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Abstract

Fluoride is an environmental toxin prevalent in water, soil, and air. A fluoride transporter called Fluoride EXporter (FEX) has been discovered across all domains of life, including bacteria, single cell eukaryotes, and all plants, that is required for fluoride tolerance. How FEX functions to protect multicellular plants is unknown. In order to distinguish between different models, the dynamic movement of fluoride in wildtype (WT) and fex mutant plants was monitored using [18F]fluoride with positron emission tomography. Significant differences were observed in the washout behavior following initial fluoride uptake between plants with and without a functioning FEX. [18F]Fluoride traveled quickly up the floral stem and into terminal tissues in WT plants. In contrast, the fluoride did not move out of the lower regions of the stem in mutant plants resulting in clearance rates near zero. The roots were not the primary locus of FEX action, nor did FEX direct fluoride to a specific tissue. Fluoride efflux by WT plants was saturated at high fluoride concentrations resulting in a pattern like the fex mutant. The kinetics of fluoride movement suggested that FEX mediates a fluoride transport mechanism throughout the plant where each individual cell benefits from FEX expression.

Key Message

Using positron emission tomography (PET) to monitor the movement of F? in wild type and Fluoride EXporter mutants revealed the inability of the mutant to keep F? out of cells.

Original abstract and full-text article online at https://link.springer.com/article/10.1007/s11103-023-01413-w

Excerpt:

Introduction

Fluoride (F–) is abundant in the environment, but is toxic to plant and animal life. Fluoride is naturally released into the biosphere by weathering of fluoride-containing minerals and from volcanoes and marine aerosols (Symonds et al. 1988; Weinstein and Davison 2004). Fluoride is released by coal burning and manufacturing processes involving metal smelting or chemical reduction of fluoride-containing minerals. Fertilizers used to enhance growth conditions for crops contain fluoride, initially from fluorapatite-rich phosphate rock, which results in fluoride accumulation in the soil (Anbuvel et al. 2014; Ramteke et al. 2018). As a result, plants are exposed to fluoride through the air, water, and soil. Different plant species vary in their fluoride uptake, accumulation, and tolerance. Some plants, such as Camilla sinensis (tea), can tolerate high levels of fluoride without ill effect, but most plants are sensitive at concentrations of less than 20 µg F/g dry weight (Jacobson et al. 1966). Other than a few species that produce toxic organofluorides, fluoride is not required biologically and is harmful to plants.

Fluoride from the soil enters the root apoplast mainly by passive diffusion (Garrec and Letourneur 1981; Mackowiak et al. 2003). Cell walls are the first barrier to cell entry, in part because the Ca2+ found there complexes with the negative fluoride ion and forms insoluble CaF2 (Miller et al. 1986; Ruan et al. 2004; Cai et al. 2014). In addition to the cell wall, the negatively charged cell membrane repels fluoride ions. However, the cell membrane is susceptible to HF, the conjugate acid of fluoride, prevalent at lower pH (pKa = 3.4). To enter the transpiration stream, fluoride must either travel through the symplast to the vasculature or pass through the root endodermal barrier. However, the root endodermis does have discontinuities through which fluoride can enter the transpiration stream (Davison et al. 1985; Takmaz-Nisancioglu and Davison 1988). The fluoride concentration found in the leaves directly reflects the concentration of fluoride in the growth medium and the amount of water flow through the plant, consistent with the ability of fluoride to by-pass the endodermis (Takmaz-Nisancioglu and Davison 1988; Banarjee and Roychoudhury, 2019). Once in the xylem, fluoride has been shown to move with the transpiration stream and accumulate at the tips of leaves (Elloumi et al. 2005; Hong et al. 2016).

Fluoride toxicity is manifest in a variety of ways, including complexation with cations like Ca2+ and Mg2+, cell wall disintegration, enzyme inactivation, and photosynthesis inhibition (Weinstein and Davison 2004; Banariee and Roychoudhury, 2019). Fluoride can also directly react with and damage proteins and cell membranes, interfere with phosphorylation, and depolarize membranes (Hong et al. 2016; Sharma and Kaur 2018; Gadi et al. 2021). Detrimental levels ultimately cause chlorosis, leaf burn, and tissue necrosis. Every stage of plant growth and tissue can be harmed by fluoride (Hong et al. 2016).

Despite being prevalent and toxic, the biochemical basis of fluoride resistance was only recently discovered. A fluoride transporter, originally found in bacteria (fluc- FLUoride Channel) and yeast (FEX, Fluoride EXporter), is an important mechanism of fluoride tolerance in plants (Li et al. 2013; Stockbridge et al. 2013; Berbasova et al. 2017). Both fluc and FEX specifically and rapidly efflux fluoride ions to keep the concentration within cells at nontoxic levels. For example, a yeast strain lacking FEX is 1000-fold more sensitive to fluoride in the growth media resulting in fluoride sensitivity at concentrations commonly found in municipal drinking water (60 uM; Li et al. 2013). Fluc is at least 1000-fold selective for F– over Cl–, the next closest halide (Stockbridge et al. 2013) and FEX from yeast or Arabidopsis is also highly selective (Tausta et al. 2021).

FEX homologs are found in all plants for which sequence information is available. Plant FEX homologs from different species were able to rescue a yeast FEX mutant grown in fluoride. This established that the putative FEX proteins from plants that are as diverse as moss and angiosperms are active fluoride transporters (Berbasova et al. 2017; Song et al. 2020; Tausta et al. 2021).

