Silicon isotope fractionation and uptake dynamics of three crop plants: laboratory studies with transient silicon concentrations

Silicon has been recognized an important element in global biogeochemical cycles for a long time. Recently, its relevance for global crop production gains increasing attention. Silicon is beneficial for plant growth and is taken up in 10 considerable amounts by crops, likewise rice or wheat. The incorporation of silicic acid from the soil solution into the plants is accomplished by a variety of strategies (rejective, passive and active) that are subject to an intense debate. To forge a new perspective on the underlying processes, we investigated how the silicon stable isotope fractionation during plant growth depends on uptake strategy, transpiration, water use, and Si transfer efficiency. Crop plants with a rejective (tomato, Solanum lycopersicum and mustard, Sinapis alba) and active (spring wheat, Triticum aestivum) uptake were hydroponically grown for 15 6 weeks. Using inductively coupled plasma mass spectrometry, the silicon amounts and the isotopic composition of the nutrient solution, the roots, and the shoots were determined. Wheat revealed the highest Si transfer efficiency from root to shoot followed by tomato and mustard. All three species preferentially incorporated light Si, with a fractionation factor 1000∙ln(α) of -0.33 ‰ (tomato), -0.55 ‰ (mustard) and -0.43 ‰ (wheat). Even though the rates of active and passive Si root uptake differ, the physico-chemical processes governing Si uptake and stable isotope fractionation do not, they are governed by a diffusion 20 process. In contrast, the transport of silicic acid from the roots to the shoots depends on the preceding precipitation of silicic acid in the roots and the presence of active transporters at the root endodermis. Plants with a significant biogenic silica precipitation in roots (mustard, and wheat), preferentially transport silicon enriched in Si into their shoots, whereas the transport in tomato is governed by a diffusion process and hence preferentially transports light silicon Si into the shoots.

diffusion, and precipitation) may be accompanied by a shift in an element's stable isotope ratios -so-called mass-dependent 65 isotope fractionation (Poitrasson, 2017). This isotope fractionation either entails an equilibrium isotope effect, where the isotopes are partitioned between compounds according to bond strength, or a kinetic isotope effect, where the isotope fractionation depends on the relative rate constants of reactions involving the different isotopologues. For stable Si isotope fractionation in aqueous media, both equilibrium effects (He et al., 2016;Stamm et al., 2019) and kinetic effects (Geilert et al., 2014;Oelze et al., 2015;Poitrasson, 2017;Roerdink et al., 2015) have been observed. Previous studies on stable Si 70 fractionation in higher plants focused on rice (Ding et al., 2008a;Köster et al., 2009;Sun et al., 2008Sun et al., , 2016bSun et al., , 2016a, banana (Delvigne et al., 2009;Opfergelt et al., 2006Opfergelt et al., , 2010, bamboo (Ding et al., 2008b) and cucumber (Sun et al., 2016b) and most of these studies show the preferential incorporation of lighter Si isotopes. Importantly, in most of these studies, Si concentrations in the growth media were held constant by frequently replenishing the nutrient solution. This imparts the disadvantage that the dynamics (temporal evolution) of the Si isotope fractionation during uptake cannot be derived from the 75 isotope shift recorded by the nutrient solution over the course of the experiment, nor does the provision of constant Si amounts allow additional constraints to be placed on Si uptake mechanisms employed by plants.
In this study we elucidated the mechanisms of Si uptake using crop species that differ significantly in their Si uptake capacity and the presence of specific Si transporters. To do so, we combined the measurement of physiological plant performance ratios 80 with observations of the shifts in the Si isotope ratios due to mass dependent isotope fractionation. Three crops -tomato, mustard and wheat -were grown in a hydroponic system, with a finite nutrient supply during the experiment, allowing direct quantification of the dynamics of isotopic fractionation from the temporal evolution of the nutrient solutions' isotopic composition. With the combination of the physiological plant performance ratios and isotope chemical parameters we develop new insights to the mechanisms underlying the different Si uptake and translocation strategies. 85 2 Materials and Methods

