The Small GTPase OsRac1 Forms Two Distinct Immune Receptor Complexes Containing the PRR OsCERK1 and the NLR Pit.

Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we revealed that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane (PM). Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the PM and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity.

Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice.

Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.

TFL1-Like Proteins in Rice Antagonize Rice FT-Like Protein in Inflorescence Development by Competition for Complex Formation with 14-3-3 and FD.

Hd3a, a rice homolog of FLOWERING LOCUS T (FT), is a florigen that induces flowering. Hd3a forms a ternary 'florigen activation complex' (FAC) with 14-3-3 protein and OsFD1 transcription factor, a rice homolog of FD that induces transcription of OsMADS15, a rice homolog of APETALA1 (AP1), which leads to flowering. TERMINAL FLOWER 1 (TFL1) represses flowering and controls inflorescence architecture. However, the molecular basis for floral repression by TFL1 remains poorly understood. Here we show that RICE CENTRORADIALIS (RCN), rice TFL1-like proteins, compete with Hd3a for 14-3-3 binding. All four RCN genes are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex to antagonize florigen activity and regulate inflorescence development. The antagonistic function of RCN to Hd3a is dependent on its 14-3-3 binding activity. Our results suggest a molecular basis for regulation of the balance between florigen FT and anti-florigen TFL1.

The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance.

Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

FT-like proteins induce transposon silencing in the shoot apex during floral induction in rice.

Floral induction is a crucial developmental step in higher plants. Florigen, a mobile floral activator that is synthesized in the leaf and transported to the shoot apex, was recently identified as a protein encoded by FLOWERING LOCUS T (FT) and its orthologs; the rice florigen is Heading date 3a (Hd3a) protein. The 14-3-3 proteins mediate the interaction of Hd3a with the transcription factor OsFD1 to form a ternary structure called the florigen activation complex on the promoter of OsMADS15, a rice APETALA1 ortholog. However, crucial information, including the spatiotemporal overlap among FT-like proteins and the components of florigen activation complex and downstream genes, remains unclear. Here, we confirm that Hd3a coexists, in the same regions of the rice shoot apex, with the other components of the florigen activation complex and its transcriptional targets. Unexpectedly, however, RNA-sequencing analysis of shoot apex from wild-type and RNA-interference plants depleted of florigen activity revealed that 4,379 transposable elements (TEs; 58% of all classifiable rice TEs) were expressed collectively in the vegetative and reproductive shoot apex. Furthermore, in the reproductive shoot apex, 214 TEs were silenced by florigen. Our results suggest a link between floral induction and regulation of TEs.

Dual role of tree florigen activation complex component FD in photoperiodic growth control and adaptive response pathways.

A complex consisting of evolutionarily conserved FD, flowering locus T (FT) proteins is a regulator of floral transition. Intriguingly, FT orthologs are also implicated in developmental transitions distinct from flowering, such as photoperiodic control of bulbing in onions, potato tuberization, and growth cessation in trees. However, whether an FT-FD complex participates in these transitions and, if so, its mode of action, are unknown. We identified two closely related FD homologs, FD-like 1 (FDL1) and FD-like 2 (FDL2), in the model tree hybrid aspen. Using gain of function and RNAi-suppressed FDL1 and FDL2 transgenic plants, we show that FDL1 and FDL2 have distinct functions and a complex consisting of FT and FDL1 mediates in photoperiodic control of seasonal growth. The downstream target of the FT-FD complex in photoperiodic control of growth is Like AP1 (LAP1), a tree ortholog of the floral meristem identity gene APETALA1. Intriguingly, FDL1 also participates in the transcriptional control of adaptive response and bud maturation pathways, independent of its interaction with FT, presumably via interaction with abscisic acid insensitive 3 (ABI3) transcription factor, a component of abscisic acid (ABA) signaling. Our data reveal that in contrast to its primary role in flowering, FD has dual roles in the photoperiodic control of seasonal growth and stress tolerance in trees. Thus, the functions of FT and FD have diversified during evolution, and FD homologs have acquired roles that are independent of their interaction with FT.

Potato Tuber Induction is Regulated by Interactions Between Components of a Tuberigen Complex.

Photoperiod-regulated flowering and potato tuber formation involve leaf-produced mobile signals, florigen and tuberigen, respectively. The major protein component of florigen has been identified as the FLOWERING LOCUS T (FT) protein. In rice, an FT-like protein, Heading date 3a (Hd3a), induces flowering by making the florigen activation complex (FAC) through interactions with 14-3-3 and OsFD1, a rice FD-like protein. In potato, StSP6A, an FT-like protein, was identified as a major component of tuberigen. However, the molecular mechanism of how StSP6A triggers tuber formation remains elusive. Here we analyzed the significance of the formation of a complex including StSP6A, 14-3-3 and FD-like proteins in tuberization. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays showed that StSP6A and StFDL1, a potato FD-like protein, interact with St14-3-3s. StSP6A overexpression induced early tuberization in a 14-3-3-dependent manner, and suppression of StFDL1 delayed tuberization. These results strongly suggest that an FAC-like complex, the tuberigen activation complex (TAC), comprised of StSP6A, St14-3-3s and StFDL1, regulates potato tuber formation.

