Introduction

Proper timing of flowering (or “heading date” in cereals) is very important for a plant to complete its life cycle, and is controlled by both environmental signals, such as temperature, day length (or photoperiod) and availability of nutrients (Brambilla and Fornara 2013; Putterill et al. 2004), and internal signals, such as flowering suppressors and activators (Jack 2004; Jarillo and Piñeiro 2011). SUPPRESSOR OF OVEREXPRESSION OF CO1/AGAMOUS-LIKE 20 (SOC1/AGL20) has been identified in Arabidopsis thaliana through screening of the suppressor mutants that function in early flowering (Onouchi et al. 2000). As a well-studied activator, SOC1 encodes a MADS box protein (Borner et al. 2000; Lee et al. 2000) that regulates the flowering time, floral patterning, and floral meristem determinacy by integrating multiple flowering pathways derived from photoperiod, temperature, hormones, and age-related signals (Liu et al. 2009; Lee and Lee 2010; Melzer et al. 2008).

Early studies indicated that FLOWERING LOCUC (FLC) gene involves in the vernalization pathway and represses the expression of SOC1 and FLOWERING LOCUC T (FT) by binding to their promoter regions (Helliwell et al. 2006; Hepworth et al. 2002). FT and SOC1 may act in parallel pathways downstream of CONSTANS (CO), and their mutations partially suppress the early flowering phenotype of 35S::CO plants (Samach et al. 2000). Meanwhile, activation of SOC1 by CO is dependent on FT under long-day condition (Yoo et al. 2005). APETALA1 (AP1, class A gene) is not only essential for the determination of flower meristem identity at early stage, but also for the initiation and development of floral organs at late stage (Bowman et al. 1993). SOC1, AGAMOUS-LIKE 24 (AGL24), and SHORT VEGETATIVE PHASE (SVP), which are repressed by the binding of AP1 to their promoters, act redundantly to maintain shoot identity (Lee and Lee 2010) and prevent class B and C genes from precociously expressing in the emerging floral meristem. Nevertheless, SEPALLATA 3 (SEP3) is also required for the repressions in the process (Liu et al. 2009). Taken together, SOC1 regulates the flowering time via the mediation of FLC, FT and CO, and the floral development via manipulation of the class A, B and C genes.

SOC1-like genes have been widely studied in both eudicots and monocots, and found to have similar expression patterns but possibly play different functions, depending on the plant species (Lee and Lee 2010). In addition to their roles in floral development and flowering time (Ferrario et al. 2004; Lee and Lee 2010; Ruokolainen et al. 2011), SOC1-like genes may have special functions (Cseke et al. 2003; Kimura et al. 2015; Melzer et al. 2008). Recently, a pair of SOC1 homologues have been found in some plant species including Oryza sativa (OsMADS50 and OsMADS56), Hordeum vulgare (HvSOC1-like1 and HvSOC1-like2), Glycine max (GmGAL1 and GmGAL2), Citrus sinensis (CsSL1 and CsSL2) and Chrysanthemum (CISOC1-1 and CISOC1-2) (Fu et al. 2014; Papaefthimiou et al. 2012; Ryu et al. 2009; Tan and Swain 2007; Zhong et al. 2012). The pair of SOC1 homologues usually do not show opposite functions but the pair of OsMADS50 and OsMADS56 from rice functions antagonistically in the regulation of LD-dependent flowering by controlling expression of OsLFL1 and Ehd1 (Ryu et al. 2009). Ectopic expression of OsMADS50 causes early flowering (Lee et al. 2004; Tadege et al. 2003), but overexpression of OsMADS56 results in delayed flowering (Ryu et al. 2009). These data indicate that individual SOC1-like genes may play different role in different species, even in the same species.

The majority of bamboo species have a long and unpredictable period of flowering that varies from a few years to 120 years (Janzen 1976; Sharma 1994). They flower after a long vegetative growth phase, and are often followed by the death of flowered clumps (Keeley and Bond 1999). This brings difficulties for disclosing the phenomenon of bamboo flowering. To overcome them, scientists took sequencing approach to dissect the genome of Phyllostachys edulis (Phyllostachys heterocycla) and the transcriptome of P. edulis, Bambusa oldhamii, Bambusa edulis, Dendrocalamus latiflorus, and identified numerous genes that could play important roles in bamboo flowering (Gao et al. 2014; Lin et al. 2010; Peng et al. 2013; Shih et al. 2014; Zhang et al. 2012; Zhao et al. 2014). Novel miRNAs were also identified in P. edulis and D. latiflorus, and verified to play significant regulatory roles in bamboo flowering (Gao et al. 2015; Zhao et al. 2015). Most recently, proteomic approach has been applied to reveal the veil of bamboo flowering. It turned out that the sporadic bloom of bamboo was associated with stress elements, mobile genetic elements and signal transduction cross-talk elements (Louis et al. 2015). Undoubtedly, these results provide critical starting points for evaluating the regulatory roles of the genes in bamboo flowering, but the functions have to be verified experimentally.

