Introduction

The timing of the transition from vegetative to reproductive growth is important for the reproduction and productivity of flowering plants (Putterill et al. 2004). The regulatory pathway controlling flowering time has been extensively investigated in monocots and in dicot model plants (Trevaskis et al. 2006; Kim et al. 2009; Andres and Coupland 2012; Sun et al. 2012; Liu et al. 2013). The proposed genes or pathways regulating flowering time in response to external and endogenous signals also function in controlling the transition to flowering (Jaya et al. 2011; Manzano et al. 2011; Wahl et al. 2013).

MADS-box transcription factors, which have a conserved DNA-binding domain, play diverse roles during plant development, with particularly prominent roles in floral organogenesis in some plant species (Kater et al. 2006). In Arabidopsis, SVP is expressed in the early-stage floral primordia (Hartmann et al. 2000; Lee et al. 2007) and has been identified as a flowering repressor. In Arabidopsis, overexpression of AtSVP caused floral meristem indeterminacy by promoting the development of new ectopic floral meristems and aberrant floral morphology, such as flowers with shoot-like structures and sepaloid petals (Masiero et al. 2004; Liu et al. 2007; Gregis et al. 2008). In addition, AtSVP interacted with a flowering repressor, FLOWERING LOCUS C, to directly repress a floral pathway’s integrator gene, FLOWERING LOCUS T, in the leaves and the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 gene in the shoot apical meristem (Li et al. 2008), which suggested that AtSVP played a central role in the flowering regulatory network. Furthermore, AtSVP also interacted with the floral meristem identity genes AGAMOUS-LIKE 24 (AGL24) and APETALA1 (AP1) to repress class B and C floral homeotic genes (Yu et al. 2002; Gregis et al. 2009). A recent study by Fernandez et al. (2014) demonstrated that AtSVP was one of the necessary genes for floral program initiation in vegetative organs. Moreover, AtSVP could also mediate the ambient temperature signal to modulate the timing of the developmental transition to the flowering phase in Arabidopsis (Lee et al. 2007). Further, post-translational regulation of the SVP protein(s) was implicated as a major mechanism for thermoregulation during flowering (Hwan Lee et al. 2014). These results suggested that the versatile AtSVP gene plays crucial and persistent roles during floral transition, meristem development, flower development and flowering time control.

The functions of SVP-like genes from various plant species in the regulation of flowering time and floral organs appear to be conserved, but their functions differ in some cases (Fornara et al. 2008; Li et al. 2010; Wu et al. 2012; Jaudal et al. 2014). For example, overexpression of Medicago SVP genes caused floral defects and delayed flowering in Arabidopsis but affected only floral development in Medicago (Jaudal et al. 2014). Accordingly, ectopically expressing a kiwifruit SVP-like gene in Arabidopsis resulted in morphological abnormalities in inflorescences and floral structures (Wu et al. 2012). However, in tomato, JOINTLESS was shown to be involved in abscission layer formation (Mao et al. 2000). In maize, ZMM19 was responsible for the formation of the large glumes encapsulating the maize kernels (He et al. 2004). In addition to their involvement in regulating the development of floral organs, SVP-like genes also function in controlling floral transition. For example, ectopic expression of the SVP-like gene OsMADS22 induced spikelet meristem indeterminacy in Arabidopsis (Fornara et al. 2008). PkMADS1, an SVP ortholog from Paulownia kawakamii, promoted vegetative growth in Paulownia (Prakash and Kumar 2002). Overexpression of PtSVP prolonged reproductive growth and affected floral development rather than flowering time in transgenic tobacco plants (Li et al. 2010). These results suggested that SVP-like genes have conserved roles in determining inflorescence architecture but diverse roles in regulating flowering time.

Soybean [Glycine max (L.) Merr.] is an economically important legume. Although a gene network regulating flowering has been described in the model plant Arabidopsis (Blazquez 2005) and genes responsible for flowering loci, E1, E2, E3 and E4, have been characterized in soybean (Liu et al. 2008; Watanabe et al. 2009, 2011; Xia et al. 2012), the molecular control of flowering and floral identity in soybean remains unclear. In the present study, we report the molecular and functional characteristics of GmSVP1, a gene that encodes a MADS-box family protein. We demonstrated that GmSVP1 is intensely expressed in floral organs and is responsive to abiotic stresses. Furthermore, ectopic expression in transgenic Nicotiana caused abnormal petal and pistil development and slightly accelerated the flowering time.