Nutrient Solution
The nutrient solution was prepared from technical grade salts following the recipe after Schilling et al., 1982;and Mühling & Sattelmacher, 1995. Silicon was added in the form of NaSiO4 to an initial starting concentration of 49.5 µg•g -1 . Details can be found in supplementary methods S1. 90

Plant species
Three species were chosen based on their silicon uptake characteristics, the ability to grow in hydroponic environments, and previous knowledge about their Si transporter. Tomato (Solanum lycopersicum cultivar MICRO TOM) and mustard (Sinapis alba) are both rejective of Si, while spring wheat (Triticum aestivum cultivar SW KADRILJ) actively takes up Si (Hodson et al., 2005;Takahashi et al., 1990). The two Si excluder species differ in the presence of the NOD26-like-instrinsic proteins (orthologues of Lsi1, homologous gene sequence of low silicon rice 1) which are associated with the transport of Si. In the family of Brassicaceae (mustard) these are absent (Sonah et al., 2017), whereas for tomato the Lsi1 homologue seems to be present but inactive (Deshmukh et al., 2016(Deshmukh et al., , 2015. Conversely, the alleged active Si efflux transporter (Lsi2-like) are present in the family of Brassicacea (Sonah et al., 2017), but not in tomato.

Plant germination and growth conditions 100
Plant seeds were germinated in Petri dishes with half-strength nutrient solution used for the later growth experiment that contained no NaSiO4. After cotyledons formed, seedlings were transferred into a foam block and grown for a further two weeks in the same half-strength nutrient solution. Four plants each were then transferred into one experimental container that was filled with fresh nutrient solution including NaSiO4, and each species was replicated in three containers. Plants were germinated and grown in a growth chamber under controlled climate conditions. Each week the transpired water was replenished with 105 ultrapure water. The temperature in the growth chamber during the day and night was maintained at 18 °C for 14 h and at 15 °C for 10 h, respectively, and the daylight intensity at the top of the container was adjusted to 350 µE•m -2 •s -1 ) at the start of the experiment. The relative humidity was maintained at approximately 65 %. Details of the plant germination and growth conditions are provided in supplementary methods S2.

Sampling 110
The nutrient solutions were sampled at the start of the experiment and then every seven days until harvesting. For sampling, 40 mL were taken after replenishing water loss via transpiration loss and mixing of the solution. All sampled nutrient solutions were stored until analysis in precleaned PP vials in darkness at 4 °C. The 280 mL sample taken over the course of 6 weeks corresponds to 3.5 % of the initial nutrient solution. After 6 weeks the plants were harvested, and stem and leaves were separated from the roots. The roots were immersed multiple times in ultrapure water to remove potential extracellular Si 115 deposits. The plant parts were dried at 104 °C to constant weight.

Determination of concentrations and isotope ratios
The chemical compositions of the growth solution and the digested plant samples were measured using an axial inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian 720-ES, instrument settings are reported in Table S1).
Samples and standard were doped with an excess of CsNO3 (1 mg g -1 ) to reduce matrix effects that are likely to be caused 120 from the high nitrogen content of the samples. The relative analytical uncertainties are estimated to be below 10% and agreed with the nominal concentration of the starting solutions. For details of the analytical method and an extended verification see 'S1 Description of analytical methods' in Schuessler et al., 2016Schuessler et al., . https://doi.org/10.5194/bg-2020 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License.

Nutrient solution
After the concentration measurements an aliquot of each nutrient solution containing approximately 1000 µg Si was dried 125 down in silver crucibles on a hotplate at 80-95 °C. Crucibles were then filled with a solution containing 400 mg NaOH (prepared from Merck pellets, p.a. grade, previously checked for low Si blank levels) in ultrapure water and dried down. A blank containing ultrapure water and NaOH was processed together with the samples.

