A leaf-emanated signal orchestrates grain size and number in response to maternal resources.

In plants, variations in seed size and number are outcomes of different reproductive strategies. Both traits are often environmentally influenced, suggesting that a mechanism exists to coordinate these phenotypes in response to available maternal resources. Yet, how maternal resources are sensed and influence seed size and number is largely unknown. Here, we report a mechanism that senses maternal resources and coordinates grain size and number in the wild rice Oryza rufipogon, a wild progenitor of Asian cultivated rice. We showed that FT-like 9 (FTL9) regulates both grain size and number and that maternal photosynthetic assimilates induce FTL9 expression in leaves to act as a long-range signal that increases grain number and reduces size. Our findings highlight a strategy that benefits wild plants to survive in a fluctuating environment. In this strategy, when maternal resources are sufficient, wild plants increase their offspring number while preventing an increase in offspring size by the action of FTL9, which helps expand their habitats. In addition, we found that a loss-of-function allele (ftl9) is prevalent among wild and cultivated populations, offering a new scenario in the history of rice domestication.

Omics Profiles of Non-GM Tubers from Transgrafted Potato with a GM Scion.

"Transgrafting" is a grafting procedure whereby a transgenic plant body is grafted to a non-transgenic plant body. It is a novel plant breeding technology that allows non-transgenic plants to obtain benefits usually conferred to transgenic plants. Many plants regulate flowering by perceiving the day-length cycle via expression of FLOWERING LOCUS T (FT) in the leaves. The resulting FT protein is translocated to the shoot apical meristem via the phloem. In potato plants, FT is involved in the promotion of tuber formation. Here we investigated the effects of a genetically modified (GM) scion on the edible parts of the non-GM rootstock by using potato plants transformed with StSP6A, a novel potato homolog of the FT gene. Scions prepared from GM or control (wild-type) potato plants were grafted to non-GM potato rootstocks; these were designated as TN and NN plants, respectively. After tuber harvest, we observed no significant differences in potato yield between TN and NN plants. Transcriptomic analysis revealed that only one gene-with unknown function-was differentially expressed between TN and NN plants. Subsequent proteomic analysis indicated that several members of protease inhibitor families, known as anti-nutritional factors in potato, were slightly more abundant in TN plants. Metabolomic analysis revealed a slight increase in metabolite abundance in NN plants, but we observed no difference in the accumulation of steroid glycoalkaloids, toxic metabolites found in potato. Finally, we found that TN and NN plants did not differ in nutrient composition. Taken together, these results indicate that FT expression in scions had a limited effect on the metabolism of non-transgenic potato tubers.

Multifunctional chemical inhibitors of the florigen activation complex discovered by structure-based high-throughput screening.

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.

Crystal structure of potato 14-3-3 protein St14f revealed the importance of helix I in StFDL1 recognition.

In potato (Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.

Characterization of Frond and Flower Development and Identification of FT and FD Genes From Duckweed Lemna aequinoctialis Nd.

Duckweeds (Araceae: Lemnoideae) are aquatic monocotyledonous plants that are characterized by their small size, rapid growth, and wide distribution. Developmental processes regulating the formation of their small leaf-like structures, called fronds, and tiny flowers are not well characterized. In many plant species, flowering is promoted by the florigen activation complex, whose major components are florigen FLOWERING LOCUS T (FT) protein and transcription factor FD protein. How this complex is regulated at the molecular level during duckweed flowering is also not well understood. In this study, we characterized the course of developmental changes during frond development and flower formation in Lemna aequinoctialis Nd, a short-day plant. Detailed observations of frond and flower development revealed that cell proliferation in the early stages of frond development is active as can be seen in the separate regions corresponding to two budding pouches in the proximal region of the mother frond. L. aequinoctialis produces two stamens of different lengths with the longer stamen growing more rapidly. Using high-throughput RNA sequencing (RNA-seq) and de novo assembly of transcripts from plants induced to flower, we identified the L. aequinoctialis FT and FD genes, whose products in other angiosperms form a transcriptional complex to promote flowering. We characterized the protein-protein interaction of duckweed FT and FD in yeast and examined the functions of the two gene products by overexpression in Arabidopsis. We found that L. aequinoctialis FTL1 promotes flowering, whereas FTL2 suppresses flowering.

Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein, NanoLuc.

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice.

The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.

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.

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.

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.

Florigen signaling.

Florigen is a systemic signal that promotes flowering. Its molecular nature is a conserved FLOWERING LOCUS T (FT) protein that belongs to the PEBP family. FT is expressed in the leaf phloem and transported to the shoot apical meristem where it initiates floral transition. In the cells of the meristem, FT binds 14-3-3 proteins and bZIP transcription factor FD to form the florigen activation complex, FAC, which activates floral meristem identity genes such as AP1. The FAC model provides molecular basis for multiple functions of FT beyond flowering through changes of its partners and transcriptional targets. The surface of FT protein includes several regions essential for transport and functions, suggesting the binding of additional components that support its function. FT expression is under photoperiodic control, involving a conserved GIGANTEA-CONSTANS-FT regulatory module with species-specific modifications that contribute variations of flowering time in natural populations.

