Heterologous Expression of OsPDR Enhances The Tolerance to Cobalt in Yeast
TIAN Si-Qi1, QIAO Kun1, WANG Fan-Hong1, LIANG Shuang1, WANG Hong1, CHAI Tuan-Yao1,2     
1. College of Life Science, University of The Chinese Academy of Sciences, Beijing 101408, China;
2. Southeast Asia Biodiversity Research Institute, Chinese Academy of Science, Yezin, Nay Pyi Taw 05282, Myanmar
*This work was supported by grants from The National Natural Science Foundation of China (U1632111, 61672489), the Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences (Y4ZK111B01) and the Chinese Academy of Sciences (KJRH2015-001)
** Corresponding author: WANG Hong. Tel: 010-88256343, E-mail: hwang@ucas.ac.cn
CHAI Tuan-Yao. E-mail:tychai@ucas.ac.cn
Received: September 11, 2018 Accepted: October 31, 2018
Abstract: A cadmium-responsive metal ion transporter OsPDR, which was highly upregulated in the shoot of rice (Oryza sativa ssp. japonica cv. Nipponbare) at 24 h after treatment with 50 μmol/L Cd, was identified in cadmium-responsive transcriptome profiles analyzed by RNA-Seq. In this study, we isolated OsPDR from rice (Oryza sativa cv. Nipponbare) and the metal ions transport activity of OsPDR was analyzed. The results of metal tolerance experiments showed that overexpression of OsPDR can enhance yeast's tolerance to Co, but not to Zn, Ni or Cd, and higher accumulation of Co was observed compared to the empty vector-transformed yeast as determined by inductively coupled plasma mass spectrometry (ICP-MS). The EGFP-OsPDR fusion protein was localized to the vacuolar membrane using confocal microscopy. These data suggested that OsPDR might play an important role in Co homeostasis. Additional studies are needed to understand the function of OsPDR in plants.
Key words: OsPDR     cobalt     transport     yeast    
田思琪1, 乔坤1, 王凡红1, 梁爽1, 王红1 , 柴团耀1,2     
1. 中国科学院大学生命科学学院,北京 101408;
2. 中国科学院东南亚生物多样性研究中心,缅甸 耶津 内比都 05282
摘要: 在通过RNA-Seq技术得到的镉响应转录组图谱中,用50 μmol/L Cd处理24 h后,一个镉响应金属离子转运蛋白OsPDR被鉴定出其在水稻(Oryza sativa ssp. japonica cv. Nipponbare)茎中的表达量显著上调.本研究中,从水稻(Oryza sativa cv. Nipponbare)中分离了OsPDR基因,并对其金属离子转移活性进行了分析.金属耐受性实验结果表明,过表达OsPDR能提高酵母对Co的耐受性,但对Zn、Ni和Cd的耐受性不强,并且经电感耦合等离子体质谱法(ICP-MS)测定Co含量后,与空载体转化酵母相比,过表达OsPDR的酵母中Co的积累更高.利用共聚焦显微镜观察发现,EGFP-OsPDR融合蛋白定位于液泡膜上.这些数据表明OsPDR可能在Co稳态中起着重要作用.OsPDR在植物中的作用,还需要进一步的研究.
关键词: OsPDR          转运     酵母    

Cobalt (Co) is an essential trace element for plant growth and is a component of vitamin B12. Low concentrations of Co can promote plant growth properly, but if the concentration of Co in the soil solution reaches 10 mg/L, it inhibits the growth and development of plants or even causes death, which would have a significant effect on human health[1-3]. With the development of modern industry, an increasing number of cultivated rice fields are polluted by the heavy metal Co. Co, once entering the soil, is stored in the cultivated layer by adsorption, precipitation and complexation[4-5]. Additionally, Co can be highly transported and is easily absorbed through the roots of plants and, in turn, accumulates in the vegetative and reproductive organs of plants, damaging not only the yield and quality of crops but also endangering human health by entering the human body through the food chain[6-7].

The ATP-binding cassette (ABC) transporter subfamily, a family of membrane proteins with a strong metal transport function, is mainly located in the plasma membrane, endoplasmic omentum, mitochondrial membrane and vacuolar membrane[8-11]. All of the members of this subfamily contain 4 or 6 highly hydrophobic transmembrane domains and ATP binding and/or nucleoside binding domains (NBDs) in the peripheral cytoplasm[8]. The ABC transporter proteins in yeast can be classified into subfamilies, from ABCB to ABCG, according to their sequence similarity to mammalian NBDs[12].

