Introduction

Plants have evolved a complex network of signal transduction pathways to cope with extracellular stimuli. Ca2+, as a universal second messenger, plays an important role in a variety of eukaryotic signal transduction pathways. In plants, calcium-signaling mechanisms mediate a multitude of responses to external biotic and abiotic stimuli, including drought, high salinity, low temperature, hormones, and pathogens (Harper 2001; Knight and Knight 2001). Both the temporal and spatial parameters of Ca2+ signals, such as frequency, magnitude, duration, and subcellular localization of Ca2+ transients, contribute to the specificity required for the efficient coupling of the stimulus response (Allen et al. 2001). In addition to complex Ca2+ signatures, regulation in calcium signaling is also achieved via the function of calcium-binding proteins (Sanders et al. 2002). Such proteins sense changes in the local calcium concentration and relay the information to downstream responses, such as phosphorylation cascades, protein translocation process, and finally regulation of gene expression (Sanders et al. 2002; Luan et al. 2002). To date, several families of Ca2+ sensors have been identified in higher plants, such as the Calmodulin (CaM), calmodulin like proteins (CAML), Calcium-Dependent Protein Kinases (CDPK), and calcineurin B-like proteins (CBL) (Luan et al. 2002; Zielinski 1998; Kudla et al. 1999; McCormack and Braam 2003; Asano et al. 2005).

CBL-type Ca2+ sensors are a family of plant-specific proteins originally identified by their significant similarity to calcineurin B (CNB) and to neuronal calcium sensors (NCS) from animals and yeast (Kudla et al. 1999; Luan 2009). They are characterized by four common helix-loop-helix structural motifs (the EF-hand) that could act as a calcium-binding site (Kolukisaoglu et al. 2004; Cheong et al. 2007). CBL proteins have no enzymatic activity and function by interacting with and regulating a group of serine-threonine protein kinases called CBL-interacting protein kinases (CIPK) (Luan et al. 2002). To date, genes encoding CBLs and CIPKs have been found in all sequenced higher plant species (Yu et al. 2007; Zhang et al. 2008). Genome-wide analysis has revealed ten CBL members in Arabidopsis, rice, and poplar (Zhang et al. 2008). Different CBLs might response to particular stimuli in plants. For example, AtCBL1 is strongly induced by drought, high-salinity, cold, wounding, and the Abscisic acid (ABA) (Cheong et al. 2003), while AtCBL2 and AtCBL3 are induced by light (Nozawa et al. 2001). Loss-of-function analysis and over-expression analysis indicated that CBL1 functions as a positive regulator of salt and drought responses, but a negative regulator of cold response, whereas CBL9 was identified as a regulator for both ABA and stress response in the early development of plants (Pandey et al. 2004). Function analysis of CBL genes from woody plant and comparative analysis of their functions with Arabidopsis are fundamentally important for further understanding of calcium-signaling regulatory networks in higher plant.

Ammopiptanthus mongolicus is an evergreen epibiotic shrub of the desert region of China. It is considered as a relic population adapted to the dry and hot environment in Middle Asia after the withdrawal of subtropical evergreen broadleaved forest to the south (Zhao and Wang 2002). Furthermore, A. mongolicus is able to resist high solar radiation, heat, cold, and drought stresses, due to its evolution under harsh environments for a long period. In this paper, we report a novel plasma membrane localized CBL homologous gene from A. mongolicus, AmCBL1, whose expression pattern under multiple abiotic stresses such as drought, high salinity, cold, heat, and ABA was analyzed. Its overexpression in tobacco (Nicotiana tabacum L.) plants confers increased tolerance to salinity and temperature stresses, indicating its potential for improving multiple-stress tolerance in plants.

Materials and methods

Plant material and stress treatments

Seeds of A. mongolicus were obtained from Alashan district of Inner Mongolia of China. The seeds were surface-sterilized in 75% (V/V) ethanol for 1 min and then in a solution of 0.5% (V/V) sodium hypochlorite for 22 min. The surface-sterilized seeds were washed with sterile deionized water five times. The seeds were then grown on hormone-free Murashige and Skoog (MS) medium in a culture room at 25°C day/22°C night under long day conditions (day 16-h-light/8-h-dark cycle). A. mongolicus seedlings with four true leaves were used in this study. For drought treatment, seedlings grown in the MS medium were carefully removed and dehydrated on filter paper. For cold treatment, seedlings were transferred to a 4°C cold room under white light. For NaCl and ABA treatments, 300 mM NaCl or 200 μM ABA was added to the seedlings in the liquid MS medium (without agar). For CaCl2 treatment, the seedlings were treated by watering with a 50 mM CaCl2 solution. For heat treatment, the seedlings were treated in a hybridization oven at 42°C. Under all the treatments, A. mongolicus seedlings were collected after 0, 4, 12, 24 and 48 h. Seedlings taken at the respective time points were immediately frozen in liquid nitrogen, and stored at −80°C until use.

