The fluoride transporter FLUORIDE EXPORTER (FEX) is the major mechanism of tolerance to fluoride toxicity in plants

Abstract

Fluoride is everywhere in the environment, yet it is toxic to living things. How biological organisms detoxify fluoride has been unknown until recently. Fluoride-specific ion transporters in both prokaryotes (Fluoride channel; Fluc) and fungi (Fluoride Exporter; FEX) efficiently export fluoride to the extracellular environment. FEX homologues have been identified throughout the plant kingdom. Understanding the function of FEX in a multicellular organism will reveal valuable knowledge about reducing toxic effects caused by fluoride. Here we demonstrate the conserved role of plant FEX (FLUORIDE EXPORTER) in conferring fluoride tolerance. Plant FEX facilitates the efflux of toxic fluoride ions from yeast cells and is required for fluoride tolerance in plants. A CRISPR/Cas9-generated mutation in Arabidopsis thaliana FEX renders the plant vulnerable to low concentrations (100 µM) of fluoride at every stage of development. Pollen is particularly affected, failing to develop even at extremely low levels of fluoride in the growth medium. The action of the FEX membrane transport protein is the major fluoride defense mechanism in plants.

Figure 1

Any plant FEX rescues the yeast FEX DKO. A, DKO yeast were transformed with cDNA sequences of FEX homologs from the indicated plants and 10-fold dilutions of yeast cultures at OD₆₀₀ of 1 were tested on plates with increasing amounts of NaF. B, Relative positions of the conserved N in yeast and A. thaliana sequences. Blue lettering highlights conserved amino acid residues. C, Growth assay of yeast DKO transformed with N to A substitutions. Rescue of yeast DKO fails only when A is substituted for N in TM7/Pore II. D, Model of FEX in cellular membrane based on bacterial crystal structure and yeast modeling. Cylinders and numbers denote transmembrane domains. A conserved N (blue circle) is positioned in both Pore I and II. In yeast, Pore II is a functional conduit for fluoride ions and Pore I is not.

Figure 2

Intracellular fluoride measurements show higher accumulation in yeast cells without a functioning FEX. DKO yeast and DKO with empty vector grown in 50-μM NaF for 14 h accumulated fluoride. DKO transformed with yeast FEX (ScFEX1) or plant FEX (AtFEX, NtFEX, SiFEX) recovered the ability to export fluoride. Substitution of a conserved N in Pore II (N373A), but not Pore I (N186A) impaired fluoride efflux. Points represent biological replicates, bars are the mean, and error bars are SD. One-way ANOVA (Dunnett) results between a and b are P < 0.0001, and P = 0.0428 between a and c.

Figure 3

AtFEX, CsFEX, and ScFEX1 efflux F⁻ but not Cl⁻. A, Model of in vitro assay using proteoliposomes with an inside concentration of 150-mM KF (pH 7) and an outside concentration of 1-mM KF (pH 7). Valinomycin (V) was added at time 0 (arrow), creating a chemical gradient and efflux of ions. B, Relative fluoride efflux by ScFEX1 (black), AtFEX (blue), CsFEX (brown), or no FEX (orange), as detected with a fluoride ion selective electrode-based probe. The Y-axis represents negative voltage versus time, where the negative voltage has been transformed into nanomoles of ions based on a calibration with an ion standard and then compared with how many nanomoles of fluoride ions there were in all liposomes. C, Instead of F⁻, Cl⁻ ions were loaded into proteoliposomes and efflux was detected with a chloride specific electrode–base probe. Arrowheads indicate the approximate time of β-OG addition to break open proteoliposomes and release trapped ions.

Figure 4

Promoter GUS expression indicates AtFEX is expressed in young tissues, veins, and hydathodes. Promoter constructs in two different vectors (pKGWFS7, 8 lines and pGWB3, 7 lines) were analyzed with similar results. Pictures are from the pGWB3 lines. A, Widespread staining in seedling. B and C, Staining in older leaves reveals expression in hydathodes and veins. D, Flowers exhibit differential staining depending on the developmental stage. E, Magnification of flowers in (D) showing staining in pollen and sepals in more mature flowers. F, Silique from heterozygous parent with staining in funiculi. Scale bars are 1 mm.

