Magnesium Stable Isotope Fractionation on a Cellular Level Explored

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Environmental Processes

Magnesium stable isotope fractionation on a cellular level explored by cyanobacteria and black fungi with implications for higher plants Rasesh Pokharel, Ruben Gerrits, Jan A. Schuessler, Patrick J. Frings, Roman Sobotka, Anna A. Gorbushina, and Friedhelm von Blanckenburg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02238 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Environmental Science & Technology

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Magnesium stable isotope fractionation on a cellular level explored by

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cyanobacteria and black fungi with implications for higher plants

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Rasesh Pokharela,b,*, Ruben Gerritsc,d, Jan A. Schuesslera, Patrick J. Fringsa, Roman Sobotkae

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Anna A. Gorbushinab,c,d, Friedhelm von Blanckenburga,b

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a

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Potsdam, Germany

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b

Institute of Geological Sciences, Freie Universität Berlin, 12249 Berlin, Germany

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c

BAM Federal Institute for Materials Research & Testing, Department 4, Materials & Environment, 12205 Berlin,

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Germany

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d

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e

GFZ German Research Centre for Geosciences, Section 3.3, Earth Surface Geochemistry, Telegrafenberg, 14473

Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, 14195 Berlin, Germany

Institute of Microbiology, Centre Algatech, 379 81 Třebon, Czech Republic

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Abstract *

Corresponding author phone: 0049 331 288-28964; fax: 0049 331 288-2852; email: [email protected]

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ACS Paragon Plus Environment


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In a controlled growth experiment we found that the cyanobacterium Nostoc punctiforme has a

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bulk cell 26Mg/24Mg ratio (expressed as δ26Mg) that is -0.27‰ lower than the growth solution at a

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pH of ca. 5.9. This contrasts with recently published δ26Mg value that was 0.65‰ higher than

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growth solution for the black fungus Knufia petricola at similar laboratory conditions, interpreted

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to reflect loss of

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chlorophyll extract we inferred the δ26Mg value of the main Mg compartments in a cyanobacteria

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cell: free cytosolic Mg (-2.64‰), chlorophyll (1.85‰), and the non-chlorophyll-bonded Mg

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compartments like ATP and ribosomes (-0.64‰). The lower δ26Mg found in Nostoc punctiforme

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would thus result from the absence of significant Mg efflux during cell growth in combination

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with either a) discrimination against 26Mg during uptake by desolvation of Mg or transport across

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protein channels; or b) discrimination against

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The model predicts the preferential incorporation of

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and the absence of isotope fractionation in those high in Mg, corroborated by a compilation of

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Mg isotope ratios from fungi, bacteria, and higher plants.

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Mg during cell growth. By a mass balance model constrained by δ26Mg in

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Mg in the membrane transporter during efflux. 26

Mg in cells and plant organs low in Mg,

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TOC/Abstract art

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Introduction 2


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Magnesium (Mg) is a macronutrient for all living organisms and has a wide range of cellular

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functions. It plays a vital role as a cofactor in many enzymes, is responsible for ATP production

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and is also a requirement for protein synthesis in ribosomes.1,2,3 Further, it is essential for

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photosynthesis as Mg is bound as a central atom in the porphyrin ring of the chlorophyll

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molecule.4,5 Intracellular Mg exists either as free (cytosolic) Mg2+, or as Mg bonded in organic

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compounds like ATP and ribosomes.6,7 The level of free cytosolic Mg2+ is kept within narrow

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limits through cellular transport mechanisms, but a clear picture of the regulation process

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involved has not yet emerged.8,9 The stable isotopes of Mg offer a new approach to exploring

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cellular Mg metabolism. Mg has three stable isotopes:

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abundances of approximately 79, 10 and 11%, respectively. The relative mass differences (ca. 8%

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between

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fractionation) that can be resolved by mass-spectrometric measurements. It is now well

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established that biological processes induce Mg isotope fractionation, but the detailed cellular

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mechanisms

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identified.10,11,12,13,14,15,16,17,18,19,20,21,22

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Mg and

24

Mg,

25

Mg and

26

Mg, with relative

24

Mg) cause subtle variations in the Mg isotope ratios (i.e. isotope

causing

the

observed

isotopic

shifts

have

not

yet

been

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Cell Mg concentrations and isotope ratios measured in previous studies require that the bonded

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and free Mg compartments are in chemical and isotopic equilibrium.15,23 Mg incorporating cell

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components like ATP, ribosomes or chlorophyll - with strong covalent or highly coordinated

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bonds - tends to bind the heavier

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Mg2+.15 The bonded Mg compartment is therefore enriched in the heavier Mg isotopes. The

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bonded Mg compartment also dominates total cellular Mg, amounting to >95% of total cellular

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Mg.23,8,24,25 The enrichment of heavier Mg isotope in the dominant portion of total intracellular

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Mg suggests that in a system where Mg in the cell freely exchanges with its growth environment,

26

Mg isotope preferentially over

3


24

Mg from the free cytosolic


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bulk cell Mg should be isotopically heavier than the Mg in the growth solution. In reality, this is

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not always observed. For example, the Mg isotopic composition of the bulk ectomycorrhizal

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fungi measured by Fahad et al. (2016) shows slight enrichment in the lighter Mg isotopes relative

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to the substrate Mg (about -0.15‰ lower in

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that measured in a non-mycorrhizal fungi in the same study did show the expected enrichment of

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26

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within higher plants show significant variability.10,11,12 In general, roots are isotopically heavier

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than shoots. Assessments of Mg isotope fractionation between chlorophyll and the growth

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solution have also yielded variable results. For example, Black et al. (2006) demonstrated

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enrichment of lighter Mg isotopes with respect to the growth solution in chlorophyll extracted

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from a cyanobacterium (0.71‰ lighter in 26Mg/24Mg), whereas Ra and Kitagawa (2007) showed

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that the chlorophyll of marine red algae incorporates heavier Mg isotopes from the growth

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solution (0.58‰ heavier in

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extracted from a cultured coccolithophore (Emiliania huxleyi) is slightly depleted in heavier Mg

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isotopes than the growth solution, although their observations were complicated by a dependence

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on the simultaneous intracellular production of coccoliths.28

Mg (0.3‰ higher in

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Mg/24Mg than that of the growth solution), while

26

Mg/24Mg than that of the growth solution).14 Mg isotope compositions

26

Mg/24Mg).26,27 Ra et al. (2010) further showed that chlorophyll

85 86

The aim of this study is to apply the stable Mg isotope system to investigate the relationship

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between cell physiology and Mg cellular cycling and to identify mechanisms that explain the

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differing Mg isotope compositions of various biological systems. To this end we investigated Mg

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isotope fractionation by a model phototrophic cyanobacterium Nostoc punctiforme ATCC 29133

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in a well-controlled laboratory growth experiment. N. punctiforme is an aerobic, diazotrophic,

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gram-negative, filamentous and motile cyanobacterium that is ubiquitous at the land surface and

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that has been intensively studied to understand microbial carbon and nitrogen dynamics.29,30 It is 4


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important in ecological studies as it forms symbiotic relationships with plant and fungal species

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in a wide variety of environmental conditions and across large temperature and pH

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gradients.31,32,33. In symbiotic relationships with fungi, N. punctiforme provides carbohydrates

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produced by photosynthesis to fungi that in turn supply nutrients and ensure structural support to

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the Nostoc cells.33,29,

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vegetation-free environments and is successfully used in laboratory biofilm and weathering

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studies.34,35 In this study we determined the Mg isotope composition of the bulk cell N.