Cloning of the AmCBL1 using 5′ and 3′ rapid amplification cDNA ends (5′ and 3′ RACE)

For AmCBL1 cloning, total RNA was isolated from drought-treated A. mongolicus using the CTAB method (Chang et al. 1993). cDNA synthesis was carried out by a reverse transcription kit (Promega). According to the sequence of AtCBL1 and conserved EST regions of tomato, pine, and rice, a pair of degenerate primers (JBF 5′-ARACNGCNTTYAGNGTNAGT-3′; JBR 5′-AGNGCDATNAACATYTGYTTNACC-3′) was designed. Polymerase chain reaction (PCR) amplification using 100 ng of cDNA as the template was performed as follows: one cycle at 95°C for 5 min; 31 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min. Based on the sequence information of the amplified fragment, gene specific primers were designed for RACE to obtain the full length sequence of the gene. Primers for 3′ RACE were: H2, 5′-CTCAATGTCTCCACCCAAATGC-3′ and hyaR, 5′-GGCCACGCGTCGACTAGTAC-3′. Three specific primers GSP0, GSP2 and GSP3 were designed as follows: GSP0, 5′-CTATGACAGAACTGCTGATGC-3′; GSP2, 5′-GTCTGTGATGCAAGAATTACTG-3′; GSP3, 5′-GTCTCCTAACCTTAGAGTTGAAGCAC-3′. 5′ RACE was performed according to the instruction manual of the 5′ RACE System (Invitrogen Version 2.0). The full-length cDNA of AmCBL1 was amplified by PCR using the primers F-248 (5′-GCGTTGTCAACTTCCC TTCACC-3′) and R-248 (5′-GAAAGAGTAGGAAATGACCG -3′). The resulting PCR product was cloned into pGEM-T Easy vector (Promega) and the nucleotide sequence of the insert was confirmed by DNA sequencing.

Bioinformatics analysis of AmCBL1

Multiple sequence alignment was obtained using the DNAman program. To predict the isoelectric point (pI), molecular weight, and signal peptides of deduced proteins, ExPasy (http://www.expasy.org/tools) and SoftBerry (http://www.softberry.com) programs were used. For phylogenetic analysis, the deduced amino acid sequence of AmCBL1 was aligned manually with the sequences of CBL homologs from Arabidopsis, poplar, and rice. The sequences used for phylogenic analysis were as follows: AtCBL1(AF076251), AtCBL2(AF076252), AtCBL3(AF076253), AtCBL4(AF192886), AtCBL5(AF192885), AtCBL6(AF192884), AtCBL7(AF290434), AtCBL8(AF411957), AtCBL9 (AF411958), and AtCBL10(AF490607) of Arabidopsis; PeCBL1 (DQ337190), PeCBL2 (DQ907707), PeCBL3 (DQ907709), PeCBL4 (DQ907706), PeCBL5 (DQ907708), PeCBL6 (DQ907710), PeCBL8 (DQ907711), PeCBL9 (DQ337189), and PeCBL10 (DQ899956) of Populus euphratica; and OsCBL1 (DQ201195), OsCBL2(DQ201196), OsCBL3(DQ201197), OsCBL4(DQ201198), OsCBL5 (DQ201199), OsCBL6(DQ201200), OsCBL7(DQ201201), OsCBL8(DQ201202), OsCBL9(DQ201203), and OsCBL10(DQ201204) of Oryza sativa. The phylogenetic tree was constructed using the Clustal X program by the Neighborhood Joining Bootstrap method.

