Introduction

Abiotic stress limits crop productivity [1, 2], and plays a major role in determining the distribution of plant species across different types of environments. Drought and salt stress are important factors that limit crop production. And many abiotic genes had been identified [3–5].

Dehydrin is one of the most abundant plant proteins produced during late embryogenesis or in response to drought, low-temperature, salinity, and ABA [6]. Dehydrins have been found in many plants including Arabidopsis, wheat, barley, and rice [7]. They are thought to act as chaperons, and thus to stabilize vesicles, proteins, and membrane structures in stressed plants [6, 8, 9], although their exact function remains uncertain.

Ammopiptanthus mongolicus (Maxim.) Cheng f., an ancient relic of the tertiary period, is a rare and endangered species of Fabaceae in Mid-Asia desert and has been listed as one of primary protection plants by the Chinese government. As a kind of typical drought-resisting resources plant, it mainly grow in the desert and semi-desert area in the northwest of China, which displays prominently characteristics of draught resisting, cold resisting and salt and alkali resisting. Because of its high value of study and ecological usefulness in defending desert, this plant has been called ‘live fossil’ [10]. A. mongolicus distinctively distributed in the northwestern desert area of China, where is marked by seasonally extreme drought and temperatures (over 40 °C in the summer and under −30 °C in the winter).

Previous studies have demonstrated that some dehydrin proteins play important roles in drought and salt resistances. As mentioned above, dehydrin proteins play important roles in response to stresses. If we clone dehydrin protein gene from A. mongolicus and over-express it in other plants, we might improve the ability of other plants to tolerate various stresses to support the development of agriculture.

A SSH cDNA library induced by salt stress had been constructed and some abiotic genes had been screened by reverse Northern blot methods. An expressed sequence tag (EST) of AmDHN gene was one of these differential expression sequences. In the present study, the full-length dehydrin cDNA, designated AmDHN, from A. mongolicus was obtained with rapid amplification of cDNA ends (RACE). Its expression patterns were analyzed with RT-PCR. When overexpressing this gene in Arabidopsis thaliana and tobacco, stress-resistances were improved. The characterization of AmDHN might provide insight into the physiological processes of stress response in higher plants.

Materials and methods

Plant materials and treatments

Seeds of A. mongolicus (Maxim.) Cheng f. and A. thaliana (ecotype Col-0) were obtained from Chinese Academy of Agricultural Sciences. Seeds of A. mongolicus were gown on 1/2 MS agar media. Seeds were placed on soil and germinated in the dark at 25 °C for 1 day, then were grown under a regime of 16 h light and 8 h dark at 25 °C. For growth on the soil, Arabidopsis seeds were sown on soil and incubated at 22 °C under LD (16 h light and 8 h dark) conditions. For growth on the agar medium, Arabidopsis seeds were sown on a 1/2 MS plate and incubated at 22 °C under continuous light.

Amplification of Full-length AmDHN cDNA

An EST of 432-bp was isolated from a SSH cDNA library of A. mongolicus induced by PEG6000. RACE was performed to amplify its unknown 3′ and 5′ ends. Total RNA was isolated from A. mongolicus seedlings of 40-day-old by a Trizol extract method. Any contaminated genomic DNA was removed by incubating the total RNA with RNase-free DNase (Promega) at 37 °C for 30 min. Total RNA was used to synthesize 5′-RACE-Ready-cDNA and 3′-RACE-Ready-cDNA according to the manufacturer’s recommendation of SMARTTM RACE cDNA Amplification Kit (Clontech, USA). Based on the EST sequence already obtained, gene-specific primers: 96F and 96R (seen in Table 1) were used to amplify the 3′-cDNA end and 5′-cDNA end, respectively, with AdvantageTM 2 PCR Enzyme Kit (Clontech). Thermocycling was performed at 35 cycles with 94 °C for 30 s, 68 °C for 30 s and 72 °C for 3 min, and an additional polymerization step at 72 °C for 5 min. PCR product was separated by electrophoresis on a 1 % agarose gel stained with ethidium bromide, purified using the DNA gel extraction kit (TaKaRa, Janpan). The products were cloned into the pMD-18T (TaKaRa, Japan) vector and then transformed into Escherichia coli DH5α. Recombinant plasmids were sequenced by Beijing Genomics Institute (Beijing, China).

