Selection for Cd Pollution-Safe Cultivars of Chinese Kale (Brassica

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Selection for Cd pollution-safe cultivars of Chinese kale (Brassica alboglabra L. H. Bailey) and biochemical mechanisms of the cultivar-dependent Cd accumulation involving in Cd subcellular distribution Jing-Jie Guo, Xiao Tan, Hui-Ling Fu, Jing-Xin Chen, Xiao-Xia Lin, Yuan Ma, and Zhongyi Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05123 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Journal of Agricultural and Food Chemistry

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Selection for Cd pollution-safe cultivars of Chinese kale

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(Brassica alboglabra L.

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mechanisms of the cultivar-dependent Cd accumulation

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involving in Cd subcellular distribution

Bailey)

and

biochemical

Jing-jie Guo a . Xiao Tan a . Hui-Ling Fu a . Jing-Xin Chen ab . Xiao-Xia Lin a .

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H.

Yuan Ma a . Zhong-Yi Yang a*

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Jing-jie Guoa . Xiao Tana . Hui-Ling Fua . Jing-Xin Chenab . Xiao-Xia Lina . Yuan

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Maa . Zhong-Yi Yanga *

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a

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University, Xingang Xi Road 135, Guangzhou, 510275, China.

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b

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Technology, Dongguanzhuang Road 110, Guangzhou, 510000, China.

State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-Sen

The Fifth Electronics Research Institute of the Ministry of Industry and Information

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* Corresponding Author:

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Zhong-Yi Yang* (Corresponding Author)

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Mail address: Xingang Xi Road 135, Guangzhou, 510275, China.

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Email: [email protected]

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Tel: +86 2084113220

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Fax number: +86 2084113220

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ACS Paragon Plus Environment


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Abstract

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Two pot experiments were conducted to compare and verify Cd accumulation

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capacities of different cultivars under Cd exposures (0.215, 0.543 and 0.925 mg kg-1

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in Exp-1 and 0.143, 0.619 and 1.407 mg kg-1 in Exp-2) and Cd subcellular

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distributions between low- and high-Cd cultivars. Shoot Cd concentrations between

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the selected low- and high-Cd cultivars were 1.4-fold different and the results were

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reproducible. The proportions of Cd-in-cell-wall of shoots and roots were all higher in

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a typical low-Cd cultivar (DX102) than in a typical high-Cd cultivar (HJK), while

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those of Cd-in-chloroplast or Cd-in-trophoplast and Cd-in-membrane-and-organelle

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were opposite. The proportions of Cd-in-vacuoles-and-cytoplasm of roots in DX102

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were always higher than in HJK under Cd stresses, while there was no clear pattern in

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those of shoots. These findings may help to reduce health risk of Cd from Chinese

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kale consumption and explained biochemical mechanisms of cultivar-dependent Cd

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accumulation among the species.

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Keywords:

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Pollution-safe cultivars, subcellular distribution.

Brassica alboglabra L. H. Bailey, Cadmium (Cd), Cultivar variation,

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Introduction

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Heavy metal pollution has become a pressing environmental issue that is closely

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related to public health.1 Cadmium (Cd), as a non-essential element for plants and

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humans, is one of the most toxic heavy metals 2 and can be taken up by crops which

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threaten human health due to the biological amplification via food chain.3 It was

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reported that more than 70% Cd intake by humans originate from vegetables

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consumption.4 Therefore, it is urgent to reduce the potential health risks by

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minimizing Cd accumulation in edible parts of vegetable crops.5,6

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Although there are various technologies to remediate soil Cd contamination, it is

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still difficult to put them into practice in farmland due to many limited conditions

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such as long-term fallow, high cost and potential risk of secondary pollution.7,8 In

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recent years, proper cultivar selection has been proposed as a practical eco-friendly

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approach to restrict Cd uptake and accumulation in crops and finally limits the

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movement of Cd into the human diet.9 The strategy of pollution-safe cultivars (PSCs),