Plant samples
The oven-dried samples were homogenised by milling the plant parts in a tungsten carbide planetary ball mill (Pulversiette 7, 130 Fritsch). 50-800 mg of plant material, depending on the estimated Si concentration, was weighed into Ag crucibles and combusted overnight (2h at 200 °C, 4h at 600 °C, then cooled to room temperature) in a furnace (LVT 5/11/P330, Nabertherm).
A blank (empty crucible) was processed together with the samples. After cooling the loss of ignition was determined and 400 mg NaOH (TraceSELECT, Sigma-Aldrich) added.

Fusion and chromatography 135
The crucibles containing the sample (nutrient solution or plant material) and NaOH were placed in a high temperature furnace at 750 °C for 15 min. The fusion cake was dissolved in ultrapure water and 0.03 M HCl, and the pH was adjusted to 1.5.
Approximately 60 µg Si was chromatographically separated using cation exchange resin (Georg et al., 2006;Zambardi & Poitrasson, 2011;Schuessler & von Blanckenburg, 2014). The purity and Si yield of the fusion procedure and the column chemistry was determined by ICP-OES. See Methods S3 for more details. 140

Silicon isotope ratio measurements
The purified solutions were acidified to 0.1 M HCl and diluted to a concentration of 0.6 µg•g -1 . Sample and standard were both doped with 0.6 µg•g -1 Mg and the 25 Mg/ 24 Mg ratio used as a monitor of mass bias drift and to ensure stable measurement conditions during the analysis (Oelze et al., 2016). The solutions were introduced using an ESI ApexHF desolvator and a PFA nebuliser (measured uptake 140 µL min -1 ) into the MC-ICP-MS (Neptune, equipped with the Neptune Plus Jet Interface, 145 Thermo Fisher Scientific; instrument settings are given in Table S1). Measurements were made in dynamic mode (magnet jump) alternating between Si and Mg isotopes, each for 30 cycles with 4 s integration time. ERM-CD281 and BHVO-2 were analysed together with the nutrient and plant samples to ensure complete fusion, dissolution and chromatographic separation.
ERM-CD281 resulted in δ 30 Si = -0.34 ± 0.20 ‰, 2s, n=13 and BHVO-2 in δ 30 Si = -0.29 ± 0.09 ‰, 2s, n=40, in line with literature values. The results of reference materials are reported in the supplementary information in Table S2, and the results 150 of growth solutions and plants in Table S3 and Table S4. All δ 29/28 Si and δ 30/28 Si are reported in delta notation relative to NBS28 (NIST SRM8546) unless stated otherwise (Coplen et al., 2002;Poitrasson, 2017). An isotopic difference between two compartments is expressed as Δ 30 Si, calculated following Eq. (1): where the subscripts plant, root and shoot refer to the bulk plant, and roots and shoots, respectively, and M is the mass of silicon incorporated into the roots or shoots of the plant. 160

Plant performance ratios
In order to compare the plant species with respect to their water uptake as well as Si uptake and transfer the following performance ratios were calculated at the end of the experiments: 1. Water use efficiency: total phytomass (g) divided by the amount of transpired water (L), calculated separately for 165 each pot.
2. Si uptake efficiency: total Si mass (mg) in plants divided by the amount of transpired water (L), calculated separately for each pot.
3. Si transfer efficiency: Si mass (mg) in plant shoots divided by the amount of transpired water (L), calculated separately for each pot. 170 The uptake characteristics were classified based on the ratio of measured and theoretical Si uptake. A ratio of greater than 1 indicates an active uptake mechanism, a ratio much smaller than 1 a rejective strategy, and a ratio of 1 indicates passive uptake. The theoretical Si uptake was calculated based on the amount of transpired water and the nutrient solution Si concentration.

