Metallomics Integrated Biometal Science Accepted Manuscript

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This article can be cited before page numbers have been issued, to do this please use: A. Babst- Kostecka, W. J. Przybyowicz, A. van der Ent, C. G. Ryan, C. C. Dietrich and J. Mesjasz-Przybyowicz, Metallomics, 2019, DOI: 10.1039/C9MT00239A.

Volume 10 Number 4 This is an Accepted Manuscript, which has been through the April 2018 Pages 515-652 Royal Society of Chemistry peer review process and has been Metallomics accepted for publication. Integrated biometal science Accepted Manuscripts are published online shortly after acceptance, rsc.li/metallomics before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available.

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PAPER Bin He, Ligang Hu et al. shall the Royal Society of Chemistry be held responsible for any errors Antibacterial mechanism of silver nanoparticles in Pseudomonas aeruginosa: proteomics approach or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.

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1 2 3 1 Endosperm prevents toxic amounts of Zn from accumulating in the seed embryoView Article Online 4 DOI: 10.1039/C9MT00239A 5 2 6 – an adaptation to metalliferous sites in metal-tolerant Biscutella laevigata 7 3 8 9 4 10 1 2,3 4,5 6 11 5 Alicja Babst-Kostecka , Wojciech J. Przybyłowicz , Antony van der Ent , Chris Ryan , Charlotte 12 6 Dietrich1, Jolanta Mesjasz-Przybyłowicz3 13 14 7 15 16 8 1 W. Szafer Institute of Botany, Polish Academy of Sciences, Department of Ecology, Lubicz 46, 31- 17 18 9 512 Krakow, Poland. 19 10 20 21 11 2AGH University of Science and Technology, Faculty of Physics & Applied Computer Science, 22 23 12 al. Mickiewicza 30, 30-059 Kraków, Poland. 24 25 13

26 14 3Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Manuscript 27 28 15 Matieland 7602, South Africa. 29 30 16 31 17 4Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, 32 33 18 The University of Queensland, Australia. 34 35 19 36 Accepted 5 37 20 Laboratoire Sols et Environnement, Université de Lorraine, France. 38 21 39 40 22 6CSIRO, Mineral Resources, Australia. 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 23 43 44 24 45 25 46 47 26 48 Metallomics 49 27 Corresponding author: 50 28 51 52 29 Alicja Babst-Kostecka 53 54 30 Tel: +48 12 424 17 04, Fax: +48 12 421 97 90 55 56 31 e-mail: [email protected] 57 32 58 59 33 60

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1 2 3 View Article Online 4 34 Table of contents entry DOI: 10.1039/C9MT00239A 5 6 35 7 36 The pseudometallophyte Biscutella laevigata adapts to metalliferous soils by allocating excess 8 9 37 metal(loid)s to the endosperm (E) of seeds to protect embryonic tissues and improve reproductive 10 11 38 success. 12 39 13 40 14 MiSceroedscpohpoytoSmeiecrdogPriacptuhre SSeeeeddSschematiicc XFXMFM SeseededPmicatupre 15 0.6 16 Ca 0.5 17 E 0.4 18 Hi 19 C 0.3 20 0.2 21 T R 22 0.1 Hy 23 1000 μm wt% 24 41 25

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1 2 3 42 ABSTRACT View Article Online 4 DOI: 10.1039/C9MT00239A 5 43 Seed germination represents the first crucial stage in the life cycle of a plant, and the seed must 6 7 44 contain all necessary transition elements for the development and successful establishment of the 8 45 seedling. Problematically, seed development and germination are often hampered by elevated 9 10 46 metal(loid) concentrations in industrially polluted soils, making their revegetation a challenging task. 11 12 47 Biscutella laevigata L. (Brassicaceae) is a rare perennial pseudometallophyte that can tolerate high 13 14 48 concentrations of trace metal elements. Yet, the strategies of this and other plant species to ensure 15 49 reproductive success at metalliferous sites are poorly understood. Here we characterized several 16 17 50 parameters of germination and used synchrotron X-ray fluorescence microscopy to investigate the 18 19 51 spatial distribution and concentration of elements within B. laevigata seeds from two metallicolous 20 21 52 and two non-metallicolous populations. We find that average germination time was shorter and the 22 53 seed weight was lower in the metallicolous compared to the non-metallicolous populations. By 23 24 54 allowing for at least two generations within one growth season, relatively fast germination at 25 55 metalliferous sites accelerates microevolutionary processes and likely enhances the potential of 26 Manuscript 27 56 metallicolous accessions to adapt to environmental stress. We also identified different strategies of 28 29 57 elemental accumulation within seed tissues between populations. Particularly interesting patterns 30 31 58 were observed for zinc, which was found in 6-fold higher concentrations in the endosperm of 32 33 59 metallicolous compared to non-metallicolous populations. This indicates that the endosperm protects 34 60 the seed embryo from accumulating toxic concentrations of metal(loid)s, which likely improves 35 36 61 reproductive success. Hence, we conclude that elemental uptake regulation by the seed endosperm is Accepted 37 38 62 associated with enhanced metal tolerance and adaptation to metalliferous environments in B. 39 40 63 laevigata. 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 64 42 43 65 Significance to Metallomics statement 44 45 66 Plants can only establish in metalliferous environments, if their reproduction is successful. Seeds 46 47 67 must therefore be protected from intoxication by excess metal(loid)s that are abundant in soils at Metallomics 48 68 industrial legacy sites. This study visualizes, quantifies, and compares the distribution of various 49 50 69 elements in Biscutella laevigata seeds from metalliferous and natural habitats. Interestingly, we 51 52 70 found that this species has developed a strategy to allocate zinc and other elements to non-harmful 53 54 71 sections of the seed, using them as a barrier to prevent the intoxication of sensitive parts. This 55 72 enhances our knowledge of how plants can adapt to and tolerate soil pollution. 56 57 58 59 60

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1 2 3 74 1. INTRODUCTION View Article Online 4 DOI: 10.1039/C9MT00239A 5 75 The global industrial revolution has led to an unprecedented release of toxic substances into 6 1 7 76 the environment . The far-reaching consequences of this pollution include soil contamination with 8 77 hazardous waste, which threatens environmental and human health around the world. Among 9 10 78 pollutants, trace metal elements (including arsenic, As, cadmium, Cd, zinc, Zn, lead, Pb, and 11 12 79 thallium, Tl) are of major concern. Negative impacts can arise from direct contact with polluted soil 13 14 80 or ground water, or from ingestion via the food chain (soil-plant-human or soil-plant-animal-human), 15 81 reduction in food quality, and food insecurity resulting from reduced soil fertility and agricultural 16 17 82 production 2-4. Unlike organic contaminants, trace metal elements do not undergo microbial or 18 19 83 chemical degradation and may persist at elevated concentration in soils for a long time after their 20 5 21 84 dissemination . This is particularly problematic in soils in the vicinity of metalliferous mining and 22 85 smelter sites, where trace elements are continuously accumulated upon release 6. Hence, there is a 23 24 86 growing demand for cost-effective and environmentally friendly technologies to remediate 25 87 contaminated sites 7. Revegetation has drawn special attention as a promising “green and clean 26 Manuscript 27 88 technology” for intervention to toxic exposures 8. Plant establishment on mine tailings not only 28 29 89 mitigates hazards associated with wind dispersal of local contaminated dust, but every square meter 30 31 90 of vegetation can effectively remove up to 1 kg of dust per year from the air that moves across the 32 9 33 91 planted region . A lasting plant cover also helps immobilizing contaminants in the ground, with 34 92 positive effects on ground water quality 10, and provides important ecosystem services such as 35 36 93 carbon sequestration, intensified water cycling, and habitat for numerous species. Problematically, Accepted 37 38 94 most plants are sensitive to high soil trace metal element concentrations, which often inhibit seed 39 11-13 40 95 germination and plant growth on mine tailings . 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 96 Only a limited number of vascular plant species called ‘metallophytes’ have developed the 42 43 97 ability to survive and reproduce in toxic metalliferous environments 14, 15. This unusual characteristic 44 16, 17 45 98 has been defined as metal tolerance . While non-metallophyte species may to some extent also 46 99 tolerate elevated metal concentrations in soils, only metallophytes possess physiological mechanisms 47 48100 that allow them to cope with very high concentrations that cause toxicity. Two contrasting Metallomics 49 50101 physiological strategies have thereby evolved: plants are either ‘excluders’ that restrict trace metal 51 52102 element allocation to aerial parts by limiting root uptake and/or transport through the stem, or they 53103 are ‘(hyper)accumulators’ that allocate extraordinarily large amounts of trace metal elements to their 54 55104 shoots. Plants that pursue a third, intermediate strategy are called ‘indicators’ and seek a proportional 56 14, 18 57105 relation between elements in soil and in plants . 58106 While trace metal element allocation to foliage has been extensively studied in metallophytes, 59 60107 only a very limited number of investigations have focussed on the elemental distribution in their