Table 1 Primers used in this study
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To obtain the full-length of the gene, the 3′-RACE cDNA was used as temple. The cDNA template was then amplified using gene-specific primer DHN1 and UPM (Universal Primer A Mix) (seen in Table 1).

Bioinformatics analysis

The overlapping and assembly of cDNA fragments were done by the tool of “align two sequences” (bl2seq) in GenBank. Sequences homology analysis was against nucleotide and protein database of GenBank using BLAST tools. Conserve sequences was analyzed with ExPaSy ScanProsite (http://www.expasy.org/tools/scanprosite/). Potential signal peptide cleavage site was identified using Signal P 4.0 (http://www.cbs.dtu.dk/services/SignalP/). And pI/MW were predicted on the ExPaSy website (http://us.expasy.org/tools/pi tool.htm). Amino acid sequences of AmDHN and its related proteins were obtained from the NCBI database. The neighbor-joining trees were generated using the MEGA version 5.0 software, with bootstrap values obtained from 1,000 replications.

Expression analysis

In order to investigate the expression pattern of AmDHN in different tissues including dry seeds, roots, stems, leaves and flowers of A. mongolicus, semi-quantitative RT-PCR was carried out. Expression profiles of AmDHN under different kinds of abiotic conditions including NaCl, PEG6000, drought and ABA were also investigated. Seedlings of 40-day-old were grown on soil were used to extract RNA. All RNA templates were digested with DNase I (RNase-free). Aliquots of total RNA (0.5 μg) extracted were used as templates in one-step RT-PCR with the forward primer DHN-RT-f and reverse primer DHN-RT-r (seen in Table 1) specific to coding sequence of AmDHN. Meanwhile the RT-PCR reaction for the housekeeping gene (Actin gene, which is highly conserved in plants) using specific primers Actin-1 and Actin-2 (seen in Table 1) designed according to the conserved regions of plant Actin genes were performed to estimate if equal amounts of RNA among samples were used as an internal control. Amplifications were performed under the following condition: 94 °C for 3 min followed by more than 25 cycles of amplification (94 °C for 58 s, 60 °C for 30 s and 72 °C for 40 s). RT-PCR images were captured using a UVP transilluminator.

Subcellular localization of AmDHN

AmDHN-GFP (green fluorescent protein) fusion was constructed and transformed into onion epidermal cells to express the fusion protein. To amplify the coding sequence of AmDHN, two primers were designed, one primer D-Gf with Xho I restriction site and another primer D-Gr with Spe I restriction site (seen in Table 1). The PCR product was digested with Xho I and Spe I, and ligated with the vector pA7-GFP which was digested with the same restriction enzymes. The fusion was then transformed into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed GFP-AmDHN fusion was detected by a confocal laser scanning microscope.

Plasmid construction

For the overexpression experiments, the 35S promoter was used to drive the expression of the AmDHN gene. The whole CDS of AmDHN gene was amplified by PCR with the primer pair pBI-DHN-f and pBI-DHN-r (seen in Table 1). The PCR products were digested by Xba I and BamH I and used to insert into the enzyme sites of pBI121 (Clontech, Palo Alto, CA) to generate 35S::AmDHN, which was used to generate transgenic tobaccos and transgenic A. thaliana.

Generation of transgenic plants

Agrobacterium GV3101, which had the expression vector pBI121 with the full-length cDNA of AmDHN gene, was infected to Arabidopsis using the floral dip method [11]. Transformed Arabidopsis seeds were selected on a 1/2 MS medium containing 50 mg L−1 kanamycin and 0.8 % agar.

Stresses response assay

The germination response to the salt was measured by placing Arabidopsis seeds on plates with 1/2 MS containing 100 mM NaCl. After incubation at 4 °C for 2 days, seeds were transferred to room temperature (22 °C) and the germination rate was scored every day.

