Zinc, iron, and copper isotopic fractionation in Elsholtzia splendens Nakai: A study of elemental uptake and (re)translocation mechanisms

Journal Pre-proofs Zinc, iron, and copper isotopic fractionation in Elsholtzia splendens Nakai: a study of elemental uptake and (re)translocation mechanisms Shi-Zhen Li, Xiang-Kun Zhu, Long-Hua Wu, Yong-Ming Luo PII: DOI: Reference:

S1367-9120(20)30002-X https://doi.org/10.1016/j.jseaes.2020.104227 JAES 104227

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Journal of Asian Earth Sciences

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19 November 2019 30 December 2019 3 January 2020

Please cite this article as: Li, S-Z., Zhu, X-K., Wu, L-H., Luo, Y-M., Zinc, iron, and copper isotopic fractionation in Elsholtzia splendens Nakai: a study of elemental uptake and (re)translocation mechanisms, Journal of Asian Earth Sciences (2020), doi: https://doi.org/10.1016/j.jseaes.2020.104227

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Zinc, iron, and copper isotopic fractionation in Elsholtzia splendens Nakai: a study of elemental uptake and (re)translocation mechanisms

Shi-Zhen Lia,, Xiang-Kun Zhua,*, Long-Hua Wub, Yong-Ming Luob

a

MNR Key Laboratory of Isotope Geology, MNR Key Laboratory of Deep-Earth Dynamics, Institute of Geology,

Chinese Academy of Geological Sciences, Beijing 100037, China b Key

Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of

Sciences, Nanjing 210008, China

*Corresponding author E-mail address: [email protected] (X.-K. Zhu),

Abstract Zinc, Fe and previously reported Cu isotopic compositions were applied in a systematic investigation of uptake and translocation in Cu-tolerant Elsholtzia splendens Nakai. They exhibit characteristic isotopic fractionation during uptake: non-specific uptake for Zn, direct uptake as Fe(Ш)-complexes as strategy II species and a prerequisite of Cu(II) reduction to Cu(I) before uptake of Cu. There are, however, differences during translocation from stem and branch to leaf and flower. Light Zn and Fe isotopic compositions of flowers are dependent on their phloem pool, with enrichment in branch and stem. The similarity of Zn and Fe isotopic compositions in flowers, regardless of height, implies uniform mobility in phloem. Cu isotopic composition displays marked variations in leaves and flowers with height. Re-translocation from old tissue to new tissue has a significant influence on metal isotopic compositions in leaves and flowers during the late life stage. Light Zn and Fe isotopes are exported from leaves by transporters, leaving heavy isotopes in the remaining fraction. Cu readily forms complexes, and its heavy isotope is retranslocated preferentially and then light isotope by transporter proteins from old leaves dependent on the degree of remobilization. The remobilization efficiency follows the trend Zn < Fe < Cu, opposite to the trend of metal mobility in xylem and phloem (Zn > Fe > Cu).

Keywords: Zn isotopes; Fe isotopes; Cu isotopes; uptake; translocation; remobilization

1. Introduction

Nutrient elements Zn, Fe, and Cu play vital roles in the metabolism and growth of plants over their entire life cycle. Isotopic fractionation of Zn, Fe, and Cu has been found to occur during plant growth (Weiss et al., 2005; Guelke and von Blanckenburg, 2007;Weinstein et al., 2011；Li et al., 2016). This process has been well studied in recent years and is used as a tool in elucidating translocation mechanisms involved in plant metabolism in bio-geosphere cycles (e.g., Viers et al., 2007; Aucour et al., 2011, 2016; Guelke and von Blanckenburg, 2012; Jouvin et al., 2012; Tang et al., 2012, 2016; Moynier et al., 2013; Arnold et al., 2015; Li et al., 2016; Garnier et al., 2017; Araújo et al., 2018; Dinis et al., 2018; Khoshgoftarmanesh et al., 2018; Kříbek et al., 2018). Zn exists as Zn(II) in plants and is the only metal present in all six major classes of enzymes, which have catalytic, regulatory, and structural roles (Coleman, 1992; Vallee and Auld, 1992). It is also involved in the regulation of DNA transcription and transduction of intra- and inter-cellular signaling (Broadley et al., 2007; Maret, 2013), whereas Fe exists as Fe(II) and Fe(III) in plants, mainly in chloroplasts, and is involved in photosynthesis, heme biosynthesis, and Fe−S cluster assembly (Marschner, 1995). Cu exists as Cu(I) and Cu(II) in plants and participates in protein synthesis, membrane activity, and photosynthesis (Burkhead et al., 2009). The study of Zn, Fe, and Cu isotopic fractionation should advance our understanding of elemental homeostasis in oganism and interactions between transition biometals (Zhu et al., 2002). Isotopic compositions of Zn, Fe, and Cu in individual tissues have been reported

during the plant growth process. Roots accumulate heavy isotopes of Zn (Weiss et al., 2005; Viers et al., 2007; Caldelas et al., 2011; Jouvin et al., 2012; Couder et al., 2015; Tang et al., 2016) and light isotopes of Fe and Cu (Guelke and von Blanckenburg, 2012; Jouvin et al., 2012; Moynier et al., 2013; Ryan et al., 2013; Li et al., 2016) in most plants relative to soil. In the stem, no isotopic fractionation occurs during upward transport of Zn in bamboo (Moynier et al., 2009), Cu in E. splendens Nakai (Li et al., 2016), or Fe in kentucky bluegrass and johnsongrass (Moynier et al., 2013). Leaves and shoots are isotopically light in Zn compared with the rest of the plant, including hyper-accumulating plants at growth stages with Zn sufficiency or deficiency (Weiss et al., 2005; Viers et al., 2007, 2015; Moynier et al., 2009; Caldelas et al., 2011; Jouvin et al., 2012; Tang et al., 2012, 2016; Deng et al., 2014; Couder et al., 2015). The greater their distance from the root, the lighter the Zn isotopic composition of leaves, with the degree of fractionation being correlated with height in bamboo and reeds (strategy-II plants, which rely on the chelation of metals rather than reduction during uptake; i.e., graminaceous monocotyledon species; Moynier et al., 2009; Caldelas et al., 2011). Fe and Cu isotopic compositions of leaves relative to the rest of the plant vary with species and growth conditions. There is no or slight Fe or Cu isotopic fractionation during uptake and translocation in strategy-II species (Guelke and von Blanckenburg, 2007, 2012; Weinstein et al., 2011; Jouvin et al., 2012; Moynier et al., 2013). This contrasts with strategy-I species (dicotyledons and non-grass monocotyledons), which excrete H+ via plasmalemma H+–ATPase to acidify the rhizosphere, increasing the solubility of metals (Moynier et al., 2013; Ryan

