Different strategies for lead detoxification in dwarf bamboo tissues

Different strategies for lead detoxification in dwarf bamboo tissues

Ecotoxicology and Environmental Safety 193 (2020) 110329 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal ho...

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Ecotoxicology and Environmental Safety 193 (2020) 110329

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Different strategies for lead detoxification in dwarf bamboo tissues

T

Mingyan Jiang, Xinyi Cai, Jiarong Liao, Yixiong Yang, Qibing Chen, Suping Gao, Xiaofang Yu, Zhenghua Luo, Ting Lei, Bingyang Lv, Shiliang Liu∗ College of Landscape Architecture, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Antioxidant compounds Lead detoxification Lead species Sasa argenteostriata Subcellular compartmentalization

Dwarf bamboo Sasa argenteostriata (Regel) E.G. Camus is considered as potential plants for metal phytoremediation in previous filed observations. However, the mechanisms of lead (Pb) detoxification has not been described. The objective of this study was to explore the difference strategies or mechanisms of Pb detoxification in plant tissues. In this regard, four Pb treatments with hydroponics including 0 (control), 300, 600, and 900 mg L−1 were conducted to examine subcellular compartmentalization, Pb accumulation/species and antioxidant-assisted chelation. Our findings showed the retention of Pb by the whip-root system is one of its detoxification mechanisms to avoid damage the shoots. In addition, the cell wall retention is the dominant detoxification strategy of whips, new roots, old roots and new/old stems, while vacuolar compartmentalization is for new/old leaves. Interestingly, four low-mobility/-toxicity Pb species (i.e., FNaCl, FHAc, FHCl and FR) are distributed in roots, whips and stems, while two high-mobility/-toxicity Pb species (FE and FW) in leaves. The conversion of Pb to low-toxicity/-migration is a Pb-detoxification strategy in roots, whips and stems but not in leaves. Besides, the new/old roots and leaves can alleviate Pb damage through the synthesis of non-protein thiol, glutathione and phytochelatins. Among these, phytochelatins play a leading role in the detoxification in new/old roots, while glutathione is in new/old leaves. This study provides the first comprehensive evidence regarding the different strategies for Pb detoxification in dwarf bamboo tissues from physiological to cellular level, supporting that this plant could be rehabilitated for phytoremediation in Pb-contaminated media.

1. Introduction Nowadays, rapid urbanization and industrialization have caused an exceeding increase in environmental metal-pollution worldwide and even threatening human health through the food chain (Liu et al., 2015a,b,c, 2016; Jiang et al., 2019). Among these, lead (Pb) is considered to be the second most toxic pollutant of all dangerous metals after arsenic, which usually induce plant cells to produce large amounts of active oxygen (Phang et al., 2011; Pallavi et al., 2012; Chandana and Joseph, 2019), destroy cell structure and membrane stability (Figlioli et al., 2019), irreducible damage to organelles (Kaur et al., 2013; Monteiro et al., 2015) and interfering normal physio-biochemical metabolism (Tang et al., 2008). Therefore, it has become a current priority to use effective and feasible techniques to reduce the Pb content in the medium to an acceptable level. To deal with the toxicity of metals, plants have evolved several defense strategies including Pb avoidance, Pb biochemical-tolerance

and Pb detoxification strategy (Sharma and Dubey, 2005; Wójcik and Siebielec, 2014). Among these, the Pb biochemical-tolerance strategy refers to the regulations of the secondary stress-defense systems, including the antioxidant system, ascorbate–glutathione cycle and glyoxylate pathway (Hasanuzzaman et al., 2018; Xie et al., 2018). For example, antioxidant compounds, e.g., non-protein thiol (NPT-SH), glutathione (GSH) and phytochelatins (PCs), combine with Pb ions owing to the inclusion of abundant metal-related sulfhydryl (-SH) and/ or cysteine residues (Cys) (Yuan et al., 2015). As a precursor of PCs synthesis, GSH is capable of promoting the synthesis of PCs as well (Gupta et al., 2010). Most importantly, PCs as the transporter of Pbcomplexes can realize the cyclic detoxification of loading and transporting in cytoplasm-vacuoles (Gisbert et al., 2003). Previous studies have stated that PC2 and PC3 levels were markedly increased in Ceratophyllum demersum L. leaves for reducing the Pb detoxification (Mishra et al., 2006). On the other hand, the Pb detoxification strategy represents plants reduce the mobility and toxicity of Pb-absorbed by



Corresponding author. E-mail addresses: [email protected] (M. Jiang), [email protected] (X. Cai), [email protected] (J. Liao), [email protected] (Y. Yang), [email protected] (Q. Chen), [email protected] (S. Gao), [email protected] (X. Yu), [email protected] (Z. Luo), [email protected] (T. Lei), [email protected] (B. Lv), [email protected] (S. Liu). https://doi.org/10.1016/j.ecoenv.2020.110329 Received 26 September 2019; Received in revised form 7 February 2020; Accepted 12 February 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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2019). However, the detoxification mechanism of Pb regarding on the subcellular compartmentalization, Pb-species transformation and antioxidant-assisted chelation under aqueous-media have not been described. Based on these backgrounds, the primary objectives of this controlled-study were to investigate the differences in Pb detoxification strategies in the tissues of S. argenteostriata plants and combining the clonal characteristics to provide theoretical basis for the Pb-defense mechanism of bamboos. The results of this study are expected to provide some new ideas for phytoremediation of large-scale areas of heavy metal (especially in lead) contaminated soils worldwide.

