EARLY FLOWERING3 Gene Confers Earlier Flowering and Enhancement of Salt Tolerance in Woody Plant Osmanthus fragrans

Abstract

Osmanthus fragrans Lour. is popular in landscaping and gardening in Asia. In recent years, growing attention has been given to evergreen tree flowering and adaptation. EARLY FLOWERING3 (ELF3) plays an essential role in plant flowering regulation and abiotic stress tolerance. However, there is very little known about how the ELF3 gene affects flowering time and salt tolerance in O. fragrans. To elucidate the potential role of the flowering-related gene ELF3 in responding to salt tolerance, a significantly upregulated gene OfELF3 was obtained by RNA sequencing (RNA-seq) after salt treatment in O. fragrans. Our results showed that OfELF3 is a nuclear protein, which did not have a transcriptional activation ability. OfELF3 accumulation was determined in different tissues and the differentiation process of floral buds by qRT–PCR, and the gene was also significantly induced by salt stress treatment. In addition, overexpression of OfELF3 accelerated the flowering time of transgenic Arabidopsis lines, and an increase in the expression of flowering integrators such as AtFT, AtSOC1, and AtAP1 was investigated. Moreover, OfELF3 overexpression significantly improved the salt tolerance of transgenic plants, seed germination and root length of transgenic plants and was superior to those of the wild type (WT) under NaCl treatment at 4 days post-germination and the 5-day-old seedling stage, respectively. Similarly, phenotype and physiological indexes (REL, MDA and soluble protein) of 3-week-old transgenic plants were superior to the WT plants as well. Together, our results suggest that OfELF3 is not only a positive regulator in the regulation of flowering but is also involved in the salt tolerance response in O. fragrans.

1. Introduction

In a plant’s life history, the behavior of flowering plays a critical role in reproductive success and plant development. The majority of evidence shows that the circadian clock involved in photoperiodic measurement not only regulates plant flowering processes, but also responses to environmental changes. The genes of circadian components have a great impact on how a plant reacts to environmental challenges such as heat, drought, and salt [1]. Among them, it is well known that salt stress has been demonstrated, which is an important abiotic stress factor that affects many areas in the world, resulting in an early arrest of floral development and reproductive failure [2].

EARLY FLOWERING 3 (ELF3), as a negative regulator of core circadian clock components, is responsible for the regulation of photoperiodic flowering [3]. In Arabidopsis thaliana, elf3 mutants showed a photoperiod-insensitive phenotype and early flowering in long-day (LD) or short-day (SD) conditions [4]. Most research has revealed that ELF3 binds the E3 ubiquitin ligase Constitutively Photomorphogenic 1 (COP1) to regulate GIGANTEA (GI), leading to CONSTANS (CO) and FLOWERING LOCUS T (FT) reduction at the transcript level to inhibit the flowering process [5]. ELF3 also binds to the MADS-box-type transcription factor SHORT VEGETATIVE PHASE (SVP) and inhibits FT expression, resulting in late flowering [6]. Additionally, endogenous hormone gibberellin (GA) content and FT expression are increased in an elf3 mutant, leading to the early flowering phenotype under noninductive photoperiods in spring barley (Hordeum vulgare) [7]. However, ELF3 exerts the opposite function in the regulation of flowering in some species. In the japonica rice cultivar ‘Gimbozu’, an Ef7 gene was identified and encoded an ELF3-like protein that promotes flowering by repressing Grain number, plant height, and heading date 7 (Ghd7) expression under both short-day (SD) and long-day (LD) conditions [8]. The roles of OsELF3-1 in rice (Oryza sativa) have been demonstrated, whereby OsELF3-1 induces Ehd1 expression to promote flowering under SD conditions, whereas OsELF3-1 inhibits Ghd7 expression to indirectly promote flowering under LD conditions [9]. In soybean (Glycine max), the gene GmELF3a/J, as the ortholog of AtELF3, directly inhibits the key legume-specific flowering repressor E1 and thus induces FT expression, promoting soybean flowering under short-day conditions [10]. All of the results indicated that ELF3, as a key zeitnehmer, plays different roles in the regulation of flowering in different species. In addition to the regulation of flowering, ELF3 is also involved in responding to abiotic stress in plants. The expression of LcELF3 can be induced by drought treatment, which indicates that the gene may be involved in resistance to drought stress in Lens culinaris [11]. Sakuraba showed that AtELF3 could enhance A. thaliana salt tolerance by suppressing the expression of Phytochrome interacting factor 4 (PIF4) [12]. Similarly, it was reported that the J gene positively regulates the expression of GmWRKY12 and GmSIN1 to enhance salt tolerance in soybean [13].