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1 2 3 108 seeds (e.g. ref. 19-24 ) and most studies to date have focussed on nickel (Ni), Zn View and Article Cd Online 4 DOI: 10.1039/C9MT00239A 5 109 hyperaccumulator plants 25-30. Yet, more research of this kind is urgently needed because plant 6 7 110 establishment at post-mining sites (and elsewhere) critically depends on successful seed germination 8 111 and early seedling growth 31. 9 10112 A general presumption is that metallophytes must keep their seeds free of toxic trace metal 11 12113 concentrations to provide their offspring with a ‘fresh start’ on metalliferous soils 32. However, some 13 14114 transition elements are necessary in the seed to ensure the development of the seedling. Trace metal 15115 element uptake, distribution and concentration must therefore be carefully regulated to reduce the 16 17116 toxicity risk. Importantly, it becomes more and more evident that not only environmental conditions, 18 19117 but also the species demographic history and pre-adaptation of plant populations play a role in plant 20 21118 tolerance and sensitivity to unfavourable soil conditions during these first two crucial life stages. 22119 Accordingly, plant populations originating from specific habitats may perform better or worse on a 23 24120 given metalliferous site than those from other habitats 33-36. This calls for comprehensive, 25 121 quantitative investigations of the variation in the elemental distribution in the seeds of metallophytes 26 Manuscript 27122 to gain insight into plant adaptation to metal contaminated soils. Pseudometallophytes, i.e. taxa with 28 29123 populations both on and off metalliferous soils, are of particular research interest because extreme 30 31124 environmental conditions promote rapid differentiation between their metallicolous (M) and non- 32 37-39 33125 metallicolous (NM) populations . 34126 Biscutella laevigata L. (Brassicaceae) is a perennial pseudometallophyte, that is widespread 35 36127 across Central and Western Europe 40, 41. Its distribution range reaches its northern limit in Poland, Accepted 37 38128 where the species is restricted to only a few known localities, mostly on non-metalliferous sites in 39 40129 the Western Tatra Mts, but also on calamine waste heaps in the Olkusz region. Metal tolerance is 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 130 present in all known Polish B. laevigata populations and is further enhanced in metallicolous 42 43131 populations in response to stress from high trace metal element concentration in soils 42. Thus, 44 45132 populations from natural and anthropogenic locations from Southern Poland have adapted to 46133 different environmental conditions and have thereby genetically diverged 38. Due to these clear 47 48134 divergence patterns, Polish metallicolous and non-metallicolous populations of B. laevigata Metallomics 49 50135 represent particularly interesting material to study plant adaptation to trace metal element polluted 51 52136 environments. In terms of elemental allocation patterns in B. laevigata seeds, metallicolous plants 53137 were reported to strictly and actively select elements and their amounts taken up by different seed 54 55138 tissues 20. However, because no comparison with seeds from unpolluted sites is yet available, the 56 57139 association of these interesting patterns to adaptation to metalliferous environments remains 58140 unclear. 59 60

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1 2 3 141 In this study, we investigated two metallicolous and two non-metallicolousView Article B. Online 4 DOI: 10.1039/C9MT00239A 5 142 laevigata populations that have previously been identified as the most and the least trace metal 6 42 7 143 element tolerant accessions from Southern Poland . We examined several reproductive traits (e.g. 8 144 germination rate, average germination time, seed weight) and investigated the spatial distribution and 9 10145 concentration of elements within B. laevigata seeds from these four populations in the context of 11 12146 adaptation to metalliferous environments. For the latter, we employed synchrotron X-ray 13 14147 fluorescence microscopy (XFM). The XFM approach offers several unique capabilities that are of 15148 interest to plant scientists, including the highly sensitive detection of most trace elements and a fine 16 17149 spatial resolution 24, 43. The specific aims of this study were to i) characterize the parameters of 18 19150 germination, ii) investigate the elemental composition of B. laevigata seeds, and iii) compare 20 21151 patterns of elemental distribution and concentration among populations growing on anthropogenic 22152 and natural habitats. This is to verify, if metallicolous plants have evolved a strategy to combat 23 24153 trace metal element stress at the seed level. By addressing these points, our study provides new 25 154 insight into plant adaptation to restrictive environments. At the same time, we aim to provide a better 26 Manuscript 27155 understanding of the mechanisms that underlie the physiology of trace metal element tolerance at the 28 29156 seed developmental stage. 30 31157 32 33158 MATERIALS AND METHODS 34159 35 36160 Study sites and plant material Accepted 37 38161 The sampling included two metalliferous (M_PL2 and M_PL6) and two non-metalliferous 39 40162 (NM_PL8 and NM_SK14) locations of B. laevigata at its north-eastern distribution limit. Both 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 163 metalliferous sites are located on lowland waste heaps and dust deposits in the heavily contaminated 42 43164 post-mining area of Olkusz in Southern Poland. Total and extractable Zn soil concentration at site 44 -1 45165 M_PL2 was 16 580 and 34 200 µg g , respectively. At site M_PL6, the corresponding values were 46166 51 140 and 5920 µg g-1 (Table 1). The NM_PL8 site is located in a meadow at the Tatra Mts 47 48167 foothills, whereas NM_SK14 grows on a calcareous slope of the Western Tatra Mountains. The Metallomics 49 50168 metal concentration in non-metalliferous soils was considerably lower, ranging between 50 and 100 51 -1 -1 52169 µg g for total Zn and 20-70 µg g for extractable Zn. At each site, ripe seeds from ten B. laevigata 53170 mother plants were sampled separately at a minimum distance of 3 m to avoid clonal repetition 44. 54 55171 56 57172 Germination test and reproductive traits 58173 Thirty seeds were randomly selected from each population and germinated in plastic 59 60174 containers (25 x 15 x 5 cm) filled with a sterile, mixed perlite:vermiculite (4:1, v:v) substrate. Seeds