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i.e., the cultivars in which the concentration of certain pollutant in edible parts is low

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enough for safe consumption (≦0.2 mg kg-1 FW, according to the Codex ML of Cd),

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when growing in contaminated soil, has been elicited as a cost-effective way to

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minimize Cd accumulation in crops.10,11

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Cd absorption, accumulation and distribution in crops are quiet different not only

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among species but also among genotypes within the same species.12,13 The

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intra-specific differences were well studied in man crops,12 such as maize (Zea mays 3



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L.),14 wheat (Triticum sestivum L.),15 pakchoi (Brassica rapa chinensis L.)16 and

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asparagus bean (Vigna unguiculata subsp. sesquipedalis).17 Plants have developed a

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series of mechanisms against the toxicity of Cd and numerous evidence has indicated

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that Cd compartmentalization in specific tissues may be highly associated with Cd

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tolerance, translocation and detoxification.18 Thus, to investigate subcellular

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distribution of Cd in different plant tissues can help to understand the mechanisms

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relating to Cd uptake and accumulation in plants.19

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Chinese kale (Brassica alboglabra L. H. Bailey), one of the popular leafy

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vegetables, is widely consumed in China, Southeast Asia and Mediterranean areas.20

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The leafy vegetable species of brassica have been indicated to be Cd accumulative

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12,21

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however, there are few studies on Cd accumulation capacity and its mechanisms in

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Chinese kale. Accordingly, targets of the present study are (i) to study the Cd

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accumulation among Chinese kale cultivars and screen the cultivars with low Cd

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accumulation capacity or Cd-PSCs and (ii) to investigate the biochemical mechanisms

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involving in the cultivar variation of Cd accumulation through Cd subcellular

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distribution analysis. Considering the observations obtained from some previous

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studies on other leafy vegetables,16,18,22 it is hypothesized that (i) cultivar variation in

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Cd accumulation capacity of Chinese kale should be great enough to identify the

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Cd-PSCs and (ii) the cultivar variation would be associated with the cultivar

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difference in subcellular distribution of Cd.

and Cd in Chinese kale is thus probably a potential health issue. As we know,

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Materials and methods

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The tested cultivars

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Two pot experiments were conducted in a greenhouse. First, twenty-eight

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cultivars of Chinese kale currently cultivated in China were used in the Exp-1 (Table

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1). Second, in Exp-2, four low Cd accumulative cultivars (low-Cd cultivars) and four

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high Cd accumulative cultivars (high-Cd cultivars) selected from the Exp-1 were used

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to verify their Cd accumulation capacities. Then, two verified typical cultivars with

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low and high Cd accumulation capacity, respectively, were used to compare

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differences in Cd subcellular distribution.

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Soil preparation and treatments

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The control soil (CK) was collected from a farm and was air-dried, ground and

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passed through a 5 mm sieve. The artificial Cd soils in two experiments were

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prepared by adding the solutions containing CdCl2. The final Cd concentrations were

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0.215 mg kg-1 (CK), 0.543 mg kg-1 (LCd) and 0.925 mg kg-1 (HCd) in Exp-1, and

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0.143 mg kg-1 (CK), 0.619 mg kg-1 (LCd) and 1.407 mg kg-1 (HCd) in Exp-2,

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respectively. The soil Cd concentrations under LCd and HCd treatments all exceeded

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the maximum limitation for Cd when compared to the Farmland Environmental

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Quality Evaluation Standards for Edible Agricultural Products (HJ 332-2006) (0.3 mg

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kg-1, DW basis). The prepared soils were watered in a two-week duration for

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equilibrium and then thoroughly mixed for further experiment.