Here, we isolated a SOC1 homolog from Phyllostachys violascens and named it as PvMADS56. The gene was characterized by transferring into the wild-type A. thaliana. The flowering time and transcriptional levels of the floral-related genes were determined. On the basis that PvMADS56 gene promoted flowering, and regulated the development of floral organs, leaves and culms, while its promoter activity was upregulated by abscisic acid (ABA) and methyl jasmonate (MeJA), we proposed that SOC1-like gene PvMADS56 may regulate bamboo flowering by responding to ABA and MeJA.

Materials and methods

Plant materials and growth conditions

Bamboo samples used for gene cloning and quantitative real-time PCR (RT-qPCR) were collected in the Bamboo Garden of Zhejiang Agriculture and Forestry University. To analyze the tissue-specific expression of PvMADS56, the samples including different tissues (young leaves, mature leaves, culms, bamboo shoots, rhizome roots and flowers) of the flowering and non-flowering P. violascens plants were collected on 12 April, 2014. P. violascens usually flower from later of March to mid of April every year. Thus, the leaves and flowers of the flowering plants at different stages were harvested for the gene expression analysis every 2 weeks from 15 March to 12 April, 2014, while the leaves of non-flowering plants were also collected. The sampling time was selected according to the structural change of floral organs (Lin et al. 2012): T1, the time when the floral bud formed and switched from the vegetative phase into reproductive stage (15 March); T2, the time when the inner organs of flower began to form, which was examined by anatomy under stereomicroscope (29 March); T3, the bloom stage when the anther was outcropped from palea (12 April).

All A. thaliana including the wild-type and soc1 mutant (SALK_138131C) (from ABRC) in Columbia-0 (Col-0) background were planted under long days (16 h light/8 h dark) at 22 °C.

Isolation of PvMADS56 and its promoter

Total RNA was isolated using Trizol method. First-strand cDNA was synthesized according to the manufacturer’s recommendation of Reverse Transcriptase M-MLV (TAKARA Company). The sequence of OsMADS56 (a SOC1 homolog from rice) ORF was blasted on the P. edulis (affinis species of P. violascens) transcriptome database (Peng et al. 2013) by BioEdit software. A sequence (ID: PH01000059G1270) with the highest identity to OsMADS56 was identified, which was actually annotated as a homolog of OsMADS56 in the database. Then the corresponding genomic sequence (PH01000059) was extracted from the genome database of P. edulis (Peng et al. 2013), and used to design the primers for amplifying PvMADS56 gene and its promoter. A pair of primers was designed to assure the accuracy and integrity of the full-length ORF (Table S1), and a 780 bp cDNA fragment containing a 666 bp open reading frame (ORF) was thus obtained. The PvMADS56 promoter was amplified by PCR using the primers (Table S1) designed on the basis of the 5′ flanking sequence of PH01000059. A sequence of 1880 bp in length was obtained.

Quantitative real-time PCR

The RT-qPCR primers were designed based on the full-length ORF sequence of PvMADS56. Here, PheUBC18 was used as the internal control gene (Qi et al. 2013) (Table S1). PCR amplification was carried out using SYBR Premix Ex Taq II mix (Takara) and CFX96™ Real-Time PCR Detection System (Bio-Rad). The amplification was 95 °C for 3 min, followed by 40 cycles of amplification (95 °C for 10 s, 60 °C for 20 s). Reactions were performed in 20 µL mixtures consisting of 10 µL 2 × SYBR Premix Ex Taq II mix, 0.5 µL each of forward or reverse primer (Table S1), and 1 µL cDNA template. The sample was made up with water to a final volume of 20 µL. Data were analyzed by the 2−∆∆Ct method (Livak and Schmittgen 2001).