Expression characteristics of AmCBL1

To investigate the tissue specific expression of AmCBL1, cotyledons, hypocotyls, radicle, leaf, stem, and root materials were harvested. After RNA isolation and reverse transcription, semi-quantitative PCR was carried out. The 25-μl reaction system contained 1 μl of cDNA, 0.5 μl forward primer (10 μmol/l), 0.5 μl reverse primer (10 μmol/l), and 0.125 μl ExTaq DNA polymerase (TaKaRa). PCR amplification (30 s at 95°C, 30 s at 58°C and 1 min at 72°C) was performed for 27 cycles. PCR products were separated by agarose gel electrophoresis. The expression level of AmCBL1 was normalized to that of the AmActin gene (NCBI accession no. HM209307).

The expression level of AmCBL1 under different treatments was determined by quantitative real-time PCR (qRT-PCR) analysis using ABI StepOnePlus (Applied Biosystems, CA, USA) and SYBR green as a fluorescent dye. Each reaction contained 10 μl of power SYBR Green PCR Master Mix, 0.3 μl of 10 μmol/l gene-specific primers, and 50 ng of first-strand cDNAs. The gene-specific primers used in semi-quantitative PCR and qRT-PCR analysis were as follows: AmCBL1, 5′-GCCAAGAGGTCAAGCAAATGT-3′ (sense) and 5′-TCAGGTCAGCATCCAAGAAAGT-3′ (antisense); AmActin, 5′-GTCCTCTTCCAGCCATCTC-3′ (sense) and 5′-TTCGGTCAGCAATACCAGG-3′ (antisense). The thermal program for qRT-PCR was 10 min at 72°C, 45 cycles of PCR for 15 s at 95°C and 1 min at 60°C. Serial dilutions of total RNA from cultured cells were used to construct standard curves. Each PCR assay for the same tissue samples was carried out for three biological replicates and each replicates three technological repeats in separate experiments.

Subcellular location of AmCBL1

To study subcellular accumulation patterns of the AmCBL1 protein, we constructed chimeric genes with the AmCBL1 cDNA fused to green fluorescent protein (GFP) gene. The fusion constructs were introduced into onion epidermal cells using a helium biolistic device (Bio-Rad PDS-1000) under the control of the CaMV 35S promoter. A 35S-GFP vector was also introduced as a control. GFP expression was observed using a confocal laser-scanning microscope (Carl Zeiss LAM510) after incubation for 24 h in the dark.

Generation of transgenic plants

AmCBL1 gene was cloned into the plasmid pBI121 by replacing the GUS fragment that could be excised using SmaI and SacI, yielding the p35S-AmCBL1 construct. The plasmid was introduced into Agrobacterium tumefaciens EHA105. Tobacco plants were transformed by the Agrobacterium-mediated method. Primary transformants were screened on MS agar medium containing 50 mg/L kanamycin.

The determination of nptII and AmCBL1 in tobacco lines was analyzed by PCR from leaf tissue samples. PCR was carried out on 20 ng of genomic DNA isolated from 4-week-old greenhouse-grown tobacco transgenic plants, using 0.4 μM each of nptII primers or AmCBL1 gene-specific primers and one unit of ExTaq in a final volume of 25 μl. The gene-specific primers used were: 5′-TCGACGTTGTCACTGAAGCGCG-3′ (sense) and 5′-GCGGTCAGCCCATTCGCCGCC-3′ (antisense) for NtpII; 5′-CGTCTTCCACCCAAATGC-3′ (sense) and 5′-AGTTGTTATGTCCCTCAGATAGG-3′ (antisense) for AmCBL1.

Segregation and gene expression analysis

Transgene copy number was determined by segregation analyses. For segregation analysis, tobacco seeds were germinated on MS medium containing 100 mg/l kanamycin. Homozygous T1 lines with respect to the T-DNA loci were selected by determining the frequency of their antibiotic-resistant T0 seeds after self-pollination.

The detection of transgene expression was carried out by RT-PCR method. Total RNA was extracted by the CTAB method from leaves of 2-month-old transgenic plants grown under standardized greenhouse conditions. First-strand cDNA was synthesized from 1 μg of total RNA using the D6110A kit (TaKaRa) according to the manufacturer’s instructions. PCR assays were performed with AmCBL1-specific primers and 50 ng of cDNA, using NtActin gene as a house-keeping control (Yang et al. 2008).