Element budgets 175
The digested plant samples and nutrient solutions were analysed prior to the column purification by ICP-OES, and the concentrations of major elements (Ca, Fe, K, Mg, P, S and Si) and the retrieval was determined using Eq. (3): where Msolution, end is the mass of the element X in the solution at the end of the experiments, MPlants is the mass of the element X in the plants, and MSolution, start the mass of the element X in the solution at the beginning of the experiment. 180 https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License.

Silicon isotope budget
A simple test of whether incomplete recovery of Si or analytical artefacts in the Si isotope composition measurements are affecting the results is offered by an isotope budget. The concept is that the summed Si isotope composition of the remaining growth solution at the end of the experiment and the Si taken up by plants should be identical to the Si isotope composition of the initial growth solution. The Si total isotope composition at harvest is estimated using Eq. (4)

Plant dry mass and transpiration
Substantial differences are apparent in the growth rate between and within all three plant species. During the six-week period mustard formed the greatest amount of dry biomass, with an average of 7 g per plant (range: 0.7 -16.6 g). Spring wheat produced on average 4 g (range: 1.9 -5.6 g), and tomato produced the lowest amount of biomass per plant with an average of 3 g (range: 0.2 -8.7 g, see Table 1 and Table S4 for the individual results). No dependence of replicated growth experiments 195 on pot placement or proximity to the venting system was apparent. The amount of water transpired by the plants during the growth period is correlated with the biomass formed (rSpearman Rank = 0.95, p-value <0.001). In contrast, no differences between plant species were observed in terms of the shoot-root ratios (5.4 -6.5 g•g -1 , Table 2).

Dynamics of water, Si and other nutritive elements uptake
The three plant species revealed very different transpiration dynamics during the 6 weeks of plant growth. After a lag phase 200 of two weeks, differences in transpiration between mustard and the other two species became apparent. Figure 1 shows the cumulative transpiration for the three replicate growth experiments and species. Mustard showed the highest, wheat intermediate and tomatoes the lowest cumulative transpiration. The water use efficiency of tomato was significantly higher (3.8 g•L -1 ) than that of the other two plant species (2.4 -2.6 g•L -1 , Table 2).

205
Based on the temporal evolution of Si concentrations in the nutrient solutions ( Figure 1) spring wheat exhibited the highest total Si uptake, mustard an intermediate amount, and tomato the lowest total Si uptake and the Si contents of bulk plants reflect this sequence (Table 1): spring wheat as Si accumulator took up the most Si (448 mg), followed by mustard (150 mg). Tomato took up the least amount (95 mg). Considering only roots, the highest Si concentrations and Si amounts were found in mustard, https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License.
while spring wheat and tomato were significantly lower. In contrast, considering only plant shoots, the highest Si mass were 210 found in wheat while Si concentrations in mustard and tomato were similar, but more than an order of magnitude lower (Table   1). Spring wheat also showed a much higher Si uptake efficiency than the other two plant species, which resemble each other (Table 2 and Figure 1). The same trend holds for the Si mass ratio between roots and shoots (Table 2). Moreover, wheat shows a much higher efficiency of Si transport into the shoot per mass of transpired water than the other two plant species. In contrast to the Si uptake efficiency, the Si mass ratio between root and shoot for mustard was lower than for tomato (Table 2). For the 215 calculation of Si uptake rates, we assume there is no back diffusion or efflux of Si out of the plant roots. Such a process has not been reported in the literature and would be driven against the concentration difference between the root and the nutrient solution Si concentration and against the water flow direction (Raven, 2001).
After 6 weeks of growth, some nutrients were fully consumed, and the first mustard plants showed signs of deficiency in the 220 form of chlorosis in young and old leaves. Mustard, forming the largest biomass, had also the largest demand for Ca (mean ~644 mg per container), Mg (~140 mg), P (~205 mg) and S (~209 mg). Fig. S1 in the supplement shows the temporal evolution of the other nutrient concentrations for the three plant species.

