Overexpression of OsFTL10 induces early flowering and improves drought tolerance in Oryza sativa L.

Vector construction and rice transformation

To obtain the OsFTL10 overexpression construct, a fragment containing the complete OsFTL10 cDNA (Os05g0518000) was amplified with the primer set Fw-OsFTL10-EcoRI/Re-OsFTL10-EcoRI (Table S1), and cloned into the EcoRI site in binary expression vector pCAMBIA1390. To make the RNAi construct, the cDNA fragment was amplified with the primer set Fw-OsFTL10-RNAi/ Re-OsFTL10-RNAi, and cloned into the pTCK303 vector (Wang et al., 2004). To examine the promoter activity in rice, the OsFTL10 promoter (pFTL10), spanning about 2,000 bp upstream of the ATG start codon of OsFTL10 was amplified using PCR. The CaMV35S promoter for β-glucuronidase (GUS) expression in pCAMBIA1301 was replaced with pFTL10. For subcellular localization studies of OsFTL10, its cDNA fragment was amplified by PCR and fused with EGFP in the binary vector pCAMBIA1390. The binary vector for expression of Hd3a was from a previous work (Li et al., 2011a; Li et al., 2011b). The above constructs were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation (Li et al., 2011a; Li et al., 2011b). Agrobacterium-mediated transformation of rice (Oryza sativa L. ssp. japonica., Zhonghua 11) was performed as described (Li et al., 2011a; Li et al., 2011b).

Plant materials and growth conditions

Mature seeds of Zhonghua 11 (Oryza sativa L. ssp. japonica.) were collected from WT and homozygous T2 transgenic plants. Seeds were germinated for 2 days at 28 °C in the dark, after which they were planted in soil and grown under greenhouse conditions to maturity (12-h light at 30 °C/12-h dark at 22 °C). Mature leaves from 30 day old plants were collected at 10 O’clock in the morning, and RNAs were extracted for qRT-PCR analysis of the expression of flowering related genes. The major agronomic traits including heading date, plant height, grain weight and number of flowers per panicle of the homozygous plants were investigated.

FTL10 sub-cellular localization and BiFC analysis

The subcellular localization of FTL10 and GF14c, as well as BiFC analysis characterizing the interaction of FTL10 and GF14c, were carried out according to (Lin et al., 2017). For subcellular localization studies of FTL10 and GF14c, the coding regions of both FTL10 and GF14c were amplified with primers (Table S1) and fused with EGFP, respectively. For BiFC analysis, GF14c and OsFTL10 were amplified by PCR and restriction-cloned (Bam HI and/or Eco RI) into the p35S::cYFP and p35S::nYFP vectors, respectively. Negative controls constructs were made by replacing GF14c or OsFTL10 with GUS (Taoka et al., 2011). The above vectors were transformed into rice protoplasts for live cell imaging. Images were captured using a LSM 710 confocal microscope (Zeiss, Jena, Germany) (Lin et al., 2017).

Yeast two-hybrid assays

Yeast two-hybrid (Y2H) assays were performed using the Matchmaker™ Gold Yeast Two-Hybrid Systems (Clontech, Mountain View, CA, USA) according to Lin et al. (2017). The open reading frames of OsFTL10 and 14-3-3s were inserted into pGBKT7 and pGADT7 vectors with proper restriction enzyme sites indicated in the primers to create bait and prey, respectively.

GUS staining of transgenic rice plants

Homozygous transgenic lines carrying the pFTL10::GUS were randomly chosen for the study. Seeds were briefly sterilized and germinated, then planted in soil and grown under greenhouse conditions. Immature and mature leaf, roots and other tissues were collected from the plants grown at different stages, GUS staining of the plant materials was performed according to procedures described by Jefferson (1987).

Growth regulators and abiotic stress treatments

WT seedlings germinated for 2 weeks on half strength MS medium were collected and incubated in ddH₂O containing either of the following components: 5 mmol/L IAA, 1 mmol/L 6-BA, 0.1 mmol/L MeJA, 10 mmol/L GA₃, 100 µmol/L ABA, 1 mmol/L SA, 200 mmol/L Mannitol, 200 mmol/L NaCl, respectively. Treatments with IAA and BA were performed for 0, 0.5, 1 and 4 h, respectively. Treatments with ABA, SA, MeJA and GA were performed for 0, 1, 4 and 12 h, respectively. Treatments with mannitol and NaCl were performed for 0, 6, 14 and 48 h, respectively. All the treatments were started from about 10 O’clock in the morning. After treatment for the indicated times, total RNAs were extracted and reversely transcribed as the template for qRT-PCR.

