Introduction

Black locust (Robinia pseudoacacia L.) is native to the Appalachian Mountains of the southeastern United States. This species was introduced to China in the late 1700s, and it is cultivated as a popular ornamental in mild regions throughout the world (Isely and Peabody 1984). Because of its adaptability to various climates, black locust was introduced to China in the early twentieth century (Requena et al. 2001). Its flowers, bark and roots are rich in many types of flavonoids, which can be used for drug development, the treatment of disease and in the food industry (Garlock et al. 2011). In addition, black locust grows quickly and has a pleasant fragrance, which makes it a useful component of secondary forests in gardens and on roads. It has been suggested that black locust can tolerate abiotic stresses, such as salt, low temperature, high CO2 concentrations and drought (Leal et al. 2012). However, knowledge of the response to salt stress at the level of gene expression is limited.

Currently, the worldwide population of black locust is growing rapidly. Saline soils are becoming increasingly common, directly affecting the growth and reproduction of crops. Research has shown that salt stress causes three chief harmful effects on plant growth and development: the generation of damaging reactive oxygen species, osmotic stress and ion toxicity (Hasegawa et al. 2000; Jayaraman et al. 2008; Munns and Tester 2008). To adapt to salt stress, signaling and regulatory pathways are altered via cellular and metabolic reprogramming. The SOS signaling pathway promotes plant salt tolerance by controlling ion homeostasis, and it has an important regulatory function in salt stress. The Salt Overly Sensitive (SOS) signaling pathway controls the dynamic balance between Na+ and K+ and is triggered by Ca2+. Additionally, members of the SOS protein family have been shown to regulate Na+ transport. To understand the mechanisms by which salt tolerance is regulated in plants, a detailed molecular-level study of the salt stress response is indispensable (Zhang et al. 2013). Generally, the identification of genes involved in salt stress is the first essential step toward understanding the molecular genetic mechanisms of abiotic stress responses (Kang et al. 2010; Zou et al. 2011).

Several techniques are currently available for transcriptomic analysis, including representational difference analysis (RDA), cDNA microarray and suppression subtractive hybridization (SSH) (Breyne and Zabeau 2001). Additionally, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis is widely used to isolate genes from various plant species because it is highly sensitive, efficient and reproducible. Compared with mRNA fingerprinting, cDNA-AFLP analysis is a less labor-intensive method for determining differential gene expression. cDNA-AFLP analysis has also been applied in many studies to evaluate genetic diversity (Jena et al. 2015).

In this work, to characterize TDFs in salt-stressed black locust, we used cDNA-AFLP analysis to detect and isolate the genes involved in salt tolerance in black locust. Based on this analysis, we can understand the mechanism of salt stress responses and improve plants’ tolerance to salinity.

Materials and methods

Plant materials and salt stress treatments

Black locust plants were obtained from the Beijing Forest University and were cultivated in plastic pots (21 cm in both length and width). When the plants were 2 months old, they were treated with 500 mM NaCl. To prevent salt shock, the NaCl concentration was gradually increased by a predetermined concentration, and the plants were irrigated (500 mL/pot) every 3 days. As a control (CK), a set of plants was irrigated with the same amount of nutrient solution without salt. After 0, 5, 10 and 15 days of exposure to the NaCl treatment, leaves were randomly cut and stored at −80 °C.

RNA extraction

Total RNA from 500 mg of leaf tissue was extracted according to the method described by Pawlowski (Pawlowski et al. 1994) and purified with RNA purification reagent (Life Technologies, Carlsbad, CA, USA). The quality of the RNA was checked via 0.8 % agarose gel electrophoresis with 0.5 μg/mL EB. The RNA concentration was estimated by measuring the absorbance at 260 and 280 nm with a spectrophotometer (UV-1800, Japan).

cDNA synthesis and AFLP analysis

The AFLP protocol and reaction conditions were adapted from previous reports with minor modifications. The cDNA-AFLP procedure was conducted using an M-MLV RTase cDNA Synthesis Kit (TaKaRa, Kyoto, Japan) for second-strand synthesis. The template for the AFLP reaction was prepared using an AFLP Template Preparation Kit (TaKaRa) according to the manufacturer’s instructions. First, 150 ng of cDNA was digested with EcoRI (Invitrogen, USA) for 4 h at 37 °C, after which it was digested with Mse I at 65 °C for 20 min. Then, the digested products were ligated to double-stranded EcoRI and MseI adapters at 20 °C for 2 h.