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1 2 3 175 were sown to a depth of ~1 cm and the distance among the seeds was 5 cm. Three containersView Article per Online 4 DOI: 10.1039/C9MT00239A 5 176 population were randomly arranged in a controlled growth chamber with the following conditions 42: 6 7 177 photoperiod: 13 h day and 11 h night; temperature: 20 °C day and 15 °C night; relative humidity: 8 178 65%; and irradiance: 300 μmol photons m−2s−1. Seed germination was determined by visual detection 9 10179 of seedling emergence and was recorded daily until no seeds germinated for 3 days, which was the 11 12180 case after 27 days. The fresh biomass of seedlings was measured 5 days after germination. 13 14181 Additionally, from each population the seed weight [mg] was determined for 30 independent seeds 15182 from two randomly selected B. laevigata mother plants (n = 30 seeds x 8 plants). One seed from each 16 17183 of the eight mother plants was then selected for X-ray fluorescence microscopy and longitudinal 18 19184 sections of the middle parts of air-dried ripe seeds were adhered to Kapton (polyimide) tape mounted 20 21185 on specimen holders. 22186 23 24187 Synchrotron X-ray Fluorescence Microscopy (XFM) 25 188 The X-ray fluorescence microscopy (XFM) beamline of the Australian Synchrotron employs 26 Manuscript 27189 an in-vacuum undulator to produce a brilliant X-ray beam of 4.1–20 keV with a focus down to 28 29190 ~1000 nm. An Si(111) monochromator and a Kirkpatrick-Baez (K/B) pair of mirrors delivers a 30 31191 monochromatic focused beam onto the specimen 45. The beamline is equipped with a Maia detector, 32 33192 which uses a large detector array to maximize the detected signal and count-rates for efficient 34193 imaging. Maia enables high overall count-rates and uses an annular detector geometry, where the 35 36194 beam passes though the detector and strikes the sample at normal incidence 46, 47. This enables a large Accepted 37 38195 solid-angle (1.2 steradian) to be achieved in order to maximize the detected signal and consequently 39 48 40196 to reduce the dose and potential damage to a specimen . Maia is designed for event-mode data 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 197 acquisition, where each detected X-ray event is recorded, tagged by detector number in the array, 42 43198 position in the scan, and other metadata (Ryan et al. 2014). 44 45199 46200 Data processing and statistics 47 48201 The differences in mean germination rate, germination time, and seed weight were assessed Metallomics 49 50202 using the Kruskal-Wallis test. The XFM data was quantitatively processed using the Dynamic 51 49 52203 Analysis method . This method generates elemental images, which are (i) overlap-resolved, (ii) 53204 with subtracted background and (iii) quantitative, i.e. in μg g-1 dry weight units. The Compton scatter 54 55205 maps were used to correct for local areal density variations. Elemental concentrations from these 56 -1 57206 areas are also reported in μg g dry weight. The error estimates were extracted from the error matrix 58207 generated in the fit and the minimum detection limits (MDL) were calculated using the Currie 59 60208 equation 50. Maps were complemented by data extracted from arbitrarily selected regions of interest

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1 2 3 209 (ROIs) within scanned seeds, representing specific seed parts. Six ROIs were selected basedView on Article the Online 4 DOI: 10.1039/C9MT00239A 5 210 seed morphological structures (Figure 1). Accordingly, besides the whole seed section, we 6 7 211 distinguished the testa, hilum, endosperm, radicle, hypocotyl, and cotyledon. For few results below 8 212 the MDL, one-half of the reported limit was used for the purpose of statistical analysis 51. The 9 10213 differences in mean concentration of elements between ecotypes at the whole-seed cross-section 11 12214 level and for individual ROIs were assessed using the Wilcoxon signed-rank test. At the ecotype 13 14215 level, the differences in mean concentration of elements between seed tissues were assessed using 15216 the Kruskal-Wallis test. 16 17217 18 19218 RESULTS & DISCUSSION 20 21219 The adaptation of plants to extremely harsh conditions at metalliferous sites affects 22220 reproductive traits and eventually leads to the evolution of metal tolerant ecotypes 52-54. Our study 23 24221 identified important differences between metallicolous and non-metallicolous B. laevigata ecotypes 25 222 that may contribute to this process and greatly impact the colonization of and subsequent survival at 26 Manuscript 27223 metalliferous sites. 28 29224 The germination rate varied broadly between populations, with the lowest values in 30 31225 NM_SK14 (53.3%) and the highest values in M_PL6 (76.6%; Table 2 and Figure 2). All populations 32 33226 showed average germination times above 180 hours (Table 2). Population M_PL2 was characterized 34227 by more rapid and uniform germination than the other three populations. The average germination 35 36228 time is considered to be a good measure of the speed with which a species can occupy a certain Accepted 37 38229 environment. It can be classified into three general categories: rapid (< 120 hours), intermediate 39 55 40230 (between 120 and 240 hours), and slow (> 240 hours) . Accordingly, the germination time of B. 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 231 laevigata seeds from metallicolous accessions falls into the intermediate category, whereas 42 43232 germination of seeds from non-metallicolous plants is slow. Recent studies have indicated that slow 44 45233 and heterogeneous germination times may be associated with species that occur in alpine 46234 environments with spatially variable and temporally unstable conditions56, 57. Specifically, seeds that 47 48235 are dispersed in such habitats do not always find favourable conditions immediately, due to e.g. low Metallomics 49 50236 temperatures and short growing seasons. Thus, slow and temporally distributed germination of B. 51 52237 laevigata seeds in mountain habitats is likely part of a germination strategy that promotes successful 53238 recruitment of new individuals in an unpredictable climate. By contrast, relatively fast germination 54 55239 and more stable climatic conditions at the investigated metalliferous sites provide these lowland B. 56 57240 laevigata accessions with at least two generations within one growing season (A. Babst-Kostecka, 58241 personal observation). A similar strategy has been reported for A. arenosa, Silene vulgaris, and 59 60242 Rumex dentatus, and can be associated with greater potential of metallicolous accessions to adapt to

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1 2 3 243 environmental stress at metalliferous sites, i.e. through an increased rate of microevolutionaryView Article Online 4 DOI: 10.1039/C9MT00239A 5 244 processes 58-60. 6 7 245 Seed mass is another important parameter that ensures effective and prolific reproduction of 8 246 plants in metalliferous soils. Being positively correlated with seedling survival, this trait is 9 10247 considered to be critical for the adaption of plants to unfavorable environments 61. Indeed, seed mass 11 12248 is the most realistic measure of plant generative processes in response to metal exposure 62. In plants 13 14249 that colonize metalliferous sites, reproduction often increases at the expense of vegetative 15250 development 37, 63, 64. Thus, vegetative parts are minimized, but flowers and seeds are usually normal 16 17251 in size or even bigger than in plants at non-metalliferous sites. Accordingly, heavier seeds in 18 19252 metallicolous compared to non-metallicolous populations have been reported, e.g., in natural 20 58 65 21253 populations of A. arenosa and Nocceae caerulescens . In the present study, we found that B. 22254 laevigata populations significantly differed in seed weight, however, non-metallicolous plants had on 23 24255 average 1.7-fold heavier seeds (0.029 ± 0.007 mg) than metallicolous plants (0.017 ± 0.004 mg; 25 256 Figure 3). This lower reproductive biomass allocation in metallicolous B. laevigata can be associated 26 Manuscript 27257 with costs of tolerance. Indeed, given that metal tolerance in B. laevigata is constitutive 42, our 28 29258 findings are consistent with the theory that elevated concentration of trace metal elements in soil 30 31259 increases maintenance costs because an organism needs to spend energy to counterbalance their 32 66 33260 potentially toxic effects . Such a trade-off between trait and environment can increase survival 34261 under stress conditions, but leaves less energy for growth, reproduction and/or other processes 4, 36, 39. 35 36262 Importantly, B. laevigata can reproduce via vegetative propagation and thereby occupy habitats with Accepted 37 38263 genetically identical individuals that are best suited for local conditions. Our results suggest that the 39 40264 latter strategy may play an important role in the colonization of and establishment at unfavourable 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 265 metalliferous sites by B. laevigata. While the trade-offs between investment in vegetative vs sexual 42 43266 reproduction at metalliferous sites may limit resource allocation to seed production, new genotypes 44 67 45267 still need to be introduced into the population to ensure diversity of the genetic pool . Thus, a 46268 sufficient amount of seeds needs to be produced to enable populations to cope with environmental 47 48269 heterogeneity and ensure species survival. In this context, knowledge of the elemental composition Metallomics 49 50270 of seeds is essential for better understanding the processes and strategies that underlie successful 51 68 52271 sexual reproduction in plants . 53272 The XFM analysis detected the following macro- and micro-nutrients in B. laevigata seeds: 54 55273 Zn, sulphur (S), chlorine (Cl), potassium (K), calcium (Ca), manganese (Mn), iron (Fe) and copper 56 57274 (Cu) (Table 3). Other elements whose concentrations did not reach the minimum detection limit in 58275 some tissues, e.g. chromium (Cr), cobalt (Co), Ni, and As, are shown in Supplementary Table S1. 59 60276 Strong and diverse patterns of elemental distribution and concentrations were observed at the