Osmanthus fragrans Lour. is well known for its unique fragrance and is one of the ten most well-known traditional plants in China, with a history of more than 2500 years. This species is important in landscaping and gardening, and it has also been extensively cultivated in Asia [14]. Previous studies have mainly focused on the flower fragrance in O. fragrans [15,16]. The flower is often used as an important component of perfumes, fragrances, and cosmetics because it is rich in aromatic compounds [17,18]. However, there are few reports on the regulation of flowering time and salt tolerance. In this study, we aimed to understand the role of OfELF3 in regulation on flowering and salt tolerance in O. fragrans, and our results provide important information for achieving the molecular mechanism of between flowering regulation and salt resistance in O. fragrans.

2. Materials and Methods

2.1. Materials

The obtained material, O. fragrans ‘Yanhonggui’, was kept in the O. fragrans resource garden of Zhejiang Agriculture and Forestry University. Based on the anatomical characteristics of floral bud differentiation [19], six stags were collected in the flowering process, including the flower bud undifferentiated stage (0 d), inflorescence differentiation stage (20 d), floret differentiation stage (30 d), flower bud differentiation stage (40 d), stamen differentiation stage (50 d), and pistil degeneration stage (60 d). The different tissues were sampled, including the leaves, flower buds, stems, roots, and flowers. For the salt treatment, the uniform cut-branches of O. fragrans were subjected to a 250 mM NaCl solution, and leaf samples were collected after 0, 3, 6, 12, and 24 h for further OfELF3 expression analysis. Three biological replicates were prepared for this experiment. All samples were stored at −80 °C for gene cloning, RNA sequencing, and expression analysis.

2.2. RNA Extraction and RNA Sequencing

Total RNA was extracted from 0.5 μg fresh leaves after 0 and 12 h of salt treatment with 250 mM NaCl using the RNAiso Plus reagent (TaKaRa, Dalian, China), following the manufacturer’s protocol. Then, 0.5 μL RNA was employed for RNA-sequencing, and the produced sequences using BLAST software were assigned functions and lodged in the databases of the O. fragrans genome [20]. In addition, the differential expression of each gene was analyzed by the reads per kilobase of exon per million reads mapped (RPKM).

2.3. First-Strand Complementary DNA Synthesis and OfELF3 Gene Cloning

First-strand complementary DNA (cDNA) was obtained with PrimeScript™ RT Master Mix (TAKARA, Dalian, China). The system contained 2 µL 5 × PrimeScript™ RT Master Mix (Perfect Real Time), 1 µL total RNA, and water, to a final volume of 10 µL. The reaction was carried out at 37 °C for 15 min, 85 °C for 5 s, and finally 4 °C to stop the reaction. The full-length cDNA of OfELF3 was amplified from the cDNA of flower buds. Then, the PCR amplification products were ligated into the pMD18-T vector (TAKARA, Dalian, China), and the recombinant plasmid was transformed into the DH5α strain for sequencing. The cloned sequences were then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 17 March 2022) under accession number: UPI49081.

2.4. OfELF3 Sequence Analysis

Multiple sequence alignment was carried out using DNAMAN software (https://www.lynnon.com/dnaman.html, accessed on 19 March 2022). The open reading frame (ORF) and coding amino acids of OfELF3 gene were analyzed using TBtools software [21]. The physicochemical properties of OfELF3 protein were further analyzed using ExPASy (https://web.expasy.org/protparam/, accessed on 19 March 2022), and the transmembrane region was predicted using TMHMM (https://services.healthtech.dtu.dk/, accessed on 19 March 2022). In addition, the secondary structure of the protein was predicted using SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/, accessed on 20 March 2022). Gene sequence homology and the phylogenetic tree were analyzed using the NCBI BLAST tool (https://blast.ncbi.nlm.nih.gov/, accessed on 20 March 2022) and MEGA 7.0 software [22], respectively. All primers were designed by primer premier6.0 (http://www.premierbiosoft.com/primerdesign/, accessed on 20 March 2022), and the information of sequences is listed in Table S1.

