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Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar
Accepted Manuscript Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar Liping Sun, Yifeng Ma, Huihong Wang, Weipeng Huang, Xiaozhu Wang, Li Han, Wanmei Sun, Erqin Han, Bangjun Wang PII:
S0006-291X(18)30364-4
DOI:
10.1016/j.bbrc.2018.02.133
Reference:
YBBRC 39498
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 13 February 2018 Accepted Date: 15 February 2018
Please cite this article as: L. Sun, Y. Ma, H. Wang, W. Huang, X. Wang, L. Han, W. Sun, E. Han, B. Wang, Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.02.133. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Overexpression of PtABCC1 contributes to mercury tolerance
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and accumulation in Arabidopsis and poplar
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Liping Sun1, Yifeng Ma1, Huihong Wang, Weipeng Huang, Xiaozhu Wang,
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Li Han, Wanmei Sun, Erqin Han, Bangjun Wang*
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Key Laboratory of Eco-Environments in Three Gorges Reservoir Region
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(Ministry of Education), College of Life Sciences, Southwest University,
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Chongqing 400715, China
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These authors contributed equally to this work.
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* Corresponding author.
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E-mail address: [email protected] (B. Wang).
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Abstract
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Mercury (Hg) is a highly biotoxic heavy metal that contaminates the environment.
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Phytoremediation is a green technology for environmental remediation and is used to
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clean up Hg contaminated soil in recent years. In this study, we isolated an ATP-binding
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cassette (ABC) transporter gene PtABCC1 from Populus trichocarpa and overexpressed
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it in Arabidopsis and poplar. The transgenic plants conferred higher Hg tolerance than wild
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type (WT) plants, and overexpression of PtABCC1 could lead to 26-72% or 7-160%
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increase of Hg accumulation in Arabidopsis or poplar plants, respectively. These results
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demonstrated that PtABCC1 plays a crucial role in enhancing tolerance and accumulation
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to Hg in plants, which provides a promising way for phytoremediation of Hg contamination.
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Key words
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Hg, poplar, PtABCC1, tolerance, accumulation
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1. Introduction
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Mercury (Hg) is a highly toxic pollutant in the environment. Hg is toxic to living
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organisms even at very low level and affects ecological systems [1-3]. Consumption of
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Hg-contaminated foods could lead to Minamata, a neurological syndrome disease,
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causing global concern in recent years [4]. The main concerns are the food safety
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problem from what we consume and Hg may constantly flow in food chain system with the
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intake of Hg pollutant. Hence, it is essential for us to tackle the Hg contamination problem.
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Phytoremediation is a plant-based approach to clean up polluted environments. Hg2+ is
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the most predominant form of mercury for plants to take up [5], thus phytoremediation
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could be a desirable way to reduce Hg pollution in the soils.
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Plants can tolerate heavy metal stress to a certain extent with the help of some
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transporters. The multidrug resistance-associated proteins (MRP, ABCC) from the
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superfamily of ATP-binding cassette (ABC) transporters have been proved to transport
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heavy metals in yeast and plants [6-8]. Previous studies found that a yeast ABCC-like
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heavy metal transporter ScYCF1 was involved in cadmium (Cd) detoxification in yeast
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and increased Cd tolerance and uptake in Arabidopsis [9-10]. A rice ABCC transporter
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OsABCC1 was involved in the arsenic (As) detoxification and reduced As accumulation in
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the rice grains [11]. With the identification of more heavy metal transporters, many of them were used in
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phytoremediation. Hg tolerance and accumulation in plants are crucial to their
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phytoremediation capacity. Overexpression of the bacterial heavy metal transporter genes
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merC and merT enhanced tolerance to Hg and merC increased Hg accumulation in
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transgenic Arabidopsis [12-14]. In addition, the phytochelatin transporters AtABCC1 and
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AtABCC2 were found to contribute to Hg and Cd resistance in Arabidopsis [15].
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Woody plants have higher biomass comparing to Arabidopsis and candidate trees for
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more efficient phytoremediation might be found. Populus deltoides expressed with the
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bacterial mercury reductase genes MerA and MerB showed more tolerance to Hg [16-17].
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In China, the Chinese white poplar Populus tomentosa has been widely cultivated for a
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long history, but little is known about its Hg tolerance or accumulation. The engineering of
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Populus tomentosa with higher Hg accumulation will provide a promising application for
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phytoremediation of Hg contaminated soil in China and other countries.
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In this study, we cloned a new ABC transporter gene PtABCC1 (GenBank accession: MG791913)
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overexpressed it in Arabidopsis thaliana and Populus tomentosa, then examined the Hg
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tolerance and Hg accumulation in transgenic plants. The results demonstrated that
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overexpression of the PtABCC1 is a effective way for enhancing Hg tolerance and
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accumulation in transgenic plants, which provides a new option for phytoremediation of
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Hg pollution.
the
genome-sequenced
poplar
(Populus
trichocarpa),
and
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2. Materials and methods
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2.1. Bioinformatic and phylogenetic analysis
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The structure of PtABCC1 protein was predicted and analyzed using bioinformatic tool
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(http://smart.embl-heidelberg.de). Some homologous protein sequences in plants were
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obtained from NCBI (http://www.ncbi.nlm.nih.gov/Blast.cgi) and aligned using DNAMAN
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8.0 (Lynnon Biosoft, USA). A neighbor-joining (NJ) phylogenetic analysis of the PtABCC1
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transporter was generated with MEGA 5.0 software [18]. Bootstrap replication was used to
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determine statistical support for the nodes of the phylogenetic tree.