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Main agronomic properties of the CK, LCd and HCd soils (Table 2) were

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measured using routine analytical methods for agricultural soils. 23 The soil samples

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(0.2g, DW basis) were digested with 6 mL HCl (36-38% m/m, Guaranteed reagent

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(G.R.), Guangzhou, China), 2 mL HNO3 (65% v/v, G.R., Guangzhou, China) and 1

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mL H2O2 (30% v/v, G.R., Guangzhou, China) in a microwave digestion device

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(Microwave digestion Topex, Shanghai PreeKem Scientific Instruments Co., Ltd.,

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China). The digested samples were cooled at room temperature and diluted to a tube

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of 10 mL with deionized water and then concentrations of total Cd were determined

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by a flame atomic absorption spectrophotometer (FAAS, Shimadzu AA-7000, Japan;

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bought from Shenzhen, China).

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Seeds were surface sterilized for 10 min with 2% H2O2 and rinsed three times

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with deionized water. After that, the seeds were soaked in deionized water at 25 ± 1℃

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in the dark for 24 hours, and then 10 seeds were sown in each pot. Three pots for each

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cultivar under each treatment were conducted as replicate. All pots (28 cultivars × 3

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treatments × 3 replicates = 252) were randomly placed in a greenhouse with daytime

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(14 h) temperature about 25°C-30°C and nighttime (10 h) temperature about 18-20℃.

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Within 7-15 days after germination, seedlings were gradually thinned till 4 plants left

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in each pot finally.

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Plant sampling

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After two-month growth, shoots and roots of each plant were harvested

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separately. The shoots were fully rinsed with tap water and washed twice with

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deionized water. The roots were soaked in 0.5 mmol L-1 CaCl2 solution for 30 min to

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remove the Cd adsorbed on root surfaces, and then washed with deionized water for

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three times. Fresh weight (FW) of whole shoots and whole roots for each pot were

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measured separately. Afterwards, all samples for Exp-1 and a part of the samples for

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Exp-2 were then oven-dried at 105℃ for 15min and then at 70°C to constant weight.

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Dry weight (DW) of the dried samples were recorded and then the samples were

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ground to pass through a 100-mesh sieve (149 micron). Another part of the fresh

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samples for Exp-2 was reserved and frozen in liquid nitrogen (N2) immediately and

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stored at -80°C for analysis of Cd subcellular distribution.

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The frozen samples were preprocessed to separate subcellular fractions based on

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the method of Wu et al. (2005)24. Each sample was homogenized by a pestle in a

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chilled mortar with pre-chilled extraction buffer (50 mM Tris-HCl, 250 mM sucrose,

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1.0 mM DTE (C4H10O2S2) and 5.0 mM ascorbic acid, pH 7.50). The homogenate was

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then sieved through a 180-mesh nylon cloth (80 micron), and the residue on the nylon

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cloth was washed twice with the extraction buffer. This first filter residue left mainly

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constituted cell walls and cell wall debris, and was designated as the fraction 1 (F1).

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The filtrate was centrifuged (Beckman JA-25.50 rotor, Fullerton, CA, USA) under

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two different centrifugation programs at 4℃ to obtain other fractions. The pellet

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retained after 1500 ×g for 10 min (root sample, 2500 ×g for 20 min) was designated

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as fraction 2 (F2), containing chloroplast (for shoot) or trophoplast (for root). The

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supernatant was then centrifuged at 15000 ×g for 35 min. The filter residue was

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designated as fraction 3 (F3), including membrane and organelle, and the final

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supernatant was designated as fraction 4 (F4) which mainly consist of the the vacuole

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and its contents.

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Analyses of Cd concentration in plant

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The plant samples (0.20 g for each, DW basis) and the subcellular fractions were

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digested with 5 mL HNO3 (65% v/v, G.R., Guangzhou, China) and 2 mL H2O2 (30%

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v/v, G.R., Guangzhou, China) in a microwave digestion device (Microwave digestion

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Topex, Shanghai PreeKem Scientific Instruments Co., Ltd., China). The digested

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samples were cooled at room temperature and diluted to a tube of 10 mL with

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deionized water and then their Cd concentrations were analyzed by a flame atomic

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absorption spectrophotometer (FAAS, Shimadzu AA-7000, Japan; bought from

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Shenzhen, China). A Certified Reference Material (CRM) of plant (GBW-07603,

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provided by the National Research Center for CRM, China) was used to evaluate the

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precision of the Cd analytical procedure.

