Introduction

To produce progeny and perpetuate the species, flowering plants must flower in time, where a successful initiation of flowering is critical and known controlled by multiple endogenous signal pathways under the influences of environmental factors. The well-studied pathways include photoperiod, autonomous, vernalization and gibberellin-dependent signaling (Johanson et al. 2000; Kin et al. 2009). Although they interact with each other and form complex regulatory networks, they converge onto a handful of floral integrator genes including SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), FLOWERING LOCUS (FT), LEAFY (LFY) and FLOWERING LOCUS C (FLC) (Boss et al. 2004; Amasino and Michaels 2010; Fornara et al. 2010).

SOC1 is a crucial integrator that aggregates signals from different flowering pathways and a flowering activator in Arabidopsis (Lee et al. 2000; Samach et al. 2000; Moon et al. 2003). In the photoperiod pathway, SOC1 is regulated by CO through FT, causing an early flowering (Yoo et al. 2005). Under short days, GA can activate the expression of SOC1 (Moon et al. 2003). As a strong repressor of flowering, FLC is the merging point of the autonomous and vernalization pathways (Moon et al. 2003), and can inhibit SOC1 expression directly and indirectly by binding to the SOC1 regulatory region (Hepworth et al. 2002). In addition to control flowering time, SOC1 can regulate floral meristem determinacy and floral patterning by activating LFY and AP1 (Liu et al. 2009; Melzer et al. 2008; Lee and Lee 2010). To date, SOC1-like genes have been cloned and studied from many plants including Oryza sativa (OsSOC1), Triticum aestivum (WSOC1), Zea mays (ZmMADS1/3), Vigma unguiculata (VuSOC1), and Vitis vinifera (VvSOC1). The results demonstrate that they are widely expressed in various tissues such as roots, leaves, shoot meristems and floral organ primordia (Nakamura et al. 2005; Papaefthimiou et al. 2012). Additionally, SOC1-like genes promote flowering when overexpressed, similar to SOC1, but some involve in the floral development as well. Although SOC1 genes are conserved and have similar expression patterns among plant species, their functions can be divergent in angiosperms (Lee and Lee 2010; Zhong et al. 2012). Ectopic expression of UNSHAVEN (UNS), a SOC1-like gene of Petunia hybrid, leads to ectopic trichome formation on floral organs and conversion of petals into organs with leaf-like features accompany by early flowering phenotype (Ferrario et al. 2004; Zhong et al. 2012). A homolog of SOC1 in Prunus armeniaca, ParSOC1, may be involved in the regulation of dormancy break of the vegetative shoots (Trainin et al. 2013). Moreover, SOC1 also involves in stomatal regulation (Kimura et al. 2015). Thereby, a detail study may be required to uncover its functional conservation and divergence for a specific plant.

Bamboo belongs to grass family but differs from many other members in the kingdom; it has elusive characteristics in flowering. The fact that it has an unpredictable and long vegetative period before flowering, and undergoes death after flowering brings challenges to investigate its flowering molecular mechanisms. Additionally, numerous factors, such as biological clock, nutrition, temperature and soil conditions, and endogenous hormones (CTK, GA and ABA), all are reported to affect bamboo flowering (Janzen 1976; Keeley and Bond 1999). However, the flowering mechanism of bamboo still remains to be elucidated.

SOC1-like genes have been identified and found to play a conserved role as a key flowering gene in eudicot and monocot plants, but SOC1 homologs in bamboo have not been reported. In our study, a SOC1-like gene was isolated, referred to as PvSOC1, from Phyllostachys violascens (Poaceae: Bambusoideae), and characterized its biological properties. The gene was analyzed through its sequences structure, expression pattern in bamboo, and transgenic expression in Arabidopsis and rice. The results showed that the gene is conservative in the promotion of bamboo flowering and regulation of floral organs development, but different from most known SOC1-like genes, it might additionally regulate leaf development.

