Introduction

Salinization and alkalization in soils have detrimental effects on the growth, development and differentiation of plants and cause lower productivity of agricultural crops and grasses (Clark and Zeto 1996). Due to the existence of alkaline salts (Na2CO3 and NaHCO3), in various plant species, plants growing at high soil pH (>8.0) were more vulnerable than those with soil salinization (Yang et al. 2007, 2008a, b). The high pH environment surrounding the roots can have serious influences on root cells such as direct damage of the structure and function (e.g., membrane selectivity), precipitation of Ca2+, Mg2+ and H2PO4 −, and disruption of the ion homeostasis (Takahashi et al. 2001; Yang et al. 2007). Plants have evolved complex mechanisms of signal perception and transduction that allow them to perceive the incoming stresses and rapidly regulate their physiology and metabolism to cope with them. In recent years, tremendous progress was made in understanding plant response to external stimuli, such as the MAP kinase signal transduction pathway of osmotic stress and SOS signal transduction pathway of ionic stress (Nakagami et al. 2005; Zhu 2002). Research was mainly focused on salt stress, and limited progress was made with regards to alkali stress.

Ubiquitination plays an important role in various cellular responses in plants, such as regulation of the cell division cycle, hormonal signaling and stress responses (Seo et al. 2012). S-phase kinase-associated protein 1 (SKP1) is a component of a SKP1-Cullin1-F-box (SCF) complex that facilitates ubiquitin-mediated protein degradation in eukaryotes (Hotton and Callis 2008). The SCF-type E3 ligase is composed of four major subunits: Cullin (CDC53 in yeast), SKP1, a RING finger protein (RBX1/HRT1/ROC1) and an F-box protein. Among them, SKP1 functions as an adaptor between F-box and Cullin1 (CUL1). SKP1 containing an N-terminal structural motif interacts with multiple F-box subunits, which specifically recognize different target proteins via a variable C-terminal domain (Bai et al. 1996; Connelly and Hieter 1996). In Arabidopsis, 21 SKP1 homologs have been identified. They are collectively called Arabidopsis-SKP1-like (ASK) (Kong et al. 2007). Arabidopsis SKP1-like 1 (ASK1) was the first SKP1 homolog to be isolated, and its interaction with several kinds of F-box proteins such as TIR1 and COI1 has been reported (Gray et al. 1999; Ji et al. 2006; Li et al. 2012). The ASK1 gene is required for homolog separation in male meiosis and is also involved in auxin response. The ASK2 protein, which has the most similar sequence to ASK1, was able to substitute for ASK1 during male meiosis (Li et al. 2012). Moreover, the ask1 ask2 double mutant showed a developmental retardation during embryogenesis and lethality at the seedling stage (Shu et al. 2011). T-DNA-insertion mutants of ASK11 and ASK12 showed no obvious phenotypic changes. The phenotypes of ASK14 and ASK18 Ds transposon-insertion mutants were not obvious either. Unlike typical ASKs, ASK20 and ASK21 are distinguishable from other ASKs and clustered into another clade according to phylogenetic analysis of ASK proteins (Ji et al. 2006). It has been reported that ASK20A, ASK20B protein cannot interact with CUL1 in yeast two or three hybrid systems, and may prevent SCF complexes from forming by competing with other ASK proteins in their role of adaptors in the SCF complex (Ogura et al. 2008). In spite of the progress made in characterizing SKPs, their biochemical and physiological functions in plants are limited. Some reports investigating the role of SKPs in stress showed that the overexpression of Triticum aestivum SKP1 (TSK1) in Arabidopsis resulted in enhanced tolerance to drought stress (Li et al. 2006). The effects of overexpressing SKPs on alkali stress tolerance have to be further explored.

The phytohormone abscisic acid (ABA) is a vital plant hormone and a central regulator that protects plants against abiotic stresses such as drought and salinity (Bhalerao et al. 1999; Hu et al. 2013; Santiago et al. 2009). In the presence of ABA, they bind to protein phosphatase 2C (PP2C), release the PP2C-mediated inhibition of SNF1-related kinase 2 (SnRK2) and subsequently activate downstream transcription factors (Komatsu et al. 2009). RCARs/PYLs were identified as the ABA receptors that can bind to ABA and interact with group A protein PP2C to inhibit the activity of the phosphatase (Gong et al. 2013; Wang et al. 2009). ASK1 and ASK2 were recently reported to be involved in ABA signaling. The ask1/ask1 ASK2/ask2 seedlings exhibited reduced ABA sensitivity; overexpression of ASK1 and ASK2 in the abi5-1 mutant can rescue or partially rescue the ABA insensitivity of the abi5-1 mutant, respectively (Farrás et al. 2001). Although it was reported that ASK1/SKP1 interacted with the PRL1-binding C-terminal domains of SnRKs and that PRL1 reduced the interaction of ASK1/SKP1 with SnRKs (Farrás et al. 2001), direct evidence for SKPs in ABA response is limited.