Materials and methods

Plant materials and growth conditions

Soybean cultivar “Nannong 86-4” plants were grown in an experimental field at Nanjing Agricultural University. Wild-type and transgenic tobacco plants were grown in the greenhouse under the same conditions with uniform management.

The soybean seedlings with three fully opened trifoliate leaves were used for abiotic stress treatments. Heat and cold treatments were conducted by transferring soybean seedlings into artificial climate boxes at temperatures of 45 and 4 °C, respectively. For salt stress, soybean seedlings were carefully removed, washed to remove excess soil, and then transferred to Hoagland’s solution supplemented with NaCl solution (150 mM). For wounding treatment, compound leaves were scratched with a blade at the beginning of the trial.

For tissue-specific investigation, samples of the tested tissues (roots, stems, leaves, floral organs, developing seeds and pod shell) were collected from soybean plants at the vegetative and reproductive stages. All of the tissues were sampled from three individuals and then immediately frozen in liquid nitrogen and stored at −80 °C until use.

Phylogenetic and structure analysis

The known SVP-like genes were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/). The protein sequences were aligned using ClustalX software (v. 2.0.10) (Altschul et al. 1998). Alignment was visualized using GeneDoc software (v. 2.0) (Nicholas et al. 1997). A neighbor-joining phylogenetic tree was constructed using MEGA (v. 4) with 1000 bootstrap replicates (Kumar et al. 2008).

RNA extraction and PCR conditions

Total RNA was isolated using TRIzol (Promega, USA), and first-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s instructions. Specific primers (GmSVP1-1F and GmSVP1-1R, Table S1) for GmSVPs cloning were designed according to the soybean reference genome (Glyma.Wm82.a1.v1). PCR was performed with TaKaRa LA polymerase (TaKaRa, China) in a total volume of 50 μl. The PCR products were purified with a Gel Extraction Kit (Axygen, USA), subcloned into a pMD19-T vector (TaKaRa, Japan), and confirmed by sequencing.

The gene expression levels were analyzed by quantitative real-time PCR (qPCR) using the ABI 7500 Sequence Detection System (ABI, USA). The specific primers (Invitrogen, USA) were designed to distinguish GmSVP1 and the highly identical GmSVP2 (Table S1). Three technical replicates were used for each sample. The soybean Tubulin gene (AY907703.1, Table S1) and the tobacco 18S gene (Table S1) were used as reference genes for quantitation of gene expression in soybean and tobacco, respectively.

Vector construction and transformation

The open reading frame (ORF) of GmSVP1 was cloned into a binary vector, pMDC83, using the Gateway Technology with Clonase™ II Kit (USA) according to the manufacturer’s protocol. The 2×CaMV 35S::GmSVP1 vector was transferred into Agrobacterium tumefaciens strain EHA105 via the freeze-thaw method (Chen et al. 1994). Tobacco transformation was performed using the leaf-disk method as previously described (Hoekema et al. 1983). The transgenic tobacco plants were initially screened using 50 mg/L hygromycin B after regeneration. Positive transgenic tobacco plants were identified by both regular PCR using two pairs of combined primers (GmSVP1-1F + GmSVP1-1R and 35S-F + GmSVP1-1R, Table 1) and qPCR. For the phenotypic evaluation, the flowering time and flowering duration were assessed. The flowering time was defined as the period from transplanting to flowering of the first flower on top of the tobacco inflorescence as described by Li et al. (2010), and the flowering duration was defined as the number of days from the blooming of the first flower to the formation of the last flower. Thirteen plants were selected for scoring, and the data from ten tobacco plants were analyzed using Microsoft Excel.