All primers were synthesized by Shanghai Sangon Biotech (Shanghai, China). The digested DNA was ligated to the oligonucleotide adaptors using T4 DNA ligase (TaKaRa, China) at 4 °C overnight. The ligated products were amplified using their corresponding pre-amplification primers. The pre-amplification reaction was performed with the following conditions: a 30 s preliminary denaturation at 94 °C; 30 cycles of a 30 s denaturation at 94 °C, 30 s annealing at 56 °C, and a 1 min elongation at 72 °C; with a final 10 min extension at 72 °C. The pre-amplification products were diluted 20-fold with ddH2O, and 5 μL of the diluted products was used for subsequent selective amplification. The selective amplification used three steps, including an initial denaturation (94 °C for 3 min), a touchdown program including a reduction of the annealing temperature starting at 65 °C (reduction of 0.7 °C per cycle), and the final elongation (72 °C for 10 min). The amplified fragments were detected on a 6 % polyacrylamide sequencing gel containing 8 mol/L urea. The fragments were electrophoresed at 1500 V until the bromophenol blue reached the bottom of the gel, and the DNA bands were visualized with silver staining.

Marker fragment cloning and sequence analysis

The polymorphic TDFs were separated from the agarose gel based on their presence, absence, or differential intensity using a Silver Beads Kit (TaKaRa, China) and cloned into the pMD18-T vector (TaKaRa, China). Then, the TDFs were propagated by introducing the recombinants into E. coli cells (DH5α TOP10). Sequencing was performed by the Bioasia Co. (Shanghai, China).

Real-time PCR analysis

To detect the expression of the TDFs, real-time PCR was performed using total RNA samples from leaf tissue taken after four different periods of NaCl treatment (0, 5, 10, and 15 days). To perform the qRT-PCR, a SYBR Premix Ex Taq Kit (TaKaRa, Kyoto, Japan) with SYBR Green II was used, along with the primers. Each reaction was performed in a 20 μL volume containing 0.2 μL of SYBR Ex Taq premix (5 U/μL, Takara, Dalian, China), 1 μL each of 4 μM forward and reverse primers, 2 μL of cDNA and 6 μL of sterile H2O. The amplification was performed as follows: 95 °C for 1.5 min; 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s; and 79 °C for 1 s. The Ct values for the transcripts of each gene were normalized to the Ct value of an internal control gene, actin, and the transcript levels of each gene were calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001).

Table 1 BLASTn alignment of differentially expressed R. pseudoacacia fragments
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Sequence analysis of the TDFs

Homology analysis of the TDF sequences was performed using the BLAST function of the National Center for Biotechnology Information (NCBI) database. The sequences were manually curated for their putative functions based on the analyses of scientific documents and information reported by the Gene Ontology consortium.

Results

cDNA-AFLP analysis

A total of 256 primer combinations were screened, and sixty combinations successfully produced scorable, high-intensity, reproducible and relatively highly polymorphic bands. cDNA-AFLP analysis of the four different stages with these sixty primer pairs yielded 3000 reproducible fragments, with an average of 63 fragments per primer combination. Based on their presence, absence, or differential intensity in the gels (data not shown), a total of seventy-four TDFs were unequivocally expressed, and they ranged in size from 100 to 2000 bp. The results indicate the existence of different bands under different treatments.

TDF identification and analysis

The seventy-four TDFs were cloned and sequenced, then analyzed using the GenBank database. Based on this analysis, the TDF sequences were assigned an assumed biological function. Thirty TDFs showed high homology with known genes, and based on BLAST, they could be largely classified into 6 groups: metabolism-related factors (18 %), defense-related proteins (4 %), transcription factors (1 %), stress and signal transduction-related factors (4 %) and energy-related factors (13 %) (Fig. 1). The accession numbers and DNA sequences of the TDFs are shown in Table 1.