segregating it in the low-mobility region of plant cells, and/or converse into Pb-detoxification chemical forms (Sharma et al., 2005). Among these, Pb detoxification strategy is closely related to the subcellular compartmentalization of Pb ions (López-Orenes et al., 2018). Previous publications have shown that the negatively charged groups of the cell wall combine with Pb and form Pb-complexes, then bind with the cell wall, which delay the transmembrane transportation of Pb ions (Tian et al., 2010; Krzesłowska et al., 2016). Similar results have been reported in a Zn/Cd/Pb hyperaccumulator Sedum alfredii Hance plants (Tian et al., 2010), valerian (Potamogeton crispus L.) (Li et al., 2017), and two hybrid aspen plants (Krzesłowska et al., 2016). Furthermore, the intracellular vacuole equipped with S groups, can effectively bind with metals forming complexes (e.g., metal-phytochelatins/PCs) (Kopittke et al., 2008), which are then transported to the interstitial and stored in vacuoles (Meyers et al., 2008; Piwowarczyk et al., 2018). Previous studies cited that several plants such as Brassica chinensis L. (Wu et al., 2013), Potamogeton crispus L. (Qiao et al., 2015), and Neyraudia reynaudiana (Kunth) Keng (Zhou et al., 2016) could transfer large amount of Pb2+ into the vacuoles, reducing the Pb toxicity of their central organelles. However, the accurate detoxification mechanism in Pb-induced plants is still not well described. The integration of Pb species into low-mobility and -toxicity forms is also a typical defense-strategy for plants to achieve self-detoxification. In general, Pb in environmental medium has six chemical forms, whose concentration and distribution influence the ecotoxicity of Pb directly (Piwowarczyk et al., 2018). Among them, ethanol (EtOH) and water (H2O2) extractive are easily to penetrate into the cytoplasm and/or attaching the organelles due to its highly mobility, causing serious toxicity to plants cells. Besides, the other four Pb species, i.e., sodium chloride (NaCl), sodium acetate (HAc), hydrochloric acid (HCl) extractive and residual state are also important Pb-detoxification forms in plants, which can reduce the toxicity and mobility of Pb by forming protein-bound or adsorbing pectin, oxalic acid and Pb-phosphate (Weng et al., 2012; Wu et al., 2013). Studies verified that NaCl and HAc extractive are the main Pb chemical forms in Camellia sinensis L. (Xu et al., 2011) and Arenaria orbiculate L. plants (Zu et al., 2015), while the insoluble Pb-phosphate and oxalate-bound are the dominant Pb forms in Triarrhena sacchariflora (Maxim.) Nakai (Kumar et al., 2012) and Talinum fruticosum L. plants (Dalcorso et al., 2013). In our previous study, with the addition of ethylenediaminetetraacetic acid (EDTA), we found that the levels of residual fraction and reducible fraction of Pb species were reduced, but the weak acid-soluble fraction was increased in two dwarf bamboos (Jiang et al., 2019). However, few reports have focused on the toxicity and mobility of Pb species in bamboos. Bamboo (Bambusoideae) is one of the most valuable plants and is widely distributed throughout the world (Bamboo Phylogeny Group et al., 2012; Jiang et al., 2019). In the Cu- (Chen et al., 2015a,b), Cd- (Li et al., 2016) and Pb-contaminated (Zhong et al., 2017) soil-medium, moso bamboo (Phyllostachys pubescens Mazel) showed higher tolerance and larger biomass, suggesting that some large bamboos may be provided as new materials for remediation. Similarly, dwarf bamboo is also considered to be a potential plant for effective phytoremediation (Chen et al., 2015a,b; Jiang et al., 2019). Sasa argenteostriata (Regel) E.G. Camus is a widely used dwarf bamboo due to its high biomass, rapid growth and strong adaptability (Jiang et al., 2019), as well as the typical advantage of clonal plants. Several studies proposed, in the heterogeneous environment, clonal plants can utilize physiological integration and transport materials/energy between clonal parts in both directions, which ensure the daughter ramets can maintain normal physiological growth for adapting to environmental changes (Zhao et al., 2015; Yu et al., 2016; Guo et al., 2017). The other possible reason is that the whole clonal plant could share the stress-risks by transporting metals among the ramets (Xu and Zhou, 2016; Luo et al., 2017; Quan et al., 2018). A recent study showed that S. argenteostriata employed biochemical-tolerance strategies for coping with Pb stress, such as osmotic adjustment and antioxidation system regulation (Jiang et al.,