Ectopic expression of PvMADS56 in Arabidopsis

To accomplish the constitutive expression of PvMADS56, its full-length ORF was cloned into binary vector pCAMBIA1301 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis including wild-type and soc1 mutant were transformed according to the floral dip method as described by Clough and Bent (1998). Transformed Arabidopsis seeds were selected on 1/2 MS solid medium containing 50 mg/mL kanamycin, and the survival plants were further confirmed by genomic PCR. The flowering time of PvMADS56 overexpressing plants was measured by counting the number of rosette leaves and days when the first bolt reached to 1-cm-long inflorescence. Total RNA was extracted from the seedlings of transgenic Arabidopsis expressing 35S::PvMADS56 and wild type, and analyzed for the expression levels of PvMADS56 as well as several other genes associated with flowering including FT, FLC, AP1, AP3, PISTILLATA (PI) and AGAMOUS (AG) by RT-qPCR using gene-specific primers (Table S1). A SAND family gene (TAIR ID: AT2G28390) was used as the control to normalize the amount of cDNA (Hong et al. 2010).

Yeast two-hybrid assays

To construct the plasmids for yeast two-hybrid assays, the ORFs of PvMADS56, PvAP1 (also named as PpMADS1), and PvSEP3 were amplified using the corresponding primer pair (Table 1). PCR fragments were ligated into the vectors pGBKT7 (BK, bait) or pGADT7 (AD, pray) provided by the Matchmaker™ Gold Yeast Two-Hybrid System (Clontech). All clones were verified by sequencing. The bait and prey plasmids were transformed into yeast strains Y2H Gold and Y187, respectively, by the lithium acetate method (Gietz et al. 1992) for growth on SD/-Trp or SD/-Leu solid medium. The positive bait and prey were mated together in yeast peptone dextrose adenine (YPDA) media, and then spread out on SD/-Leu/-Trp solid medium. Finally, positive interactions were confirmed by plating on the SD/-Leu/-Trp/-His/-Ade with X-α-gal. PvMADS56, PvAP1, PvSEP3 in pGBKT7 or pGADT7 were tested for autoactivation activity. Yeast containing a vector combination of pGBKT7-53 and pGADT7-T that activates the expression of the reporter gene was served as a positive control, and that of pGBKT7-Lam and pGADT7-T as a negative control.

Table 1 Phenotypes of 35S::PvMADS56 Arabidopsis plants
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Subcellular localization of PvMADS56

The ORF sequence of PvMADS56 without the stop codon was cloned into CaM 35S-gfp vector to generate a PvMADS56-GFP fusion protein for the subcellular localization study. The particle bombardment method was adopted for the transient expression assays (Wang et al. 1988). Empty vector was used as control. The onion epidermal cells were visualized and recorded by confocal laser scanning microscopy (LSM510, Zeiss, Germany).

Agrobacterium-mediated transient expression for promoter activity assay in efr-1 Arabidopsis seedlings subject to exogenous ABA and MeJA treatments

A fragment of 1852 bp in the flanking region of PvMADS56 (PvMADS56p-1852) before the start codon was cloned into the pBI101 vector with gusA gene using the ClonExpress®II One-Step Cloning Kit (Vazymem™, C112-01). The sequences of the primers are listed in Table 1. The construct was transformed into Agrobacterium tumefaciens strain C58C1 by electroporation. Transient expression experiment was carried out in efr-1 Arabidopsis seedlings by AGROBEST (Agrobacterium-mediated enhanced seedling transformation) method (Wu et al. 2014). Three-day-old seedlings were treated with ABA (100 µM) or MeJA (100 µM) for 24 h before Agrobacterium infection. Ten seedlings grown in each well were infected and three biological repeats were performed in each independent experiment.

For detection of GUS activity, Arabidopsis plants were vacuum-infiltrated (three times for 1–2 min each) and incubated for 24–48 h at 37 °C in X-Gluc solution (50 mM Na2PO4, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6·3H2O, 0.1 % Triton-X-100, and 0.5 mg ml−1 X-Gluc) and subsequent destaining of the tissue in 95 % ethanol. RT-qPCR was used to estimate the expression of GUS gene using the primers (Rusconi et al. 2013), and the ACTIN 2 (At3g18780) was used as an internal control (Wu et al. 2014) (Table S1). Three biological and technical replicates were used to assess GUS gene expression.

Bioinformatics and statistical analysis

Primers were designed by the Vector NTI and Premier Primer 5. Related proteins were obtained by BLAST analysis from the NCBI database. Phylogenetic analysis was conducted using neighbor-joining method of MEGA 5.0 with default parameters and a bootstrap of 1000 replicates (Tamura et al. 2011). The software ProtParam from ExPASy (http://exPasy.org) was used to analyze the physical and chemical properties of the protein. Subcellular localization of the protein was predicted with WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html). Cis-acting regulatory elements of the promoter were predicted using PlantCARE data (Lescot et al. 2002). Statistical analysis was carried out using SPSS 11.5. Differences were analyzed with one-way ANOVA followed by Tukey’s test. Significance was accepted at the level of p < 0.05.

