Introduction

Post-transcriptional control of gene expression plays a vital role in the normal growth and development of eukaryotes. This level of regulation includes pre-mRNA splicing, capping, polyadenylation, mRNA transport, stability, and translation (Burd and Dreyfuss 1994; Dreyfuss et al. 2002). The regulation is accomplished either directly by RNA-binding proteins (RBPs) or indirectly via the interaction between RBPs and other regulatory factors. The superfamily of plant RBPs is extensive compared with other eukaryotes (Peal et al. 2011). In fact, RBPs are characterized by conserved domains in the protein sequence. These domains include RNA recognition motif (RRM, also known as the consensus-sequence RNA-binding domain (cs-RBD)) or ribonucleo-protein motif (RNP domain), K homology motif, zinc finger domain, DEAD/DEAH box, RGG box, and serine-arginine-rich motif, among others. RBPs containing RRMs are the most common among eukaryotes. Structurally, RRM is comprised of 80–90 amino acids and occurs in one or more copies in various RBPs. The RRM consists of a highly conserved octamer or RNP-1 amino acid sequence and a less well-conserved hexamer or RNP-2 amino acid sequence. The RRMs are conserved structurally rather than sequentially (Lorkovic and Barta 2002).

Due to the lack of suitable plant-derived systems in vitro for studying post-transcriptional events, RBPs have received much less attention in plants than in other organisms (Lorkovic 2009), despite the extensive superfamily of RBPs. In plants, the only RBP database available is POGs/PlantRBP (Walker et al. 2007), which contains predicted plant RBPs that are identified by sequence similarity. The genome of Arabidopsis contains 279 RBPs (Anantharaman et al. 2002) compared with 257 RBP genes identified in Oryza sativa L. (Morris et al. 2011), 100 in Caenorhabditis elegans (Crittenden et al. 2002), and 117 in Drosophila (Lasko 2000). However, the functions of only a few plant RBPs have been reported thus far. Interestingly, plant RBPs are emerging with increasing frequency as the regulatory factors in floral transition, floral patterning, abscisic acid (ABA) signaling, stress responses, circadian rhythms, and chromatin modification (Lorkovic 2009; Lu and Fedoroff 2000; Staiger 2001).

Indeed, recent identification and analyses of plant RBPs have elucidated their important role in regulating diverse developmental processes and environmental stresses (Lorkovic 2009). For example, the expression of GR-RBP4, one of the eight GR-RBP family members in Arabidopsis thaliana, increased markedly with cold stress, while dehydration and salt stress strongly increased GR-RBP1 transcription (Kwak et al. 2005). Three UBA2 genes encoding hnRNP-type nuclear RNA-binding proteins were shown to be involved in a novel wound signal transduction pathway (Bove et al. 2008). Recent work found that SaRBP1, an RNA-binding protein from a salt-treated halophyte Suaeda asparagoides, enhanced tolerance to osmotic, freezing, and heat shock stresses when overexpressed in yeast (Ayarpadikannan et al. 2012). HYL1 encoding a nuclear dsRNA-binding protein in the Arabidopsis was associated with ABA-regulation at the transcriptional or post-transcriptional level (Lu and Fedoroff 2000). Similarly, ABH1 encodes the Arabidopsis homolog of a nuclear mRNA-cap-binding protein, and modulated ABA signaling (Hugouvieux et al. 2001). atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions (Kim et al. 2007). These reports support the role of RBPs in stress response.

Alfalfa (Medicago sativa L.) is a perennial forage legume. It is widely distributed in irrigated arid and semi-arid regions worldwide. Water deficit and salinity severely restrict alfalfa growth. In our previous study, we constructed an alfalfa cDNA library induced by salt stress using suppression subtractive hybridization (SSH) technology (Jin et al. 2010). In this study, we cloned an RNA-binding protein gene (MsRBP) from alfalfa based on an EST sequence (GenBank Accession No. FE896907) in the SSH library. We also investigated the subcellular location of MsRBP and its expression patterns under different stresses by using real-time RT-PCR. Furthermore, we overexpressed MsRBP in Arabidopsis and examined the effect on seed germination and seedling growth of transgenic plants under salt stress.

