Trapping of lead (Pb) by corn and pea root border cells

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1 Plant Soil (2018) 430: REGULAR ARTICLE Trapping of lead (Pb) by corn and pea root border cells David A. Huskey & Gilberto Curlango-Rivera & Robert A. Root & Fushi Wen & Mary Kay Amistadi & Jon Chorover & Martha C. Hawes Received: 29 January 2018 /Accepted: 12 June 2018 /Published online: 29 June 2018 # Springer International Publishing AG, part of Springer Nature 2018 Abstract Aims Most plants produce a root tip extracellular matrix that includes viable border cell populations programmed to disperse into soil. Like neutrophils, border cells export structures that trap pathogens and prevent root tip Responsible Editor: Fangjie Zhao. D. A. Huskey : G. Curlango-Rivera : R. A. Root : F. Wen : M. K. Amistadi : J. Chorover : M. C. Hawes (*) Department of Soil, Water and Environmental Science, University of Arizona, 429 Shantz Building, #38, 1177 E. Fourth St., POB , Tucson, AZ , USA mhawes@ .arizona.edu D. A. Huskey dahuskey@ .arizona.edu G. Curlango-Rivera curlango@ .arizona.edu R. A. Root robroot.az@gmail.com F. Wen fwen@ .arizona.edu M. K. Amistadi amistadi@ag.arizona.edu J. Chorover chorover@ .arizona.edu M. K. Amistadi : J. Chorover Arizona Laboratory for Emerging Contaminants, University of Arizona, 1040 E 4th St, Gould-Simpson 828, Tucson, AZ 85721, USA infection. Border cells also trap metals. The goal of this study was to determine if border cells trap Pb. Methods Border cell responses to Pb were observed microscopically. Border cell impact on Pbinduced injury to roots was assessed using root growth assays. Pb removal from solution was measured using inductively coupled plasma mass spectrometry (ICP-MS). Speciation of Pb associated with border cells was evaluated by synchrotron X-ray absorption spectroscopy (XAS). Results Increased border cell trap size and number occurred within minutes in response to Pb but not silicon (Si). Transient immersion of root tips into Pb after border cells were removed resulted in growth inhibition. Immersion of root tips and border cells into Pb solution resulted in significant removal of Pb. Si levels in the presence of root tips remained unchanged. The Pb speciation, measuredwithpbl III XAS, altered when reacted with border cells, indicating that direct binding by extracellular traps occurred. Conclusions Border cells can trap Pb and prevent damage to the root tip. Keywords Border cells. Extracellular DNA traps. Rhizosphere. Root cap. Rhizofiltration Abbreviations exdna Extracellular DNA ANOVA Analysis of variance DI Deionized Pb Lead

2 206 Plant Soil (2018) 430: ICP-MS Si Introduction Inductively coupled plasma-mass spectrometry Silicon Newly generated, nonlignified plant cells are extremely vulnerable to parasitic invasion and damage by environmental toxins, and yet such cells of the growing root tip generally escape infection and injury (Hawes et al. 2012). This is despite the fact that, as Lynch and Whipps (1990) expressed it, BMicrobial growth in the rhizosphere is stimulated by the continual input of readily assimilable organic substrates from the root.^ The basis for this paradox was a long-standing mystery (Jones et al. 2009; Knudson1919; Odell et al. 2008; Rogers et al. 1942). Studies have now revealed that the organic substrates from the root include living cells, termed root border cells, that can attract and stimulate growth of some microorganisms but stop the movement andgrowthofothers(hawesetal. 