Control of flowering and storage organ formation in potato by FLOWERING LOCUS T.

Seasonal fluctuations in day length regulate important aspects of plant development such as the flowering transition or, in potato (Solanum tuberosum), the formation of tubers. Day length is sensed by the leaves, which produce a mobile signal transported to the shoot apex or underground stems to induce a flowering transition or, respectively, a tuberization transition. Work in Arabidopsis, tomato and rice (Oryza sativa) identified the mobile FLOWERING LOCUS T (FT) protein as a main component of the long-range 'florigen', or flowering hormone, signal. Here we show that expression of the Hd3a gene, the FT orthologue in rice, induces strict short-day potato types to tuberize in long days. Tuber induction is graft transmissible and the Hd3a-GFP protein is detected in the stolons of grafted plants, transport of the fusion protein thus correlating with tuber formation. We provide evidence showing that the potato floral and tuberization transitions are controlled by two different FT-like paralogues (StSP3D and StSP6A) that respond to independent environmental cues, and show that an autorelay mechanism involving CONSTANS modulates expression of the tuberization-control StSP6A gene.

14-3-3 proteins act as intracellular receptors for rice Hd3a florigen.

'Florigen' was proposed 75 years ago to be synthesized in the leaf and transported to the shoot apex, where it induces flowering. Only recently have genetic and biochemical studies established that florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved in higher plants. Nonetheless, the exact function of florigen during floral induction remains poorly understood and receptors for florigen have not been identified. Here we show that the rice FT homologue Hd3a interacts with 14-3-3 proteins in the apical cells of shoots, yielding a complex that translocates to the nucleus and binds to the Oryza sativa (Os)FD1 transcription factor, a rice homologue of Arabidopsis thaliana FD. The resultant ternary 'florigen activation complex' (FAC) induces transcription of OsMADS15, a homologue of A. thaliana APETALA1 (AP1), which leads to flowering. We have determined the 2.4 Å crystal structure of rice FAC, which provides a mechanistic basis for florigen function in flowering. Our results indicate that 14-3-3 proteins act as intracellular receptors for florigen in shoot apical cells, and offer new approaches to manipulate flowering in various crops and trees.

Hd3a promotes lateral branching in rice.

Accumulating evidence indicates that the FLOWERING LOCUS T (FT) protein is the mobile floral signal known as florigen. A rice FT homolog, Heading date 3a (Hd3a), is transported from the phloem to shoot apical cells, where it interacts with 14-3-3 proteins and transcription factor OsFD1 to form a florigen activation complex (FAC) that activates a rice homolog of the floral identity gene APETALA1. Recent studies showed that florigen has roles in plant development beyond flowering; however, the exact nature of these roles is not well understood. It is not clear whether FT is transported to organs outside the shoot apex, and whether FAC formation is required for processes other than flowering. We show here that the Hd3a protein accumulates in axillary meristems to promote branching, and that FAC formation is required. Analysis of transgenic plants revealed that Hd3a promotes branching through lateral bud outgrowth. Hd3a protein produced in the phloem reached the axillary meristem in the lateral bud, and its transport was required for promotion of branching. Moreover, mutant Hd3a proteins defective in FAC formation but competent with respect to transport did not promote branching. Finally, we show that Hd3a promotes branching independently from strigolactone and FC1, a transcription factor that inhibits branching in rice. Together, these results suggest that Hd3a functions as a mobile signal for branching in rice.

Heterotrimeric G proteins control stem cell proliferation through CLAVATA signaling in Arabidopsis.

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gß mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.

Crosstalk of Signaling Mechanisms Involved in Host Defense and Symbiosis Against Microorganisms in Rice.

Rice is one of the most important food crops, feeding about half population in the world. Rice pathogens cause enormous damage to rice production worldwide. In plant immunity research, considerable progress has recently been made in our understanding of the molecular mechanisms underlying microbe-associated molecular pattern (MAMP)-triggered immunity. Using genome sequencing and molecular techniques, a number of new MAMPs and their receptors have been identified in the past two decades. Notably, the mechanisms for chitin perception via the lysine motif (LysM) domain-containing receptor OsCERK1, as well as the mechanisms for bacterial MAMP (e.g. flg22, elf18) perception via the leucine-rich repeat (LRR) domain-containing receptors FLS2 and EFR, have been clarified in rice and Arabidopsis, respectively. In chitin signaling in rice, two direct substrates of OsCERK1, Rac/ROP GTPase guanine nucleotide exchange factor OsRacGEF1 and receptor-like cytoplasmic kinase OsRLCK185, have been identified as components of the OsCERK1 complex and are rapidly phosphorylated by OsCERK1 in response to chitin. Interestingly, OsCERK1 also participates in symbiosis with arbuscular mycorrhizal fungi (AMF) in rice and plays a role in the recognition of short-chitin molecules (CO4/5), which are symbiotic signatures included in AMF germinated spore exudates and induced by synthetic strigolactone. Thus, OsCERK1 contributes to both immunity and symbiotic responses. In this review, we describe recent studies on pathways involved in rice immunity and symbiotic signaling triggered by interactions with microorganisms. In addition, we describe recent advances in genetic engineering by using plant immune receptors and symbiotic microorganisms to enhance disease resistance of rice.