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1 2 3 277 ecotype and population levels (Table S2), and considerable differences were also observed Viewbetween Article Online 4 DOI: 10.1039/C9MT00239A 5 278 specific seed sections (Figures 4 and 5; Table S3). The most notable results were observed for Zn 6 7 279 (Figure 4). Zinc is an essential element that needs to be transported to the developing seeds; 8 280 however, the mechanisms regulating its allocation to specific seed tissues are largely unknown. In 9 10281 our study, the highest Zn concentrations were found in the endosperm of seeds from metallicolous 11 12282 populations (up to 584 µg g-1 in M_PL6). These concentrations were significantly (p = 0.029; 13 14283 Supplementary Table S2) – on average 6-fold – higher than the concentrations in the endosperm of 15284 seeds from non-metallicolous populations. The second region that exhibited differences between the 16 17285 two ecotypes in terms of accumulated Zn was the hilum, with on average 4-fold higher Zn content in 18 19286 metallicolous than in non-metallicolous samples (except for one sample in NM_SK14). Other tissue 20 21287 types contained rather similar amounts of Zn in material from both edaphic origins, with the lowest 22288 values predominantly found in testa (2.2–38.0 µg g-1) and cotyledon (15.1–46.4 µg g-1). Within 23 24289 embryonic tissues, the highest Zn concentration was found in the radicle, independent of plant 25 290 edaphic type. By preventing the accumulation of toxic concentrations of metal(loid)s within the seed 26 Manuscript 27291 embryo, mother plants ensure reproductive success 32. Accordingly, the uptake of Zn is known to be 28 29292 restricted and previous studies have shown species-specific Zn distribution patterns within seeds. For 30 31293 instance, in seeds of the metal hyperaccumulator Thlaspi praecox, Zn was mainly allocated to 32 26 33294 cotyledons and epidermis . This seems to be a protective strategy for the seeds, as cotyledons are 34295 rapidly discarded by most plants at an early developmental stage. By contrast, Mesjasz-Przybyłowicz 35 36296 et al. (2001) found the highest amount of Zn in the endosperm of metallicolous B. laevigata seeds. Accepted 37 38297 The authors suggested that the endosperm acts as a barrier against the transport of toxic amounts of 39 40298 elements into embryonic parts. In metalliferous environments, due to the potential toxicity of excess 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 299 levels of metal(loid)s, carefully regulated delivery of elements from the mother plant into seeds is 42 43300 essential to prevent seed inhibition and subsequent negative effects on seed germination 69. The 44 45301 pronounced differences in Zn concentration between seeds from metallicolous and non-metallicolous 46302 B. laevigata populations revealed in our study – in particular primary Zn allocation to the endosperm 47 48303 in plants exposed to highly elevated Zn content in soil – further suggest that uptake regulation by the Metallomics 49 50304 endosperm is indeed associated with metal tolerance and adaptation to metalliferous environments. 51 52305 Moreover, Zn acquisition by different seed tissues appears to be controlled in a population-specific 53306 manner, indicating the importance of local microevolutionary processes. 54 55307 The endosperm was also a primary region of accumulation for K, Ca, Cl, Mn, Fe and Cu in 56 57308 the investigated B. laevigata seeds (Figure 6). Furthermore, we observed element-specific allocation 58309 patterns to other tissues as described in the following paragraphs: 59 60

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1 2 3 310 The range of observed K and Ca concentrations was rather narrow in radicle, hypocotylView Article and Online 4 DOI: 10.1039/C9MT00239A 5 311 cotyledon, whereas it was much broader in testa, hilum, and endosperm. For these two elements, we 6 7 312 did not observe any ecotype-specific trends. Limited differences in K and Ca concentrations between 8 313 seed tissues were previously shown for A. thaliana 70. Moreover, a remarkably small influence of 9 10314 plant type (e.g. wild type vs mutant plants) on K and Ca allocation patterns emphasized a common 11 12315 trend towards abundant storage of both elements in seeds in preparation for germination 70. 13 14316 Independent of plant origin, a clear and unique pattern was found for S, which was 15317 predominantly allocated to the radicle (29 880–40 770 µg g-1), hypocotyl (22 670–36 450 µg g-1) and 16 17318 cotyledon (19 560–26 230 µg g-1). Considerably lower S concentrations (<850–8850 µg g-1) were 18 19319 found in testa, hilum and endosperm. Accordingly, S was the only element that formed two distinct 20 21320 groups of ROIs, with no overlap between those groups. Its primary storage in embryonic tissues 22321 supports successful seedling establishment and early plant development 71. 23 24322 Several other elements revealed ecotype-specific allocation patterns. The concentration of Cl 25 323 was on average twice higher in all specific ROIs of metallicolous compared to non-metallicolous 26 Manuscript 27324 seeds, except for testa where it was even 4-times higher. Due to relatively high variability among 28 29325 samples, the differences between ecotypes were slightly above the significance level of 0.05 (p = 30 31326 0.057 for the whole seed section, radicle and cotyledon; Supplementary Table S2). In terms of Cu, 32 33327 concentrations were also higher in samples from metallicolous compared to non-metallicolous 34328 origin. The biggest differences between ecotypes appeared in endosperm, radicle, and cotyledon, 35 36329 where Cu was on average twice higher in metallicolous plants (except for one NM_SK14 sample that Accepted 37 38330 showed very high Cu in hilum, endosperm and radicle). As for Mn, we found the most interesting 39 40331 pattern in endosperm, where its concentration was 3-fold higher in metallicolous compared to non- 41 -1 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 332 metallicolous samples (p = 0.029). One particularly high value (254 µg g ) emerged from a sample 42 43333 from the M_PL6 population. Interestingly, other anomalously high values of 108 µg g-1 44 45334 characterized also the hilum of single samples from both non-metallicolous populations. These latter 46335 concentrations were 5-fold higher than the average Mn concentration in hilum of metallicolous 47 48336 samples. Manganese storage in seed tissues is interlinked with normal seedling growth, development, Metallomics 49 50337 and its vigour index 72. By increasing the Mn content in seeds, plants are able to increase grain yield 51 73 52338 . 53339 Overall, the three elements addressed above (Cu, Mn, and Cl) were predominantly allocated 54 55340 to the endosperm, with considerably lower (on average 5-, 4- and 2-fold, respectively) concentrations 56 57341 in the remaining ROIs. Exceptions were one metallicolous plant with an equally high Cl 58342 concentration in testa as in endosperm, and two non-metallicolous plants for which Mn 59 60

