Bioaccumulation，subcellular distribution and chemical formsof cadmium in Aster subulatusMichx.

LIANG Xue-lian CHEN Wei JIANG Wen-yan LIAO Jie YANG Yu-xia

WANG Hai-jun LI Hui-ling LU Wei-fan WANG Tian-shun1*，XIE Hong-zhao3*

（1Agro-products Quality Safety and Testing Technology Research Institute，Guangxi Academy of Agricultural Sciences，Nanning，Guangxi 530007，China；2Institute of Agro-products Processing Science and Technology，Guangxi Academyof Agricultural Sciences，Nanning，Guangxi 530007，China；3Guangxi Association for Standardization，Nanning，Guangxi 530009，China）

Abstract：【Objective】Through analyzing the bioaccumulation capacity，subcellular distribution and chemical forms of cadmium（Cd）in Aster subulatusMichx.，this study was to provide reference for revealing the Cd tolerance mecha-nismofA.subulatusMichx.【Method】After cultured for 24 d under the action of Hoagland nutrient solution and gradient Cd concentrations（0，30，60 and 90 mg/L），A.subulatusMichx.were harvested，and its leaf，stem and root were treated by differential centrifugation，chemical reagent extraction，and digested with graphite digester，respectively，then the Cd content in the root，stem and leaf were determined by atomic absorption spectroscopy.【Result】The experimental results indicated that the bioaccumulation capacity of Cd in A.subulatusMichx.was root>stem>leaf，and the maximum Cd con-centration in the root，stem and leaf of A.subulatusMichx.were 130.74，78.69 and 56.62 mg/kg（fresh matter），respec-tively.Most of Cd stored in the cell wall and the soluble fractions of the root and leaf ofA.subulatusMichx.，with only a smaller portion Cd in organelle fraction.Analysis result oA5n9fnACtBv5Z3/YLhYndg==f subcellular Cd content showed that 52.27%-58.61%of Cd for root was mainly stored in the soluble fraction，but 42.10%-63.28%of Cd for leaf was mainly stored in the cell wall frac-tion.The concentration of pectates and protein integrated-Cd was higher in the root and leaf compared to other chemical forms Cd.Pectates and protein integrated-Cd was the main chemical forms Cd in the root and leaf ofA.subulatusMichx.，and their percentages were 68.91%-74.80%and 57.38%-83.80%，respectively.Cd treatment could significantly increase the proportion of water-soluble organic acid Cd from 13.64%to 22.72%in root and undissolved phosphate Cd from 10.02%to 32.78%in leaf with increasing Cd concentration in the culture medium.【Conclusion】The root，stem and leaf of A.subulatusMichx.has strong bioaccumulation capacity to Cd，Cd is primarily stored in the soluble fractions of the root and cell wall fractions of the leaf，and less toxic pectates and protein integrated-Cd is the main chemical forms Cd in the root and leaf ofA.subulatusMichx.，this might be the main mechanism of Cd tolerance in A.subulatusMichx.

Key words：AstersubulatusMichx.；cadmium；bioaccumulation；chemical form；subcellular distribution