Results

Isolation of PvMADS56 and analysis of its expression pattern

The SOC1-like gene from P. violascens was successfully isolated using the specific primers (Table S1). Its ORF contained 666 bp encoding a protein of 221 aa (GenBank Accession No. KX241616) (Fig. S1). A phylogenetic tree was constructed to determine its relationship with SOC1-like proteins derived from other plant species. As shown in Fig. 1, this protein belonged to the family of SOCl homologs from monocots and was closely related to MADS56-like proteins from O. sativa, Setaria italica, Triticum urartu and Brachypodium distachyon. PvMADS56 shared high identity with OsMADS56 (75.65 %), OsSOC1 (65.95 %) and SOC1 (55.16 %) (Fig. S2). Thereby, the name PvMADS56 was assigned.

Fig. 1
figure 1

Phylogenetic analysis of PvMADS56 protein. The phylogenetic tree was generated using MEGA 5.0 and displayed as a phylogram with the branch lengths proportional to the distances. Bootstrap values for 1000 replicates are provided to indicate reliability at each node. The scale bar indicates the branch length

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To determine the expression level of PvMADS56 in different bamboo tissues in both flowering and non-flowering P. violascens, we performed RT-qPCR using young and mature leaves, culms, bamboo shoots, rhizome roots, and flowers. PvMADS56 transcripts were detected in all the tested tissues (Fig. 2a). Except bamboo shoots, PvMADS56 expression in all tissues of the non-flowering plants was higher than that in the flowering plants, especially in the young leaves and culms. Within flowering plants, the highest transcript level of PvMADS56 was found in flowers.

Fig. 2
figure 2

Spatial and temporal expression of PvMADS56 in the flowering and non-flowering P. violascens. a Relative expression level of PvMADS56 was detected in different tissues of flowering and non-flowering plants. b Relative expression level of PvMADS56 mRNA was detected in the leaves of non-flowering (VL), flowering plants (FL) and flowers (FF) of the flowering plants during flower development. T1, T2 and T3 represented the sampling time points. T1 the time when the floral bud formed and switched from the vegetative phase into reproductive stage (15 March); T2 the time when the inner organs of flower began to form, which was examined by anatomy under stereomicroscope (29 March); T3 the bloom stage when the anther was outcropped from palea (12 April). Error bars on each column indicate the standard deviations from three replicates

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The temporal expression experiment showed that PvMADS56 expression gradually decreased in the leaves of flowering (FL) and non-flowering (VL) plants from T1 to T3 (Fig. 2b). Despite PvMADS56 transcripts declined significantly in the leaves of flowering plants at the early stage, it remained stable in flower (FF) during the flower development (Fig. 2b).

PvMADS56 promoter activity was upregulated by ABA and MeJA in efr-1 Arabidopsis

A fragment of 1852 bp located at upstream of the start codon (GenBank Accession No. KX241617) in PvMADS56 gene was obtained. Its sequence shared a high similarity (90.9 %) with the reference sequence from P. edulis genomic database. To explore potential regulatory elements, PvMADS56 promoter was analyzed using PlantCARE web tool. Several putative cis-regulatory elements were deciphered (Table S2). Besides a typical TATA box and a CAAT-box, the promoter also contained cis-acting elements including 3-AF1 binding site, ACE, Box I, CAAT-box, G-Box, G-box, GT1-motif involving light responsiveness, and ABRE and CGTCA-motif responding to hormones (Fig. S3). The ABRE, and CGTCA-motif are regulated by ABA and MeJA, respectively (Fig. S3). The other types of cis-acting elements including CCAAT motif (an MYBHv1 binding site), GC motif (an enhancer-like element) involving anoxic induction, GCN4_motif mediating endosperm expression and O2 site regulating zein metabolism were also found in the PvMADS56 promoter region (Table S2).