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punctiforme, and of the chlorophyll molecules extracted from them. These data and experimental

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results from the growth experiments were combined with mass balance models to predict the

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parameters that control isotope fractionation in a cyanobacterial cell. Finally, we explored the

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applicability of these predictions using and analysing a compilation of published Mg isotope data

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from higher plants and fungi. We thereby aim to identify the unifying mechanisms underlying

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stable Mg isotope fractionation in cells.

33

N. punctiforme also acts as a biological weathering agent even in

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Material and Methods

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Nostoc punctiforme ATCC 29133

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N. punctiforme used in this study was supplied by J.M. Meeks (University of California, Davis,

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CA, USA). As described in Seiffert et al. (2016) the axenic cultures were pre-grown in a BG-11

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media at pH 7.5 25°C and 90 µmole photons of photosynthetically active light m2 s−1 for 24 h

112

d−1 and shaking (160 rpm).35 They were transferred weekly by diluting 1:100 into fresh media.

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After having been grown for 2 weeks, cultures were harvested and used in our Mg uptake

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experiments.

115

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Mg uptake experiments and analytical methods

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In this paper we use the same experimental conditions, growth media, sampling procedures and

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analytical techniques as described in detail in Pokharel et al. (2017).15 The major difference is the

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exchange of the fungus Knufia petricola for the cyanobacterium N. punctiforme. Further, the

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experiments were performed only under pH-buffered conditions since the acidic conditions

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employed in the Knufia petricola study are not favourable for the growth of N. punctiforme. A

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brief description of the experimental setup, sample strategies and the analytical procedure is

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given below; more details can be found in Pokharel et al. (2017).15

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All growth experiments were conducted in triplicates to verify repeatability. Experiments were

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conducted in an aerobic climate chamber (Percival LT-36VL, Percival, USA) under constant

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temperature (25°C) and constant light source (90 µmol photons/m2s). Acid-cleaned 500 mL

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Erlenmeyer flasks were used to perform the growth experiments. The growth solution was a

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modified BG-11 media buffered to pH of 5.9 using the MES buffer (Table S1). About 0.002 g

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(dry weight) pre-grown biomass of N. punctiforme (as described above) was inoculated in these

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media under aseptic conditions. An abiotic control experiment (without N. punctiforme) was run

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in parallel with the biotic experiments. The Erlenmeyer flasks with N. punctiforme biomass were

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shaken at 150 rpm in the aerobic climate chamber and the experiments were ran for 10 days.

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Periodic sampling during the experiment was done with a volumetric pipette while manually

135

shaking to mix the growing biomass with the supernatant. The aliquots obtained were then

136

filtered through sterile 0.2µm filters (MF-Millipore, Merck) to separate the supernatant from the

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biomass. The filtered supernatant solution was used for pH measurements, multi-element

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concentration and Mg isotope analysis and the biomass for growth quantification at the time of

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sampling. At the end of the experiments the entire biomass of N. punctiforme was separated by 6


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centrifugation and analyzed for biomass growth. Elemental concentrations and Mg isotope

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compositions of the final biomass were determined after its dissolution in HNO3/H2O2/HCl/HF in

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screw-top PFA vials on a hotplate at 150°C for >24 hours. The dissolution process was repeated

143

several times until the biomass was completely dissolved.

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The growth of N. punctiforme cells at different sampling intervals was determined by extracting

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DNA followed by quantitative PCR (qPCR) analysis.36 Analytical protocols for elemental

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concentrations and Mg isotopes are identical to those described in Pokharel et al., (2017).15

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Briefly, elemental concentrations (Mg, Fe, K, and P) in the supernatant solution and in the

149

dissolved biomass were analyzed with an Inductively Coupled Plasma Optical Emission

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Spectrometer (ICP-OES, Varian 720-ES). The analysis was performed following the procedure

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described in Schuessler et al. (2016).37 Mg isotope analysis was achieved with Multicollector

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Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS, Thermo Fisher Scientific

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Neptune) after Mg purification by column chromatography (cation exchange resin Bio Rad AG-

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50W-X12 200-400 mesh). Analytical results are expressed in the δ-notation as the parts per

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thousand (‰) deviation of the

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DSM3, δxMg = [((xMg/24Mg)sample / (xMg/24Mg)DSM3) ̶ 1 × 1000], where x = 26 or 25. Based on

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repeated measurements of reference materials processed together with the samples, the

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uncertainty in δ26Mg is estimated at ± 0.1‰ (2SD) and ± 0.06‰ (2SD) in δ25Mg. Further details

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on sample preparation and instrumental setup for qPCR, ICP-OES and MC-ICPMS along with

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quality control results on reference materials and blanks are provided in Pokharel et al. (2017)

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and their Supporting Information.

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The Mg isotope fractionation by chlorophyll molecules in N. punctiforme was determined in a

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separate growth experiment conducted in triplicate. The experimental conditions were identical to

26

Mg/24Mg ratio from the international measurement standard

7



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those of the experiment described above, with the only difference being that no periodic sampling

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of the supernatant was undertaken. At the end of the experiment after 10 days, the N. punctiforme

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biomass was centrifuged. Since the amount of chlorophyll-bound Mg is low in cells, the biomass

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from the three experiments was combined and stored at -20°C before chlorophyll extraction. A

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detailed description of the chlorophyll pigment extraction procedure, separation by HPLC, and

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Mg isotope analysis is provided in the Supporting Information.

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Results and Discussion

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Evolution of pH, cell growth, and uptake of nutrients by N. punctiforme

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As expected under the buffered conditions, the pH of the experiment remained stable and the

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acids excreted during the growth of N. punctiforme were buffered by the MES at a pH of ca. 5.9

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(Figure 1b). This circum-neutral condition is favorable for growth of N. punctiforme as evidenced

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by the increase in biomass during the experiment (Figure 1a). During the experiment N.

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punctiforme consumed about 21 % of Mg from the initial solution (Figure 1e). The measured

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amount of Mg per gram of dry cells of N. punctiforme was 3004 µg/g (Table 1). The major

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nutrients in the growth solution, including K and P, were also consumed during the growth of N.

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punctiforme (Figure 1d and 1f). All Fe initially contained in the growth solution was precipitated

180

as iron (III) oxyhydroxide (Figure 1f), the stable form of Fe at the pH of ca. 5.9, and the fully

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oxygenated conditions of the growth solution.