Materials and methods

Plants and stress treatments

Seeds of alfalfa (Medicago sativa L. cv. Zhongmu-1, a salt-tolerance alfalfa cultivar) were sown on MS medium and germinated in the dark at 25 °C for 1 day, followed by growth under alternating 16-h light and 8-h darkness at 25 °C. We transferred 6-day-old seedlings into Hoagland’s nutrient solution. The solution was changed every 5 days. We transferred 2-week-old seedlings into Hoagland’s nutrient solution supplemented with 20 % PEG6000, 100 μM ABA, and 300 mM NaCl, respectively. PEG6000 was used to simulate drought stress. ABA was used to simulate endogenous ABA after salt stress. Arabidopsis thaliana ecotype Col-0 was used as a recipient host for plant transformation. Arabidopsis plants were grown in a greenhouse at a constant temperature of 22 °C with 16-h light and 8-h darkness.

Isolation of MsRBP gene from alfalfa

Based on one EST sequence (GenBank Accession No. FE896907) from an alfalfa salt-stress-induced SSH cDNA library, two gene-specific primers (GSP) were designed to amplify the 3′- and 5′-ends of MsRBP. P1: 5′-AGTTTATTAGCCGTGCTGGTGCTGAGA-3′ was used to amplify the 3′-RACE end, and P2: 5′-AACAGCAACATCAACAACAACCACCCT-3′ was used to amplify the 5′-RACE end with Advantage™ 2 PCR Enzyme kit (Clontech, USA). Total RNA was isolated from 2-week-old alfalfa seedlings by TRIZOL reagent (Invitrogen, USA) according to the manufacturer’s instructions. Any contaminated genomic DNA was removed by incubating the total RNA with RNase-free Dnase I (Promega, USA) at 37 °C for 30 min. The total RNA was used to synthesize 3′-RACE-Ready-cDNA and 5′-RACE-Ready-cDNA according to the manufacturer’s recommendation of SMART™ RACE cDNA amplification kit (Clontech, USA). Thermocycling was performed for 30 cycles at 95 °C for 3 min, 95 °C for 30 s, 68 °C for 30 s, and 72 °C for 2 min, and an additional polymerization step at 72 °C for 10 min. RACE products were separated by electrophoresis on 1 % agarose gel stained with ethidium bromide and extracted using a DNA gel extraction kit (Takara, Japan). The products were cloned into the pMD-19T vector (Takara, Japan) and then transformed into Escherichia coli DH5α. Recombinant plasmids were sequenced by Biomed Company (Beijing, China). By comparing and aligning the sequences of the known EST, the 5′- and 3′-end sequences, the full-length sequence of MsRBP was obtained.

Bioinformatics analysis

Overlapping and assembly of sequences were accomplished with DNAMAN software (http://www.lynnon.com/). Homology searches were performed with BLAST algorithms (http://blast.ncbi.nlm.nih.gov/) to confirm sequence identity. The Open Reading Frame Finder (http://blast.ncbi.nlm.nih.gov/) was used to search for ORF of MsRBP. The theoretical isoelectric point, molecular weight, and hydrophobic structure of the putative protein were predicted on ExPASy website (http://us.expasy.org). Secondary structure of the putative protein was predicted in PBIL LYON-GERLAND database (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html). The subcellular localization of MsRBP was analyzed by ProtComp version 9.0 software (http://linux1.softberry.com/berry.phtml). Amino acid sequence alignment was carried out by ClustalX 2.0 (http://www.clustal.org/), and the phylogenetic tree of MsRBP and RNA-binding proteins with triple RNA recognition motifs of some other plants was constructed by MEGA 5.0 software with the neighbor-joining method (Saitou and Nei 1987).

Subcellular localization

To observe the subcellular localization of MsRBP, a MsRBP-GFP fusion was constructed and transiently expressed in onion epidermal cells. Two primers P3 and P4 were designed to amplify the coding sequence of MsRBP, one with Xho I restriction site (P3: 5′-TACTCGAGATGATGCAACCAGGAGGACCAG-3′) and another with Spe I restriction site (P4: 5′-GCACTAGTCTATATCCTAATTGCTGTTGCT-3′). The PCR product was digested with Xho I and Spe I and ligated with the pA7-GFP vector (Supplementary Figure 1), which was digested with the same restriction enzymes. The pA7-GFP vector was used as the control. The pA7-MsRBP-GFP vector was then transformed into onion epidermal cells using a particle gun (Bio-Rad, USA) and visualized under laser scanning confocal microscopy (Nikon, Japan).