2. Materials and METHODS 2.1. Plant materials and growth conditions All experiments were conducted at Sichuan Agricultural University, Chengdu, Sichuan, Southwestern China. In this study, biennial ramet seedlings of S. argenteostriata plants were purchased from Anji County, Zhejiang, Eastern China in November 2017. To avoid geographical differences and growth constraints, all studied bamboo seeds were sown in plastic pots (Jiang et al., 2019). In July 2018, fifteen bamboo seedlings with similar size were selected and transplanted to the opaque buckets (10 L) in 5 L modified half-strength Yoshida nutrient solution (Yoshida et al., 1976). The mother liquor solution includes 134.83 g L−1 of Ca(NO3)·4H2O, 44.30 g L−1 of CaCl2, 81.00 g L−1 of MgSO4·7H2O, 20.15 g L−1 of NaH2PO4·2H2O, 35.70 g L−1 of K2SO4, 0.75 g L−1 of MnSO4·H2O, 0.037 g L−1 of (NH4)4Mo7O24·4H2O, 0.467 g L−1 of H3BO3, 0.0175 g L−1 of ZnSO4·7H2O, 0.0155 g L−1 of CuSO4·5H2O, 5.95 g L−1 of C6H8O7·H2O, and 0.005 M of NaFeEDTA, pH 5.0–5.5 and diluent with 2000 times. To ensure uniform biomass, appropriate pruning of ramets was allowed before transplanting. During the experiment, all plants are placed in a growing greenhouse under controlled conditions, and watered with tap water (lead-free) (Jiang et al., 2019). After 30 days, Pb-induced experiments were carried out after all the bamboo seedlings presenting more than 50 of new roots. Based on our pre-test and previous study (Zhang et al., 2011), four Pb treatments, including 0 (control/CK without external Pb added), 300, 600, and 900 mg L−1 were employed as being calculated by the amount of pure Pb [added as Pb(NO3)2]. Because the differences of nitrogen concentration in culture solution, we therefore selected NH4NO3 to balance the differences. In the experiment, the culture solution was replaced by fresh solution as each five days. The fallen bamboo leaves were collected throughout the experiment. At 14 days after treatments (DAT), these bamboos were harvested and then divided into new root, old root, whips, new stem, old stem, new leaves and old leaves. For parameters requiring fresh mass measurements, individual plants were powdered using liquid nitrogen at −80 °C. To analyze quantitatively the significance, the entire experiment included three biological replicates (no technical replicates). Data were evaluated using a one-way ANOVA followed by Tukey's test (SPSS Inc., Chicago, IL, USA) at P < 0.05. 2.2. Determination of Pb and antioxidants levels To examine Pb level in plant tissues, 0.2 g of samples were completely dried and grinded through a 50-mesh sieve, and then digested into HNO3:HClO4 mixture (v:v, 5:1). Pb concentrations in plant tissues were determined by atomic absorption spectrophotometer (Shimadzu AA-7000, Kyoto, Japan; Jiang et al., 2019). Furthermore, the chemical forms of Pb were successively extracted by designated solutions according to the previous published protocols (He et al., 2013; Wang et al., 2015) as the following order: (1) 80% ethanol, extracting inorganic Pb including nitrate, nitrite, and aminophenol Pb (FE); (2) Deionized water (d-H2O), extracting water soluble Pb-organic acid complexes and Pb (H2PO4)2 (FW); (3) 1 M NaCl, extracting Pb associated with pectates and proteins (FNaCl); (4) 2% HAc, extracting 2

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The underground parts had significantly higher Pb concentrations than those of the aerial part. The Pb concentrations in new/old leaves and new/old roots were form ca. 11%–17% and form ca. 26%–37%, respectively. In new/old tissues, a big difference in Pb level of aerial and underground parts was recorded. The Pb concentrations in new roots were up to 1.78-fold than those of old roots, while the Pb concentrations in old stem and old leaves were up to 2.12- and 1.63-fold than those of new stem and new leaves, respectively. The Pb proportion in F1 and F2 components showed an opposite trend of increasing/decreasing among the new/old tissues of roots, while presenting the same trend of that in stems and leaves (Table 1, Fig. 2). Bedsides, the Pb proportion in F3 components showed a similar trend among new/old tissues of stems and leaves. The majority of Pb ions were found in F1 component in new root, old root, whips, new stem and old stem. Their distribution proportion was up to 93.7%, 89.0%, 76.2%, 82.8% and 82.8% of total Pb content, respectively (Fig. 2A–C). In leaves, the F1 component proportions decreased with increasing Pb doses, while F3 components increased. The F3 proportion in new/old leaves were up to 52.7% and 60.7% of total Pb content, respectively (Fig. 2D). In addition, the Pb content of subcellular components in roots, whips and stems were consistent with the changes of Pb level in tissues (P < 0.05). However, in leaves, a significant positive correlation between the F1 and F3 components was observed, while the Pb level in F2 components have no related with them significantly (Table 2). To investigate the Pb particles in the subcellular fractions, the tissues under Pb treatments were analyzed using the TEM (Figs. 3 and 4). Compared to CK treatment, in roots, the cell wall structure treated by Pb900 was distorted, and the intercellular space increased to some extent (Fig. 3A–D). Further, lots of granular, acicular black substances high electron deposits were distributed along the cell wall and plasma membrane. Similarly, in whips and stems, black sediment group substances were observed distributing on the cell wall, plasma membrane and cellular vacuoles (Fig. 3E–H, 4 A-D). Disparately, no black deposits were observed in leaves under Pb treatment except for the osmiophilic granules (Fig. 4E–H), but only minor destructive changes of organelle morphological was found in leaves.