et al., 2013). Enrichment in heavy Cu isotope (in contrast to light Fe isotopes) occurs in leaves (Guelke and von Blanckenburg, 2012; Ryan et al., 2013), whereas Cu retained in roots is enriched in the light isotope. Cu and Zn isotopic compositions of leaves are correlated with stem height in hairy-leaved sedge (strategy I) in all growth stages (Weinstein et al., 2011; Jouvin et al., 2012; Moynier et al., 2013). Fe isotopic compositions of leaves are not correlated with stem height in oats, johnsongrass, or kentucky bluegrass (strategy II; Guelke and von Blanckenburg, 2012; Moynier et al., 2013), and there are no Fe isotopic data yet for stems of strategy-I species. For seeds, enrichment in light isotope occurs with Cu (relative to leaves) in hairy-leaved sedge (Weinstein et al., 2011) and Fe in beans, rape, amaranth, and soybean (Guelke and von Blanckenburg, 2012), but there is no Fe fractionation between seeds and leaves in oats or wheat (strategy II; Guelke and von Blanckenburg 2012; Moynier et al., 2013). Fe isotopic compositions of new-grown seeds in johnsongrass are slightly heavier than in the rest of the plant (Moynier et al., 2013). Fe and Cu isotopic compositions of seeds are not correlated with height (Weinstein et al., 2011; Moynier et al., 2013). There are no Zn isotopic data for seeds yet. Together, these findings indicate that Zn, Fe, and Cu isotopic fractionation during plant growth provides insights into the uptake, translocation, and incorporation of these elements into and within plant biological systems, and that fractionation is influenced by plant type, growth conditions, and other micronutrients. Studies of multi-element isotopic fractionation in a single plant are required to improve our understanding of the fractionation of biometals and their interactions with other micronutrients. Furthermore, physiological

variations (i.e., seedling, growth, florescence, and seed stages) throughout the life of a plant influence isotopic fractionation of biometals. It is therefore necessary to determine the degree of biometal isotopic fractionation at different stages to develop a complete picture of translocation and remobilization processes or mechanisms. However, studies of isotopic fractionation in the blooming stages have not yet been reported, and such study is necessary for a comprehensive understanding of Zn, Fe, and Cu isotopic fractionation in a single plant. Copper isotopic fractionation in E. splendens Nakai has been reported earlier (Li et al., 2016). As a strategy-I species with relatively high Zn, Fe, and Cu concentrations and strong isotopic fractionation, it is an ideal plant for such a study (Yang et al., 1998, 2002; Jiang et al., 2003). The aim of this study is to systematically investigate the Zn and Fe isotopic fractionations associated with plant growth in the soil–plant system using the Cu-tolerant grass species E.splendens Nakai, and to compare these results with those for Cu isotopes obtained in a previous study (Li et al., 2016). These Zn, Fe, and Cu isotopic data are then used to investigate elemental uptake, transport, translocation, and retranslocation processes in E. splendens Nakai.

2. Materials and methods

2.1 Plant growth and sampling

Plant growth was conducted in a natural field environment within a few kilometers of abandoned Pb–Zn mines in the suburbs of Hangzhou City, Zhejiang

Province, China. The yellow (fluvio-marine) alluvial soil used in this experiment was formed under oxidizing conditions and has a pH of 6.3. The plants grew for approximately nine months until the later stages of anthesis, and seeds had begun to form, with plants and corresponding soil samples being collected on 20 November 2015. Plant samples were separated into roots, stems, branches, leaves, and flowers using ceramic scissors, with samples divided into height intervals of 0–20 cm and 20–40 cm. The samples were washed sequentially in deionized and ultrapure water, and then enzymes were deactivated by heating at 105°C for 30 min. The samples were then dried at 80°C for 48 h and ground to powders using the same protocols as described by Li et al. (2016).

2.2 Chemical purification and Zn–Fe isotopic measurements

2.2.1 Chemical purification

Soil samples were dispersed by “immersion of samples” in vials in an ultrasonic bath for 16 h at room temperature, followed by centrifugation. 40 mL 0.43 M acetic acid (HOAc) (i.e., the phytoavailable pool) was added to each soil sample (1 g) in a Teflon vial following previously reported procedures (Ure et al., 1993; Quevauviller, 1998). The supernatant phyto-available component and residue were transferred to PTFE vials respectively and evaporated to dryness. The plants were ground to powders, and 50–900 mg of sample were placed in PTFE vials. Samples were digested in 2 mL HCl + HNO3 (3:1 mixture of 12 M and 16 M acids, respectively) and 0.1 mL of concentrated HF in a sealed Teflon bomb at 120°C for 12 h. The digest

solution was evaporated to dryness, then re-digested in 0.2 mL 11.7 M HClO4 at 150°C for 12 h. The solution was evaporated again, then re-dissolved in 1 mL of 16 M HNO3 to drive off HClO4 (150°C for 12 h), with this procedure being repeated three times following Li et al. (2008). Samples were finally dissolved in 6 M HCl for Zn and Fe purification following the methods of Zhu et al. (2002) and Tang et al. (2006) using AG-MP-1 anion-exchange resin (BioRad, USA). Zn and Fe eluate fractions were collected in PTFE vials and evaporated to dryness on a hotplate. Samples were then dissolved in 0.1 M HNO3. Matrix element (including Na and Al) contents were measured using a Nu Plasma HR multi-collector–inductively coupled plasma–mass spectrometer (MC–ICP–MS) to determine the suitability of Zn and Fe samples for determination of Zn and Fe isotopic ratios.

2.2.2 Zn and Fe isotopic and concentration measurements

Zinc and Fe isotopic ratios were determined using a Nu Plasma HR MC–ICP–MS with correction of the mass bias via a standard−sample bracketing approach. Isotope ratios are expressed as δ66Zn values relative to standard IRMM-3702 Zn, and δ56Fe values relative to IRMM-014 Fe isotopic reference materials. (66Zn 64Zn)sample

δ66Zn (‰) = [(66Zn 64Zn)IRMM - 3702 – 1] × 1000 (56Fe 54Fe)sample

δ56Fe (‰) = [(56Fe 54Fe)IRMM - 014 – 1] × 1000

(1) (2)

The long-term reproducibility of δ66Zn and δ56Fe measurements was better than 0.05‰ at the 95% confidence level. Mass spectrometry methods have been described elsewhere (Zhu et al., 2002, 2008; Li et al., 2008). Natural standard reference

materials GBW07105 and CAGSR were analyzed for quality control. The δ66ZnIRMM3702

and δ56Fe IRMM-014 values were determined as 0.12‰ ± 0.07‰ (2SD, n = 4) and

0.12‰ ± 0.06‰ (2SD, n = 4), respectively, consistent with previously reported values for δ56Fe of 0.15‰ ± 0.02‰ (Craddock and Dauphas, 2011), 0.13‰ ± 0.09‰ (Dong et al., 2017), and 0.15‰ ± 0.05‰ (Li et al., 2019); and for δ66Zn of 0.17‰ ± 0.11‰ (Li et al., 2019). Zinc and Fe isotopic compositions in plant tissue samples were calculated using the following mass balance equation: δxMwhole plant = iδxMi × Fi

(3)

where Fi is the fraction of Zn and Fe contents in a given tissue sample i relative to the whole plant, and δxMi (‰) is the isotopic composition of Zn and Fe in the sample. Isotopic fractionation between two components i and j is defined as follows: ∆xMi–j = (δxM)i – (δxM)j (M = Zn, Fe; x = 66, 56)

(4)

Two-sigma (2σ) uncertainties for δ66Zn and δ56Fe were determined by replicate analyses, and the uncertainty for a given sample was calculated by error propagation using the Zn and Fe isotopic compositions of individual fractions (Kusonwiriyawong et al., 2016; Li et al., 2016): Uncertainty =

𝑛

∑𝑖 (Fi × 2i)2

(5)

where Fi is the Zn and Fe content in sample i relative to the total in several samples, and the experimental analytical uncertainty (2σ) is given as two standard deviations for sample i.