qRT-PCR for gene expression analysis

Total RNAs were extracted using a TRIzol kit according to the user’s manual (Invitrogen, Carlsbad, CA, USA). For each sample, 1 µg of total RNAs was reverse-transcribed as cDNA template using a kit (TaKaRa, Kusatsu, Japan). qRT-PCR was carried out in a mixture of 20 µL consisting of cDNA, gene specific primers (Table S1) and 10 µL 2X mix (TaKaRa), water was added to the mixture to a final of 20 ul. qPCR was performed on an ABI 7900 real-time PCR machine according to the manufacturer’s instruction (Applied Biosystems). For each sample, three biological replicates were performed and cDNA were amplified in triplicate by quantitative PCR. The relative expression values were determined by using rice Ubiquitin gene as reference and the comparative Ct method (2- ΔΔCt).

Drought stress treatment

Drought stress treatment of WT and transgenic plants was performed according to Li et al. (2011a); Li et al. (2011b). Seeds of WT, overexpression transgenic lines ox2, ox5, and ox8 and RNAi lines i1,i2 and i5 (T3, homozygous seeds) were germinated, and in the same pot containing vermiculite were soaked with distilled water. Eight days later, water was withheld from the seedlings. After another 20 (for RNAi lines) or 25 days (for overexpression lines), the plants were re-watered for recovery. Three days later, plants with green, healthy leaves were regarded as viable, having survived. For study of drought related gene expression in transgenic plants, seedlings germinated for 2 weeks were incubated in a solution containing 200 mmol/L mannitol for 2, 4, 8, and 24 h, respectively. Total RNA was extracted for qRT-PCR.

Expression pattern of OsFTL10 in rice

OsFTL10 encodes a protein consisting of 174 amino acid residues. To investigate the temporal and spatial gene expression pattern of OsFTL10 in rice, we quantified the OsFTL10 expression level in different tissues including immature and mature leaves, stem, young panicle and floral organs from the wild-type plants grown under SD conditions. OsFTL10 expression was detected in all the above tissues. The highest expression level was observed in immature leaves with a lower level of expression found in floral organs (Fig. 1A).

The expression pattern of OsFTL10 was also investigated by GUS staining assay in stable transgenic plants. The promoter region of OsFTL10 was cloned and a construct was made by placing the GUS reporter under the OsFTL10 promoter. The construct was transformed into rice cell and stable transgenic plants were obtained. GUS expression was found in immature leaf, mature leaf, flower, young panicle, pollen and shoot tip (Figs. 1B–1H). Strong GUS staining was found in immature leaf tissue and very faint blue staining was found in the pollen (Fig. 1G).

Expression of OsFTL10 under growth regulators and abiotic stress treatments

To determine whether OsFTL10 expression is influenced by environmental factors, qRT-PCR was performed to study the expression of OsFTL10 under different growth regulators and abiotic stress treatments. Seedlings germinated for 2 weeks on half strength MS medium were collected and treated with different growth regulators and abiotic stresses for different time intervals, and total RNAs were extracted for qRT-PCR. Amongst the treatments of different growth regulators, ABA at 100 µM stimulated the expression of OsFTL10 to nearly two-fold after 4 hrs (Fig. 2C). The addition of GA₃ at 10 mM greatly increased the expression of OsFTL10 to more than ten-fold its basal level (Fig. 2F). Treatments with SA and MeJA also showed slightly increased expression of OsFTL10 (Fig. 2D, 2E), whilst treatment with IAA or BA reduced the expression level of OsFTL10 (Figs. 2A, 2B). Abiotic stress treatment was performed using 200 mM mannitol or 200 mM NaCl. Treatment with 200 mM mannitol resulted in increased OsFTL10 expression to four-fold basal levels at 6 hrs, and the expression dropped after 24 h (Fig. 2G). 200 mM NaCl slightly increased the expression of OsFTL10 which dropped after 24 h (Fig. 2H). In accordance with the above results, some cis elements involved in drought stress and ABA response were found in OsFTL10 promoter (Table S1). As ABA and mannitol are related to drought stress, we further studied the drought tolerance of OsFTL10 transgenic plants in addition to its function in flowering.