undissolved Pb-phosphate complexes including PbHPO4 and Pb3(PO4)2 (FHAc); and (5) 0.6 M HCl, extracting Pb-oxalate (FHCl). The concentration of NPT-SH was measured following the protocol of De Vos et al, (1992) using GSH as standard, while the level of GSH was quantified using a reduced glutathione assay lit (A006-1; Jiangcheng, Nanjing, China; Liu et al., 2016). The concentration of PCs was calculated by the subtractive subtraction method following the formula (Estrella-Gómez et al., 2009):

PCs concentration = [NPT − SH] concentration − GSH concentration [1] 2.3. Extraction of Pb in subcellular fractionations Briefly, 0.2-g of fresh samples were mixed in buffer including 0.25 mM of sucrose, 0.05 mM of Tris-HCl (pH = 7.5), and 0.001 M of C4H10O2S2. The homogenate was centrifuged at 1000×g for 15 min, and the deposition was the cell wall fraction (F1). The supernatant solution was further centrifuged at 12,000×g for 30 min, the resultant deposition and the supernatant were organelle-containing fraction (F2) and soluble fraction (F3), respectively (Huang et al., 2018; Li et al., 2019). All the steps were performed at 4 °C. The separation of the components were dried using evaporation, and then digested with the HNO3: HClO4 mixture (v:v, 5:1) and diluted to designated volume (Lai, 2015). 2.4. Ultrastructural analysis of TEM in tissues To understand the Pb-induced inter-structure in bamboo tissue, an ultrastructural analysis of transmission electron microscopy (TEM) was employed according to the previous protocols (Hussain et al., 2019; Jiang et al., 2019) with minor modifications. Briefly, the sample was prefixed with a mixed solution of 3% glutaraldehyde, then fixed in 1% osmium tetroxide, continuously dehydrated with acetone, and then infiltrated/embedded in Epox. Semi-thin sections were stained with methylene blue and ultrathin sections, then stained with uranyl acetate and lead citrate, and observed using an TEM (Hitachi, H–600IV, Kyoto, Japan).

3.2. Impacts of Pb induce on Pb chemical forms in plant tissues 3. Results In the present study, four low-mobility/-toxicity Pb chemical forms (i.e., FNaCl, FHAc, FHAc, and FR) were predominant forms of Pb in roots, whips and stems and occupied 82.6%, 72.2%, 72.6%, 73.1%, and 70.2% of total Pb in new root, old root, whips, new stem and old stem, respectively (Fig. 5). However, the proportion of two high-mobility/toxicity (i.e., FE and FW) appeared smaller (Fig. 5A–C). In contrast, the two forms were predominant in leaves, occupying 57.2% and 77.7% of total Pb in new and old leaves, respectively (Fig. 5D). Additionally, the distribution proportions of low-toxicity/-mobility forms of new tissues

3.1. Impacts of Pb induce on concentration/distribution of Pb To investigate the concentration and distribution of Pb ions in bamboo tissues, we thus examined the Pb concentrations, Pb subcellular distribution and corresponding TEM observation under different Pb treatments (Fig. 1). The Pb concentrations showed a trend of decreasing from source to sink in the tissues of S. argenteistriatus plants under Pb treatments, presenting as roots > whips > stems > leaves.

Fig. 1. Impacts of different Pb dosages on Pb concentration in different tissues of Sasa argenteistriatus E.G. Camus plants. Pb300, Pb600, and Pb900 indicate that the Pb provided concentration is 300, 600 and 900 mg L−1 (being calculated by the amount of pure Pb), respectively. Vertical bars represent the SDs of the mean (n = 3). Bars within each tissue in different treatments labeled by the same numbers are not significantly different according to the Tukey's test (P = 0.05); bars with each treatment in different tissue labeled by the same letters are not significantly different according to the Tukey's test (P = 0.05).

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Table 1 Impacts of Pb treatments on the subcellular distribution of Pb in different tissues of Sasa argenteostriata (Regel) E.G. Camus plants. Pb300, Pb600, and Pb900 indicate that the Pb concentration are 300, 600 and 900 mg L−1 (being calculated by the amount of pure Pb), respectively. Values represent the SDs of the mean (n = 3). F1, cell wall fractions; F2, cellular organelles fraction; F3, soluble component. Plant tissue

New leaf

Old leaf

New stem

Old stem

New root

Old root

Whip

Treat.