To determine the regulation of PvMADS56 promoter activity, we used transient expression by agroinfiltrated in Arabidopsis seedlings. The efficiency of transient expression by infection with C58C1 using AGROBEST was higher in the efr-1 mutant seedlings than in the wild-type Arabidopsis (Col) (Wu et al. 2014). The PvMADS56p-1852 was fused with GUS reporter gene in pBI101 vector. Then the PvMADS56p-1852:GUS chimeric construct was used to infect Arabidopsis seedlings by AGROBEST method, while the pBI101 vector was served as a control. A histochemical assay showed that strong GUS activity was mainly found in roots (Fig. 3a, b), indicating PvMADS56p-1852 was functional in Arabidopsis. Furthermore, ABA and MeJA treatments for 24 h induced GUS expression driven by PvMADS56p-1852, and GUS activity became stronger in the treated plants when compared with the untreated plants (Fig. 3c, d). Based on RT-qPCR, GUS expression in Arabidopsis was also significantly increased under ABA and MeJA treatments (p < 0.05) (Fig. 3e).

Fig. 3
figure 3

PvMADS56 promoter-GUS transient expression in Arabidopsis under treatments with ABA and MeJA. a Control plants carrying the pBI101 vector. b Control plants carrying PvMADS5p-1852:GUS construct without treatments. c Plants carrying PvMADS5p-1852:GUS construct were treated with ABA (100 µM) for 24 h. d Plants carrying PvMADS5p-1852:GUS construct were treated with MeJA (100 µM) for 24 h. e RT-qPCR analysis of GUS expression in response to ABA (100 µM) and MeJA (100 µM). Scale bars represent 1 mm. Data are expressed in mean ± SD. Error bars on each column indicate the standard deviation from three replicates. Asterisks indicate significant differences (*P < 0.05)

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Ectopic expression of PvMADS56 caused early flowering and abnormal floral organs in Arabidopsis thaliana

To examine the function of PvMADS56 in the regulation of flowering, we generated transgenic Arabidopsis plants in which PvMADS56 was overexpressed. A total of 13 independent T1 transgenic lines were obtained based on Kanamycin selection and genomic DNA PCR. An early flowering phenotype (Fig. 4a, b) and abnormal floral organs (Fig. 5) were observed in 12 transgenic lines, whose flower sizes were all smaller than the wild-type plant. They were classified into four groups based on the abnormal severity of the floral organs with least abnormality in Group I and most abnormality in Group IV (Table 1; Fig. 5). As shown in Fig. 6 and Table 1, 35S::PvMADS56 transgenic plants also showed abnormal leaves. The rosette leaves of all the transgenic lines became smaller (Fig. 6a) and were easier to enter senescence than those in the wild-type plants. The fifth rosette leaves of the transgenic lines (Groups III and IV) began to be inflexed (Fig. 6a, h), meanwhile the inflexed phenotype was also observed in the cauline leaves (Fig. 6c). However, cauline leaves in the transgenic lines of Groups I and Group II were bigger and wider than those in the wild-type Arabidopsis plants (Fig. 6d). Besides, 35S::PvMADS56 transgenic plants became dwarfs (data not shown) and had shorter capsules (Fig. 5g) with reduced fertility (Groups I and II), or even without fertility (Groups III and IV).

Fig. 4
figure 4

Phenotypic differences between transgenic plants and WT Arabidopsis thaliana. Overexpression of PvMADS56 enhanced flowering in WT Arabidopsis. a Days to flowering and the counts of rosette leaves for T1 35S::PvMADS56 transgenic Arabidopsis plants (n = 13); b Flowering phenotype of PvMADS56 transgenic Arabidopsis plants under LD conditions. c Early flowering phenotype of PvMADS56 transgenic Arabidopsis plants under LD conditions; d Days to flowering and rosette leaves counts for T1 35S::PvMADS56 transgenic Arabidopsis plants (n = 15). Data are mean ± SD. The scale bar represents 2 cm. Error bars on each column indicate standard deviations from three replicates. Asterisks indicate significant differences (**P < 0.01)

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Fig. 5
figure 5

Flower phenotypes of WT and soc1 Arabidopsis overexpressing PvMADS56 under LD conditions. a Flowers of WT Arabidopsis. b, c, d, e Flowers of WT Arabidopsis overexpressing PvMADS56. f Flowers of soc1 Arabidopsis overexpressing PvMADS56. g Siliques of 35S::PvMADS56 transgenic Arabidopsis. h Siliques of WT Arabidopsis. All the scale bars represent 2 mm

