Characterization of a Type 1 Metallothionein Gene from the Stresses-Tolerant Plant Ziziphus jujuba

Plant metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, and metal-binding proteins, which play an important role in the detoxification of heavy metal ions, osmotic stresses, and hormone treatment. Sequence analysis revealed that the open-reading frame (ORF) of ZjMT was 225 bp, which encodes a protein composed of 75 amino acid residues with a calculated molecular mass of 7.376 kDa and a predicated isoelectric point (pI) of 4.83. ZjMT belongs to the type I MT, which consists of two highly conserved cysteine-rich terminal domains linked by a cysteine free region. Our studies showed that ZjMT was primarily localized in the cytoplasm and the nucleus of cells and ZjMT expression was up-regulated by NaCl, CdCl2 and polyethylene glycol (PEG) treatments. Constitutive expression of ZjMT in wild type Arabidopsis plants enhanced their tolerance to NaCl stress during the germination stage. Compared with the wild type, transgenic plants accumulate more Cd2+ in root, but less in leaf, suggesting that ZjMT may have a function in Cd2+ retension in roots and, therefore, decrease the toxicity of Cd2+.

gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into Arabidopsis thaliana genomes by the Agrobacterium-mediated transformation method. Transgenic plants showed tolerance to NaCl and CdCl2 stresses, and the Cd 2+ was accumulated in roots and showed decreased accumulation in leaves.

ZjMT Encodes a Protein with a Metallothionein (MT) Domain
ZjMT (GenBank No. AB513130) was obtained by screening jujube full-length cDNA libraries. The ZjMT cDNA is 225 bp in length and encodes a polypeptide of 74 amino acid residues and with a predicted molecular mass of 7.376 kDa. The deduced amino acid sequence analysis indicated that ZjMT contains highly conserved cysteine-rich domains in its N-and C-terminal respectively and a cysteine-free region between them, which was the common feature of the Type 1 MT proteins reported in other plants. With the BLASTN search from the NCBI database, the deduced amino acid sequence showed homology with counterpart Type I MT family members from other plant species ( Figure 1A). Phylogenetic analysis revealed that ZjMT was clustered in the same clade with Mangifera indica, but distinct from Pisum sativum ( Figure 1B). The proteins used in the alignment and phylogenetic tree all had an MT domain and were obtained by database searching in NCBI.

ZjMT Is a Potential Stress-Related Gene
To identify whether ZjMT could be induced by heavy metal or other abiotic stresses, the expression profiles of ZjMT in Z. jujuba young seedlings under CdCl2, NaCl and PEG treatments were investigated using quantitative RT-PCR. ZjMT expression was significantly activated by CdCl2, NaCl and PEG stresses. The transcripts level of ZjMT increased at 0.25 h after CdCl2 treatment, reached a peak at 24 h, and then declined at 48 h ( Figure 2A). ZjMT transcript level reached a peak at 0.75 h when the young seedlings were under 50 and 100 mM NaCl treatments, however at 0.5 h, it reached the peak under 200 and 300 mM NaCl treatments ( Figure 2B). Similarly, the ZjMT transcript level reached a peak at 0.25 and 0.75 h under 1.2 MPa PEG treatments and 0.5 and 0.8 MPa PEG treatments, respectively ( Figure 2C). Six-week-old Z. jujuba young seedlings were treated with 100 mM CdCl2, 50, 100, 200 and 300 mM NaCl, and 10% PEG 6000 under different conditions at indicated time points. Different letters (a-d) indicate statistically significant differences between means at p < 0.05 (Student's t-test). N/A: Not applicable. Standard errors were calculated from three biological replicates in which ZjH3 (an actin gene, accession number EU916201) transcripts were used as internal controls. The 2 −ΔΔCt method was used to measure the relative expression levels of the target gene in stressed and non-stressed leaves. Error bars represent standard error.

Subcelluar Localization of ZjMT
To investigate the localization of ZjMT, the 35S:ZjMT-YFP plasmid was constructed and transformed into Arabidopsis by the floral dipping method. Homozygous transgenic lines were used for localization analysis. Firstly, we analyzed the localization of the ZjMT-YFP fusion protein in epidermal cells; it was primarily localized in the cytoplasm of the stomata guard cells ( Figure 3A). In addition, the fluorescence could also be detected in the cytoplasm and nucleus in stem and roots, respectively ( Figure 3B-D).