Pb300 Pb600 Pb900 Pb300 Pb600 Pb900 Pb300 Pb600 Pb900 Pb300 Pb600 Pb900 Pb300 Pb600 Pb900 Pb300 Pb600 Pb900 Pb300 Pb600 Pb900

Pb concentration (mg·kg−1 FW) F1

F2

F3

Total

235.24 ± 10.03 345.32 ± 26.55 438.02 ± 60.22 258.41 ± 20.06 426.44 ± 10.03 449.61 ± 10.03 652.40 ± 24.65 2448.53 ± 516.90 3369.77 ± 327.95 750.89 ± 120.16 2906.24 ± 137.93 5745.28 ± 237.28 2299.65 ± 93.92 4859.96 ± 160.43 8033.45 ± 27.41 1166.32 ± 67.88 3158.72 ± 139.62 6931.86 ± 104.12 398.62 ± 8.73 1091.00 ± 41.05 2235.27 ± 100.80

101.98 ± 10.05 113.57 ± 10.03 119.36 ± 20.06 115.30 ± 8.53 113.57 ± 10.03 101.98 ± 10.05 101.97 ± 20.08 136.73 ± 20.06 165.71 ± 20.08 78.79 ± 2.74 171.50 ± 53.11 224.05 ± 11.46 85.71 ± 12.19 472.72 ± 15.00 1122.94 ± 100.24 165.71 ± 16.49 351.19 ± 24.22 422.00 ± 31.35 107.77 ± 30.11 171.50 ± 40.14 234.37 ± 48.35

49.83 ± 1.39 351.11 ± 9.06 617.63 ± 46.58 140.07 ± 4.77 663.99 ± 77.27 860.98 ± 160.56 174.83 ± 2.64 322.15 ± 40.14 536.52 ± 59.27 246.83 ± 52.15 478.58 ± 52.49 982.66 ± 253.28 69.04 ± 7.16 333.73 ± 30.11 813.07 ± 10.20 44.58 ± 9.21 116.89 ± 4.76 438.02 ± 49.31 46.49 ± 7.16 177.29 ± 30.11 464.13 ± 57.04

387.04 ± 17.44 809.99 ± 40.08 1175.01 ± 54.54 513.78 ± 24.43 1204.00 ± 96.92 1412.56 ± 171.48 929.20 ± 42.73 2907.41 ± 577.09 4071.99 ± 406.09 1076.51 ± 169.88 3556.32 ± 217.29 6951.99 ± 497.11 2454.40 ± 92.86 5666.41 ± 132.17 9969.46 ± 102.08 1376.61 ± 51.96 3626.81 ± 131.79 7791.88 ± 169.59 552.88 ± 46.00 1439.80 ± 108.03 2933.76 ± 155.85

Fig. 2. Subcellular fraction of Pb distribution proportion in Sasa argenteostriata (Regel) E.G. Camus tissues. Along the abscissa of each graph, Pb300, Pb600, and Pb900 indicate that the Pb concentration are 300, 600 and 900 mg L−1 (being calculated by the amount of pure Pb), respectively. The relative values in the vertical bars with different padding mean the percentage of Pb concentration in each subcellular fraction and total Pb concentration in the corresponding fraction. Vertical bars represent the SDs of the mean (n = 3). F1, cell wall fractions; F2, cellular organelles fraction; F3, soluble component.

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Table 2 Correlation analysis between Pb subcellular fractions and Pb treatment concentration in Sasa argenteostriata (Regel) E.G. Camus plants. * means significantly correlated at 0.05 level, while ** means significantly correlated at 0.01 level. F1, cell wall fractions; F2, organelle fractions; F3, soluble component. Bamboo tissue

F1–Pb F2–Pb F3–Pb

New root

Old root

Whip

New stem

Old stem

New leaf

Old leaf

0.981** 0.964** 0.958**

0.938** 0.961** 0.871**

0.967** 0.828** 0.930**

0.780* 0.718* 0.914**

0.916** 0.943** 0.856**

0.911** 0.493 0.968**

0.813** −0.495 0.858**

with Pb to form an insoluble Pb-phosphate precipitate, resulting in reducing Pb migration and bioavailability and hindering its upwardtransportation (Islam et al., 2008). Intriguingly, Pb2+ in the whips is present in the vertical transport of the stem and lateral transport within the tissues due to clonal integration and directional diffusion growth characteristics of whips (Saitoh and Nishiwaki, 2002). As a higher level of lignification, roots and whips have a greater proportion of negatively charged groups on the cell wall to increase the Pb binding sites (Krzes, 2011) and facilitate Pb mainly distribute in cell wall (Fig. 2). Furthermore, based on TEM observations and correlation analysis, our study proved that cell wall retention plays a leading detoxification role in subcellular regionalization (as showed in Fig. 3−4; Table 1–2). Therefore, the maximum retention of lead in the root-whips system is considered to be one of the tissue detoxification mechanisms of S. argenteistriatus plants to reduce Pb stress. In the aerial part, Pb level in stems and leaves were higher than those in old tissues (Fig. 1). One possible explanation is that the physiological integration of the clonal ramets attenuate the Pb damage on the daughter ramet, which facilitates the adaptation of the entire clone to Pb stress (Gruntman et al., 2017; Roiloa and Rubén, 2012). In a homogeneous environment, self-cloning achieves a two-way transportation and differential distribution of Pb between mother and daughter ramet through the whips, which makes the old tissues of the mother ramets more likely to endure the risk of Pb stress and thus successfully reduce ecotoxicity in new tissue (Gruntman et al., 2017; Guo et al., 2017). Similarly, cell walls are the major Pb sequester region in stems (Fig. 2). One explanation is that stem cell walls containing lots of lignin could bind with the effectively of Pb (Marmiroli et al., 2005). Simultaneously, cell wall immobilization reduces the translation of Pb from stems to leaves, thereby reducing the toxicity of Pb to the leaves.