Transcriptional analysis by real-time PCR

To investigate the expression patterns of MsRBP, 2-week-old alfalfa seedlings were transferred into Hoagland’s solution with NaCl (300 mM), ABA (100 μM), and PEG 6000 (20 %), respectively. Plants were sampled at 0, 2, 4, 10, and 24 h after starting each treatment. The 0-h seedlings were used as control. The roots and shoots were harvested separately. The total RNA was reverse transcribed, and the synthesized cDNA was used as template in real-time PCR. P5: 5′-CCGATGAAGTCAAAACTCTCTGGAT-3′ and P6: 5′-GCACGGCTAATAAACTCAAGAAACC-3′ were used for the amplification of MsRBP. Actin gene was used as a housekeeping gene to normalize the target gene quantities. P7: 5′-TGGGCTGCCACAGAACATTTGA-3′ and P8: 5′-GCTGTGGTTGCTTTTTTGGTGTCTC-3′ were used for the amplification of actin gene. The real-time PCR was performed following the instructions of SYBR Premix Ex Taq™ (Takara, Japan) using the fluorescent quantitative PCR amplification instrument (ABI7500, USA). The relative expression level of MsRBP was calculated by –ΔΔCt method.

Transformation and selection of transgenic Arabidopsis lines

Based on the cDNA sequence, the following two gene-specific primers were designed to amplify the coding sequence, with Xba I and Sma I restriction sites: P9: 5′-TATCTAGAATGATGCAACCAGGAGGACCAG-3′ and P10: 5′-ATCCCGGGCTATATCCTAATTGCTGTTGCT-3′, P9 and P10 were used to amplify the coding sequence of MsRBP without stop codon and fuse with the MsRBP-GUS for overexpression. The amplification product was subcloned into pBI121 vector (Supplementary Figure 1). The pBI121-MsRBP vector was transformed into Arabidopsis with a floral-dipping method by Agrobacterium GV3101 and expressed under the control of CaMV 35S promoter. The transgenic Arabidopsis plants were selected on MS medium supplemented with kanamycin (50 mg/L) and verified by PCR, RT-PCR. The tissue expression pattern of MsRBP was analyzed by GUS staining.

Salt stress regulation in transgenic Arabidopsis

Wild-type seeds and T3 generation transgenic Arabidopsis seeds were sterilized by 0.1 % HgCl2 for 8 min and then rinsed with sterile deionized water. Germination assays were carried out with 40–50 seeds in each MS medium agar plates. The plates were placed at 4 °C for 3 days in the dark and then transferred to normal growth conditions. Each plate was divided into two parts, one each for wild-type and the T3 transgenic Arabidopsis seeds. To determine the effect of salt stress on germination and seedling growth, the medium was supplemented with 0, 75, 125, or 175 mM NaCl. Germinating seeds were identified by radicle protrusion from seed coat. To determine the effect of treatment on seedling growth, we placed the plates vertically in a growth chamber and measured root length every day.

Results

Cloning of a cDNA encoding RBP

An 1,127-bp fragment was isolated by using 3′-RACE PCR, and a 424-bp fragment was obtained by using 5′-RACE PCR. A full-length of 1,551 bp cDNA sequence was assembled by the 1,127 bp 3′-end sequence, 424 bp 5′-end sequence, and the known EST sequence (Supplementary Figure 2). A 1,230-bp open reading frame (ORF) encoding a protein of 409 amino acids was predicted by ORF Finder. Sequence analysis indicated that the theoretical pI of the putative protein was 6.31 and its Mw was 45.6 kDa. Secondary structure analysis of the putative protein indicated α-helix, extended strand, and random coil. The functional site prediction showed three RRMs and a Glu-Pro-rich domain at the C-terminal ends of the protein. It was tentatively named MsRBP (GenBank Accession No. JN986878). A phylogenetic tree was constructed using neighbor-joining method. It was based on the amino acid sequence of MsRBP (GenBank Accession No. AEW68341) as well as RNA-binding proteins containing triple RNA recognition motifs. The result revealed that MsRBP was homologous with RNA-binding protein in other plants, such as XP_003610417 (Medicago truncatula) and XP_004507695 (Cicer arietinum) (Supplementary Figure 3).