182 183

Mg isotope fractionation

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All isotope measurements reported follow the expected mass-dependent isotope fractionation line

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in a three-isotope (δ26Mg vs δ25Mg) plot (Figure S1), demonstrating the absence of any

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resolvable mass-independent isotope effect, or polyatomic interferences during measurement. 8


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Hence, we discuss all further Mg isotope data in terms of δ26Mg values. Because the growth

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solution differs in its Mg isotope composition from the reference material DSM3 (used as isotope

189

measurement standard during MC-ICP-MS analysis, Table S3), we report and discuss all

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measured sample data as net isotopic difference to δ26Mg of the initial growth solution:

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∆26Mgsample

̶ solution

= (δ26Mgsample ̶ δ26Mginitial solution) (‰)

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Here, sample can refer to the growth solutions at different sampling times, N. punctiforme

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biomass, or the extracted chlorophyll. N. punctiforme cells at the end of the experiment was

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enriched in the lighter Mg isotopes relative to the growth solution. The calculated

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∆26MgN.punctiforme ̶ solution is -0.27 ± 0.14‰ (2SD, n = 3) (Figure 1g, Table 1, and Table S3). No

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significant changes in isotopic composition of the supernatant solution was observed despite 21%

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Mg uptake (Figure 1e and 1g). This observation is consistent with a Rayleigh isotope

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fractionation model: Mg uptake of 21% and a fractionation of ∆26MgN.punctiforme ̶ solution of -0.27‰

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predicts an increase in δ26Mg of the growth solution by only +0.07‰, which is analytically not

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resolvable. The absence of changes in the composition of the growth solution in both the abiotic

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control experiments and the N. punctiforme growth experiments demonstrates that any potential

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Mg isotope fractionation induced during Mg co-precipitation with iron oxides is not analytically

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resolvable. Mg isotope measurements of chlorophyll extracted from the N. punctiforme biomass

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resulted in a ∆26Mgchlorophyll-solution value of 1.85 ± 0.14‰ (2SD) (Figure 1g, Table 1, and Table

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S4), where the uncertainty derives from error propagation of individual measurements analyzed

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by MC-ICP-MS. The observation of chlorophyll Mg being distinctly heavier than both the

207

growth solution and total cell Mg supports the assumption in Pokharel et al. (2017) that Mg-

208

bearing organic compounds inside the cell preferentially incorporate the heavier Mg isotopes.15 A

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similar trend of heavy Mg isotope incorporation in chlorophyll molecules extracted from leaves

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is consistent with previous laboratory experiments and theoretical calculations.38,18 9



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The question thus arises of why bulk N. punctiforme has lower δ26Mg than the bonded Mg

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compounds (chlorophyll, ATP, or ribosome), when these compounds are the dominant Mg

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carriers in the cell. In the following we use a mass balance model to explain the overall low

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δ26Mg of the N. punctiforme and identify parameters controlling the Mg isotope composition of

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fungi and higher plants. The mass balance equation for the isotope composition of the total cell

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is:

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MCell × δ26MgCell= Mfree × δ26Mgfree + Mchlo × δ26Mgchlo + MATP × δ26MgATP

(1)

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Where M is a mass of Mg and the subscripts Cell, free, chlo and ATP refer to Mg in the total cell,

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as free cytosolic Mg2+, in chlorophyll and in other non-chlorophyll compounds, respectively, and

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δ26Mg their respective isotope composition. The implicit assumption is that these three pools are

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an adequate representation of the complexities of intracellular Mg physiology. By replacing the

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values of δ26Mgchlo and δ26MgATP in eq 1 we get,

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MCell× δ26MgCell = Mfree × δ26Mgfree + Mchlo × (δ26Mgfree + εchlo-free) + MATP × (δ26Mgfree + εATP(2) free)

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Where εtarget-source is a fractionation factor (in ‰) between a target and a source compartment. The

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measured isotopic composition of bulk N. punctiforme cell (δ26MgN.punctiforme) is -0.27‰ and

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δ26Mgchlo of extracted chlorophyll is 1.85‰. Solving eq 2 for δ26Mgfree by assuming that εATP-free

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= 2‰ (Pokharel et al., 2017) and using the respective compartment’s mass fractions (Table 2) a

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δ26Mgfree value of -2.64‰ is obtained.15 From this value and εATP-free of 2‰, δ26MgATP and εchlo-

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free

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range of the value calculated by Moynier et al. (2017) and Black et al. (2007) using ab initio

of -0.64‰ and 4.5‰, respectively, can be calculated. The value for

10


εchlo-free is within the

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models.18,39 To satisfy the conditions that (i) the bulk N. punctiforme cell has a composition

235

of -0.27 ‰ and (ii) the measured δ26Mgchlo to be 1.85‰, our calculation requires that

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nearly double that of εATP-free. The various intracellular Mg compartments of N. punctiforme cells

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and their respective Mg isotopic compositions resulting from these considerations are

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summarized in Table 2.

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We display the relationships among the δ26Mg values of the three Mg compartments that emerge

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from eq. (2) in a closed-reactor type isotope exchange diagram in which the measured bulk cell

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δ26Mg is -0.27‰ lower than the growth solution (Figure 2). While this model explains the

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measured isotope partitioning between the compartments given assumed mass fractions (Table

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2), it does not explain the overall lower δ26Mg of N. punctiforme. This low isotope ratio can

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potentially result from either a) a net negative fractionation during uptake (i.e. discriminating

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against 26Mg) during Mg desolvation of hydrated Mg2+(aq) prior to or during transport across the

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protein channel; or b) a positive fractionation associated with the efflux of Mg from the cell

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through the membrane transporter, leaving the residual cellular Mg pool with lower δ26Mg. Ra et

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al. (2010) indicated that the Mg isotope composition of a coccolithophore cell may be influenced

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by a preferential supply of light Mg isotopes during cellular uptake due to a kinetic isotope effect

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and leakage of heavy Mg isotopes across the membrane.28 For Ca, the discrimination against the

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heavier isotopes during desolvation and transport through the protein channels has already been

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identified.40,41,42 To our knowledge, no work on Mg isotope fractionation in membrane

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transporters has been done. However, Mg equilibrium isotope fractionation factors between

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various aqueous Mg species have been determined by Schott et al. (2016) using ab-initio

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calculations.43 Within the context of magnesite precipitation experiments they predict the δ26Mg

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of solvated Mg to be 0.3 to 0.7‰ higher in δ26Mg than desolvated Mg2+ - which is compatible 11


εchlo-free be


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with explanation a) above: isotope fractionation towards lower δ26Mg before membrane transport

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into the cell.