Analysis of transgenic plants overexpressing AmCBL1

To observe the growth performance of transgenic tobacco plants under different stresses, seeds from wild-type (WT) and T0 transgenic lines were surface sterilized and germinated under standard conditions on MS agar Petri-plates (9-cm diameter) with or without 100 mg/L Kanamycin. Subsequently, WT and kanamycin-positive T1 transgenic seedlings were used for various abiotic stress treatments.

To induce salinity stress during germination, surface-sterilized seeds were sown on agar plates containing MS medium with different concentrations of NaCl (0, 100, 150, and 200 mM). Tobacco seedlings were grown with a day/night cycle of 16/8 h at 25°C and the conditions of seed germination were determined after the 7th day. The seedlings of WT and transgenic lines (uniformly developed with four true leaves) were also selected from MS agar medium and transferred to soil to perform salt resistance assay. After 20 days of treatment by watering with 200 mM NaCl at three-day intervals, their growth conditions were measured. For freezing stress, 15-day-old seedlings growing in soil were exposed to −2°C and maintained under continuous light for 48 h. After the freezing stress, plants were allowed to recover for 8 days at 25 ± 2°C. Thirty seeds were sown for each heat-tolerance test, and seedlings that germinated within 7 days after sowing in the selective medium were transferred to MS agar medium. After 2 days of growth, they were pre-incubated at 37°C for 3 h and then treated at 42°C for 20 h in a hybridization oven. After the incubation period, the plants were grown under normal conditions for 1 week.

Photographs were taken after all treatments and plants’ growth in response to various stresses was determined by measuring fresh weight and dry weight of plants/seedlings challenged by a stress. The fresh weight of plants/seedlings was measured immediately after the harvest while the dry weight was measured after drying the material for 48 h at 80°C. Chlorophyll content was determined by spectrophotometric method. Photochemical efficiency was monitored using a Dual-PAM-100, P700, and chlorophyll fluorescence measuring system (Walz, Effeltrich, Germany) at room temperature as previously described (Chen et al. 2009). Leaf injury was assessed by electrolyte leakage method (Zwiazek and Blake 1990).

Results

Cloning of a full length cDNA of A. mongolicus CBL homolog

A core fragment of 309 bp was first isolated from leaves of A. mongolicus. To obtain the full length cDNA, we employed 5′ and 3′ RACE to extend both ends of the putative CBL1 homolog. Finally, a 1,053 bp full-length cDNA sequence with a 27 bp polyA tail was obtained (Fig. 1). The corresponding gene was named AmCBL1, and it has been submitted to GenBank under the accession no. AY902246. The AmCBL1 contains a 642 bp open reading frame (ORF), a 124 bp 5′ untranslated region, and a 288 bp 3′ untranslated region. It encodes a protein of 213 amino acids with a predicted molecular mass of 24,415 and a theoretical isoelectric point (pI) of 4.6.

Fig. 1
figure 1

cDNA and deduced amino acid sequence of AmCBL1. The non-coding region is shown by a grey-background. The boxed sequence harbors a typical myristoylation site (MGXXXSK). The underlined sequence represents the cDNA fragment first identified by PCR (see “Materials and methods”). An in-frame stop codon (TAA) is immediately upstream of the ATG starting codon

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Sequence analysis of the deduced amino acid revealed four putative EF-hand motifs responsible for binding Ca2+. The first EF-hand of AmCBL1 exhibits an amino acid substitution (D is altered to S) as well as AtCBLs (Fig. 2a). A conserved myristoylation site (MGCXXSK/T) was found at the short-N terminus (Fig. 2a), which might play important roles in protein-membrane attachment and protein–protein interactions in the CBL signaling system (Batisticˇ et al. 2008; Weinl and Kudla 2009). The structural basis for Ca2+ binding resides in the four EF-hand domains in all CBL proteins and they are arranged with completely invariant spacing within the protein (Kolukisaoglu et al. 2004). Consequently, their amino acid variation is mainly accounted for the divergence of their N- and C-terminal domains (Batisticˇ et al. 2010). Phylogenic analysis of currently known CBL-related proteins showed that AmCBL1 shared a high homology with other CBL1 proteins (Fig. 2b). Among them, AmCBL1 is closely related to Populus euphratica PeCBL1 (NCBI accession no. DQ337190).