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1 2 3 343 concentrations in the hilum exceeded those in endosperm). By comparison, in T. praecoxView Article and Online 4 DOI: 10.1039/C9MT00239A 5 344 Arabidopsis thaliana seeds, Cu was most abundant in radicle, cotyledon and seed coat 26, 74, 75 6 7 345 Distinct distribution patterns were observed for Fe. While in radicle, hypocotyl and 8 346 cotyledon, Fe concentration was rather similar in all samples, extremely high values were observed 9 10347 in testa (114.0 and 151.0 µg g-1), hilum (279 µg g-1) and endosperm (297 µg g-1) of individual seeds 11 12348 from non-metallicolous populations. Overall, this element was present in all samples and structures, 13 14349 mostly in the embryonic parts, and its distribution was clearly linked to the provascular network 15350 (Figure 5). Similar Fe distribution patterns were reported for Silene vulgaris 21 and A. thaliana seeds 16 17351 75-77. Transport of Fe into the provascular network has been shown to be mediated by VIT1, an 18 19352 ortholog of the vacuolar metal transporter CCC1 in yeast, based on complete loss of Fe enrichment 20 78 21353 around provascular strands in vit1 knock out mutants . Allocation of Fe to provascular tissues 22354 allows rapid mobilization of this element for the growing parts of the seedling during germination, 23 24355 thus it supports metabolic processes such as photosynthesis 79, 80. In germinating seeds, the staining 25 356 of Fe around provascular strands disappears during the first three days and thereafter, root Fe 26 Manuscript 27357 acquisition takes over 81. 28 29358 30 31359 CONCLUSION 32 33360 The seed is simultaneously the final sink, as well as the first source of nutrients in the life 34361 cycle of annual plants and their offspring. Whereas roots and foliage of mature plants have been 35 36362 widely studied, elemental transportation to the seed and allocation patterns within the seed are still Accepted 37 38363 poorly understood. In this study, we visualized and quantified a range of elemental allocation 39 40364 patterns in the metal-tolerant pseudometallophyte B. laevigata from both metalliferous and non- 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 365 metalliferous sites. The patterns that we observed – especially the enhanced function of endosperm 42 43366 as a barrier against Zn excess in the embryonic parts of seeds on polluted sites – provides deep 44 45367 insight into plant adaptation to metalliferous environments and, by extent, the evolution of metal 46368 tolerance at the seed developmental stage. Yet, several open questions remain regarding the selective 47 48369 pressure(s) that drive the evolution of metal tolerance. Answering these questions will require Metallomics 49 50370 transplant experiments to investigate, how tolerance affects the fitness in heterogeneous 51 52371 environments. The mechanistic understanding of processes underlying the optimization of 53372 metal(loid) concentration and localization in seeds is relevant for further investigations on the 54 55373 remediation of polluted mine tailings, and for the improvement of plant stress tolerance in general. 56 57374 58375 ACKNOWLEDGEMENTS 59 60

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1 2 3 376 This research was undertaken on the X-Ray Fluorescence Microscopy beamline of the AustralianView Article Online 4 DOI: 10.1039/C9MT00239A 5 377 Synchrotron, Victoria, Australia. We thank Martin de Jonge (ANSTO) and Hugh Harris (University 6 7 378 of Adelaide) for support during the synchrotron experiment, Aneta Słomka (Jagiellonian University) 8 379 for discussions on seed anatomy, and Kamila Murawska and Szymon Miszczak (Institute of Botany 9 10380 PAS) for technical support. This work was supported by the Multi-modal Australian ScienceS 11 12381 Imaging and Visualisation Environment (MASSIVE), the POWROTY/REINTEGRATION 13 14382 programme of the Foundation for Polish Science co-financed by the European Union under the 15383 European Regional Development Fund (POIR.04.04.00-00-1D79/16-00), and statutory funds from 16 17384 the W. Szafer Institute of Botany PAS. W.J. Przybylowicz and J. Mesjasz-Przybylowicz are 18 19385 recipients of the South African National Foundation incentive grants No 114693 and 114694, 20 21386 respectively. The Tatra National Park granted permits to collect samples within the park boundaries. 22387 23 24388 AUTHOR CONTRIBUTIONS 25 389 ABK planned and designed the research. AVDE, JM-P and WJP performed the measurements. CR, 26 Manuscript 27390 JM-P, WJP, CD processed the image analyses. ABK, JM-P and CD analysed the data. ABK wrote 28 29391 the manuscript with contributions from all authors. 30 31392 32 33393 REFERENCES 34394 1. Millennium Ecosystem Assessment, Ecosystems and Human Well-being: Synthesis, Island 35 36395 Press, Washington, DC, 2005. Accepted 37396 2. M. J. McLaughlin, B. A. Zarcinas, D. P. Stevens and N. Cook, Soil testing for heavy metals, 38397 Communications in Soil Science and Plant Analysis, 2000, 31, 1661-1700. 39398 3. W. Ling, Q. Shen, Y. Gao, X. Gu and Z. Yang, Use of bentonite to control the release of 40399 copper from contaminated soils, Soil Research, 2007, 45, 618-623. 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 400 4. A. Kabata-Pendias, Trace elements in soils and plants, CRC Press, Boca Raton, Florida, 42 43401 Fourth edn., 2011. 44402 5. P. Maslin and R. M. Maier, Rhamnolipid-Enhanced Mineralization of Phenanthrene in 45403 Organic-Metal Co-Contaminated Soils, Bioremediation Journal, 2000, 4, 295-308. 46404 6. H. B. Bradl, Heavy metals in the environment: origin, interaction and remediation, 47405 Elsevier Academic Press, Neubrucke, Germany, 2005. Metallomics 48406 7. G. M. Pierzynski, J. T. Sims and G. F. Vance, Soils and environmental quality / by Gary M. 49 50407 Pierzynski, J. Thomas Sims, and George F. Vance, Lewis Publishers, Boca Raton, 1994. 51408 8. J.-L. Morel, G. Echevarria and N. Goncharova, Phytoremediation of metal-contaminated 52409 soils, Springer Science & Business Media, 2006. 53410 9. J. Gil-Loaiza, J. P. Field, S. A. White, J. Csavina, O. Felix, E. A. Betterton, A. E. Sáez and R. M. 54411 Maier, Phytoremediation Reduces Dust Emissions from Metal (loid)-Contaminated Mine 55412 Tailings, Environmental Science & Technology, 2018, 52, 5851-5858. 56 57413 10. M. T. Gómez-Sagasti, I. Alkorta, J. M. Becerril, L. Epelde, M. Anza and C. Garbisu, 58414 Microbial monitoring of the recovery of soil quality during heavy metal 59415 phytoremediation, Water, Air, & Soil Pollution, 2012, 223, 3249-3262. 60