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Data statistics

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To estimate growth response to Cd exposure of the tested cultivars, a value of biomass response to stress (BRS) are calculated as follow: 18 BRS (%) = (BCd - BCK)/BCK×100%

(1)

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where BCd and BCK are the shoot biomass under LCd or HCd and CK treatments,

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respectively.

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To examine Cd transporting capacity from roots to shoots, the translocation factor (TF) was calculated as follows: 5

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TF = Cshoot/Croot where Cshoot and Croot are the Cd concentrations (mg kg-1 DW) in shoot and root.

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(2)

To estimate Cd uptake from soil to shoot, the bioaccumulation factor (BF) was calculated as follows: 16 BF = Cshoot/Csoil

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(3)

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where Cshoot is the Cd concentration (mg kg-1 FW) in shoot, and Csoil is the total Cd

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concentration in the corresponding soil (mg kg-1 DW ) or solution (mg L-1).

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Data obtained were statistically analyzed with the independent samples t-test and

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least significant difference (LSD) test based on two-way ANOVA using Excel 2015

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and SPSS 22.0 (SPSS, Inc., Chicago, IL, USA).

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Results

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Plant growth and cultivar variation in Cd concentration in Exp-1

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Biomass response to Cd exposure

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No visual Cd toxic symptoms, such as stunted growth, chlorosis and leaf rolling,

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mean values of shoot fresh weights (FW) of all cultivars under CK, LCd and HCd

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treatments were 12.470±3.276, 12.830±3.223 and 12.570±4.315 g pot-1 (mean±

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SD), respectively, and the differences were not significant (p > 0.05). According to

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the results of BRS (Figure 1), most cultivars (16 under LCd and 19 under HCd) had

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the similar shoot biomass to CK (without significant difference), indicating that the

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Cd exposure lower than 1 mg kg-1 would not restrict growth of Chinese kale. Among

were observed in all the tested Chinese kale cultivars under any Cd treatment. The

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all of the tested cultivars, only the cultivars DX104, TWDX and GS had significantly

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higher shoot biomass and the cultivar SC had significantly lower one under both LCd

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and HCd treatments when compared with that under CK (p < 0.05).

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Cd concentration

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The result of two-way ANOVA showed that shoot Cd concentrations were

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significantly affected by Cd treatment, cultivar and interaction between treatment and

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cultivar (p < 0.05) (Table S1). Averages of the shoot Cd concentrations (Table S2)

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were 0.032 mg kg-1 (0.015-0.059 mg kg-1), 0.592 mg kg-1 (0.298-0.782 mg kg-1) and

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0.993 mg kg-1 (0.609-1.751 mg kg-1) (Figure 2), and maximal differences in shoot Cd

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concentrations among the cultivars were 3.82-, 2.62- and 2.87-fold under CK, LCd

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and HCd treatments, respectively. Compared to CK, soil Cd concentration of LCd and

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HCd treatments only increased two and four times, while the average shoot Cd

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concentrations under LCd and HCd treatments were 15- and 31-fold higher than that

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of CK. It is indicated that Chinese kale should be a crop with high capacities of Cd

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uptake and translocation. According to the calculation of bioaccumulation factor (BF),

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the values in Exp-1 were in a range of 0.071-0.272 for CK, 0.549-1.441 for LCd and

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0.658-1.893 for HCd.

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The average Cd translocation factors (TFs, FW) were 0.307 (0.134-0.511), 0.458

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(0.214-0.720) and 0.566 (0.194-1.216) for CK, LCd and HCd treatments, respectively.

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The differences between any two treatments were all significant (p < 0.05), indicating

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that Cd translocation from roots to shoots was more effective under higher Cd stress.