Fig. 2
figure 2

Deduced amino acid sequence of AmCBL1, its alignment with related sequences, and phylogenic analysis. a Deduced amino acid sequence of AmCBL1, PeCBL1, AtCBL1, and OsCBL1. A myristoylation site and four predicted EF-hand motifs (EF1-4) are indicated. Black and colored shading indicate identical and conserved amino acid residues, respectively. b Phylogenetic analysis of AmCBL1 with other representative CBL proteins. The phylogenetic tree was constructed using Clustal X by the Neighborhood Joining Bootstrap method (Bootstrap analysis with 1,000 replicates), based on the multiple alignments of the full-length amino acid sequences of CBLs from different plant species

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AmCBL1 is expressed in all tissues examined and induced by multiple-stresses

Members of the CBL family are calcium sensors with an established function in regulating ion homeostasis, exhibit a predominant expression in roots, and contribute to ion uptake or exclusion from roots (Xu et al. 2006). To investigate the tissue-specificity of AmCBL1 expression, the AmCBL1 mRNA levels in different plant organs were examined using semi-quantitative PCR analysis. The results indicated that AmCBL1 is expressed in all tissues examined, including cotyledons, hypocotyls, radicle, leaves, stem and roots of the plant, although the expression levels appeared to be somewhat different (Fig. 3a). We also produced and analyzed transgenic plants harboring AmCBL1 promoter–GUS fusion constructs, and found that the GUS activity was associated with vascular bundles, especially phloem of the leaf vein, stem, and roots (Guo et al. 2010).

Fig. 3
figure 3

Organ-specific expression of AmCBL1 and effects of stress treatments. a Expression analysis of AmCBL1 and Actin in cotyledons, hypocotyls, radicle, leaf, stem, and root. b Real-time PCR analysis of the AmCBL1 gene in A. mongolicus under different abiotic stresses. The relative expression was calculated using Actin gene as an internal reference. The unstressed expression level was assigned a value of 1. Values are given as mean ± SD from three replicates

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To gain an insight into possible functions of AmCBL1, the expression patterns of AmCBL1 under various abiotic stresses was examined by qRT-PCR analysis. The results showed AmCBL1 expression peaked 4 h after drought or heat treatment, and returned to near starting levels within 48 h. For salt treatment, the AmCBL1 transcript level peaked at 4 h and was maintained at the high level until 48 h. AmCBL1 transcript levels were also significantly upregulated in response to CaCl2 treatment. However, when exposed to ABA or cold treatment, AmCBL1 only showed weak induction (Fig. 3b).

AmCBL is localized at the plasma membrane

We investigated the distribution of GFP fluorescence after transient expression in onion epidermal cells (Fig. 4). Green fluorescence signals were observed throughout the cells transformed with the control plasmid 35S-GFP, whereas the GFP::AmCBL1 fusion protein was observed to be distributed at the plasma membrane in ~90% of the analyzed cells. The results suggest that AmCBL1 is a plasma membrane-localized protein.

Fig. 4
figure 4

Subcellular localization of AmCBL1. The 35S::GFP (top) and 35S::GFP-AmCBL1 fusion gene (middle and bottom) were transiently expressed in onion epidermal cells. The GFP-AmCBL1 fusion protein was exclusively expressed in the plasma membrane

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Expression of AmCBL1 in transgenic tobacco lines

The stress-inducible expression suggested a possible role of AmCBL1 gene in abiotic stress signaling. To examine the function of the AmCBL1 gene, tobacco was transformed by the Agrobacterium-mediated method. Fourteen independent transgenic lines were established, transferred into soil, and grown in a greenhouse to generate T1 seeds. Six independent transgenic lines (L1–L6) were selected for transient analysis. Under normal conditions, AmCBL1 mRNA of L1–L6 was detected in the leaves but not in the WT plants (Fig. 5).

Fig. 5
figure 5

Analysis of AmCBL1 expression in transgenic tobacco plants. Total RNA was isolated from greenhouse-grown transgenic plants. AmCBL1 gene-specific primers and NtActin gene-specific primers were used in the PCR reactions

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The gene copy number of the construct in the transformed plants was further confirmed by segregation analysis of the ntpII gene in the T1 seedlings. Segregation analysis suggested that the L3 and L5 lines appeared to have an integrated T-DNA locus on a single chromosome, as 75% of their T1 segregating seedlings were resistant to kanamycin (Table 1). Therefore, they were selected for further analysis.