3. Results

3.1 Zn and Fe concentrations in soil and plants

Zinc and Fe concentrations in soil samples are listed in Table 1 together with previously reported Cu concentrations (Li et al., 2016). Total Zn, Fe, and Cu concentrations in bulk soils calculated using phyto-available and residual metal concentrations are 1170, 12,700, and 121 µg g–1, respectively (Table 1). Unpolluted soils worldwide contain 10–100 µg g–1 Zn, 10,000–50,000 µg g–1 Fe, and 2–109 µg g– 1

Cu (Baize, 2000; Schulte, 2004; Kabatas-Pendias, 2011). Zn, Fe, and Cu

concentrations in phyto-available components are 880, 20, and 50 µg g–1, respectively. Metal concentrations of the present study, particularly Zn, are higher than those of unpolluted soils. This indicates that the trial field is a polluted site affected by Zn contamination due to Pb–Zn mining (Chen et al., 2013). Elemental concentrations and biomass are unevenly distributed in plants (Table 2). Cu concentrations (Li et al., 2016) are given in Table S2. The results are presented in Figs 1 and 2. Excluding the root, the biomass in the 0–20 cm height interval is greater than that in the 20–40 cm height interval. Leaves from the 20–40 cm height interval have a minimum biomass of 0.9 g, and the stem in the 0–20 cm interval has a maximum biomass of 15.3 g (Table 2; Fig. 1). Zinc concentrations in E. splendens Nakai range from 97 µg g–1 in flowers at 20−40 cm height interval to 446 µg g–1 in leaves at 20−40 cm height interval (Table 2), higher than those for most plants (20–100 µg g–1) grown in unpolluted soil

(Mengel and Kirkby, 1987; Viers et al., 2007; Weiss et al., 2007; Arnold et al., 2015; Couder et al., 2015), and lower than those in Zn-tolerant plants (e.g., 500–48,900 µg g–1 for rape, ryegrass, and Phragmites australis; Caldelas et al., 2011; Couder et al., 2015; Tang et al., 2016). Fe concentrations in E. splendens Nakai range from 161 µg g–1 in stems at the 0–20 cm height interval to 1520 µg g–1 in leaves at the 20−40 cm interval (Table 3), within the range of Fe concentrations in most plants (58–2020 µg g–1; Kiczka et al., 2010; Garnier et al., 2017), but much less than in Fehyperaccumulating plants such as Imperata cylindrica (1.0%–2.3%; Rodríguez et al., 2005; Fuente et al., 2016). Cu concentrations range from 7 µg g–1 in stems at the 0−20 cm height interval to 60 µg g–1 in the root (Table 4; Li et al., 2016), within the range of previously reported Cu concentrations in E. splendens Nakai (1.7–215 µg g–1; Song et al., 2004; Wu et al., 2007) and other Cu-tolerant plants including Silene fortune, S. dioica, and Trifolium repens (29–567 µg g–1; Brooks and Crooks, 1980; Nishizono et al., 1987; Zhang and Young, 2006; Tang et al., 1999; Wang et al., 2008; SánchezPardo and Zornoza, 2014.) As a Cu-tolerant plant, E. splendens Nakai displays good growth in polluted soils, with high Zn and Cu concentrations relative to most crops, although concentrations may be below the threshold values defined for hyperaccumulation (Zn, Fe, and Cu: 10,000, 10,000, and 1,000 µg g–1, respectively; Brooks, 1998; Rodríguez et al., 2005; Fuente et al., 2016).

3.2 Zn, Fe, and Cu isotopic compositions of soils and plants

Zinc and Fe isotopic compositions in soil samples are presented in Table 1, whereas those in the plants are listed in Table 2. The isotopic fractionations between plants and soils are illustrated in Figs. 3 and 4. Cu isotopic results that were previously obtained for the same samples (Li et al., 2016) are also listed in supplementary material Table S2. Zn, Fe, and Cu isotopic fractionations between plant and soil were calculated based on mass balance using Eqs (3) and (4), and uncertainties were calculated using Eq. (5) (Table 3; Fig. 5). Zn and Fe isotopes display similar degrees of fractionation in the same tissue at different intervals, but there is variation between different tissues. δ66Zn and δ56Fe values of bulk soil are – 0.68‰ and +0.08‰, respectively, within the global range for unpolluted soils (δ56FeIRMM-014 = –0.15‰ to +0.60‰; Thompson et al., 2007; Wang and Zhu, 2013; Arnold et al., 2015) or slightly below the global range (δ66ZnJMC 3−0749L = –0.53‰ to +0.76‰; Viers et al., 2007; Bigalke et al., 2010; Fekiacova et al., 2015). Zinc, Fe and Cu (Li et al., 2016) isotopic compositions vary during uptake process, although the variation is small. Zn isotopic fractionation relative to soil ranges from –0.08‰ in flowers to +0.27‰ in leaves at the lower height interval, within the range of Zn isotopic fractionation in most plants (Viers et al., 2007, 2015; Couder et al., 2015; Tang et al., 2016). Fe isotopic fractionation relative to soil ranges from –0.02‰ in branches to +0.41‰ in roots, within the range of isotopic fractionation in grass species (Guelke et al., 2007, 2012; Kiczka et al., 2010; Moynier et al., 2013; Arnold et al., 2015).

Table 1. Zinc, Fe and Cu isotopic compositions and concentrations in soils. Sample

δ66Zn IRMM-3702 (‰) 2s.d. δ56Fe IRMM-014 (‰) 2s.d. δ65Cu (‰) 2s.d. Zn(µg g-1) Fe(µg g-1) Cu(µg g-1)

Total soil

−0.98

0.07

0.08

0.06

−0.12

0.04

1170

12700

121

Phytoavailable component −1.01

0.09

−0.26

0.06

0.18

0.07

884

22

54

Residue

0.05

0.09

0.03

−0.28

0.04

284

12700

67

−1.00

*Copper data are from Li (et al., 2016) Table 2. Zinc and Fe isotopic compositions and concentrations in E. splendens Nakai. Sample

δ66ZnIRMM-3702 ∆66Znx−soil

2 s.d. n

δ56FeIRMM-014 ∆56Fex−soil

2 s.d. n

Zn

Fe

Dry biomass(g)

Zn

Fe

−0.05

0.01

2 0.49

0.41

0.20

(µg g–1) 2 250 1020 2.8

−0.79

0.19

0.15

2 0.23

0.15

0.10

2 110

161

15.3

17.0

12.1

Branches −0.91

0.07

0.04

2 0.26

0.16

0.04

2 336

509

7.9

26.9

19.7

Leaves

−0.71

0.27

0.10

2 0.31

0.23

0.14

2 442

1523 1.4

6.5

10.8

Flowers

−1.06

−0.08

0.07

2 0.13

0.05

0.06

2 97

406

5.9

12.0

Root

(‰) −1.03

(‰)