2.5. Quantitative Real-Time PCR (qRT-PCR) Expression Analysis

The qRT–PCR system was composed of the following: 10 μL SYBR Premix Ex Taq (Vazyme, Nanjing, China), 2 μL cDNA, 0.8 μL primer, and double-distilled water up to a total volume of 20 μL; three biological replicates were prepared for each sample. The thermocycler program consisted of 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s; 95 °C for 5 s; and 60 °C for 1 min. Three technical replicates of each sample were analyzed, and the 2^−ΔΔCt formula was used to calculate their expression [23]. OfACT and AtACT2 were used as reference genes in O. fragrans and Arabidopsis [24], respectively. All primers were designed using Primer Premier 6.0 software, and the sequences are shown in Table S1.

2.6. Vector Construction

For subcellular localization and transgenes, the coding sequence (CDS) of OfELF3, except for the termination codon, was amplified and inserted into the pORE-R4 vector using the XhoI and ClaI sites, which contains the CaMV35S promoter to activate gene expression, and was ligated using T4 DNA Ligase (TAKARA, Dalian, China). The system contained 6 µL of DNA fragment, 1 µL of vector DNA, 1 µL of T4 DNA Ligase, and 1 µL of 10 × T4 DNA Ligase Buffer, and the reaction was carried out for 30 min at 16 °C. To create pGBKT7-OfELF3 constructs for the transcription activation assays, the BamHI and EcoRI sites were used to insert the CDS of OfELF3 into the pGBKT7 vector. The reaction system and procedure were similar to the construction of the pORE-R4 vector. The primers for generating the above constructs are shown in Table S1.

2.7. Subcellular Localization and Transcription Activation Assays

To explore the subcellular localization of OfELF3, a control vector (pORE-R4) and a fusion construct (35S::OfELF3-GFP) were transformed into Agrobacterium rhizogenes strain GV3101. 35S::OfELF3-GFP and 35S::GFP were co-injected into Nicotiana benthamiana leaves with nuclear marker (35S::D53-RFP) [25]. After 3 days, subcellular localization was observed with a confocal microscope (Olympus Corporation, Tokyo, Japan). For transcription activation analysis, the plasmid containing the fusion protein was transformed into yeast strain AH109, and the transformed strain was grown in SD/-Trp, SD/-Trp-Leu-Ade, and SD/-Trp-Leu-Ade+X-α-gal media broth and incubated at 30 °C for 2–4 days for observation. The detailed protocol followed that of the Matchmaker™ Gold Yeast Two-Hybrid System (Clontech).

2.8. Overexpression and Phenotypic Observation in Arabidopsis

The constructed pORE-R4-OfELF3 vector was introduced into A. thaliana by the inflorescence dip method [26]. Positive transgenic plants were selected on a Murashige and Skoog (MS) culture medium containing kanamycin (50 mg/L). Then, genomic DNA was extracted from T1 transgenic plants and detected by PCR amplification. The obtained transgenic positive seedlings were cultured to T3 generation, and then the harvested seeds were sterilized along with A. thaliana ecotype Columbia (Col-0) seeds by immersion in 75% ethanol (V/V) for 5 min. All of the seeds were rinsed three to five times with sterile distilled water for 1 min each, spotted on MS culture medium, incubated for 72 h at 4 °C in the dark, and then transferred to 23 °C incubation under long-day conditions (16-h light/8-h dark). In addition, the overexpressed OfELF3 seedlings were transferred into soil at four leaf stages, and the blotting time, rosette leaves, and flowering time were investigated.

2.9. Investigation of Germination and Root Length in Arabidopsis

The obtained seeds of transgenic and WT plants were sterilized and sown on an MS medium with 0, 50, and 100 mM NaCl, and incubated for 72 h at 4 °C in the dark. The germination rate was determined after 4 days. Then, transgenic and WT seedlings were transferred to MS medium containing 0, 50, and 100 mM NaCl under long-day conditions, and root length was recorded after 7 days. Six plants of each transgenic lines were employed for investigation of root length at three technical replications per repeat.

