on three rice (Oryza sativa) YABBY genes, TONGARI-BOUSHI (TOB1, TOB2, TOB3), which are closely related to Arabidopsis (Arabidopsis thaliana) FILAMENTOUS FLOWER (FIL). To elucidate the function of these YABBY genes, we employed a reverse genetic approach. TOB genes were expressed in bract and lateral organ primordia, but not in meristems. RNAi knockdown of TOB2 or TOB3 in the tob1 mutant caused abnormal spikelet development. Furthermore, simultaneous knockdown of both TOB2 and TOB3 in tob1 affected not only spikelet, but also inflorescence development. In severe cases, the inflorescences comprised naked branches without spikelets. Analysis of inflorescence development at an early stage showed that the observed phenotypic defects were closely associated with a failure to initiate and maintain reproductive meristems. These results indicate that the TOB genes regulate the maintenance and fate of all reproductive meristems. It is likely that the function of FIL/TOB clade YABBY genes has been conserved between Arabidopsis and rice to maintain the proper function of meristems, even though these genes are expressed in lateral organ primordia.

Genetic Enhancer Analysis Reveals that FLORAL ORGAN NUMBER2 and OsMADS3 Co-operatively Regulate Maintenance and Determinacy of the Flower Meristem in Rice.

Meristems such as the shoot apical meristem and flower meristem (FM) act as a reservoir of stem cells, which reproduce themselves and supply daughter cells for the differentiation of lateral organs. In Oryza sativa (rice), the FLORAL ORGAN NUMBER2 (FON2) gene, which is similar to Arabidopsis CLAVATA3, is involved in meristem maintenance. In fon2 mutants, the numbers of floral organs are increased due to an enlargement of the FM. To identify new factors regulating meristem maintenance in rice, we performed a genetic screening of mutants that enhanced the fon2 mutation, and found a mutant line (2B-424) in which pistil number was dramatically increased. By using a map-based approach and next-generation sequencing, we found that the line 2B-424 had a complete loss-of-function mutation (a large deletion) in OsMADS3, a class C MADS-box gene that is known to be involved in stamen specification. Disruption of OsMADS3 in the fon2 mutant by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology caused a flower phenotype similar to that of 2B-424, confirming that the gene responsible for enhancement of fon2 was OsMADS3. Morphological analysis showed that the fon2 and osmads3 mutations synergistically affected pistil development and FM determinacy. We also found that whorl 3 was duplicated in mature flowers and the FM was enlarged at an early developmental stage in severe osmads3 single mutants. These findings suggest that OsMADS3 is involved not only in FM determinacy in late flower development but also in FM activity in early flower development.

Polar patterning of the spikelet is disrupted in the two opposite lemma mutant in rice.

Angiosperms produce diverse flowers and the pattern of floral symmetry is a major factor for flower diversification. Bilaterally symmetric flowers have evolved multiple times in different angiosperm lineages from radially symmetric ancestors. Whereas most monocots produce radially symmetric flowers, grasses such as rice (Oryza sativa) and maize (Zea mays) generate bilaterally symmetric flowers and spikelets. In this paper, we focused on the two opposite lemma (tol) mutant, which displays a pleiotropic phenotype in the spikelet. Close morphological examination revealed that a typical spikelet phenotype of the tol mutant was principally based on the mirror image duplication of the lemma-side half of the spikelet. Other spikelet phenotypes can be explained as the derivation from the spikelet with this duplication. A polar pattern of organ formation along the lemma-palea axis was disrupted by this duplication. Accordingly, tol mutation seems to change the spikelet from bilateral symmetry (monosymmetry) to disymmetry. Thus, the tol mutant provides good genetic material to investigate the regulation of spikelet symmetry in rice.

Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

Generation of artificial drooping leaf mutants by CRISPR-Cas9 technology in rice.

CRISPR-Cas9 technology, which uses an RNA-guided nuclease, has been developed as an efficient and versatile genome-editing method to induce mutations in genes of interest. To examine the feasibility of this method in developmental studies of a model monocot, rice (Oryza sativa), we introduced the construct gDL-1, which produced a guide RNA targeting the DROOPING LEAF (DL) gene. DL regulates midrib formation in the leaf and carpel specification in the flower. Because loss of function of DL causes the drooping leaf phenotype in regenerated seedlings, the effect of gene disruption should be easily detected. In transgenic plants carrying gDL-1, the DL gene was disrupted at high efficiency: seven out of nine plants examined had bi-allelic mutations. All transgenic plants with the bi-allelic mutation showed the drooping leaf phenotype. Observation of cross sections of the leaf blade clearly indicated that these transgenic plants failed to make midrib structures, and were comparable to the severe dl mutant dl-sup1. Thus, CRISPR-Cas9 technology can be a useful and efficient tool in developmental studies in rice.