Element and Si isotope budgets
The biomass amounts, concentrations, and isotope compositions used to calculate element and Si isotope budgets are reported 225 in the supporting information Table S4. The element retrievals are shown in Table 3. All three plant species showed less than complete retrieval, with variable deficits between elements. For Si the retrieval amounted to between 83% (mustard) and 90% (wheat). For the other nutrients (Ca, Fe, K, Mg, P and S, see Table 3) the retrievals were between 70% and 110%. S in mustard was an exception, with a retrieval of only 50%, which we attribute to the loss of volatile S species during drying and charring, leading to the low retrieval (Blanck et al., 1938). The results for the Si isotope budget are shown in Table 4. Within uncertainty,230 there is no significant difference between the isotopic composition of the starting solution and the weighted average isotopic composition of the different compartments at the end of the experiment. Thus, we conclude that all significant pathways that fractionate Si isotopes are accounted for.

Dynamics of isotope fractionation between the nutrient solution and plants
The average initial δ 30 Si composition of the nutrient solution is -0.21 ± 0.07 ‰ (2 s, relative to NBS28; individual results are 235 reported in Table S3). The temporal evolution of the nutrient solution and the individual Si isotopic composition of the roots, shoots and the entire plants are shown in Figure 2 (reported as Δ 30 Si relative to the nutrient solution). All three plant species preferentially incorporated the lighter silicon isotope ( 28 Si), leaving the nutrient solution enriched in heavier silicon ( 30 Si).
After an initial lag phase for all three species, in which the nutrient solution's Si isotope composition does not vary, its isotopic composition becomes increasingly enriched in 30  Our experimental setup allows us to determine the Si isotope fractionation factors into bulk plants directly from the temporal evolution of the Si isotope composition of the nutrient solution. This approach differs from previous studies of Si isotope fractionation by plants, in which the Si pool in the nutrient solution was frequently replenished (Ding et al., 2008a;Sun et al., 260 2008Sun et al., 260 , 2016b. Evaluating the temporal evolution of wheat nutrient solution ( Figure 3) and assuming no back-diffusion, a Rayleigh like fractionation can be fitted using Eq. (5) (Mariotti et al., 1981): where fsolution is the fraction of Si in the remaining solution, R0 the initial 30 Si/ 28 Si isotope ratio, R the 30 Si/ 28 Si isotope ratio of the product, and α the fractionation factor. A best fit to the data, minimising the root-mean-square-deviation, results in αPlant-  (Table 5). Within uncertainty, there is no significant difference in the bulk fractionation factor between active 270 and rejective uptake species. The best fit through all results, across the three plant species from this study, results in a fractionation factor 1000•ln(α) of -0.41 ± 0.09 ‰ (1 s) at an initial Si concentration of 49.5 µg•g -1 (ca. 1.76 mM).
If we assume the uptake of Si to be governed by diffusion through cell membranes and Si permeable transporters (Ma et al., , 2007Ma and Yamaji, 2015;Mitani et al., 2009;Zangi and Filella, 2012) and the diffusion of Si is non-quantitative, the https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License. lighter isotopes will be enriched in the target compartment (Sun et al., 2008;Weiss et al., 2004). To a first approximation, the 275 difference between the diffusion coefficient of isotopologues 28 Si(OH)4 and 30 Si(OH)4 sets the theoretical upper limit of observable isotopic fractionation in a system dominated by diffusion. The diffusion coefficient ratio approximated by Eq. (6) corresponds to the fractionation factor in an idealised system consisting of pure water and silicic acid only (Mills and Harris, 1976;Richter et al., 2006).