in roots, stems and leaves were greater than those of old tissues, while the two high-toxicity/-mobility forms proportions were greater in old tissues. These findings suggested that new tissue had a stronger Pb detoxification ability than the old ones. 3.3. Impacts of Pb induce on antioxidant compounds in tissues Levels of NPT-SH and PCs significantly enhanced in new root, new leaves and old leaves with increasing Pb dosage (Fig. 6A and B). In new/old roots, the highest increasing at Pb900 was markedly greater by 223.2% and 636.3% than those at CK treatment, respectively. GSH levels in new/old root decreased with increasing Pb dosage, while an opposite trend was reported in leaves, which significantly increased by 193.9% and 122.0% in comparison with CK respectively (Fig. 6C). Moreover, the proportions of PCs in NPT-SH were greater than those of GSH in roots, however showing opposite in leaves (Fig. 6D). Interestingly, the proportion of PCs increased in new root but greater than old root at Pb900, while the proportion of GSH in new leaves showed higher level than that in old leaves. 4. Discussion 4.1. Subcellular compartmentalization contributes to lead detoxification in tissues Similar to most Pb non-hyperaccumulator plants (Gupta et al., 2009; Tian et al., 2010; Chandana et al., 2019), our studied bamboo retained majority of the Pb-uptake (total Pb) in roots under Pb induction. Among them, the highest Pb level was recorded in new root (Fig. 1). One possible explanation is that the phosphates of root-secreted combine

Fig. 3. Cell ultrastructure observation in new roots (A–D) and whips (E–H) of Sasa argenteostriata (Regel) E.G. Camus plants. Fig. 3A/E and 3C/G obtained from 5000 times observation under CK and Pb900 treatments, respectively. Fig. 3B/F and 3D/H obtained from 10,000 times observation under Pb and Pb900 treatments, respectively. N, nucleus; CW, cell wall; CM, cell membrane; PM, plasma membrane; Chl, chloroplast; M, mitochondria; V, Vacuole; PD, plasmodesma; ICS, intercellular space; GOS, osmiophilic granules. Bars in the above figures present 1 μm. 5

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Fig. 4. Cell ultrastructure observation in new stems (A–D) and new leaves (E–H) of Sasa argenteostriata (Regel) E.G. Camus plants. Fig. 4A/5 E and 4C/G obtained from 5000 times observation under CK and Pb900 treatments, respectively. Fig. 4B/5 F and 4D/H obtained from 10,000 times observation under Pb and Pb900 treatments, respectively. N, nucleus; CW, cell wall; CM, cell membrane; PM, plasma membrane; Chl, chloroplast; M, mitochondria; V, Vacuole; PD, plasmodesma; ICS, intercellular space; GOS, osmiophilic granules. Bars in the above figures present 1 μm.

salts, organic acids and organic bases in the vacuolar component (Sharma et al., 2010; Zhou et al., 2016; Zhao et al., 2015). In our study, there was no significant correlation between F2 composition and tissue Pb content (Table 2), indicating that the leaves reduce the toxicity of Pb to the active central region of the cell by decreasing the distribution of

Interestingly, cell wall immobilization initially played a major role in the leaves under low-concentration Pb treatment, but vacuolar division become a dominant role with increasing Pb dose (Gupta et al., 2013; Fei et al., 2016). The explanation may be that the Pb-binding sites on the cell wall were saturated and Pb-entering mainly related with inorganic

Fig. 5. Impacts of Pb treatments on the proportion of different Pb chemical forms in Sasa argenteostriata (Regel) E.G. Camus tissues. Along the abscissa of each graph, Pb300, Pb600, and Pb900 indicate that the Pb provided concentrations are 300, 600 and 900 mg L−1 (being calculated by the amount of pure Pb), respectively. Vertical bars represent the SDs of the mean (n = 3). FE, ethanol extraction state; FHAc, acetic acid extraction state; FHCl, hydrochloric acid extraction state; FNaCl, sodium chloride extraction state; FR, residual state; FW, deionized water extraction state. 6

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Fig. 6. Impacts of different Pb dosages on the levels of NPT-SH content (A), PCs content (B) and GSH content (C), and the proportion of PCs/GSH in NPT-SH (D) in Sasa argenteistriatus E.G. Camus tissues. CK, Pb300, Pb600, and Pb900 indicate that the Pb provided concentration are 0, 300, 600 and 900 mg L−1 (being calculated by the amount of pure Pb), respectively. Vertical bars represent the SDs of the mean (n = 3). Bars within each tissue in different treatments labeled by the same numbers are not significantly different according to the Tukey's test (P = 0.05); bars with each treatment in different tissue labeled by the same letters are not significantly different according to the Tukey's test (P = 0.05).