2.10. Physiological Determination

Physiological indexes were identified on 3-week-old transgenic plants under 250 mM NaCl treatment to confirm transgenic salt tolerance. The relative electrolyte leakage (REL) was measured by the methods of Wang et al. [27]. After rinsing with distilled water, 0.2 g of the leaves were sheared and placed in 10 mL of deionized water for 12 h, and the initial conductivity (R1) was measured. After 30 min of boiling, the final conductivity (R2) was measured. The REL was calculated using the formula REL = (R1/R2) × 100%. MDA content was measured according to Hu et al. [28], whereby 0.2 g of leaves were ground in 5 mL of 10% trichloroacetic acid (TCA) solution, homogenized, and centrifuged. In addition, a volume of 2 mL of supernatant from the centrifugation was added to 0.67% Thiobarbituric acid (TBA). Finally, the MDA content was measured by monitoring the absorbance at 450, 532, and 600 nm after mixing and incubating in boiling water for 30 min. In addition, the content of soluble protein was measured by the Coomassie brilliant blue G250 method [29]. Three replicates were conducted for each measurement.

2.11. Statistical Analysis

Data analysis was conducted using Excel 2016 (Microsoft, Redmond, WA, USA) and IBM SPSS 20 software (IBM, Armonk, NY, USA), and significant differences were determined by Student’s t test at significance levels of p < 0.001 (***), p < 0.01 (**) and p < 0.05 (*).

3. Results

3.1. RNA-Sequencing and OfELF3 Sequence Analysis

To investigate the possible function of the ELF3 gene in plant salt stress responses of O. fragrans, three OfELF3-like potential fragments and expression were identified by comparing the transcriptome profiles of O. fragrans (

Table 1. Analysis of differently expressed OfELF3-like genes after salt treatment.

Gene_id	Expression		Fold Change	p Value	Annotation
	CK	Salt
gui0033520.1	10.00	33.54	1.75	1.12 × 10−31	EARLY FLOWERING 3
gui0278630.1	34.87	46.70	0.42	8.64 × 10−4	EARLY FLOWERING 3-like1
gui0155440.1	15.66	21.31	0.44	1.40 × 10−4	EARLY FLOWERING 3-like2

). The results showed that a OfELF3-like gene gui0033520.1 was significantly upregulated by salt treatment, so we named is OfELF3 (accession number: UPI49081). Then, we further isolated the gene sequence with a 1986 bp ORF in length from O. fragrans ‘Yanhonggui’, which encoded 661 amino acids with three highly conserved domains. The molecular formula was estimated to be C₃₁₉₁H₄₉₉₉N₉₀₉O₉₈₆S₂₂, with an isoelectric point of 7.97 and hydrophobicity of −0.648. Phylogenetic tree analysis revealed that OfELF3 has the closest evolutionary relationship (89.39%) with the OeELF3 protein from Olea europaea (Figure 1A), and the OfELF3 protein has a high similarity with OeELF3. In a comparison of OfELF3 with other plant species, three conserved structural domains were also identified, including phyB and COP1 binding, ELF4 binding, and nuclear localization (Figure 1B). In addition, there is no transmembrane region in the OfELF3 protein, which has a higher ratio of random coils (77.46%) (Figure S1).

3.2. Analysis of Subcellular Localization and Transcription Activation

The subcellular localization of OfELF3 was identified by the construct 35S::OfELF3-GFP, and 35S::D53-RFP was used as a nuclear marker. As shown in Figure 2A, the green fluorescence produced by OfELF3-GFP was consistent with the red fluorescence produced by D53-RFP. This result exhibited that OfELF3 was a nuclear-localized protein. In order to determine whether OfELF3 has transcriptional activity, the plasmid pGBKT7-OfELF3 was transferred into the yeast strain AH109 (Figure 2B). During the experiment, the positive group grew well on an SD/-Trp-Leu-Ade medium and turned blue on an SD/-Trp-Leu-Ade+X-α-gal medium, whereas the experimental and negative groups could not, indicating that OfELF3 does not autonomously activate reporter genes in the absence of prey proteins.