1998, 2011, 2016a, 2016b). Border cell populations are programmed to disperse from the root cap as a component of a root tip extracellular matrix, also termed root cap slime (Paull and Jones 1975), root exudates (Baetz and Martinoia 2014), root mucilage (Read and Gregory 1997) and mucigel (Greaves and Darbyshire 1972). This active process can yield up to 98% of the carbon-based exudates from the root (Griffin et al. 1976). However, it is a tightly regulated process in which a genotype-specific number of cells is produced by a dedicated meristem that is distinct from the apical meristem (Hamamoto et al. 2006; Hawes and Pueppke 1986). Once the programmed delivery of cells is complete, cell production ceases and the cells remain appressed to the root surface in the absence of free water, even at 99% humidity. In response to a drop of free water, the matrix swells immediately, the border cells disperse into the root cap slime covering the tip, and renewed cell production begins within minutes (Hawes et al. 2012). A continuous film of free water also is required for movement of microbes toward root tips, but studies have revealed that border cells do not provide a nonspecific source of nutrients for microbial growth (Brigham et al. 1998; Hamamoto et al. 2006). Van Egeraat (1975) was the first to document that the metabolic profile of products from the root tip differs from those of other root tissues. This reflects the fact that border cells exhibit specialized patterns of gene expression that are distinct from progenitor cells of the root cap (Wen et al. 2007; Watson et al. 2015). As with human neutrophils (Brinkmann and Zychlinsky 2004; Papayannopoulos 2017), root border cells are programmed to secrete an array of proteins and extracellular DNA (exdna) that together function as traps to attract and immobilize diverse pathogens in a hostmicrobe specific manner (Hawes et al. 2015, 2016a; Tran et al. 2016; Wen et al. 2009, 2017). As a result, root tips generally escape infections that inhibit root growth by damaging the root meristem (Hawes et al. 2011). When root tips are treated with DNase to degrade exdna traps at the time of inoculation with pathogens, root rot increases from 0 to 100% overnight (Wen et al. 2009). Border cells also trap aluminum (Cai et al. 2008; Hawes et al. 2016b; Miyasaka and Hawes 2001). Removing snapbean border cells from root tips prior to immersion in aluminum results in cessation of root growth and altered root development. Extracellular traps produced by border cells expand in a dosage-dependent manner in response to aluminum. Use of the stain lumogallion in vitro revealed that the metal remained within the mucilage and that uptake into the root was inhibited. Resistant cultivars produced a larger capsule than sensitive cultivars, suggesting that border cells may contribute to quantitative resistance to aluminum damage. Recent studies in animal systems have revealed that neutrophil extracellular traps also are induced in response to aluminum (Stephen et al. 2017). Border cells of diverse plant species now have been reported to react with arsenic, copper, cadmium, iron, nickel, and zinc as well as aluminum (Hawes et al. 2016b; Li et al. 2017; Peng et al. 2016; Srimake and Miyasaka 2015; Sun et al. 2015; Yangetal.2016). Lead (Pb) is among the more toxic and abundant of soil contaminants (Ashraf et al. 2015; Huang et al. 2009; Patra et al. 2004). The primary damage of this toxic metal is inhibition of root growth due to damage to the apical meristem (Fahr et al. 2013). Lead nitrate was found to accumulate in the outer root cap and slime covering the root and cap surface of barley and maize seedlings, but the role of border cells in this phenomenon was not examined (Sobotik et al. 1998). Here we examined predictions of the hypothesis that border cells can protect vulnerable root tips by producing traps that immobilize Pb and thereby prevent its uptake. Pb-root