Recent research has revealed that ABC transporters can export harmful substances, extracellular toxins and targeted membrane modules, including substances that are related to resistance to biotic and abiotic stress in plants[13]. The known ABC genes can be classified into three subfamilies in plants: multidrug resistance (MDR), multidrug resistance- associated protein (MRP) and pleiotropic drug resistance (PDR)[14-15]. MRP genes play a role in many processes, such as detoxification and the transport of vacuolar flavonoid[16-17]. The MRP and MDR subfamilies are mainly related to Cd transportation in plants[18-20]. MRPs may be involved in the transport of the PC-Cd or GS-Cd complex on the vacuolar membrane[21-23]. PDR proteins, which are the subject of this study, have been found in plants that can respond to abiotic and biotic stress[24]. cDNA of SpTUR2 was cloned as the first PDR gene in Spirodella polyrhiza and encodes a PDR5-like ABC transporter[25]. The expression of PDR genes is induced by pathogens, and PDR proteins can transport products to the cell surface[15]. PDR proteins are widely found in the plant cell membrane and can transport many cell substrates, such as metal ions[26-27]. Furthermore, studies have shown that the absence of some ABCG/PDR transporters on the plasma membrane of rice can increase its sensitivity to heavy metals[28]. Several PDR genes have been found in plants. NtPDR3 was induced under iron-deficiency in Nicotiana tabacum[29-30]. The expression of AtPDR8 in Arabidopsis thaliana increased, and the tolerance of transgenic plants with overexpression of AtPDR8 to Cd and Zn also increased[31-32]. Further studies showed that the expression of OsABCG43/PDR5 had effects on the distribution of Cd in yeast cells[33]. OsPDR9 can be induced by Cd and Zn in rice[34]. Finally, the expression of AtPDR9 can detoxify the herbicide 2, 4-D in Arabidopsis thaliana[35]. However, there are few reports of PDR genes that are related to the transport of Co.

To analyze the function of OsPDR involved in heavy metal transport in yeast, confocal microscopy was used to observe the subcellular localization of OsPDR. Changes in the phenotype of yeast with OsPDR over-expressed were observed and the heavy metals content of OsPDR expressed in yeast was also measured to analyze the transport functions of OsPDR. The mechanism for regulating the Co content was defined, and this mechanism may underlie a theoretical foundation to support the breeding of rice with low Co accumulation in the future.

1 Materials and methods 1.1 Plant materials and growth conditions

Wild-type rice (Oryza sativa cv. Nipponbare) was used as the experimental material for gene cloning. The rice seeds were germinated in sterile water in glass dishes and were kept at 37℃ for 2-3 d in the dark and then were transferred to 1/2 Hoagland's solution that was renewed every 2 d[36]. The materials were cultured in a greenhouse at 25℃ under a 16 h/8 h light/dark cycle[37].

1.2 Gene cloning and plasmid construction

The OsPDR gene was obtained from the transcriptome profiles of cadmium-responsive metal ion transporters identified in rice[38]. Total RNA was isolated from seedlings of wild-type rice (21-day-old) using RNAsio Plus (TaKaRa, Japan). The cDNA was synthesized by the HiScript 1st Strand cDNA Synthesis Kit (Vazyme, China). The PCR products were cloned into the pEASY®-Blunt Zero Cloning Vector (Transgen biotech, China) and were used as templates. The full-length sequences of OsPDR, OsPDR-EGFP and EGFP were cloned into the pYES2 vector between Hind Ⅲ and EcoRⅠrestriction sites using the ClonExpress® ⅡTM One-Step Cloning Kit (Vazyme), and the primers are listed in Table 1.

Table 1 List of primers used to amplify OsPDR sequences
1.3 Yeast strains and growth conditions

The yeast strains used in this study included BY4741 (his3D1, leu2D0, met15D0, ura3D0, mating type α) and YK44 mutant strains (ura3-52, his3-200, ZRC, Cdt1, mating type α), which were received from Euroscarf (Frankfurt, Germany). The LiOAc/PEG method was used for yeast transformation, and the positive clone was selected from synthetic dropout medium without uracil (SD-Ura)[39]. The Yeast Extract Peptone Dextrose Medium (YPD) was used for the growth assay and glucose (Glu) in YPD medium was replaced by 2% (w/v) galactose (Gala) (YPG) was used for inducing the expression of genes.