Materials and methods

Plant materials and growth conditions

All Ph. violascens samples for isolation and quantitative real-time PCR were collected in the campus of Zhejiang A & F University. Wild-type and soc1 mutant (SALK_138131C) Arabidopsis thaliana in Columbia-0 background as well as 35S::PvSOC1 transgenic Arabidopsis plants of the same background were cultivated under the condition of 16 h light/8 h dark at 22 °C. All rice of transgenic lines and wild-type plants used here were from O. sativa cv. Zhonghua11, which were cultivated in the paddy field of Lin’an (Hangzhou, north latitude 30°14′ and east longitude 119°42′).

Isolation of PvSOC1 cDNA and its promoter from Ph. violascens

Isolation of total RNA from Ph. violascens was performed using a Trizol Method. To isolate a SOC1-like gene from Ph. violascens, we compared the amino acid sequences of SOC1 homologs from grass family plants including O. sativa, T. aestivum and Zea mays (Vandenbussche et al. 2003; Nakamura et al. 2005), and designed the primers (S1 and S2 in Table 1) from the conserved regions. Specific SOC1-like cDNA fragment (approximately 250 bp) was amplified using the pair of primers (S1 and S2). The cDNA fragment was then ligated into the pMD18-T vector, and confirmed as partial sequence of a SOC1-like gene base on sequencing. To acquire the full-length sequence of this cDNA, 3′-RACE (Rapid Amplification of cDNA ends) was performed with the gene-specific primers (3′GSP1 and 3′GSP2, Table 1) base on the manufacturer’s recommendation (Invitrogen Company). The 5′ sequence of the gene was obtained by sequence alignment against the known PvSOC1 partial sequence and the RNA-seq database of Ph. violascens. Eventually, the full-length cDNA sequence was obtained by the primers (FGSP1 and FGSP2) based on the known 5′ and 3′ sequences. Genomic DNA was isolated from the collected leaves using modified CTAB method. Two primers (WGSP1 and WGSP2, Table 1) were designed according to the known 5′UTR sequence. The 800 bp fragment was isolated from the DNA using BD GenomeWalker kit following the manual instruction. A sequence of 1496 bp was then obtained by the primers S3 and S4 that were designed by using the acquired sequence and Phyllostachys edulis transcriptome database (Peng et al. 2013).

Table 1 Primers used in this study
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Bioinformatics analysis

The proteins sequences of homologs were obtained through BLAST searches in the NCBI database. The phylogenetic tree was built using neighbor-joining method of MEGA version 5.0 software with bootstrap values obtained form 1000 replications (Tamura et al. 2011). The molecular weights (MW) and theoretical isoelectric point (pI) of the protein was analyzed using the software ProtParam from ExPASy (http://exPasy.org).

Gene expression assay

Total RNAs from collected samples of the non-flowering and flowering Ph. violascens was extracted. The gene-specific primers were designed in accordance with the PvSOC1 ORF while PheUBC18 was used as a reference (Qi et al. 2013) (Table 1). RT-qPCR reactions were run on the instrument of CFX96TM Real-Time PCR Detection System (Bio-Rad) following the 2 × SYBR Premix Ex Taq II mix manuals. 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 1), and 1 µL cDNA template (50 ng/µL). The sample was made up with water to a final volume of 20 µL. The data was analyzed by the 2−∆∆Ct method (Livak and Schmittgen 2001).

Plasmid construction and transformation

The full-length ORF of PvSOC1 gene was ligated into the binary vector pCAMBIA1301 to generate the plasmid 35S::PvSOC1. The plasmid was transferred into wild-type Arabidopsis plants (Col-0) using the floral dipping method mediated by Agrobacterium tumefaciens strain GV3101 (Clough and Bent 1998). All transgenic Arabidopsis seeds were picked out on 1/2 MS solid medium containing 50 mg/L hygromycin. The same construct was also transformed into A. tumefaciens strain EH105 and then into wild-type rice plants (Zhonghua 11) according to Agrobacterium-mediated co-cultivation methods (Lee et al. 1999). Positive transgenic lines were confirmed by genomic PCR.

GA treatment

The experiment of GA treatment was performed as described by Liu et al. (2016). GA3 treatment was performed by ejecting the leaves of Ph. violascens seedlings with the GA3 solution (40 mg L−1 GA3, 0.02 % Silwett-77) or water as mock solution once a day for 10 days. GA3 treatment was carried out with two biological replicates. Then total RNA of the leaves was extracted for further detecting the PvSOC1 transcript level.