3 Plant Soil (2018) 430: tip interactions were observed microscopically and were compared with responses to Si, which is classified as a beneficial plant micronutrient. The impact on root growth when root tips with or without border cells were exposed to Pb was measured. Inductively coupled plasma-mass spectrometry (ICP-MS) has been used to measure uptake of lead and other metals into plant tissues including pea roots grown in hydroponic culture (Hanc et al. 2016; Nguyenetal.2017). We used ICP- MS to test the possibility that trapping can be quantified based on removal of metals from solution. In preliminary tests, up to 83% of Pb was removed from a 1 mm solution after one hour in the presence of a single cotton or corn root (Huskey et al. 2017). In the current study, significant removal of Pb from solution occurred in the presence of border cells, and altered speciation of Pb occurred in association with border cells. Materials and methods Plant material Maize seeds (Zea mays L.) cv Golden Bantam (Victory Seeds) were surface sterilized by immersion in 95% ethanol for 5 min followed by 10 min in 0.5% sodium hypochlorite. Seeds were rinsed six times and then soaked for 1 h in sterile deionized water. Pea seeds (Pisum sativum L, cv Little Marvel, Meyer Seed Co., Baltimore MD, USA) were immersed into 95% ethanol for 10 min followed by 30 min in 6.15% sodium hypochlorite, as described (Curlango-Rivera et al. 2010). Seeds with cracks in the seed coat and internal microbial contamination absorb water more quickly than intact seeds and float to the surface where they can be selected and discarded. The remaining seeds were rinsed 5 times with sterile deionized water and then imbibed for 6 h in sterile water. Individual seeds were placed onto 1% water agar overlaid with sterile germination paper (Anchor Paper Company Hudson, WI, USA) and incubated at room temperature (ca 25 o degrees C). Seedlings were used in experiments when radicles were mm in length. Si concentration A 16.6 mmol/l stock solution of Si solution was made following Gao et al. (2013).Briefly,1.0gsilica (SiO2(s), 99.8% Sigma-Aldrich) was added to a 0.1 mol/l NaOH solution, and then rotated end over end for 24 h at 7 rpm. The final ph was adjusted to 8.0 by dropwise addition of 0.1 M HCl. The stock solution was diluted for use in experiments in deionized water (18.2 MΩ, Milli-Q Barnstead). Pb concentration The Pb solution was made from reagent grade Pb(OH) 2 (MP Biochemicals) (Hayes et al. 2012). A 10 mmol stock solution was made by dissolving g of Pb(OH) 2 in 1 L of de-ionized water (18.2 MΩ,Barnsted NanoPure), confirmed to be free of bicarbonate and chloride by carbon and ion chromatography analysis (Shimadzu TOC-L, Columbia, MD; Dionex ICS-1000, Sunnyvale, CA). Aqueous samples and blank solutions were analyzed for dissolved Pb concentrations by inductively coupled plasma-mass spectrometer (ICP-MS; Perkin Elmer Elan DRC-II). Analytical blanks and NIST reference samples were analyzed with the experimental samples. Multi-point standard calibration curves were constructed using NIST traceable premixed standards prior to each analytical run. Correlation coefficients for standard curves were or better and verified against a NIST traceable second source standard with less than ±10% relative difference. Chemical analyses were carried out in the Arizona Laboratory for Emerging Contaminants (ALEC), University of Arizona, Tucson AZ. Border cell responses to Pb and Si Dynamic changes in border cell extracellular structures were measured as described in previous studies (Cai et al. 2008; Curlango-Rivera et al. 2013; Hawes et al. 2012; Miyasaka and Hawes 2001). Individual root tips were immersed in 0.1 ml of sterile distilled water for 2 3 min and border cells were dispersed by gentle agitation prior to adding 0.1 ml Pb (1 mm) or Si (16.6 mm). After incubation for 5 min to 2 h, 10 μlofindiainkwas added and a cover slip was added to the sample. Changes in the number and size of traps on individual cells within populations of at least 100 cells from each of four individual roots in at least four replicate experiments (n 1600 cells) were monitored using an Olympus BX60 F5 compound microscope and a Zeiss Stemi SV8 stereoscope. ANOVA was used to assess variation.