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2.2. Plant materials and growth conditions Arabidopsis seeds of WT (Columbia-0), abcc1 (a loss-of-function mutant of
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Atabcc1-3, Salk_017431) [19] and PtABCC1 transgenic plants (OE-1 and OE-2) were
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grown on half-strength MS agar medium plates containing 1.5% sucrose [19] in a
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controlled environment with a 16-h day length with 120 µmol m-2 s-1 light intensity. Day and
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night temperatures were 22°C and 18°C, respectively.
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Populus trichocarpa Torr. & Gray and Populus tomentosa Carr. (clone 741) (Chinese
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white poplar) were obtained from Institute of Resources Botany, Southwest University,
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China and were grown in the greenhouse (day/night temperature: 30/25 °C; relative air
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humidity: 40-50%; natural light). Transgenic poplar plants were cultured on woody plant
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medium (WPM) in a controlled-growth chamber (14 h light at 25°C/10 h dark at 23°C;
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relative humidity: 50-60%; lighting condition: 45 µmol m-2 s-1). To obtain more transgenic
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seedlings, the leaf-disc cuttings of transgenic line were used for regeneration and
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subculture. After 2-3 months, the rooted transgenic plantlets were transferred to soil and
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grown in a greenhouse.
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2.3. Plant transformation
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The PtABCC1 cDNA was amplified from Populus trichocarpa and ligated into the plant
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binary vector pCX-SN [20] to produce a PtABCC1 expression construct under the 35S
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promoter (35S-PtABCC1). The primers used were listed in Supplementary Table S1.
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35S-PtABCC1 was transformed into the Agrobacterium tumefaciens strain GV3101 and
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the suspension of Agrobacterium recombinant was used for plant transformation.
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Transgenic Arabidopsis plants were generated according to the floral dip method [21].
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Selection of Arabidopsis transformants was on half-strength MS medium containing 40
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mg/L hygromycin and 200 mg/L cefotaxime. Further PCR analysis were used to confirm
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putative Arabidopsis transformants and homozygous T3 lines were selected for further
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analysis.
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Agrobacterium-mediated leaf disc method [22]. Putative transgenic poplar plants were
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Transformation
of
Populus
tomentosa
was
performed
via
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generated and selected on WPM medium supplemented with 9 mg/L hygromycin and 400
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mg/L cefotaxime, and further confirmed by PCR analysis (Supplementary Fig. S1).
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2.4. Stress tolerance analysis in transgenic plants On one hand, WT, abcc1, and transgenic Arabidopsis lines (OE-1 and OE-2) were
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selected for testing Hg tolerance. Sterilized Arabidopsis seeds grew vertically on
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half-strength MS medium with or without 50 µM glutathione (GSH, TCI, Japan) and 50 µM
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DL-buthionine sulfoximine (BSO, Sigma, USA) [23] in the absence or presence 20 µM
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HgCl2 for 12 days. Meanwhile, untreatment was used as reference. Subsequently, the root
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length of seedlings was measured and the root tips were photographed under a
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fluorescent microscope (Olympus BX53, Japan). On the other hand, poplar seedlings
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were exposed to WPM hydroponic culture solution containing 20 µM HgCl2 for two weeks,
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blank culture was used as a control at the same time. Then, they were harvested for Hg
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content analysis. Each experiment was repeated three times.
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2.5. Determination of Hg level
Plant materials were washed extensively with distilled water, oven-dried, ground into
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powder and stored under vacuum conditions. For Hg level determination, 50 mg of power
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was used and processed as reported method [24] to convert all forms of Hg to Hg2+. SnCl2
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was then added, making Hg2+ reduced to elemental Hg. Finally, diluted by milliQ water
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and analyzed with cold-vapour atomic fluorescence mercury measurement instrument
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(Tekran® Model 2500 CVAFS Mercury Detector, USA).
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2.6. Statistical analysis
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Statistical analyses were performed by the statistical software SPSS 9.0. One-way
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analysis of variance (ANOVA) with Duncan's multiple range tests was considered as
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significance test. Different letters represented significant differences (P<0.05). Data
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showed mean value with standard deviation.
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3. Results
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3.1. Sequence and phylogenetic analysis
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We cloned the full-length cDNA of PtABCC1 (4875 bp) from Populus trichocarpa
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encoding a polypeptide of 1624 amino acids, and we submitted the sequence to GenBank
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PtABCC1 protein contains two putative transmembrane domains (TMD) and two putative
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nucleotide binding domains (NBD) (Fig. 1A). Each NBD domain has about 200 amino acid
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residues, and it contains a Walker A motif (GXXGXGKS/T), a Walker B motif (hhhhD) and
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an ABC signature motif (LSGGQQ/R/KQR) [25]. To date, ABCCs subfamily transporters
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have been identified in many plant species, including Arabidopsis thaliana, Oryza sativa,
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Glycine max and Vitis vinifera. The two NBD domains are highly conserved among Vitis
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vinifera, Glycine max and Arabidopsis thaliana (Fig. 1A).
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PtABCC1 had 82.1%, 77.5%, 78.2% and 85.3% amino acid sequence identity to
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GmABCC2 (XP_003542944), AtABCC1 (NP_174329), AtABCC2 (NP_181013), and
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VvABCC6 (XP_002280819), respectively. Phylogenetic analysis revealed that PtABCC1
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was very close to the ABCC proteins from dicots such as Glycine max and Arabidopsis, as
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well as monocots such as Oryza sativa (Fig. 1B).