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Fig. 6
figure 6

Leaf phenotypes of WT and soc1 Arabidopsis overexpressing PvMADS56 under LD conditions. Rosette leaf phenotypes of WT and transgenic Arabidopsis plants (a, f, g, h). a Rosette leaf phenotypes of WT (right) and overexpressing PvMADS56 in WT Arabidopsis (middle and left) when they grew for 45 days; f WT Arabidopsis; g 35S::PvMADS56 transgenic plants; h 35S::PvMADS56 soc1 transgenic plants. Cauline leaf phenotypes of WT and transgenic Arabidopsis plants (b–e). b Cauline leaf phenotypes of WT Arabidopsis; c, d cauline leaf phenotypes overexpressing PvMADS56 in WT Arabidopsis; e cauline leaf phenotypes overexpressing PvMADS56 in soc1 Arabidopsis. Scale bars in a, f, g, h represent 5 mm, in b–e represent 2 mm

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To further recapitulate its function, PvMADS56 was overexpressed in soc1 mutant. It turned out that PvMADS56 completely rescued the late-flowering phenotype of soc1 mutant (Fig. 4c, d). Similar to 35S::PvMADS56 transgenic Arabidopsis, 35S::PvMADS56 soc1 transgenic plants also displayed abnormal floral organs (Fig. 5f) and leaves (rosette and cauline leaves) (Fig. 6e, g) and low fertility.

To explore if the abnormal phenotype of the floral organs in the transgenic plants was associated with the expression level of PvMADS56, its transcripts were tested in representative samples from the four individual groups of 35S::PvMADS56 transgenic Arabidopsis plants (Table 1). The RT-qPCR results showed that the expression level of PvMADS56 was gradually increased with the abnormal severity of the floral organs (Fig. 7).

Fig. 7
figure 7

Expression analysis of PvMADS56 and some flowering-related genes in transgenic plants overexpressing PvMADS56 in WT Arabidopsis. Data are mean ± SD. Error bars on each column indicate the standard deviations from three replicates. Blue columns represent PvMADS56 gene; red columns are for Flowering Locus T (FT) gene; green columns for Flowering Locus C (FLC) gene; purple columns for APETALA1 (AP1) gene; black columns for APETALA3 (AP3) gene; yellow columns for PISTILLATA (PI) gene and gray columns for AGAMOUS (AG) gene

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Taken together, our data indicated that PvMADS56 gene could regulate the development of floral organs and cause early flowering.

Induction of flowering-related genes in 35S::PvMADS56 transgenic Arabidopsis plants

Genes such as FLC, FT, AP1, AP3, PI and AG are involved in floral initiation and development in Arabidopsis. We assumed that overexpression of PvMADS56 affects the expression of these genes in transgenic Arabidopsis plants, resulting in early flowering and abnormal floral organs. To test this hypothesis, we determined the expression of FLC, FT, AP1, AP3, PI, and AG in transgenic plants using RT-qPCR.

In 35S::PvMADS56 transgenic Arabidopsis plants, the level of FT (an activator) was significantly increased (mean ≈5.4-folds more than that in wild-type control plants), while the level of FLC (a repressor) was dramatically decreased (mean ≈4.4-folds less than that in wild-type control plants) (Fig. 7). The phenotypic alterations of flowering organs in the 35S::PvMADS56 transgenic plants were also observed. According to the ABC model, the related genes such as AP1 (A-function gene), AP3/PI (B-function gene) and AG (C-function gene) were selected for analysis. The expression levels of AP1, AP3/PI and AG were downregulated in the four individual representatives from Groups I to IV and the downregulation levels correlated with the abnormal severity of the floral organs (Fig. 7). These results indicated that PvMADS56 involves in the development of floral organs by regulating the expression of AP1, AP3/PI and AG in Arabidopsis.

PvMADS56 is a nuclear protein and interacts with PvAP1 and PvSEP3

To determine the subcellular location of PvMADS56 protein, we adopted particle bombardment method. Green fluorescent protein (GFP) was used as the marker in the in vivo targeting experiment. As shown in Fig. 8, the fusion protein PvMADS56-GFP was located in the nucleus of the onion epidermal cells, whereas the control GFP was uniformly distributed in the whole onion cell.