Glycine soja (G. soja), the wild ancestor of cultivated soybean Glycine max (G.max), belongs to the same Subgenus Soja as the cultivated soybean. Because of the close genetic relationship and strong abiotic resistance, G. soja supplies valuable genetic resources for the cultivation of transgenic plants with improved abiotic tolerance (Phang et al. 2008). G. soja 07256 is an ideal plant candidate for isolating alkali-tolerance-related genes, as the seeds of this wild soybean can germinate in sodic soils of pH 9.02 and continue to survive in nutrient solutions containing 50 mM NaHCO3 (Ge et al. 2010, 2009). In our laboratory, the sequencing transcriptome of G. soja 07256 under alkali and salt stresses showed that GsSKP21 was a putative alkali-response gene. Compared with thesalt stress profile, GsSKP21 exhibited more abundant expression under alkali stress by screening protein expression in G. soja. Here, we characterized GsSKP21, a putative novel SKP1-like family gene with a conserved SKP domain bearing the highly conserved amino acid pattern of ASK21, which is involved in plant response to alkali stress and ABA treatment. Expression levels of GsSKP21 were greatly and rapidly induced by high alkali levels as evidenced by quantitative real-time PCR (qRT-PCR). Overexpression of GsSKP21 in Arabidopsis conferred enhanced tolerance to alkali stress and downregulated expression levels of stress-responsive marker genes. Furthermore, GsSKP21 overexpression decreased plant sensitivity to ABA and altered expression levels of ABA signal-related genes. Subcellular localization studies using an enhanced green fluorescent protein (eGFP) fusion protein showed that GsSKP21 is localized in the nucleus. All the data presented illustrate the important role of GsSKP21 as a regulator of alkali stress tolerance and ABA signaling in plants.

Materials and methods

Plant material, growing conditions and stress treatments

Seeds of G. soja 07256 and G. soja 50109 were obtained from the Jilin Academy of Agricultural Sciences (Changchun, China). G. max cultivar Suinong 28 and G. max cultivar Hefeng 55 were obtained from the Chinese Crop Germplasm Information System. For gene expression analysis, seedlings of G. soja 07256 were grown in a culture room with the following settings: 60 % relative humidity, 24 °C and a light regime of 16 h light/8 h dark. The light source SON-T ARGO 400 W generated constant illumination of 30,000 lx. Before sowing, seeds of G. soja 07256 were shaken for 10 min in 98 % sulfuric acid. Subsequently, seeds were washed five times with sterile water. Nineteen days after sowing, seedlings in the stress treatment group were transferred into 1/4 strength Hoagland’s solution with 200 mM NaCl for salt stress and 50 mM NaHCO3 for alkali stress, respectively. Equal amounts of leaves and roots were sampled at eight time points, 0, 1, 3, 6, 9, 12, 24 and 48 h.

For GsSKP21 expression analysis in different soybean varieties, 20 seeds of G. soja 07256, G. soja 50109, G. max Suinong 28 and G. max Hefeng 55 were placed on each petri dish to accelerate germination for 2 days. Germinated seedlings were then transferred into 1/4 strength Hoagland’s solution. Nineteen days after sowing, seedlings in the stress treatment group were transferred into 1/4 strength Hoagland’s solution with 50 mM NaHCO3. Equal amounts of roots were sampled at 0 and 1 h.

Arabidopsis thaliana ecotype Columbia (Col-0), used for transformation, was grown in a greenhouse under controlled environmental conditions (21–23 °C, 100 μmol photons/m2 s, 60 % relative humidity, 16 h light/8 h dark cycles). For the expression analysis of alkali stress and ABA response marker genes, seeds from wild-type (WT) and GsSKP21 overexpression (OX) lines were sown on filter paper saturated with 1/2 MS solution. After 14 days of growth, seedlings were saturated with water (control), NaHCO3 (50 mM) or ABA (100 μM). Rosette leaf samples were collected from three biological replicates at 0 and 6 h after treatment.

Quantitative real-time PCR

Total RNA was extracted from G. soja or Arabidopsis seedlings, and cDNA synthesis was performed using the SuperScript™ III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) with the Oligo(dT)18 reverse primer. Prior to qRT-PCR assays, cDNA quality was assessed by PCR using specific primers for GADPH (glyceraldehyde-3-phosphate dehydrogenase, accession no. DQ355800) to exclude genomic DNA contamination. The qRT-PCR was performed using the SYBR Premix ExTaq™ II Mix (TaKaRa, Shiga, Japan) on an ABI 7500 sequence detection system (Applied Biosystems, USA). One microliter of each synthesized cDNA (diluted 1:5) was used as template. Amplifications of GAPDH in G. soja and ACTIN2 in Arabidopsis were used as controls. Relative intensities were calculated and normalized as described previously (Willems et al. 2008).