1.4 Subcellular localization in yeast cells by fluorescence microscopy

pYES2-EGFP and pYES2-OsPDR-EGFP were transformed into BY4741 and YK44 and were used to observe the subcellular localization of OsPDR in yeast. Transformants were pre-cultured on SD-Ura medium and then on YPG for induction[40]. The transformed yeast cells were selectively stained with FM4-64, a marker of vacuolar membranes[41-42]. The yeast cells were incubated with 5 μmol/L FM4-64 for 30 min at 30℃ in the dark, washed three times with YPG medium, cultured with 3 ml YPG for 2 h and then washed three times with 0.05 mol/L PBS (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na2HPO4 10 mmol/L, KH2PO4 2 mmol/L, pH 7.4) before observation with a confocal laser scanning microscope (LSM 710 NLO; Carl Zeiss, Jena, Germany)[43].

1.5 Metal tolerance experiments

For the complementation assays, pYES2-OsPDR and pYES2 plasmids were transformed into yeast BY4741 and yeast YK44 on YPD medium and YPG medium containing 500 μmol/L or 1 mmol/L CoCl2 using the lithium acetate method[44]. The transformed yeast cells were cultured overnight at 30℃ until reaching an A600 value of 0.5 and then were successively diluted to A600 values of 0.5, 0.05, 0.005 and 0.0005. Diluted yeast cultures (4.5 μl) were used for the drop-test experiments with metal solutions (500 μmol/L and 1 mmol/L ZnSO4, 50 μmol/L and 100 μmol/L CdSO4, 500 μmol/L and 1 mmol/L NiCl2, and 500 μmol/L and 1 mmol/L CoCl2) added to the solid medium. Three biological replicates were analyzed for all of the drop-test experiments.

1.6 ICP-MS experiments

To detect the metal content in yeast cells, OsPDR expressed in the yeast strains BY4741 and YK44 were pre-cultured on SD-Ura medium at 30℃ for 30 h and grown for 12 h on the induction medium to induced the expression of OsPDR by a dilution of culture broth (1: 1 000). The A600 was diluted to 0.8 and subsequently incubated on the induction medium supplemented with 150 μmol/L CoCl2 and 0 μmol/L CoCl2 (control) for 48 h. Ethylenediamine tetra-acetic acid (EDTA, 10 mmol/L) was used to absorb metal ions bound to the precipitate after centrifugation, rinsed with deionized water three times, then stored at 50℃ for 3 d. The concentration changes of Co in yeast were determined after microwave digestion (Milestone, Italy) by inductively coupled plasma mass spectrometry (ICP-MS).

2 Results 2.1 Isolation and phylogenetic analysis of the OsPDR

Cadmium-upregulated transcriptome profiles were identified in rice roots and shoots under Cd treatment by RNA-Seq analysis[38]. We focused on one gene that was highly responsive under Cd stress identified by the Cufflinks program[38]. The open reading frame of OsPDR (chr07: 20207865..20213557) was 900 bp and analyzed using DNASTAR Lasergene v7.1 software[45]. OsPDR can be classified as a member of the ABCG/PDR subfamily through a phylogenetic analysis (Figure 1a). The OsPDR gene was 77% identical to TuPDR5 at the amino acid level using sequence alignment and encoded a deduced protein of 299 amino acids (Figure 1b)[25]. OsPDR has 3 transmembrane domains (TMs) predicted by TMHMM with an extracellular N terminus (http://www.cbs.dtu.dk/services/TMHMM/) (Figure 1c, d)[46-47]. SWISS-MODEL provides the information of the model, such as the oligomeric state, ligands and cofactors. The modelling process and reliability of the model was estimated by the global quality estimation score (GMQE:0.55) and the local composite scoring function (QMEAN.:-5.23). The combined quality estimate showed that the resulting GMQE was 0.20, which combines the QMEAN with the GMQE acquired from the alignment between target and template. The tertiary structure of OsPDR credible predicted by the SWISS MODEL was generated based on the template of the ATP-binding cassette subfamily G member 2 (ABCG2), which has 33% sequence similarity, 23.29% sequence identity with ABCG2 and the coverage was 0.98 (Figure 2).