4 208 Plant Soil (2018) 430: Visualization of root tip extracellular matrix interaction with Pb Corn roots were removed from agar plates, and root tips were immersed into water for 60 s without agitation to allow hydration of the matrix. Roots were then gently transferred into 1 mm Pb solution and observed for 2 h using an Olympus BX60 F5 compound microscope and a Zeiss Stemi SV8 stereoscope. To observe the appearance of Pb-treated roots without border cells, root tips were immersed into water for 2 min, and border cells were dispersed by agitation. Their removal was confirmed by microscopic observation. The roots were removed from the water sample and root tips were immersed into Pb solution. Inhibition of root growth by Pb CYG seed germination pouches (Mega International, Newport MN) were used to monitor growth of roots nondestructively, as described in previous studies (Curlango-Rivera et al. 2010; Gunawardena et al. 2005; Hawes et al. 2011; Wen et al. 2007, 2009). Root tips were immersed into water for 2 min to allow hydration of the root tip extracellular matrix. Control roots with border cells were gently transferred to microfuge tubes containing 150 μl of 1 mm Pb solution and incubated for 15 min prior to transfer to growth pouches. Alternatively, border cells were dispersed from root tips by gentle agitation prior to immersion in Pb for 10 min, and then transferred to growth pouches. Root growth was monitored by direct observation for 3 days. ANOVA was used to assess variation. Measurement of Pb and Si in solution Levels of Pb in solutions before and after interaction with roots and border cells were measured using Perkin Elmer Elan DRC II inductively coupled plasma mass spectrometry (ICP-MS) (Shelton, CT) analysis. Quality control solutions were acquired from High Purity Standards (Charleston, SC). Pb speciation Speciation of the Pb in aqueous solution, border cell traps and Pb-bearing reference minerals were investigated with Pb L III edge (E 0 = 13,035 ev) extended X-ray absorption fine structure (EXAFS) spectroscopy at the Stanford Synchrotron Radiation Lightsource (SSRL) on beam line Measurements were made using a liquid nitrogen (LN 2 ) cryostat sample stage operating at ~ 76 K, with a double-crystal Si(220) monochromator and a 100- element germanium fluorescence detector, and 2 mm vertical slits. Three experimental samples were compared: i) unreacted Pb solution control (1 mm), and Pb + border cell traps from ii) 10 corn roots per ml and iii) 10 pea roots per ml. Scans (n = 4) were deadtime corrected and averaged using the SIXpack software package (Webb 2005), and spectra were processes (normalized, background removed, spline fit to the post edge oscillations using Athena and fit using Artemis software suite (Ravel and Newville 2005) by non-linear least square methods on individual atomic shells in k-space using the k-range 2 8. Theoretical phase-shift and amplitude functions were calculated using ATOMS and FEFF9 (Rehr et al. 2009) using atomic clusters of 256 atoms taken from the cerussite structure. Owing to the quality of data and the complexity of Pb L III EXAFS, fits were limited to single scattering paths and fitting ranges were k(å 1 )=2 8and R + Δ(Å) = The minimum resolvable distance between neighboring shells is Δr=π/2k max, therefore the degeneracy of the Pb-O shells <0.19 Å apart were fit as a converged distance, and N ± 33%. Based on fits to known Pb reference compounds the estimated errors in ligand distance were R ± 0.02 Å. The R-factor reported as a goodness of fit parameter had values above 0.02, indicative of the noisy Pb spectra at the L III edge. Results Altered extracellular trap formation in response to Pb As with exdna in bacterial biofilms, exdna-based traps surrounding border cells can be visualized using India ink, which does not penetrate the matrix (Curlango-Rivera et al. 2013; Miyasaka and Hawes 2001). In at least four independent experiments using >100 cells from three different root tips for each replicate, sterile control corn border cells (viability 97 ± 3%) produced traps with an average diameter of 4 ± 2 μm in diameter, with up to 20% having traps of 8 10 μm(9± 4 μm) in diameter (Fig. 1a, b, c). Within 5 min of adding 1 mm Pb to border cells immersed in water, traps expanded markedly (Fig. 2a-d). Up to 80% of cells produced traps >30 μm in diameter(43 ± 10 μm). Cell viability was not significantly different from that of controls (95 ± 4%). Similar results occurred in pea