There is one FEX gene in Arabidopsis, which is expressed at a low level in most tissues ((BAR-Arabidopsis eFP browser; Schmid et al. 2005; Winter et al. 2007; Tausta et al. 2021). Arabidopsis with a fex knock-out mutation cannot tolerate even small amounts (~1 µM) of fluoride in the growth substrate resulting in yellowing leaves, stunted growth, and infertile flowers (Tausta et al. 2021). Higher concentrations of fluoride lead to necrosis and death of the plants. When grown in the presence of fluoride, the flowers at the apex of the mutant plant were shown to accumulate higher concentrations of fluoride when compared to wild type (WT) FEX flowers (Tausta et al. 2021). Conversely, overexpression of FEX resulted in fluoride tolerance at elevated concentrations (Zhu et al. 2019; Song et al. 2020; Tausta et al. 2021). Thus, FEX is necessary and sufficient for fluoride resistance in Arabidopsis.

This raises the question of how FEX functions to protect plants from fluoride toxicity. A few different possibilities can be imagined. One model is that fluoride ions could be detoxified by localization to the vacuole, a mechanism called ‘vacuole detoxification’, for which there is some evidence in the hyperaccumulator tea (Camilla sinensis; Gao et al. 2014). Many toxins and excess ions are shunted to the plant vacuole where they can be stored or detoxified. For instance, Cl– is localized into barley vacuoles (Martinoa et al. 1986) as are heavy metals such as cadmium and zinc (Sharma et al. 2016; Clemens and Ma 2016). A second model is that FEX functions in the plant roots to exclude fluoride from the transpiration stream, a strategy termed ‘avoidance’. It is a known strategy for metal tolerance (Tognacchini et al. 2020). A third possibility involves FEX expression in specific cell types in such a way that it directs fluoride to expendable tissues, such as the plant leaves, a model termed ‘selective tissue targeting’. For example, zinc accumulates in the leaf trichomes of Arabidopsis thaliana and A. halleri (Zhao et al. 2000; Ricachenevsky et al. 2021). A fourth model is that each cell utilizes FEX to efflux accumulated fluoride out of its own cytoplasm, thus continually moving fluoride into and through the transpiration stream in the plant. Fluoride could then be exuded through the hydathodes and/or released at the stomata with water. This model is effectively ‘each cell for itself’ and is most similar to what happens in single-celled systems like yeast (Li et al. 2013; Stockbridge et al. 2013). These four mechanisms are not necessarily exclusive of each other and other variations can be imagined. The FEX knockout mutation in Arabidopsis provides an opportunity to distinguish between these models and to explore how the fluoride transporter protects a multicellular organism from fluoride toxicity.

Fluoride has a short-lived isotope, [18F]fluoride, that is a positron emitter with a half-life of 110 min. This makes it possible to use positron emission tomography (PET) imaging to monitor fluoride kinetics within a plant in real time during the first few hours of fluoride exposure. PET was first employed for the study of fluoride movement in soybean using planar coincidence detectors of [18F]fluoride (McKay, 1988). Subsequent work with a more advanced positron-emitting tracer imaging system (PETIS) advanced these findings to show that [18F]fluoride transport in soybean was faster than [15O]H2O, and, thus, may not be equivalent to water transport (Nakanishi et al. 2001). Currently, PET scanners have developed sufficient sensitivity to provide three-dimensional measurements of radiolabeled tracers in the thinner tissues of plants (Converse et al. 2015; Fatangare and Svatos, 2016; Schmidt et al. 2020; Mincke et al. 2021). Specifically, monitoring [18F]fluoride via PET has confirmed the ability of plants to transport fluoride in the transpiration stream (McKay et al. 1988; Hubeau and Steppe 2015).

We set out to determine how FEX affects fluoride uptake, movement, and accumulation by analyzing wildtype (WT) and mutant FEX (fex) Arabidopsis plants using PET. We did this under a variety of experimental conditions to understand the role FEX plays in protecting the plant from fluoride toxicity. These observations provide experimental evidence to distinguish between several roles for FEX in fluoride resistance. The results reported here support the ‘each cell for itself’ model of fluoride movement within the plant. We found that the presence of FEX did not greatly affect initial fluoride uptake into the plant, but fluoride movement in the transpiration stream was severely inhibited in the FEX mutant.

References

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Funding

This work was supported by the National Science Foundation 1953903.

Author information

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06510, USA

    S. Lori Tausta & Scott A. Strobel

  2. Institute of Biomolecular Design and Discovery, Yale University West Campus, West Haven, CT, 06516, USA

    S. Lori Tausta & Scott A. Strobel

  3. Department of Radiology and Biomedical Imaging, Yale School of Medicine, 330 Cedar Street, New Haven, CT, 06519, USA

    Kathryn Fontaine & Ansel T. Hillmer

  4. Yale PET Center, Yale University, 801 Howard Avenue, New Haven, CT, 06510, USA

    Kathryn Fontaine & Ansel T. Hillmer

  5. Department of Biomedical Engineering, Yale University, 17 Hillhouse Avenue, New Haven, CT, 06511, USA

    Ansel T. Hillmer

  6. Department of Psychiatry, Yale School of Medicine, 300 George Street, New Haven, CT, 06510, USA

    Ansel T. Hillmer

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