CsFEX, a Fluoride Export Protein Gene

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Biotechnology and Biological Transformations

CsFEX, a fluoride export protein gene from Camellia sinensis, alleviates fluoride toxicity in transgenic Escherichia coli and Arabidopsis thaliana Jiaojiao Zhu, Anqi Xing, Zichen Wu, Jing Tao, Yuanchun Ma, Bo Wen, Xujun Zhu, Wanping Fang, and Yuhua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00509 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Agricultural and Food Chemistry

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CsFEX, a fluoride export protein gene from Camellia sinensis,

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alleviates fluoride toxicity in transgenic Escherichia coli and

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Arabidopsis thaliana

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Jiaojiao Zhu1, Anqi Xing1, Zichen Wu1, Jing Tao1, Yuanchun Ma1, Bo Wen1, Xujun Zhu1,

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Wanping Fang1, Yuhua Wang1*

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1 College

of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

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E-mail:

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Jiaojiao Zhu: [email protected]

Anqi Xing: [email protected]

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Zichen Wu: [email protected]

Jing Tao: [email protected]

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Yuanchun Ma: [email protected]

Bo Wen: [email protected]

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Xujun Zhu: [email protected]

Wanping Fang: [email protected]

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Corresponding author

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Yuhua Wang

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College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

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Tel: +86-25-84395182

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E-mail: [email protected]

Fax: +86-25-84395182

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1

ACS Paragon Plus Environment


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ABSTRACT: A Fluoride export gene (CsFEX) was newly found and isolated from

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Camellia sinensis and its functions in detoxifying F were investigated in transgenic

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Escherichia coli and Arabidopsis thaliana. CsFEX contains two crcB domains, which

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is the typical structure in plants. The expression of CsFEX in C. sinensis is tissue

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specific and related to maturity of leaves, and its expression is significantly induced

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by F treatments in different tissues of C. sinensis, particularly in leaves. Additionally,

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the growth of C. sinensis, E. coli, and A. thaliana can all be inhibited by F treatment.

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However, the growth of CsFEX-overexpression E. coli was increased with lower F

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content under F treatment compared to the control. Similarly, the germination and

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growth of CsFEX-overexpression A. thaliana were enhanced with lower F content

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under F treatment compared to wild type. CsFEX relieves F toxicity in the transgenic

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E. coli and A. thaliana by alleviating F accumulation.

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KEYWORDS: Camellia sinensis, Fluoride Export Protein, F tolerance/detoxication,

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Transgenic Escherichia coli, Transgenic Arabidopsis thaliana

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INTRODUCTION

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As one of the main halogens, fluorine is mainly present in the environment as

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fluoride (F) compounds 1. With human activities 2, industrial pollution 3, and

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application of chemical phosphate fertilizer 4, the content of F in soil has gradually

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increased. Studies have found that F is a common phytotoxin in plants via inhibition

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of enzyme activity

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slowing of growth 9. Furthermore, F can be absorbed from both the soil by plant roots

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or the air by leaves 10-11 and then transported and distributed throughout the plant 12.

5

and photosynthesis 6, resulting in the visible damage

7-8

and

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Accumulating reports have shown that the F content of mature leaves of C.

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sinensis is 871-1,337 mg.kg-1, while the F content of old leaves can exceed 2,000

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mg.kg-1 13, which is 10-100 times higher than that of other plants grown in the same

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environmental conditions

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of F without exhibiting any toxic symptoms, indicating that C. sinensis is a

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hyperaccumulator with high tolerance to F and has specific mechanisms driving F

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accumulation/detoxification 17. At present, there are three mechanisms that explain the

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accumulation and detoxification of F in C. sinensis. Cell wall precipitation and

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vacuolar compartmentalization prevent F from entering the physiological metabolic

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center of the protoplast without affecting the normal metabolic activities of C.

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sinensis cells, which may be one of the mechanisms of the F tolerance observed in C.

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sinensis

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free aluminum 21, magnesium, calcium 10, 22-23, or tea polysaccharides allows F to exist

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in C. sinensis in the form of stable fluoride-ligand complexes, thereby reducing F

18-20.

14-16.

Interestingly, tea seedlings accumulate large amounts

An alternative mechanism is the chelation of F ions in C. sinensis with

3



1, 18, 24.

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damage to C. sinensis

Additionally, recent reports reveal that F as a signal

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triggers the response of the defense gene (RLK) on the plasma membrane, and then

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activates the Ca2+ ATPase, the activated Ca2+ ATPase subsequently promotes the

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absorption and transport of F, this finding indicates that Ca2+ signaling pathway might

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involve in F accumulation/detoxification in C. sinensis 25. However, the complicated

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molecular mechanisms governing F accumulation/detoxification in C. sinensis remain

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unclear.