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Predicting cell δ26Mg with a flux model

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In contrast to the model developed here for N. punctiforme, the substantial enrichment of heavy

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26

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efflux of free cytosolic Mg low in δ26Mg after bonding of heavy Mg into ATP.15 To explore

264

possible mechanisms by which bulk cells obtain δ26Mg that can be either higher or lower than

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that of the growth solution, Pokharel et al. (2017) proposed an equation that relates the net

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isotope fractionation to the relative mass fluxes of Mg into and out of a fungal cell.15 Their

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derivation was based on fluxes of Mg in and out of the cell, and is strictly valid only for a cell

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growing at a constant rate with a constant ratio of bonded and free Mg compartments. The net

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Mg isotopic difference between bulk cell and growth solution was defined as a function of

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cellular Mg fluxes:

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Mg observed by Pokharel (2017) in the fungus Knufia petricola was interpreted to be due to

Δ26Mgbulk cell-solution = εbonded-̶ free [fbonded ̶ (Rbonded /Rin)]

(3)

272

where Rin is the input rate of Mg into the cell [moles sec-1], Rbonded is the net bonding rate of Mg

273

into intracellular Mg bonding organic compounds [moles sec-1];

274

δ26Mgfree is the lumped isotope fractionation between free Mg2+ and Mg bonded (ATP,

275

ribosomes, chlorophyll) and fbonded is the fraction of total cell Mg present in a bonded form

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compartment in the cell (i.e. Mribo+chlo+ATP/Mcell).

12


εbonded ̶free = δ26Mgribo+ATP+chlo ̶

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By replacing Rbonded = Rin – Rout - Rfree 15, where Rout is the rate of Mg efflux from the cell and

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Rfree is the rate of Mg transferred into the free cytosolic compartment [moles sec-1] in eq 3, we

279

obtain

280

Δ26Mgcell ̶solution = εbonded ̶ free [fbonded ̶ (Rin – Rout -Rfree)/Rin]

281

Δ26Mgcell ̶solution = εbonded ̶ free [fbonded ̶ [1 – (Rout/Rin) –(Rfree /Rin)]

(4)

(5)

282

Since, Rfree/Rin gives a very small number (Rin>>Rfree; see Supporting Information for detailed

283

information) the Rfree /Rin term can be neglected and we obtain an expression relating

284

Δ26Mgcell ̶solution to Rout,

285

Δ26Mgcell ̶solution = εbonded ̶ free [fbonded ̶ [1 – (Rout/Rin)]

(6)

286

The complete derivation of this expression is given in the Supporting Information. Equations 3

287

and 6 suggest that the isotopic composition of cells is governed by fbonded and Rbonded/Rin (or

288

alternatively Rout/Rin), assuming that the fractionation between bonded and cytosolic free Mg is

289

constant. It is well established that intracellular mechanisms tightly regulate the partitioning

290

between free and bonded Mg, and that the bonded Mg is the dominant Mg pool in cells.44,45,8 Past

291

investigations on bacterial and yeast cells show that the Mg compartment sizes in these species

292

have a restricted range of fbonded values, ranging from about 0.95 to 0.997 under normal growth

293

conditions.23,8,46 From equation 3, we can deduce that when Rbonded/Rin is less than fbonded,

294

∆26Mgbulkcell ̶ solution is positive. The process by which this comes about is thought to be light Mg in

295

the cell being continuously replaced by unfractionated Mg from fresh intake of bulk growth

296

solution that is isotopically identical to the initial growth solution (∆26Mg = 0‰). The cell can be

297

thus considered to be an open reactor with continuous exchange of Mg. Figure 3 shows the

13



298

resulting isotopic composition for K. petricola cells in which Mg is heavier than the growth

299

solution.

300

We can secondly predict from equation 3 that when Rbonded/Rin is greater than fbonded then

301

∆26Mgbulkcell ̶ solution obtains a value close to zero or slightly lower than the growth solution. In this

302

system Mg enters the cell and is partitioned into 95% of bonded heavy Mg and 5% of free light

303

Mg, but with minimal efflux of light free Mg, unlike in the open system case of K. petricola

304

where the efflux is high. We thus speculate the N. punctiforme cell behaves as an almost closed

305

system where Rout is minimal. Thus although the Mg-bonding organic compounds contain 95% of

306

the cellular Mg in the N. punctiforme and the Mg extracted from the chlorophyll molecule had a

307

distinctly higher δ26Mg value than the growth solution, the bulk cell is close to zero or slightly

308

lower. In this regard it is important to note that N. punctiforme incorporated 10 times more Mg

309

(3004 µg/g of dry cell) than the K. petricola cell. Attaining large intracellular Mg inventories is

310

consistent with all Mg entering into the cell being utilized to make Mg-bonded compounds and

311

no significant efflux of Mg from the cell. Isotope mass balance requires that if there is no efflux

312

of isotopically fractionated Mg the cell must take on the isotopic composition of the growth

313

solution, plus any fractionation associated with influx. The slight negative value of -0.27‰

314

measured in N. punctiforme might be due to fractionation prior to Mg entering the cytoplasm. As

315

mentioned above, lighter isotopes may be preferred during interaction of different Mg species in

316

solution, desolvation, or transfer across the protein channel, for example. If present, a slight

317

preference towards uptake of lighter Mg isotopes was not detectable in the K. petricola cell as it

318

behaved more like open system containing a dominant compartment being isotopically heavy in

319

Mg.

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To summarize, K. petricola cells behave as an open system with low Rbonded and small

321

intracellular Mg inventories that result in high Rout and a high ∆26Mgbulkcell ̶

322

Conversely, N. punctiforme cells behave as a closed system with high Rbonded and large

323

intracellular Mg quotas due to low Rout, resulting in ∆26Mgbulkcell ̶

324

Figure 4 shows the dependence of ∆26Mgbulkcell ̶ solution (for both K. petricola and N. punctiforme)

325

on Rbonded /Rin within the possible range of fbonded values from 0.95 to 0.997. It can be seen from

326

figure 4 that for the measured Mg isotope composition of K. petricola, Rbonded/Rin is close to 0.7,

327

assuming a εbonded ̶ free value of 2.5‰. However, the measured value of N. punctiforme is beyond

328

the lower boundary limit of ∆26Mgbulkcell ̶

329

supporting our speculation of Mg isotope fractionation during Mg entering or leaving the

330

cytoplasm of N. punctiforme.