Subcellular localization

The online ProtComp version 9.0 program was used to predict the subcellular localization. Based on the integral prediction method, the MsRBP was most likely localized in the nucleus. A strong fluorescence signal was observed in the nucleus of onion epidermal cells transformed with pA7-MsRBP-GFP vector under a confocal laser scanning microscope (Fig. 1a–c). In contrast, the fluorescence signal was distributed throughout the onion epidermal cells transformed with pA7-GFP control vector (Fig. 1d–f). The results indicated that MsRBP was a nuclear-localized protein.

Fig. 1
figure 1

Subcellular localization of MsRBP in onion. The pA7-MsRBP-GFP and pA7-GFP vectors were transformed into onion epidermal cells using a particle gun. The fluorescence signal was examined by a confocal laser scanning microscope. GFP fluorescence from cells expressing MsRBP-GFP fusion protein (a–c). GFP fluorescence from cells expressing GFP protein (d–f). a, d Superposition of the bright and dark vision. b, e Dark field vision. c, f Bright light vision

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Expression analysis of MsRBP mRNA

In order to determine the expression pattern of MsRBP, real-time RT-PCR was carried out to examine the transcriptional levels of MsRBP in alfalfa seedlings under various stresses including high salinity, drought, or abscisic acid (ABA) treatments. The seedlings treated at 0 h were used as control. As shown in Fig. 2, the expression of MsRBP was affected differently depending on stress conditions. The transcriptional level of MsRBP in roots increased up to 5–6-fold after salt stress for 10 h and then decreased marginally afterward, while the MsRBP transcript level in shoots was slightly upregulated by NaCl (Fig. 2a). The transcriptional level of MsRBP was increased sharply after the first 2 h of ABA treatment and then gradually decreased afterward, but still higher than control at 24 h (Fig. 2b). When treated by PEG6000, the relative transcriptional level of MsRBP reached the highest at 4 h in roots, but at 2 h in shoots (Fig. 2c).

Fig. 2
figure 2

Expression level of MsRBP in alfalfa after 300 mM NaCl treatment (a), 100 μM ABA treatment (b), and 20 % PEG treatment (c). The y-axis indicates the increased expression level of relative fold compared with control (0 h), and the expression level of control was set to 1. Vertical bars indicate the mean ± SE of three independent experiments

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Characterization of transgenic Arabidopsis plant

The 35S::MsRBP transgenic plants of T0 generation were screened using kanamycin (50 mg/L) and confirmed further by genomic PCR, RT-PCR. The presence of MsRBP in transgenic plants was confirmed by genomic PCR (Supplementary Figure 4). Thirty-seven independently transformed T0 transgenic plants were selected and grown to maturity to obtain homozygous lines. Three T3 homozygous lines, T31, T33, and T35, were randomly selected and used throughout the study. The MsRBP gene was amplified with cDNA of T3 transgenic plants using P9/P10 primers. The results showed that MsRBP had integrated into transgenic Arabidopsis plants genome and expressed at the transcription level by RT-PCR (Supplementary Figure 4). GUS staining analysis result indicated that MsRBP-GUS fusion was expressed in almost all of the transgenic Arabidopsis (Fig. 3).

Fig. 3
figure 3

GUS staining of wild-type Arabidopsis thaliana and MsRBP-overexpressing lines. a–d The GUS staining of wild-type Arabidopsis thaliana, and overexpression lines T31, T33, and T35

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We examined the effect of MsRBP in germinating seed and seedling growth of Arabidopsis under salt stress. Under normal growth condition, no significant differences in seed germination and seedling growth were observed between the wild-type and overexpressed lines (Fig. 4a). However, a large variation in germination rate and root growth was observed under high-salinity stresses. Under enriched NaCl conditions, increased inhibition of germination was seen. In the presence of 175 mM NaCl, the wild-type seeds germinated significantly earlier than transgenic seeds (Fig. 4). These results suggested that MsRBP negatively affected seed germination under salt stress. We tested the effect of MsRBP gene on seedling growth under salt stress, by measuring the root length of plants grown in 125 mM NaCl MS medium (the root length data in 75 mM and 175 mM NaCl treatments were not shown. In 75 mM NaCl, the difference of root length between transgenic plants and wild-type plants was not significant. In 175 mM NaCl, transgenic plants and wild-type plants can only germinate but not grow). Results indicated that transgenic seeds displayed retarded root growth, compared with wild type (Fig. 5). These results demonstrated that MsRBP played a negative role in seed germination and root growth of Arabidopsis under salt stress.

