Phylogenetic Analysis of Rice PEBP Genes

The rice genome contains 19 PEBP genes, including 13 FT-like genes (OsFTL1 to OsFTL13), 4 RCN genes (RCN1 to RCN4), and 2 MFT genes (OsMFT1 and OsMFT2). The evolutionary relationship between the 19 PEBP genes in rice and the six PEBP genes in Arabidopsis was investigated (Chardon and Damerval 2005). To clarify the relationship between OsFTL4 and 76 functional PEBP proteins in other species, a neighbor-joining tree was generated after aligning the functionally annotated PEBP proteins from 11 monocotyledonous species (e.g., rice, maize, and onion) and 12 dicotyledonous species (e.g., Arabidopsis and soybean) (Fig. 1). The phylogenetic tree included one TFL1-like clade, one MFT-like clade, and five FT-like clades. Most of the TFL1 homologous proteins in the TFL1-like clade repress flowering. The MFT-like clade contained four MFT proteins that vary in terms of their functions. The FT-like clade Ia comprised OsFTL1, OsFTL2/Hd3a, and OsFTL3/RFT1 from rice as well as AtFT from Arabidopsis, suggesting that the characteristics of this family in plants developed before the dicot–monocot divergence. Furthermore, most of the FT-like genes encode flowering inducers. Although all of the FT-like proteins in FT-like clade Ib are from dicotyledonous species, they have diverse functions. Notably, the FT-like proteins in FT-like clade III, including OsFTL8 and OsFTL10 from rice, ZCN8 and ZCN12 from Zea mays, and HvFT3 from Hordeum vulgare, are all from monocotyledonous species. Although PaFTL1 and PaFTL2 from Picea abies were the only two members of FT-like clade IV in the phylogenetic tree, they delay flowering and appear to be functionally similar to TFL1 (Karlgren et al. 2011). Within clade FT-like II, OsFTL4 and TgFT3, which are highly homologous proteins, were associated with OsFTL5, OsFTL6, OsFTL7, and OsFTL11; however, only TgFT3 has been functionally characterized. The various FT-like proteins from the same species were distributed in different FT-like clusters, indicative of divergence.

Fig. 1
figure 1

Phylogenetic analysis of PEBP family members in Oryza sativa and other plant species. Sequence alignment of OsFTL4 and other FT(-like) and TFL1(-like) proteins in Arabidopsis thaliana, Oryza sativa, Glycine max, Nicotiana tabacum, Helianthus annuus, Beta vulgaris, Tulipa gesneriana, Malus domestica, Hordeum vulgare, Narcissus tazetta, Gentia natriflora, Oncidium ‘Gower Ramsey’, Vitis vinifera, Populus nigra, Zea mays, Picea abies, Allium cepa, Pisum sativum, Ipomoea nil, Ananas comosus, Populus spp., Solanum tuberosum, Lolium perenne, Cymbidium spp., Saccharum spp., and Triticum aestivum