Figure 5

Germination and fluoride sensitivity of the AtFEX mutant. A, Germination of seeds from either FEX/FEX (WT) (filled circle) or FEX/fex heterozygous (half-filled circle) plants on 0 NaF or NaF-containing plates. Germination on 0 NaF was set to 100% for each genotype. Each data point represents 40–50 seeds. ***P < 0.001 as determined by one-way ANOVA (Dunnett’s) between WT and heterozygous plants. B, Germination of seeds from a FEX/fex plant on lower concentrations of NaF. Arrested represents seeds where the seed coat was broken and the root emerged, but further growth stalled. Sample size was at least 200 per fluoride treatment. C, Germination of seeds from plants with either WT (closed circle) or OX (closed square) levels of FEX and mutant seeds rescued with AtFEX (half-filled circle). Data points represent replicates of 40–70 seeds each. There was no difference among genotypes at each fluoride concentration as determined by ANOVA (Dunnett’s). D, Germinated seedlings from FEX/fex plants transferred to plates containing 0.25-mM NaF were phenotyped after 10 d and then genotyped. A total of 230 plants were analyzed and the percentage of healthy versus chlorotic for each genotype is represented. In all cases, error bars are SD.

Figure 6

FEX overexpression protects from fluoride toxicity. A, Chlorophyll a concentrations from either WT (FEX/FEX) or OX (FEX/FEX (AtFEX)) 10 d plants grown on media with increasing amounts of NaF. Data points represent a replicate of at least 10 seedlings. One-way ANOVA (Tukey) analysis P-value results between the two genotypes are indicated. Error bars are SD. B, Fresh weights of the WT and OX seedlings. Statistical analysis as in A. C, WT and OX seedlings on media with or without NaF.

Figure 7

Root growth in fluoride is impaired in mutants and restored by AtFEX. A, Seeds from FEX/fex plants, mutant plants (fex/fex), and rescued mutant plants (fex/fex (AtFEX)) were plated on no and low fluoride. The root lengths of 7-d seedlings were measured and then genotyped. B, Root lengths of plants overexpressing AtFEX (line 14 data are the first open box followed by line 31) were compared with those of FEX/FEX (WT) plants. In both panels, one-way ANOVA (Tukey: calculated P-values are **** and ** indicating <0.0001 and <0.01, respectively). Both graphs represent one biological replicate of three total replicates with N ≥ 10. Bars on box plots indicate min and max measurements and the horizontal line indicates the mean.

Figure 8

FEX mutant flowers are sensitive to fluoride and cannot produce viable seed. A, Flowering plants grown on regular soilless substrate Fafard Growing mix #2. Heterozygote FEX/fex plants appear WT and homozygote fex/fex plants have shorter flower stalks. B, Heterozygote flowers were phenotypically identical to WT. C, fex/fex flowers at a later time point were dead. D, Flower sepals on fex/fex mutant were chlorotic. E, Chlorosis in fex/fex cauline leaf tip. F, Stage 13 FEX/fex flower grown under low fluoride conditions (∼2-μM) opened to reveal pollen-shedding stamen. G, Stage 13 fex/fex flower opened to reveal stunted stamen with no pollen shed. Scale bars are 1 mm.

Figure 9

Mutant stamen and pollen show defects from fluoride. A, C, and E show KI-stained pollen from heterozygote, mutant, and rescued plants, respectively. Scale bar is 10-μm. B, D, and F show stamen stained with Alexander’s stain. Viable pollen stains pink and inviable pollen stains blue/green. Scale bar is 50-μm. Each panel (A)–(F) was adjusted to give a uniform black background.

Figure 10

FEX mutant plants accumulate fluoride in the flowers. A, Samples from plants growing on growth substrate with low fluoride (∼2-μM) exhibited continued fluoride accumulation in the flowers of mutants. The top of the bar indicates the mean and error bars are SD. B, A comparison of flowers of plants on two growth substrates Growing mix #2 (∼2-μM fluoride; G) and Nursery mix (∼0.6-μM fluoride; N) demonstrated accumulation in mutants in proportion to the fluoride in the substrate. The horizontal bar indicates the mean. A and B, Each point represents one biological replicate. Lower case letters above data points indicate statistically significant differences P < 0.0001 as calculated by one-way ANOVA (multiple comparisons, Tukey).