Constitutive Expression of ZjMT in Arabidopsis Enhances Their High Salinity Salt Tolerance
We examined the role of ZjMT in NaCl stress responses, under normal conditions, no significant difference was observed between transgenic and wild type plants (data not shown). Although the cotyledon greening rate was similar between transgenic and wild type plants, the radicle emergence of transgenic seedlings was slightly higher than wild type in the presence of 50 mM NaCl medium. Furthermore, the radicle emergence of transgenic seedlings increased significantly compared to those of the wild type plants on the medium containing 100 mM NaCl, and the wild type seedlings were failed to develop radicle on the medium containing 200 mM NaCl. The wild type and transgenic seeds were both failed to germinate when the NaCl concentration reach to 300 mM ( Figure 4). These results indicate that constitutive expression of ZjMT leads to enhanced tolerance in transgenic seedlings under salt stress and that ZjMT might act as a positive regulator to salt stress.

Cd 2+ Accumulation and Distribution in Transgenic Plants
To explore whether constitutive expression of ZjMT influences endogenous Cd 2+ content, the concentrations of Cd 2+ in transgenic plants and wild type plants were measured. Although the concentration of Cd 2+ in leaves of transgenic plants and wild type plants gradually increased after CdCl2 treatment, the transgenic plant accumulated less Cd 2+ in leaves compared to wild type ( Figure 4C). However, the root Cd 2+ concentrations for transgenic plants were higher than those of WT plants ( Figure 4B). In addition, similar results were observed after various concentrations of CdCl2 treatment ( Figure 4D,E).

Discussion
Heavy-metal contamination is a great environmental concern globally, and the risk posed to humans is increasing. Metallothioneins (MTs) are Cys-rich proteins, which are involved in the metal tolerance of diverse living organisms. Although many studies have revealed the roles of MTs in plants in response to diverse metal stresses, the function of plant MTs remain poorly understood [38].
In this study, the ZjMT cDNA was cloned from Ziziphus jujube full-length cDNA libraries and determined as type-I MT based on the protein sequence alignment. Phylogenetic analysis also revealed that ZjMT shared high similarity of cysteine residue levels with other species (Figure 1). Previous research results of MT subcellular location showed that BjMT2 was localized in the cytoplasm of tobacco leaf cells, and AtMT4a and AtMT4b were both localized in cytoplasm, nucleus and membrane of Arabidopsis hypocotyls cells [39,40]. In our work, we found that the ZjMT was located in cytoplasm and nucleus (Figure 3). Microarray analysis indicated that MT transcripts were significantly up-regulated under salt and drought conditions in rice and barley [41,42]. In our study, the expression of ZjMT was generally induced after CdCl2 stress (Figure 2A), and the transcripts levels were also influenced by NaCl and PEG treatments ( Figure 2B,C). To gain additional insight into the function of ZjMT in these stress responses, we evaluated the effect of salt stress on the growth of transgenic seedlings. On MS medium supplemented with NaCl, the wild type seedlings grew slowly, and failed to germination on the medium containing 200 mM NaCl, while the transgenic seedlings still develop radicles ( Figure 4A). In addition, under CdCl2 stress, the transgenic plants exhibit increased accumulation of Cd 2+ in roots and decreased the accumulation in leaves, whereas the accumulation of Cd 2+ were increased both in roots and leaves in wild type plants ( Figure 4B-E). In previous study, Cd 2+ is taken up by plant roots and caused growth retardation [4], however further studies are needed to answer the underlying mechanisms of Cd 2+ accumulation in different tissues. Furthermore, overexpression of plant MT genes increased Cd 2+ , Cu 2+ and Zn 2+ accumulation in transgenic plants [43], and type-I MT genes were more abundantly expressed in roots [20], according to these results we propose that ZjMT likely has a function in retention of Cd 2+ in roots and decreased the Cd 2+ toxic to leaves.