Silencing OsFTL4 Induces Earlier Flowering in Rice

To functionally characterize OsFTL4, which is one of the homologs of Hd3a, OsFTL4 knockout mutants with the Guangluai 4 (GLA 4) genetic background (i.e., an early flowering indica variety) were generated using the CRISPR/Cas9 system. A total of 10 transgenic lines were obtained. Two homozygous mutants (osftl4-1 and osftl4-2) were isolated from two different transformants and confirmed by sequencing. Compared with the wild-type OsFTL4 sequence, the osftl4-1 and osftl4-2 mutant sequences had a 1-bp insertion and a 2-bp deletion in the target site, respectively (Fig. 2a, b). An amino acid sequence alignment revealed that the changes in the osftl4-1 and osftl4-2 sequences were frame-shift mutations that resulted in the introduction of a premature stop codon and the translation of a protein with a truncated PEBP domain (Fig. 2c). The heading date of the two osftl4 mutants was 9.6 and 5.8 days earlier than that of GLA 4 under natural short-day (NSD) and natural long-day (NLD) conditions, respectively (Fig. 2d, j, k). The osftl4-1 and osftl4-2 plants had short panicles (Fig. 2e, l) and a semi-dwarf phenotype at maturity (Fig. 2f, i). Further analyses revealed that the semi-dwarf phenotype of the mutants was due to a decrease in the internode length (Fig. 2g, h). Yield-related parameters were also quantitatively analyzed, including panicle number per plant (PN), grain number per panicle (GN), 1000-grain weight (TGW), and grain yield per plant (GYP) (Fig. 2m–p, Additional file 2: Table S1). The two osftl4 mutants produced more tillers at the tillering stage (Fig. 2d). At maturity, the PN was 29.4% and 38.0% higher for osftl4-1 and osftl4-2, respectively, than for the wild-type control (Fig. 2m). In contrast, the GN of the mutants decreased significantly (Fig. 2n). Additionally, the TGW of osftl4-1 and osftl4-2 decreased by 4.25% and 6.62%, respectively (Fig. 2o). Moreover, there was a significant decrease in the GY of the osftl4-1 and osftl4-2 plants (Fig. 2p). Considered together, these findings suggest that OsFTL4 expression delays flowering and substantially influences multiple agronomic traits in rice.

Fig. 2
figure 2

Knockout of OsFTL4 affects the heading date and other agronomic traits in rice. a A specific target guide RNA located in the first exon was used to edit OsFTL4 via the CRISPR/Cas9 system. The PAM region and guide sequence are marked in red and blue, respectively. The OsFTL4 mutation sites in the two mutants (osftl4-1 and osftl4-2) are indicated. b Mutations were confirmed by sequencing. Mutated bases are marked with red boxes. c OsFTL4 protein structures in GLA 4, osftl4-1, and osftl4-2. The PEBP domain is marked in yellow. d Morphology of GLA 4, osftl4-1, and osftl4-2 plants at heading stage. Scale bars, 10 cm. e Panicle phenotype of GLA 4, osftl4-1, and osftl4-2. f Morphology of GLA 4, osftl4-1, and osftl4-2 plants at maturity satge. Scale bars, 10 cm. g Panicle and internode at maturity. Scale bars, 10 cm. h Length of four internodes below the spike. Heading date for GLA 4, osftl4-1, and osftl4-2 in Lingshui j and Yangzhou k. Comparison between GLA 4 and the mutants in terms of the plant height i, panicle length l, panicle number per plant m, grain number per panicle n, 1,000-grain weight o, and grain yield per plant p. Data are provided as the mean ± SD from 20 individual plants. Significant differences were determined according to Student’s t-test (**, p < 0.01)

OsFTL4 Expression Pattern and Subcellular Localization of the Encoded Protein

To examine the temporal and spatial expression patterns of OsFTL4, we performed a quantitative real-time PCR (qRT-PCR) analysis to examine OsFTL4 expression levels in various tissues collected from GLA 4 plants grown under NLD conditions. The qRT-PCR data indicated that OsFTL4 was expressed in all examined tissues, but especially in the node and sheath (Fig. 3b). The tissue-specific expression was analyzed using OsFTL4 promoter-GUS transgenic plants. Consistent with our qRT-PCR results, the GUS signal was detected in the panicle, node, root, sheath, and leaf blade (Fig. 3c–h).

Fig. 3
figure 3

OsFTL4 expression profile. a Diurnal OsFTL4 expression pattern under SD and LD conditions. b OsFTL4 transcript levels in different tissues. c OsFTL4 promoter-GUS activity was detected in the leaf. d Sheath. e Node. f Stem. g Root. h Mature spikelet before fertilization. Scale bars: c 0.5 mm; e 5 mm; d, fh 1 mm. i Subcellular localization of OsFTL4 in Nicotiana benthamiana leaf epidermal cells and rice protoplasts. Scale bars, 50 μm in N. benthamiana and 20 μm in rice