Florigen in rice: complex gene network for florigen transcription, florigen activation complex, and multiple functions.

Regulation of flowering time directly influences successful rice grain production; thus, the long history of domestication and breeding has improved the genetic network of flowering. Recent advances using molecular genomic approaches have revealed the targets of these modifications and the underlying molecular mechanism for flowering. These efforts contributed to identifying the molecular nature of the systemic floral signal 'florigen' and have shown how florigen functions, how florigen expression is controlled, and how regulatory pathways are diversified. In this review, we summarize the advances in our understanding of the detailed molecular and genetic mechanisms that allow rice plants to produce flowers at the proper time to ensure grain production.

Structure and function of florigen and the receptor complex.

In the 1930s, the flowering hormone, florigen, was proposed to be synthesized in leaves under inductive day length and transported to the shoot apex, where it induces flowering. More recently, generated genetic and biochemical data suggest that florigen is a protein encoded by the gene, FLOWERING LOCUS T (FT). A rice (Oryza sativa) FT homolog, Hd3a, interacts with the rice FD homolog, OsFD1, via a 14-3-3 protein. Formation of this tri-protein complex is essential for flowering promotion by Hd3a in rice. In addition, the multifunctionality of FT homologs, other than for flowering promotion, is an emerging concept. Here we review the structural and biochemical features of the florigen protein complex and discuss the molecular basis for the multifunctionality of FT proteins.

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.

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.

Regulation of flowering in rice: two florigen genes, a complex gene network, and natural variation.

Photoperiodic control of flowering time consists of a complicated network that converges into the generation of a mobile flowering signal called florigen. Recent advances identifying the protein FT/Hd3a as the molecular nature responsible for florigen activity have focused current research on florigen genes as the important output of this complex signaling network. Rice is a model system for short-day plants and recent progress in elucidating the flowering network from rice and Arabidopsis, a long-day plant, provides an evolutionarily comparative view of the photoperiodic flowering pathway. This review summarizes photoperiodic flowering control in rice, including the interaction of complex layers of gene networks contributed from evolutionarily unique factors and the regulatory adaptation of conserved factors.

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

FLOWERING LOCUS T protein may act as the long-distance florigenic signal in the cucurbits.

Cucurbita moschata, a cucurbit species responsive to inductive short-day (SD) photoperiods, and Zucchini yellow mosaic virus (ZYMV) were used to test whether long-distance movement of FLOWERING LOCUS T (FT) mRNA or FT is required for floral induction. Ectopic expression of FT by ZYMV was highly effective in mediating floral induction of long-day (LD)-treated plants. Moreover, the infection zone of ZYMV was far removed from floral meristems, suggesting that FT transcripts do not function as the florigenic signal in this system. Heterografting demonstrated efficient transmission of a florigenic signal from flowering Cucurbita maxima stocks to LD-grown C. moschata scions. Real-time RT-PCR performed on phloem sap collected from C. maxima stocks detected no FT transcripts, whereas mass spectrometry of phloem sap proteins revealed the presence of Cm-FTL1 and Cm-FTL2. Importantly, studies on LD- and SD-treated C. moschata plants established that Cmo-FTL1 and Cmo-FTL2 are regulated by photoperiod at the level of movement into the phloem and not by transcription. Finally, mass spectrometry of florally induced heterografted C. moschata scions revealed that C. maxima FT, but not FT mRNA, crossed the graft union in the phloem translocation stream. Collectively, these studies are consistent with FT functioning as a component of the florigenic signaling system in the cucurbits.

Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation.

Overall shoot architecture in higher plants is highly dependent on the activity of embryonic and axillary shoot meristems, which are produced from the basal adaxial boundaries of cotyledons and leaves, respectively. In Arabidopsis thaliana, redundant functions of the CUP-SHAPED COTYLEDON genes CUC1, CUC2, and CUC3 regulate embryonic shoot meristem formation and cotyledon boundary specification. Their functional importance and relationship in postembryonic development, however, is poorly understood. Here, we performed extensive analyses of the embryonic and postembryonic functions of the three CUC genes using multiple combinations of newly isolated mutant alleles. We found significant roles of CUC2 and CUC3, but not CUC1, in axillary meristem formation and boundary specification of various postembryonic shoot organs, such as leaves, stems, and pedicels. In embryogenesis, all three genes make significant contributions, although CUC3 appears to possess, at least partially, a distinct function from that of CUC1 and CUC2. The function of CUC3 and CUC2 overlaps that of LATERAL SUPPRESSOR, which was previously shown to be required for axillary meristem formation. Our results reveal that redundant but partially distinct functions of CUC1, CUC2, and CUC3 are responsible for shoot organ boundary and meristem formation throughout the life cycle in Arabidopsis.