CLC number：S567.21 Document code：A Article：2095-1191（2024）08-2386-10

摘要：【目的】分析鎘（Cd）在鉆葉紫菀中的生物富集能力、亞細(xì)胞分布及化學(xué)形態(tài)，為揭示鉆葉紫菀對(duì)Cd的耐受機(jī)制提供參考依據(jù)。【方法】在Hoagland營養(yǎng)液及不同濃度（0、30、60和90 mg/L）Cd作用下培養(yǎng)鉆葉紫菀，24 d后收獲鉆葉紫菀。鉆葉紫菀葉、莖和根經(jīng)差速離心、化學(xué)試劑萃取及石墨消解處理后，利用原子吸收光譜儀測定其Cd含量?！窘Y(jié)果】鉆葉紫菀Cd生物富集能力表現(xiàn)為根>莖>葉，鉆葉紫菀根、莖和葉中的最大Cd含量分別為130.74、78.69和56.62 mg/kg（鮮重）。鉆葉紫菀根和葉中大部分Cd儲(chǔ)存在細(xì)胞壁和細(xì)胞可溶性組分，僅有少量Cd儲(chǔ)存在細(xì)胞器組分。鉆葉紫菀根中細(xì)胞可溶性組分Cd占根亞細(xì)胞組分Cd的52.27%～58.61%，而鉆葉紫菀葉中細(xì)胞壁組分Cd占葉亞細(xì)胞組分Cd的42.10%～63.28%。鉆葉紫菀根與葉中果膠和蛋白質(zhì)結(jié)合形態(tài)Cd含量高于其他化學(xué)形態(tài)Cd含量。果膠和蛋白質(zhì)結(jié)合形態(tài)Cd是鉆葉紫菀根和葉中存在的主要化學(xué)形態(tài)Cd，占比分別為68.91%～74.80%和57.38%～83.80%。隨著培養(yǎng)基Cd濃度的增加，鉆葉紫菀根中水溶性有機(jī)酸Cd占比由13.64%增至22.72%，同時(shí)葉中不溶性磷酸Cd占比由10.02%增至32.78%。【結(jié)論】鉆葉紫菀根、莖和葉對(duì)Cd有較強(qiáng)生物富集能力，Cd主要儲(chǔ)存在鉆葉紫菀根細(xì)胞可溶性組分和葉細(xì)胞壁組分，且根和葉中的化學(xué)形態(tài)Cd以毒性弱的果膠和蛋白質(zhì)結(jié)合形態(tài)Cd為主，這可能是鉆葉紫菀耐受Cd的主要機(jī)制。

關(guān)鍵詞：鉆葉紫菀；鎘；生物富集；化學(xué)形態(tài)；亞細(xì)胞分布

0 Introduction

【Research significance】Hyperaccumulator plantspecies have high metal absorption and accumulation ability（Baker and Brooks，1989；Reeves et al.，2018b）and thus can tolerate and accumulate nonessential heavy metal ions，such as cadmium（Cd），chromium（Cr），lead（Pb），etc.（Zeng et al.，2017；Zheng et al.，2019）.These plants have high stress resistance and high biomass production.So far，approximately 720 metal hyperaccumulators have been identified（Reeves et al.，2018a），forming an alternative in situ technologyto clean up toxic metals，which is cost-effective and eco-friendly（Ali et al.，2013；Manzatu et al.，2015；Bello et al.，2018）.Human activities such as industrial effluent discharge，mining，and application of sewagesludge，pesticides and phosphate fertilizers are the major sources of Cd（Gallego et al.，2012；Daud et al.，2013；Shi et al.，2016；Dong et al.，2019）.The metal exerts its toxic effects on plants cells in many ways，including inhibiting the DNA repair system，damaging genomic DNA and chloroplast，and inducing cell death（?uki?-?osi?et al.，2020）.Cd can also be enriched in the human body through the diet chain，resulting in renal insufficiency，lung cancer，and car‐diovascular diseases（Nawrot etal.，2010）.Therefore，the Cd polluted environment needs to be remedied urgently，and the Cd hyperaccumulator Aster subula-tus Michx.is a choice for phytoremediation.A.subula-tus Michx.is a newly discovered Cd hyperaccumula‐tor，it belongs to the family Compositae，found in Guangxi，China（Chen et al.，2023）.A.subulatusMichx.has the super-accumulation ability and hightolerance to Cd since the Cd concentrations in itsshoot reached 47.6-133.6 mg/kg（Chen et al.，2022；Chen et al.，2023）.【Research progress】Plants have evolved various techniques to withstand Cd toxicity，including vacuolar compartmentation，regionalization of cell wall deposition and isolation in vacuoles（Zhang et al.，2013）.However，Cd resistance mecha‐nism in Cd hyperaccumulators has not been fully stu-died（Zhou et al.，2016）.Different chemical forms of Cd might affect its toxicity in plants（Zhang et al.，2014；Shi et al.，2017）；for example，water-soluble Cd and inorganic Cd exhibited the highest toxicity，fo-llowed by insoluble phosphates Cd，and protein and pectate-integrated Cd，thenoxalate Cd and residual Cd had the weakest toxicity（Xu et al.，2018）.These chemical forms also affected the transportation and accumulation of Cd in plants.Therefore，convertingthe highly toxic chemical forms of Cd might be a key mechanism for detoxifying Cd pollutants（Zhao et al.，2015）.Some scholars also suggested that Cd subcellu‐lar distribution and chemical morphology affected its accumulation and toxicity in plants（Luka?ováet al.，2013；Meyer et al.，2015；Parrotta et al.，2015）.Pre-vious research by the research team had shown that Cd had little impact on the physiology of A.subulatusMichx.，and it could grow normally，showed strongtolerance and accumulation ability to Cd stress under 2.5-30.0 mg/LCd stress treatments.A.subulatusMichx.could adapt to Cd stress habitat by regulating the accu‐mulation and translocation of Cd in their body.More‐over，chelators in a suitable dose which could promotethe absorption and accumulation of Cd in A.subulatusMichx.（Wang et al.，2020）.【Research breakthrough point】A.subulatusMichx.，found in Guangxi，China，could accumulate more than 100 mg/kg Cd in its aboveground shoot and has been suggested as a poten‐tial detoxifier of Cd-contaminated sites（Chen et al.，2022；Chen et al.，2023）.But there have been a few reports on the bioaccumulation，subcellular distribu‐tion，and localization of different chemical forms of Cd in subcellular components of A.subulatusMichx.under Cd stress.Moreover，Cd accumulation andresis‐tance mechanisms of A.subulatusMichx.have not known yet.Identifying these mechanisms is important in understanding the Cd superaccumulation and detoxification abilities of A.subulatusMichx.【Sol-ving problems】Through sand-based hydroponic experi‐ment in greenhouse，taking A.subulatus Michx.as the experimental material，the present study was to investi‐gate the bioaccumulation characteristic of Cd in A.subulatusMichx.，to estimate the distinguishing fea‐ture of Cd subcellular distribution in A.subulatusMichx.，and to study the different Cd chemical forms affecting its distribution in A.subulatusMichx.This study can provide data support and a theoretical refe-rence for the application of A.subulatus Michx.as in situ phytoremediation of Cd polluted ecological envi-ronment.