(%) Content ratio 7.1

14.0

0−20 cm height Stem

6.0

20−40 cm height Stem

−0.78

0.20

0.15

2 0.21

0.13

0.10

2 314

440

7.0

22.1

15.0

Branches −0.83

0.15

0.04

2 0.06

–0.02

0.08

2 185

201

3.3

6.3

3.3

Leaves

−0.84

0.14

0.10

2 0.31

0.23

0.23

2 446

1168 0.9

4.2

5.3

Flowers

−1.05

−0.07

0.07

2 0.17

0.09

0.14

2 99

396

4.1

7.9

4.1

Table 3 Zinc, Fe, and Cu isotopic fractionations, calculated from Equations (3) and (4), in E. splendens Nakai relative to bulk soil. Copper data come from Li (et al., 2016) ∆66Znx−soil (‰)

2sd

∆56Fex−soil (‰)

2sd

∆65Cux−soil (‰)

2 sd

–0.05

0.01

0.41

0.20

–0.23

0.11

Leaves+flowers

0.10

0.09

0.14

0.07

–0.80

0.06

Branches+leaves+flowers

0.08

0.05

0.15

0.04

–0.73

0.09

Stem+branches+leaves+flowers 0.11

0.06

0.15

0.08

−0.70

0.07

0.16

0.15

0.12

–0.86

0.06

Root 0−20 cm height plant

20−40 cm height plant Leaves+flowers

0.04

Branches+leaves+flowers

0.09

0.12

0.11

0.10

–0.75

0.09

Stem+branches+leaves+flowers 0.15

0.10

0.12

0.07

–0.65

0.05

Stem

0.20

0.13

0.13

0.07

–0.56

0.03

Branches

0.09

0.04

0.15

0.04

–0.68

0.12

Leaves

0.22

0.10

0.23

0.12

–1.16

0.19

Flowers

–0.07

0.05

0.07

0.07

–0.77

0.04

Aboveground

0.13

0.06

0.14

0.06

–0.68

0.05

Whole plant

0.12

0.05

0.18

0.06

–0.65

0.04

*Copper data are from Li (et al., 2016)

Fig. 1. Biomasses of E. splendens Nakai.

Fig. 2. Zn, Fe, and Cu concentrations in E. splendens Nakai. Copper data are from Li (et al., 2016)

Fig. 3. Zn, Fe and Cu isotope compositions in E. splendens Nakai and soil. Copper data are from Li (et al., 2016)

Fig. 4. Zn, Fe, and Cu isotopic fractionation calculated for E. splendens Nakai. Error bars represent the 2σ uncertainty of analytical procedures (Table 3). Copper data are from Li (et al., 2016)

Fig. 5. Zn, Fe, and Cu isotopic fractionations calculated for E. splendens Nakai. Error bars represent the 2σ uncertainties of our analytical procedures (Tables 2–4). Copper data are from Li (et al., 2016)

4. Discussion

4.1 Zn and Fe isotopic fractionation during uptake

4.1.1 Fractionation between the phyto-available component and soil

Plants take up mineral elements from soil solutions. Thus, the knowledge of metal isotopes in the soil solution (phyto-available component) is needed for discussing the isotopic fractionation during uptake. HOAc extracts represent the phyto-available component (PC) of heavy metals in soils (Ure et al., 1993; Quevauviller, 1998; LagoVila et al., 2014). In natural yellow soil, Fe is present mainly in aluminosilicate minerals, iron oxyhydroxides (goethite), and amorphous states (99.8%) (Diao and Wei, 1999), and its phyto-available content is very low (<0.2%). Zn exists mainly in mineral-bound forms (95%–99%), with little in phyto-available acid-extractable forms (0.16%–0.39%) and with total concentrations of 62–65 μg g–1 (Wei et al., 2005). Acid

extractable ratios in soils were calculated using equation (6). The acid-extractable Zn content of yellow soils of Hangzhou City is relatively high (6.5%–25.5%, with total concentrations of 54–305 μg g–1; Fu et al., 2004). Pollution from the nearby Pb–Zn mining area leads to high total Zn concentrations and phytoavailable fraction in soil, while Fe is more structural in yellow soil (total Zn and Fe concentrations of 1168 and 12,706 μg g–1; acid extractable ratios: 75.7% and 0.17%, respectively using Eq. 6). Acid extractable ratio (ACR) =

[Metal content]HOAc [Metal content]bulk soil

(6)

The ∆66Zn(phytoavailable fraction)−(bulk soil) value (−0.03‰) is within analytical error (0.06‰).The low δ66Zn values in polluted soils may be attributed to atmospheric emissions and long-range transport from Pb−Zn mining areas (e.g., Cloquet et al., 2006; Mattielli et al., 2009). The ∆56Fe(phytoavailable fraction)−(bulk soil) value is −0.34‰. Heavy Fe isotopes are preferentially enriched in Fe(III) species in which Fe is more strongly bound. The light isotopes enter solution more readily due to dissolution of selected mineral phases with isotopically light signatures (Wiederhold et al., 2006; Chapman et al., 2009; Kiczka et al., 2010). Fe(II) is thus the main species in the HOAc extract (Masscheleyn et al., 1993) with enrichment in light Fe isotopes. It is suitable for HOAc extract to discuss Fe isotopic fractionation during uptake in E. splendens Nakai, as a strategy-I plant, which take up Fe(II) after the reduction of Fe(III).

4.1.2 Zn and Fe isotopic fractionation during uptake

Mineral nutrient elements are taken up by a plant via the root surface (root

apoplast) from rhizospheric soil, and then loaded into the root tissue and translocated throughout the plant. Concentrations and isotopic compositions of the whole plant reflect the isotopic compositions taken up by the roots from phyto-available component of the soil. Zinc is always found in the +2 valence state under physiologically relevant conditions, and is mainly complexed to carboxylic groups (Zn(II)−O) in the root (Straczek et al., 2008). The root tissue of E. splendens Nakai adsorbs Zn as Zn−humic acid rather than free Zn2+ in solution at pH = 6.3 (Jouvin et al., 2009), similar to this study. Heavy Zn isotopes are enriched in Zn−humic acid due to equatorial Zn–O bonds being shorter than those in Zn aquo complexes (Xia et al., 1997; Korshin et al., 1998; Harding, 1999; Sheals et al., 2001). Thus, the Zn adsorbed on the root cell walls of E. splendens Nakai must be excluded (Straczek et al., 2008; Jouvin et al., 2012) from discussion of isotopic fractionation during uptake. However, the Zn isotopic signature of the adsorbed component in the root can be ignored because the fraction in the total root relative to the total plant is minimal, based on Eq. (7) (Zn Contentroot/i Zn Contentplant = 7%). The instability of Fe(III) organic complexes adsorbed in root cell walls (Brantley et al., 2004) and the lack of isotopic fractionation of Fe(II) during in vivo adsorption experiments (Icopini et al., 2004) mean that Fe adsorption on root cell walls of E. splendens Nakai do not need to be considered. Zinc and Fe isotopic fractionation between whole plant and phyto-available component (PAC) in soil thus represents Zn and Fe isotopic fractionation during uptake, which displays similar isotopic fractionation (Δ66Znwhole plant−PAC = +0.14‰;