Table 1 Expression of kanamycin resistance and salt stress tolerance in the T1 generation of transgenic tobacco plants
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Transgenic tobacco harboring AmCBL1 are tolerant to salt stress

To perform salt tolerance analysis, transgenic T1 seeds expressing the AmCBL1 gene, as well as wild-type seeds, were allowed to germinate in MS media containing different concentrations of NaCl. Under normal conditions (0 mM NaCl) both wild-type and T1 transgenic seeds germinated into seedlings with similar frequency. Differences of germination efficiency became apparent at 100 mM NaCl and were statistically significant at 150 mM NaCl (Fig. 6). While the germination efficiency of WT seeds was 30% reduced at 150 mM, L3 and L5 transgenic lines displayed almost normal germination frequencies. At a higher concentration (200 mM) of NaCl, about half of the AmCBL1-overexpressing seeds could germinate with healthy cotyledons, but very few of the control seeds could germinate (Fig. 6). For the L3 and L5 independent transgenic lines, the salt tolerant germination phenotype at 200 mM NaCl was found to be linked to the kanamycin gene because it segregated with the same ratio as the kanamycin-resistant phenotype (Table 1).

Fig. 6
figure 6

AmCBL1 transgenic lines exhibit increased tolerance to salinity during germination. Transgenic (L3 and L5) and control (WT) seeds were germinated on MS agar plates containing the indicated concentrations of NaCl. The data are expressed as a percentage of 100 seeds germinated from each indicated line and the values are given as mean ± SD (n = 3) from three independent experiments

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Phenotypic analysis of salt tolerance was also performed on soil-grown plants; the seedlings of WT and transgenic lines (uniformly developed with four true leaves) were selected from MS agar medium and transferred to soil. They were subjected to salt stress by irrigation with a 200 mM NaCl solution at three-day intervals for 10 days. As a result, both tobacco transgenic lines accumulated significantly higher fresh weight, and root elongation was also observed to be significantly faster in all the transgenic seedlings compared with the wild type under high salt stress after 10 days (Fig. 7a–c). These data indicated that AmCBL1 enhanced salt stress tolerance during seed germination and early seedling development.

Fig. 7
figure 7

Overexpression of AmCBL1 confers salt tolerance in transgenic tobacco plants. Each transgenic line and WT plants were watered with 200 mM NaCl for 10 days from the seedling stage. a Representative plants of wild-type and transgenic tobacco under high salt stress. b Fresh weight. c Root elongation. Results depicted in (b) and (c) are means ± SE (n = 30 seedlings) of three independent experiments

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Transgenic tobacco plants show improved tolerance to temperature stresses

Freezing stress tolerance was tested in 15-day-old seedlings of transgenic and wild-type tobacco plants. Soil-growing seedlings were exposed to freezing at −2°C for 48 h and then returned to normal conditions (25 ± 2°C) for recovery. After eight days, photosynthetic efficiency, fresh weight and electrolyte leakage rate were examined (Fig. 8a–d). Transgenic seedlings were observed to grow better than the wild-type and they accumulated significantly more fresh weight, higher Fv/Fm, and lower membrane damage over the wild type. These observations indicated that the AmCBL1 conferred some degree of freezing stress tolerance on the transgenic tobacco plants.

Fig. 8
figure 8

Freezing tolerance assay of AmCBL1 transgenic tobacco plants. a Representative plants of untreated plants (CK), wild-type, and transgenic tobacco grown under normal conditions for 8 days after freezing treatment. b Fresh weights of plants in (a). c Photosynthetic efficiency (Fv/Fm) of plants in (a). d Electrolyte leakage rate of plants in (a). Each bar value represents the mean of ± SD (n = 10 plants) of triplicate experiments

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To determine whether transgenic plants overexpressing AmCBL1 had an improved tolerance to heat stress, we investigated the thermotolerance of the 35S: AmCBL1 plants. Seven-day-old seedlings germinating on selective agar plates were transferred to MS agar plates. After 2 days of growth, WT and transgenic plants were pre-incubated at 37°C for 2 h and then treated at 45°C for 20 h. Subsequently, young seedlings were transferred to a greenhouse for 1 week. As shown in Fig. 9a, although all the plants subjected to the heat treatment appeared to be developmentally delayed in comparison with the plants growing under normal conditions, the two transgenic lines showed better performance than wild-type plants. The transgenic lines showed higher fresh weight accumulation, higher chlorophyll content and higher Fv/Fm over the WT plants (Fig. 9b–c), which indicated that the transgenic plants had been injured to a lesser extent than the WT plants. The damage of cell membrane for transgenic seedlings due to heat treatment was also lower than the WT (Fig. 9d). These results indicated that the transgenic tobacco plants have better ability to tolerate heat-stress than control (untransformed) plants.