1 Material and Methods

1.1 Plant materials and Cd treatments

Seeds of A.subulatusMichx.，with Cd concentra‐tions of 47.6-133.6 mg/kg in shoot，were collected from an abandoned ore-processing plant in Yulin，Guangxi，China（Chen et al.，2022；Chen et al.，2023）.The seeds were sterilized with 70%ethanol for 3 min and planted in cadmium-free soil.After attaining six fully-open leaves，the young seedlings were rinsed 3 times in ultrapure water and transplanted into plastic pots（16 cm top diameter，11 cm height，and 9 cm bo-ttom diameter）containing 2.5 kg of sand in a net shed.Subsequently，1 L of Hoagland nutrient solution was applied to each pot，and the treatments were repeated 10 d later.At 15 d of the nutrient solution treatment，1 L of Cd solution（CdCl2·2.5H2O）was applied to the pots at 4 different concentrations，0，30，60 and 90 mg/L and labeled as T0，T T2 and T3 respectively.The pots were then randomly arranged in the shed，and 1 L of different Cd concentrations solution was applied repeatedly every 3 d during the 24 d culture period.Each treatment had 6 replicates，with 4 plants per replicate.

After 24 d of culture，the plants were collectedand rinsed 3 times in deionized water，and their root were soaked in 20 mmol/L Na2-EDTA solution for 20 min according to Xu and Wang（2013）.The samples were then grouped based on different Cd treatments and categorized in triplicate into leaf，stem，and root.One batch of the triplicated samples was oven-dried at 105℃for 30 min，then oven-dried at 65℃for 72 h to constant weight，and their dry weight was weighed.Subsequently，leaf and root from the other batch were immediately frozen in liquid N2 and stored at-80℃until use.The frozen leaf（1.0 g）and root（0.5 g）were then homogenized with mortar and pestle in ste-rile distilled water，then digested with a mixture of HNO3 and H2O2（3∶ v∶v）on a graphite digest fur-nace.