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Recently, many reports have indicated that the resistance to F toxicity is

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promoted by the family of F exporters in bacteria and eukaryotes. The exporters are

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divided into two phylogenetically unrelated categories: the first is a variant in the

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voltage-gated chloride channel (CLC) superfamily, namely CLCF. The CLCF genes

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encode F-/H+ antiporters in eubacteria

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family, Fluc (formerly crcB) in bacteria 28 and FEX2 in eukaryotes 29. For example, a

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recent study showed that FEX proteins (including FEX1 protein and FEX2 protein) in

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Saccharomyces cerevisiae function as ion channels, that select fluorides over

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chlorides, and FEX proteins can be constitutively expressed in yeast plasma

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membranes

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efflux of fluoride ions 30. Ji et al. (2014) believe that microorganisms have developed

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into a highly selective F channel (Fluc) to cope with the presence of F stress in the

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environment, which alleviates microbial toxicity by exporting inhibitory F in the

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cytoplasm to outside of the cell

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family in prokaryotes and lower eukaryotes would alleviate the damage caused by

30.

26-27.

The second is a more broadly distributed

In addition, studies have shown that FEX protein acts directly in the

31.

Nicholas et al. (2016) concluded that the Fluc

4


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32.

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fluorosis

Interestingly, the crcB homologs in eukaryotes (such as Neurospora

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crassa, S. cerevisiae, and Candida albicans) play a role in the export of F, so Li et al.

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(2013) renamed these genes as fluoride export genes (FEX), and this study directly

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shows that FEX and Fluc are homologous genes in different organisms. Based on

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these studies about F exporters in bacteria and eukaryotes, we speculate that F

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exporters might also be present in C. sinensis. Nonetheless, to the best of our

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knowledge, no F exporter gene or protein has yet been identified from C. sinensis.

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Therefore, this study focused on identifying whether C. sinensis contains a F

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export gene or protein, conferring its high F tolerance. Interestingly, a fragment

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containing two crcB domains was identified from our previous reported

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transcriptomic database (PRJNA315669) 33, named CsFEX, which encoded a putative

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CsFEX protein. The expression of CsFEX in different tissues and different maturity of

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C. sinensis leaves was analyzed and its response to exogenous F treatment was

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detected in C. sinensis. Furthermore, CsFEX was cloned from C. sinensis and

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transformed into E. coli and A. thaliana to detect the response of transgenic E. coli

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and A. thaliana to F treatments, thereby investigating the functions of CsFEX in F

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resistance. This study suggests that the CsFEX gene plays an important role in the F

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tolerance in C. sinensis. To the best of our knowledge, CsFEX is an F export gene

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which is newly found in C. sinensis in this study.

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MATERIALS AND METHODS

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Growth conditions, F treatment, and sample collection 5



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The one-year-old cutting seedlings of C. sinensis cv. LongjingChangye were

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collected from Nanjing Ya Run Tea Co., Ltd., China. The tea seedlings with uniform

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growth were selected, rinsed with double distilled water, and used as experimental

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materials. The seedlings were placed in three plastic containers (four C. sinensis

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seedlings per plastic container) containing 1 L of 1/2 complete nutrient solution for 2

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weeks in a light chamber with a photoperiod of 12-h light (25 ± 2oC)/12-hour dark (20

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± 2oC), and 70 ± 10% relative water content. Then the seedlings were pre-cultured in

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complete medium for 4 weeks before F treatment. The complete nutrient solution was

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prepared following the method of Ghanati et al. (2005) 34. The nutrient solutions were

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refreshed every 5 days, and the pH value was adjusted to 5.0 by 1 M HCl or 1 M

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NaOH solution every day.

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The pre-cultured C. sinensis seedlings were treated with 0 mM (control), 0.42

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mM, and 0.84 mM NaF for 0 d, 1 d, 2 d, and 20 d. The seedlings treated by 0 or 0.84

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mM F for 20 days were photographed to record the growth status. The young leaves

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and new roots were collected after 2 days of treatment at 0 mM, 0.42 mM, and 0.84

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mM F and dried at 80°C for determination of F accumulation. The young leaves (the

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first and second leaves from the top of plants), old leaves (the first and second leaves

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from the bottom of plants), roots, flowers, fruit, pollen, and stems of C. sinensis were

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collected and stored at -80 °C to detect the difference in the expression of CsFEX in C.

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sinensis. The young leaves, old leaves, roots, and stems of C. sinensis treated with

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0.84 mM NaF for 0 d, 1 d, 2 d were collected for expression analysis. In addition, the

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C. sinensis leaves with different maturity (buds, the first leaf, the second leaf, the third 6


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leaf, the fourth leaf, the fifth leaf and the old leaf) were collected respectively for

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expression analysis.

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Isolation and cloning of the CsFEX gene Based on the conserved crcB domain, the CsFEX gene was detected from 33.

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transcriptome data (PRJNA315669) reported by Pan et al. (2016)

The ORF of

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CsFEX was acquired by amplification of PCR using the primer pairs

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(CsFEX-ORF-F/R) listed in Table 1. The amplification product was then purified and

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cloned into the cloning vector pDONR201 for sequencing. Subsequently, the 5' and 3'

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untranslated region (UTR) sequences of CsFEX were amplified using the specific

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primers CsFEX-5'GSP1/5'GSP2 and CsFEX-3'GSP1/3'GSP2 (Table 1).

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Analyses of sequences and phylogenetic relationships

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The secondary structure of the CsFEX protein was predicted using the online

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software SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa), and the model of the

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tertiary structure of the CsFEX protein was constructed by Swiss-Model

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(https://www.swissmodel.expasy.org). The homologous sequence was aligned by

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DNAMAN software. A phylogenetic tree of CsFEX in C. sinensis and FEX in other

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organisms was constructed using the neighbor-joining method in MEGA6 software.