Δ56Fewhole plant−PAC = +0.52‰). However, this is not consistent with light-Zn isotopic enrichment in plants such as Arabidopsis species (Δ66Znplant–solution = −0.19‰ to −0.05‰; Aucour et al., 2011) and Thlaspi arvense (Δ66Znplant–solution = −0.26‰; Tang et al., 2016). Assuming Zn isotopic fractionation is +0.24‰ between free Zn(II) and Zn(II) complexes above pH 6 (Jouvin et al., 2009), and that the fraction of Zn adsorbed relative to the Zn content in root is 30% as in Thlaspi arvense (Tang et al., 2016), δ66Znroot symplast should be −1.14‰ based on Eq. (8). The Δ66Znroot symplast−PAC value is expected to be −0.13‰, with a slight preference for light Zn isotopic enrichment in the root symplast. However, this is not consistent with the Δ66Znwhole plant–PAC

value, which indicates that even light Zn isotopes must have entered the plant

during absorption, with some other transport mechanism influencing δ66Zn values of Zn taken up by the plant (Sections 4.4.1 and 4.4.2). Nonspecific uptake pathways could occur due to high free-Zn(II) concentrations in the phyto-available component, with the diffusion of Zn ion or Zn–complex with a low bond strength leading to enrichment of light Zn isotopes in root symplast (Jouvin et al., 2012; Weiss et al., 2005). FZn adsorbed on root × δ66Znadsorbed on root + FZn in root symplast (taken up) × δ66Znroot symplast = δ66Znwhole root

(8)

where FZn adsorbed on root = 30%, δ66Znadsorbed on root = −0.77‰ (−1.01‰ + 0.24‰), FZn in root symplast (taken up)

= (100%–30%) = 70%, δ66Znwhole root = −1.03‰.

The degree and direction of Fe isotopic fractionation in this experiment (+0.52‰; i.e., rich in iron oxyhydroxide, goethite) is consistent with that between goethite and

Fe(Ш)–PS complexes (+0.21‰, Brantley et al., 2004; +0.6‰, Dideriksen et al., 2008). The slight enrichment in the heavy Fe isotopes is consistent with Fe isotopic fractionation caused by Fe(III) complex as for all investigated strategy-II plants (+0.20‰; Guelke and von Blanckenburg, 2007, 2012; Arnold et al., 2015). This indicates that, as strategy-I plant, E. splendens Nakai takes up Fe(III) directly. Fe(III) complexes are considered to enter the root symplast via plasma membrane transporters of the oligopeptide transporter (OPT) family (Lubkowitz, 2011), similar to ZmYS1 in maize, HvYS1 in barley, and OsYSL15 in rice (Murata et al., 2006; Inoue et al., 2009; Ueno et al., 2009; Suzuki et al., 2012), whereas Fe(II) exists mainly in HOAc extractant. HOAc is therefore unsuitable for use in examining Fe isotopic fractionation in plants that take up Fe(III) complex directly. This demonstrates that although E. splendens Nakai is a strategy-I plant, it takes up Fe as Fe(Ш) complexes (e.g., PS) as strategy II species (Römheld and Marschner 1981; Orera et al. 2010).

4.2 Zn and Fe isotopic fractionation between root and above-ground tissues

Isotope fractionation between above-ground tissues and root are +0.18‰ and −0.27‰ for Δ66Zn and Δ56Fe respectively (Table 3), indicating that heavy Zn and light Fe isotopes are preferentially translocated from the root. After absorption, the mineral nutrients flow into the apoplast, symplast, and then into the xylem in root and stem, from where they flow upward into various tissues. For Zn, it seems that heavy isotopes flow into above-ground tissues. However, the Zn isotopic fractionation between above-ground tissues and root is not consistent with the Δ66Znroot symplast−root

value (−0.11‰ = (−1.14‰) − (−1.03‰)) (Section 4.1.2). Combining the two datasets indicates that light Zn isotopes are preferentially translocated into the xylem in stems from the root symplast during growth, and then preferentially exported from old tissues (i.e., leaves) during senescence (Section 4.4.2). Remaining leaves would thus be enriched in heavy Zn isotopes at the late life stage. Fe exists in the apoplast as Fe(III) complexes (PS or phosphates), in symplast as Fe(II)–nicotinamine (NA), and in xylem as Fe(III) citrate (Rellán-Alvarez et al., 2008; Rellán-Alvarez et al., 2010). However, the weak Fe isotopic fractionation indicates that no redox change takes place at the root symplast–xylem interface, as observed for Fe in oats (Guelke and Von Blanckenburg, 2012) (such a redox change would cause ~+3‰ isotopic fractionation). Oxidation thus occurs before Fe enters the xylem from the symplast, or after it enters the xylem. Heavy Fe isotopes would be enriched in the xylem relative to the symplast, because Fe(III)–citrate would be isotopically heavier than Fe(II)–NA (Moynier et al., 2013), although this not consistent with present results. The origin of the slightly light Fe isotopic compositions of above-ground parts would be the root symplast. Fe(III) complexes in apoplast have a higher bond strength than Fe(II)–NA in symplast (Moynier et al., 2013). The differences in complexation and reduction processes would lead to the enrichment of light Fe isotopes in root symplast and further cause slight enrichment of light Fe isotopes in above-ground tissues relative to the root.

4.3 Zn and Fe isotopic fractionation through stem

There is no significant Zn or Fe isotopic fractionation through the stem (Δ66Zn(stem 20–40 cm)−(stem 0–20 cm)

= +0.01‰ ± 0.10‰; Δ56Fe(stem 0–20 cm)−(stem 20–40 cm) = −0.02‰ ±

0.07‰). Zn and Fe concentrations in the stem at the 20−40 cm height interval (314 and 440 μg/g, respectively) are higher than those of the 0−20 cm height interval (110 and 161 μg/g, respectively; Tables 2–4). It shows that convective transpiration flow controls transport of all three elements (Lorenz et al., 1994; Barberon and Geldner, 2014; Couder et al., 2015; Li et al., 2016) through the stem and branches. The absence of such enrichment in light isotopes due to diffusion (Rodushkin et al., 2004) is consistent with previous studies which reported that most Zn and Fe exist in plants as metal complexes (van der Mark et al., 1982; Clark et al., 1986; Schmidke and Stephan, 1995; von Wiren et al., 1999; Rellán-Álvarez et al., 2008, 2010; Nishiyama et al., 2012; Sinclair and Krämer 2012; Hazama et al., 2015). Zinc and Fe display similar isotopic fractionation between branches+leaves+flowers and stem with increasing height (Δ66Zn 0–20 cm = −0.11‰ ± 0.06‰; Δ66Zn 20–40 cm = −0.11‰ ± 0.10‰; Δ56Fe 0–20 cm = +0.01‰ ± 0.08‰; Δ56Fe 20– 40 cm

= −0.01‰ ± 0.07‰). Although these fractionations are very weak, their

similarity suggests that the same mechanism may occur for mineral nutrient transport between stem and branches. Convective transpiration flow would not cause Zn or Fe isotopic fractionation in the stem, so the weak fractionation must occur during convective transpiration flow from stem to branches+leaves+flowers.

4.4 Zn and Fe isotopic fractionation between branches and leaves and flowers

4.4.1 Effect of mobility on the elemental distribution in leaves and flowers

E. splendens Nakai leaves exhibit the highest Zn and Fe concentrations of all the tissues. Although there are apparent differences in elemental concentrations in branches at different height intervals, the Zn concentration shows no variation with height in leaves and flowers when uncertainties are considered (± 9%) (~1% for leaves and ~3% for flowers; 100  ([Zn]0−20 cm interval − [Zn]20−40 cm interval)/[Zn]0−20 cm interval).