Palmitoylation-dependent membrane localization of the rice resistance protein pit is critical for the activation of the small GTPase OsRac1.

Nucleotide binding domain and leucine-rich repeat (NLR)-containing family proteins function as intracellular immune sensors in both plants and animals. In plants, the downstream components activated by NLR family proteins and the immune response mechanisms induced by these downstream molecules are largely unknown. We have previously found that the small GTPase OsRac1, which acts as a molecular switch in rice immunity, is activated by Pit, an NLR-type resistance (R) protein to rice blast fungus, and this activation plays critical roles in Pit-mediated immunity. However, the sites and mechanisms of activation of Pit in vivo remain unknown. To clarify the mechanisms involved in the localization of Pit, we searched for consensus sequences in Pit that specify membrane localization and found a pair of potential palmitoylation sites in the N-terminal coiled-coil region. Although wild-type Pit was localized mainly to the plasma membrane, this membrane localization was compromised in a palmitoylation-deficient mutant of Pit. The palmitoylation-deficient Pit displayed significantly lower affinity for OsRac1 on the plasma membrane, thereby resulting in failures of the Pit-mediated cell death, the production of reactive oxygen species, and disease resistance to rice blast fungus. These results indicate that palmitoylation-dependent membrane localization of Pit is required for the interaction with and the activation of OsRac1 and that OsRac1 activation by Pit is vital for Pit-mediated disease resistance to rice blast fungus.

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Plasma membrane localization is essential for Oryza sativa Pto-interacting protein 1a-mediated negative regulation of immune signaling in rice.

Oryza sativa Pto-interacting protein 1a (OsPti1a), an ortholog of tomato (Solanum lycopersicum) SlPti1, functions as a negative regulator of innate immunity in rice (Oryza sativa). In ospti1a mutants, the activation of immune responses, including hypersensitive response-like cell death, is caused by loss of the OsPti1a protein; however, it is as yet unclear how OsPti1a suppresses immune responses. Here, we report that OsPti1a localizes to detergent-resistant membrane fractions of the plasma membrane through lipid modification of the protein's amino terminus, which is highly conserved among Pti1 orthologs in several plant species. Importantly, mislocalization of OsPti1a after deletion of its amino terminus reduced its ability to complement the mutant phenotypes, including hypersensitive response-like cell death. Furthermore, complex formation of OsPti1a depends on its amino terminus-mediated membrane localization. Liquid chromatography-tandem mass spectrometry analysis of OsPti1a complex-interacting proteins identified several defense-related proteins. Collectively, these findings indicate that appropriate complex formation by OsPti1a at the plasma membrane is required for the negative regulation of plant immune responses in rice.

Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes.

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function.

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice.

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.

Functional diversification of FD transcription factors in rice, components of florigen activation complexes.

Florigen, a protein encoded by the FLOWERING LOCUS T (FT) in Arabidopsis and Heading date 3a (Hd3a) in rice, is the universal flowering hormone in plants. Florigen is transported from leaves to the shoot apical meristem and initiates floral evocation. In shoot apical cells, conserved cytoplasmic 14-3-3 proteins act as florigen receptors. A hexameric florigen activation complex (FAC) composed of Hd3a, 14-3-3 proteins, and OsFD1, a transcription factor, activates OsMADS15, a rice homolog of Arabidopsis APETALA1, leading to flowering. Because FD is a key component of the FAC, we characterized the FD gene family and their functions. Phylogenetic analysis of FD genes indicated that this family is divided into two groups: (i) canonical FD genes that are conserved among eudicots and non-Poaceae monocots; and (ii) Poaceae-specific FD genes that are organized into three subgroups: Poaceae FD1, FD2 and FD3. The Poaceae FD1 group shares a small sequence motif, T(A/V)LSLNS, with FDs of eudicots and non-Poaceae monocots. Overexpression of OsFD2, a member of the Poaceae FD2 group, produced smaller leaves with shorter plastochrons, suggesting that OsFD2 controls leaf development. In vivo subcellular localization of Hd3a, 14-3-3 and OsFD2 suggested that in contrast to OsFD1, OsFD2 is restricted to the cytoplasm through its interaction with the cytoplasmic 14-3-3 proteins, and interaction of Hd3a with 14-3-3 facilitates nuclear translocation of the FAC containing OsFD2. These results suggest that FD function has diverged between OsFD1 and OsFD2, but formation of a FAC is essential for their function.