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The bootstrap method was used with 1,500 replicates; other parameters of the

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phylogenetic tree were set according to Wang et al. (2016) 35.

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RNA extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

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analysis

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Total RNA of C. sinensis was extracted using a Rapid RNA Isolation Kit

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(TaKaRa, Japan), and then the quality of RNA was assessed using the ONE DropTM

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OD-1000+ spectrophotometer (ONE Drop, USA). cDNA was obtained with the

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PrimeScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) following the

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manufacturer’s instructions. QRT-PCR was performed following the method reported

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by Wang et al. (2014)

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performed by 2−ΔΔCT method

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CsGAPDH. All experiments were repeated three times with independent RNA

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samples, and the primers were listed in Table 1.

36,

and the relative expression analysis of the data was 37.

The internal reference gene used in this study was

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Construction of prokaryotic expression vector and F tolerance of transgenic E.

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coli cells

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The ORF of CsFEX was subcloned from the cloning vector pDONR201, and

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then ligated into the pET-28a expression vector. The pET-CsFEX recombinant

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plasmid and the empty vector were used to transform E. coli ROSETTA cells, and

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proper insertion was confirmed by enzyme digestion and sequencing.

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To examine the effect of overexpressing CsFEX on the growth of E. coli strains,

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the empty vector and CsFEX transformed E. coli strains were cultured in LB medium

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until the OD600 value reached 0.8, and then five-fold serial dilutions were inoculated

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in solid LB media containing different concentrations of F (0 mM, 5 mM, 50 mM, and 8


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100 mM), and the growth state of the two strains was observed after 12 h. Meanwhile,

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the transformed E. coli strains with an OD600 value of 1 were added to LB liquid

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containing different concentrations of F (0 mM, 5 mM, 50 mM, and 100 mM) at a

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ratio of 1:1000 and then cultured in an oscillating incubator with shaking at 220 rpm

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at 37 °C. The OD600 value was measured by a spectrophotometer at different times (2

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h, 4 h, 8 h, 10 h, and 12 h), and the curves were plotted with GraphPad Prism software.

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In addition, to detect the F content in the two strains, the cells were collected after

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culturing for 10 h under treatment with different concentrations of F.

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Transformation of CsFEX into A. thaliana

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To further confirm the function of CsFEX, CsFEX was amplified using

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CsFEX-gate-F/R (Table 1) and cloned into pDONR201. It was then recombined into

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pK7FWG2 to generate the 35S:CsFEX:EGFP construct. Then, the construct was

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introduced into the Colombia ecotype of A. thaliana by the floral dip method

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Transgenic A. thaliana was screened on 1/2 MS agar media containing 50 μg·mL-1

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kanamycin. An RT-PCR assay was conducted to verify the integration of CsFEX in

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positive transgenic A. thaliana using gene-specific primers (CsFEX-gate-F/-R, Table

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1), and the AtACTIN2 (AT3G18780) gene in Table 1 was used as a control. All A.

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thaliana were grown at 22 ± 2 °C in a light incubator with a 16-h light (220

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μmol·m−2·s−1)/8-h dark cycle.

38.

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Phenotype analysis of transgenic A. thaliana and subcellular localization of 9



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CsFEX

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WT and transgenic A. thaliana seeds (WT, L-2, L-4，and L-8) were plated on

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1/2 MS solid medium with various concentrations of F (0 mM, 4 mM, 6 mM, or 8

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mM). After 2 days of vernalization at low temperature (4 oC), the plates were

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transferred to a light incubator with a 16-h light (220 μmol·m−2·s−1)/8-h dark cycle for

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7 days. The germination rate was calculated. The phenotypes of A. thaliana were

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observed and photographed with a camera after cultured for 14 days with various

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concentrations of F (0 mM, 4 mM, 6 mM and 8 mM). Meanwhile, A. thaliana grown

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for 14 days on 1/2 MS solid media with different concentrations of exogenous F (4

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mM and 6 mM) were harvested for F content detection.

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To confirm the subcellular localization of CsFEX, signals of CsFEX-EGFP in

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roots of WT and transgenic A. thaliana were observed by laser confocal microscopy

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(CarlZeiss LSM710, Germany).

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Extraction and determination of F in samples

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To collect bacterial samples, the bacterial liquid was centrifuged at 5000 rpm for

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15 min, and then resuspended 3 times with ddH2O. To collect plant samples, C.

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sinensis and A. thaliana samples were collected and weighed. The above bacterial

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samples and plant samples were placed into 50 mL centrifugal tubes that contained 30

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mL of ddH2O, and then extracted at 100 °C for 30 min in water bath. The extraction

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mixtures were cooled to room temperature and centrifuged at 5000 g for 15 min, then

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the supernatant was collected to determine the F content using the 9609BNWP 10


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fluoride ion selective electrode and 096019 stirrer probe following the description in

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Gao et al., (2013) 39.

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Statistical analyses

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All data in this study were analyzed using SPSS version 17.0, all data were

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presented as mean ± standard deviation (SD). Significant differences between the

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experimental data were tested with ANOVA and Duncan’s test and were marked by

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different letters (P < 0.05).