Arabidopsis with 35S::AmDHN and wild-type plant seeds were planted on 1/2 MS plates with 100 mM NaCl and 15 % PEG6000 under long-day conditions. After incubation at 4 °C for 3 days, seeds were transferred to room temperature and representative pictures were taken 5 days after PEG6000 treatment.

Real-time PCR assays of abiotic genes in transgenic plants

Transgenic Arabidopsis with 35S::AmDHN were selected to analyze relative expression level of abiotic genes compared with Arabidopsis with 35S::GUS. Total RNA was isolated from 10-day-old seedlings, and total RNA preps were then treated with DNase (TaKaRa, Japan) for 30 min at 37 °C. Quantitative analysis of gene expression was performed by Realtime RT-PCR using ABI 7500 apparatus and SYBR Green I detection. For the quantification of gene expression the following primers were used (seen in Table 1). Actin was used as a control to normalize the amount of cDNA.

Results

Isolation of the full-length cDNA of AmDHN and sequence analysis

Base on the sequence of 432 bp isolated from a SSH cDNA library of A. mongolicus, two primers were designed to obtain 5′-cDNA end and 3′-cDNA end. A 519-bp fragment was isolated by using 3′ RACE and a 483- bp fragment was obtained by 5′ RACE. The 1,008-bp full-length cDNA of AmDHN was amplified from cDNA with primers DHN1 and UPM, tentatively designated AmDHN.

Sequence analysis indicated that the full-length cDNA contained an open reading frame of 549-bp encoding a putative protein of 183 amino acids. Ahead of the original code ATG and after the stop code TAA, there were a 56-bp 5′-UTR and a 400-bp 3′-UTR. Conserve sequence was a K fragment at the C-terminal end of the protein, which sequence was EKKGIMNKIKEKLPG (Fig. 1). Its amino acid sequence was used to search the protein databank and showed that it could be aligned with other dehydrin proteins from different species (Fig. 2). Its theoretical pI and MV were 5.78 and 18.4 kDa. The signal peptide analysis showed that the sequence had no signal peptide (Fig. 3).

Fig. 1
figure 1

The nucleotide sequence of AmDHN cDNA and the deduced amino acid sequence. One K fragment is boxed

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Fig. 2
figure 2figure 2

Comparison of amino acid sequences of AmDHN with other reported dehyrin proteins in NCBI database. Ten dehyrin proteins from other plants were compared with Ammopiptanthus mongolicus dehydrin, including Ammopiptanthus nanus, Cicer pinnatifidum, Corylus mandshurica, Codonopsis lanceolata, Trifolium repens, Pisum sativum, Glycine max, Galega orientalis, Helianthus annuus

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Fig. 3
figure 3

Evolutionary relationships of taxa. AmDHN is boxed

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Expression analysis of AmDHN gene

Transcripts of AmDHN were found in all organs tested, including seeds roots, stems, leaves and flowers, and the expression in seeds was the highest (Fig. 4a). The expression of AmDHN could be improved under different abiotic conditions including salt, PEG6000, ABA and drought treatments (Fig. 4b–e). This expression pattern implies that AmDHN might play a role in the response of plants to abiotic stresses.

Fig. 4
figure 4

Expression pattern of AmDHN. AmDHN expression level was detected by RT-PCR. The Ammopiptanthus mongolicus Actin gene was used as an internal control. (a) RT-PCR analysis of seeds (Sd), stems (Sm), leaves (Lf), roots (Rt) and flowers (Fr). (b) RT-PCR analysis of 20-day seedlings treated with 30 % PEG6000 for 0, 1, 3, 6, 12 and 24 hours (h). (c) RT-PCR analysis of 20-day seedlings treated with 150mM NaCl for 0, 1, 6, 12, 24 and 48 h. (d) RT-PCR analysis of 20-day seedlings treated with 100 μM ABA for 0, 0.5, 1, 3, 6 and 12 h. (e) RT-PCR analysis of leaves from mature plants without water for 0, 1, 3, 6, 9 and 12 days (d)

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AmDHN localizes in nucleus

To examine the subcellular localization of AmDHN, the fusion AmDHN-GFP was constructed. The recombinant DNA and pA7-GFP vector were transformed into onion epidermal cells by a gene gun. When the cultured onion epidermal cells with AmDHN-GFP fusion proteins were examined by epifluorescence microscropy, a strong fluorescence signal was observed only in the nucleolus (Fig. 5d). In contrast, the GFP signal distributed throughout the onion epidermal cells with control pA7-GFP vector (Fig. 5a). Taken together, these results indicated that AmDHN is a nuclear-localized protein.