1.2 Extraction of subcellular Cd from the sam-ples

Frozen leaf（1.0 g）and root（0.5 g）were homo-genized with precooled pestle and mortar in 20 mL of the prechilled extraction buffer containing 1.0 mmol/L DL-dithioerythritol，50 mmol/L Tris-HCl buffer solu‐tion（pH 7.5）and 250 mmol/L sucrose.Thereafter，leaf and root were divided into 3 fractions，the organe-lle fraction，the soluble fraction，and the cell wall fraction respectively，using a modified centrifugation method（Weigel and J?ger，1980）.Briefly，the mix‐ture were centrifuged at 400×g for 20 min，and the pre‐cipitate was the cell wall fraction，while the soluble and cell organelle fraction formed the supernatant.The supernatant was again centrifuged at 20000×g for 45 min，forming soluble fraction as the supernatant and cell organelle fraction as the precipitate.All opera‐tion was conducted at 4℃，and all component obtained by centrifugation was evaporated on a graphi-te digest furnace at 100℃to a constant volume，fo-llowed by digestion in HNO3 and H2O2（3∶ v∶v）mixed solution system.

1.3 Extraction of Cd chemical forms

Chemical forms of Cd were extracted via succes‐sive steps using various extractants（Weng et al.，2012；Zhang et al.，2014）.These extractants included（1）80%ethanol for extracting inorganic Cd（F1），（2）deionized water（dH2O）for extracting water-soluble organic acid Cd（F2），（3）1 mol/L NaCl for extrac-ting pectates and protein integrated Cd（F3），（4）2%acetic acid（HAc）for extracting undissolved phos‐phate Cd（F4），and（5）0.6 mol/L HCl for extractingoxalic Cd（F5）.Briefly，1.0 g of the frozen leaf and 0.5 g of the frozen root was homogenized with mortar and pestle in appropriate extractant solutions，diluted at the ratio of 1∶35（w/v）in 50 mL centrifuge tube，and incubated in a rotary shaker for 22 hat 25℃.The mixture was then centrifuged at 5000×g for 20 min，and the obtained supernatant was transferred into digestion tube.Meanwhile，the precipitate was re-extracted twice in appropriate extractants，incubated on a rotary shaker for 2 h at 25℃，and centrifuged under the same condition.All obtained supernatant was pooled together and evaporated with the final resi‐dues（F6）on a graphite digest furnace at 100℃to a constant volume，followed by digestion in a mixture of HNO3 and H2O2（3∶ v∶v）.

1.4 Cd concentration and quality assuranceyAUoHIyArSzpd/286JlBC5YCYgaZd1WTjt5QHiW/6Cc=/quality control analysis

The concentration of Cd in each digestion solu‐tion was determined using the flame and graphite fur‐nace atomic absorption spectrophotometer（PE900T）.The quality assurance/quality control（QA/QC）of the process were verified using certified reference mate‐rial（citrus leaf，GBW10020，National Research Cen‐ter for Standard Materials in China）.Replicate analy‐sis showed that the Cd content of the reference mate‐rial was 0.16±0.03 mg/kg（dry weight），consistent with the given standard value of 0.17±0.02 mg/kg（dry weight）.

1.5 Statistical analysis

The experimental data were expressed as means±standard deviations（SD）.SPSS 22.0 was used to per‐form the statistical analysis.Differences in the res-ponse of various treatments were determined by analy‐sis of variance（One-way ANOVA）and means sepa‐rated by Duncan’s multiple range test at the signifi‐cance level of P<0.05.The independent sample t-test was used to determine the significant difference in the resulting values（P<0.05）.

2 Results and Analysis

2.1 Plant biomass and Cd concentration

Biomass of A.subulatusMichx.under different Cd treatments were shown in Table 1.The leaf，stem，root，and total biomass of A.subulatusMichx.decreased with increasing Cd concentration in the cul‐turemedium.However，there were no significant dif‐ferences between T1 and T0（P>0.05，the same below），and the leaf，stem，root，and total biomass ofA.subula-tusMichx.respectively decreased by 1.8%，0.6%，2.7%and 1.6%under T1 compared to T0.But the leaf，stem，root，and total biomass significantly reduced by 36.6%，16.0%，38.2%and 27.8%respectively under T3 compared to the T1（P<0.05，the same below）.

The Cd concentrations in leaf，stem，and root of A.subulatusMichx.were shown in Table 2.The Cd content in leaf，stem and root of A.subulatusMichx.gradually increased with increasing Cd concentration in the culture medium，and there were significant dif‐ferences between the Cd-treated groups and T0.The maximum values of Cd concentrations in the root，stem，and leaf of A.subulatusMichx.were 130.74，78.69，and 56.62 mg/kg respectively.Moreover，the Cd translocation factor in stem and leaf increased ini‐tially and then decreased with increasing Cd concen‐tration in the culture medium；however，that of the leaf increased again after decreasing.Compared to the T the largest stem translocation factor of plants culturedin T2 increased by 34.04%，while the smallest leaf translocation factor of plants exposed to T2 decreased by 16.22%.