331

Mg cycling in higher plants

332

Both laboratory and field experiments have shown that bulk higher plants are enriched in the

333

heavier Mg isotopes over the source from which they obtain their Mg.10,11,12,16,17,22,21 Further, the

334

Mg isotope composition of different parts of higher plants shifts due to Mg isotope fractionation

335

during Mg translocation within the plants.11,10 Studies by Bolou-Bi et al. (2010 and 2012) show

336

that the leaves of plants typically obtain high Mg concentrations and are enriched in the lighter

337

Mg isotopes relative to their roots that have lower Mg concentrations and are enriched in the

338

heavier Mg isotopes.11,12 In order to assess whether a relationship between concentration and Mg

339

isotope composition is universal we compiled all Mg isotope available data on compartments of

340

higher plants (roots, leaves and needles) in addition to fungi, and cyanobacteria (this study), and

341

their Mg sources, measured in both laboratory and field experiments (Figure 5 and Table S2). We

342

excluded Mg isotope data on wood because wood serves as a pathway of Mg from roots to leaves

solution

15

solution

solution

value.

values close to zero.

values calculated using equation 3, further



343

and also as a store of Mg that was resorbed from leaves, for example during autumnal

344

senescence.11,19 Given that translocation and precipitation for storage may entail additional

345

isotope fractionation, Mg from wood likely does not reflect the isotope compositions of the

346

cellular processes we explore here. In Figure 5 an inverse relationship between Mg concentration

347

and the Mg isotope composition becomes apparent from both laboratory growth experiments and

348

ecosystem samples. The scatter in the relationship partially reflects discrepancies in identifying

349

the exact composition of the Mg source of species sampled in natural ecosystems. Both

350

laboratory and field data show that low Mg concentrations amounts to high ∆26Mgspecies ̶source

351

values, whereas at high Mg concentrations, ∆26Mgspecies ̶

352

relationship also holds for what we observed for K. petricola and N. punctiforme cells. With this

353

relationship we can now explain the observation of Fahad et al. (2017), namely that non-

354

mycorrhizal fungi with low Mg concentration features high positive ∆26Mgspecies ̶source values

355

whereas a mycorrhizal fungi with high Mg concentration yields ∆26Mgspecies ̶ source close to zero or

356

even slightly negative.14 In terms of our mass balance model, high Mg concentrations and low

357

δ26Mg correspond to low Rout/Rin or high Rbonded/Rin.

358

The Mg metabolic pathway that governs the isotopic composition of K. petricola and N.

359

punctiforme cells, respectively, may thus serve as a model for roots and leaves of higher plants.

360

Within the framework suggested by Pokharel et al. (2017) we suggest that Mg enters the root

361

cell, possibly with a minor isotope fractionation favoring the lighter Mg isotopes during

362

desolvation or in transport channels, that heavy Mg isotopes are incorporated into the 95% of Mg

363

contained in the root cells’ bonded compounds (as seen for K. petricola), and that the effluxed

364

free Mg thus depleted in heavy Mg isotopes is preferentially transported through the xylem into

365

the leaves.15 There it becomes further fractionated with preference of light Mg isotopes during

366

desolvation or transport across the protein channel of the leaf cells similar to the fractionation 16

source


tends to be closer to zero. This

Page 16 of 28

Page 17 of 28


367

observed in N. punctiforme cells. Inside the leaf cells, most of the Mg is partitioned into Mg-

368

ATP, ribosomes and the chlorophyll molecule and thus due to the high requirements of Mg by

369

these bonding compounds there is little efflux of light free Mg2+ from the leaf. During growth the

370

leaf thus behaves as a closed system and is thus depleted in

371

model can explain why the root cells exhibit elevated δ26Mg, while leaf cells show low δ26Mg.

372

During autumnal senescence some of the Mg present in the leaves is withdrawn into the plants

373

and remobilized during growth seasons, whereas the remaining is returned to the soil as plant

374

litter fall.47,19,17 We speculate that both the fraction of Mg withdrawn from the leaves during

375

resorption and the fraction returned to soil as litter fall determine the Mg isotope composition of

376

the bulk plants.

26

Mg compared to the roots. This

377 378 379 380

Environmental Implications

381

Cyanobacteria are ubiquitous colonizers of natural rock surfaces and building stones. They

382

acidify their environment by releasing organic acids or locally increase the pH by photosynthesis

383

and hence could stimulate weathering of the substrates they inhabit. They take the nutrients

384

released from minerals, use them for photosynthesis and transfer them to fungi and higher plants

385

through symbiotic relationships. Together with fungi they form lichens that cover about 8% of

386

Earth’s terrestrial surface.48 Given that large fluxes of mineral nutrients pass through this

387

bacterial and fungal pool, methods that fingerprint the partitioning of nutrients through both

388

species might deliver crucial information for nutrients cycling studies in ecosystems.49,50 Mg

389

stable isotope ratios serve to track these cycles. Knowing the fractionations factors and the 17



390

environmental conditions that set them is an essential prerequisite to their application. We found

391

a fractionation factor of -0.27‰ for uptake of Mg into Nostoc punctiforme when grown at

392

circum-neutral conditions. In contrast, uptake of Mg into a black fungus as explored in a previous

393

study involves a fractionation factor of +0.65 and +1.1‰, depending on pH.15 These results show

394

that in general Mg stable isotope fractionation in ecosystems depend on species and on

395

environmental conditions, including pH, that determine the fluxes of Mg into root cells or

396

symbionts.

397

More broadly, we inferred that it is the rate of Mg bonding into intracellular compartments

398

relative to the rate of Mg intake into and efflux from the cell that governs the Mg isotopic

399

composition of cells, and here we present a mathematical expression that describes this

400

relationship. A data compilation of Mg isotope fractionation in bacteria, fungi, lichens and higher

401

plants suggests that this relationship holds for a broad range of species, given that Mg isotope

402

fractionation factors are inversely proportional to cellular Mg concentrations. This study thus

403

provides an internally consistent framework of Mg cycling through different types of cells and

404

supplies an explanation for preferred intake of heavy Mg isotopes into plant organs low in Mg,

405

and the apparent absence of isotope fractionation during intake into plant organs high in Mg.

406

Clearly, there are still parameters, pathways and processes that need further attention. For

407

example, the possibility of Mg isotope fractionation during cell influx or efflux, and intracellular

408

fractionation occurring during Mg translocation and resorption mechanism are important next

409

steps to explore.

410 411

Supporting Information

18


Page 18 of 28

Page 19 of 28


412

Methods: Chlorophyll extraction, HPLC separation and derivation of cellular Mg flux model.

413

Tables: Experimental growth solution composition (Table S1), compilation of published data on

414

Mg concentration and isotopes analysis in plants and growth experiments (Table S2),

415

concentration and isotope analysis of individual biomass and growth solution samples (Table S3),

416

and Mg isotope analysis of growth experiment for chlorophyll extraction (Table S4). Figure S1:

417

Three isotope plot.

418 419

Acknowledgements

420

This research is the contribution of the ISONOSE Marie Curie Research training network funded

421

from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework

422

Programme FP7/2007-2013/ under REA grant agreement no (608068). Dr, Roman Sobotka was

423

supported by the Czech Ministry of Education (project LO1416). We gratefully acknowledge

424

Jutta Schlegel and Josefine Buhk for help during clean lab sample preparation and Mg isotope

425

measurements in the Helmholtz Laboratory for the Geochemistry of the Earth Surface (HELGES)

426

at GFZ Potsdam. Comments by three anonymous reviewers were highly appreciated and helped

427

to improve the clarity of the manuscript.