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3.2. Overexpression of PtABCC1 enhanced Hg tolerance in Arabidopsis and
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poplar
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PtABCC1 overexpression construct (35S-PtABCC1) was made and transformed into
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Arabidopsis and poplar. The positive lines were identified by PCR for further analysis
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(Supplementary Fig. S1). To determine the effect of the PtABCC1 gene on Hg tolerance in
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Arabidopsis, the root growth of WT, abcc1 and transgenic lines (OE-1 and OE-2) was
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analyzed. Four different Arabidopsis plants were treated with HgCl2 (0, 10, and 20 µM) for
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12 days, and they displayed different tolerance to Hg. There is no detectable growth
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difference when Hg is absent (Fig. 2A). In the presence of 10 or 20 µM HgCl2, the growth
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of Arabidopsis was remarkably inhibited. The abcc1 mutants had the shortest primary
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roots, and transgenic Arabidopsis plants (OE-1 and OE-2) displayed longer primary root
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than WT (Fig. 2B and C), indicating that PtABCC1 mediated tolerance to Hg. In contrast,
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the roots of transgenic lines (OE-1 and OE-2) were longer than WT root under 10 µM
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HgCl2 (16% and 15% longer, respectively) and 20 µM HgCl2 (24% and 31% longer,
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respectively) (Fig. 2D). Furthermore, the effect of GSH (an ions chelator) and BSO (an
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inhibitor of GSH biosynthesis) on Hg tolerance was also observed in Arabidopsis
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ACCEPTED MANUSCRIPT (Supplementary Fig. S2). There was no obvious difference among WT, abcc1 and
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transgenic Arabidopsis lines without Hg treatment (Supplementary Fig. S2A-C). When
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plants were treated with 20 µM Hg, GSH could promote primary root growth and relieve
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the root inhibition of abcc1 by Hg, while BSO inhibited root growth, and transgenic
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Arabidopsis showed better growth condition than WT and abcc1 (Fig. S2E-F). Together,
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the results suggested PtABCC1 enhanced tolerance to Hg in Arabidopsis.
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To further study the root tip morphology under Hg stress, we randomly selected ten
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seedlings for microscopic examination. The swelling phenomenon was observed in the
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root tips under HgCl2 treatment (Fig. 2B and C) but not in the untreated plants (Fig. 2A).
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There was no obvious difference with regard to root tip morphology between 0 and 10 µM
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HgCl2 treatments (Fig. 2A and 2B). The abcc1 plants had significant toxicity damage under
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20 µM HgCl2, with significantly shorter meristematic zone and elongation zone (Fig. 2C),
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indicating that ABCC1 gene may be involved in the Hg stress response of root tips.
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Furthermore, the toxic effect of Hg was examined in WT and transgenic poplar lines.
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HgCl2 treatment led to damaged phenomenon compared with the untreated plants (Fig.
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3A). As shown in Fig. 3B, WT had visible foliar damage such as chlorosis and necrosis,
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whereas less leaf necrosis could be observed in transgenic plant lines, indicating that
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poplars transformed with PtABCC1 gene displayed lower level of Hg damage. In other
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words, overexpression of PtABCC1 contributes to enhanced Hg tolerance in poplar.
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These data demonstrated that the PtABCC1 transgenic plants exhibited higher Hg
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tolerance than WT plants, and introducing of the PtABCC1 gene can enhance Hg
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tolerance in both Arabidopsis and poplar.
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3.3. Overexpression of PtABCC1 enhanced Hg accumulation in Arabidopsis
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and poplar
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In order to investigate the Hg accumulation function of PtABCC1, we measured the
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Hg content in the Arabidopsis transgenic lines. As shown in Fig. 4A, the amount of Hg
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accumulated in OE-1 and OE-2 were higher than WT in the presence of 10 µM HgCl2 (60%
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and 72% higher, respectively) and in the presence of 20 µM HgCl2 (26% and 67% higher,
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respectively). In particular, Hg level was as high as 200.69 mg/kg (under 10 µM HgCl2) and
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344.95 mg/kg (under 20 µM HgCl2) in OE-2. However, abcc1 plants were at the lowest Hg
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content level and not more than half of Hg content of WT neither under 10 nor 20 µM HgCl2
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treatment. To determine whether the increased Hg tolerance corresponded to an increase in Hg
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accumulation in transgenic poplar lines, total Hg contents of WT and transgenic poplar
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plants were analyzed in the roots, stems and leaves. According to Fig. 4B, the amount of
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Hg accumulated in the roots of the transgenic poplar plants (OX-1, OX-2 and OX-3) were
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significantly higher (136%, 76% and 53% higher, respectively) than that in WT plants. The
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Hg content levels of the transgenic poplar plants (OX-1, OX-2 and OX-3) were higher
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(31%, 15%, and 7% higher, respectively) in the leaves and higher (160%, 58% and 26%
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higher, respectively) in the stems than those in WT plants, indicating PtABCC1 was able
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to participate in the transport of Hg from root to shoot. It's worth noting that the levels of
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Hg accumulated in the leaves were at least twice as those in the stems in poplar.
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In short, PtABCC1 transgenic Arabidopsis or poplar could lead to 26-72% or 7-160%
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increase of Hg accumulation comparing to WT, respectively, suggesting the engineering
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Arabidopsis and poplar could apply for phytoremediation of Hg pollution.
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4. Discussion
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Hg is one of the hazardous and non-biodegradable pollutants in soils. Excessive Hg
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causes physiological disorders and inhibits plant growth. Hg induced oxidative stress and
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impaired seedling growth in Cucumis sativus and Medicago sativa [26-27]. In addition, Hg
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treatment resulted in a visible root swelling in the root tips [28]. Similar to this result, the
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swelling phenomena was observed in the root tip under Hg treatment (Fig. 3B and C).
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Exogenous GSH conferred higher Hg tolerance and alleviated Hg toxicity against Hg
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stress in Arabidopsis [23]. In our study, GSH conferred higher Hg tolerance and showed
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better growth condition (Supplementary Fig. S2).