2.2 Subcellular Cd distribution in leaf and root of A.subulatusMichx.

The subcellular Cd distribution in A.subulatusMichx.was presented in Table 3.A large proportion of Cd stored in the cell wall and the soluble fractions of leaf and root of A.subulatusMichx.，with only a small portion of Cd in the organelle fraction.Moreover，the Cd concentration in 3 subcellular fractions gradually increased with increasing Cd concentration in the cul-turemedium.Under T0，T T and T3，the Cd con-centration in the soluble fraction of leaf were 0.10，0.95，2.7 and 10.55 mg/kg respectively，while those in the root were 1.73，15.90，35.67，and 71.37 mg/kg respectively.The Cd concentration in root cell wall components increased from 0.45 to 38.20 mg/kg，and that in leaf cell wall component increased from 0.28 to 17.67 mg/kg，with the increasing Cd concentration in the culture medium.

Analysis result of subcellular Cd content indi-cated that Cd was mostly located in the cell wall frac-tion（42.10%-63.28%），followed by cell organellesfraction（18.89%-32.76%），and then soluble fraction（17.84%-25.14%）；additionally，the Cd content of theroot was highly distributed in the soluble fraction（52.27%-58.61%），followed by the cell wall fraction（29.12%-37.22%），and then cell organelle（10.51%-12.53%）；most of the Cd in the leaf（67.24%-84.11%）and root（87.47%-89.49%）were in the cell wall andthe soluble fraction，while a small portion of Cd accu-mulated in the organelle（Fig.1）.

2.3 Chemical forms of Cd in leaf and root of A.subulatusMichx.

The concentration of Cd chemical forms was evi-dently different among the Cd-treated and the control plants（Table 4，Table 5）.The concentrations of the different Cd chemical forms gradually increased in the leaf and root，with increasing Cd concentration in the culture medium.

The concentrations of F3 were the highest in the leaf of all treated plants，followed by F4.The concen-trations of F F F5 and F6 were relatively low.Moreover，the percentage of F4 increased，but that of F3 decreased in the leaf，with the increasing Cd con-centration in the culture medium.F3 accounted for the highest percentage（57.38%）of the total Cd，followed by F4（32.78%），and F6（0.26%）had the smallest percentage in the leaf under T3.Furthermore，com-pared to T the percentage of F4 increased by 2.27 times，and that of F3 decreased by 31.53%in leaf under T3.

The concentrations of F3 were the highest in the root across all treated plants，followed by F while those ofF F5，F(xiàn)4 and F6 were relatively low.More-over，the Cd treatments increased the concentration of F2 in the root，and the concentration of F2 increased by 5.28 times in root under T3 compared to the T1.F3 accounted for the highest percentage（68.91%）of the total Cd，followed by F2（22.72%），and F6（0.11%）had the smallest percentage in the root under T3.Fur-thermore，the percentage ofF2 increased by 66.57%in roots under T3 compared to T1.

The concentration of pectates and protein inte-grated Cd was higher in the root and leaf compared to other chemical forms Cd.Pectates and protein inte-grated Cd was the main chemical forms Cd in the root and leaf of A.subulatusMichx.，and their percentages were 68.91%-74.80%and 57.38%-83.80%，respec-tively.Cd treatment could increase the proportion of water-soluble organic acid Cd from 13.64%to 22.72%in root and undissolved phosphate Cd from 10.02%to 32.78%in leaf with increasing Cd concentration in the culture medium.

3 Discussion

A.subulatusMichx.has the super-accumulation ability and high tolerance to Cd，this may be due to the unique Cd sequestration and translocation mecha-nismofA.subulatusMichx.Therefore，there is a high potential for Cd-contaminated areas phytoremediation using A.subulatusMichx.because of its high bioaccu-mulation ability of Cd，high biomass production and rapid growth.In this study，a slight reduction in the dry weight ofA.subulatusMichx.plants was observed after exposure to Cd treatments for 24 d.