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RESULTS

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Accumulation of F in C. sinensis

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The phenotype of tea seedlings was investigated after 20 days of treatment with

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0.84 mM of F. Long-term F treatment caused the growth of tea seedlings to slow,

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which was manifested by necrosis of new roots, chlorosis, and scorching of the young

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leaves (Figure 1A, Figure 1B, Figure 1C and Figure 1D). Furthermore, the effects of

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different concentrations of F on the accumulation of F in new roots and young leaves

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(the first leaf and the second leaf) were also investigated. The results showed the F

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content of new roots and young leaves of C. sinensis in fluorine-free culture medium

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was 6.71 mg.kg-1 and 315.85 mg.kg-1, respectively. Then it increased to 241.97

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mg.kg-1 and 335.45 mg.kg-1 after treated with 0.42 mM F for 2 days. It reached to

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427.57 mg.kg-1 and 356.45 mg.kg-1 after treated with 0.84 mM F for 2 days. These

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results indicate that the F content of young leaves and new roots of C. sinensis 11



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increased significantly after 2 days of F treatment; the increase in F content in new

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roots was significantly greater than that of young leaves (Figure 1E).

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Cloning and characterization of CsFEX

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The full-length of the CsFEX cDNA sequence is 2,205 bp (Figure 2), which

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contains a 1,443-bp open reading frame (ORF) (Figure 3), encodes 480 amino acids

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(predicted molecular weight 52.391 kDa), and has a theoretical isoelectric point of 6.5.

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Through SOPMA prediction, CsFEX is composed of 38.96% α-helices, 15.21% of the

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extended backbone, 4.79% of β-sheets, and 41.04% of random coils. The tertiary

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results of FEX protein are presented in Figure S1. The blast alignment showed that

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the sequence of CsFEX exhibits a high homology to FEX in plants. The phylogenetic

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analysis of FEX in the organism showed that FEXs are mainly divided into two

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categories, and the evolutionary relationship of FEXs between C. sinensis and other

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plants is very close (Figure 4A). Sequence analysis by Pfam showed that FEXs in C.

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sinensis, Actindia chinensis, Ipomoea nil, and Sesamum indicum contain two

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conserved crcB-like protein structures (Figure 4B), which is the typical structure in

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plants.

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Expression analysis of CsFEX in C. sinensis

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To test the expression profile of CsFEX in C. sinensis, the expression levels of CsFEX

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were compared in roots, stems, young leaves, old leaves, fruits, flowers and pollen,

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and the results showed that the expression level of CsFEX in the pollen and young 12


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leaves of C. sinensis was significantly higher than that of other organs (Figure 5A),

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which indicates that the expression of CsFEX in C. sinensis is tissue specific.

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Furthermore, as shown in Figure 5B, the expression of CsFEX in different tissues of C.

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sinensis under F treatment was increased with the increase of F treatment time. The

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CsFEX expression level increased 7.93-fold in the young leaves of C. sinensis in

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response to 0.84 mM F for 2 days compared to plants grown under the control

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conditions, and the CsFEX expression levels of other organs also had slightly

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increased after F treatment (Figure 5B). Moreover, the expression profiles of CsFEX

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in C. sinensis leaves (bud, 1st, 2nd, 3rd, 4th, 5th and old) with different maturity was

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detected and the results showed that the relative expression level of CsFEX was

266

lowest in the buds and first leaf, peaked in the second leaf and then it decreased with

267

the increase of leaf maturity (Figure 5C), indicating that the expression of CsFEX in C.

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sinensis is related to the leaf maturity.

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Enhanced F tolerance in E. coli cells overexpressing CsFEX

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Reverse transcription polymerase chain reaction (RT-PCR) was performed to

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obtain the correct overexpressed CsFEX strain and empty vector strain (Figure S2).

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The growth of both control and overexpressing strains were inhibited by F treatment

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in a dose-dependent manner. Furthermore, the strain overexpressing CsFEX showed

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better growth after treatment with 0 mM, 5 mM, 50 mM, and 100 mM F compared to

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the control (Figure 6A). Simultaneously, the results also showed that the strain

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overexpressing CsFEX had a higher survival rate when treated with 5 mM, 50 mM, 13



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and 100 mM F compared to the empty vector (Figure 6B). In addition, the results

279

showed that the strain overexpressing CsFEX accumulated lower levels of F than the

280

control, indicating that CsFEX functions in enhancing the tolerance of E. coli to F

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(Figure 7).

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Enhanced F tolerance in A. thaliana overexpressing CsFEX

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As a C. sinensis transgenic system is currently difficult to establish, the model

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plant A. thaliana was employed to overexpress CsFEX to further study its biological

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functions in the accumulation of F. As shown in Figure 8A, CsFEX overexpression

287

was detected in transgenic A. thaliana lines (L-2, L-4, and L-8), but not in wild type

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(WT) A. thaliana. The germination rate, growth status and F accumulation of WT and

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transgenic lines were detected to investigate whether overexpression of CsFEX in A.