Results

Isolation of PvSOC1 gene

To isolate SOC1-like gene in bamboo, a cDNA fragment (approximately 250 bp) was obtained by amplification using primers designed based on the conserved regions of the gene in the known grass family plants. The 5′ sequence was obtained by blasting the 250 bp sequence and Ph. violascens RNA-seq database (data not shown), while the 3′ sequence was amplified by 3′-RACE. The full-length cDNA of 1025 bp was cloned by aligning the 5′ and 3′ sequences, referred to as PvSOC1, which contains a 681 bp ORF that encoding a protein of 226 amino acids. Analysis of PvSOC1 amino acid sequence showed the theoretical values pI/Mw of 8.99/25653.3. Like other MADS-box proteins, PvSOC1 also contains the more conserved M and K domain as well as the less conserved I domain and the divergent C-terminal region (Fig. 1). Phylogenetic comparison of the PvSOC1 protein with its orthologs in other plants showed that PvSOC1 was closely related to these from the monocots rather than eudicots, especially the monocot Ph. edulis and O. sativa (Fig. 2).

Fig. 1
figure 1

Alignment of the amino acid sequences of PvSOC1 and its orthologs from other plant species. The regions of MADS domain, K domain and the specific SOC1 motif are underlined

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Fig. 2
figure 2

Phylogenetic analysis of PvSOC1 and its orthologs from various plant species. The phylogenetic tree was generated with the neighbor-joining algorithm. Bootstrap values (>50 %) in 1000 replicates are indicated next to the nodes. The PvSOC1 is underlined

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Expression pattern of PvSOC1

To characterize the expression pattern of PvSOC1, tissue-specific expression analysis was carried out by RT-qPCR using aerial tissues. Although PvSOC1 transcripts were detectable almost in all tested organs (Fig. 3), including culm, leaf bud, young leaf, mature leaf, as well as floral bud, they were mainly expressed in leaves (leaf bud, young and mature leaf). Comparison of PvSOC1 expression in non-flowering and flowering plants showed that the expression levels of PvSOC1 exhibited no remarkable change in culms but significant change in leaf buds, young or mature leaves. In the non-flowering plants, the expression level of PvSOC1 decreased with differentiation and development of leaf from leaf bud, young leaf to mature leaf but increased with leaf development in the flowering Ph. violascens plants. In addition, PvSOC1 expression level in the floral bud was medium among other tissues.

Fig. 3
figure 3

Relative expression level of PvSOC1 in different aerial tissues of flowering and non-flowering Ph. violascens

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Comparison of the promoter sequences for PvSOC1, SOC1 and OsSOC1

To analyze the correlation between the expression patterns of PvSOC1, AtSOC1 and OsSOC1 genes and the regulation of their promoters, their promoter sequences were assessed. A 1496 bp 5′ flanking sequence of PvSOC1 gene was cloned, and the promoter sequences of AtSOC1 and OsSOC1 in the same length were extracted from NCBI database. PvSOC1 shared 36.26 and 45.51 % similarity with SOC1 and OsSOC1 by sequence alignment, respectively. Using PlantCARE, we predicted the existence of potential cis-acting regulatory elements. The typical CAAT-box, TATA-box core elements, and other elements associated with light responsive and hormone such as G-box and Sp1 were commonly found in these three promoters (Fig. 4). However, the promoter sequences of PvSOC1 and OsSOC1 shared more of the same cis-acting elements, such as A-box, CCGTCC-box, MBS and TCA element, while G-Box, LTR, P-box and dOCT were only found in the promoter of PvSOC1 (Table S1).