428

References

429 430 431 432 433 434 435 436 437 438

1. Maguire, M.; Cowan, J., Magnesium chemistry and biochemistry. BioMetals 2002, 15, (3), 203210. 2. Moomaw, A. S.; Maguire, M. E., The Unique Nature of Mg(2+) Channels. Physiology (Bethesda) 2008, 23, (5), 275-285. 3. Pasternak, J.; Kocot, J.; Horecka, A., Biochemistry of magnesium. J. Elementol. 2010, 15, (3), 601616. 4. Katz, J. J., Coordination Properties of Magnesium in Chlorophyll from IR and NMR Spectra. In Developments in Applied Spectroscopy: Selected papers from the Eighteenth Annual Mid-America Spectroscopy Symposium Held in Chicago, Illinios May 15–18, 1967, Baer, W. K.; Perkins, A. J.; Grove, E. L., Eds. Springer US: Boston, MA, 1968; pp 201-218.

19



439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486

5. Bohn, T.; Walczyk, T.; Leisibach, S.; Hurrell, R. F., Chlorophyll-bound Magnesium in Commonly Consumed Vegetables and Fruits: Relevance to Magnesium Nutrition. J. Food Sci. 2004, 69, (9), S347S350. 6. Silver, S., Active transport of magnesium in Escherichia coli Proc. Natl. Acad. Sci. U.S.A 1969, 62, (3), 764-771. 7. Quamme, G. A., Molecular identification of ancient and modern mammalian magnesium transporters. American Journal of Physiology-Cell Physiology 2010, 298, (3), C407-C429. 8. Alatossava, T.; Jütte, H.; Kuhn, A.; Kellenberger, E., Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187. J. Bacteriol. 1985, 162, (1), 413-419. 9. Grubbs, R. D., Intracellular magnesium and magnesium buffering. BioMetals 2002, 15, (3), 251259. 10. Black, J.; Emanuel Epstein; William D. Rains; Yin, Q.-z.; Casey, W. H., Magnesium-Isotope Fractionation During Plant Growth. Environ. Sci. Technol. 2008, 42, (21), 7831-7836. 11. Bolou-Bi, E. B.; Poszwa, A.; Leyval, C.; Vigier, N., Experimental determination of magnesium isotope fractionation during higher plant growth. Geochim. Cosmochim. Acta 2010, 74, (9), 2523-2537. 12. Bolou-Bi, E. B.; Vigier, N.; Poszwa, A.; Boudot, J.-P.; Dambrine, E., Effects of biogeochemical processes on magnesium isotope variations in a forested catchment in the Vosges Mountains (France). Geochim. Cosmochim. Acta 2012, 87, 341-355. 13. Schmitt, A.-D.; Vigier, N.; Lemarchand, D.; Millot, R.; Stille, P.; Chabaux, F., Processes controlling the stable isotope compositions of Li, B, Mg and Ca in plants, soils and waters: A review. C. R. Geosci. 2012, 344, (11–12), 704-722. 14. Fahad, Z. A.; Bolou-Bi, E. B.; Köhler, S. J.; Finlay, R. D.; Mahmood, S., Fractionation and assimilation of Mg isotopes by fungi is species dependent. Environ. Microbiol. Rep. 2016, 8, (6), 956-965. 15. Pokharel, R.; Gerrits, R.; Schuessler, J. A.; Floor, G. H.; Gorbushina, A. A.; von Blanckenburg, F., Mg Isotope Fractionation during Uptake by a Rock-Inhabiting, Model Microcolonial Fungus Knufia petricola at Acidic and Neutral pH. Environ. Sci. Technol. 2017, 51, (17), 9691-9699. 16. Tipper, E. T.; Gaillardet, J.; Louvat, P.; Capmas, F.; White, A. F., Mg isotope constraints on soil pore-fluid chemistry: Evidence from Santa Cruz, California. Geochim. Cosmochim. Acta 2010, 74, (14), 3883-3896. 17. Uhlig, D.; Schuessler, J. A.; Bouchez, J.; Dixon, J. L.; von Blanckenburg, F., Quantifying nutrient uptake as driver of rock weathering in forest ecosystems by magnesium stable isotopes. Biogeosciences 2017, 14, (12), 3111-3128. 18. Moynier, F.; Fujii, T., Theoretical isotopic fractionation of magnesium between chlorophylls. Sci. Rep 2017, 7, (1), 6973. 19. Kimmig, S. R.; Holmden, C.; Bélanger, N., Biogeochemical cycling of Mg and its isotopes in a sugar maple forest in Québec. Geochim. Cosmochim. Acta 2018, 230, 60-82. 20. Chapela Lara, M.; Buss, H. L.; Pogge von Strandmann, P. A. E.; Schuessler, J. A.; Moore, O. W., The influence of critical zone processes on the Mg isotope budget in a tropical, highly weathered andesitic catchment. Geochim. Cosmochim. Acta 2017, 202, 77-100. 21. Opfergelt, S.; Burton, K. W.; Georg, R. B.; West, A. J.; Guicharnaud, R. A.; Sigfusson, B.; Siebert, C.; Gislason, S. R.; Halliday, A. N., Magnesium retention on the soil exchange complex controlling Mg isotope variations in soils, soil solutions and vegetation in volcanic soils, Iceland. Geochim. Cosmochim. Acta 2014, 125, 110-130. 22. Mavromatis, V.; Prokushkin, A. S.; Pokrovsky, O. S.; Viers, J.; Korets, M. A., Magnesium isotopes in permafrost-dominated Central Siberian larch forest watersheds. Geochim. Cosmochim. Acta 2014, 147, 76-89. 23. Nierhaus, K. H., Mg(2+), K(+), and the Ribosome. J. Bacteriol. 2014, 196, (22), 3817-3819.