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Higher Hg tolerance is important for plants to protect tissues from the serious toxic
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effect caused by Hg. In this study, we found that both Arabidopsis and poplar
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PtABCC1-overexpressing lines showed enhanced Hg tolerance compared to WT (Fig. 2
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level in the leaves of transgenic poplar (Fig. 3B and Fig. 4B). We do not yet know the
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detoxification mechanism of PtABCC1, but vacuole might be responsible for the uptake
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and detoxification of Hg. Vacuolar compartmentalization is indispensable for heavy metals
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detoxification in plants [29]. Some intriguing studies have shed light on how AtABCC1,
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AtABCC2 and AtABCC3 in Arabidopsis transported cadmium into the vacuole where Cd
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was stored in the form of PC-Cd (II) [15,30]. Moreover, AtABCC1 and AtABCC2
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transported PC-Hg(II) and PC2-As(III) into the vacuole for detoxification, which improved
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tolerance to these heavy metals [15,19]. The additional evidence suggested that
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VvABCC1 was a vacuolar membrane-localized transporter from Vitis vinifera [31].
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Together, ABCCs may be considered as vacuole membrane transporters for importing
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heavy metal chelates into the vacuole for detoxification.
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Importantly, some previous studies also showed that heavy metal tolerance is closely
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related to heavy metal accumulation. For example, the ScYCF1-overexpressing plants
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had enhanced heavy metal tolerance and accumulation to heavy metals such as Cd and
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lead (Pb) [10,32-33]. Our data showed that overexpression of PtABCC1 also improved the
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Hg accumulation in Arabidopsis and poplar. It is likely that exogenous Hg is readily taken
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up by plant roots, accumulated in roots, and then translocated to the aerial parts and
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accumulated [34-35]. We found that the transgenic poplar accumulated twice to three
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times as much Hg in roots and stems as the WT poplar. In addition, the aboveground
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biomass is considered a key measure when phytoremediation effectiveness is evaluated.
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Compared with herbaceous plants, poplar has a larger biomass, thus potentially higher
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Hg accumulation. Poplar is a deciduous tree, and its application for Hg cleanup in soil
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must take into consideration the proper disposal of fallen leaves to avoid secondary
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pollution.
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The ability of heavy metal tolerance and accumulation is key for phytoremediation.
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Our study highlighted that PtABCC1 conferred Hg tolerance and accumulation in
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Arabidopsis and poplar, and we could potentially put these transgenic poplar lines into
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practical use for phytoremediation of Hg. Up to now, more and more Hg tolerance genes
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such as ScYCF1, MerC-H and AtABCC1,2 have been identified, and we might
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co-transform
these
genes
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phytoremediation in the future.
or
their
homologs
into
poplar
for
high-efficiency
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Competing financial interests The authors declare that they have no conflict of interest.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant
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No. 31370317 and 31571584), the Ministry of Science and Technology of China (Grant No.
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2016YFD0100504), the Natural Science Foundation of Chongqing (Grant No.
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cstc2013jcyjA80016), and Fundamental Research Funds for the Central Universities
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(XDJK2013B032).
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Zarrouk, S. Vialet, T. Marlin, M.M. Chaves, E. Martinoia, R. Nagy, ABCC1, an ATP binding
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47 (2009) 2761–2767.
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ACCEPTED MANUSCRIPT Fig. 1. Amino acid sequence alignment and phylogenetic analysis. (A) Structure analysis
2
and amino acid multi-alignment of the NBD domains of different plant species. ABCC
3
domains are marked as two yellow and green blocks. TMD, transmembrane domain; NBD,
4
a nucleotide-binding domain; walker A, walker B and ABC signature are NBD associated
5
motifs. Blue indicates identical amino acids, red indicates similar amino acids. (B)
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Phylogenetic analysis of ABCC proteins from Populus trichocarpa (PtABCC1, MG791913);
7
Arabidopsis thaliana (AtABCC1, NP_174329; AtABCC2, NP_181013; AtABCC3,
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NP_187915); Oryza sativa (OsABCC1, XP_015635452); Glycine Max (GmABCC2,
9
XP_003542944);
Vitis
vinifera
(VvABCC1,
XP_002267650;
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and
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VvABCC6,
XP_002280819); Zea mays (ZmABCC1, NP_001105942); Eucalyptus grandis (EgABCC5,
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XP_010036147).
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ACCEPTED MANUSCRIPT Fig. 2. Hg tolerance analysis in Arabidopsis. (A-C) Arabidopsis seeds were grown on
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half-strength MS medium containing 0, 10 or 20 µM HgCl2 for 12 days; then root tip
20
morphology and primary root length (D) were analyzed. WT, wild type; abcc1, Atabcc1-3
21
mutant; OE-1 and OE-2, transgenic Arabidopsis lines. White bars= 15 mm, Black
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bars=200 µm. Error bars indicate standard deviation (n=3). Different letters indicated
23
significant differences (P<0.05).
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Fig. 3. Hg tolerance analysis in poplar. The growth condition (A) and leaf damage analysis
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(B) of two-month-old wild type poplar (WT) and transgenic poplar (OX) seedlings in glass
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bottle treated with 0 or 20 µM HgCl2 for two weeks.
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ACCEPTED MANUSCRIPT Fig. 4. Hg accumulation analysis in Arabidopsis and poplar. Arabidopsis plants were
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grown on half-strength MS agar with 10 or 20 µM HgCl2 for two weeks (A) and poplar
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plants were grown in WPM hydroponic medium containing 20 µM HgCl2 for two weeks (B),
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then they were harvested for Hg content analysis. WT, wild type; abcc1, Atabcc1-3 mutant;
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OE-1 and 2, transgenic Arabidopsis lines; OX-1, 2, and 3, transgenic poplar lines. Error
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bars indicate standard deviation (n=3). Different letters indicated significant differences
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(P<0.05).