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It should be surprising that there was a cultivar, i.e. HJK, which performed very high

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Cd translocation capacity from roots to shoots (with TFs higher than 1) under HCd

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treatment.

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The correlation of shoot Cd concentrations between LCd and HCd was

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statistically significant (p < 0.01; Figure 3a), suggesting that the Cd accumulation

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differences of Chinese kale is a stable phenotype. According to the difference in shoot

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Cd concentrations among the tested cultivars under both LCd and HCd treatments,

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cultivars DX104, JX, JST and DX102 were selected as candidates of low-Cd cultivars

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and HJK, TWDX, HKBT and HCD as high-Cd cultivars for further verification.

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According to the correlation (a=1.046b+0.171, r=0.997, p < 0.05) between the

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soil Cd concentrations (a) and the average shoot Cd concentrations (b) of the 4

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low-Cd cultivars (Figure 3b), soil Cd concentrations can not exceed 0.38 mg kg-1 in

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order to get the products with Cd concentration being lower than the standard for safe

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consumption (Cd ≤ 0.2 mg kg-1 according to the Codex standard). When considering

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the correlation (a=0.522b+0.182, r=0.998, p < 0.05) based on the high-Cd cultivars,

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however, the safe soil Cd concentration had to be lower than 0.29 mg kg-1 (Figure 3c).

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Verification of the eight cultivars and selection of Cd-PSCs in Exp-2

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Biomass

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The average biomass of the 8 selected cultivars ranged from 2.913 to 4.880 g

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pot-1, and aboveground biomasses under LCd treatment (mean 4.063±0.503 g pot-1)

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were significant (p < 0.05) higher than that under CK (3.656±0.583 g pot-1) and HCd

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(3.445±0.376 g pot-1) treatments, indicating a Cd induced growth when soil Cd

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concentration was at the level of about 0.6 mg kg-1. According to the results of BRS

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(Figure 4), the profile of the biomass responses to Cd stresses for the tested cultivars

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were entirely the same to those observed in Exp-1, implying a reproducible growth

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response to Cd in the tested cultivars.

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Cd concentration

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The result of two-way ANOVA of shoots Cd concentrations was shown in Table

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S3. The averages of shoot Cd concentrations in the 8 cultivars were 0.038 mg kg-1

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(0.017-0.057 mg kg-1), 1.053 mg kg-1 (0.684-1.320 mg kg-1) and 1.787 mg kg-1

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(1.269-2.719 mg kg-1), with the maximal differences of 3.48-, 2.07- and 2.14-fold

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under CK, LCd and HCd, respectively (Figure 5). Compared with the shoot Cd

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concentrations in the plants under CK, those exposed to LCd and HCd treatments

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amazingly increased by 28- and 48-fold although the increments in soils were only 4-

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and 10-folds, which is consistent with the observations in Exp-1. According to the

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calculation of the BFs of Cd, the differences between the average BFs of low- and

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high-Cd cultivars were 1.90-, 1.45- and 1.43-fold under CK, LCd and HCd,

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respectively.

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The root Cd concentrations were much higher than those in shoots (Figure 5),

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with averages of 0.073 mg kg-1 (0.030-0.122 mg kg-1), 2.817 mg kg-1 (1.205-5.135 mg

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kg-1) and 3.686 mg kg-1 (2.329-5.767 mg kg-1) under CK, LCd and HCd, respectively.

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Unlike in shoots, the root Cd concentrations of the high-Cd cultivars were mostly

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significantly lower (p < 0.05) than those of the low-Cd cultivars under LCd treatment

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except for HJK, and similar cultivar dependent Cd accumulation pattern was also

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observed under HCd treatment except for JST.

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The average TFs for Cd were 0.407 (0.206-0.640), 0.441 (0.172-0.943) and

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0.511 (0.220-0.805) under CK, LCd and HCd and the differences between averages of

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low- and high-Cd cultivars were 1.85-, 2.45- and 1.92-fold, respectively (Figure 6).