20


Page 20 of 28

Page 21 of 28

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533


24. Romani, A.; Scarpa, A., Regulation of cell magnesium. Arch. Biochem. Biophys 1992, 298, (1), 112. 25. Froschauer, E. M.; Kolisek, M.; Dieterich, F.; Schweigel, M.; Schweyen, R. J., Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica. FEMS Microbiol. Lett. 2004, 237, (1), 49-55. 26. Black, J. R.; Yin, Q.-z.; Casey, W. H., An experimental study of magnesium-isotope fractionation in chlorophyll-a photosynthesis. Geochim. Cosmochim. Acta 2006, 70, (16), 4072-4079. 27. Ra, K.; Kitagawa, H., Magnesium isotope analysis of different chlorophyll forms in marine phytoplankton using multi-collector ICP-MS. J. Anal. At. Spectrom. 2007, 22, (7), 817-821. 28. Ra, K.; Kitagawa, H.; Shiraiwa, Y., Mg isotopes in chlorophyll-a and coccoliths of cultured coccolithophores (Emiliania huxleyi) by MC-ICP-MS. Mar. Chem. 2010, 122, (1), 130-137. 29. Meeks, J. C.; Elhai, J.; Thiel, T.; Potts, M.; Larimer, F.; Lamerdin, J.; Predki, P.; Atlas, R., An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium. Photosynthesis Res. 2001, 70, (1), 85-106. 30. Lindberg, P.; Schütz, K.; Happe, T.; Lindblad, P., A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133. Int. J. Hydrog. Energy 2002, 27, (11), 1291-1296. 31. Meeks, J. C., Symbiosis between Nitrogen-Fixing Cyanobacteria and Plants. Bioscience 1998, 48, (4), 266-276. 32. Sand-Jensen, K., Ecophysiology of gelatinous Nostoc colonies: unprecedented slow growth and survival in resource-poor and harsh environments. Ann. Bot. 2014, 114, (1), 17-33. 33. Ekman, M.; Picossi, S.; Campbell, E. L.; Meeks, J. C.; Flores, E., A Nostoc punctiforme Sugar Transporter Necessary to Establish a Cyanobacterium-Plant Symbiosis. Plant Physiol. 2013, 161, (4), 1984-1992. 34. Gorbushina, A. A.; Broughton, W. J., Microbiology of the Atmosphere-Rock Interface: How Biological Interactions and Physical Stresses Modulate a Sophisticated Microbial Ecosystem. Annu. Rev. Microbiol. 2009, 63, (1), 431-450. 35. Seiffert, F.; Bandow, N.; Kalbe, U.; Milke, R.; Gorbushina, A. A., Laboratory Tools to Quantify Biogenic Dissolution of Rocks and Minerals: A Model Rock Biofilm Growing in Percolation Columns. Front Earth Sci 2016, 4, (31). 36. Martin-Sanchez, P. M.; Gorbushina, A. A.; Kunte, H.-J.; Toepel, J., A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae. Biofouling 2016, 32, (6), 635-644. 37. Schuessler, J. A.; Kämpf, H.; Koch, U.; Alawi, M., Earthquake impact on iron isotope signatures recorded in mineral spring water. Journal of Geophysical Research: Solid Earth 2016, 121, (12), 85488568. 38. Black, J. R.; Yin, Q.-z.; Rustad, J. R.; Casey, W. H., Magnesium Isotopic Equilibrium in Chlorophylls. J. Am. Chem. Soc. 2007, 129, (28), 8690-8691. 39. Black, J.; Yin, Q.-Z.; Casey, W. H., Magnesium isotopic fractionation in chlorophyll-a. 2006; Vol. 70. 40. Gussone, N.; Eisenhauer, A.; Heuser, A.; Dietzel, M.; Bock, B.; Böhm, F.; Spero, H. J.; Lea, D. W.; Bijma, J.; Nägler, T. F., Model for kinetic effects on calcium isotope fractionation (δ44Ca) in inorganic aragonite and cultured planktonic foraminifera. Geochim. Cosmochim. Acta 2003, 67, (7), 1375-1382. 41. Gussone, N.; Langer, G.; Thoms, S.; Nehrke, G.; Eisenhauer, A.; Riebesell, U.; Wefer, G., Cellular calcium pathways and isotope fractionation in Emiliania huxleyi. Geology 2006, 34, (8), 625-628. 42. Mejía, L. M.; Paytan, A.; Eisenhauer, A.; Böhm, F.; Kolevica, A.; Bolton, C.; Méndez-Vicente, A.; Abrevaya, L.; Isensee, K.; Stoll, H., Controls over δ44/40Ca and Sr/Ca variations in coccoliths: New perspectives from laboratory cultures and cellular models. Earth Planet. Sci. Lett. 2018, 481, 48-60.

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534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556

43. Schott, J.; Mavromatis, V.; Fujii, T.; Pearce, C. R.; Oelkers, E. H., The control of carbonate mineral Mg isotope composition by aqueous speciation: Theoretical and experimental modeling. Chem. Geol. 2016, 445, 120-134. 44. Gardner, R. C., Genes for magnesium transport. Curr. Opin. Plant Biol. 2003, 6, (3), 263-267. 45. Moncany, M. L. J.; Kellenberger, E., High magnesium content ofEscherichia coli B. Experientia 1981, 37, (8), 846-847. 46. Beeler, T.; Bruce, K.; Dunn, T., Regulation of cellular Mg2+ by Saccharomyces cerevisiae. Biochim. Biophys. Acta 1997, 1323, (2), 310-318. 47. Proe, M. F.; Midwood, A. J.; Craig, J., Use of Stable Isotopes to Quantify Nitrogen, Potassium and Magnesium Dynamics in Young Scots Pine (Pinus sylvestris). New Phytol. 2000, 146, (3), 461-469. 48. Chen, J.; Blume, H.-P.; Beyer, L., Weathering of rocks induced by lichen colonization — a review. CATENA 2000, 39, (2), 121-146. 49. Landeweert, R.; Hoffland, E.; Finlay, R. D.; Kuyper, T. W.; van Breemen, N., Linking plants to rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol. Evol. 2001, 16, (5), 248-254. 50. Gadd, G. M., Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology 2010, 156, 609-643. 51. Utkilen, H. C., Magnesium-limited Growth of the Cyanobacterium Anacystis nidulans. Microbiology 1982, 128, (8), 1849-1862. 52. Marschner, H., 8 - Functions of Mineral Nutrients: Macronutrients. In Mineral Nutrition of Higher Plants (Second Edition), Academic Press: London, 1995; pp 229-312. 53. Schuessler, J. A.; von Blanckenburg, F.; Bouchez, J.; Uhlig, D.; Hewawasam, T., Nutrient cycling in a tropical montane rainforest under a supply-limited weathering regime traced by elemental mass balances and Mg stable isotopes. Chem. Geol. 2018, https://doi.org/10.1016/j.chemgeo.2018.08.024.

557 558 559 560 561 562 563 564 565 566

Tables

Table 1: Mg concentration and isotope composition analysis of growth media 22


Page 22 of 28

Page 23 of 28


and biomass

567 568 569 570 571 572 573 574 575

Mg mass in initial BG11 media (µg) ± 2SDa

Mg mass in initial biomass (µg)

1449 ± 72

1.27

Dry weight of final biomass (g) ± 2SDc

Mg amount in final BG11 media (µg) ± 2SDa

Mg amount in final biomass (µg) ± SDa

Mg concentration in final biomass (µg of Mg/gram of dry cells) ± 2SDa

Mg isotopic difference (∆ ∆26Mg = 26 δ Mgchlorophyll δ26Mgsolutiond) (‰) ± 2SDb

Mg isotopic difference (∆ ∆26Mg = 26 δ MgN.punctiforme - δ26Mgsolutione) (‰) ± 2SDb

0.07 ± 0.02

1142 ± 57

208 ± 44

3004 ± 190

1.85± 0.14

-0.27 ± 0.14

a

2SD represents analytical uncertainties from the triplicates measurements of each individual experiments or the uncertainties derived for individual measurements by ICP-OES, whichever was larger. b2SD represents the error propagation of individual measurements analyzed by MC-ICP-MS. c2SD represents uncertainties derived from repeatability of three independent experiments. dThe δ26Mgsolution value of –1.05 ± 0.1‰ (2SD, n=3) relative to DSM3 was measured for the input solution of the independent chlorophyll experiment (Table S4). eThe δ26Mgsolution value of -4.50 ± 0.1‰ (2SD, n=11) relative to DSM3 was measured for the initial input solution and time-series sampling confirmed that it stayed constant over the time of the experiment.