Table 1 Two SVP-like genes identified in soybean
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Results

Isolation of GmSVPs and sequence analysis

We used Arabidopsis SVP protein (AT2G22540) as a query (Hartmann et al. 2000) for BLAST analysis of the soybean reference genome (Glyma.Wm82.a1.v1). Two top-scoring hits were identified with a high identity of 84 % to AtSVP (Table 1). We subsequently designated these genes as GmSVP1 (Glyma01g02880) and GmSVP2 (Glyma02g04710) (Table 1). Sequence analyses indicated that GmSVP1 and GmSVP2 have identical genomic structures (9 exons) with a start codon (ATG) located in the second exon (Fig. 1a). A sequence comparison of the GmSVPs with other SVP homologs revealed that both GmSVPs share 91, 90, 84, 84 and 81 % identity with MtSVP (Medicago truncatula), PsSVP (Pisum sativum), SVP (Arabidopsis), AGL24 (Arabidopsis) and PtSVP (Poncirus trifoliata), respectively. Moreover, features shared by the MADS-box transcription factors in plants (Parenicova et al. 2003), such as a MADS_MEF2-like region (77 amino acids), a K-box region (63 amino acids), and a conserved domain at the C terminus, are well conserved in both GmSVP1 and GmSVP2 (Fig. 1b).

Fig. 1
figure 1

Structural analysis of GmSVPs and sequence alignment and phylogenetic relationships of SVP homologs. a Schematic representation of exon/intron organization of GmSVP1 and GmSVP2. The solid black boxes denote exons, and the lines between the boxes denote introns. ATG and TGA represent the start and termination codons, respectively. The scale bar represents 500 base pairs in length. The genomic lengths of GmSVP1 and GmSVP2 are 6209 and 6520 base pairs, respectively. b Multiple alignments of SVP-like proteins. The identical and conservative residues among them are shaded with black or gray. The region under the black solid line represents the conserved domains of MADS_MEF2-like, K-box and C-terminal regions, respectively. c A neighbor-joining tree constructed with SVP-like proteins in various plant species. The numbers indicate the bootstrap support percentages (above 50 %), and the scale bar indicates a divergence of 0.5 amino acid substitutions per site. The protein accession numbers used in the alignment and tree construction are MtSVP (AES96012, Medicago truncatula), PsSVP (AAX47170, Pisum sativum), PtSVP (AAR92206, Populus tomentosa), CtSVP (ACJ09169, Citrus trifoliata), BrSVP (ABG24233, Brassica rapa), TaAGL13 (ABF57917, Triticum aestivum), OsMADS55 (AAQ23144, Oryza sativa), ZMM19 (CAH64526, Zea mays), AGL24 (AT4G24540, Arabidopsis thaliana), SVP (ABU95408, Arabidopsis thaliana), OsMADS22 (NP_001048193, Oryza sativa), BM10 (ABM21529, Hordeum vulgare), JOINTLESS (Q9FUYU.1, Solanum lycopersicum), CaJOINTLESS (AFI49342.1, Capsicum annuum), SVP3 (AF37969.1, Actinidia chinensis), and MPF1 (AAV65498, Physalis pubescens)

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To study the relationship between GmSVPs and other reported SVP-like genes, a neighbor-joining tree was constructed. As shown in Fig. 1c, the tree was split into three major clades. The proteins GmSVPs, MtSVP PsSVP, BrSVP, CtSVP, and JOINTLESS from dicot plant species formed an independent clade I, which was distinct from the monocot plant species clade II. This finding suggested that GmSVPs might share conserved functions with other evolutionarily closer SVP homologs in the dicot clade, but they possessed possible distinct roles for some features of the SVP-like genes (TaAGL13, OsMADS55, OsMADS22, ZMM19 and BM10) in monocot plants. Meanwhile, the SVP-like proteins PtSVP, kiwifruit SVP3 and AtAGL24, and MPF1 from Physalis pubescens formed the distinct clade III, which was more evolutionarily distant from both clades I and II, suggesting the possible functional diversification of these SVP-like proteins. The molecular sequence analyses suggested that both GmSVP1 and GmSVP2 isolated from soybean Nannong 86-4 were putative SVP candidates in soybean.

Expression patterns of GmSVPs

To gain insight into the possible roles of GmSVPs during soybean development, its relative expression levels in various soybean tissues were investigated using qPCR. As shown in Fig. 2a, expression of GmSVP1 and GmSVP2 was detectable in all of the tested tissues except developing soybean seeds and pod shells. Both genes exhibited similar expression patterns in these tissues; however, GmSVP1 displayed somewhat higher expression levels than GmSVP2 in all tested tissues. In contrast with the relatively low expression in roots, stems and calyces, GmSVPs was expressed predominantly in soybean floral organs, such as in floral buds, particularly in stamens and pistils. These observations are consistent with its hypothesized role in determining meristem identity. It is also conceivable that GmSVPs might play unique roles in regulating the development of floral organs in soybean.