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The TFs of low-Cd cultivars were all significantly lower than those of high-Cd

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cultivars (p < 0.05) whatever Cd was spiked or not and the cultivar differences of TFs

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under LCd and HCd were larger than those of BFs. By the way, although the TFs of

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the 8 tested cultivars were all lower than 1, there were two cultivars (HKBT and HJK)

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that displayed the TFs for Cd higher than 0.8, implying a rather high risk of Cd

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pollution in edible part of the high-Cd cultivars of Chinese kale under where the soil

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was contaminated by Cd. Above all, the DX102 and HJK have the lowest and highest

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shoot Cd concentrations under Cd treatments and were selected as the typical low-Cd

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and high-Cd cultivars, respectively, for further study on subcellular distribution of Cd.

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Subcellular distribution of Cd in low- and high-Cd cultivars

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Total Cd

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The result of two-way ANOVA was shown in Table S4. The total Cd

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concentrations in shoots of low-Cd cultivar were significantly lower than in high-Cd

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cultivar under Cd exposures (p < 0.05 or p < 0.01), while it exhibited a opposite trend

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in roots that the total Cd concentrations in the low-Cd cultivar were significantly

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higher than those in the high-Cd cultivar under Cd exposures (p < 0.01; Figure 7).

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These results are similar to the observations in the above-mentioned experiment

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(Figure 5). The increments of the total shoot Cd concentrations of the low- and the

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high-Cd cultivars under LCd were 6.53-fold and 14.12-fold, respectively, when

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compared with that under CK, and those under HCd were 9.70-fold and 19.39-fold,

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respectively, being much mitigated when compared with the results obtained from the

274

above-mentioned experiments (Figure 5).

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Subcellular distribution of Cd

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In both shoots and roots, Cd located mainly in cell wall (F1), followed by

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vacuoles and cytoplasm fraction (F4) and then chloroplast / trophoplast fraction (F2)

278

and organelle fraction (F3) (Figure 8). The mean values of Cd in the different

279

fractions (F1, F2, F3 and F4) of HJK shoots were all higher than those of DX102

280

under the three Cd treatments and the cultivar differences were significant (p < 0.05

281

or p < 0.01) except F1 fraction under CK. For the roots, however, the results were

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distinctly different from the shoots, especially in F1 and F4 where the Cd

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concentration were significantly lower in HJK than in DX102 under the Cd treatments

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(p < 0.01). For F2 and F3 in roots under the two Cd exposures, except for F3 under

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LCd, the Cd concentrations were significantly higher in HJK than in DX102 (p < 0.05)

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which was similar to the observation in shoots although the cultivar differences in

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roots were lessened.

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Proportions of Cd in the subcellular fraction

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Average proportions of Cd in different subcellular fractions of shoots and roots

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for the two tested cultivars were shown in Figure 9. The proportions of the Cd

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combined in cell wall (F1) were the highest either in shoots or roots. The proportion

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in shoots decreased obviously with the increase of soil Cd stress, while it showed

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slight increase in roots. The proportions of F1-Cd in DX102 (low-Cd cultivar),

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wherever in shoots or roots, were higher than in HJK (high-Cd cultivar) whatever Cd

295

stress existed or not. The results, associating with the Cd concentrations in F1,

296

suggested that the cell wall played a decisive role in the low accumulation capacity of

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Cd in the low-Cd cultivar.

298

Either in chloroplast of shoots or trophoplast of roots (F2), the Cd proportion all

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increased under Cd stress, especially the trophoplast in roots of the high-Cd cultivar.

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In shoots, Cd induced increase of the proportion of Cd in membrane and organelle

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fraction (F3) in HJK was observed, especially under HCd treatment. In DX102,

302

however, the proportions of F3-Cd showed a little change and were always at a low

303

level wherever Cd exposed or not, indicating that the protection of shoot organelle in

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the low-Cd cultivar was more effective than in the high-Cd cultivars. In roots, the Cd

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induced change of F3-Cd proportion was irregular but always stayed at a relatively

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low level under the three Cd exposures. Under HCd treatment, the proportion

307

decreased obviously in low-Cd cultivar while changed slightly in high-Cd cultivar,

308

implying that protection of organelles in root cells of Chinese kale against Cd toxicity

309

is also quite effective.