Fig. 1 Sequence features of OsPDR (a) A phylogenetic tree of OsPDR was speculated by the Maximum-Likelihood method and analyzed with MEGA6. ABC(ATP-binding cassette transporter), ABCG(ATP-binding cassette subfamily G), PDR(pleiotropic drug resistance). (b) Amino acid sequence alignment between OsPDR and TuPDR5. The alignment was conducted by ClustalW. White letters in black indicate identical amino acid residues. The predicted transmembrane domains of OsPDR are denoted by TM1-TM3. (c) Transmembrane domains predicted by TMHMM. (d) Topological analysis of OsPDR by SACS HMMTOP.
Fig. 2 Tertiary structure of OsPDR predicted by the SWISS MODEL(https://www.swissmodel.expasy.org/) (a) Template of the ATP-binding cassette subfamily G member 2(ABCG2). (b) The model of OsPDR.(c) Modelling results. An analysis of the quality estimation information. The oligomeric structure (matching prediction) was a monomer without ligand and the model was built with ProMod3 Version 1.1.0. The score of GMQE was 0.55 and the score of QMEAN was -5.23. Local model quality estimates were presented as a per-reside plot by the QMEAN scoring function and the overall model quality based on global QMEAN estimates was provided as a Z-score, which the obtained values in relation to the global score caculated by a series of PDB structures with high-resolution. The combined quality estimate showed that the resulting GMQE (combines the QMEAN with the GMQE obtained from the alignment between target and template) was 0.20. The sequence identity was 23.29% and the sequence similarity was 33% between OsPDR and the template ABCG2.
2.2 OsPDR is localized to the vacuolar membrane in yeast

To confirm the subcellular localization of OsPDR, the OsPDR-EGFP fusion protein and EGFP protein were heterologously expressed in BY4741 yeast respectively. For the EGFP protein expressed in BY4741 yeast, green fluorescence was observed throughout nearly all of the cell, indicating that the protein is localized in the cytoplasmic matrix[48-49]. To further verify the subcellular localization of the fusion protein, the lipophilic dye FM4-64 was used to specifically stain the vacuolar membrane. Merging the GFP and FM4-64 images demonstrated that the EGFP-tagged OsPDR localized to the vacuolar membrane when analyzed in the stationary phase (Figure 3b).

Fig. 3 Subcellular localization of OsPDR (a) Schematic representation of pYES2-OsPDR-EGFP. (b) The localization of EGFP and OsPDR-EGFP fusion proteins. pYES2-EGFP and pYES2-OsPDR-EGFP expressed in the yeast strain BY4741. pYES2-EGFP and pYES2-OsPDR-EGFP expressed in yeast were cultured in SD-Ura until they reached the stationary phase. The cells were then induced in YPG medium overnight. From left to right, the figure shows the bright-field images, green fluorescence signals, FM4-64 fluorescence signals and the merged images with FM4-64 in red and GFP in green for pYSE2-EGFP or pYSE2-OsPDR-EGFP.
2.3 Overexpression of OsPDR in yeast confers Co tolerance

The pYES2 and pYES2-OsPDR plasmids were transformed into the wild-type yeast strain BY4741 and the yeast mutant YK44 that is sensitive to Zn, Cd, Co and Ni. Compared with pYES2 transformed into yeast, pYES2-OsPDR expressed in BY4741 and YK44 grew more efficiently in the presence of 500 μmol/L CoCl2 and 1 mmol/L CoCl2 plus Gala. The growth of pYES2 and pYES2-OsPDR in BY4741 and YK44 on the YPD medium was uniform (Figure 4b and 4c). However, there were no apparent phenotypic differences between pYES2 and pYES2-OsPDR in BY4741 and YK44 on the medium after a series of drop-test experiments were carried out with different concentrations of Zn, Cd and Ni. These results indicate that the expression of OsPDR in yeast BY4741 and YK44 can enhance the tolerance to heavy metal Co and may transport Co into vacuoles together with the result of vacuolar membrane localization.

Fig. 4 Co tolerance of OsPDR over-expressed in BY4741 and YK44 (sensitive to Zn/Cd/Co/Ni) (a) Schematic representation of pYES2-OsPDR. (b and c) Sensitivity of yeast mutants on solid medium containing heavy metals. BY4741 and YK44 were transformed with pYES2 or pYES2-OsPDR and pYES2-OsPDR-transformed BY4741 and YK44 grows more efficiently than the pYES2-transformed BY4741 and YK44 (control) in the presence of 500 μmol/L CoCl2 and 1 mmol/L CoCl2. The YPG medium supplemented with 500 μmol/L CoCl2 and 1 mmol/L CoCl2 or no additional metal (control) was uesd for and incubated for 3-5 d at 30℃.
2.4 The overexpression of OsPDR affected the content of Co in yeast

The Co content of pYES2 and pYES2-OsPDR transformed into BY4741 and YK44 strains was measured by ICP-MS. When OsPDR was expressed in BY4741 and YK44, the Co content was increased compared to that found in the pYES2-only transformants treated with 150 μmol/L CoCl2(Figure 5).