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thaliana can enhance the tolerance to F stress. The germination rate of WT, L-2, L-4

291

and L-8 all approached 100% under normal conditions, and the germination rate of

292

wild and overexpressed lines were both inhibited by F treatment in a dose-dependant

293

manner. Moreover, the germination rate was higher in transgenic lines than the WT

294

under the same concentration of F treatment. For example, the germination rates of

295

L-2, L-4 and L-8 were 3.79, 6.05 and 6.87 times that of the WT under 8 mM F,

296

respectively (Figure 8B). The growth rate of wild-type and overexpressing A. thaliana

297

was significantly inhibited by exogenous F treatment, and the reduction of growth rate

298

of the WT was more severe than transgenic A. thaliana (Figure 8C). Additionally, the

299

F accumulation increased with the increasing concentration of exogenous F in WT 14


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and transgenic A. thaliana. Interestingly, the F concentration in transgenic A. thaliana

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was lower than that of the wild type under the same F treatment conditions (Figure

302

8D).

303 304

Subcellular localization of CsFEX in the roots of A. thaliana

305

Subcellular localization prediction software WOLF PSORT28 predicts that

306

CsFEX is localized in the plasma membrane. To further validate the subcellular

307

localization of CsFEX, we constructed a CsFEX:GFP fusion protein and transformed

308

it into A. thaliana. As shown in Figure 9, the GFP signal was distributed in the plasma

309

membrane of A. thaliana root cells (Figure 9A, 9B, 9C), while no fluorescence signal

310

was detected in WT A. thaliana root cells (Figure 9D, 9E, 9F). Therefore, these

311

results are consistent with each other.

312 313

DISCUSSION

314

Recent surveys have indicated that many plants exposed to F often show some

315

morphological symptoms such as chlorosis and tip and leaf edge necrosis

316

suggesting that F is toxic to plants. It is well known that C. sinensis is a crop that

317

hyperaccumulates F, while many studies have shown this F-tolerant plants also

318

exhibit some physiological and biochemical changes in response to high concentration

319

of F, including a decrease of photosynthesis, alteration of leaf antioxidant system, and

320

disruption of the cell ultrastructure

321

concentration of exogenous F induced the new roots to turn black, the young leaves to

44-45.

40-43,

Similarly, this study showed that high

15



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gradually turn yellow, and the edges of the leaves to exhibit scorch symptoms after

323

long-term F treatment (Figure 1A). Additionally, the F content of different tissues in C.

324

sinensis exhibits certain regularity, which mainly appears as mature leaf > young leaf

325

> root/stem 13, 46. As the tissue with the strongest ability to accumulate F in C. sinensis,

326

the leaves are also the strongest tolerant to F. This study revealed that the F content in

327

tea leaves was approximately 43 times that of new roots under normal conditions,

328

indicating that C. sinensis leaves are the main tissue that accumulate F, which is

329

consistent with the previous findings of Gao et al. (2014)

330

Zhang et al. (2013) suggests that F uptake showed biphasic response patterns,

331

following saturable Michaelis-Menten kinetics in the range of low external F, while

332

increased linearly with external supply in the range of high concentrations 47. Here we

333

found that the F content increased significantly with the increase of exogenous F

334

concentration, especially in new roots of C. sinensis, indicating that the new roots can

335

quickly absorb and transfer F

336

clear that although C. sinensis is severely impacted by high concentrations of

337

exogenous F, it is still a hyperaccumulator with higher F-tolerance compared with

338

other species. However, the molecular mechanism driving F tolerance/detoxification

339

in C. sinensis still remains ambiguous.

47.

19.

According to report by

Combining previous reports and our findings, it is

340

The recent discovery of F exporters is a breakthrough in exploring the

341

mechanisms of F resistance; therefore, current research is increasingly focusing on F

342

exporters. Based on reports in prokaryotes and eukaryotes and our previous

343

transcriptome database (PRJNA315669) 33, we identified a fragment that contains two 16


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conserved crcB domains, indicating that this fragment is a typical plant FEX, so we

345

named it CsFEX. Furthermore, the expression of CsFEX in C. sinensis showed

346

tissue-specific and was highest in the young leaves, particularly in the second leaf,

347

and then it was reduced with the increase of leaf maturity. These results indicate that

348

the expression of CsFEX is related to the maturity of C. sinensis leaves which is

349

closely related to the contents of F 48. In the respect of the lower relative expression

350

levels of CsFEX in buds and the first leaf, we speculate that it may be resulted from

351

the fact that the buds and the first leaf have not formed a complete and mature leaf

352

structure compared to the mature leaves. Additionally, the expression of CsFEX in

353

different tissues of C. sinensis can be induced by high concentration of exogenous F

354

treatment in 2 days, indicating that CsFEX respond to exogenous F triggers indeed.