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26483 33. C. Gonneau, N. Noret, C. Godé, H. Frérot, C. Sirguey, T. Sterckeman and M. Pauwels, Manuscript 27484 Demographic history of the trace metal hyperaccumulator Noccaea caerulescens (J. Presl 28485 and C. Presl) F. K. Mey. in Western Europe, Molecular Ecology, 2017, 26, 904-922. 29486 34. A. Babst-Kostecka, H. Schat, P. Saumitou ‐Laprade, K. Grodzi ńska, A. Bourceaux, M. 30 31487 Pauwels and H. Frérot, Evolutionary dynamics of quantitative variation in an adaptive 32488 trait at the regional scale: The case of zinc hyperaccumulation in Arabidopsis halleri, 33489 Molecular Ecology, 2018, 27, 3257-3273. 34490 35. J. Nowak, H. Frérot, N. Faure, C. Glorieux, C. Liné, B. Pourrut and M. Pauwels, Can zinc 35491 pollution promote adaptive evolution in plants? Insights from a one-generation Accepted 36 37492 selection experiment, Journal of Experimental Botany, 2018, DOI: 10.1093/jxb/ery327, 38493 ery327-ery327. 39494 36. C. C. Dietrich, K. Bilnicki, U. Korzeniak, C. Briese, K. A. Nagel and A. Babst-Kostecka, Does 40495 slow and steady win the race? Root growth dynamics of Arabidopsis halleri ecotypes in 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 496 soils with varying trace metal element contamination, Environmental and Experimental 42497 Botany, 2019, DOI: https://doi.org/10.1016/j.envexpbot.2019.103862, 103862. 43 44498 37. G. Jimenez-Ambriz, C. Petit, I. Bourrié, S. Dubois, I. Olivieri and O. Ronce, Life history 45499 variation in the heavy metal tolerant plant Thlaspi caerulescens growing in a network of 46500 contaminated and noncontaminated sites in southern France: role of gene flow, 47501 selection and phenotypic plasticity, New Phytologist, 2007, 173, 199-215. 48502 38. A. A. Babst-Kostecka, C. Parisod, C. Gode, P. Vollenweider and M. Pauwels, Patterns of Metallomics 49503 genetic divergence among populations of the pseudometallophyte Biscutella laevigata 50 51504 from southern Poland, Plant and Soil, 2014, 383, 245-256. 52505 39. C. Dechamps, C. Lefèbvre, N. Noret and P. Meerts, Reaction norms of life history traits in 53506 response to zinc in Thlaspi caerulescens from metalliferous and nonmetalliferous sites, 54507 New Phytologist, 2007, 173, 191-198. 55508 40. J. Jalas, J. Suominen and R. e. Lampinen, Atlas Florae Europaeae. Distribution of Vascular 56509 Plants in Europe. 11. Cruciferae (Ricotia to Raphanus), The Committee for Mapping the 57 58510 Flora of Europe & Societas Biologica Fennica Vanamo, Helsinki, 1996. 59511 41. K. Tremetsberger, C. König, R. Samuel, W. Pinsker and T. F. Stuessy, Infraspecific genetic 60512 variation in Biscutella laevigata (Brassicaceae): new focus on Irene Manton's

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1 2 3 View Article Online 611 synchrotron-based multi-angle X-ray fluorescence mapping, Annals ofDOI: botany 10.1039/C9MT00239A, 2007, 4 612 100, 1357-1365. 5 6 613 77. T. Punshon, K. Hirschi, J. Yang, A. Lanzirotti, B. Lai and M. L. Guerinot, The role of CAX1 7 614 and CAX3 in elemental distribution and abundance in Arabidopsis seed, Plant 8 615 Physiology, 2012, 158, 352-362. 9 616 78. S. A. Kim, T. Punshon, A. Lanzirotti, L. Li, J. M. Alonso, J. R. Ecker, J. Kaplan and M. L. 10617 Guerinot, Localization of iron in Arabidopsis seed requires the vacuolar membrane 11 12618 transporter VIT1, Science, 2006, 314, 1295-1298. 13619 79. E. L. Bastow, V. S. G. De La Torre, A. E. Maclean, R. T. Green, S. Merlot, S. Thomine and J. 14620 Balk, Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of 15621 iron in germinating seeds, Plant physiology, 2018, 177, 1267-1276. 16622 80. V. Lanquar, F. Lelièvre, S. Bolte, C. Hamès, C. Alcon, D. Neumann, G. Vansuyt, C. Curie, A. 17623 Schröder and U. Krämer, Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is 18 19624 essential for seed germination on low iron, The EMBO journal, 2005, 24, 4041-4051. 20625 81. H. Roschzttardtz, G. Conéjéro, C. Curie and S. Mari, Identification of the endodermal 21626 vacuole as the iron storage compartment in the Arabidopsis embryo, Plant physiology, 22627 2009, 151, 1329-1338. 23 24 25

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1 2 3 TABLES 4 5 Table 1. Geographic location, soil pH and mean total (T) and extractable (E) Zn concentrations at the study sites in Southern Poland. 6 7 -1 -1 8 Site Type Location Latitude N Longitude E Elevation (m a.s.l.) pH* ZnT (µg g ) * ZnE (µg g ) ** 9 M_PL2 metalliferous Olkusz 50°17´34.45˝ 19°29´01.95˝ 304 8.2 16 580 ± 2860 3420 ± 1130 10 M_PL6 metalliferous Olkusz 50°17´06.74˝ 19°27´59.27˝ 338 7.8 51 140 ± 22 210 5920 ± 2320 11 NM_PL8 non-metalliferous Tatra Mts 49°15´05.63˝ 19°54´37.62˝ 1342 7.7 100 ± 40 70±40 12 13 NM_SK14 non-metalliferous Tatra Mts 49°13´57.22˝ 20°16´24.10˝ 1719 7.5 50 ± 25 20±13 14 15 * adapted from Babst-Kostecka et al. 2016 16 ** adapted from Babst-Kostecka et al. 2014 17 Manuscript 18 19 20 21Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 22 23

24 Accepted 25 26 27 28 29 30 31 32 33 Metallomics 34 35 36 37 38 39 40 41 42 19 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Metallomics Page 20 of 30

1 2 3 Table 2. Seed germination rate and the average germination time (mean ± SD) of metallicolousView Article (M) Online 4 DOI: 10.1039/C9MT00239A 5 and non-metallicolous (NM) populations of Biscutella laevigata from the germination test under 6 7 controlled conditions. Thirty seeds per population were sown. Values in the same column, identified 8 by different letters differ significantly (P < 0.05). 9 10 11 12 Population Germination rate (%) Germination time (h) 13 M_PL2 60.0 a 186.7 ± 65.6 a 14 15 M_PL6 76.7 b 229.6 ± 93.2 a 16 NM_PL8 53.3 c 342.0 ± 132.2 b 17 NM_SK14 70.0 d 339.4 ± 98.2 b 18 19 20 21 22 23 24 25