Fig. 5
figure 5

Subcellular localization of AmDHN-GFP fusion in onion epidermal cells. The AmDHN-GFP fusion and the pA7-GFP control plasmid were transformed into onion epidermal cells using a gene gun. The fluorescence signals were examined by a confocal laser scanning microscope. The GFP fluorescence from cells expressing AmDHN-GFP fusion protein was localized to the nucleus of the cells (d–f). The GFP fluorescence was distributed throughout the entire cells expressing GFP empty vector (a–c). The photographs were taken in dark field vision (a, d), bright light vision (c, f), and superposition of the bright and dark vision (b, e). Bar = 100 μm

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Overexpression of AmDHN enhances abiotic stresses tolerance of transgenic lines

The increased expression level of AmDHN under different abiotic stresses helped us to evaluate the gene functions, if any, of AmDHN overexpression on the salt and drought stresses response. The CDS of AmDHN under the control of the 35S promoter was transformed into wild-type plants. Based on PCR, RT-PCR and Southern blot analysis, several independent transgenic plants showing increased expression of the AmDHN transgene were selected for further investigation. We used transgenic Arabidopsis plants with 35S::AmDHN to test their germination efficiencies in the presence of NaCl. In the absence of NaCl, all plants, including those transformed with 35S::AmDHN, 35S::GUS and WT seeds germinated with similar efficiencies (Fig. 6b). When adding 100 mM NaCl, WT and 35S::GUS seeds showed reduced germination efficiencies compared with transgenic lines expressing 35S::AmDHN (Fig. 6a). To test whether overexpression of AmDHN could enhance drought resistances, Arabidopsis plants were treated with 15 % PEG6000 simulating the drought condition. Transgenic plants with AmDHN showed higher survival rate after 15 % PEG6000 treatments compared to the WT Arabidopsis (Fig. 7). These data indicated that Arabidopsis lines over expressing AmDHN are more salt and drought-resistant than WT Arabidopsis.

Fig. 6
figure 6

Seeds germination of wild-type and AmDHN transgenic tobacco strain in response to NaCl. a Germination in the presence of 100 mM NaCl; b Germination in the absence of NaCl

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Fig. 7
figure 7

Growth analysis of transgenic Arabidopsis thaliana in plates with 15 % PEG6000

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The AmDHN-overexpressing A. thaliana were then used to determine whether increased expression of AmDHN would affect the expression of other abiotic-resistant genes and, therefore, modulate the tolerance of plants to salt or other stresses. To examine the expression of other abiotic genes in transgenic plants, we carried out relative quantitative real-time RT-PCR analysis using gene-specific primers of CAT1, BADH1 and DREB2A (seen in Table 1), with cDNA from 35S::AmDHN and 35S::GUS seedlings of Arabidopsis thaliana as templates. The relative expression was calculated as:

$$ 2^{{ - \vartriangle {\text{Ct}}}} = 2^{{ - \left[ {{\text{Ct}},{\text{t}} - {\text{Ct}},{\text{r}}} \right]}} $$

Ct,t: The threshold cycle of target gene; Ct,r: The threshold cycle of housekeep gene.

As shown in Fig. 8, overexpression of AmDHN leads to accumulate expression of CAT1 and BADH1. But another major abiotic gene DREB2A was not affected with overexpression of AmDHN (Fig. 8). Taken together, these results show that overexpression of AmDHN leads the transgenic plants to become more tolerant to salt and drought stresses.