Fig. 5 ICP-MS measurement of the Co content in yeast BY4741 and YK44 with pYES2-transformed and pYES2-OsPDR-transformed (a)The content of Co in BY4741 cells grown in the presence of 150 μmol/L Co or no additional metal added to the YPG culture medium. (b) Content of Co in YK44 cells grown in the presence of 150 μmol/L Co or no additional metal added to the YPG culture medium. The error bars represent the SD of three replicates (*P < 0.05, **P < 0.01).

The Co content did not change significantly between pYES2 and pYES2-OsPDR-transformed yeast of BY4741 and YK44 under the condition of no additional metals. The content of Co in pYES2- OsPDR-transformed yeast of BY4741 was significantly increased compared to that of pYES2-only transformed yeast of BY4741 after 150 μmol/L CoCl2 treatment (Figure 5a), and the same results were obtained in yeast Yk44 (Figure 5b). The pYES2-OsPDR- transformed yeast in BY4741 and YK44 can be more tolerant to Co compared with the empty vector pYES2 expressed in yeast BY4741 and YK44. The same results were obtained in yeast strains BY4741 and YK44. The result is consistent with the results of the metal tolerance experiments in yeast, which further proved that heavy metal Co can be stored in vacuoles where can isolate Co from causing damage to the yeast cells (Figure 4).

3 Discussion 3.1 OsPDR can enhance Co tolerance in yeast

Previous studies have shown that the expression of OsPDR is upregulated in shoots of rice (43-fold) with 24 h Cd exposure and exhibits no difference in the roots between 1 and 24 h. The gene for PDR5 (the homolog of yeast OsPDR) cloned from Saccharomyces cerevisiae is linked to the excretion of cytotoxic metabolites[25]. Here, we analyzed the function of OsPDR in yeast. In this study, we found no Cd sensitivity in yeast expressing OsPDR, however, we found that heterologous expression of OsPDR in yeast BY4741 and YK44 can result in increasing tolerance to Co when we tested the transport ability of OsPDR (Figure 4). The functions of vacuoles are diverse and can involve the following points: maintaining osmotic pressure, participating in and regulating the accumulation and transportation of intracellular substances, maintaining the stability of cells and their internal environment and isolating wastes which have detoxifying effects. These data suggest that OsPDR is a vacuolar membrane-localized Co transporter that can pump Co into vacuoles for storage. Our results in yeast differ from a previous study that showed that OsPDR was highly responsive to cadmium exposure in the rice shoot. This inconsistency may be largely due to the heterologous expression of plant genes in yeast that can yield a difference from the functions of genes in rice plants. The reason that only OsPDR expressed in yeast BY4741 and YK44 showed tolerance to Co with no effect on other metals such as Zn, Ni or Cd is not clear. We hypothesize that the differences may be related to the different mechanisms for compartmentalizing metals and/or the toxicity of these metals toward yeast[50].

3.2 Overexpression of OsPDR increased the Co content in yeast under Co stress

Empty vector-transformed yeast and OsPDR- transformed yeast showed higher Co contents for both BY4741 or YK44 when treated with 150 μmol/L CoCl2 compared with other metals. This difference may be related to the greater content of Co transported from the culture medium into cells. The content of Co was almost unchanged between yeast cells (BY4741 and YK44) expressing pYES2 or pYES2-OsPDR without any Co in the medium with Gala. However, with 150 μmol/L CoCl2 supplied in the medium, the Co content was higher in OsPDR-transformed yeast compared with empty vector-transformed yeast (Figure 5), which means that the expression of OsPDR can be more tolerance to Co. The localization of EGFP-tagged OsPDR to the vacuolar membrane also supported that supposition (Figure 3). This evidence strongly suggests that the metal tolerance of yeast strains with transformed OsPDR might be due to the sequestration of Co into vacuoles through the activity of OsPDR.

Taken together, our results show that OsPDR can function as a Co influx pump that can sequester extra Co into vacuoles in yeast. To further understand the function of OsPDR, additional studies need to be carried out in plants.

Acknowledgements We thank Dr. David Eide (University of Wisconsin-Madison) for the gifts of yeast strains used for complementation analysis and Euroscarf (Frankfurt, Germany) for providing the yeast strains BY4741 and YK44.
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TIAN Si-Qi, QIAO Kun, WANG Fan-Hong, LIANG Shuang, WANG Hong, CHAI Tuan-Yao
田思琪, 乔坤, 王凡红, 梁爽, 王红, 柴团耀
Heterologous Expression of OsPDR Enhances The Tolerance to Cobalt in Yeast
Progress in Biochemistry and Biophysics, 2018, 45(12): 1259-1267
生物化学与生物物理进展, 2018, 45(12): 1259-1267


收稿日期: 2018-09-11
接受日期: 2018-10-31