3.3. Expression Analysis

The expression of the OfELF3 gene was detected by qRT-PCR in different tissues and flower bud development stages in O. fragrans. The transcript of OfELF3 can be detected in all tissues, whereas the leaf tissue exhibited the highest transcript levels, about 71-fold higher than that expressed in flower tissue (Figure 3A). In addition, the expression of the OfELF3 gene showed an overall upward trend during flowering processes, and the peak of expression was observed at the flower bud differentiation stage (Figure 3B). However, from the inflorescence differentiation stage (20 d) to the floret differentiation stage (30 d), the expression of OfELF3 showed no significant differences relative to flower bud undifferentiated stage (0 d) in O. fragrans. In addition, we found that OfELF3 was significantly induced by NaCl treatment, and the highest expression was reached at 12 h, approximately 3.5-fold higher than that at 0 h (Figure 3C).

3.4. Overexpression of OfELF3 Increases Flowering Time

OfELF3 was overexpressed using the CaMV 35S promoter to investigate the flowering time regulation in A. thaliana. Two transgenic lines (E7 and E9) with relatively high expression were employed for subsequent experiments (Figure 4A). We observed firstly the bolting time and rosette leaf in E7 and E9 plants. We found that the bolting time of E7 and E9 were earlier than that of the WT plants under long-day conditions, and lines E7 and E9 bolted at approximately 25 and 23 days, respectively (Figure 4A,B). Approximately 11 rosette leaves were produced in the two transgenic lines, fewer than that of the WT plants at the bolting stage (Figure 4C). The flowering time of all the two transgenic lines was significantly earlier than that of WT plants (Figure 4D). Furthermore, the expression of flowering integrator genes was investigated, including AtFT, APETALA1 (AtAP1), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (AtSOC1). The expression of AtFT increased 1.7-fold and 83.8-fold in E7 and E9 lines relative to WT plants, respectively (Figure 4E). Similarly, compared with WT plants, the expression of AtAP1 and AtSOC1 was upregulated 3.1-fold and 3.1-fold in E7 lines, 10.7- and 17.6-fold in E9 lines, respectively (Figure 4F).

3.5. Overexpression of OfELF3 Enhances Salt Tolerance

To investigate whether the flowering regulator OfELF3 responds to salt stress, we observed the germination rate and primary root length on an MS medium with different NaCl concentrations. In general, there was no significant difference in germination rate and primary root length between the transgenic line and WT on MS culture without NaCl. Under NaCl treatment, transgenic seeds from E7 and E9 lines showed stronger germination ability (Figure 5A), compared to the rate of seed germination of 51.67 ± 4.16% and 40.56 ± 4.04% under 50 mM and 100 mM NaCl conditions, respectively, 73.33 ± 1.53% and 62.78 ± 2.52% of the germination rate in E7 lines under 50 mM and 100 mM NaCl conditions, and 75.56 ± 3.22% and 65.00 ± 4.36% in E9 lines under 50 mM and 100 mM NaCl conditions, respectively (Figure 5B). For the primary root length, OfELF3 overexpression significantly promoted the primary root length under salt stress conditions (Figure 5C), between 3.37 ± 0.66 and 3.26 ± 0.33 cm of the primary root length in E7 and E9 lines under 50 mM NaCl conditions, respectively, whereas it was 2.48 ± 0.41 cm of the primary root length in WT (Figure 5D). Similarity, it was 2.73 ± 0.33 and 2.94 ± 0.33 cm in E7 and E9 lines under 100 mM NaCl conditions, respectively, and 2.25 ± 0.31 cm of the primary root length in WT (Figure 5D). Then, the phenotype and physiological indexes were further investigated in the 3-week-old transgenic plants by 250 mM NaCl treatment. Our results exhibited that overexpression OfELF3 also enhanced the salt tolerance in transgenic lines compared to WT plants (Figure 6A). After NaCl treatment, the electrolyte leakage and MDA content significantly decreased in E7 and E9 lines relative to WT plants, respectively (Figure 6B,C). However, the content of soluble protein was significantly accumulated in E7 and E9 lines (Figure 6D). All results indicate that OfELF3 can improve the salt tolerance of A. thaliana.

4. Discussion

Numerous plants’ ELF3 gene has been discovered and investigated, and the gene governs many growth and development processes, including flowering, hypocotyl elongation. etc [3,30], also play significant roles in the response of plants to environmental stress [12]. At present, there are few reports of the ELF3 gene on the regulation of flowering and salt tolerance in the woody plant O. fragrans.