Fig. 1
figure 1

The functional categories of transcripts found in R. pseudoacacia leaves using cDNA-AFLP analysis, as determined via gene homology

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Fig. 2
figure 2

Expression analysis of TDF #166, TDF #143, TDF #87, TDF #210, TDF #230, TDF #207, TDF #275, and TDF #288 in the leaves of salt-stressed Robinia pseudoacacia L. seedlings using qRT-PCR. The bars indicate standard error (±SE), which was calculated from three independent experiments (P ≤ 0.05)

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The TDFs represent protein-coding genes. TDF #227 was classified as being involved in energy-related processes and showed significant homology to Populus trichocarpa serine/threonine protein kinase 1 (SOS1) (Diedhiou et al. 2008). The SOS1 gene encodes a Na+/H+ antiporter, and plants that overexpress this gene show high levels of salt tolerance. TDF #276 was classified as being involved in signal transduction and resembled a calmodulin binding protein from Ricinus communis. This TDF plays an important role in the response to salt stress (Galon et al. 2010).

TDF #256 was similar to the AP2/ERF (APETALA2/ETHYLENE) transcription factor (TF), which may play crucial roles in stress responses. Identification of the unknown TDFs, which may represent novel salt-induced genes, could elucidate the mechanisms of salt stress tolerance in black locust.

Validation of differentially expressed transcripts using qRT-PCR

To verify the genes identified via cDNA-AFLP analysis, 8 clones that were likely to represent candidate genes with potential functions in plant salt stress tolerance were selected for real-time PCR studies (Fig. 2). The results showed that of the 8 genes, 6 corresponding TDF fragments were up-regulated and 2 genes were down-regulated across all four treatments. In particular, the expression levels of TDF #210 and TDF #166 were always low after salt stress. Under salt stress, the expression of TDF #143 increased to 1.3-fold that of the control. The expression of TDF #87 after 15 days of salt stress increased to approximately 1.1-fold that of the CK. The basal expression of TDF #207 after 10 days of salt stress was 1.1-fold higher than the control after 15 days. The expression of TDF #230 was very high after 5 days under salt stress. After 5 days of salt treatment, the expression level of TDF #275 increased 2.14-fold. The expression of TDF #288 after 5 days of salt stress was 1.77-fold higher than that after 10 days. These differentially regulated TDFs and their direct and indirect relations with stress responses are discussed in more detail below.

Discussion

cDNA-AFLP analysis is a differential display technique that can greatly reduce the number of false positives by using restriction enzymes to generate cDNA-specific tags, thereby allowing for high-stringency PCR conditions. Compared with other differential display techniques, the cDNA-AFLP technique is reliable, sensitive, and economical, and it has been used for many plants such as Citrullus colocynthis (Si et al. 2009), Leymus chinensis (Yu et al. 2013), and Korean Miscanthus (Yook et al. 2014). In addition, cDNA-AFLP analysis does not require prior sequence knowledge, making it the most suitable technique for our research. Our results indicate that our analytical system is an effective method to search for stress-inducible genes.

Two hundred and ten TDFs involved in plant abiotic stresses, including low temperature, drought, and salt, were identified via cDNA-AFLP analysis (Gong et al. 2014). Guo et al. (2014) used cDNA-AFLP analysis to study Ammopiptanthus mongolicus under cold, drought and heat conditions, and they selected 670 TDFs to sequence. In our work, 74 unique cDNA-AFLP fragments were identified, isolated, and sequenced, and almost half of the genes had unknown functions. Some of the 30 TDFs that were correlated with stress tolerance and/or its genetic regulation had known functions. Eight TDFs that were differentially expressed in the cDNA-AFLP analysis were randomly selected for qRT-PCR.