However, there is a variation in Fe concentrations in leaves with height (~23%;

100  ([Fe]0−20 cm interval − [Fe]20−40 cm interval)/[Fe]0−20 cm interval), but this is not evident in flowers (~3%). These results indicate that there is no obvious influence of Zn and Fe concentrations in branches on those in leaves and flowers. However, it seems that slightly more Fe is remobilized from the leaves to the flowers at the 20−40 cm interval. This is evident from the same Fleaves+flowers (41% at the 0−20 cm interval and 42% at the 20−40 cm interval; Fleaves+flowers = 100  ([Fe] leaves+flowers/[Fe] stem+branches+flowers+leaves))

at different height intervals, but with slightly lower Fleaves in

the 20−40 cm interval (16% at the 20−40 cm interval; Fleaves = 100  ([Fe]leaves/[Fe]stem+branches+flowers+leaves)) compared with the 0−20 cm interval (19%). However, the Zn distribution in different parts of the plant are similar at each height interval (Fstem+branches, Fleaves, and Fflowers = 78%, 11%, and 11% in the 0–20 cm interval, and 78%, 11%, and 11% in the 20–40 cm interval, respectively; Fstem+branches = 100  ([Zn]stem+branches/[Zn]stem+branches+flowers+leaves); Fleaves = 100  ([Zn]leaves/[Zn]stem+branches+flowers+leaves); Fflowers = 100  ([Zn]flowers/[Zn]stem+branches+flowers+leaves)). This result, combined with the similar Zn

concentrations in leaves and flowers at different height intervals, may indicate that Zn is more mobile in the xylem and phloem (Haslett et al., 2001; Clemens et al., 2013) than Fe, which has intermediate phloem mobility in E. splendens Nakai (Kochian, 1991). The extent of remobilization of Fe is also different at each height interval. In contrast with Zn and Fe, Cu concentrations in E. splendens Nakai vary with height (Li et al., 2016). The heterogeneous Cu concentrations highlight the weak phloem mobility of Cu, compared with Zn and Fe (Garrnett and Graham, 2005). As such, the mobility of nutrient elements in the xylem and phloem of E. splendens Nakai might follow the trend Zn > Fe > Cu, when there is sufficient supply of these elements. Zn, Fe, and Cu concentrations in parts of E. splendens Nakai at different growth stages indicates a need to constrain the relative degree of remobilization in the senescence phase.

4.4.2 Zn and Fe isotopic fractionation between branches and flowers

Metal isotopic compositions in flowers are a promising tool for investigating the translocation and remobilization processes of nutrient elements during florescence. The phloem has been recognized as supplying nutrient elements for flowers (Curie et al., 2009) and, as such, isotopic compositions of the nutrient elements Zn and Fe of flowers should reflect those of the phloem in the branches. A combination of the Zn and Fe elemental inputs and translocation processes into the flowers controls their Zn and Fe isotopic compositions. There are two processes that provide mineral nutrient elements to the phloem: (1) direct xylem-to-phloem

transfer involving continuous uptake from the metal pool through the stem and branches (Yamaji et al., 2008; Yamaji and Ma, 2009; Zheng et al., 2012); and (2) phloem loading after remobilization (i.e., net export of stored or recycled nutrients) from the leaves and stems at senescence (Garrnett and Graham, 2005; Li et al., 2016). In general, the more remobilization that takes place from old tissues (i.e., branches or leaves) to new tissues (i.e., flowers or seeds), the greater the elemental ratio in the new tissue as compared with the whole plant. We define the concentration ratios of Zn and Fe in flowers to those in branches+flowers+leaves as FZn and FFe, respectively (Fmetal = 100  ([metal]flowers/[metal]branches+flowers+leaves)%; FZn 0−20 cm = 9%; FZn 20−40 cm = 13%; FFe 0−20 cm = 28%; FFe 20−40 cm = 48%). Combined with FCu (FCu 0−20 cm = 22%; FCu 20−40 cm

= 60%) values from a previous study (Li et al., 2016), these results indicate

that Zn, Fe, and Cu all have a higher remobilization efficiency in the upper height interval with flag leaves being the major source of remobilized micronutrients， consistent with previous studies (Wu et al., 2010; Zhang et al., 2012; Yamaji et al., 2013); and that the difference in remobilization efficiency with height follows the trend Zn < Fe < Cu, which is negatively related to metal mobility in the phloem. In contrast with the remobilization process, continuous uptake of nutrient elements in developing tissues has a mobility trend of Zn > Fe > Cu, which is consistent with the trend of metal mobility in the phloem. Although Zn has the lowest remobilization efficiency (compared with Fe and Cu), it would have the greatest influence on Zn isotopic fractionation during growth at the late life-stage, due to its high mobility in the xylem and phloem.

In E. splendens Nakai, flowers from different height intervals have similar Zn and Fe isotopic compositions respectively (Δ66Zn(flowers 0−20 cm)−(flowers 20−40 cm) = −0.01‰ ± 0.04‰; Δ56Fe(flowers 0−20 cm)−(flowers 20−40 cm) = −0.04‰ ± 0.07‰). In addition, the flowers have slightly light Zn and Fe isotopic compositions relative to stem (Δ66Zn(flowers 0−20 cm)−(branches+leaves+flowers 0−20 cm)

= −0.23‰ ± 0.05‰; Δ66Zn(flowers 20−40

cm)−(branches+leaves+flowers 20−40 cm)

cm)−(branches+leaves+flowers 0−20 cm)

= −0.19‰ ± 0.11‰; Δ56Fe(flowers 0−20

= −0.10‰ ± 0.04‰; Δ56Fe(flowers 20−40

cm)−(branches+leaves+flowers 20−40 cm)

= −0.02‰ ± 0.08‰). The largely invariant Zn and Fe

isotopic compositions and concentrations in flowers with height further indicate Zn and Fe homogeneity of the phloem in branches at different height intervals. No Zn or Fe isotopic fractionation between flowers and branches or leaves has been reported at anthesis for other plants. However, for rice grown in anaerobic soil, the seeds that also take phloem as mineral pool (Curie et al., 2009) are isotopically lighter than the shoots (Δ66Zn(seed)−(shoot) = −0.79‰; Δ56Fe(seed)−(shoot) = −0.06‰; Arnold et al., 2015), which is consistent with results of our study (Δ66Zn(flower)−(leaf 0−20 cm) = −0.35‰; Δ66Zn(flower)−(leaf 20−40 cm) = −0.21‰; Δ56Fe(flower)−(leaf 0−20 cm) = −0.18‰; Δ56Fe(flower)−(leaf 20−40 cm) = −0.14‰). The enrichment in light Zn and Fe isotopes in flowers may be caused by two possible factors. (1) Translocation of nutrient elements from the phloem to flowers causes enrichment of light isotopes in the flowers. Previous studies have shown that phloem sap is slightly alkaline (pH = 7.3–8.5; Dinant et al., 2010), and Zn and Fe need to bind to an intracellular ligand to remain soluble, probably as Zn(II)–NA,