Transformation of Arabidopsis

The coding regions of GsSKP21 were amplified from pGEM-T-GsSKP21. The sequence was inserted into the pCAMBIA2300 under the control of the strong constitutive CaMV35S promoter with SalI and BamHI linker using primers 5′-GCGTCGACATGTCAGAAATTGACATGGCAGTTAT-3′ and 5′-CGGGATCCTCAAGCGTTTCGCCTCAGAAAACTAT-3′. The construct was introduced into Agrobaterium tumefaciens strain LBA4404, and transgenic Arabidopsis plants were generated by floral dip (Clough and Bent 1998). Transformants were selected on 1/2 MS medium containing 50 mg/l kanamycin. Seeds from each T1 plant were individually collected. Selected T2 plants were propagated, and homozygous overexpression lines were confirmed by qRT-PCR analysis.

Phenotypic analysis of transgenic Arabidopsis plants

All Arabidopsis seeds were sterilized by 5 % NaClO and stored at 4 °C in the dark for 3–7 days before use. All phenotypic experiments were performed in a controlled environmental chamber under the following conditions: 21–23 °C, 100 μmol photons/m2 s1, 60 % relative humidity and 16/8 h day-night cycles.

For germination assays, the seeds of WT and T3 transgenic Arabidopsis were sown on 1/2 MS agar medium supplemented with 7, 8 and 9 mM NaHCO3 or 0.8 and 0.9 μM ABA (Lee et al. 2004). The germination rate was recorded for 6 consecutive days after sowing. On the 10th day, pictures were taken to show the growth performance of each line, followed by measurement of the opening/greening of the leaves. Ninety seeds were used for each experiment, and all experiments were repeated three times.

To characterize stress tolerance at the early seedling stage, WT and OX Arabidopsis seeds were germinated and grown on 1/2 MS medium for 7 days, followed by a transfer to fresh medium (in the absence or presence of 8 mM NaHCO3 or 20, 30 μM ABA) for 14 days of vertical growth before being photographed, and the root length was measured (Zhu et al. 2011).

The alkaline tolerance assay at the adult stage was performed using 4-week-old plants grown in pots filled with a 1:1:1 mixture of vermiculite:peat moss:nutrition soil under controlled environmental conditions (21–23 °C, 100 μmol photons/m2 s, 60 % relative humidity, 16/8 h day-night cycles). The plants were irrigated with 150 mM NaHCO3 solution every 4 days for a total of 12 days. The photos were taken when the phenotype showed distinct differences.

Protein subcellular localization assay

For subcellular localization assays, the GsSKP21 gene was amplified with primer pairs: 5′-GCGTCGACATGTCAGAAATTGACATGGCAG-3′ and 5′-CGGGATCCAGCGTTTCGCCTCAGAAAACTA-3′. The product was then cloned into SalI/BamHI-digested pBSKII-eGFP to generate pBSKII-GsSKP21-eGFP, in which the GsSKP21 coding sequence was fused to the N-terminal of eGFP. The plasmids pBSKII-eGFP and pBSK II-GsSKP21-eGFP were then precipitated onto gold beads and transformed into onion (Allium cepa) epidermis as described. Fluorescent protein expression in the epidermis was observed using a confocal laser-scanning microscope (SP5, Leica, Germany).

Statistical analysis

All experiments with each group were performed at least in triplicate. Data were reported as mean ± SD. Data were analyzed statistically by Student’s t test. Results were considered statistically significant when P < 0.05.

Results

Cloning and sequence analysis of the gene GsSKP21

GsSKP21 was identified as a putative stress-response gene based on transcriptome sequencing of G. soja under alkali stress in our laboratory. The full-length coding region of GsSKP21 was obtained by homologous cloning with gene-specific primers designed according to the G. max cDNA sequence (Glyma09g39480).

A homology search against the Phytozome database showed that GsSKP21 was homologous to ASK21. The similarity of GsSKP21 protein and Arabidopsis ASK21 protein is 79 %. Secondary structure prediction of GsSKP21 protein revealed that the N-terminus (amino acid 1–164) of GsSKP21 shared a highly conserved SKP1 domain (PF01466) and a putative POZ domain (PF03931), which are characterized by four helical regions that are conserved among SKP1 and ASK proteins, but in its C terminus (165–364 aa), no typical domain was identified. In addition, the extended C-terminal region of the GsSKP21 protein exhibited little similarity with other SKP proteins listed in the National Center for Biotechnology Information database. We also found that like ASK21, GsSKP21 differs in those well-conserved sites in ASK1-6, 9, 11-16, 18 and 19, which were demonstrated to be important for interaction with CUL1.