TDF #227, which corresponds to the Populus trichocarpa protein serine/threonine protein kinase 1 (SOS1), belonged to energy category. The SOS signaling pathway can control the dynamic balance between Na+ and K+. Recent research suggested that plasma membrane Na+ influx can regulate responses to NaCl by reducing vascular long-distance Na+ transfer to leaves and therefore it can protect photosynthetically active tissues under salt stress. Additionally, Na+/H+ antiporters localized in the vacuolar membrane and plasma membrane can separately mediated the accumulation of salt in vacuolated cells and the active extrusion of Na+ from non-vacuolated cells, accordingly, a high-cytosolic K+/Na+ ratio is maintained.

TDF #276, which encodes a Ricinus communis Ca2+-binding protein (SOS3), was grouped into the signal transduction category. In this pathway, Ca2+ increase by Na+-induced may be sensed by a CBL4 (a calcineurin B-like protein) originally identified as SOS3. The physiologically realistic increasing of cytosolic Ca2+ may facilitate the dimerization of CBL4/SOS3 and the subsequent interaction with CIPK24 (a CBL-interacting protein kinase, originally identified as SOS2). In addition, the CBL4/CIPK24 complex is a place that is targeted to a myristoyl fatty acid chain covalently bound to CBL4/SOS3 (the plasma membrane via) enabling the phosphorylation and hence the activation of the membrane bound Na+/H+ antiporter (Ramezani et al. 2013).

According to many reports, overexpressing or knocking down MAPKKKs, MAPKKs, and MAPKs (called MKKKs, MKKs, and MPKs in Arabidopsis), which are components of MAPK (mitogen-activated protein kinase) cascades, can increase or reduce plant salt sensitivity, respectively, showing that they play key roles in regulating plant development and plant responses to salt stress (Puranik et al. 2011; Xu et al. 2008). Droillard et al. (2004) have shown that the expression of these proteins increases in salt-stressed Arabidopsis. In our experiment, functional analysis showed significant homology between TDF #275 and a MAPK from Medicago sativa. This clone was induced after 5 days of salt stress, indicating changes in signal transduction. TDF #143 showed similarity to Medicago truncatula cytochrome P450. Cytochrome P450 s have many functions in plant metabolic processes, including the synthesis of pigments, defense-related compounds, UV protectants, lignin, fatty acids and phytohormones. Mao et al. (2013) investigated the function of CYP709B3 (within the Arabidopsis genome, CYP709B1, CYP709B2 and CYP709B3 are part of the CYP709B family of cytochrome P450 s) in stress responses and found that its expression was induced by 150 mM NaCl stress. In our work, the expression of this gene was higher in salt-stressed black locust than in the control, and it may regulate the salt stress response through a novel pathway that is independent of well-characterized regulators. However, due to limited research on CYP450’s role in salt tolerance, further study is needed.

TDF #230 is similar to the plant calmodulin-binding transcription activator (CAMTA). In Arabidopsis, CAMTA expression is regulated by both biotic and abiotic stress. CAMTA also responds rapidly and differentially to cold, heat, high salt, UV, drought, wounding, H2O2, signal elicitors and phytohormones (Yang and Poovaiah 2002). The expression levels of TDF #230 increased quickly in response to 5 days of salt stress, but they showed a slight decline after 10 days. Moreover, Galon et al. (Galon et al. 2010) have shown that the expression of Arabidopsis CAMTA is up-regulated by both abiotic and biotic stress. The overexpression of CAMTA can increase tolerance to dehydration and salt stress (Singla et al. 2006). TDF #288 shows similarity to Pisum sativum zinc finger proteins. Members of the zinc finger superfamily were recently shown to regulate abiotic stress tolerance and play crucial roles in environmental stress responses (Luo et al. 2014; Wang et al. 2014). Previous research has shown that zinc finger proteins (ZFPs) are transcription factors that can regulate many aspects of plant development. Importantly, ZFPs have many active repressors (Ciftci-Yilmaz and Mittler 2008; Englbrecht et al. 2004; Kielbowicz-Matuk 2012). Under optimal growth conditions, these repressors are thought to limit the expression of stress-activated genes and to make adjustments in response to stress to avoid cellular bursting. In our work, the expression of this gene in black locust was higher in salt-stressed plants than in the control after 5 days, indicating regulation of the transcription factor. TDF #87 showed similarity to the glutaredoxins (Grxs) of Medicago truncatula. Grxs are ubiquitous, small, heat-stable disulfide oxidoreductases that play a vital role in the response to oxidative stress as well as in the regulation of plant development. Guo et al. (2010) used virus-induced gene silencing to show that SlGRX1, a novel cDNA fragment from tomato, promotes resistance to salt and oxidative stress, thereby clearly confirming that a Grx regulates plant responses to salt and drought stress. The expression of TDF #87 increased dramatically after 15 days of salt stress, indicating the expression of stress- and defense-related proteins, but these genes should be studied further. TDF #207 is similar to the F-box proteins of Arabidopsis thaliana, which are characterized by an F-box motif that has been shown to play a pivotal role in regulating various stress responses (Chen et al. 2014). The expression of EID1 (Empfindlicher im dunkelroten Licht 1)-like protein 3, an F-box protein, is rapidly induced under high salt, osmotic stress and upon application of abscisic acid (ABA) (Koops et al. 2011). Koops et al. have used a conditional knock-down mutant to demonstrate that EDL3 can regulate plant development under drought stress. Additionally, the expression of EDL3 rose sharply relative to the control after 150 mM NaCl treatment (Koops et al. 2011). In conclusion, this gene is an important, positively acting factor in salt stress. TDF #207 was repressed in this work, and we will study this gene further in various germplasms to characterize its expression in response to salt stress.