We also investigated the OsFTL4 expression pattern under SD (10-h light, 28 °C/14-h dark, 26 °C) and LD (14-h light, 28 °C/10-h dark, 26 °C) conditions in an artificial climate chamber. Diurnal rhythms in OsFTL4 transcription were detected, they differed between the controlled short-day (CSD) and controlled long-day (CLD) conditions, and OsFTL4 is upregulated during the light period under CLD and the dark period under CSD (Fig. 3a). Arabidopsis lacks a homolog of Ehd1, which encodes a B-type response regulator. Ehd1Hd3a/RFT1 is a unique flowering pathway in rice, in which Ehd1 induces Hd3a/RFT1 expression under SD and LD conditions to promote flowering (Doi et al. 2004). Ehd1 exhibited a circadian pattern in osftl4 plants under both LD and SD conditions. Additionally, there was no difference in the transcription of OsphyB and OsGI between the wild-type and osftl4 plants, indicating that OsFTL4 is expressed downstream of OsphyB and OsGI (Additional file 1: Fig. S1). Furthermore, OsFTL4 regulates flowering as part of the Ehd1 pathway, possibly via the feedback-regulated expression of Ehd1.

On the basis of the ProtComp online tool (, the OsFTL4 protein was predicted to be localized in both the cytoplasm and nucleus. To verify the predicted localization, the OsFTL4-green fluorescent protein (GFP) fusion construct under the control of the CaMV 35S promoter was generated. The recombinant 35S:: OsFTL4-GFP construct was transiently expressed in Nicotiana benthamiana leaf epidermal cells and rice protoplasts. Confocal microscopy images confirmed that OsFTL4 was localized in the cytoplasm and nucleus (Fig. 3i).

Regulatory Effects of OsFTL4 on the Heading Date

To reveal the possible OsFTL4 genetic network, we compared the expression levels of several rice genes that control flowering. Among these genes, the Sepallata (SEP) gene OsPAP2/OsMADS34 and the three AP1/FUL-like genes OsMADS14, OsMADS15, and OsMADS18 encode florigen signals in the meristem (Preston and Kellogg 2006; Kobayashi et al. 2012). The OsMADS14, OsMADS15, OsMADS18, and OsMADS34 transcription levels were higher in the two osftl4 mutants than in GLA 4 under both LD and SD conditions (Fig. 4). Accordingly, OsFTL4 may delay flowering by downregulating the expression of OsMADS14, OsMADS15, OsMADS18, and OsMADS34.

Fig. 4
figure 4

Comparison of floral meristem identity gene transcription levels of the wild-type and mutant plants. OsMADS14, OsMADS15, OsMADS18, and OsMADS34 expression levels in GLA 4 and osftl4 under LD and SD conditions. For all examined genes, the transcription level in GLA 4 was set at 1 (i.e., baseline). Each point represents three technical replicates

OsFTL4 Competes with Hd3a for the Interaction with 14-3-3 Proteins

The interaction between Hd3a and 14-3-3 proteins in the SAM generates a complex that is translocated to the nucleus, where it binds to OsFD1. The resulting ternary FAC complex induces the transcription of OsMADS15, which leads to flowering (Taoka et al. 2011). To elucidate the mechanism underlying the inhibitory effects of OsFTL4 on flowering, the interaction of OsFTL4 with 14-3-3 proteins and OsFD1 was analyzed by conducting a yeast two-hybrid (Y2H) assay. Specifically, OsFTL4 was inserted into pGBKT7, whereas sequences encoding eight 14-3-3 isoforms (GF14a to GF14h) and OsFD1 were inserted into pGADT7. All combinations of recombinant plasmids were used to transform Y2HGold yeast cells. The Y2H assay confirmed that OsFTL4 can interact with GF14a, GF14b, GF14c, GF14d, GF14e, GF14f, GF14h, and OsFD1 in yeast cells (Fig. 5a). However, there was no interaction between OsFTL4 and GF14g. We also examined the interactions in a luciferase complementation imaging (LCI) assay involving N. benthamiana leaves, which produced similar results (Fig. 5b–j).