Generally，the plant root seemed to be thereposi-tories of heavy metals，thus alleviating the toxicity ofheavy metals in plants（Zhang et al.，2014）.In thisstudy，the distribution of Cd in A.subulatusMichx.was uneven，with the root exhibiting the highest Cd accumulation.Similar results were attained in spinach（Yin et al.，2016）and ryegrass varieties（Feng et al.，2019）subjected to Cd stress.In this study，root of A.subulatusMichx.sequestered 35.40-130.74 mg/kg（fresh weight）Cd，the highest Cd concentration in the whole plant.The high Cd tolerance of A.subulatusMichx.might be due to the fixation effect of root，which intercepts a large amount of Cd，restricting the Cd migration to the stem and leaf，thus reducing Cdtoxicity to plants.The hypothesis proposed by the author was consistent with the obtained translocationfactors，which were low（0.47-0.63 for stem and 0.31-0.43 for leaf）in all treatments.This indicates that root of A.subulatusMichx.has a high Cd accumulation capacity，but the Cd translocation capacity from theroot to the shoot was low，which was consistent withthe results reported by Feng et al.（2019）.

The subcellular distribution of Cd played an important role in its migration，accumulation and detoxification in plants（Li et al.，2019）.Cd compart-mentation in the vacuole and sequestration in the cell wall might be crucial cellular mechanisms associated with its detoxification and tolerance in plants（Zhang et al.，2013）.In this study，the Cd concentration ofthree subcellular fractions significantly increased withincreasing Cd concentration in the culture medium.Wang et al.（2017）found that the soluble fraction con-tained cell vacuoles with various organic ligands，such as organic acids and sulfur-rich peptides.Moreover，in the present study，a large proportion of the Cd（52.67%-58.61%）in the root of A.subulatusMichx.was distributed in the soluble fraction.Thus，theseresults suggested that vacuole was the principal stor-age site of Cd in the root of A.subulatusMichx.，which was consistent with the previously reportedfindings in duckweed（Su et al.，2017），rice（Li etal.，2016），and Myriophyllum aquaticum（Li etal.，2020）.Li et al.（2019）demonstrated that 77.98%-84.14%of Cd was distributed in the soluble fraction of Hydrilla verticillata，similar to the report by Fu et al.（2011）that 53.7%-68.3%of Cd was deposited in the solublefraction of Phytolacca americana.More than 40%of Cd was also reported in the soluble fraction of Impa-tienswalleriana（Lai，2015）.The Cd chelation with organic ligands at the storage sites reduced the activity of free Cd ion，thereby weakening their toxic effect.The concentration of Cd in the cytosol was signifi-cantly decreased with Cd absorption into the vacuoles，reducing its concentration in cytosol，thus preventingits damage to the organelles and biochemical and physiological processes within the cells.Conversely，some studies indicated that higher Cd contents were deposited in the root cell walls of Sedum alfredii L.（Ni and Wei，2003）and Morus alba L.（Huang et al.，2018）.However，Xin and Huang（2014）reported asmall amount of Cd（8.0%-17.0%）in the root cell wall fraction of hot pepper.The results of the study also showed that only small proportions of Cd（29.12%-37.22%）were stored in the root cell wallfractions of A.subulatusMichx.under Cd stress.Thus，these conflicting results presumably occurred due to differences in species type and experimental conditions since different plants had different charac-teristics，including Cd absorption，translocation，andregionalization mechanisms.

Plant cell wall contained hemicellulose，cellulose，proteins，and pectins，which chelated Cd ions to hin-der their transport through the cell membrane andreduce their concentration in the protoplasts（Yang et al.，2018）.In this study，higher proportions of Cd（42.10%-63.28%）in leaf ofA.subulatusMichx.wasintegrated into the cell wall fraction.Zhao et al.（2015）also found that most Cd content（41.2%-79.2%）in Porphyra yezoensis was distributed in the cell wall fraction，similar to the report by Wang et al.（2008）that higher Cd proportions（48.2%-61.9%）were inte-grated into the cell wall fraction of ramie.Similarly，Ramos et al.（2002）demonstrated that 64.0%of Cd was stored in the cell wall of lettuce leaf.These find-ings indicated that the leaf cell wall was critical buffer zone which could compartmentalize Cd and therefore can be involved in Cd tolerance to protect the proto-plast from Cd toxicity.