355

To further confirm the functions of CsFEX, prokaryote E. coli was employed to

356

overexpress CsFEX. Our findings indicate that CsFEX recombinant protein confers

357

enhanced F tolerance and lower F content in transformed E. coli cells. Similarly,

358

Baker et al. (2012) reported that the growth of the crcB knockout E. coli strain was

359

significantly inhibited at micromolar concentrations of F with a minimum inhibitory

360

concentration (MIC) slightly higher than 1 mM, while the growth of WT E. coli cells

361

were significantly inhibited at 30 mM NaF, with the MIC of about 200 mM

362

Baker et al. (2012) also suggests that the crcB protein reduces F toxicity by reducing

363

the concentration of F in E. coli cells. Combining published studies with our findings,

364

we therefore speculate that CsFEX can enhance the tolerance to F toxicity in E. coli

365

cells by reducing F accumulation. 17


27,

and


366

In the respect to findings of eukaryotes, deletion of the FEX gene in three model

367

eukaryotes (S. cerevisiae, N. crassa, and C. albicans) induces a fluoride-sensitive

368

phenotype, and knock-out cells lacking the FEX gene are 200-1,000 times more

369

sensitive to F than the corresponding WT strain. This directly demonstrates the

370

importance of FEX in resisting F toxicity

371

CsFEX driving F tolerance, model plant A. thaliana was used to construct

372

overexpressing lines in this study. The results of the overexpression study revealed

373

that A. thaliana lines overexpressing CsFEX showed higher germination rates and

374

growth rates with lower F content comparing to the WT under exogenous F treatment.

375

In addition, subcellular localization experiments revealed that the CsFEX is localized

376

in the plasma membrane of A. thaliana root cells, which consists with the results in

377

yeast 30. Therefore, this study demonstrates that overexpression of CsFEX enhances F

378

tolerance in A. thaliana via reducing F content.

28.

To further confirm the mechanism of

379

In summary, this is the first study to identify and clone the F-specific export gene

380

CsFEX from C. sinensis. The expression of CsFEX in C. sinensis is tissue-specific

381

and related to the maturity of leaves; it can be triggered by exogenous F treatment.

382

Furthermore, overexpression of CsFEX in E. coli and A. thaliana confers enhanced

383

tolerance to F toxicity by alleviating F accumulation. These findings directly reveal

384

the effects of CsFEX on alleviating F toxicity. Combing the previous reports and our

385

findings, we speculate that F is passively absorbed by tea plants under high F

386

concentration (Zhang et al., 2013)47 or actively absorbed through some ion channels

387

(such as Ca2+ ATPase) (Li et al., 2017)

25,

and then the putative fluoride export

18


Page 18 of 40

Page 19 of 40


388

proteins (CsFEX, CsCLCF) are activated to excrete F from the cytoplasm into the cell

389

wall or apoplast to alleviate F toxicity. On the other way, the excess F can be

390

separated into vacuoles to reduce the toxicity (Figure 10). However, the accurate

391

molecular mechanisms of F accumulation, toxicity/detoxification, and resistance in C.

392

sinensis are warranted more attention to decipher.

393 394

Supporting Information

395

The Supporting Information is available free of charge on the ACS Publications

396

website at xxx.

397

Tertiary structure prediction results of CsFEX protein (Figure S1); RT-PCR

398

identification of CsFEX in transgenic E. coli cells (Figure S2).

399 400

Funding

401

This work was supported by the National Natural Science Foundation of China

402

(31770733), the earmarked fund for China Agriculture Research System (CARS-19)

403

and the earmarked fund for Jiangsu Agricultural Industry Technology System

404

(JATS[2018]280).

405 406

Notes

407

The authors declare no competing interest.

19



408

REFERENCES

409

1. Yang, Y.; Liu, Y.; Huang, C. F.; Silva, J. D.; Zhao, F. J. Aluminium alleviates fluoride toxicity in tea

410

( Camellia sinensis ). Plant and Soil. 2016, 402 (1-2), 179-190.

411

2. Cronin, S. J.; Neall, V. E.; Lecointre, J. A.; Hedley, M. J.; Loganathan, P. Research, G., Environmental

412

hazards of fluoride in volcanic ash: a case study from Ruapehu volcano, New Zealand. Journal of

413

Volcanology and Geothermal Research. 2003, 121 (3), 271-291.

414 415

3. Mackowiak, C. L.; Grossl, P. R.; Bugbee, B. G. Biogeochemistry of fluoride in a plant-solution system. Journal of Environment Quality. 2003, 32 (6), 2230-2237.

416

4. Loganathan, P.; Hedley, M. J.; Wallace, G. C.; Roberts, A. H. Fluoride accumulation in pasture

417

forages and soils following long-term applications of phosphorus fertilisers. Environmental

418

Pollution. 2001, 115 (2), 275-282.

419 420

5. Baroni Fornasiero, R. Fluorides effects on hypericum perforatum plants: first field observations. Plant Science. 2003, 165 (3), 507-513.

421

6. Kamaluddin, M.; Zwiazek, J. Fluoride inhibits root water transport and affects leaf expansion and

422

gas exchange in aspen (Populus tremuloides) seedlings. Physiologia Plantarum. 2010, 117 (3),

423

368-375.

424

7. Dey, U.; Mondal, N. K.; Das, K.; Datta, J. K., Dual effects of fluoride and calcium on the uptake of

425

fluoride, growth physiology, pigmentation, and biochemistry of Bengal gram seedlings (Cicer

426

Arietinum L.). Fluoride. 2012, 45 (4), 389-393.

427 428

8. Jha, S. K.; Nayak, A. K.; Sharma, Y. K., Fluoride toxicity effects in onion (Allium cepa L.) grown in contaminated soils. Chemosphere. 2009, 76 (3), 353-356.

20


Page 20 of 40

Page 21 of 40

429 430


9. Miller, G. W., The effect of fluoride on higher plants: With special emphasis on early physiological and biochemical disorders. Fluoride. 1993, 26 (1), 3-22.