Fig. 4
figure 4

The sequence of promoter from PvSOC1. Some specific elements were underlined

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Ectopic expression of PvSOC1 promotes to flower in Arabidopsis

To explore the functional role of PvSOC1, its open reading frame was fused to the pC1301 vector with CaMV 35S promoter and introduced into wild-type Arabidopsis. Two independent lines in the homozygous T3 generation were chosen for further analysis. The 35S::PvSOC1 transgenic Arabidopsis plants displayed the phenotype of earlier flowering (Fig. 5a, b) with abnormal flower organs and leaves. Although both petals and sepals were smaller in size, petals exhibited wrinkles and valgus (Fig. 5d). In addition, the leaves of the transgenic plants became wider and shorter with more oval margin (Fig. 5c). Besides, we further overexpressed PvSOC1 in soc1 mutants Arabidopsis to testify if PvSOC1 could compensate for the phenotype of delayed flowering because of the loss of SOC1. As shown in Fig. 5e, f, PvSOC1 in socl mutant completely rescued the late flowering phenotype.

Fig. 5
figure 5

Overexpression of PvSOC1 in Arabidopsis caused early flowering. a Flowering time of 35S::PvSOC1 (Line1 and Line5) (n = 20) in T3 homozygous transgenic plants under LD conditions by counting the number of rosette leaves, b 35S::PvSOC1 in wild-type Arabidopsis accelerated flowering, bar 3 cm, c Leaves phenotype 35S::PvSOC1 plants, d flower organs phenotypes of 35S::PvSOC1 plants, bar 1 cm, e flowering time (Line6, Line8 and Line9) (n > 20) in T3 homozygous transgenic plants carrying 35S::PvSOC1 in soc1 background, f bar 2 cm. **The difference was extremely significant as compared to wide type (p < 0.01)

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PvSOC1 overexpression accelerates flowering and produce shortened panicles and stems in rice

To further recapitulate its function, PvSOC1 was transformed into O. sativa (rice) that is another member in the same grass family as bamboo. Four independent lines in the homozygous T3 generation were selected to analyze the flowering time. An early flowering phenotype was observed in 35S::PvSOC1 transgenic rice plants (Fig. 6a, d). Moreover, not only overexpression of PvSOC1 in rice resulted in shortened panicles (Fig. 6c, e) and stems (Fig. 6b, d), but also smaller leaves and lower fertility (Fig. 6e).

Fig. 6
figure 6

Overexpression of PvSOC1 in rice caused early flowering. a–c Days to heading, height and panicle length of 35S::PvSOC1 (Line13, 19, 27 and 30) in T3 homozygous transgenic plants under LD conditions (n = 10), respectively; *the difference was significant as compares to wild type (p < 0.05); **The difference was extremely significant as compared to wide type (p < 0.01), d 35S::PvSOC1 promoted flowering and made the heighted reduced, e 35S::PvSOC1 displayed shortened panicle. Bar 5 cm

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GA increases PvSOC1 expression level in the leaf of Ph. violascens seedlings

Comparison of the promoter sequences among PvSOC1, SOC1 and OsSOC1 indicated that a P-box element regulated by gibberellic acid is present only in PvSOC1 promoter. To determine if GA affects PvSOC1 expression, GA3 treatment was carried out in Ph. violascens plants. The result showed that the level of PvSOC1 mRNA in the leaf was significantly higher in the GA3-treated seedlings than in the mock (Fig. 7).

Fig. 7
figure 7

Effects of GA3 on the expression level of PvSOC1 in leaf of Ph. violascens seedlings. Asterisks indicate significant differences (*p < 0.05); error indicates the standard deviation

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Discussion

We isolated and characterized a SOC1 homolog, PvSOC1, from Ph. violascens. The gene encodes a protein containing a conserved SOC1-motif that exists only in SOC1 homologs from angiosperms and gymnosperms (Vandenbussche et al. 2003; Nakamura et al. 2005). Base on phylogenetic analysis, PvSOC1 can be categorized into monocot group. Its protein sequence was more similar to SOC1-like proteins from Ph. edulis (with 99.56 % identity) and O. sativa (with 80.17 % identity) compared with homologs from other plants, suggesting that PvSOC1 is a SOC1 homolog in Ph. violascens. In Arabidopsis, SOC1 gene acts as an activator and acts a critical part in mediating flowering time (Parcy 2005). When it is overexpressed, OsSOC1 promotes flowering in Arabidopsis and rice (Tadege et al. 2003; Lee et al. 2004). Studies also show that other SOC1-like genes are conserved in promoting flowering (Shitsukawa et al. 2007; Zhong et al. 2012). In this paper, an early flowering phenotype was too observed in both 35S::PvSOC1 Arabidopsis and rice plants. Meanwhile, overexpression of PvSOC1 completely rescued the delayed flowering phenotype in soc1 mutant. These results suggest PvSOC1 is conservative in the promotion of flowering time.