Fig. 5
figure 5

Interactions among OsFTL4, 14-3-3, and OsFD1 proteins. a Y2H assay. OsFTL4 was used as bait (GAL4-BD, BD), whereas GF14a to GF14h and OsFD1 were used as prey (GAL4-AD, AD). DDO: SD/-L/-T, QDO: SD/-L/-T/-H/-A. The 3-AT concentration was 25 mM. The interaction between OsFTL4 and GF14a b, GF14b c, GF14c d, GF14d e, GF14e f, GF14f g, GF14g h, GF14h i, or OsFD1 j was determined by the LCI assay involving N. benthamiana leaf epidermal cells. Effect of OsFTL4 on the activation of OsMADS15 expression by FAC. Data are presented as the mean ± SEM of three independent experiments

OsFD1 and Hd3a do not interact in vitro, whereas the presence of interactions in the yeast system is due to the bridging role played by the yeast 14-3-3 protein BMH1(Taoka et al. 2011). In the present study, OsFTL4 could interact with OsFD1 in yeast and tobacco system. Several 14-3-3 isoforms were present in tobacco, and some of them were reported to interact with bZIP proteins (Igarashi et al. 2001). Meanwhile, these interactions were dependent on conserved residuals (Taoka et al. 2011; Igarashi et al. 2001). Protein sequence alignment revealed that Nt14-3-3a, Nt14-3-3b, Nt14-3-3e in tobacco and brain modulosignalin homolog 1 (BMH1) in yeast were highly similar to the rice 14-3-3 proteins OsGF14b and OsGF14c sharing the same conserved residuals (Additional file 1: Fig. S2). Therefore, the interaction between BMH1 with OsFTL4 and OsFD1, Nt14-3-3e with OsFTL4 and OsFD1were verified by Y2H assay and LCI assay, respectively (Fig. 6a–e). Furthermore, OsFTL4/P93L, a key site mutantion of OsFTL4, could not interact with BMH1, Nt14-3-3e and OsFD1 (Fig. 6b, c, e).

Fig. 6
figure 6

OsFTL4 competes Hd3a for formation of florigen activation complex. The interaction between OsFTL4 and Nt14-3-3e a, OsFTL4/P93L and Nt14-3-3e b, OsFTL4/P93L and OsFD1 c, OsFD1 and Nt14-3-3e d were detected by using LCI assays. Nt14e: Nt14-3-3e. e The interaction between Nt14-3-3e and OsFTL4, Nt14-3-3e and OsFTL4/P93L, OsFD1 and Nt14-3-3e were tested by using Y2H assays. f Effect of OsFTL4 on OsMADS14 activation by FAC. Values are means ± SEM from three independent experiment. Asterisks indicate the significance of differences determined by Student’s t test: **, p < 0.01. g Co-localization of Hd3a and OsFTL4. BiFC assays between Hd3a and OsFTL4 (left) and between Hd3a and OsFTL4/P93L (right) were performed. N: Nucleus. Scale bar: 20 μm

To test the hypothesis that OsFTL4 and RCN are functionally similar, i.e., RCN competes with Hd3a for the binding to 14-3-3 proteins to form FAC (Kaneko-Suzuki et al. 2018), we co-expressed Hd3a and OsFD1 in rice protoplasts. This co-expression activated the expression of OsMADS14. Meanwhile, single transformation of OsFD1 also activated the expression of OsMADS14. However, its activation was significantly diminished when OsFTL4 was co-expressed, and no reduction in OsMADS14 expression levels was observed when OsFTL4/P93L mutant was co-expressed (Fig. 6f). These results demonstrated that OsFTL4 competes with Hd3a for the interaction with 14-3-3 proteins to repress the floral transition in rice. To further explore the competitive relationship between OsFTL4 and Hd3a, we performed BiFC analysis between OsFTL4 and Hd3a. When Hd3a-nCFP, OsFTL4-cCFP fusion proteins were co-expressed with OsFD1 in rice protoplasts, cyan fluorescent signals of Hd3a-OsFTL4 interactions were observed in the nucleus. When a P93L substitution was introduced in OsFTL4, no BiFC signal was detected (Fig. 6g).