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ACCEPTED MANUSCRIPT Highlights • Overexpression of PtABCC1 enhanced Hg tolerance in Arabidopsis and poplar. • Overexpression of PtABCC1 enhanced Hg accumulation in Arabidopsis and poplar.
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• PtABCC1 was the first identified poplar ABC transporter for phytoremediation of Hg.
S0006-291X(18)30364-4
DOI:
10.1016/j.bbrc.2018.02.133
Reference:
YBBRC 39498
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 13 February 2018 Accepted Date: 15 February 2018
Please cite this article as: L. Sun, Y. Ma, H. Wang, W. Huang, X. Wang, L. Han, W. Sun, E. Han, B. Wang, Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.02.133. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Overexpression of PtABCC1 contributes to mercury tolerance
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and accumulation in Arabidopsis and poplar
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Liping Sun1, Yifeng Ma1, Huihong Wang, Weipeng Huang, Xiaozhu Wang,
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Li Han, Wanmei Sun, Erqin Han, Bangjun Wang*
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Key Laboratory of Eco-Environments in Three Gorges Reservoir Region
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(Ministry of Education), College of Life Sciences, Southwest University,
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Chongqing 400715, China
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These authors contributed equally to this work.
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* Corresponding author.
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E-mail address: [email protected] (B. Wang).
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Abstract
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Mercury (Hg) is a highly biotoxic heavy metal that contaminates the environment.
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Phytoremediation is a green technology for environmental remediation and is used to
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clean up Hg contaminated soil in recent years. In this study, we isolated an ATP-binding
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cassette (ABC) transporter gene PtABCC1 from Populus trichocarpa and overexpressed
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it in Arabidopsis and poplar. The transgenic plants conferred higher Hg tolerance than wild
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type (WT) plants, and overexpression of PtABCC1 could lead to 26-72% or 7-160%
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increase of Hg accumulation in Arabidopsis or poplar plants, respectively. These results
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demonstrated that PtABCC1 plays a crucial role in enhancing tolerance and accumulation
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to Hg in plants, which provides a promising way for phytoremediation of Hg contamination.
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Key words
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Hg, poplar, PtABCC1, tolerance, accumulation
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1. Introduction
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Mercury (Hg) is a highly toxic pollutant in the environment. Hg is toxic to living
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organisms even at very low level and affects ecological systems [1-3]. Consumption of
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Hg-contaminated foods could lead to Minamata, a neurological syndrome disease,
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causing global concern in recent years [4]. The main concerns are the food safety
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problem from what we consume and Hg may constantly flow in food chain system with the
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intake of Hg pollutant. Hence, it is essential for us to tackle the Hg contamination problem.
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Phytoremediation is a plant-based approach to clean up polluted environments. Hg2+ is
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the most predominant form of mercury for plants to take up [5], thus phytoremediation
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could be a desirable way to reduce Hg pollution in the soils.
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Plants can tolerate heavy metal stress to a certain extent with the help of some
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transporters. The multidrug resistance-associated proteins (MRP, ABCC) from the
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superfamily of ATP-binding cassette (ABC) transporters have been proved to transport
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heavy metals in yeast and plants [6-8]. Previous studies found that a yeast ABCC-like
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heavy metal transporter ScYCF1 was involved in cadmium (Cd) detoxification in yeast
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and increased Cd tolerance and uptake in Arabidopsis [9-10]. A rice ABCC transporter
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OsABCC1 was involved in the arsenic (As) detoxification and reduced As accumulation in
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the rice grains [11]. With the identification of more heavy metal transporters, many of them were used in
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phytoremediation. Hg tolerance and accumulation in plants are crucial to their
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phytoremediation capacity. Overexpression of the bacterial heavy metal transporter genes
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merC and merT enhanced tolerance to Hg and merC increased Hg accumulation in
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transgenic Arabidopsis [12-14]. In addition, the phytochelatin transporters AtABCC1 and
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AtABCC2 were found to contribute to Hg and Cd resistance in Arabidopsis [15].
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Woody plants have higher biomass comparing to Arabidopsis and candidate trees for
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more efficient phytoremediation might be found. Populus deltoides expressed with the
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bacterial mercury reductase genes MerA and MerB showed more tolerance to Hg [16-17].
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In China, the Chinese white poplar Populus tomentosa has been widely cultivated for a
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long history, but little is known about its Hg tolerance or accumulation. The engineering of
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Populus tomentosa with higher Hg accumulation will provide a promising application for
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phytoremediation of Hg contaminated soil in China and other countries.
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In this study, we cloned a new ABC transporter gene PtABCC1 (GenBank accession: MG791913)
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overexpressed it in Arabidopsis thaliana and Populus tomentosa, then examined the Hg
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tolerance and Hg accumulation in transgenic plants. The results demonstrated that
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overexpression of the PtABCC1 is a effective way for enhancing Hg tolerance and
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accumulation in transgenic plants, which provides a new option for phytoremediation of
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Hg pollution.
the
genome-sequenced
poplar
(Populus
trichocarpa),
and
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2. Materials and methods
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2.1. Bioinformatic and phylogenetic analysis
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The structure of PtABCC1 protein was predicted and analyzed using bioinformatic tool
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(http://smart.embl-heidelberg.de). Some homologous protein sequences in plants were
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obtained from NCBI (http://www.ncbi.nlm.nih.gov/Blast.cgi) and aligned using DNAMAN
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8.0 (Lynnon Biosoft, USA). A neighbor-joining (NJ) phylogenetic analysis of the PtABCC1
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transporter was generated with MEGA 5.0 software [18]. Bootstrap replication was used to
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determine statistical support for the nodes of the phylogenetic tree.