Fig. 9
figure 9

Heat tolerance of the 35S:AmCBL1 tobacco plants. a Phenotype of plants under heat stress. 30 plants were used per test, and each test was repeated three times. b Fresh weight of WT and transgenic plans under heat treatment. c Chlorophyll content of plants under heat treatment. d Photosynthetic efficiency (Fv/Fm) of plants under heat treatment. e Electrolyte leakage rate of plants under heat treatment. Each bar value in (b–d) represents the mean ± SE (n = 25)

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Discussion

To determine the function of the calcium sensor CBLs in A. mongolicus, we identified a homolog of the CBL1 gene, AmCBL1, from this evergreen shrub species. The subcellular localization analysis indicated that it predominantly fulfill its function at the plasma membrane. Characterization of AmCBL1 showed that it might provide a protective function for the plants under a wide range of stresses including salinity, freezing, and heat.

Phylogenetic analysis showed that AmCBL1 was clustered into the CBL1/CBL9 subgroup and showed high homology to PeCBL1/CBL9 from woody plant. In contrast to the previously characterized AtCBL1, AmCBL1 cannot be significantly induced by low temperature (Fig. 3b). Previous studies by loss-of-function and overexpression analyses have revealed that AtCBL1 functions as a positive regulator of salt and drought response, but a negative regulator of cold response (Albrecht et al. 2003; Cheong et al. 2003; Batistic and Kudla 2004). Moderate constitutive overexpression of AtCBL1 caused an enhancement of cold-induced marker genes, but further increases in the expression levels of AtCBL1 showed a negative feedback regulation of some genes, including CBF1 and CBF2; an inhibition of DREB1A/CBF3 was also observed in CBL1 overexpression lines according to previous reports (Albrecht et al. 2003; Cheong et al. 2003). Interestingly, the negative regulation of cold response was not observed in AmCBL1-overexpressing tobacco plants. In contrast, the AmCBL1 transgenic tobacco lines showed a slightly higher tolerance to freezing stress than the controls, indicating a different mechanism involving AmCBL1 from that involving AtCBL1 in response to freezing stress. Overexpression of AmCBL1 also conferred salt and heat tolerance on transgenic tobacco plants, which indicated that AmCBL1 might play key roles in response to these stresses in A. mongolicus. Further analysis performed on the 5′ flanking region of AmCBL1 identified a 1,683-bp sequence as the AmCBL1 promoter. Analysis of the levels of transcription of GUS-fusion constructs of segments of the promoter in different tissues under various stresses suggested that the AmCBL1 promoter is a phloem-specific and multiple-stress-inducible promoter (Guo et al. 2010). These data suggest a complicated CBL signaling network in plants and provide a solid foundation for the future use of AmCBL1 in molecular breeding as new stress-resistance gene.

As an evergreen shrub, A. mongolicus can survive under harsh environments. It can tolerate a low temperature of −30°C or a high temperature of 40°C. Previous studies on its mechanism for coping with environmental stresses mainly focused on the xenomorphic structures and certain cold-resistant proteins (Jiang et al. 1999). In this report, we discovered that AmCBL1, a positive regulator of cold and heat stresses, might be another way for A. mongolicus to adapt to harsh environment. To our knowledge, this is the first work, which demonstrates involvement of CBL genes in the heat stress response. Further studies on the mechanism of temperature response of AmCBL1 needs to be done.

Most plants cultivated worldwide are exposed to severe salt or temperature stress during their life cycle, suffering a reduction in yield. However, evolutionary pressures may have conferred distinct responses to different stresses for the desert plants like A. mongolicus. In this regard, the AmCBL1 might have advantages over AtCBL1 for molecular breeding of economically valuable to generate superior plants for harsh environments. Irrespective of whether a CBLs-CIPKs calcium signaling network exists in A. mongolicus, characterization of AmCBL1 provides an important stepping stone for further understanding of the regulation of Ca2+ signal transduction in A. mongolicus, allowing us to explore the physiological and molecular aspects of the outstanding stress tolerance of this desert plant.