Therefore, we speculate that new roots may play a leading role in the transfer of Pb, in contrast, old roots and old whips may be mainly used as a carrier for Pb storage. Similarly, the Pb-detoxification form was dominant in stems, presenting a strong capability of Pb detoxification (Fig. 5). The distribution of ethanol and H2O extractable increase when the old stems maintain a high level of NaCl extractable under low-concentration Pb stress, indicating that the old stems bear more risk of Pb stress and undertake the task of transporting Pb to the leaves. In terms of leaves, the ethanol and H2O extractable, which considered to be the two high-mobility and -toxicity extractable (He et al., 2015; Jiang et al., 2019), being dominant in the leaves (Figs. 3 and 4). Based on the TEM observation, leaves cell structure remained intact under Pb stress and no obvious deformation of organelle morphology (Fig. 2). We thus speculate that there may be other mechanisms for more effective detoxification in leaves rather than in the form of Pb transferring, although the Pb distribution of ethanol and H2O extractables is at a higher level. For Pb species, the four low-mobility/-toxicity Pb chemical extractable (i.e., FNaCl, FHAc, FHCl and FR) were the main forms of Pb in roots, whips and stems. Further, Pb distribution proportion in new roots, old roots, whips, new stems, old stems were up to 82.6%, 72.2%, 72.6%, 73.1% and 70.2%, respectively. The two high-mobility/-toxicity extractable (i.e., FE and FW) were the main forms in leaves, and the proportions were up to 57.2% and 77.7% in new leaves and old leaves, respectively. The conversion of Pb to low-toxicity and -mobility forms are the Pb detoxification mechanism of roots, whips and stems, which are not obvious existed in leaves.

Pb in the organelle, thereby stabilizing normal physiological metabolism. Further, in our study, cell wall fixation is regarded as the major Pb detoxification mechanism of new roots, old roots, whips, new stems and old stems, but vacuole separation is the dominant mechanism of new and old leaves. We therefore speculate that at the tissue and subcellular levels, the retention of Pb by the root-whips system may be one of the detoxification mechanisms of this plants to alleviate the damage of Pb to the aerial parts. 4.2. Pb species transformation plays an important role in Pb detoxification Under Pb stress, the majority of Pb in roots, whips and stems were deposited into four low-mobility chemical forms, i.e., FNaCl, FHAc, FHAc, and FR, which may be beneficial to reduce Pb ecotoxicity by promoting the immobilization of Pb on cell walls and the separation of vacuoles (Xu et al., 2011; Zu et al., 2015; Li et al., 2017). Further, we found that NaCl and HAc extractable occupied the largest proportions among the four species, retaining Pb in F1 component mainly in the forms of oxalate, Pb-phosphate, protein bound or adsorbed state (Tian et al., 2010; Xu et al., 2018) to relieve stressful damage. One explanation may be related to Pb chelation and fixation of pectin, protein and various coordination groups on cell walls and/or cell membranes (Gupta et al., 2013). In addition, NaCl and HAc extractable were evidently dominant in roots and whips, accompanied by the increase of the proportions of ethanol and H2O extractable of new roots and decrease of that in old roots and whips (Fig. 5). These findings revealed that new root can facilitate the combination of Pb with nitrate ions, chlorides, organic acids and dihydroxy phosphoric acid when transferring Pb ions into the with low-mobility/-toxicity chemical forms (He et al., 2013). 7

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distribution and chemical forms of Pb in this dwarf landscape bamboo, S. argenteostriata. Moreover, this bamboo has the potential to be applied in lead-contaminated aqueous-medium for remediation with its stronger tolerance and accumulation capacity and reasonable detoxification mechanism against lead toxicity, as showed in Fig. S1. On the one hand, most of the absorbed Pb is accumulated in the roots (esp. in new roots) under short-term Pb stress, which is similar to Pb non-hyperaccumulators. However, whip Pb is present in the vertical transport of the stem and in the lateral transport within the tissue. We therefore speculate the maximum retention of Pb in root-whips system is considered to be one of the tissue detoxification mechanisms to relieve Pb poisoning. On the other hand, four low-mobility/-toxicity Pb species (i.e., FNaCl, FHAc, FHCl and FR) are distributed in roots, whips and stems, while two high-mobility/-toxicity of Pb species (FE and FW) in leaves. The conversion of Pb to low-toxicity/-migration is a Pb-detoxification strategy in roots, whips and stems not in leaves. As a complementary, the synthesis of antioxidant substances (e.g., NPT-SH, GSH and PCs) in new/old roots and leaves contribute to Pb detoxification in plant tissues. Especially, PCs play a leading role in the Pb detoxification in new/old roots, while GSH is in new/old leaves. However, transportation/distribution of chemical forms of lead under different Pb concentrations and exposure times, and different molecular strategies for lead detoxification in dwarf bamboo tissue require further investigation. To fully understand the detoxification mechanism, an in-depth and comprehensive understanding of Pb detoxification strategies in different tissues of S. argenteistriatus bamboo from the level of genes (or genomes) requires future investigation.