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2.2. Plant materials and growth conditions Arabidopsis seeds of WT (Columbia-0), abcc1 (a loss-of-function mutant of
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Atabcc1-3, Salk_017431) [19] and PtABCC1 transgenic plants (OE-1 and OE-2) were
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grown on half-strength MS agar medium plates containing 1.5% sucrose [19] in a
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controlled environment with a 16-h day length with 120 µmol m-2 s-1 light intensity. Day and
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night temperatures were 22°C and 18°C, respectively.
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Populus trichocarpa Torr. & Gray and Populus tomentosa Carr. (clone 741) (Chinese
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white poplar) were obtained from Institute of Resources Botany, Southwest University,
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China and were grown in the greenhouse (day/night temperature: 30/25 °C; relative air
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humidity: 40-50%; natural light). Transgenic poplar plants were cultured on woody plant
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medium (WPM) in a controlled-growth chamber (14 h light at 25°C/10 h dark at 23°C;
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relative humidity: 50-60%; lighting condition: 45 µmol m-2 s-1). To obtain more transgenic
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seedlings, the leaf-disc cuttings of transgenic line were used for regeneration and
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subculture. After 2-3 months, the rooted transgenic plantlets were transferred to soil and
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grown in a greenhouse.
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2.3. Plant transformation
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The PtABCC1 cDNA was amplified from Populus trichocarpa and ligated into the plant
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binary vector pCX-SN [20] to produce a PtABCC1 expression construct under the 35S
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promoter (35S-PtABCC1). The primers used were listed in Supplementary Table S1.
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35S-PtABCC1 was transformed into the Agrobacterium tumefaciens strain GV3101 and
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the suspension of Agrobacterium recombinant was used for plant transformation.
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Transgenic Arabidopsis plants were generated according to the floral dip method [21].
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Selection of Arabidopsis transformants was on half-strength MS medium containing 40
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mg/L hygromycin and 200 mg/L cefotaxime. Further PCR analysis were used to confirm
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putative Arabidopsis transformants and homozygous T3 lines were selected for further
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analysis.
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Agrobacterium-mediated leaf disc method [22]. Putative transgenic poplar plants were
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Transformation
of
Populus
tomentosa
was
performed
via
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generated and selected on WPM medium supplemented with 9 mg/L hygromycin and 400
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mg/L cefotaxime, and further confirmed by PCR analysis (Supplementary Fig. S1).
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2.4. Stress tolerance analysis in transgenic plants On one hand, WT, abcc1, and transgenic Arabidopsis lines (OE-1 and OE-2) were
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selected for testing Hg tolerance. Sterilized Arabidopsis seeds grew vertically on
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half-strength MS medium with or without 50 µM glutathione (GSH, TCI, Japan) and 50 µM
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DL-buthionine sulfoximine (BSO, Sigma, USA) [23] in the absence or presence 20 µM
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HgCl2 for 12 days. Meanwhile, untreatment was used as reference. Subsequently, the root
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length of seedlings was measured and the root tips were photographed under a
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fluorescent microscope (Olympus BX53, Japan). On the other hand, poplar seedlings
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were exposed to WPM hydroponic culture solution containing 20 µM HgCl2 for two weeks,
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blank culture was used as a control at the same time. Then, they were harvested for Hg
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content analysis. Each experiment was repeated three times.
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2.5. Determination of Hg level
Plant materials were washed extensively with distilled water, oven-dried, ground into
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powder and stored under vacuum conditions. For Hg level determination, 50 mg of power
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was used and processed as reported method [24] to convert all forms of Hg to Hg2+. SnCl2
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was then added, making Hg2+ reduced to elemental Hg. Finally, diluted by milliQ water
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and analyzed with cold-vapour atomic fluorescence mercury measurement instrument
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(Tekran® Model 2500 CVAFS Mercury Detector, USA).
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2.6. Statistical analysis
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Statistical analyses were performed by the statistical software SPSS 9.0. One-way
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analysis of variance (ANOVA) with Duncan's multiple range tests was considered as
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significance test. Different letters represented significant differences (P<0.05). Data
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showed mean value with standard deviation.
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3. Results
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3.1. Sequence and phylogenetic analysis
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We cloned the full-length cDNA of PtABCC1 (4875 bp) from Populus trichocarpa
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encoding a polypeptide of 1624 amino acids, and we submitted the sequence to GenBank
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PtABCC1 protein contains two putative transmembrane domains (TMD) and two putative
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nucleotide binding domains (NBD) (Fig. 1A). Each NBD domain has about 200 amino acid
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residues, and it contains a Walker A motif (GXXGXGKS/T), a Walker B motif (hhhhD) and
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an ABC signature motif (LSGGQQ/R/KQR) [25]. To date, ABCCs subfamily transporters
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have been identified in many plant species, including Arabidopsis thaliana, Oryza sativa,
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Glycine max and Vitis vinifera. The two NBD domains are highly conserved among Vitis
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vinifera, Glycine max and Arabidopsis thaliana (Fig. 1A).
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PtABCC1 had 82.1%, 77.5%, 78.2% and 85.3% amino acid sequence identity to
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GmABCC2 (XP_003542944), AtABCC1 (NP_174329), AtABCC2 (NP_181013), and
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VvABCC6 (XP_002280819), respectively. Phylogenetic analysis revealed that PtABCC1
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was very close to the ABCC proteins from dicots such as Glycine max and Arabidopsis, as
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well as monocots such as Oryza sativa (Fig. 1B).