Overexpression of FTL10 induced earlier flowering in rice

As OsFTL10 is one of the homologs of Hd3a, we expected that overexpressing this gene would also promote rice flowering. We generated OsFTL10 overexpression transgenic plants and examined the transgene expression by RT-PCR (Fig. 3A). At the same time, we obtained RNAi transgenic plants for OsFTL10, and in some lines OsFTL10 expression was greatly reduced (Fig. 3B). Representative pictures of the overexpressing line ox8 (Fig. 3A) and the RNAi line (Fig. 3B) are shown. To investigate the agronomic traits of the transgenic plants, the homozygous T3 generation of the three independent overexpressing lines ox2, ox5, ox8 and RNAi lines i1, i2, i5 were grown under short day conditions, and their phenotype data compared with WT plants (Fig. 3). The average heading date of the overexpressing lines was 53 days, which was about 2 weeks less than of the WT plants (about 68 days). The RNAi lines did not show significant difference in heading time compared with the WT (Fig. 3C). The plant height for the overexpressing lines was on average 65 cm (Fig. 3D), which was about 19 cm shorter than that for the WT (84 cm). The number of grains per panicle and grain weight were also reduced significantly in the overexpressing lines, compared to that in WT (Figs. 3E, 3F). In the RNAi lines, the above traits were not significantly changed (Figs. 3C–3F). These results showed that overexpression of OsFTL10 could induce early flowering, which influenced the agronomic traits of the plants. The down-regulation of endogenous OsFTL10 exerted little effect on plant growth. When compared with Hd3a overexpression plants, we found that heading time of OsFTL10 overexpression plants were about 3 weeks longer (Table S1).

To elucidate the function of OsFTL10 on flowering induction, we first examined the subcellular localization of the OsFTL10 and GF14c protein. Constructs were made to fuse the OsFTL10 or GF14c with EGFP protein, under the CaMV35S promoter (CaMV35S::OsFTL10-EGFP). The constructs were transformed into rice leaf mesophyll protoplasts, and the EGFP signal was analyzed. The EGFP signal of OsFTL10 was observed in both the nucleus and cytoplasm, whilst the EGFP signal of GF14c was observed mainly in the cytoplasm (Fig. 4A). These results indicated that FTL10 had a similar subcellular localization to Hd3a.

Hd3a was found to interact with 14-3-3, and form a complex with OsFD1 in the nucleus for activation of the downstream flowering genes (Taoka et al., 2011). The interaction of OsFTL10 with GF14c in the rice protoplast was examined by BiFC. A strong signal was detected in the nucleus, which demonstrated the interaction of the two proteins. At the same time, the negative controls showed no obvious signal (Fig. 4B). The interactions of OsFT10 with multiple 14-3-3 s were also investigated by the yeast two-hybrid system. The results showed that OsFTL10 interacted with different 14-3-3 homologs in rice, including GF14a, GF14b, GF14c, GF14d, GF14e (Fig. 4C). These results suggested that OsFTL10 functioned similarly to Hd3a to bind with 14-3-3 s, which was a key step for entering the nucleus in the formation of the FAC complex with OsFD1 (Taoka et al., 2011).

Flowering in rice is mainly influenced by the photoperiodic pathway (Tsuji, Taoka & Shimamoto, 2011). OsGI (an orthologue of Gigantea) is an integrator of photoperiod pathway in rice (Kim et al., 2008). Under short day (SD) conditions, OsGI up-regulates the expression of Hd1 (a CONSTANS-like gene), and Hd1 will induce the expression Ghd8 (grain yield, heading date and plant height 8). Ghd8 encodes the OsHAP3 subunit of a CCAAT-box binding protein and can activate the expression of Hd3a (Yan et al., 2011). Ehd1 (Early heading date 1) is a B type response regulator, which up-regulates the expression of FT-like genes (Matsubara et al., 2011). OsGI can also up-regulate Ehd1 through activating MADS51 under SD conditions (Kim et al., 2008). To dissect the genetic networks of OsFTL10 on flowering, we performed qRT-PCR analysis of the genes that played important roles in rice flowering. The cDNA template was obtained from RNA extracted from mature leaves of WT, overexpressing lines ox2 and ox8, and the RNAi lines i2 and i5 before heading. For genes upstream of Hd3a and RFT1, GI, Hd1, Ehd1 and MADS51 were selected. qRT-PCR results showed that overexpression of FTL10 had no significant influence on the expression of GI and Hd1 in both ox2 and ox8, as their expression levels were similar to those in WT (Figs. 5A and 5B). Ehd1 was significantly up-regulated in both overexpressing lines at 1.5 and 2 fold (Fig. 5C). However, MADS51 expression was largely suppressed in the overexpressing lines (Fig. 5D). Amongst the FTL genes, Hd3a, and FTL1 were found to be up-regulated in both ox2 and ox8 (Figs. 5F, 5G), whilst the expression of RFT1 slightly decreased (Fig. 5E). For the major genes downstream of Hd3a (Taoka et al., 2011), we found that MADS15 expression was significantly increased (4 to 6 fold) in ox2 and ox8 (Fig. 5I), and MADS14 was slightly changed in these two lines (Fig. 5H). The results from the RNAi lines i2 and i5, showed similar results to those in the WT line (Fig. 5). These results suggested that overexpression of OsFTL10 induced early flowering, through direct enhancement of downstream MADS15 expression and feedback regulation of its upstream genes.