In this study, the transcript levels of TDF #166 and TDF #210 (Phaseolus vulgaris phototropin and Pisum sativum GTP binding protein, respectively), were down-regulated by salt stress. Recent reports suggest that phototropins may work cooperatively with other signaling proteins to regulate plasma membrane H+-ATPases and maintain ion homeostasis (Wang et al. 2008). Plant tolerance to salt, particularly during the recovery phase, involves the plasma membrane G protein-mediated glucose signaling pathway. Another study suggests that members of the G protein family (AtRGS1, AtGPA1, AGB1, and AGG) can mediate responses to NaCl (Colaneri et al. 2014). In Arabidopsis, Na+ activates G signaling primarily via the Regulator of G Signaling 1 (AtRGS1) protein. The mechanism of salt stress survival requires G protein activation via the release of free Gα subunits and Gβγ dimers. Further study is needed to determine whether this activation occurs directly or indirectly via increased glucose levels and whether it requires complex feedback loops. The expression levels of TDF #210 and TDF #166 were always lower under salt stress. There may be two reasons for this phenomenon. First, some genes in unique salt stress-responsive pathways may be suppressed by high concentrations of NaCl (500 mM). Second, the treatments in our experiments were long. It has been reported that RGA1(I) (Rice heterotrimeric G protein alpha subunit) is up-regulated at different time intervals (0, 1, and 2 h) after treatment with 200 mM NaCl. In our study, RGA1(I) and other genes related to this same unique pathway were down-regulated after long NaCl treatments (5, 10, and 15 days). We therefore speculate that some genes are rapidly but transiently activated. By contrast, other genes related to salt tolerance might be highly expressed for a longer period of time.

In summary, the cDNA-AFLP approach was efficiently used to identify differentially expressed genes that were relevant to salt stress. In this study, we identified 74 TDFs, most of which encode corresponding proteins, in response to salinity stress. Taken together, these genes may represent good candidates for involvement in abiotic stress and should be further investigated to determine their role in abiotic stress and their mechanisms of genetic regulation. To some extent, our work will have value as a reference for research on the mechanisms of salt stress tolerance for plant genetic engineering.

Author contribution statement

Fuling Xu developed the algorithm. Mu Peng, Qiuxiang Luo, Xiuli Zhang Mingquan Jiang, and Fuling Xu carried out most of the analyses. Fuling Xu and Fanjuan Meng participated in the design of the study and helped algorithm development. Fuling Xu, Mu Peng, and Fanjuan Meng drafted the manuscript. Fanjuan Meng and Yanjun Li conceived and coordinated the study. Xiuli Zhang Xianchun Zong and Yanjun Li had participated in English-language editing to improve the language in the manuscript. All authors have read and approved the final manuscript.