431

10. Stevens, D. P.; Mclaughlin, M. J.; Alston, A. M. J., Phytotoxicity of the fluoride ion and its uptake

432

from solution culture by Avena sativa and Lycopersicon esculentum. Plant and Soil. 1998, 200 (2),

433

119-129.

434 435

11. Ruan, J.; Ma, L.; Shi, Y.; Han, W. Uptake of fluoride by tea plant (Camellia sinensis L.) and the impact of aluminium. Journal of Science of Food and Agriculture. 2003, 83 (13), 1342–1348.

436

12. Fung, K. F.; Zhang, Z. Q.; Wong, J. W. C.; Wong, M. H. Fluoride contents in tea and soil from tea

437

plantations and the release of fluoride into tea liquor during infusion. Environmental Pollution.

438

1999, 104 (2), 197–205.

439 440

13. Shu, W. S.; Zhang, Z. Q.; Lan, C. Y.; Wong, M. H. Fluoride and aluminium concentrations of tea plants and tea products from Sichuan Province, PR China. Chemosphere. 2003, 52 (9), 1475-1482.

441

14. Weinstein, L. H.; Davison, A. Fluorides in the environment. Fluorides in the Environment. 2004.

442

15. Ruan, J. Y.; Wong, M. H. Accumulation of fluoride and aluminium related to different varieties of

443

tea plant. Environmental Geochemistry and Health. 2001, 23 (1), 53-63.

444

16. Xie, Z.; Chen, Z.; Sun, W.; Guo, X.; Yin, B.; Wang, J. Distribution of aluminum and fluoride in tea

445

plant and soil of tea garden in Central and Southwest China. Chinese Geographical Science. 2007,

446

17 (4), 376-382.

447

17. Liu, Y.; Cao, D.; Ma, L.; Jin, X.; Yang, P.; Ye, F.; Liu, P.; Gong, Z.; Wei, C. TMT-based quantitative

448

proteomics analysis reveals the response of Camellia sinensis to fluoride. Journal of Proteomics.

449

2018, 176 (30), 71-81.

21



450 451

18. Gao, H.; Zhu, X.; Panpan, L. I.; Dejiang, N. I.; Chen, Y. Research on adsorption characteristics of tea polysaccharides to fluorine. Journal of Tea Science. 2016, 36 (4): 396-404.

452

19. Gao, H. J. , Zhao, Q. , Zhang, X. C. , Wan, X. C. , Mao, J. D.,, Localization of fluoride and aluminum in

453

subcellular fractions of tea leaves and roots. Journal of Agricultural Food Chemistry. 2014, 62 (10),

454

2313-2319.

455

20. Schneider, T.; Schellenberg, M.; Meyer, S.; Keller, F.; Gehrig, P.; Riedel, K.; Youngsook, L.; Eberl, L.;

456

Martinoia, E. Quantitative detection of changes in the leaf-mesophyll tonoplast proteome in

457

dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants. Proteomics. 2010, 9

458

(10), 2668-2677.

459 460 461 462 463 464

21. Ding, R.; Huang, X. Biogeochemical cycle of aluminium and fluorine in tea garden soil system and its relationship to soil acidification. Acta Pedologica Sinica. 1991, 3. 22. Ruan, J.; Ma, L.; Shi, Y.; Han, W. The impact of pH and calcium on the uptake of fluoride by tea plants (Camellia sinensis L.). Annals of Botany. 2004, 93 (1), 97. 23. Arnesen, A. K. M. Availability of fluoride to plants grown in contaminated soils. Plant and Soil. 1997, 191 (1), 13-25.

465

24. Cai, H. M.; Peng, C. Y.; Chen, J.; Hou, R. Y.; Gao, H. J.; Wan, X. C. X-ray photoelectron spectroscopy

466

surface analysis of fluoride stress in tea (Camellia sinensis (L.) O. Kuntze) leaves. Journal of

467

Fluorine Chemistry. 2014, 158 (158), 11-15.

468

25. Li, Q. S.; Lin, X. M.; Qiao, R. Y.; Zheng, X. Q.; Lu, J. L.; Ye, J. H.; Liang, Y. R. Effect of fluoride

469

treatment on gene expression in tea plant (Camellia sinensis). Scientific Reports. 2017, 7 (1), 9847.

22


Page 22 of 40

Page 23 of 40


470

26. Stockbridge, R. B.; Lim, H. H.; Otten, R.; Williams, C.; Shane, T.; Weinberg, Z.; Christopher, M.

471

Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proceedings of the

472

National Academy of Sciences of the United States of America. 2012, 109 (38), 15289-15294.

473 474 475 476

27. Baker, J. L.; Narasimhan, S.; Zasha, W.; Adam, R.; Stockbridge, R. B.; Breaker, R. R. Widespread genetic switches and toxicity resistance proteins for fluoride. Science. 2012, 335 (6065), 233-235. 28. Stockbridge, R. B.; Robertson, J. L.; Ludmila, K. P.; Christopher, M. A family of fluoride-specific ion channels with dual-topology architecture. eLife. 2013, 2.

477

29. Li, S.; Smith, K. D.; Davis, J. H.; Gordon, P. B.; Breaker, R. R.; Strobel, S. A. Eukaryotic resistance to

478

fluoride toxicity mediated by a widespread family of fluoride export proteins. Proceedings of the

479

National Academy of Sciences. 2013, 110 (47), 19018-19023.