5 Plant Soil (2018) 430: a 209 b c Fig. 1 Control corn border cells after being immersed into water for 5 min, and then treated with India ink to reveal the dimensions of extracellular structures. a Cluster of nine cells surrounded by a Fig. 2 Pb-treated corn border cells. Border cells were dispersed into water after adding Pb. After 5 min, India ink (10 μl) was added to reveal the dimensions of extracellular structures. a Three cells surrounded by a capsule up to 20 μm in diameter; b Single cell with capsule <50 μm in diameter; c Single cell with capsule >30 μm in diameter; d Single cells with capsules >50 μm in diameter. Size markers: 20 μm capsule up to 5 μm in diameter; b Cluster of four cells with capsule <3 μm in diameter; c Two linked cells with a surrounding capsule up to 5 μm in diameter. Size markers: 20 μm a b c d

6 210 Plant Soil (2018) 430: a Fig. 3 Root tips with and without border cells. a Appearance of corn root tips after immersion into water to hydrate the extracellular matrix surrounding border cells (block arrow). Arrows reveal border cells exposed to Pb: Traps up to 10 μm in diameter (mean 6 ± 4 μm) were present on 10 15% of control populations (viability 94 ± 5%). Within 5 min of adding 1 mm Pb, the percentage of cells with traps >30 μm increased to 75 ± 9%. Extracellular traps in corn and pea border cells treated with Si were not different in size and b the boundaries of the extracellular matrix. b Absence of border cells and associated matrix after gentle agitation of the surrounding liquid to disperse the cell populations. Scale bars: 1 mm number from those in control cells (as in Fig. 1). Viability in corn and pea border cells treated with 16.6 mm Si was 96 ± 3% and 93 ± 5%, respectively, and was not significantly from water controls (95 ± 4%). No changes in viability or appearance of extracellular structures occurred after 2 h in water or in 16.6 mm silicon. a Fig. 4 Altered distribution of Pb particles surrounding root tips a with border cells (block arrow); and b without border cells and associated matrix present when inserted into Pb solution. Blue b arrows indicate the boundaries of particles blocked from entry into the matrix. Scale bars: 1 mm

7 Plant Soil (2018) 430: Root tip extracellular matrix blockage of Pb access to root caps Metal particles that pass through a 0.45-μm filter are considered by the U.S.Geological Survey ( usgs.gov/nawqa/glos.html) to be in solution. Smaller particles can be visualized microscopically, and imaging such particles has been used to localize Pb and track its movement through root systems (Fahr et al. 2013; Samardakiewicz et al. 2012). Upon immersion of root tips into water for 1 2 min,thehydratedmatrixwith enmeshed border cells is evident (Fig. 3a) but disappears in response to gentle agitation of the liquid, leaving the root tip smooth and free of border cells (Fig. 3b). When root tips with border cells were gently transferred from water into a 1 mm Pb solution (Fig. 4a), a line (arrows) of concentrated particles could be seen surrounding the matrix, with no particles within the layer. In contrast, when border cells and the associated extracellular matrix were dispersed by gentle agitation in water prior to immersing into Pb, particles were uniformly distributed up to the root tip surface (Fig. 4b). To measure the potential impact of this phenomenon in protecting the root tip, root tips with border cells (as in Fig. 3a) or without border cells (as in Fig. 3b) were immersed for 15 min in 100 μl of water (Fig. 5a, c) or Fig. 5 Border cell protection of roots from Pb-induced inhibition of root growth. Root length 24 h (a, b) and 72 h (c, d) after transient 15-min immersion of roots with border cells (a, c) and without border cells (b, d)into100μl of Pb. Arrows indicate the location of root tips at time 0 24 hours a er Pb exposure a b 72 hours a er Pb exposure c d