Fig. 8
figure 8

Confocal images of PvMADS56 in onion epidermal cells. a Fluorescence of the control GFP plasmid was distributed throughout the cell; b fluorescence of the PvMADS56-GFP fusion plasmid was strongly detected in the nucleus. Scale bars represent 100 µm

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In Arabidopsis, SOC1 regulates the flower development by mediating AP1 and SEP3, and interacts with AP1 and SEP3 both in vitro and vivo (de Folter et al. 2005; Liu et al. 2013). To test if PvMADS56 interacts with PvAP1 and PvSEP3 in P. violascens (Lin et al. 2009), a yeast two-hybrid assay was performed. The fact that PvMADS56, PvAP1 and PvSEP3 in pGBKT7 or pGADT7 did not show autoactivation activity makes them suitable to test their mutual interactions (Fig. 9a). Transformants of pairs pGADT7-PvMADS56 and pGBKT7-PvAP1, pGBKT7-PvMADS56 and pGADT7-PvAP1, pGADT7-PvMADS56 and pGBKT7-PvSEP3, and pGBKT7-PvMADS56 and pGADT7-PvSEP3 grew well on the SD/-Leu/-Trp/-His/-Ade media (Fig. 9b) and demonstrated the activity of β-galactosidase, indicating that PvMADS56 could bind to PvAP1 and PvSEP3 in vitro.

Fig. 9
figure 9

PvMADS56 protein interacted with PvAP1 and PvSEP3, respectively. Interactions between these proteins were examined by yeast two-hybrid experiment. a The positive control (PCL) turned blue color, while the PvMADS56, PvAP1 or PvSEP3 remained white whether they were served as prey or as bait; b Yeast containing dual vectors of pGBKT7-53 and pGADT7-T served as the positive control, that of pGBKT7-Lam and pGADT7-T as the negative control. SD/-Leu/-Trp/-His/-Ade medium was used for clone selection purpose. Appearance of X-α-gal activity mirrors positive interaction

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Discussion

Here, we identified and characterized a SOC1-like gene PvMADS56 from P. violascens. Amino acid sequence alignment and phylogenetic analysis showed that PvMADS56 was closely related to the OsMADS56-like SOC1 homologues in monocots. Similar to other SOC1-like genes (Lee and Lee 2010), PvMADS56 was expressed in all the examined tissues in bamboo. Its overexpression caused early flowering by upregulating FT and downregulating FLC in Arabidopsis. Consistent with many SOC1-like genes (Ferrario et al. 2004; Lei et al. 2013; Zhong et al. 2012; Ding et al. 2013), PvMADS56 complemented the delayed flowering phenotype of soc1 mutant completely. These results suggested that PvMADS56 plays an evolutionarily conserved role in the regulation of flowering time in bamboo. Both PvMADS56 and OsMADS56 shared a high identity in their protein sequences, but they performed opposite function on flowering time. This may be resulted from evolutionary diversity of the genes, leading to the different flowering characteristics between bamboo and rice.

FLC, an upstream target of SOC1 represses SOC1 by directly binding to its promoter (Lee and Lee 2010). FT could move from leaves to the meristem and initiate flowering through activation of SOC1 (Corbesier et al. 2007). These results indicate that FLC and FT can directly regulate SOC1 in Arabidopsis. In this paper, all the four representative lines displayed an early flowering phenotype, and the expression of flowering time integrators FLC and FT was, respectively, downregulated or upregulated in 35S::PvMADS56 Arabidopsis plants, suggesting that FLC and FT were possible targets for PvMADS56 in transgenic plants. Although the expression levels of FT and FLC in 35S::PvMADS56 plants were affected, they did not show a correlation with PvMADS56 expression. For example, line four displayed highest expression level for PvMADS56 but lowest for FT and lower for FLC among these four lines. These results suggested that PvMADS56 promotes flowering by regulating FT and FLC in 35S::PvMADS56 plants, however, whether the regulation on FLC and FT is indirect or direct needs further experimental evidences.

The identity genes of floral organs such as AP1 (A class), AP3/PI (B class), and AG (C class) function to produce floral organs (sepals, petals, stamens and carpels) individually or in combination (Coen and Meyerowitz 1991). SOC1, AGL24, and SVP are required for the timely activation of B and C floral genes (Liu et al. 2009). AP1 contributes to floral patterning through regulation of the repression of SEP3 by SVP, SOC1 and AGL24 (Liu et al. 2009). Moreover, these proteins such as AP1, AP3, PI, AG and SEP3 could form heterodimers (AP1-SEP3 and AP3-PI) or trimmers (AP3-PI-AP1 and AP3-PI-AP3) to specify the floral organs (Honma and Goto 2001; Pelaz et al. 2001). In our study, the 35S::PvMADS56 transgenic plants showed abnormal floral organs and significant reduced expression levels for AP1, AP3, PI, and AG. AP3/PI and AG expressions were not detectable in line 4 from Group IV with the most severe phenotype. Considering the observation that the abnormal severity of floral organs correlated with the expression levels of the aforesaid genes, we proposed that PvMADS56 might involve in the development of floral organs in a manner dependent either on AP1, AP3, PI, AG individually or on their complex in Arabidopsis.