Fig. 4
figure 4

Effect of salt stress on germination of WT (wild type) and MsRBP-overexpressing lines (T31, T33, and T35). Seeds were germinated in MS medium supplemented with 0 mM NaCl (a), 75 mM NaCl (b), 125 mM NaCl (c), and 175 mM NaCl (d). Vertical bars indicate the mean ± SE of three independent duplicate experiments. Asterisk indicates significant difference of germination rates on day 7 (P < 0.05, Student’s t test)

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Fig. 5
figure 5

Effect of salt stress on root length of WT (wild type) and MsRBP-overexpressing lines (T31, T33, and T35). Root length (mean ± SE) of plants treated with 125 mM NaCl was observed by incubating the seedlings on MS plates in the vertical orientation (a), and the photographs were taken 10 days after germination (b). Data were obtained from three independent duplicate experiments. Asterisk indicates significant difference of root length on day 10 (P < 0.05, Student’s t test)

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Discussion

RNA-binding proteins (RBPs) are important mediators of gene expression and involved in all aspects of mRNA existence. RBPs have been cloned from many plant species (Ayarpadikannan et al. 2012; Churin et al. 1999; Kwak et al. 2005; Morris et al. 2011; Wang et al. 2011). However, their regulatory importance or molecular mechanism of action is not clearly established. Evidence suggests that RNA-binding proteins also regulate plant response to environmental constraints (Ambrosone et al. 2012). RBP transcription is regulated by environmental variables, including dehydration, temperature, light, ABA, high salinity, wound, and low-oxygen stresses (Ayarpadikannan et al. 2012; Kwak et al. 2005; Ambrosone et al. 2012; Fusaro et al. 2007; Park et al. 2009; Sachetto-Martins et al. 2000; Sahi et al. 2007). However, the cloning and molecular characterization of RBP from alfalfa was not previously reported. In this report, we obtained a salt-stress -induced gene MsRBP from alfalfa on the basis of 321 bp EST in a SSH cDNA library. MsRBP contained three RRMs, which are commonly found in RBPs and sufficient for RNA binding.

Previous results showed that the expression of GR-RBP4 was downregulated by either dehydration or salt stress. The overexpressed GR-RBP4 transgenic lines displayed retarded seed germination of Arabidopsis plant under salt or dehydration stress (Kwak et al. 2005). The transcription of atRZ-1a was marginally downregulated by drought stress and was not altered by salt stress (Kim et al. 2005). However, transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth (Kim et al. 2007). While the transcription of GR-RBP2 decreased marginally under dehydration and slightly increased during high salinity, over-expressing GR-RBP2 enhanced seed germination, but not seedling growth under salt stress. GR-RBP2 played no role in seed germination or seedling growth of Arabidopsis plants under drought stress (Kim et al. 2007; Kwak et al. 2005). Therefore, we examined the expression levels of MsRBP in alfalfa by real-time quantitative RT-PCR. MsRBP was induced in alfalfa by NaCl, ABA, or PEG6000, and the expression of MsRBP was affected differently depending on stress conditions. These results also suggested that MsRBP might participate in adaptation to environment conditions in alfalfa. However, the importance of its expression pattern required additional analysis (e.g., Western blot). We constructed plant overexpression vector and transferred MsRBP gene into Arabidopsis successfully. We used 35S::MsRBP seeds of transgenic Arabidopsis plant (T3 generation) and wild type to analyze the germination and seedling growth under salt stress. The results suggested retardation in seed germination rate and root growth of T3 generation compared with wild type in response to NaCl, indicating that MsRBP negatively affected seed germination and root growth under salt stress. These studies illustrate the myriad functional roles of plant RBPs during normal plant development and in response to environmental perturbations, mediated by the structural differences between MsRBP and other RBPs.

Currently, the role of MsRBP in germination and root growth under salt stress is unknown. It has been hypothesized that GR-RBP2 and atRZ-1a may play a role as an RNA chaperone similar to bacterial CSPs (Kim et al. 2005). Probably, MsRBP influences the transcriptional levels of other stress-related genes, or MsRBP binds to the target RNA to regulate the processing, stability, and/or translation of target gene involved in germination and root growth. Our present work provides novel data related to the functional role of RBPs in response to environmental stresses. The discovery of MsRBP gene may contribute to the study of mechanisms of salt tolerance in Medicago sativa L. It also provides an additional tool for the genetic improvement of stress tolerance in crop plants.