Besides its function in regulating flowering, SOC1-like genes are found to affect flower development in some plants. Downregulation of SOC1 transcript in floral meristems is liable for the precocious development of floral organs, while its upregulation partially results in the reversion of inflorescence meristems into floral meristems (Gregis et al. 2006; Liu et al. 2007, 2009). Overexpression of GhSOC1 in gerbera messes up the development of floral organ (Ruokolainen et al. 2011). In petunia this leads to unshaven floral organs with ectopic trichome formation and leaf-like petals (Ferrario et al. 2004). In orchids, this suppresses the development of perianth organs (Ding et al. 2013). Likewise, we proved that overexpressed PvSOC1 in Arabidopsis led to smaller floral organs, wrinkled and valgus petals. Moreover, shortened panicles and low fertility were observed in 35S::PvSOC1 transgenic rice plants. These data suggest that PvSOC1 might involve in floral organs development.

SOC1-like genes from Arabidopsis, O. sativa, Hordeum vulgare and T. aestivum are highly expressed in leaves but less in flowers (Shitsukawa et al. 2007; Komiya et al. 2009; Papaefthimiou et al. 2012). For instance, rice SOC1 ortholog, OsSOC1, is scarcely expressed in the inflorescence and floral meristems (Komiya et al. 2009). Its expression pattern shows that the gene mediates flowering by manipulating other flowering regulators in leaves (Ding et al. 2013). Unlike OsSOC1, PvSOC1 was expressed in tested organs but preferentially in leaves in both flowering and non-flowering Ph. violascens. Besides, the abnormal leaves were observed in 35S::PvSOC1 transgenic Arabidopsis and rice plants. Therefore, we speculate that unlike other SOC1-like genes, PvSOC1 might involve in leaf development in addition to promoting flowering. This notion is supported by the observations that PvSOC1 expression level decreased with differentiation and development of leaves in the non-flowering Ph. violascens while it increased with leaf development in the flowering plants. Previous data suggest that compared with the leaves of non-flowering bamboo, the leaves of flowering bamboo were quite abnormal. They became smaller in size and weaker photosynthesis, and were susceptible to senescence during leaf development (Qiu et al. 2002). These data suggest that an underlying connection may exist between physiological characteristics and the different expression pattern of PvSOC1 in the flowering and non-flowering bamboo.

To clarify if the different expression patterns of PvSOC1, OsSOC1 and SOC1 were correlated with the regulation of their promoters, we compared their promoter sequences. It turned out that some regulatory elements are unique and not shared among the three promoters. Notably, a P-box, the cis-acting element involving in the GA response, exists only in PvSOC1 promoter. Our experiments show that GA can upregulate the expression level of PvSOC1 upon GA3 treatment. The flower bud of Ph. violascens is easier to differentiate under the low level of GA (Lu et al. 2012). These data suggest that GA might regulate PvSOC1 expression, which in turn mediates bamboo flowering and leaf development.

In conclusion, we found that the sequence and expression pattern of PvSOC1 are evolutionarily conserved compared to SOC1 orthologs from other plants, in particular, monocots. PvSOC1 may not only regulate flowering time but also affect flower organs as well as leaf development by responding to GA. Putting together, our data lay a solid foundation for further disclosing the flowering mechanism in bamboos.

Author contribution statement

Shinan Liu involved cultivation of Arabidopsis and rice, data analysis, and wrote the manuscript. Tengfei Ma involved in gene and its promoter cloning, and cultivation of Arabidopsis. Xinchun Lin and Luyi Ma designed the experiments and revised the manuscript. All authors have declared no conflicting interests.