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As for the proportion of Cd in vacuoles and cytoplasm (F4), the Cd induced

311

change in shoots and roots was opposite, i.e. the proportion in shoots increased while

312

that in roots decreased coping with the increased Cd stress. Moreover, in roots of

313

DX102, the proportions of F4-Cd were always higher than those in HJK as

314

responding to the two Cd stresses, in accordance with the significant difference

315

between the two cultivars in the Cd induced crease of F4-Cd concentration in the

316

roots.

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Discussion

318

Chinese kale is generally a Cd tolerant vegetable crop

319

Numerous studies

26,27

had demonstrated that there is a certain dose effect of Cd

320

on the growth of plants, described as hormesis,28 namely the lower concentrations of

321

Cd can stimulate the growth of plants. The results of this study are consistent with the

322

results above-mentioned because all the tested Chinese kale cultivars showed healthy

323

growth under Cd contaminated soils both in Exp-1 and Exp-2, and their biomass

324

showed a descent trend of LCd > HCd > CK. It is suggested that the soil Cd

325

concentration as high as 0.925 mg kg-1 (Exp-1) or 1.407 mg kg-1 (Exp-2) would not

326

restrain the growth of Chinese kale. This was similar with amaranth (Amaranthus

327

mangostanus L.),29 water spinach (Nasturtium officinale R. Br.) 30 and pakchoi.16 It is

328

thus considered that Cd contamination in soils as well as Chinese kale products would

329

be hard to be found by farmers, which would increase human health risks from taking

330

Cd contaminated products of Chinese kale. It is worth noting that the shoot biomasses 16


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of SC significantly decreased when grown under the Cd contaminated soils,

332

suggesting that this cultivar might be used as an indicator of soil Cd contamination.

333

Cd accumulation capacity of Chinese kale is cultivar dependent

334

Intraspecific variations of Cd accumulation have been extensively documented in

335

plants.5,12,18 Different Chinese kale cultivars performed much differently in the uptake

336

and accumulation of Cd and the difference was reproducible in the two pot

337

experiments, suggesting that the variation in Cd accumulation of Chinese kale is

338

cultivar dependent and is thus genetically stable. The facts that the maximum

339

difference among the 28 tested cultivars in the Exp-1 were as high as 2.87-folds

340

exhibited a high possibility to use the PSC strategy to minimize Cd pollution of

341

Chinese kale. However, there was no PSC even under LCd treatment of both Exp-1

342

and Exp-2 according to the Codex ML, and the maximal Cd concentrations in soil that

343

allowed the shoot Cd concentration being below the Codex ML of Cd were 0.38 mg

344

kg-1 for the low-Cd cultivars and 0.29 mg kg-1 for the high-Cd cultivars of Chinese

345

kale. This suggested that the use of the low-Cd cultivars would facilitate safe

346

production of Chinese kale from soil Cd contamination. It should be surprising that

347

many Chinese kale cultivars had very strong ability to absorb Cd from soil which can

348

be indicated from the amazing increase of shoot Cd concentration (31-folds from CK

349

to HCd) when compared with the designed increase of soil Cd concentration (only

350

4-folds from CK to HCd). The comparative increment of Cd concentrations between

351

plant and soil (31: 4) in Chinese kale is far greater than other vegetables such as

17



Page 18 of 46

352

Chinese flowering cabbage (Brassica parachinensis L.) (11: 10) 5 and water spinach

353

(13: 7),29 and so on. Consistently, the maximal Cd concentrations in soil that allowed

354

the shoot Cd concentration being below the Codex ML of Cd were 1.09 mg kg-1 for

355

water spinach,29 1.00 mg kg-1 for Chinese flowering cabbage

356

Chinese cabbage (Brassica perkinensis L.),31 much higher than that for Chinese kale.