Mutation to OsFTL4 Improves Rice Drought Tolerance

An examination using the PLACE online program ( suggested that the OsFTL4 promoter region contains multiple hormone-responsive elements (Additional file 3: Table S2). A previous study determined that ABA helps increase the expression of drought-responsive genes in rice (Rabbani et al. 2003). We observed that OsFTL4 expression was repressed by ABA (Additional file 1: Fig S3). To investigate whether OsFTL4 is involved in rice responses to drought stress, the drought tolerance of the osftl4 mutants was assessed. The wild-type plants and osftl4 mutants were grown under well-watered conditions for 2 weeks (Fig. 7a), after which watering was stopped. Following a 4-day exposure to drought stress, all of the wild-type GLA 4 plants were severely affected, whereas the osftl4 mutant plants exhibited less leaf rolling and wilting (Fig. 7b). After the recovery period, osftl4 mutant plants had more green leaves than the wild-type plants (Fig. 7c). Additionally, the survival rates of the two osftl4 mutants (38.7% and 40.7%) were significantly higher than that of the wild-type plants (Fig. 7d).

Fig. 7
figure 7

Phenotypes of GLA 4 as well as osftl4-1 and osftl4-2 mutants under drought conditions. ac Both mutants (osftl4-1 and osftl4-2) exhibited enhanced drought tolerance. The GLA 4, osftl4-1, and osftl4-2 seedlings (nearly 40 seedlings per genotype) with regular watering for 14 days a were subjected to drought stress for 4 days b, and recovery for 7 days c, respectively. Scale bars, 5 cm. d Survival rate calculated on the basis of the growth of a new leaf blade after 7 days of re-watering. Error bars represent the SE of three biological replicates (**, p < 0.01, by Student’s t-test). e Water loss rate of detached leaves from the wild-type and mutant lines at different time-points. Data are presented as the mean ± standard deviation (n = 20). f Stomatal conductance of the wild-type and two mutant lines under normal and drought stress conditions. Error bars represent three biological replicates (six plants per replicate). g OsNCED4, OsbZIP23, and Rab16c expression levels in wild-type and osftl4 plants under normal and drought conditions. Mean values ± SD were obtained from three technical repeats and three biological repeats. Statistical differences are labeled with different letters using LSD test (p < 0.05, one-way ANOVA)

The results of an in vitro water dissipation test involving the leaves of 2-week-old GLA 4 and osftl4 plants indicated that the rate of water dissipation was significantly lower in the mutants than in the wild-type control plants (Fig. 7e). The stomatal status is an important factor associated with plant drought responses. Thus, the stomatal conductance of the wild-type and osftl4 plants at the 4- to 5-leaf stage under normal and drought conditions was analyzed. Drought stress obviously affected stomatal closure, leading to decreased conductance, but the stomatal conductance of the osftl4 mutant plants was significantly lower than that of the wild-type plants (Fig. 7f).

To explore the possible molecular mechanisms by which OsFTL4 negatively regulates drought tolerance in rice, the expression of several stress-responsive genes under normal and drought conditions was analyzed. These genes include OsNCED4, encoding a protein involved in ABA biosynthesis (Zhu et al. 2009); OsbZIP23, encoding a basic leucine zipper protein (Zong et al. 2016); Rab16c, encoding a late embryogenesis abundant protein (LEA) (Xiao et al. 2007). Compared with their expression under normal conditions, OsNCED4, OsbZIP23, and Rab16c expression levels increased in the wild-type and mutant plants in response to drought stress. However, these genes were more highly expressed in the osftl4 mutants than in the GLA 4 plants under drought conditions (Fig. 7g). These findings imply that a mutation to OsFTL4 enhances drought tolerance by inducing drought stress-responsive gene expression in rice.

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