8 212 Plant Soil (2018) 430: Table 1 Root growth after immersion of root tips into Pb (1 mm) or water and transfer to pouches Root length (mm) Roots without border cells Roots with border cells Time after treatment Into water Into Pb Into water Into Pb 24h 27±3 6±2* 26±5 26±6 48 h 44 ± 7 14 ± 3* 48 ± 7 45 ± 5 72 h 75 ± 4 23 ± 4* 78 ± 4 76 ± 8 Each value represents the mean and standard deviation of at least 25 replicate samples in 5 independent experiments. Asterisks denote values in which significant variation (p < 0.05) from control samples was detected 1 mm Pb solution (Fig. 5b, d). Seedlings were transferred into growth pouches containing 15 ml of water and observed for 3 days. Significant growth inhibition of seedlings without border cells immersed into Pb was evident within 24 h (Fig. 4b, Table 1). No discoloration of root tips occurred (Fig. B), and growth of emerging lateral roots progressed without significant changes from water controls (Fig. D, Table 1). Growth of roots with border cells immersed into Pb was not significantly different from that of roots immersed into water only (Table 1). Growth of roots whose root tips with or without border cells were immersed into Si before transfer into pouches was not different from controls. Removal of Pb from solution Significant removal of Pb from solution occurred in all tests using ICP-MS to measure levels before and after incubation with pea or corn roots, with border cells (Table 2-4). In contrast, no removal occurred with 225 ± 23 mg/l silicon, which remained at a level (224.1 ± 10.5 mg/l) that was not significantly different after one hour. Si has been implicated as a beneficial nutrient for plants, suggesting that as with beneficial and pathogenic bacteria, fungi, and nematodes (Hawes et al. 1998), border cell trapping may be selective in response to metals (Ma et al. 2001). In pea, a higher percentage of removal occurred at higher levels of Pb (Table 2). In corn, Pb removal of 22% occurred using a single root with border cells, and 44% removal occurred when two roots with border cells were used (Table 2). Removal in the presence of root tips with border cells (Table 3) or border cells only (Table 4) yielded similar ranges (17% 34 and 10% 17%) with more variation in values. Pb speciation Of particular interest for potential applications in agriculture is exploration of the chemistry of the trapping Table 2 Removal of Pb from solution by pea or corn roots plus border cells Plant Pb control Pb + root+bc % removal Pea 1 root 13,800 ± 161 μg/l 8500 ± 270 μg/l* 38% 5370 ± ± 370* 16% Corn 1 root 8120 ± 80 μg/l 6284 ± 28 μg/l* 23% 9190 ± ± 73* 22% 8822 ± ± 75* 22% 2 roots 8822 ± ± 64* 44% Two roots (20 mm) were immersed into 1 ml of 1 mm Pb or water for 1 3 min. After dispersal of border cells into suspension by gentle agitation, an additional 1 ml of Pb or water was added (1 root/ml). The sample was incubated at room temperature for 1 h, then centrifuged at 7000 rpm for 2 min to pellet the border cells. Centrifugation was repeated if any cells remained in the supernatant, which was then transferred to 9 ml of water to provide a 1:10 dilution for ICP-MS analysis. In the final experiment (above), the same protocol was used except that 2 roots/ml were incubated for 1 h. Each value represents the mean and standard deviation of at least 3 replicate samples. Asterisks denote values in which significant variation (p < 0.05) from control samples was detected

9 Plant Soil (2018) 430: Table 3 Removal of Pb by corn root tips plus border cells Experiment # Pb control Pb + root tip/border cells % removal ± 1.4 μg/l 30.2 ± 0.4 μg/l* 17% ± ± % ± ± 2.6* 20% ± ± 0.7* 34% Two corn root tips were immersed into 100 μl of Pb or water for one minute. After observing the condition of the root tips using a stereoscope, collected border cells were transferred into a 2-ml microfuge tube containing 1900 μl water or Pb, and incubated for 1 h before centrifuging at low speedfor5s.thesupernatantwastransferredtoafreshtube,centrifuged at high speed for 15 s before collecting as much of the liquid as possible without disturbing the border cell pellet. Microscopic examination of three 10 μl samples was used to confirm that the supernatant was cell-free. If border cells were found, centrifugation was repeated before adding 1 ml of the cell-free supernatant to 9 ml of water to make a 1:10 diluted solution to submit for analysis by ICP-MS. Asterisks denote values in which significant variation (p < 0.1) from control samples was detected process and the impact on Pb movement, stability, and structure when it interacts with exdna traps in the soil environment. Here we report results using Pb X-ray spectroscopy to determine