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1 2 Table 3. Elemental composition (µg·g-1) of the individual Biscutella laevigata seed cross-sections. Errors of analysis are given in brackets. 3 Region of interest (ROI) 4 Element Population Sample Whole section Testa Hilum Endosperm Radicle Hypocotyl Cotyledon 5 M_PL2 1 38.7 (0.3) 10.1 (0.3) 65.0 (0.9) 266 (2) 67.6 (0.4) 47.0 (0.2) 22.8 (0.2) 6 2 57.4 (0.3) 34.4 (0.6) 37.9 (0.5) 100 (0.7) 154 (0.9) 115 (1) 43.7 (0.3) 7 8 M_PL6 1 31.9 (0.2) 35.7 (0.5) 24.1 (0.4) 49.8 (0.5) 106 (0.7) 80.3 (0.7) 25.9 (0.2) 9 2 74.7 (0.4) 11.0 (0.7) 32.4 (0.5) 584 (4) 103 (0.6) 97.2 (0.8) 46.4 (0.3) 10 Zn Mean 50.675 22.8 39.85 249.95 107.65 84.875 34.7 11 NM_PL8 1 36.2 (0.2) 38.0 (0.5) 16.6 (0.4) 35.3 (0.4) 78.7 (0.5) 46.8 (0.5) 33.6 (0.2) 12 2 38.5 (0.2) 33.6 (0.4) 11.8 (0.4) 48.7 (0.9) 108.7 (0.8) 99.2 (0.9) 32.1 (0.2) 13 NM_SK14 1 22.0 (0.2) 2.2 (0.1) 3.8 (0.2) 25.8 (0.4) 54.6 (0.4) 37.7 (0.4) 15.1 (0.1) 14 15 2 30.9 (0.2) 7.3 (0.4) 122 (1) 48.0 (0.5) 93.1 (0.7) 82.6 (0.5) 19.6 (0.1) 16 Mean 31.9 20.275 38.55 39.45 83.775 66.575 25.1 17 M_PL2 1 23400 (160) 2440 (290) 4200 (330) 8280 (310) 32200 (270) 27350 (420) 26170 (190) Manuscript 18 2 18220 (90) 8850 (460) 2840 (310) 4860 (230) 29880 (300) 26450 (440) 20170 (90) 19 M_PL6 1 17400 (110) 5650 (320) 3660 (310) 6630 (310) 31170 (290) 22670 (400) 19560 (110) 20 2 22870 (110) 2090 (710) 3100 (310) 7220 (260) 33890 (220) 29870 (390) 25160 (120) 21Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. S 20472.5 4757.5 3450 6747.5 31785 26585 22765 22 Mean 23 NM_PL8 1 23260 (140) 4410 (300) 3900 (330) 5070 (290) 40770 (300) 33790 (410) 26230 (140) 2 22850 (130) 5570 (270) 5110 (340) 5770 (530) 39110 (300) 36450 (440) 23450 (130) 24 Accepted 25 NM_SK14 1 18090 (150) 2150 (210) 2210 (260) 4780 (340) 33500 (220) 28710 (400) 19580 (170) 26 2 22140 (150) 425 4660 (260) 6450 (260) 38180 (240) 26180 (320) 23970 (160) 27 Mean 21585 3245 3970 5517.5 37890 31282.5 23307.5 28 M_PL2 1 1480 (18) 740 (110) 2400 (120) 3510 (110) 1540 (50) 1590 (90) 1550 (25) 29 30 2 2130 (16) 2250 (160) 2310 (120) 3330 (100) 2160 (70) 2230 (100) 2100 (23) 31 M_PL6 1 2020 (14) 4700 (130) 2720 (120) 5040 (120) 2220 (60) 2240 (110) 2200 (18) 32 2 670 (16) <400* 670 (100) 1090 (80) 750 (50) 850 (100) 770 (23) 33 Cl Mean 1575 1972.5 2025 3242.5 1667.5 1727.5 1655 Metallomics 34 NM_PL8 1 520 (19) 870 (119) 570 (100) 1005 (86) 490 (44) 590 (83) 600 (22) 35 2 660 (18) 820 (86) 1000 (110) 1510 (180) 690 (60) 870 (91) 720 (20) 36 * 37 NM_SK14 1 330 (18) <110 378 (84) 1030 (100) 500 (35) 580 ((83) 550 (21) 38 2 1290 (16) <270* 1790 (90) 3040 (93) 1270 (40) 1430 (78) 1160 (17) 39 Mean 700 470 934.5 1646.25 737.5 867.5 757.5 40 M_PL2 1 9520 (60) 3040 (50) 7180 (80) 11500 (90) 10560 (53) 10390 (73) 10130 (62) 41 42 43 21 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 View Article Online DOI: 10.1039/C9MT00239A

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1 2 2 10770 (40) 11710 (90) 11020 (120) 17340 (80) 9760 (46) 9650 (50) 10590 (35) 3 M_PL6 1 10920 (56) 21840 (130) 16090 (150) 25440 (120) 12280 (57) 12390 (80) 11420 (55) 4 2 10190 (40) 7020 (120) 9530 (100) 16480 (90) 9800 (46) 9670 (56) 9900 (34) 5 K Mean 10350 10902.5 10955 17690 10600 10525 10510 6 NM_PL8 1 9570 (50) 19470 (130) 15430 (160) 21090 (130) 9040 (50) 8130 (55) 9060 (40) 7 2 12870 (60) 21480 (110) 21160 (190) 28250 (170) 10860 (50) 10980 (67) 12590 (52) 8 9 NM_SK14 1 5100 (34) 1380 (27) 1740 (36) 3590 (40) 7390 (40) 7610 (65) 5770 (40) 10 2 8410 (52) 2500 (55) 8140 (90) 13350 (95) 9260 (43) 10480 (55) 8270 (52) 11 Mean 8987.5 11207.5 11617.5 16570 9137.5 9300 8922.5 12 M_PL2 1 3240 (15) 1600 (24) 7830 (57) 9210 (49) 2200 (12) 2570 (25) 3160 (14) 13 2 3875 (9) 3080 (34) 9980 (73) 5660 (26) 1060 (9) 1890 (17) 4170 (11) 14 M_PL6 1 1864 (8) 4890 (32) 3550 (42) 5670 (28) 1010 (7) 1350 (15) 2100 (8) 15 16 2 2628 (7) 1290 (40) 4790 (41) 10830 (47) 1830 (11) 2410 (18) 2430 (6) 17 Ca Mean 2901 2715 6537 7842 1525 2055 2965 Manuscript 18 NM_PL8 1 2380 (8) 5530 (34) 4220 (40) 5590 (32) 1430 (7) 1350 (14) 2470 (7) 19 2 2216 (7) 2770 (26) 8650 (70) 2270 (38) 1860 (10) 2300 (17) 2180 (6) 20 NM_SK14 1 1725 (10) 1750 (20) 5270 (60) 7100 (46) 1120 (7) 1430 (16) 1530 (10) 21Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 2 2790 (13) 850 (30) 10730 (80) 6320 (30) 1570 (8) 2400 (18) 2850 (13) 22 23 Mean 2277.75 2725 7217.5 5320 1495 1870 2257 M_PL2 1 8.2 (0.1) 5.1 (0.6) 42 (1) 68 (1) 9.5 (0.3) 9.0 (0.5) 3.7 (0.1)

24 Accepted 25 2 10.2 (0.1) 14.9 (0.8) 12.4 (0.6) 57.9 (0.7) 7.4 (0.4) 7.3 (0.6) 6.8 (0.1) 26 M_PL6 1 6.48 (0.08) 42.7 (0.9) 18.8 (0.8) 54.5 (0.9) 6.4 (0.3) 6.2 (0.6) 4.4 (0.1) 27 2 13.8 (0.1) 8 (1) 10.1 (0.7) 254 (2) 5.1 (0.3) 5.5 (0.5) 5.21 (0.1) 28 Mn Mean 9.67 17.7 21 108 7.1 7.0 5.0 29 30 NM_PL8 1 7.23 (0.07) 26.7 (0.8) 15.4 (0.7) 27.8 (0.7) 5.6 (0.3) 4.2 (0.5) 5.6 (0.1) 31 2 11.2 (0.1) 43.6 (0.8) 108 (2) 42 (2) 8.3 (0.4) 12.3 (0.6) 7.8 (0.1) 32 NM_SK14 1 5.0 (0.1) 5.2 (0.4) 7.2 (0.6) 30.4 (1.0) 5.4 (0.2) 5.7 (0.6) 3.7 (0.1) 33 2 8.0 (0.1) 3.7 (0.9) 108 (1) 48.3 (1.0) 7.1 (0.3) 10.2 (0.5) 4.4 (0.1) Metallomics 34 Mean 7.8575 19.8 59.65 37.125 6.6 8.1 5.375 35 M_PL2 1 31.7 (0.3) 10.4 (0.5) 44.6 (0.9) 139 (1) 42.0 (0.4) 32.7 (0.6) 29.6 (0.3) 36 2 64.1 (0.3) 25.3 (0.8) 28.1 (0.7) 111 (1) 104.8 (0.8) 88 (1) 58.7 (0.3) 37 38 M_PL6 1 33.9 (0.2) 51.9 (0.9) 23.7 (0.7) 54.2 (0.8) 63.2 (0.5) 69.8 (0.8) 35.4 (0.2) 39 2 32.5 (0.2) 13 (1) 20.7 (0.8) 122 (1) 23.8 (0.3) 39.5 (0.6) 33.9 (0.2) 40 Fe Mean 40.55 25.15 29.275 106.55 58.45 57.5 39.4 41 NM_PL8 1 54.3 (0.3) 114 (1) 26.1 (0.7) 90.5 (1.0) 85.8 (0.6) 36.9 (0.7) 49.3 (0.3) 42 43 22 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 View Article Online DOI: 10.1039/C9MT00239A