Phylogenetic analysis demonstrated that members of the SKP1-like family in angiosperms evolved at highly heterogeneous rates and were derived from a single ancestral gene (Kong et al. 2004). To determine the evolutionary relationship of GsSKP21 and SKP plant proteins, we built the phylogenetic tree of the SKP1-like gene family of 6 angiosperm species (Arabidopsis, G. soja, Populus trichocarpa, Brachypodium distachyon, Oryza sativa and Zea mays). Our results showed that members of the SKP1-like familywere divided into two groups. Those SKP1-like proteins with an extended C-terminal region such as GsSKP21, ASK20 and ASK21 were clustered into group II, whereas other SKP1-like proteins without an extended C-terminal region were clustered into group I (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree of the SKP1-like family of six species. The phylogenetic tree was constructed using full-length sequences of SKP1 homologs of Arabidopsis, G. soja, Populus, B. distachyon, O. sativa and Z. mays by the maximum-likelihood method with MEGA 5.0 and a bootstrap value of 1,000. The two major phylogenetic clades are designated as groups I and II. The bar represents the branch length equivalent to 0.05 amino acid changes per residue. Shown on the right are diagrams of representative SKP1-like proteins with information on the structure and position of the SKP domain

Full size image

Expression of GsSKP21 transcripts is induced by alkali and salinity stress in G. soja

To obtain further information on the role of GsSKP21 in response to alkali and salinity stress, qRT-PCR analysis was performed to quantify its expression levels in both leaves and roots after NaHCO3 and NaCl stress treatments. Results are shown in Fig. 2a. The tendencies of alkaline treatment on leaves and roots were similar to salt treatment. Under alkaline treatment, the relative transcript abundance of GsSKP21 in roots started immediately after treatment and reached a peak at 1 h (2.5-fold); in alkaline-treated leaves, transcript abundance increased slightly at the 1-h point, but peaked at 12 h with a threefold increase. Under salt treatment, the expression of GsSKP21 in leaves also peaked at 12 h, while in roots it rose and peaked at 3 h. These results indicate that GsSKP21 is involved in responses to a wide variety of abiotic stresses. Moreover, NaHCO3 and NaCl stress induced GsSKP21 transcript abundance with different expression patterns from roots to leaves (Fig. 2a). In leaves, the GsSKP21 transcript levels were fairly stable until 12 h after NaCl and NaHCO3 treatment, where they showed an obvious increase. In roots, we noticed that GsSKP21 transcripts showed the highest expression earlier than that in leaves for both treatments. This suggests that in the leaves of G. soja, GsSKP21 may have similar but delayed stress-response expression profiles. This is consistent with the roots being the first point of contact with the stress treatments.

Fig. 2
figure 2

Expression patterns of GsSKP21 in G. soja. a Expression levels of GsSKP21 were upregulated by salt-alkali stress in roots and leaves. Total RNA was extracted at the indicated time points from leaves and roots of 3-week-old G. soja seedlings whose roots were submerged in nutrient solution with 50 mM NaHCO3 and 200 mM NaCl, respectively. Untreated plants were used as controls. Relative transcript levels were determined by qRT-PCR using GAPDH as an internal control. The mean value from three fully independent biological repeats and three technical repeats is shown. b Expression levels of GsSKP21 in soybean varieties. Total RNA was extracted from roots of 3-week-old seedlings of four soybean varieties whose roots were submerged into nutrient solution with 50 mM NaHCO3. c Tissue-specific expression patterns of GsSKP21 in G. soja. YL young leaf, OL old leaf, YE young embryo, YC young seed coat, YS: young stem, RT root, CL epicotyl, O Old seed coat, OE: old embryo, OS old stem, HC hypocotyl, FL flower. The mean value from three fully independent biological repeats and three technical repeats is shown

Full size image

Since we screened GsSKP21 as a putative gene response to alkali stress from G. soja 07265, we want to further investigate the GsSKP21 response to alkali stress in other soybean varieties by using G. soja 07265, G. soja 50109, G. max Suinong 28 and G.max Hefeng 55. As we know, G. soja 50109 was one of the highly salt-tolerant species found to tolerate up to 0.9 % of salt during the germination stage (Ji et al. 2006). Comparing with G. soja 50109, G. soja 07256 showed stronger alkaline tolerance as evidenced by relatively less change in chlorophyll content, relative conductivity and malondialdehyde (MDA) content (Supplementary Table 1). G. max Suinong 28 and G.max Hefeng 55 were Chinese soybean cultivars that exhibited much lower adaptability to a suboptimal (i.e., stressful) natural environment compared to the wild soybean (G. soja) (Ge et al. 2009). In the four varieties, only the germination rate of G. soja 07256 can reached 100 % under 100 mM NaHCO3 treatment (Supplementary Figure 2). We determined the transcript expression level of GsSKP21 using roots of four soybean variety seedlings under NaHCO3 treatment by qRT-PCR (Fig. 2b); the results showed that, under alkali stress, the relative transcript abundance of GsSKP21 in G. soja 07256 increased the highest with threefold, G. soja 50109 increased slightly by 1.7 fold, while the expression of G. max Suinong 28 and G. max Heheng 55, which showed lower tolerance to alkali stress, were relatively unchanged. This result indicates that GsSKP21 might be an important regulating gene in alkaline-resistant soybean varieties.