Analysis of a rice fickle spikelet1 mutant that displays an increase in flower and spikelet organ number with inconstant expressivity.

In rice (Oryza sativa), floral organs develop in the spikelet, an inflorescence unit unique to grass species. The floral organs, such as carpels, stamens and lodicules, are enclosed by two spikelet organs, the palea and lemma. The number of floral organs is genetically regulated. Mutations in the FLORAL ORGAN NUMBER (FON) genes cause an increase in the number of carpels and stamens due to an enlargement of the floral meristem. The spikelet organs, such as lemma and palea, are less affected in the fon mutants. We found a mutant, fickle spikelet1 (fsp1), that displayed an increased number not only of floral organs but also of spikelet organs. Because the fsp1 spikelets showed a pleiotropic phenotype, we classified them into four types. The expressivity of the fsp1 phenotype varied from plant to plant, and also from panicle to panicle within a single plant. In addition, the frequency of each fsp1 spikelet type also varied considerably among plants and among panicles within a plant. When the fsp1 mutants were grown in a growth chamber, an extra abnormality, namely a defect in pollen development, was observed. Furthermore, the expressivity of the mutant phenotype increased dramatically in mutant plants grown in a growth chamber. Thus, the expressivity of the fsp1 phenotype seems to be strongly influenced by environmental conditions.

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1.

Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.

A role for TRIANGULAR HULL1 in fine-tuning spikelet morphogenesis in rice.

The lemma and palea, which enclose the pistil, stamens, and lodicules, are the most conspicuous organs in the rice spikelet. We isolated a mutant line (ng6569) in which the lemma and palea were narrower than those of the wild type, and found that the mutant had a defect in TRIANGULAR HULL1 (TH1), which encodes a nuclear protein with an ALOG domain. Detailed morphological analysis indicated that the th1 mutation caused a reduction in the size of tubercles, which are convex structures on the surface of the lemma and palea. This reduction was more pronounced in the apical region of the lemma than in the basal region, resulting in the formation of a beak-like spikelet. By contrast, the number of tubercle rows and their spatial distribution on the lemma were not affected in the th1 mutant. Thus, the TH1 gene seems to be involved in fine-tuning the morphogenesis of the lemma and palea. In situ hybridization analysis revealed that TH1 was highly expressed in the primordia of the lemma and palea, but only weakly expressed in the primordia of the sterile lemma and rudimentary glume. We then examined the effect of th1 mutation on the lemma-like structure formed in the long sterile lemma/glume1 (g1) and extra glume1 (eg1) mutants. The result showed that the th1 mutation strongly affected the morphology of the extra lemma of eg1, but had no significant effect on the transformed lemma of g1.

Overexpression analysis suggests that FON2-LIKE CLE PROTEIN1 is involved in rice leaf development.

Peptide signaling plays important roles in various developmental processes of plants. Genes encoding CLE proteins, which are processed into CLE signaling peptides, are required for maintenance of the shoot apical meristem and for vascular differentiation. FON2-LIKE CLE PROTEIN1 (FCP1), a member of the CLE gene family, negatively regulates meristem maintenance in both shoot and root apical meristems of rice (Oryza sativa). Here, we examined the role of FCP1 in leaf development. We found that overexpression of FCP1 affects various aspects of leaf development in shoots regenerated from calli, making it difficult to distinguish between the leaf blade and leaf sheath. Differentiation of tissues such as vascular bundle and sclerenchyma was strongly inhibited by FCP1 overexpression. Spatial expression patterns of developmental genes DROOPING LEAF (DL) and OsPINHEAD1 (OsPNH1) were severely affected in the FCP1-overexpressing shoots. Whereas DL was expressed in the central region of leaf primordia in control shoots, DL expression was expanded throughout the leaf primordia of the FCP1-overexpressing shoots in early developmental stages. By contrast, OsPNH1, which is expressed in provascular and developing vascular tissues in normal seedlings, was strongly repressed by FCP1 overexpression. Taken together, our results suggest that FCP1 is involved in the regulation of cell fate determination during leaf development.