Fig. 2
figure 2

Expression patterns of the GmSVPs. a Tissue specificity analysis of GmSVP1 and GmSVP2 in roots (R), stems (St), leaves (L), calyx (C), stamens (Sm), pistils (Pi), petals (Pt), flower buds (B), seeds (Sd) and pod shells (Ps). The data represent the mean values ± SD of three technical replicates. Expression patterns of the GmSVPs under the treatments of cold (b), heat (c), wounding (d) and NaCl (50 mM) (e) at different time points

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The SVP homolog was proposed to respond to environmental stimuli (e.g., temperature fluctuation) (Lee et al. 2007). In the present study, in addition to temperature stimuli (high and low temperatures), we also evaluated the gene expression levels of GmSVPs under wounding and salt treatments. As shown in Fig. 2b–e, the gene GmSVP1 could respond to all four abiotic stresses tested, and it exhibited more active responses to low temperature, wounding and salt treatments than to heat stress. GmSVP1 displayed a fluctuating expression pattern during the cold (Fig. 2b), wounding (after 6 h; Fig. 2d), and salt treatments (Fig. 2e). In contrast, GmSVP1 showed very low expression in response to heat during the first 12 h of treatment but dramatically increased expression at 24 h and slightly decreased expression at 48 h (Fig. 2c). In contrast, GmSVP2 maintained relatively low expression levels or no change in expression under the four abiotic stimuli throughout the test period (Fig. 2b–e).

GmSVP1 affects the development of floral organs in tobacco plants

To identify the functions of GmSVPs, GmSVP1 was ectopically expressed in tobacco. After differentiation, subculture, seedling training, transplantation, and PCR confirmation, a total of 24 T1 positive transgenic tobacco plants were confirmed and selected for further evaluation (Fig. 3). The qPCR analysis also validated the expression of GmSVP1 in each transgenic tobacco plant (data not shown). In general, no significant differences were detected during the vegetative growth stage between T1 2×35S::GmSVP1 transgenic tobacco plants and wild-type tobacco. Phenotypic variations between the transgenic and wild-type plants were observed during the reproductive stages, including the development of the petals, stamens, flower buds, and flowering time (Fig. 3). In wild-type tobacco plants, all stamens were encapsulated in the petal before flowering, and five lobate corolla limbs were observed in each petal (Fig. 3a, d). However, the flowers from two transgenic lines displayed petals with drastic limbs, the stamens of which also grew out of the dehiscent petal (Fig. 3c) rather than being enclosed in the intact petal in wild-type plants (Fig. 3d). Moreover, abnormal phenotypes in the number of stamens (Fig. 3e, f) and development of flower buds (Fig. 3b) were also observed in two 2×35S::GmSVP1 transgenic plants. The abnormalities of these flowers resulted in failures in pollination and seeding. Meanwhile, the impacts of GmSVP1 expression on tobacco flowering time were also investigated. Thirteen 35S::GmSVP1 transgenic plants displayed earlier flowering (top flower) than the wild-type plants (Fig. 3g). However, no significant difference in the duration of flowering was observed between the transgenic and wild-type plants. Collectively, these results suggest that GmSVP1 might be involved in regulating flowering time and the development of floral organs, particularly in regulating stamen and petal development.

Fig. 3
figure 3

Phenotypic analysis between wild-type and transgenic tobacco plants. A wild-type plant showed an intact top flower (TF) (a) and normal coinflorescences (Co) (d); 35S::GmSVP1 transgenic plants showed abnormal floral buds (b) and flowers containing abnormal stamens and petals (c), fused stamens (e), or six stamens (f). Arrows indicate the phenotypic variations. g Transgenic tobacco plants showed earlier flowering than wild-type plants. Double asterisks represents a significant difference at the level of 0.01

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Discussion

Generally, genes that cluster evolutionarily into the same sub-clade in phylogenetic trees often share conserved functional features. In this study, a close evolutionary relationship between GmSVPs and other Papilionoideae subfamily members (Goldblatt 1981) suggested the possibly conserved roles of SVP-like genes within this subfamily (Jaudal et al. 2014). The conserved roles of GmSVP1 in regulating floral organs and flowering time were identified in the present study and also observed in the Arabidopsis and Medicago plants expressing MtSVPs (Jaudal et al. 2014).