Fe(III)–NA or –PS (von Wiren et al., 1999; Nishiyama et al., 2012; Hazama et al., 2015). Zn(II)–NA, and Fe(III)–NA or –PS complexes move from the phloem into the transfer cells, from where it can be transported into developing tissues. However, the translocation process would not fractionate Zn and Fe isotopes, as these are biomacromolecules (Tauris et al., 2009; Guelke-Stelling and von Blanckenburg, 2011). (2) The phloem pool that provides nutrient elements to the flowers is rich in light Zn and Fe isotopes. However, Zn and Fe in the phloem pool also come from two different “upstream pools”. Firstly, the xylem→phloem transfer to the phloem pool occurs during continuous uptake. The amino acids histidine (Kozhevnikova et al., 2014) and NA (Cornu et al., 2015) for Zn, and citrate for Fe(III) complexation (Rellán-Alvarez et al., 2008, 2010) have been proposed to play a role in metal binding in the xylem. Metal binding to the cell walls (as hydroxyl and carboxyl groups) lining the xylem vessels might take place and, as such, light isotopes would be enriched in the xylem sap from where it can be transferred to phloem via ion exchange. Moreover, xylem sap is an acidic environment (pH ~5.5) in which Zn(H2O)n2+ and Fe(H2O)n2+ enriched in light isotopes might remain in solution (Wilkinson et al., 1998). Secondly, the leaves→phloem transfer to the phloem pool during remobilization. Excess Zn is stored in the vacuoles of leaves as complexes with citrate, malate, or NA (Aucour et al., 2011; Tang et al., 2012), and excess Fe is oxidized to Fe(III) and stored as phytoferritin in the plastids of plant cells (which also have acidic characteristics) (van der Mark et al., 1982; Schmitt and Weaver, 1984; Starks and Johnson, 1985; Khan and Weaver, 1989). The vacuole has a pH of ~5.2

(Shen et al., 2013) and Zn may remain soluble as the Zn(II) ion (for transport in the cytosol). The non-quantitative sequestration of Fe in phytoferritin complexes favors the accumulation of isotopically heavy Fe(III) in the phytoferritin, leaving a pool of isotopically light Fe(II) in the cytoplasm available for export to the phloem (van der Mark et al., 1982). Furthermore, the equatorial metal–O/N or phosphate bonds with the organic and amino acids are shorter than those in metal aquo complexes (Xia et al., 1997; Korshin et al., 1998; Harding, 1999; Sheals et al., 2001), and the formation of Zn and Fe aquo complexes would likely favor the accumulation of heavy Zn and Fe isotopes, leaving an isotopically light Zn(II) and Fe(II) pool available to the phloem (Yamaji et al., 2013). This could also generate light Zn and Fe isotopic enrichment in the phloem, irrespective of the transport proteins. Light Zn and Fe isotopic enrichment in flowers could thus be attributed to the phloem pool, rather than translocation processes at branch–phloem−flowers. The weak isotopic fractionation indicates that no redox changes take place during xylemto-phloem transfer and remobilization.

4.4.3 Zn and Fe isotopic fractionation between branches and leaves

Slight

enrichment

in

heavier

Zn

isotopes

in

leaves

relative

to

branches+leaves+flowers has been found in E. splendens Nakai (Δ66Zn(0−20 cm) = +0.19‰ ± 0.08‰; Δ66Zn(20−40

cm)

= +0.05‰ ± 0.08‰). This is also consistent with isotopic

fractionation between leaves and root (Δ66Zn(leaves 0−20 cm)−(roots) = +0.32‰ ± 0.08‰); Δ66Zn(leaves 20−40 cm)−(roots) = +0.19‰ ± 0.15‰). These findings are consistent with results

for Lasimorpha senegalensis (+0.06‰; Viers et al., 2007) and metal-tolerant Silene vulgaris at the fruiting stage (0‰ to +0.09‰; Tang et al., 2012). Enrichment in light Zn isotopes in leaves relative to roots has been observed in trees (Δ66Zn(leaves)–(roots) = −1.42‰ to −0.24‰) (Viers et al., 2007) and herbaceous species (Δ66Zn(leaves)–(roots) = −1.06‰ to −0.04‰) in the non-blooming and seed stages, and N. caerulescens in fruiting stages (Caldelas et al. 2011; Weiss et al., 2005; Viers et al., 2007, 2015; Moynier et al., 2009; Aucour et al., 2011; Tang et al., 2012, 2016; Couder et al., 2015). Slight enrichment in heavier Fe isotopes in leaves relative to branches+leaves+flowers has been found in E. splendens Nakai (Δ56Fe(0−20 cm) = +0.08‰ ± 0.08‰; Δ56Fe(20−40 cm) = +0.12‰ ± 0.08‰), and it fits in with the values in strategy II plants (Guelke et al., 2012). This is contrary to isotopic fractionation between leaves and root (Δ56Fe(0−20 cm) = −0.18 ‰± 0.08‰; Δ56Fe(20−40 cm) = −0.18‰ ± 0.08‰), which is consistent with the isotopic fractionation results in strategy-I species which display enrichment of lighter Fe isotopes in leaves relative to root (−1.28‰ to −0.59‰; Guelke et al., 2007, 2012; Kiczka et al., 2010; Moynier et al., 2013). Zinc has been considered as being translocated from branches to leaves via transporters AtIRT3 and OsZIP4 (Ishimaru et al. 2005; Lin et al. 2009), which would lead to enrichment in light Zn isotopes in leaves for most herbaceous species. Light Zn isotopes should be preferentially transferred to the leaves relative to stem (branches+leaves+flowers) and root during the growth stage, with the enrichment in heavy Zn isotopes observed in root symplast. It remains unclear whether active Zn translocation processes occur for a few herbaceous species at branch–leaf interfaces.

This indicates that these species have a particular interior Zn homeostasis, with E. splendens Nakai being a Zn-tolerant plant, based on its Zn concentration and isotopic composition. Light Zn isotopes are expected to be more readily exported than Fe from old tissues to new ones such as flowers and seeds (Garrnett and Graham, 2005), the root, and rhizospheric soil through the phloem (Haslett et al., 2001; Page et al., 2006). Thus, the remaining Zn in leaves would be enriched in heavy Zn isotopes at the late life-stage. This leads to increasingly positive Δ66Zn(leaves)–(roots) (+0.32‰ ± 0.08‰ and +0.19‰ ± 0.15‰) and Δ66Zn(whole plant)–(soil) (+0.10‰) values, as discussed in Section 4. 4.1. The small degree of Fe isotopic fractionation indicates that no oxidation occurs between stem and leaves. Fe isotope compositions in leaves might be caused by two potential factors: i) The enrichment in light Fe isotopes in xylem pool play an important role in that in leaves despite of the transport protein (Section 4.2). The remaining would be enriched in heavy Fe isotopes due to the preferentially remobilization of light Fe isotopes from old leaves (Section 4.2.1). But the degree of Fe remobilization is smaller relative to Zn, it would not vary the Fe isotopic fractionation trend between root and leaves. ii) Active translocation processes would lead to enrichment in heavy isotopes in leaves at the branch–xylem−leaves cytoplasm interface, involving Fe(III) transporter complexes with greater bond strength in leaf cytoplasm than Fe(III)–citrate in xylem (Moynier et al., 2013; von Wiren et al., 1999). In that case, heavy Fe isotopes should preferentially be transferred to the leaves relative to stem (branches+leaves+flowers). But the degree of isotopic fractionation caused by the variation in complex species is

not enough to enrich the heavy Fe isotopes in leaves relative to root. Thus, Δ56Feleaves – root

is still negative. However, it is difficult to discriminate which factor lead to Fe

isotopic fractionation in this study.