Fig. 8
figure 8

Transcript levels of CAT1, BADH1 and DREB2A were detected by real-time PCR in 35S::GUS and 35S::AmDHN Arabidopsis plants of 10-day old. The data are mean ± SE from three independent experiments. Asterisk indicates P < 0.001 compared with 35S::GUS

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Discussion

In this study, we isolated the full-length cDNA of AmDHN from Ammopiptanthus mongolicus on the basis of 432-bp EST fragment from the SSH cDNA library. Sequence analysis of AmDHN indicated that it contained one K fragment, which amino acid sequence is EKKGIMNKIKEKLPG (Fig. 1). The K fragment is an obvious character of dehydrin protein. And this sequence has been found in many dehydrin proteins from other plants (Fig. 2). The expression patterns of AmDHN in different A. mongolicus organs were examined using RT-PCR analysis. In all tissues tested, the expression of AmDHN could be observed, whereas AmDHN is expressed highest in seeds and lowest in flowers.

Expression of AmDHN is highly induced by various stresses (Fig. 4). AmDHN is strongly and continuously expressed in response to ABA stress. Many dehydrin genes could be induced under ABA conditions [12–15]. A dehydrin gene RAB21, from Arabidopsis, could be induced with ABA [16]. And some researches show that, the longer plants under stresses conditions, the higher expression level of dehydrin could be detected in wheat, barley and populus species [17–21].

High-salt and drought stresses are major adverse environmental conditions that affect plant growth and development and crop yield. So far, some dehydrin proteins in the salt stress response pathway in plants have been identified. We have demonstrated that the transcriptional expression of gene of AmDHN was induced by salt, drought and other stresses. And the gene encoding AmDHN was obtained from a SSH cDNA library induced by PEG6000. Transgenic plants overexpression of AmDHN, are improved in salt and drought stresses.

We used the 35S::AmDHN constructs described above to transform Arabidopsis plants and test their germination efficiencies in the presence of 100 mM NaCl. In the absence of NaCl, all plants, including those transformed with 35S:: GUS and WT seeds germinated with similar efficiencies (Fig. 6b). When adding 100 mM NaCl, WT and 35S::GUS seeds show reduced germination efficiencies compared with transgenic lines expressing 35S::AmDHN (Fig. 6a).

To identify drought resistance in transgenic plants with AmDHN gene, transgenic Arabidopsis with 35S::AmDHN (T1, T2 and T3) were transferred to plates with 15 % PEG6000. Transgenic plants with AmDHN showed higher survival rate after 15 % PEG6000 treatment compared to the WT plants (Fig. 7). These data indicated that Arabidopsis lines over expressing AmDHN are more PEG6000- tolerant than control lines. These results imply that the AmDHN may be involved in the salt, drought and other stresses response in A. mongolicus. Previous studies have demonstrated that some dehydrin proteins play important roles in drought and salt resistances [6, 17, 22–25]. A wheat dehydrin DHN-5 play important roles in salt and drought resistances, and overexpression of DHN-5 enhances tolerance to salt and osmotic stress in A. thaliana [17]. Overexpression of the barley and wheat LEA protein genes, HVA1 and PMA1959 increased tolerance to drought and salt stress in transgenic rice [26, 27].

In transgenic Arabidopsis, expression level of CAT1, BADH1 was improved, but the expression level of DREB2A was not obviously changed. Those results may display that overexpression of AmDHN in Arabidopsis causes high expression level of some abiotic genes, including CAT1 and BADH1. AmDHN gene may act downstream of DREB, so the expression level of DREB2A is not changed when overexpression of AmDHN in Arabidopsis. Previous experiment may support this view. Expression of OsDhn1 is highly up-regulated in CBF1 transgenic rice, indicating that OsDhn1 is a target of CBF/DREB1 signaling [28].The identification of the genes will help to understand how AmDHN act in the regulation of the salt, drought or other stresses response.

In conclusion, a abiotic stresses-induced dehydrin protein AmDHN was isolated from A. mongolicus. The putative protein localized in the nucleus, and it could be induced in different stresses conditions. There is one K fragment at the C-terminal ends of the protein. AmDHN has function in salt and drought resistances.