Reliability of the combined element and isotope ratio approach
In contrast to previous studies, we added a finite nutrient amount to growth solutions and replenished only the transpired water.
The combination of plant physiological ratios (water use efficiency, element budgets and biomass production) with stable isotope ratio measurements allows us to explore the temporal evolution of Si uptake and translocation. Several aspects of our 290 data attest to the reliability of our approach and results. Concerning Si uptake dynamics, Si recovery rates of >80% (see Table   3) corroborate the reliability of our results. The same is observed for the isotope budgets. There is no significant difference between the isotopic composition of the starting solution and the weighted average of the isotopic compositions of the different compartments at the end (see Table 4). This implies all significant pathways that fractionate Si isotopes have been accounted for. The Si retrieval rate between 83 and 90% is likely not caused by a single systematic analytical uncertainty or unaccounted 295 sink of Si, but rather a combination of container wall absorption (up to 0.1%), root washing procedure (up to 1%), the weekly sampling (up to 3.5%) and analytical uncertainties (up to 10%). Guttation (Yamaji et al., 2008) and litter fall were not observed during the experiment.

Si uptake strategies
The ratio between measured Si uptake and the theoretical Si amount that would have entered the plant in a purely passive 300 uptake mechanism (see section plant performance ratios), shows that wheat accumulates Si and mustard and tomato both reject Si ( Figure 1 and Table 2). The accumulation of Si in wheat can be explained by the cooperation of an influx transporter (Lsi1-https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License. like) into the roots and the presumed presence of an efflux transporter (Lsi2-like) from the roots into the xylem. Closely related cereals have such transporters, therefore we expect them to be present in wheat too (Ma and Yamaji, 2015). In rice, mutants with either defective Lsi1 or Lsi2 transporter lead to significantly lower Si accumulation (Köster et al., 2009). The direct 305 comparison between both mutants revealed that Lsi1 carries a larger share of Si incorporation, thus a defective Lsi2 can partially be compensated (Köster et al., 2009).
Our experiments show a striking similarity in Si uptake characteristics between mustard and tomato. Considering the differences in ontogenesis between the plant species, this may be a fortuitous coincidence. In particular, the relatively low 310 temperatures may have inhibited the growth of the more thermophilic tomato, while the conditions were closer to optimal for mustard and summer wheat. Tomatoes have the genetic capacity to accumulate Si, since an orthologue of Lsi1 is present in the genes. An insertion in the amino acid sequence however, lead to a loss of the Si uptake functionality (Deshmukh et al., 2016(Deshmukh et al., , 2015, and thus tomato like mustard, rejects Si.

315
With our experimental approach we also detect significant differences between the crop species in Si transfer from the root to the shoot (Table 2). Wheat, which probably has a metabolically active efflux transporter (Lsi2-like) at the root-xylem interface, has the highest Si transfer efficiency per water mass (49.3 ± 8.4 mg shoot Si•L -1 ). The transfer efficiency for tomato is significantly higher than mustard (3.5 ± 0.4, and 2.4 ± 0.3 mg shoot Si•L -1 , respectively), which is not readily explainable by differences in root Si efflux pathways since tomato does not contain the active efflux transporter orthologue Lsi2 while mustard 320 does (Ma & Yamaji, 2015;Sonah et al., 2017). Phytolith formation, which was observed in mustard roots (data not shown) could explain the lower Si transfer efficiency of mustard. A similar immobilization of silica in roots has already been observed in wheat (Hodson and Sangster, 1989) and other grasses (Paolicchi et al., 2019). Other possible reasons for this phenomenon will be discussed based on the results on Si isotope fractionation.