Root sequestration is a significant resistance mechanism of plants against Cd stress.In the study，pectates and protein integrated Cd（F3）was the mainform of Cd highly accumulated in the root and leaf of A.subulatusMichx.，implying that most of the Cd inthe root and leaf might have interacted with pectates and proteins.Similar findings have been reported in Bechmeria nivea（Wang et al.，2008），Brassica para-chinensis（Qiu etal.，2011），P.yezoensis（Zhao et al.，2012）and H.verticillata（Li et al.，2019）.However，the results of the present study differed from thosereported by Su et al.（2014）and Zhao et al.（2015）that the inorganic Cd forms were predominant in plantroot and that Cd was chelated by carboxyl or hydroxyl groups to form a non-toxic complex.The higher pro-portion of Cd extracted by NaCl may help plants adaptto high Cd stress and alleviate its toxicity.Further-more，this study showed that the content of the water-soluble organic acid Cd（F2）in root of A.subulatusMichx.increased with the increasing Cd concentrationin the culture medium，enabling translocation of more Cd from root to aboveground shoot.The content of undissolved phosphate Cd（F4）in the leaf alsoincreased，reducing the Cd toxicity on the leaf.Li et al.（2019）also found that undissolved phosphate Cd extracted by 2%acetic acid increased in H.verticillata with the increasing Cd concentration.This was alsoreported by Cd chemical forms studies in Capsicum annuum（Xin and Huang 2014），Impatiens walleriana（Lai，2015）and Oryza sativa（Li etal.，2016）.Thesestudies suggested that forming an insoluble Cd-phosphate complex might be a detoxification strategy，particularly at higher Cd concentrations.Similarly，the present study demonstrated that the high contents ofthe undissolved Cd-phosphate complex in leaf mightbe associated with the Cd resistance in A.subulatusMichx.

Many reports have indicated that pectates and proteins reduced the toxicity of heavy metals（Li et al.，2011；Ye et al.，2018）.The present study reported similar results，suggesting that a larger proportion of pectates and protein integrated Cd（F3）were involved in Cd detoxification in the root（68.91%-74.55%）and leaf（57.38%-83.80%）of A.subulatusMichx.More‐over，the auxiliary detoxification strategy of organic acids in root and phosphate in leaf was also found in A.subulatusMichx.under T T2 and T3.Especially for F2 fraction in root and F4 fraction in leaf，the pro‐portion of water-soluble organic acid Cd（F2）and undissolved phosphate Cd（F4）increased signifi‐cantly（from 13.64%to 22.72%in root and from 10.02%to 32.78%in leaf），with the increase of Cd content from leaf to root.This was similar to the fin-ding by Li etal.（2020），showing that Cd in M.aquati-cum was high in pectates and proteins integrated Cd forms（51.76%-91.15%in leaf），followed by undis‐solved phosphate Cd（5.17%-22.42%in leaf）.These studies indicated that Cd stress could spontaneously induce the auxiliary detoxification strategy of organic acids in root and phosphate in leaf.Previous studies on the distribution of Cd chemical forms demonstrated that the formation of pectates and protein-bound Cd was a Cd detoxification mechanism in plants（Wang and Wang，2008；Li et al.，2011）.The findings of this study also suggested that pectates and protein poten‐tially participate in Cd detoxification in A.subulatusMichx.，and could be important heavy metal chelators.The organic acids in the root and phosphates in the leaf might also be an auxiliary detoxification strategy.

4 Conclusion

In summary，the findings demonstrate that A.subulatusMichx.has a high accumulation and tole-rance ability for Cd，and Cd is primarily stored in the soluble fraction of the root and cell wall fraction of the leaf.The concentration of pectates and protein inte‐grated Cd was higher in the root and leaf compared to other Cd chemical forms.The involvement of the water-soluble organic acid Cd in the root and undis‐solved phosphate Cd in the leaf in the auxiliary detoxi‐fication respectively suggest a significant detoxifica‐tion mechanism in A.subulatusMichx.Consequently，intracellular mechanisms of Cd detoxification in A.subulatusMichx.are mainly through the formation of less toxic chemical forms of Cd and storage of high Cd contents in the cell wall of the leaf and soluble fraction of the root.

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