4.3. Tissue antioxidant substances assist to chelate lead to alleviate ecotoxicity NPT-SH level in roots and leaves showed a significant increase (Fig. 6), which reduce the mobility and toxicity of Pb owing to its high affinity for metals (Qiao et al., 2014; Barbara, 2002). In the present study, the GSH proportion of NPT-SH in roots was lower than that in leaves, which may be due to that GSH participates in the synthesis pathway of PCs in roots, by consuming GSH to ensure the high concentration of PCs in cells (Liu et al., 2016; Jiang et al., 2019), and thus promoting the detoxification strategies of Pb chelation by PCs (Seth et al., 2011). Previous studies indicated that level of PCs is related to the concentration of metals (Gisbert et al., 2003; Estrella-Gómez et al., 2009), which was also observed in the present study. Meanwhile, NPTSH and PCs content were significant positively correlated with F1 and F3 components (Table S1), suggesting that NPT-SH and PCs synergistically involved in cell wall retention and vacuolar compartmentalization of new root (Dai et al., 2012; Sun and Luo, 2018). However, the synthesis of GSH was inhibited by Pb stress and did not participate in Pb detoxification. Moreover, levels of NPT-SH, PCs and GSH in old roots decreased with the increase of Pb added dosage, which was related to the risk of Pb stress shared by the mother ramets (Guo et al., 2017; Gruntman et al., 2017; Luo et al., 2017). Furthermore, correlation analysis showed that NPT-SH and PCs levels were extremely significant positively correlated with all subcellular components Pb contents in new roots, while GSH content was negatively correlated with them. Levels of NPT-SH, PCs and GSH were extremely significant negatively correlated with them in old roots. Moreover, NPT-SH and GSH levels significant positively correlated with F1 and F3 component Pb contents, while no significant correlation between PCs level and subcellular components in new leaves was observed. Additionally, levels of NPT-SH, PCs and GSH in old leaves were significant positively correlated with F1 and F3 components, but no significant correlated with F2 component (Table S1). These findings suggested that the synthesis of plant cell chelation substances in roots and leaves were affected by subcellular regionalization, which might be related to the subcellular distribution (Krzesłowska et al., 2016; Hasanuzzaman et al., 2018). With respect to leaves, we also observed no significant correlation between GSH and Pb in all subcellular component. One possible explanation is that GSH may act more as an antioxidant and participate, involving in resisting ROS antioxidant pathways to protect the stability of plasma membrane under Pb stress (Neyi et al., 2012; Wang et al., 2010). On the other hand, the proportion of PCs in NPT-SH is not large, but the content also maintains a high level. We thus speculate that PCs may have actively participated in the Pb cell chelation in leaves, forming a stable thiopeptide complex and stored in vacuoles (Estrella-Gómez et al., 2009; Huang and Wang, 2010). Furthermore, our study showed that a significant (P < 0.05) correlation between the PCs and F1 and F3 components Pb content both in roots and leaves was observed (Tables 1 and 2). This possible explanation is that PCs content, which mainly distributed in the cell wall and vacuole region of roots/leaves cells, is closely related to regional distribution of Pb at the subcellular level (Marmiroli et al., 2005; Krzes, 2011; Krzesłowska et al., 2016). For antioxidant system, the roots and leaves in S. argenteistriatus plants can alleviate Pb stress damage by massively synthesis of NPT-SH, GSH and PCs, which is consistent with our previous conclusions (Liu et al., 2016; Jiang et al., 2019). More importantly, PCs play a vital role of Pb detoxification in new roots and old roots, while GSH mainly plays a leading role in new and old leaves (Fig. 6). Therefore, this evidence supports our hypothesis that the antioxidant substances contribute to the detoxification strategy of lead in different tissues of this studied bamboo.

Funding This research was supported in part by the National Natural Science Foundation of China (NSFC, Grant No. 31700541) and the Sichuan Science and Technology Program (Grant No. 2017NZ0008). CRediT authorship contribution statement Mingyan Jiang: Conceptualization, Methodology, Writing - original draft, Funding acquisition, Project administration. Xinyi Cai: Formal analysis, Investigation, Visualization, Writing - original draft. Jiarong Liao: Formal analysis, Investigation, Visualization. Yixiong Yang: Formal analysis, Investigation, Visualization. Qibing Chen: Methodology, Writing - review & editing. Suping Gao: Methodology, Writing - review & editing. Xiaofang Yu: Visualization, Writing - review & editing. Zhenghua Luo: Writing - review & editing. Ting Lei: Visualization, Data curation. Bingyang Lv: Visualization, Data curation. Shiliang Liu: Conceptualization, Writing - original draft, Writing review & editing, Supervision. Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements Special thanks are given to the ultrastructural analysis of TEM in Institute of West China Medical Testing at Sichuan University. The authors are also grateful to all of editors and anonymous reviewers for their comments and suggestions on an earlier draft of the manuscript. Appendix A. Supplementary data

5. Conclusion Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110329.

This study provides the first information on the subcellular 8

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