PvMADS56 was widely expressed in the tested tissues in both flowering and non-flowering P. violascens. In non-flowering plants, its expression level was different from the other SOC1-like genes (Ferrario et al. 2004; Lei et al. 2013; Zhong et al. 2012), higher in culms and young leaves than in other tissues. In both flowering and non-flowering plants, the level of PvMADS56 mRNA gradually decreases in leaves along with leaf development, also was different from SOC1 and OsSOC1, whose transcripts gradually increased with leaf development (Lee et al. 2000, 2004). 35S::PvMADS56 transgenic Arabidopsis plants exhibited phenotypes of dwarfism and abnormal leaves (rosette and cauline leaves), suggesting that PvMADS56 involves in the development of culms and leaves.

GA-responsive elements exist in SOC1 promoter region in Arabidopsis and GA could activate SOC1 expression under short days (8 h light/16 h dark) (Moon et al. 2003). To further explore if the expression pattern is associated with the regulation of PvMADS56 promoter, we analyzed the cis-regulatory elements within the promoter. It turned out that the promoter contains no GA-responsive element but ABRE and CGTCA-motif (cis-acting elements), which respond to ABA and MeJA, respectively. To testify if ABA and MeJA affect the activity of PvMADS56 promoter, exogenous phytohormonal treatments in Arabidopsis were performed using Agrobacterium-mediated transient expression. Histochemical staining showed that GUS activity driven by PvMADS56 promoter was significantly increased after ABA and MeJA treatments. Consistent with the role of endogenous ABA which activates flowering in several species including the key floral gene FT in Arabidopsis (Conti et al. 2014), early study has shown that higher level of ABA promoted flower bud differentiation in P. violascens (Lu et al. 2012). MeJA also affected flowering time and floral structure in some species (Diallo et al. 2014). Thus, ABA and MeJA might affect bamboo flowering by regulating PvMADS56 expression. In addition, ABA was gradually increased with leaf senescence in bamboo (Xie 2004). In our study, the leaves of transgenic Arabidopsis overexpressing PvMADS56 were apt to enter senescence. Therefore, we concluded that PvMADS56 might involve in flowering time, development of floral organs and leaves in bamboo by responding to ABA and MeJA.

SOC1-like proteins are located in cytoplasm, but SOC1 can move into nucleus from cytoplasm when it combines with AGL24 (Lee et al. 2008, 2013; Zhong et al. 2012). The particle bombardment assays showed PvMADS56 locates only in the nucleus, suggesting that the protein might be different in function. SOC1 expression is regulated in emerging floral meristem by AP1 which binds to SOC1 promoter (Liu et al. 2007, 2009). SOC1 can interact with SEP3 as demonstrated by yeast two-hybrid assay (de Folter et al. 2005). In this aspect, PvMADS56 was similar to SOC1 and interacted with PvSEP3 in the yeast two-hybrid assay. Interestingly, PvMADS56 also interacted with bamboo PvAP1, different from SOC1 whose promoter does interact with AP1. These data implied that PvMADS56 might differ from Arabidopsis SOC1 in signal transduction pathways. Overexpression of AP1 or SEP3 both causes early flowering besides abnormal floral organs (Mandel et al. 1992; Wuest et al. 2012). In deed, PvAP1 and DlMADS8 (an ortholog of PvSEP3) exhibit the phenotype of early flowering in Arabidopsis when they are overexpressed (Lin et al. 2009; Tian et al. 2006). Thus, PvMADS56 might be more similar to SOC1 in the aspect of functioning on flowering time than in the aspect of regulating flower development by regulating PvAP1 and PvSEP3 in bamboo.

In conclusion, our data suggested that PvMADS56 in association with PvAP1 and PvSEP3 may involve in regulation of flowering time and development of floral organs and leaves in bamboo through responding to ABA and MeJA. PvMADS56 shared high sequence similarity with while functions differently from many other SOC1 homologs. Our results promote a better understanding of the underlying mechanism of bamboo flowering.

Author contribution statement

Shinan Liu conducted RT-qPCR assays, promoter cloning, activity measurement, Arabidopsis cultivation, phenotype observation, subcellular location, yeast two-hybrid assay, analyzed data and wrote the manuscript. Tiantian Qi and Jingjing Ma performed the cultivation of Arabidopsis. Tengfei Ma carried out gene cloning. Xinchu Lin and Luyi Ma designed the experiments and revised the manuscript.