Stress Treatments and Real-Time Polymerase Chain Reaction (PCR) Analyses
Z. jujuba Mill "Hupingzao" was used in this study and its seeds were germinated and grown in a greenhouse under controlled conditions: temperature at 25 ± 1 °C, a relative humidity of 65%-70%, and light density of ~2500 Lux at 12:12 h dark/light circle. For CdCl2 treatment, the six-week-old seedlings were transferred to ½ MS liquid medium (pH = 6.0) containing 100 mM CdCl2. For NaCl treatment, the seedlings were transferred to ½ MS liquid medium containing 50, 100, 200 or 300 mM NaCl. For PEG treatment, seedlings were transferred to ½ MS liquid medium containing 20% PEG (molecular weight 6000) under 0.5, 0.8, or 1.2 MPa [44]. The leaves of seedlings were harvested at indicated time points, and were snap-frozen in liquid nitrogen and stored at −80 °C before RNA isolation. ZjH3, an actin gene (accession number EU916201) transcripts were used as internal controls. The relative level of gene expression was detected using the 2 −ΔΔCt method.

Sequence Analysis of ZjMT
The conserved domains of MT from Z. jujube, G. max, P. trichocarpa x P. deltoids, B. platyphylla, N. nucifera, Sdrummondii, V. radiate, V. angularis, F. sylvatica and P. trichocarpa were aligned using the ClustalX program (version 1.83) with default parameters. The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA (version 5.05) [45]. Bootstrap analysis was performed using 1000 replicates in MEGA to evaluate the reliability of different phylogenetic groups.

Subcellular Localization
The open reading frame (ORF) of ZjMT was cloned into pEarleyGate-103 vector [46], which contained the yellow fluorescent protein (YFP) reporter gene, to generate a ZjMT-YFP fusion construct under the control of the CaMV 35S promoter. Arabidopsis plants were transformed by Agrobacterium-mediated floral dip. YFP fluorescence was observed under a confocal laser scanning system (Nikon, Tokyo, Japan), and examined at 514 nm (excitation) using an argon laser with an emission band of 515-530 nm.

Generation of Transgenic Plants
ZjMT cDNA was cloned into the vector under the control of CaMV 35S promoter. The construct was introduced into Agrobacterium tumefaciens GV3101 strain cells and then transferred into wild type Arabidopsis (ecotype Columbia) plants by floral infiltration [47]. The seeds of T0 generation were harvested and sown in soil, and 10-day-old seedlings of T1 plants were screened by spraying with 0.05% (v/v) phosphinothricin (ppt) solution. The survival transformants (T1) were confirmed by PCR amplification of ZjMT. The T2 seeds were planted on MS [48] agar medium containing 10 mg/L ppt and the transgenic lines with a 3:1 (resistant:sensitive) segregation ratio were selected to produce T3 seeds. The T3 lines displaying 100% ppt resistance were considered homozygous and used for further experiments.

Stress Treatments
For salt assays on plates, wild type and transgenic lines were planted on MS agar medium with various concentrations of NaCl for three days before being placed at a controlled environment. After 10 days, the phenotypes of the plants were examined and pictures were taken.
For CdCl2 experiments, two-week-old transgenic and wild type plants were planted in MS medium with various concentrations of CdCl2 for 24 h and then measured the Cd 2+ content of roots and leaves.

Measurement of Cd 2+ Content
For measurement of Cd 2+ content in plant tissues, the seedlings of Arabidopsis wild type and transgenic plants were planted. After 14 days, seedlings were treated with 0.1 mM CdCl2 for 1, 6, 24, and 48 h. Roots and rosette leaves were excised carefully to determine their Cd 2+ content. After 24 h at 105 °C, the dry weight was measured. The resulting dry matter was dissolved in nitric and perchloric acid (4:1) on a muffle furnace at 175 °C for 3 h. When the liquid became limpid, the Cd 2+ content of the samples was determined with an atomic absorption spectrophotometer.

Conclusions
In conclusion, overexpression of ZjMT in Arabidopsis positively has a function in retention of Cd 2+ in roots and decreased the Cd 2+ toxic to leaves and enhances the salt tolerance of Arabidopsis. Therefore, ZjMT can be used as a candidate gene to improve stress tolerance by genetic transformation in crops.