Dynamics of Si isotope fractionation during uptake 325
The plant performance parameters show that there are two distinctly different Si uptake mechanisms present: an active strategy in wheat, and a rejective strategy in tomato and mustard. Despite these different Si uptake mechanisms, we find preferential uptake of light Si isotopes observed in all three species with the average 1000•ln(α) of -0.41 ± 0.09 ‰ (1 s). We can only speculate on the reasons for the plants' preference for 28 Si over 30 Si. Si is taken up (actively facilitated) through Si permeable channels (orthologues of Lsi1 in rice, maize and barley) and passively with the water flow. Nowhere along these pathways 330 does a change in the coordination sphere of silicic acid occur (Ma et al., , 2007Mitani et al., 2009) which could lead to the preferential incorporation of the heavy Si isotope in the fraction taken up. Thus we speculate that both pathways favour the light isotopologue because of its greater diffusion coefficient (Sun et al., 2008;Weiss et al., 2004), a process for which a predicted maximum isotope fractionation of -1.6‰ (based on Eq. (6)) is expected. While the processes of active and rejective processes governing Si uptake, which induce the stable isotope fractionation, are identical at a given initial concentration in the nutrient solution.

Silicon fractionation between the roots and shoots
The presence or absence of the efflux (Lsi2-like metabolically active) transporter allows to explore its influence on isotope fractionation in the root and during further transport. (1) If Lsi2 has a similar functionality as Lsi1, a preference for the light 28 Si as caused by diffusion should emerge which would be indistinguishable from the passive diffusion in the absence of Lsi2.
(2) Alternatively, the presence of Lsi2 could also induce equilibrium isotope fractionation during a change in the speciation of 350 silicic acid, causing the preferential transport of either 28 Si or 30 Si. (3) The third possibility are indirect effects in the roots such as precipitation of silicic acid in the roots which enrich the remaining silicic acid which is transported into the shoots in heavy 30 Si.
The three crop species show large differences in their root Si isotopic composition. Mustard and spring wheat preferentially 355 store light 28 Si in their roots ( Mustard Δ 30 Siroots -0.77 ± 0.15 ‰, Wheat Δ 30 Siroots -1.04 ± 0.34 ‰, relative to the nutrient solution) whereas tomato does not show a preference for either the lighter or heavier silicon isotopes ( Tomato Δ 30 Siroots -0.01 ± 0.16 ‰).
The further transport of Si from the roots into the xylem seems not be driven by a diffusion process through Lsi2. Thus, hypothesis (1), that Lsi2 has a similar functionality as Lsi1 and transports Si in a diffusive process, is not applicable. For mustard and wheat orthologues of Lsi2 have been shown to be involved in the Si transport (Deshmukh et al., 2016;Sonah et 360 al., 2017). The current understanding of the molecular functionality of Lsi2 however, provides not enough evidence for an equilibrium process where a preferential transport of 30 Si over 28 Si into the xylem would be expected (hypothesis 2).
The isotopic difference between the Si in the shoots and in the roots ( 30 ΔRoot-Shoot) for mustard and wheat, amounts to -0.72 and -0.98 ‰, respectively, and could be explained by precipitation reactions in the roots (for wheat mineral depositions in the 365 roots have been observed see Hodson & Sangster, 1989, supporting hypothesis 3). Precipitation of biogenic silica would enrich the mobile silicon pool in the root in heavy 30 Si, which is then transported into the shoots. Köster et al., 2009, showed that rice mutants with a defective Lsi2 lead to an additional (compared to non-mutants) preferential transport of heavy 30 Si into the https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License.
straw. This could be explained by an oversaturation in the roots due to the missing efflux transporter (Lsi2), leading to additional biogenic silica precipitation in the roots. The positive 30 ΔRoot-Shoot of +0.37 ‰ for tomato, where Lsi2 is absent, 370 indicate that the pool of Si in the roots was depleted in 28 Si by a preferential diffusion process of the lighter isotope.