480

30. Smith, K. D.; Gordon, P. B.; Rivetta, A.; Allen, K. E.; Berbasova, T.; Slayman, C.; Strobel, S. A. Yeast

481

Fex1p is a constitutively expressed fluoride channel with functional asymmetry of its two

482

homologous domains. Journal of Biological Chemistry. 2015, 290 (32), 19874.

483 484 485 486

31. Ji, C.; Stockbridge, R. B.; Miller, C. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. Journal of General Physiology. 2014, 144 (3), 257-261. 32. Last, N. B.; Kolmakova-Partensky, L.; Shane, T.; Miller, C. Mechanistic signs of double-barreled structure in a fluoride ion channel. eLife. 2016, 5.

487

33. Pan, J.; Wang, W.; Li, D.; Shu, Z.; Ye, X.; Chang, P.; Wang, Y. J. B. G. Gene expression profile

488

indicates involvement of NO in Camellia sinensis pollen tube growth at low temperature. BMC

489

Genomics. 2016, 17 (1), 809.

490 491

34. Ghanati, F.; Morita, A.; Yokota, H. Effects of aluminum on the growth of tea plant and activation of antioxidant system. Plant and Soil. 2005, 276 (1-2), 133-141. 23



492

35. Wang, Y.; Shu, Z.; Wang, W.; Jiang, X.; Li, D.; Pan, J.; Li, X. CsWRKY2 , a novel WRKY gene from

493

Camellia sinensis, is involved in cold and drought stress responses. Biologia Planterum. 2016, 60

494

(3), 1-9.

495

36. Wang, W.; Wang, Y.; Du, Y.; Zhao, Z.; Zhu, X.; Jiang, X.; Shu, Z.; Yin, Y.; Li, X. Overexpression of

496

Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco. Plant Cell

497

Reports. 2014, 33 (11), 1829-1841.

498 499 500 501

37. Livak, K. J.; Schmittgen, T. D., Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001, 25 (4), 402-408. 38. Clough, S. J.; Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal. 2010, 16 (6), 735-743.

502

39. Gao H, Zhang Z, Wan X. Influences of charcoal and bamboo charcoal amendment on soil-fluoride

503

fractions and bioaccumulation of fluoride in tea plants. Evironmental Geochemistry and Health.

504

2012, 34 (5), 551-562.

505

40. Fornasiero, R. B. Phytotoxic effects of fluorides. Plant Science. 2001, 161 (5), 979-985.

506

41. Kamaluddin, M.; Zwiazek, J. J. Fluoride inhibits root water transport and affects leaf expansion and

507

gas exchange in aspen (Populus tremuloides) seedlings. Physiologia Plantarum. 2010, 117 (3),

508

368-375.

509 510 511 512

42. Jha, S. K.; Nayak, A. K.; Sharma, Y. K. Fluoride toxicity effects in onion (Allium cepa L.) grown in contaminated soils. Chemosphere. 2009, 76 (3), 353-356. 43. Barbier, O.; Arreola-Mendoza, L.; Razo, L. M. D. Molecular mechanisms of fluoride toxicity. Chemico-Biological Interactions. 2010, 188 (2), 319-333.

24


Page 24 of 40

Page 25 of 40


513

44. Cai, H. M.; Dong, Y. Y.; Li, Y. Y.; Li, D. X.; Peng, C. Y.; Zhang, Z. Z.; Wan, X. C. Physiological and

514

cellular responses to fluoride stress in tea (Camellia sinensis) leaves. Acta Physiologiae Plantarum.

515

2016, 38 (6), 144.

516 517

45. Li, C.; Zheng, Y.; Zhou, J.; Xu, J.; Ni, D. Changes of leaf antioxidant system, photosynthesis and ultrastructure in tea plant under the stress of fluorine. Biologia Plantarum. 2011, 55 (3), 563-566.

518

46. Fung, K. F.; Zhang, Z. Q.; Wong, J. W.; Wong, M. H. Aluminium and fluoride concentrations of three

519

tea varieties growing at Lantau Island, Hong Kong. Environmental Geochemistry Health. 2003, 25

520

(2), 219-232.

521 522 523 524

47. Zhang, L.; Li, Q.; Ma, L.; Ruan, J. Characterization of fluoride uptake by roots of tea plants (Camellia sinensis (L.) O. Kuntze). Plant Soil Research. 2013, 366 (1-2), 659-669. 48. Feng, M. L.; Yun, R. J.; Zhi, S. Y. Study on accumulation characteristics of fluorine in tea plants. Acta Agriculturae Zhejiangensis. 2004, 16 (2), 96-98.

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525

Figure legends

526

Figure 1 The effects of F treatment on C. sinensis growth. Changes of young leaves

527

and new roots of C. sinensis treated with different concentrations of F (0 mM control

528

(A, C), 0.84 mM treatment (B, D), respectively) for 20 days. (E) Differences in F

529

content between young leaves and new roots of C. sinensis treated with different

530

concentrations of F for 2 days. Data are presented as mean ± SD. Letters indicate

531

significant differences at P

CsFEX, a Fluoride Export Protein Gene

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