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1 2 2 53.3 (0.3) 151 (2) 81 (1) 297 (3) 86.9 (0.7) 136 (1) 41.1 (0.3) 3 NM_SK14 1 26.1 (0.2) 11.2 (0.4) 19.3 (0.6) 50.5 (1.0) 44.0 (0.4) 70 (1) 25.7 (0.2) 4 2 37.5 (0.3) 10.9 (0.8) 279 (3) 82 (1) 58.8 (0.5) 62.6 (0.8) 29.4 (0.2) 5 Mean 42.8 71.775 101.35 130 68.875 76.375 36.375 6 M_PL2 1 4.82 (0.07) 0.8 (0.2) 8.7 (0.4) 39.6 (0.6) 5.6 (0.1) 3.9 (0.2) 2.95 (0.07) 7 2 3.37 (0.05) 2.2 (0.3) 1.7 (0.2) 5.8 (0.2) 6.2 (0.2) 5.1 (0.3) 3.14 (0.06) 8 9 M_PL6 1 2.55 (0.04) 8.2 (0.3) 3.1 (0.3) 11.7 (0.3) 4.7 (0.1) 3.5 (0.3) 2.28 (0.05) 10 2 4.05 (0.07) 2.0 (0.6) 3.3 (0.3) 19.2 (0.6) 4.3 (0.2) 3.3 (0.3) 3.2 (0.06) 11 Cu Mean 3.6975 3.3 4.2 19.075 5.2 3.95 2.8925 12 NM_PL8 1 0.82 (0.03) 2.4 (0.3) 1.0 (0.2) 2.8 (0.2) 1.2 (0.1) 0.9 (0.3) 0.79 (0.04) 13 2 1.81 (0.03) 5.0 (0.2) 2.3 (0.3) 5.1 (0.5) 3 (0.2) 2.8 (0.2) 1.43 (0.04) 14 NM_SK14 1 1.44 (0.04) 1.0 (0.2) 1.1 (0.2) 5.1 (0.3) 2.6 (0.1) 2.4 (0.3) 1.07 (0.04) 15 16 2 3.44 (0.05) 0.9 (0.4) 8.0 (0.3) 45.7 (0.5) 6.4 (0.1) 4.2 (0.3) 1.45 (0.05) 17 Mean 1.8775 2.325 3.1 14.675 3.3 2.575 1.185 Manuscript 18 * Note that for results below errors of analysis one-half of the reported limit was used to calculate the mean. 19 20 21Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 22 23

24 Accepted 25 26 27 28 29 30 31 32 33 Metallomics 34 35 36 37 38 39 40 41 42 43 23 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Metallomics Page 24 of 30

1 2 3 FIGURES View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 MiSceroedscpohpoytoSmeiecrdogPriacptuhre SSeeeeddSschematiicc XFXMFM SeseededPmicatupre 7 0.6 8 Ca 0.5 9 E 0.4 10 Hi 11 C 0.3 12 0.2 13 R T 0.1 14 Hy 15 1000 μm wt% 16 17 18 Figure 1. Photomicrograph, schematic and quantitative elemental map of a longitudinal section of a 19 Biscutella laevigata seed and schematic representation of tissue parts used in the interpretation of 20 21 XFM results. T – testa, Hi – hilum, E – endosperm, R – radicle, Hy – hypocotyl, C – cotyledon. 22 23 24 25

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1 2 3 View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 7 8 9 10 11 12 13 14 15 16 17 Figure 2. Cumulative germination rate over time for Biscutella laevigata seeds from two 18 metallicolous (M, red) and two non-metallicolous (NM, blue) populations (30 seeds per population). 19 20 21 22 23 24 25

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1 2 3 View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Figure 3. Seed weight (mean ± SD, n = 30) of metallicolous (M, red) and non-metallicolous (NM, 22 23 blue) Biscutella laevigata. The box represents the interquartile range of the data, the median is 24 25 indicated by the horizontal line. Different letters indicate statistically significant differences at

26 P≤0.05. Manuscript 27 28 29 30 31 32 33 34 35 36 Accepted 37 38 39 40 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 43 44 45 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

26 Manuscript 27 28 Figure 4. Quantitative elemental maps showing the distribution of Zn in Biscutella laevigata seed 29 cross-sections. Seeds originated from two metallicolous (M) and two non-metallicolous (NM) 30 31 populations. 32 33 34 35 36 Accepted 37 38 39 40 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 43 44 45 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

26 Manuscript 27 28 29 30 31 32 33 34 35 36 Accepted 37 38 39 40 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 43 44 45 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60 Figure 5. Quantitative elemental maps in Biscutella laevigata seed cross-sections. Seeds originated from two metallicolous (M) and two non-metallicolous (NM) populations. 28 Page 29 of 30 Metallomics

1 2 3 View Article Online 4 DOI: 10.1039/C9MT00239A 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

26 Manuscript 27 28 29 30 31 32 33 34 35 Figure 6. Elemental composition of the seed cross-sections from metallicolous (red) and non- 36 Accepted 37 metallicolous (blue) Biscutella laevigata ecotypes (mean ± SD, n=4). Different letters indicate 38 39 statistically significant differences at P ≤ 0.05 between seed tissues at the ecotype level (red and blue 40 font for metallicolous and non-metallicolous samples, respectively). Significant differences between 41 Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 the ecotypes are shown with * for P ≤ 0.05; ▪ shows differences at 0.1 ≤ P ≤0.05. T – testa, Hi – 43 44 hilum, E – endosperm, R – radicle, Hy – hypocotyl, C – cotyledon. 45 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Endosperm prevents toxic amounts of Zn from accumulating in the seed embryo – anView Article Online DOI: 10.1039/C9MT00239A 4 adaptation to metalliferous sites in metal-tolerant Biscutella laevigata 5 6 1 2,3 4,5 6 7 Alicja Babst-Kostecka , Wojciech J. Przybyłowicz , Antony van der Ent , Chris Ryan , 8 Charlotte Dietrich1, Jolanta Mesjasz-Przybyłowicz3 9 10 1 W. Szafer Institute of Botany, Polish Academy of Sciences, Department of Ecology, Lubicz 11 46, 31-512 Krakow, Poland. 12 2AGH University of Science and Technology, Faculty of Physics & Applied Computer 13 14 Science, 15 al. Mickiewicza 30, 30-059 Kraków, Poland. 16 3Department of Botany and Zoology, Stellenbosch University, Private Bag X1, 17 Matieland 7602, South Africa. 18 4Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, 19 The University of Queensland, Australia. 20 5Laboratoire Sols et Environnement, Université de Lorraine, France. 21 6 22 CSIRO, Mineral Resources, Australia. 23 24 25 Table of contents entry

26 Manuscript 27 28 29 The pseudometallophyte Biscutella laevigata adapts to metalliferous soils by allocating 30 excess metal(loid)s to the endosperm (E) of seeds to protect embryonic tissues and improve 31 32 reproductive success. 33 34 35 36 MiSceroedscpohpoytoSmeiecrdogPriacptuhre SSeeeeddSschematiicc XFXMFM SeseededPmicatupre Accepted 0.6 37 Ca 38 0.5 39 E 0.4 40 Hi 0.3 41 C Published on 07 November 2019. Downloaded by University of Queensland 11/10/2019 10:30:58 PM. 42 0.2 R 43 T 0.1 44 1000 μm Hy 45 wt% 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60