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3.2. Overexpression of PtABCC1 enhanced Hg tolerance in Arabidopsis and
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PtABCC1 overexpression construct (35S-PtABCC1) was made and transformed into
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Arabidopsis and poplar. The positive lines were identified by PCR for further analysis
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(Supplementary Fig. S1). To determine the effect of the PtABCC1 gene on Hg tolerance in
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Arabidopsis, the root growth of WT, abcc1 and transgenic lines (OE-1 and OE-2) was
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analyzed. Four different Arabidopsis plants were treated with HgCl2 (0, 10, and 20 µM) for
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12 days, and they displayed different tolerance to Hg. There is no detectable growth
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difference when Hg is absent (Fig. 2A). In the presence of 10 or 20 µM HgCl2, the growth
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of Arabidopsis was remarkably inhibited. The abcc1 mutants had the shortest primary
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roots, and transgenic Arabidopsis plants (OE-1 and OE-2) displayed longer primary root
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than WT (Fig. 2B and C), indicating that PtABCC1 mediated tolerance to Hg. In contrast,
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the roots of transgenic lines (OE-1 and OE-2) were longer than WT root under 10 µM
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HgCl2 (16% and 15% longer, respectively) and 20 µM HgCl2 (24% and 31% longer,
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respectively) (Fig. 2D). Furthermore, the effect of GSH (an ions chelator) and BSO (an
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inhibitor of GSH biosynthesis) on Hg tolerance was also observed in Arabidopsis
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ACCEPTED MANUSCRIPT (Supplementary Fig. S2). There was no obvious difference among WT, abcc1 and
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transgenic Arabidopsis lines without Hg treatment (Supplementary Fig. S2A-C). When
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plants were treated with 20 µM Hg, GSH could promote primary root growth and relieve
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the root inhibition of abcc1 by Hg, while BSO inhibited root growth, and transgenic
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Arabidopsis showed better growth condition than WT and abcc1 (Fig. S2E-F). Together,
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the results suggested PtABCC1 enhanced tolerance to Hg in Arabidopsis.
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To further study the root tip morphology under Hg stress, we randomly selected ten
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seedlings for microscopic examination. The swelling phenomenon was observed in the
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root tips under HgCl2 treatment (Fig. 2B and C) but not in the untreated plants (Fig. 2A).
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There was no obvious difference with regard to root tip morphology between 0 and 10 µM
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HgCl2 treatments (Fig. 2A and 2B). The abcc1 plants had significant toxicity damage under
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20 µM HgCl2, with significantly shorter meristematic zone and elongation zone (Fig. 2C),
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indicating that ABCC1 gene may be involved in the Hg stress response of root tips.
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Furthermore, the toxic effect of Hg was examined in WT and transgenic poplar lines.
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HgCl2 treatment led to damaged phenomenon compared with the untreated plants (Fig.
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3A). As shown in Fig. 3B, WT had visible foliar damage such as chlorosis and necrosis,
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whereas less leaf necrosis could be observed in transgenic plant lines, indicating that
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poplars transformed with PtABCC1 gene displayed lower level of Hg damage. In other
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words, overexpression of PtABCC1 contributes to enhanced Hg tolerance in poplar.
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These data demonstrated that the PtABCC1 transgenic plants exhibited higher Hg
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tolerance than WT plants, and introducing of the PtABCC1 gene can enhance Hg
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tolerance in both Arabidopsis and poplar.
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3.3. Overexpression of PtABCC1 enhanced Hg accumulation in Arabidopsis
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and poplar
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In order to investigate the Hg accumulation function of PtABCC1, we measured the
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Hg content in the Arabidopsis transgenic lines. As shown in Fig. 4A, the amount of Hg
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accumulated in OE-1 and OE-2 were higher than WT in the presence of 10 µM HgCl2 (60%
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and 72% higher, respectively) and in the presence of 20 µM HgCl2 (26% and 67% higher,
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respectively). In particular, Hg level was as high as 200.69 mg/kg (under 10 µM HgCl2) and
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344.95 mg/kg (under 20 µM HgCl2) in OE-2. However, abcc1 plants were at the lowest Hg
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content level and not more than half of Hg content of WT neither under 10 nor 20 µM HgCl2
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treatment. To determine whether the increased Hg tolerance corresponded to an increase in Hg
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accumulation in transgenic poplar lines, total Hg contents of WT and transgenic poplar
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plants were analyzed in the roots, stems and leaves. According to Fig. 4B, the amount of
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Hg accumulated in the roots of the transgenic poplar plants (OX-1, OX-2 and OX-3) were
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significantly higher (136%, 76% and 53% higher, respectively) than that in WT plants. The
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Hg content levels of the transgenic poplar plants (OX-1, OX-2 and OX-3) were higher
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(31%, 15%, and 7% higher, respectively) in the leaves and higher (160%, 58% and 26%
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higher, respectively) in the stems than those in WT plants, indicating PtABCC1 was able
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to participate in the transport of Hg from root to shoot. It's worth noting that the levels of
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Hg accumulated in the leaves were at least twice as those in the stems in poplar.
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In short, PtABCC1 transgenic Arabidopsis or poplar could lead to 26-72% or 7-160%
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increase of Hg accumulation comparing to WT, respectively, suggesting the engineering
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Arabidopsis and poplar could apply for phytoremediation of Hg pollution.
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4. Discussion
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Hg is one of the hazardous and non-biodegradable pollutants in soils. Excessive Hg
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causes physiological disorders and inhibits plant growth. Hg induced oxidative stress and
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impaired seedling growth in Cucumis sativus and Medicago sativa [26-27]. In addition, Hg
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treatment resulted in a visible root swelling in the root tips [28]. Similar to this result, the
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swelling phenomena was observed in the root tip under Hg treatment (Fig. 3B and C).
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Exogenous GSH conferred higher Hg tolerance and alleviated Hg toxicity against Hg
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stress in Arabidopsis [23]. In our study, GSH conferred higher Hg tolerance and showed
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better growth condition (Supplementary Fig. S2).