Implications for terrestrial Si isotope cycling
Several field based studies have investigated the isotope fractionation induced by plants (Cornelis et al., 2011;Ding et al., 2005;Frings et al., 2014;Opfergelt et al., 2010;White et al., 2012) In contrast to field based studies, the stable silicon isotope that the fractionation between roots and shoots is variable in direction and is controlled by internal plant processes, which are likely also being present within subparts of the roots and shoots.
This implies that Si which is liberated through weathering reactions, may be recycled multiple times through plants, redissolved into soil solution and precipitated into secondary minerals before being exported from the ecosystem as biogenic 385 silica, secondary clays, or as dissolved Si. The relative magnitude between these fluxes depends however on the environmental conditions (Frings et al., 2016;Sommer et al., 2006Sommer et al., , 2013. The isotope composition of the dissolved Si in river water shows almost exclusively a heavier silicon isotope signature than the bedrock they drain (Frings et al., 2016;Opfergelt and Delmelle, 2012). To close the Si isotopic mass balance therefore requires an isotopically light solid counterpart (Bouchez et al., 2013).
The plant and phytolith data aggregated by Frings et al., 2016 suggest that biogenic silica is unlikely one of the main export 390 flux of Si from catchments. Plants are an important factor for the internal ecosystem element cycling (Uhlig and von Blanckenburg, 2019), but not for the particulate Si export.

Conclusion
We have confirmed that Si uptake into crop plants is species-specific and complex, involving rejective, passive and active processes in varying proportions. Regardless of the uptake strategy (active and rejective) all three crop species preferentially 395 incorporate light silicon ( 28 Si) with a fractionation factor 1000•ln(α) for tomato -0.33 ‰, for mustard -0.55 ‰ and for wheat -0.43 ‰. Within uncertainty, the fractionation factors between these species are indistinguishable. This similarity indicates that the physico-chemical processes governing Si uptake, whether active or passive, or with Lsi1-like transporters present or absent, are identical. The incorporation and fractionation of stable Si isotope ratios at the root epidermis is likely https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License. governed by the preferential diffusion of the lighter homologue of silicic acid. In contrast at the root endodermis, for species 400 with the Lsi2-like transporter (wheat and mustard), the further transport of silicic acid from the roots into the xylem and shoots is not controlled by the preferential diffusion of light 28 Si. A likely change in the chemical environment in the roots results in the precipitation of biogenic silica, which is enriched in 28 Si over 30 Si. The remaining silicic acid which is transported and deposited in the shoots is thus enriched in 30 Si. For plant species where the biogenic silica precipitation is absent, the transport is governed by a diffusion process and hence preferentially light silicon 28 Si is transported into the shoots. A full description 405 of the isotope and elemental fractionation during transport of silicic acid and precipitation of biogenic silica requires a better understanding of the biological processes and molecules involved in the dehydration of silicic acid to amorphous silica (He et al., 2015;Leng et al., 2009). Here, the toolbox of isotope geochemistry is well-poised, e.g. temporal isotope-spiking experiments during a short period of the plant growth and ripening can deliver insights into the mobility and pathways of newly and formerly acquired silicic acid. 410

Author Contribution
All authors designed the study, D. A. F. and R. R. have grown the plants, D. A. F. has analysed the samples and evaluated the data, prepared the figures. All authors have contributed to the discussion, interpretation and writing of this manuscript.  https://doi.org/10.5194/bg-2020-66 Preprint. Discussion started: 9 April 2020 c Author(s) 2020. CC BY 4.0 License.  Transpiration [L pot -1 ] Pot 11.0 ± 0.3 6.8 ± 1.5 3.2 ± 0.6  Table 2: Ecophysiological performance ratios for mustard, wheat and tomato (means ± standard deviation based on 3 pots with 4 plants each). The uptake classification is based on the ratio of measured and theoretical Si uptake. A ratio of greater than 1 indicates an active uptake mechanism, a ratio much smaller than 1 a rejective strategy and a ratio of 1 is passive uptake. Start -0.20 0.08 -0.25 0.10 -0.23 0.02 End -0.09 0.19 -0.11 0.31 -0.14 0.31 Table 4: Silicon isotope budget (calculated using Eq. (4)) for mustard, wheat and tomato at the start of the experiment (based on the isotopic composition of the nutrient solution) and the end (based on the plants and nutrient solution isotopic composition).