the bulk speciation of Pb in experimental samples (Fig. 6). All samples were determinedtobepb 2+ and no change in valence was observed after interaction of 1 mm Pb solution with pea and corn border cells or roots. Quantitative fits of the Pb L III EXAFS supply information about the average near neighbor atomic bonding environment around Pb. The Pb was initially present as a dissolved divalent cation (Pb 2+ (aq)), and was fit to a split first shell of oxygen indicative of apically (R Pb-O =2.36 Å, N =2) and equatorially (R Pb-O =2.70Å,N =5.2)coordinatedPb-O ligands (Table 5). The Pb-O coordination was assumed based on the coordination of Pb and oxygen in cerussite, which has aragonite structure. The EXAFS spectrum of 1.0 mm aqueous Pb solution differed from the corn and pea samples, and indicated that the bonding environment was altered by interaction with the border cells. Table 4 Removal of Pb by corn border cells Experiment# Pb control BC + Pb % removal ± 0.3 μg/l 27.6 ± 1.5 μg/l* 10% ± ± % ± ± % Two root tips were immersed into 100 μl of water for one minute. After observing the condition of the root tip the sample was agitated with a pipette to disperse border cells. Root tips were removed and cell suspensions were transferred into a 2-ml microfuge tube containing 1900 μl of Pb or water for 1 h, then removed prior to submission of the solution for ICP-MS analysis. Asterisks denote values in which significant variation (p <0.1) from control samples was detected Previous results showed similar Pb speciation transformation, observed with linear combination fits of Pb L III EXAFS, upon interaction with rhizosphere organic matter during phytostabilization of Pb contaminated soil with Fagopyrum esculentum (buckwheat) (Hashimoto et al. 2011). Spectral differences resulted from border cell interaction with dissolved Pb (Fig. 6, arrows). The corn-root sample showed two first shell oxygen ligands, an apical Pb-O (R Pb-O =2.42 Å, N = 3) and equatorial Pb-O (R Pb-O =2.64 Å, N = 6.1) coordination, which were slightly elongated in the apical and contracted in the equatorial distances relative to the aqueous solution. The pea-root sample had a similar but slightly shorter first shell oxygen coordination, with apical Pb-O (R Pb- O =2.35 Å, N = 2.6) and equatorial Pb-O (R Pb-O = 2.58 Å, N = 6.5) coordination. The EXAFS determined Pb-O distances in cerussite were longer (R Pb-O =2.57Å, N=2.6andR Pb-O = 2.70 Å, N = 5.2) than in the aqueous and border cell reacted samples. The larger amplitude observed for Pb-O in the Fourier transform is attributed to constructive interference and greater uniformity of the Pb-O distance, FEFF R Pb-O calculated for cerussite = with coordination of N =9. At distances greater than the first shell oxygen backscatters (R > 2.85 Å), border cell samples (and cerussite) showed backscattering attributed to C and Pb, with the Pb-C and Pb-Pb ligands for the border cell samples fit at Å (R Pb-C ) and Å (R Pb-Pb ). Cerussite second shell fits for Pb-C and Pb-Pb were in good agreement with crystal structure models (R Pb-C = 3.06 Å, crystal structure R Pb- C = Å, R Pb-Pb = 4.17 Å, crystal structure R Pb-C = Å). Direct bonding of Pb- C and Pb-Pb is not observed in cerussite and is not predicted in the border cell samples, rather

10 214 Plant Soil (2018) 430: Fig. 6 Lead LIII edge EXAFS and Fourier transforms (FTs) of border cell trap experiments. Solid lines are data; dashed lines are nonlinear least-squares fits (numerical fit results provided in Table 5). Arrows indicate spectral changes caused by the interaction with border cell traps coordination of Pb to the cells/traps through a shared O atom is expected. Best fits to the Pb EXAFS spectra indicated that the Pb is trapped by complexation with an oxygen ligand from an as-yet undetermined R-CO group with a coordination environment different form crystalline cerussite (PbCO 3 ). Discusson Border cell trapping responses to Pb reported here are similar to results in previous studies with aluminum and other metals (Hawes et al. 2016b). The observations implicate border cells in protection of roots as they Table 5 Results of Pb L III edge EXAFS fits Sample Atom N R σ 2* ΔE 0 S 2 * 0 R (Å) (Å 2 ) (ev) factor PbCO 3(s) Pb-O Pb-O Pb-C Pb-Pb Pb 2+ (aq) Pb-O Pb-O Pb-corn (aq) Pb-O Pb-O Pb-C Pb-Pb Pb-pea (aq) Pb-O Pb-O Pb-C Pb-Pb Due to the