Tissue expression pattern of GsSKP21 in G. soja

In Arabidopsis, the diverse expression patterns of ASK genes suggest that they may regulate different developmental and physiological processes (Gao et al. 2011; Ge et al. 2010). In order to explore tissue-specific expression patterns of GsSKP21 in G. soja, the expression levels in different tissues were determined by qRT-PCR analysis. As shown in Fig. 2c, GsSKP21 was expressed in most vegetative tissues and reproductive organs. The GsSKP21 expression levels were high in the flower, young leaf, young stem and hypocotyl. Among all tissues, the highest expression was observed in the hypocotyl, and the lowest level was detected in the old embryo. These results were consist with the expression pattern of ASK21, which also showed high expression levels in the flower, young leaf and young stem (Dezfulian et al. 2012).

Overexpression of GsSKP21 resulted in enhanced tolerance to alkali stress

To further characterize the function of GsSKP21 in alkali stress, we investigated whether GsSKP21 overexpression affects plant tolerance to alkali stress. We compared the germination and growth of OX lines with WT by the NaHCO3 gradient concentration. In the absence of NaHCO3, GsSKP21 overexpression does not affect plant germination and development under normal conditions, as shown by the similar performance of the WT and OX lines. However, in the presence of NaHCO3, seeds from the GsSKP21 OX lines were able to develop healthy cotyledons subsequent to seed coat breakage and radicle emergence with higher germination rates (Fig. 3a, b). In addition, on the 10th day after germination, OX lines had a much higher percentage of seedlings with open and green leaves and seedlings with at least four leaves than WT (Fig. 3c). For example, in the presence of 9 mM NaHCO3, the percentage of WT seedlings with fully opened cotyledons was <20 % compared with approximately 40 % for the GsSKP21 OX seedlings.

Fig. 3
figure 3

The improved alkaline tolerance due to overexpression of GsSKP21. a Growth performance of WT and OX seedlings on 1/2 MS medium with 7, 8 and 9 NaHCO3. Photographs were taken 10 days after stratification. b NaHCO3 dose response of germination. Experiments were performed at least three times. The bars represent standard errors. The data show means (±SE) of three replicates. *P < 0.05, **P < 0.01 by Student’s t test. c Quantitative analysis of the leaf opening and greening rate from the WT and OX lines. d Phenotypes of WT and OX seedlings grown in 1/2 MS medium with and without 8 mM NaHCO3. e Measurements of primary root lengths of seedlings under normal and alkali stress. The data show means (±SE) of three replicates. *P < 0.05, **P < 0.01 by Student’s t test

Full size image

GsSKP21 OX also improved plant tolerance to alkali stress at the seedling stage. Seven-day-old seedlings of WT and OX were transferred to 1/2 MS medium supplemented with 8 mM NaHCO3. As shown in Fig. 3d, the growth and development were significantly inhibited in WT plants compared with those of GsSKP21 overexpression under NaHCO3 treatment. OX seedlings had longer primary roots than WT under alkali stress (Fig. 3e).

Similarly, when 3-week-old soil-grown plants were irrigated with 150 mM NaHCO3 for 2 weeks, the transgenic plants displayed greater alkali stress tolerance than the WT (Fig. 4a). Without NaHCO3 treatment, there was almost no difference in growth and total chlorophyll content among the WT and two transgenic lines. However, after exposure to 150 mM NaHCO3 stress, the total chlorophyll content decreased in both the transgenic and WT plants, and the extent of this decline in transgenic plants was less than that observed for WT (Fig. 4b). These results indicate that GsSKP21 OX reduces the effects of high alkali stress on chlorophyll formation and enhances the alkaline tolerance of transgenic plants. It is generally accepted that under conditions of abiotic stress, the level of MDA produced during peroxidation of membrane lipids is often used as an indicator of oxidative damage (Kotchoni et al. 2006; Weber et al. 2004). Therefore, we measured the MDA content in the transgenic and WT plants under alkali stress conditions and found that the WT accumulated significantly higher levels of MDA than GsSKP21 OX lines (Fig. 4c). Together, the data indicate that overexpression of GsSKP21 enhances alkali stress tolerance in Arabidopsis.