In this study, the differentially expressed OfELF3 gene was screened by RNA-Seq analysis in O. fragrans. Then, the gene was isolated and characterized, from which a novel 661 amino acid protein was encoded. Phylogenetic analysis showed that OfELF3 was structurally similar to the OeELF3 protein (Figure 1A). Multiple sequence alignment analysis showed that the OfELF3 protein also had three domains (Figure 1B), and the N-initial domain of ELF3 is responsible for binding to PhyB and COP1, the middle domain is responsible for binding to ELF4, and the C-terminal domain is responsible for nuclear localization, and the structures as conserved as in other species [9,31,32]. This result suggests that OfELF3 was conserved during their evolution. In A. thaliana and O. sativa, ELF3 was reported to be located in the nucleus [32,33]. Similarly, subcellular localization analysis also showed that OfELF3 was a nuclear-localized protein (Figure 2A), and transcriptional activity analysis revealed that OfELF3 does not have transcriptional activation activity, which speculates that it may regulate downstream genes by interacting with other proteins [34]. Furthermore, ta issue-specific expression analysis revealed that OfELF3 was expressed in all tissues and exhibited the highest expression levels in leaves among all tissues (Figure 3A). An examination of different periods of flower bud differentiation showed that the expression of OfELF3 increased sharply when the flower buds were in the bud differentiation and stamen differentiation stages (Figure 3B). OfELF3 may be involved in the flower bud differentiation process in O. fragrans. For our interest, salt stress significantly induced OfELF3 after 3 h treatment, and the highest level of expression was reached after 12 h (Figure 3C). It is indicated that OfELF3 might be involved in the regulation of salt tolerance in O. fragrans.

Most of the previous studies have concluded that ELF3 functions as a flowering repressor that delays flowering by affecting the regulation of the circadian clock, such as in A. thaliana, wheat (Triticum aestivum), and barley [35,36,37]. However, the opposite function occurs in some species, such as rice and soybean [8,9,10]. An elf3 mutant exhibited late heading and flowering in rice, and the flowering phenotype is consistent with the overexpression of OfELF32 in A. thaliana. Compared with WT plants, OfELF3 overexpression resulted in significantly earlier bolting and flowering time and a significant reduction in the number of rosette leaves in the transgenic A. thaliana lines E7 and E9 (Figure 4). Three major flowering integrator genes were further analyzed in the transgenic lines, including AtFT, AtAP1, and AtSOC1, and the expression of these integrator genes was significantly increased in the E7 and E9 relative to WT (Figure 4). All of these preliminary results confirmed that OfELF3 might be a flowering activator promoting flowering time in the evergreen tree O. fragrans. Furthermore, OfELF3 may promote flowering time by influencing the expression of FT and other integrator genes (like AP1, or SOC1) in a direct or indirect manner. On the other hand, ELF3 has an important role in the plant response to environmental stresses in most species [12]. It is reported that AtELF3 enhanced salt resistance of A. thaliana by directly repressing the expression of AtPIF4, which in turn upregulates the expression of JUNGBRUNNEN1 (JUB1) [38]. The J gene indirectly regulates the expression of the GmWRKY and GmNAC genes to positively regulate the salt resistance pathway in soybean [13,39,40]. In the evergreen tree O. fragrans, we investigated the germination and root length of transgene lines under 50 and 100 mM NaCl conditions. The seed germination rate of both transgenic and WT lines was inhibited by salt stress treatment; however, the seed germination rate was significantly higher in the seeds of both transgenic lines compared to the corresponding rate of WT seeds. In addition, primary root length is important for the adaptability of the root system to environment. We found that the primary root length of transgenic lines was also longer than that of WT plants (Figure 5), which indicated that the heterologous expression of OfELF3 could improve the adaptation of A. thaliana under salt stress conditions. Meanwhile, the same phenotype was identified on 3-week-old plants as well (Figure 6). All the results demonstrated that OfELF3 plays an essential role in responding to salt tolerance and improving environmental adaptability in O. fragrans.

5. Conclusions

In conclusion, a differently expressed OfELF3 gene was identified and characterized, and the gene contributes to earlier flowering and enhancement of salt tolerance in O. fragrans. Our results provide valuable information for understanding the relationship between flowering and salt tolerance in the evergreen plant O. fragrans.