5. Comparison of Zn, Fe, and Cu isotopic fractionations in E. splendens Nakai

Zinc 、 Fe and Cu concentrations and their isotopic fractionations during plant growth processes have been investigated from a single plant E. splendens Nakai growing in the same condition. This provides a precious opportunity to understand Zn, Fe, and Cu isotopic fractionations in similar and characteristic uptake and translocation processes. During uptake, non-specific uptake pathways for Zn take place in E. splendens Nakai based on the Δ66Znroot symplast−PAC (−0.13‰) and high Zn concentration in phytoavailable component. However, the preferential remobilization of light Zn isotopes out of plant lead to enrichment in heavy Zn isotopes in the remaining, which further lead to the enrichment in heavy Zn isotopes in plant relative to PAC. A slight positive Fe isotopic fractionation between plant and PAC displays direct uptake processes of Fe(III) complexes. Whereas, a lager negative isotopic fractionation displays a prerequisite of Cu(II) reduction to Cu(I) before uptake of Cu. During translocation from root to above-ground tissues, heavy Zn and light Fe and Cu isotopes are preferentially translocated from the root. The translocation of mineral biometal from root to above-ground tissues occur at symplast−xylem interface. The small Fe isotopic fractionation indicate no change in redox state occurs at

symplast−xylem interface. Because E. splendens Nakai uptake Fe (III) complex directly, and transported to apoplast as Fe (III), and then transported to symplast as Fe (II), the enrichment of light Fe isotopes in root symplast and further cause slight enrichment of light Fe isotopes in above-ground tissues relative to the root. In contrast, the large negative Cu isotopic fractionation indicates change in redox state occurs at symplast−xylem interface. For Zn, based on the Δ66Znroot symplast−root value (−0.11‰), light Zn isotopes should be translocated from symplast in root to xylem in stem, but remobilization process would influence the isotopic fractionation. It is difficult to discriminate the two factors in plant at a growth stage. During transport through the stem and branches, Zn, Fe, and Cu undergo no significant isotopic fractionation through the stem due to metal–complex formation in xylem and phloem. But a small but systematic enrichment in light Zn, Fe and Cu isotopes in branches+leaves+flowers relative to stem occur regardless of height. Diffusion process of light isotopes in “downwards” (i.e., branches) or deposition of heavy isotopes in “upwards” (i.e., stem) at the “nodes” would occur. Further investigations are needed to verify this. During translocation processes at branch–flower interfaces, the similar enrichment in Zn and Fe lighter isotopes in flower relative to branches+leaves+flowers regardless of height, but the inverse variation of isotope compositions for Cu with height, occur in E. splendens Nakai. In principle, the same translocation process should occur at branch– flower interface in different interval, which would lead to the same biometal isotopic fractionation for one element. The biometal isotope compositions in phloem as “pool”

for leaves would play an important role in that in flowers other than translocation processes. During translocation at branch–leaf interfaces, E. splendens Nakai displays the similar

size

of

enrichment

in

heavier

isotopes

between

leaves

and

branches+leaves+flowers parts regardless height-intervals for Zn and Fe, but the inverse variation for Cu contrast to that in flowers. Zn, Fe and Cu isotope compositions in branches+leaves+flowers parts in different height-interval are the same. Zn and Fe isotope compositions in leaves are also the same. But the inverse variation in Cu isotope compositions in leaves is contrast to that in flowers in one interval. It shows that recycled nutrients interiorly (remobilization) play an important role in biometal isotope compositions in old leaves in E. splendens Nakai. Overall, the isotope compositions of flowers seemingly being dependent on that in their “pools” in the phloem that provide mineral nutrient elements for flowers. Accordingly, metal isotope compositions in leaves are dependent on the elements remaining after remobilization to the phloem in the late life stage. Remobilization thus plays characteristic roles in the isotopic compositions of leaves and flowers for different elements and heights despite translocation during the late life stage. Metal remobilization efficiency follows the trend Zn < Fe < Cu, which is opposite to the mobility in the xylem and phloem (Zn > Fe > Cu). The degrees of remobilization of Zn and Fe from old to new parts of the plant are higher in the upper height interval, as for Cu. Light isotopes for Zn and Fe, and heavy isotope of Cu are remobilized from old tissues (i.e., leaves, branches and stem) to the phloem, and then to new tissues (i.e.,

flowers and seeds), while light Cu isotopes subsequently have higher degrees of remobilization. Accordingly, the remaining tissues (i.e., leaves) are enriched in heavy isotopes for Zn and Fe, and light isotope of Cu, with more remobilization of heavy Cu occurring subsequently due to different Cu species stored in leaves (Li et al., 2016). Zn mobility in the phloem might affect Zn isotopes throughout the plant, with light Zn isotopes being exported from old tissues (e.g., leaves) to new tissues (e.g., flowers and seeds), the root, and rhizospheric soil through the phloem. This would lead to heavy Zn isotopic enrichment in the old tissues relative to the new ones, and in the whole plant relative to soil. Cu remobilization from old tissues plays an important role in controlling the isotopic composition of the phloem, particularly for tissues at flag intervals, compared with Zn and Fe. As a strategy-I species, E. splendens Nakai displays similar uptake and translocation modes to those of strategy-II species, which are influenced by the Cu-sufficient state in soil. This indicates that Cu and Fe are interrelated during uptake processes within the plant.

6. Conclusions

Overall, Zn, Fe, and Cu undergo isotopic fractionation into and within plants, providing a potential tool for investigations of the mechanisms of uptake and translocation. Furthermore, Zn, Fe, and Cu isotopic fractionation between plant and soil would be a good methodology to understand biometals cycle of the soil–plant, i.e. biosphere–lithosphere, system.

Acknowledgements

This publication was made by funding from the National Natural Science Foundation of China (Grant Nos. 41430104 and 41203005) and China Geological Survey (DD20190002). We thank Zhu Li for assistance with fieldwork and plant culture experiments, and Jin Li, Zhihong Li, Bin Yan, Jian-Xiong Ma, Suo-Han Tang, Yao Shi, Chenxu Pan, and Kan Zhang for their meticulous guidance in the CAGS laboratories. We thank the editors and official reviewers for their helpful comments and suggestions, which have improved the manuscript significantly.

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Highlights:

•Zn, Fe and Cu exhibit their characteristic isotopic fractionation during

uptake and translocation through the plant, but with no vertical fractionation in the stem. • Remobilization plays characteristic roles in the isotopic compositions of leaves and flowers for different elements and heights at the late life stage. •The remobilization efficiency follows the trend Zn < Fe < Cu, opposite to the trend of metal mobility in xylem and phloem (Zn > Fe > Cu).

Credit Author Statement Shizhen Li: Formal analysis; Writing - Original Draft Xiangkun Zhu: Conceptualization; Investigation LonghuaWu: Methodology; Writing - Review & Editing Yongming Luo: Validation; Review & Editing