covariance of coordination numbers (N), the Debye-Waller term (σ 2 ), and the scale factor (S 2 0 ), σ2 and S 2 0 were determined based on fits to pure crystallographic cerussite (PbCO 3 ). N and interatomic distances (R, Å) were iteratively varied, the energy shift parameter (ΔE 0 ) was varied for each sample and fixed for each shell in the least-squares fits, and σ 2 and S 2 * 0 were assigned and 0.7 respectively. Parameter fixed in least-squares fit using value from fits to cerussite. The R-factor is a goodness of fit parameter where R = Σ(data-fit) 2 / Σ(data) 2. The degrees of freedom in the signal were determined by N idp =2ΔkΔR/π, where k = 2 8(Δk = 6) and R =1 4.5 (ΔR = 3.5); the number of variables was not allowed to exceed the degrees of freedom, N idp was 13 and fits were constrained to 10 or fewer variables

11 Plant Soil (2018) 430: move through soil, by immobilizing and preventing uptake of toxic metals including Pb. This could in part explain why efforts to use plants for soil remediation by uptake into root systems have yielded mixed results (Barrow 2017; Fahr et al.2013; Gramss et al.2016; Huang et al. 1997; Iqbal et al. 2015; Kreuzeder et al. 2018; Krzeslowska et al. 2011; Saifullah et al. 2009; Solis-Dominguez et al. 2012; Trebolazabala et al. 2017). The function of border cells in protecting the root tip from toxic chemicals as well as pathogens is of interest in understanding and controlling soil health. Molecular mechanisms underlying host-microbe specificity in trapping of bacteria, fungi and nematodes remain unknown (Curlango-Rivera et al. 2010; Hawes et al. 1998, 2012, 2015, 2016a). Specificity is implicated by the observation that Si does not induce traps and is not removed from solution in the presence of border cells. The potential for using metals as a model to define the mechanism of trapping and its specificity is of interest. Defining such functions could facilitate understanding variable results from studies of Pb uptake into roots (Fahr et al. 2013). For example, species that have been designated as hyperaccumulators based on uptake of metals into the aboveground parts include Arabidopsis, which does not produce populations of viable border cells (Hamamoto et al. 2006). Uptake of arsenic into garden vegetables is inversely correlated with number of border cells produced by different species (Hawes et al. 2016b). Border cell production is a tightly controlled process in which a specific number of cells is produced and remains tightly appressed to the root surface in the absence of free water (Brigham et al. 1998; Hawes et al. 1998, 2016a). As soon as a drop of water is available, cells begin to disperse within seconds at the same time that movement of microbes and metals can begin. Renewed cell production begins immediately under controlled conditions, but factors other than genetics including ph, carbon dioxide levels, compost, and plant metabolites can alter the numbers and dispersal of border cells (Curlango-Rivera et al. 2010; Tollefson et al. 2015; Zhaoetal.2000). Conclusions The ability to disperse border cells into suspension without causing cell death provides a convenient tool to study cellular responses to pathogens and toxins (Hawes and Pueppke 1986; Hawes et al. 1998; Wen et al. 2017). However, agitation and centrifugation to disperse and collect the cells are disruptive tools that may introduce variables that do not reflect dynamics as they occur in the soil. These differences could underlie variability in trapping observed in some experiments in which agitation was used to disperse the cells (Table 2-4), and might reflect a significantly altered potential for Pb removal in the absence of agitation. Further studies to measure in situ changes in border cell responses to metals and microbes as they occur in the soil environment and to determine how they may influence rhizosphere population dynamics are needed (Honeker et al. 2016; Knoxetal.2007; Pengetal.2016; Radmer et al. 2012; Ruangdech et al. 2017). Understanding root defense mechanisms against toxic metals may facilitate efforts to localize and remove them from contaminated soils. Acknowledgements Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The authors thank the reviewers of the manuscript for helpful suggestions to improve the presentation. 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