Fig. 4
figure 4

Enhanced tolerance of transgenic plants to alkaline stress at the adult stage. a Phenotypes of WT and OX plants in response to alkali stress. b The total chlorophyll content of WT and OX plants. c The total MDA content of WT and OX plants. For the alkaline tolerance test at the adult stage, 3-week-old plants were irrigated with 150 mM NaHCO3 solution every 4 days for a total of 12 days. Photos were taken on the 12th day after initial alkali treatment

Full size image

GsSKP21 overexpression decreased plant ABA sensitivity at both the seed germination and early seedling stages

It has been reported that overexpression of a homologous gene TSK1 from wheat can change the Arabidopsis response to ABA (Shu et al. 2011). To gain insights into the possible roles of GsSKP21 in ABA signaling, we examined the responses of GsSKP21 OX lines to exogenous 0.8 and 0.9 μM ABA. In the absence of ABA, both GsSKP21 OX lines and WT seeds showed identical germination behavior; however, in the presence of ABA, seed germination was significantly inhibited in WT compared with that in transgenic lines (Fig. 5a). Likewise, in the presence of 0.8 μM ABA, the seed germination rate was 70 % in transgenic lines compared to 40 % in the WT (Fig. 5b). Transgenic lines had more open and green leaves than WT after 12 days (Fig. 5c). These results suggest that GsSKP21 overexpression interferes with the response to ABA.

Fig. 5
figure 5

GsSKP21 overexpression in Arabidopsis resulted in decreased ABA sensitivity. a Growth performance of WT and OX seedlings on 1/2 MS medium without or with 0.8 and 0.9 μM ABA. Photographs were taken 10 days after stratification. b ABA dose response of germination. Experiments were performed at least three times. The bars represent standard errors. c Quantitative evaluation of the leaf opening and greening rate. d Phenotypes of WT and OX seedlings grown in 1/2 MS medium with and without 20 and 30 μM ABA. Ten-day-old seedlings grown on normal 1/2 MS medium were transferred to new solid agar plates supplemented with 20 and 30 μM ABA. Photographs were taken after 7 days. e Measurements of primary root lengths of seedlings under normal and ABA treatment. The data show means (±SE) of three replicates. *P < 0.05, **P < 0.01 by Student’s t test

Full size image

Different concentrations of ABA can inhibit both seed germination and seedling growth of Arabidopsis. The ABA sensitivity of root growth was further investigated. Seven-day-old seedlings grown on 1/2 MS medium were transferred onto vertical agar plates containing 1/2 MS medium supplemented with 20 μM and 30 μM ABA. When grown under the influence of 20 μM ABA, WT root growth was more inhibited than in GsSKP21 OX lines (Fig. 5d). The roots of the GsSKP21 OX lines were significantly longer than those of WT plants (Fig. 5e). These results indicate that GsSKP21 OX transgenic Arabidopsis was less sensitive to exogenous ABA treatment during seed germination and root growth.

GsSKP21 overexpression altered expression patterns of stress responsive and ABA signal-related genes

The induction of numerous stress-inducible marker genes is a hallmark of stress adaptation in plants (Zhu et al. 1997). The alkali phenotype of the GsSKP21 OX lines suggests that the expression of some stress-responsive genes might be altered in the GsSKP21 OX lines. To verify this possibility, expression of some stress inducible genes (RD29A, RAB18, COR15A, COR47, KIN1 and NCED3) was examined (Hu et al. 2013; Kong et al. 2004; Santiago et al. 2009; Shinozaki et al. 2003). In the presence of alkali stress treatment, the expression of all of these stress-inducible genes was upregulated in both GsSKP21 OX lines and WT plants. RD29A, RAB18, COR15A and KIN1 were upregulated in transgenic lines as compared with WT plants (Fig. 6). COR47 and NCED3 of GsSKP21 OX lines showed higher transcript levels than the WT; however, they exhibited no statistically significant differences. These indicated that the overexpression of GsSKP21 in Arabidopsis resulted in an alteration in the expression of stress-responsive genes.