The structural analysis demonstrated that the nine-exon organization of GmSVPs was identical to those of AtSVPs (Hartmann et al. 2000) and BrSVP (Lee et al. 2007) but different from the eight-exon organization of PtSVP (Li et al. 2010). In addition, the start codons of GmSVPs and PtSVP were located in the second exon, whereas those of BrSVP and AtSVP were present in the first exon. In turn, a variation in the position of the ATG site of SVP genes might cause a transcriptional regulatory difference. These trans-acting factors were reported to bind to the region between the first exon and the initiation codon (Jeong et al. 2006), which may result in the functional diversity that regulates differences in plant development.

In Arabidopsis, the expression of AtSVP could be detected in roots and leaves, but it was nearly undetectable in advanced flowers (Hartmann et al. 2000). In contrast, GmSVPs were highly expressed in floral organs and some vegetative tissues in our study, which was consistent with the findings of Libault et al. (2010). Our results were also in accordance with the high expression of an SVP-like gene, MPF1, in the petals and stamens of Physalis floridana (He et al. 2010). In addition, the absence of expression of GmSVPs in the developing pod shell and seeds reported here and by Severin et al. (2010) indicated that GmSVPs might function in regulating floral organ development during the early reproductive stages but might not be responsible for the formation of soybean seeds. Similar patterns of SVP expression in those tissues were also observed by He et al. (2010) and Hartmann et al. (2000). The dynamic expression of GmSVPs during the temperature and wounding treatments also suggested that the role of SVP-like genes in ambient temperature perception is functionally conserved, as observed in Arabidopsis SVP (Lee et al. 2007).

Tobacco has been extensively used for characterizing genes related to flowering control (Shin et al. 2011; Huang et al. 2014), such as SVP-like genes (Li et al. 2010; Wu et al. 2014). In this study, the ectopic expression of GmSVP1 in tobacco demonstrated the conserved roles of SVP homologs regarding the regulation of floral transition. In contrast with floral reversion or inhibition phenotypes in Arabidopsis expressing BM1, BM10, or PtSVP (Trevaskis et al. 2006; Li et al. 2010), the tobacco expressing GmSVP1 did not exhibit any inhibition in floral transition other than causing floral organ defects. Our findings are consistent with the observations of Arabidopsis expressing MtSVP (Jaudal et al. 2014). These studies demonstrate that the downstream genes in the SVP-involved pathway are important for floral development (Ciannamea et al. 2006). For instance, SEPALLATA3, a protein regulating floral organ identity, is a common regulatory target shared by SVP and AP1; the latter two proteins are involved in the regulation of flowering time and the determination of floral organs (Chi et al. 2011; Gregis et al. 2013). In addition, functional diversification of SVP-like genes in controlling time to flower has also been observed (Fornara et al. 2008; Li et al. 2010; Wu et al. 2012; Jaudal et al. 2014). In this study and a previous study (He et al. 2010), both GmSVP1 and MPF1 promoted flowering. However, ectopic expression of PtSVP and LpMADS10 did not significantly affect the flowering time in tobacco plants (Ciannamea et al. 2006); AtSVP was regarded as a flowering repressor (Hartmann et al. 2000). Here, epistasis between ectopically expressed GmSVP1 and tobacco native SVP-like genes (Sierro et al. 2014) might occur, influencing the phenotypic variations in transgenic tobaccos. This possibility requires further investigation.

The expression of SVP or other flowering-regulating genes is tightly controlled by various environmental and endogenous signals (Lee et al. 2007; Hwan Lee et al. 2014). Coordinated regulatory events mediated by these regulatory genes allow plants to initiate flowering and to develop appropriate inflorescence architectures ensuring successful reproduction (Teo et al. 2014). Soybean is a typical photoperiod-sensitive crop. The alleles of GmSVP1 from various soybean germplasm resources with diverse genetic backgrounds might contribute differently to flowering time (Xu et al. 2013). The specific effects of GmSVP1 on dehiscent petals may be applied for soybean improvement through genetic engineering, which will facilitate molecular breeding in self-pollinated soybean crops. Our future work will be devoted to elucidating the detailed roles of GmSVP1 in the regulatory network of floral identity.