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Higher Hg tolerance is important for plants to protect tissues from the serious toxic
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effect caused by Hg. In this study, we found that both Arabidopsis and poplar
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PtABCC1-overexpressing lines showed enhanced Hg tolerance compared to WT (Fig. 2
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level in the leaves of transgenic poplar (Fig. 3B and Fig. 4B). We do not yet know the
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detoxification mechanism of PtABCC1, but vacuole might be responsible for the uptake
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and detoxification of Hg. Vacuolar compartmentalization is indispensable for heavy metals
246
detoxification in plants [29]. Some intriguing studies have shed light on how AtABCC1,
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AtABCC2 and AtABCC3 in Arabidopsis transported cadmium into the vacuole where Cd
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was stored in the form of PC-Cd (II) [15,30]. Moreover, AtABCC1 and AtABCC2
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transported PC-Hg(II) and PC2-As(III) into the vacuole for detoxification, which improved
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tolerance to these heavy metals [15,19]. The additional evidence suggested that
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VvABCC1 was a vacuolar membrane-localized transporter from Vitis vinifera [31].
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Together, ABCCs may be considered as vacuole membrane transporters for importing
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heavy metal chelates into the vacuole for detoxification.
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Importantly, some previous studies also showed that heavy metal tolerance is closely
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related to heavy metal accumulation. For example, the ScYCF1-overexpressing plants
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had enhanced heavy metal tolerance and accumulation to heavy metals such as Cd and
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lead (Pb) [10,32-33]. Our data showed that overexpression of PtABCC1 also improved the
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Hg accumulation in Arabidopsis and poplar. It is likely that exogenous Hg is readily taken
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up by plant roots, accumulated in roots, and then translocated to the aerial parts and
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accumulated [34-35]. We found that the transgenic poplar accumulated twice to three
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times as much Hg in roots and stems as the WT poplar. In addition, the aboveground
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biomass is considered a key measure when phytoremediation effectiveness is evaluated.
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Compared with herbaceous plants, poplar has a larger biomass, thus potentially higher
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Hg accumulation. Poplar is a deciduous tree, and its application for Hg cleanup in soil
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must take into consideration the proper disposal of fallen leaves to avoid secondary
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pollution.
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The ability of heavy metal tolerance and accumulation is key for phytoremediation.
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Our study highlighted that PtABCC1 conferred Hg tolerance and accumulation in
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Arabidopsis and poplar, and we could potentially put these transgenic poplar lines into
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practical use for phytoremediation of Hg. Up to now, more and more Hg tolerance genes
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such as ScYCF1, MerC-H and AtABCC1,2 have been identified, and we might
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co-transform
these
genes
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phytoremediation in the future.
or
their
homologs
into
poplar
for
high-efficiency
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Competing financial interests The authors declare that they have no conflict of interest.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant
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No. 31370317 and 31571584), the Ministry of Science and Technology of China (Grant No.
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2016YFD0100504), the Natural Science Foundation of Chongqing (Grant No.
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cstc2013jcyjA80016), and Fundamental Research Funds for the Central Universities
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(XDJK2013B032).
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ACCEPTED MANUSCRIPT Fig. 1. Amino acid sequence alignment and phylogenetic analysis. (A) Structure analysis
2
and amino acid multi-alignment of the NBD domains of different plant species. ABCC
3
domains are marked as two yellow and green blocks. TMD, transmembrane domain; NBD,
4
a nucleotide-binding domain; walker A, walker B and ABC signature are NBD associated
5
motifs. Blue indicates identical amino acids, red indicates similar amino acids. (B)
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Phylogenetic analysis of ABCC proteins from Populus trichocarpa (PtABCC1, MG791913);
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Arabidopsis thaliana (AtABCC1, NP_174329; AtABCC2, NP_181013; AtABCC3,
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NP_187915); Oryza sativa (OsABCC1, XP_015635452); Glycine Max (GmABCC2,
9
XP_003542944);
Vitis
vinifera
(VvABCC1,
XP_002267650;
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and
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VvABCC6,
XP_002280819); Zea mays (ZmABCC1, NP_001105942); Eucalyptus grandis (EgABCC5,
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XP_010036147).
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ACCEPTED MANUSCRIPT Fig. 2. Hg tolerance analysis in Arabidopsis. (A-C) Arabidopsis seeds were grown on
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half-strength MS medium containing 0, 10 or 20 µM HgCl2 for 12 days; then root tip
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morphology and primary root length (D) were analyzed. WT, wild type; abcc1, Atabcc1-3
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mutant; OE-1 and OE-2, transgenic Arabidopsis lines. White bars= 15 mm, Black
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bars=200 µm. Error bars indicate standard deviation (n=3). Different letters indicated
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significant differences (P<0.05).
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Fig. 3. Hg tolerance analysis in poplar. The growth condition (A) and leaf damage analysis
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(B) of two-month-old wild type poplar (WT) and transgenic poplar (OX) seedlings in glass
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bottle treated with 0 or 20 µM HgCl2 for two weeks.
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ACCEPTED MANUSCRIPT Fig. 4. Hg accumulation analysis in Arabidopsis and poplar. Arabidopsis plants were
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grown on half-strength MS agar with 10 or 20 µM HgCl2 for two weeks (A) and poplar
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plants were grown in WPM hydroponic medium containing 20 µM HgCl2 for two weeks (B),
35
then they were harvested for Hg content analysis. WT, wild type; abcc1, Atabcc1-3 mutant;
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OE-1 and 2, transgenic Arabidopsis lines; OX-1, 2, and 3, transgenic poplar lines. Error
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bars indicate standard deviation (n=3). Different letters indicated significant differences
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(P<0.05).
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ACCEPTED MANUSCRIPT Highlights • Overexpression of PtABCC1 enhanced Hg tolerance in Arabidopsis and poplar. • Overexpression of PtABCC1 enhanced Hg accumulation in Arabidopsis and poplar.
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• PtABCC1 was the first identified poplar ABC transporter for phytoremediation of Hg.