Fig. 6
figure 6

Expression patterns of stress-induced marker genes in WT and GsSKP21 transgenic Arabidopsis seedlings in response to alkali stress. The induction of stress-responsive genes RD29A, RAB18, COR47, COR15A, KIN1 and NCED3 were measured by qRT-PCR analysis. Expression of ACTIN2 was used as an internal control. Values represent the means of three fully independent biological replicates and three technology replicates for each. *P < 0.05, **P < 0.01 by Student’s t test

Full size image

To further investigate the effect of GsSKP21 in ABA signaling, we used qRT-PCR to determine the expression level of various ABA-responsive genes in two independent homozygous T3 transgenic lines, line 5 (L5) and line 28 (L28) (Fig. 7). ABI1 and ABI2 encode PP2Cs and negatively regulate ABA signaling (Hu et al. 2013; Weber et al. 2004). The qRT-PCR assay showed that the relative expression of ABI1 was increased by 5.7- and 2.3-fold in L5 and L28, respectively, compared with that in WT. ABI2 gene expression was increased by more than 15-fold in both L5 and L28 after ABA treatment. SnRK2s are positive regulators of ABA signaling, and their activities are inhibited by PP2C under normal conditions. SnRK2 genes showed stable expression under ABA stress. Our results showed that the expression levels of SnRK2.2 and SnRK2.3 were stable in both WT and OX plants, which is consistent with the results of previous studies. We also detected ABF4 and ABI5, key transcription factors in the ABA signaling pathway (Bhalerao et al. 1999). ABF4 and ABI5 in GsSKP21 OX lines were significantly lower than those in WT, suggesting that the expression of ABI5 and ABF4 was negatively regulated by GsSKP21 in response to ABA signaling. Furthermore, Pyrabactin Resistance 1 (PYR1), a membrane-localized ABA receptor, can be strongly downregulated by ABA treatment (Park et al. 2009), and the expression of PYR1 detected in OX plants was lower than in WT. Taken together, the findings consistently show that GsSKP21 regulates the expression of some key ABA signaling regulators.

Fig. 7
figure 7

Expression of ABA-responsive genes in GsSKP21 overexpressing transgenic lines. Transcription levels of ABA-responsive genes were determined by qRT-PCR with total RNA from 2-week-old seedlings. ACTIN2 was used as an internal control. Each value represents the means of three fully independent biological replicates, three technology replicates for each. *P < 0.05, **P < 0.01 by Student’s t test

Full size image

GsSKP21 protein targeted to the nucleus in the onion epidermal cells

To better understand the functions of GsSKP21, we examine the subcellular distribution of the GsSKP21 protein. The GsSKP21-coding sequence was fused in-frame at the 5′ end to eGFP. Confocal imaging showed that the GsSKP21-eGFP fusion protein was localized exclusively in the nuclei of onion (A. cepa) epidermal cells in a transient expression assay (Fig. 8). As a control, the eGFP protein alone was found in both the nucleus and cytoplasm.

Fig. 8
figure 8

Subcellular localization of GsSKP21. Subcellular localization assay of the GsSKP21 protein. Images showing onion epidermis cells expressing the eGFP (upper lane) or the GsSKP21-eGFP fusion protein (bottom lane) examined under fluorescent-field illumination to examine GFP fluorescence (left); under bright-field illumination (middle); by confocal microscopy (right) for an overlay of bright and fluorescent illumination

Full size image

Discussion

The Western Songnen Plain of China is severely influenced by the effects of alkalization and has become one of the three largest sodic-saline areas in the world (Jin et al. 2006; Wang et al. 2009, 2010). Alkalized soil is one of the major environmental challenges limiting crop growth and productivity globally (Takahashi et al. 2001). Mining alkali-resistant genes and understanding the molecular basis of the plant response to alkali stress will therefore facilitate biotechnological efforts to breed crop plants with enhanced tolerance to high levels of alkali. The wild soybean used in this study can survive in nutrient solutions that contain 50 mM NaHCO3 (pH 8.5) and is an ideal organism for the identification of carbonate stress-response genes (Ge et al. 2010; Zhu et al. 2011). Up to now, only a few of the alkali stress-response genes have been functionally analyzed (Kotchoni et al. 2006). In our previous study, GsSKP21 was identified as a putative alkaline tolerance gene and was isolated from G. soja, providing us with very useful clues for the functional characterization of plant alkali stress adaptation and signal transduction.

Arabidopsis-SKP1-like genes are expressed in a variety of tissues, and they may regulate different developmental and physiological processes (Komatsu et al. 2009; Li et al. 2012; Marrocco et al. 2003). In our study, we observed ubiquitous expression of GsSKP21 in both vegetative tissues and reproductive organs using qRT-PCR (Fig. 2c). The tissue expression pattern of GsSKP21 is similar to ASK20 and ASK21, which were found to be expressed in a large number of tissues (Farrás et al. 2001; Gao et al. 2011). GsSKP21 was identified as a putative alkali response gene in G. soja 07256. Therefore, we investigated the expression pattern of GsSKP21 under alkali treatment. The qRT-PCR results showed that GsSKP21 was strongly induced by alkali stress (Fig. 2a). The response of GsSKP21 to alkali stress was further confirmed in different soybean varieties. As we speculated, the highest expression change was observed in G. soja 07256. These results suggested GsSKP21 was involved in the plant response to alkali stress.