Overexpression of FTL10 improved drought tolerance in rice

As OsFTL10 expression can be influenced by different growth regulators and abiotic stresses such as ABA and mannitol treatments, the drought stress tolerance in transgenic plants was investigated. Transgenic and WT seedlings grown under normal conditions with similar vitality were used for treatment (Figs. 6A, 6D). When water was withheld for 25 days from the seedlings of the OsFTL10 overexpressing plants (ox2, ox5, and ox8) and the WT plants, obvious drought stress phenotypes were observed (Fig. 6B). Compared with the WT plants, leaf rolling and browning were delayed in the OsFTL10 overexpressing plants. Three days after re-watering, most of the OsFTL10 overexpressing plants survived, whereas the WT plants withered and died (Fig. 6C). The average survival rate for WT, ox2, ox5 and ox8 plants were 6%, 84%, 88% and 91%, respectively (Fig. 6G). Similar treatment was performed on OsFTL10 RNAi lines (i1, i2, and i5) and the WT plants, except that water withholding time was reduced to 20 days (Fig. 6E). The OsFTL10 RNAi plants were found to be more sensitive to drought stress, and after re-watering, most of the WT remained viable, whereas the RNAi plants died (Fig. 6F). The survival rate for WT, i1, i2 and i5 plants were 92%, 14%, 6% and 8%, respectively (Fig. 6H). These results suggested correlation of drought tolerance with OsFTL10 expression. In transgenic rice plants overexpressing OsFTL10 acquired significantly drought tolerance, whilst the suppression of OsFTL10 resulted in drought sensitivity.

Influence of OsFTL10 on the expression of drought stress-related genes

Environmental stresses and other external signals can induce complex responses in plants including changes in gene transcription, protein synthesis and metabolism. Studies in different plant species have revealed key genes and proteins involved in stress signaling and transcriptional regulation (Ito et al., 2006; Shinozaki & Yamaguchi-Shinozaki, 2007; Molina et al., 2008; Nakashima et al., 2011; Rai & Penna, 2013; Janiak, Kwaśniewski & Szarejko, 2016). To elucidate the mechanisms of OsFTL10 on drought tolerance, the expression of stress-related transcription factors in stable transgenic plants under drought stress was revealed. Seedlings from WT, i5 and ox8 were collected for drought stress treatment, and RNAs were extracted for qRT-PCR analysis.

OsDREB2A is a drought inducible transcription factor found in rice, which shows increased expression upon drought stress treatment and constitutive expression of this gene results in drought tolerance of transgenic plants (Ito et al., 2006). The expression of this gene was rapidly induced in WT plants and ox8 upon drought stress, a response which continued for 24 h. However, the expression level increased significantly after 8 h of treatment in ox8 compared to WT plants. In i5, the expression of OsDREB2A decreased after 8 h of treatment (Fig. 6I).

bZIP23, a transcription factor that confers ABA-dependent drought resistance in rice (Xiang et al., 2008), has also been shown to be induced by drought stress. Overexpression of this gene resulted in drought tolerance in transgenic plants. The expression of bZIP23 was rapidly induced in WT, ox8 and i5 plants under drought stress, whilst its expression decreased after 8 h in both WT and i5 plants. In ox8, the high expression level of this gene was maintained for 24 h (Fig. 6J).

SNAC1 gene belongs to the stress-related NAC superfamily of transcription factors. SNAC1 expression was induced rapidly in WT, i5 and ox8 plants. In ox8, the expression of this gene showed higher expression levels after induction which were maintained after 24 h. Together, these results suggested that overexpression of OsFTL10 improved drought stress tolerance in rice by modulating the expression of stress responsive genes (Fig. 6K).