576 577 578 579

580 581 582 583 584 585 586 587 588 589 590 591 592

Table 2: Intracellular Mg compartments with their size and respective Mg isotopic compositions. Mass Compartment Name Isotope Composition (δ26Mg) Fraction Free Mg

0.05a

δ26Mgfreed

Chlorophyll

0.19b

(δ26Mgfree + εchlo-freee) = 1.85 ± 0.14 (2SD)

ATP

0.76c

(δ26Mgfree + εATP-freef)

a

Mass Fraction of free Mg in the cell, assuming that the fraction of bonded (chlorophyll- and ATP-bound) Mg is 0.95. b Chlorophyll-bound Mg fraction; i.e. 20% of bonded Mg (20% of 0.95).51,52 c Fraction of non-chlorophyll bonded Mg compartment (ATP, and ribosomes) (80% of 0.95). d Mg isotopic composition of intracellular free Mg2+. e Mg isotope fractionation factor between chlorophyll and free Mg in ‰. f Mg isotope fractionation factor between ATP and free Mg, i.e. εATP-free = 2‰, similar to the assumption made by Pokharel et al. (2017).15

Figures

23



593 594 595 596 597 598 599 600 601 602 603 604 605 606 607

Figure 1: Documentation of parameters of the growth experiment as a function of run time. Open circles represent parameters of the solution in the abiotic experiments, the green circles represent solution parameters of N. punctiforme experiments, the red square represents the difference in δ26Mg between N. punctiforme biomass and growth solution, and the yellow diamond the difference in δ26Mg between chlorophyll extracted from N. punctiforme and the growth solution. All uncertainty bars represent 2SD. The uncertainty of the graphs a, b, c, d, e, and f was derived from the analytical uncertainty or the repeatability of independent growth experiments, whichever was larger. The uncertainty on the graph g represent the error propagation of individual measurements analyzed by MC-ICP-MS. Detailed information on both types of uncertainty on all data points are given in the Supporting Information (Table S3 and S4). In graph g, the ∆26Mgsample−solution is the isotopic difference between either the biomass of N. punctiforme or the growth solution at respective sampling time or the chlorophyll extracted and the initial growth solution. For plots where no uncertainty is shown, the uncertainty is smaller than the symbols.

24


Page 24 of 28

Page 25 of 28


608 609 610 611

Figure 2: Isotope exchange diagram assuming a closed reactor for cyanobacterium N. punctiforme of which the measured bulk cell δ26Mg is -0.27‰ relative to the growth solution (grey dashed line). The solid, blue, orange, and green lines represent Mg isotope values of free-, ATP/ribosome-, and chlorophyll-

612

bonded Mg, respectively (expressed relative to the initial growth solution as ∆26Mgcell-solution). Here εchlo-free

613 614 615 616

is 4.5‰, and εATP-free is 2‰. The black dashed arrows indicate the resulting Mg isotope values of free, ATP/ribosome-bound, and chlorophyll-bound Mg if the mass fraction of bonded Mg is 0.95 (5% free Mg and 95% bonded Mg in cells).

617 618

25



619 620 621 622 623 624 625 626 627 628 629

Figure 3: Isotope exchange diagram assuming an open reactor for fungus K. petricola in which bonded and free Mg exchange at chemical and isotopic equilibrium. The solid blue line represents the Mg isotope values of free Mg, the solid orange line that of bonded Mg (expressed relative to the initial growth solutions as ∆26Mgcell-solution).. A fractionation factor εbonded-free of 2‰ is assumed between free and bonded. The grey dashed line is the measured isotope fractionation between bulk K. petricola cell and the initial growth solution measured by Pokharel et al. (2017) (∆26Mgk.petricola-solution = 0.65‰).15 The green arrow shows the direction of the Mg isotopic shift of free Mg that is continuously being replaced by unfractionated Mg from fresh intake of bulk growth solution that is isotopically identical to the initial growth solution (∆26Mg = 0‰). Black arrow shows the resulting isotopic composition of free and bonded Mg, respectively, at a fraction of bonded Mg of 0.95.

630 631 632

26


Page 26 of 28

Page 27 of 28


633 634 635

Figure 4: Dependence of ∆26Mgcell ̶ solution on Rbonded/Rin following eq (3). The area under the oblique grey line shows this dependence for fbonded values of 0.95 to 0.997, the range typically observed in fungal and

636

cyanobacterial cells.

637 638 639 640 641 642 643 644 645 646

free of 2‰. The blue dashed line represents the measured isotopic composition of bulk N. punctiforme cell with respect to the growth solution (∆26MgN.punctiforme-solution = -0.27‰), and the black dashed line that of K. petricola, respectively (∆26MgK.petricola-solution =0.65‰) measured in this study and by Pokharel et al. (2017).15 The red arrows denote the range of Rbonded/Rin values derived from the intersection of the measured ∆26Mg K.petricola-solution with the grey model line. The measured value of ∆26Mg N.punctiforme-solution does not intersect with the oblique grey lines indicating a very high Rbonded/Rin and minor Mg isotope fractionation during Mg entering or leaving the cytoplasm of N. punctiforme, respectively (denoted by a green arrow).

εbonded-free of 2.5‰ was derived by weighing individual fractionation factors (εbondedfree) by their respective Mg mass fractions: 20% chlorophyll with εchlo-free of 4.5‰ and 80% ATP with εATP-

647 648

27



649 650 651 652 653 654 655 656 657 658 659 660 661 662

Figure 5: Compilation of Mg isotope composition relative to the Mg source (∆26Mgspecies ̶source) measured in fungal and cyanobacterium cells and the organs of plants with respect to the concentration of Mg in the species from both laboratory growth experiments (red circles) and field studies (blue diamonds). Data are compiled from Bolou-Bi et al. (2010): rye grass and clover (leaves, stem, and shoots); Bolou-Bi et al. (2012): Norway spruce and hair grass (needles, roots, and leaves); Black et al. (2008): wheat (root, leaves and shoot); Uhlig et al. (2017): ponderosa pine, jeffrey pine, manzanita, whitethorn (needles, root, and leaves); Opfergelt et al. (2014): grass; Kimmig et al. (2018): sugar maple (root, leaf); Mavromatis et al. (2014): sphagnum, shrub (leaves), lichen, larch (roots and needles); Fahad et al. (2017): mycorrhizal and non-mycorrhizal fungi; Schuessler et al. (2018): Eurya japonica, Maesa indica, Neolitsea fuscata, Cestrum aurantiacum, Ixora calycina, Arundinaria debilis (leaves); Pokharel et al. (2017): Knufia petricola and melanin-mutant; this study (Nostoc punctiforme).11,12,10,17,21,19,22 14,15,53 Mg isotope data from woody plant parts was not used as it may be affected by secondary isotope fractionation. Details on the data sources and Mg source compositions is provided in Table S2 in the Supporting Information.

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Magnesium Stable Isotope Fractionation on a Cellular Level Explored

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