It has been concluded from only a handful of reports that SKP genes are involved in plant tolerance to abiotic stresses. The overexpression of a wheat SKP1 homolog resulted in an enhanced stomatal closure response to ABA and enhanced drought tolerance in transgenic plants (Li et al. 2006). As a component of E3 ubiquitin ligases, SKP1 has been reported to regulate abiotic stress signal transduction in Arabidopsis (Lee and Kim 2011); however, little is known about the function of SKPs in alkali stress. In our study, we demonstrated that overexpression of GsSKP21 improved plant tolerance to alkali stress by comparing the growth performance. The results of plate germination assays demonstrated that transgenic plants exhibited much better growth performance than WT with much higher germination rates and more green, open leaves (Fig. 3a–c); GsSKP21 transgenic lines showed longer primary roots at the seedling stage (Fig. 3d, e); GsSKP21 overexpression enhanced plant alkali resistance at the adult seedling stage with higher chlorophyll content (Fig. 4a, b). In addition, the ability of GsSKP21 to upregulate RD29A, RDAB18, COR15A and KIN1 (Fig. 6) suggested that it may be an important regulator of alkali resistance and play a key role in coordinating the expression of many aspects of plant stress-related marker genes.

ABA plays a pivotal role in coordinating the adaptive response to abiotic stress (Hartung et al. 2005). It has been reported that ASK1 and ASK2 genes participate in the ABA signaling pathway. ASK1/2 overexpression rescued ABA sensitivity in abi5-1 mutants (Shu et al. 2011). In this study, we found that GsSKP21 overexpression also led to a decrease in plant sensitivity to ABA regarding the seed germination and root growth (Fig. 5). Moreover, GsSKP21 overexpression altered the expression patterns of ABA-responsive genes. The expression levels of the basic leucine zipper (bZIP) transcription factor genes ABI5 and ABF4 are decreased in GsSKP21 overexpressing Arabidopsis. Meanwhile, the expression of ABI1 and ABI2, two negative regulators of the ABA signaling pathway, is induced. Moreover, ABA treatment significantly decreased the expression level of PYR1, which is a membrane-localized ABA receptor and can be strongly downregulated by ABA treatment (Santiago et al. 2009); the expression of PYR1 in GsSKP21 OX plants was obviously decreased compared to WT. The expression patterns of these genes are consistent with the ABA decreased sensitivity phenotype of GsSKP21 transgenic Arabidopsis lines. It has been reported that SKP1/ASK1 recognized homologous C-terminal segments of Arabidopsis SnRKs that are also implicated in the binding of the SnRK inhibitor PYR1/PRL1 WD protein. Moreover, PYR1/PRL1 reduced the interaction of SKP1/ASK1 with SnRKs, and SKP1-SnRK protein kinase interactions mediate the proteasomal binding of a plant SCF ubiquitin ligase. It can be speculated that GsSKP21 might mediate the ABA pathway by protein ubiquitination and degradation (Bhalerao et al. 1999; Farrás et al. 2001). The overexpression of GsSKP21 results in a constitutively enhanced tolerance to alkali stress, and decreased sensitivity to ABA implies that GsSKP21 may regulate plant alkali stress tolerance by promoting the expression of alkali defense genes within the ABA signaling pathway.

In addition, we noticed that the effect of GsSKP21 on the ABA pathway differs from that of ASK1 and ASK2, possibly because GsSKP21 contains changed conserved amino acid sites and extra C terminal regions (Fig. 1). In Arabidopsis, the N-terminal regions of these ASK proteins and SKP1 are postulated to be CUL1-binding sites, and the N-terminal regions of the GsSKP21 proteins showed low levels of similarity with those of other ASK proteins, with the exception of ASK20, 21. Crystal structural analysis of the human SCF complex revealed that the last four α-helices in the C-terminal domain of SKP1 were directly attached to the F-box protein (Schulman et al. 2000). The GsSKP21 protein, with extra C terminus regions, may prevent certain F-box proteins from forming SCF complexes by competing with other ASK proteins that are able to act as components of the SCF complex (Ogura et al. 2008). It may be speculated that GsSKP21 and ASK1 compete with certain F-box proteins because of their difference in amino acids. The end effect would be the different sensitivities to ABA between GsSKP21- and ASK1-overexpressing lines.

In summary, we have cloned and characterized an SKP1-like homolog, SKP21, from G. soja. We found that it can modify plant alkali stress tolerance and ABA signaling responses. It will be interesting to discover in detail how GsSKP21 regulates plant stress tolerance and ABA signal transduction. Further investigations should elucidate the relationship between alkali stress and the ABA signaling pathway as well as the role of GsSKP21 in the crosstalk of ABA and ABA-mediated signaling systems during plant stress responses.