357

It is suggested that Chinese kale has much high Cd accumulation capacity in edible

358

parts and studies on strategies and technologies to minimize the Cd pollution risk in

359

Chinese kale are thus an important and urgent task.

5

and 1.25 mg kg-1 for

360

Because it is difficult to obtain Cd-PSC only by screening currently using

361

cultivars, breeding technologies of the Cd-PSCs must be developed. There have been

362

some low-Cd cultivars that was obtained via breeding process, such as “Wilwash”, a

363

potato (Solanum tuberosum L.) cultivar;32 “Strongfield”, a durum wheat (Triticum

364

durum Desf.) cultivar,15 and a new cultivar of water spinach.33 According to our

365

recent study, breeding of Cd-PSCs for vegetables could be assisted by molecular

366

markers which had been suggested in water spinach,34 Chinese flowering cabbage

367

and pakchoi.36 For Chinese kale, it is considered that the typical cultivars DX102

368

(low-Cd) and HJK (high-Cd) would be used for developing the molecular assisted

369

breeding methods of the PSCs.

370

The high Cd accumulation capacity of Chinese kale is mainly decided

371

by its high Cd uptake ability

18


35

Page 19 of 46


372

Cd accumulation in plants is connected with physiological processes including

373

Cd uptake, translocation and detoxification. It has been widely recognized that uptake

374

of heavy metals from soil occurs either passively with the mass flow of water into the

375

roots or through active transport across the plasma membrane of root epidermal

376

cells.37,38 Cd distribution in shoots and roots of a plant is determined by Cd uptake,

377

translocation, chelation and compartmentalization.38 The TF and BF reflect the ability

378

of Cd immigrated via root-to-shoot process and soil-to-plant process, respectively,

379

which play a critical role in the shoot Cd accumulation differences between low- and

380

high-Cd cultivars. These two factors are thus frequently employed as major

381

quantitative indicators in assessing heavy metal-resistant crop varieties.39 Compared

382

to other vegetables (Table 3), 30, 35, 40,41 the Cd BFs (DW) of Chinese kale were higher

383

than most others. However, the TFs (DW) were much lower (0.27-1.86 under soils

384

with Cd concentrations of 0.22-0.93 mg kg-1) when compared to Chinese flowering

385

cabbage (3.78-29.39 under soils with Cd concentrations of 0.11-1.13 mg kg-1)

386

celery (Apium graveolens L.) (4.12-11.32 under soil with Cd concentration of

387

0.35-0.67 mg kg-1)

388

absorb Cd from soil but the translocation from roots to shoots is impeded. It is

389

impressed that when soil Cd concentration slightly increased (e.g. about 4-fold

390

increase), shoot Cd concentration increased amazingly in Chinese kale (e.g. 31-fold

391

increase), which is rarely observed in other crops. It is considered that the excessive

392

migration of Cd toward plant from soil can cover the deficiencies in Cd translocation

5

and

42

. It is suggested that Chinese kale has rather high ability to

19



393

ability from roots to shoots and thus easily causes the risk of Cd pollution in Chinese

394

kale. Therefore, for the improvement of Chinese kale to reduce its Cd accumulation

395

capacity using breeding methods, it shall be firstly targeted to modify its high Cd

396

uptake ability.

397

The cultivar different Cd subcellular distributions well explained the

398

mechanisms of cultivar dependent Cd accumulation of Chinese kale

399

Previous researchers have demonstrated that the plant tissues have a barrier for

400

Cd translocation, which can inhibit the transfer of Cd from roots to shoots. This

401

process is mainly regulated by the loading capacity of xylem in root, the content of

402

long-distance carriers, such as PCs, and the compartmentalization of root cell wall

403

and vacuole.6,22 Although the BF of Cd decided that Chinese kale is a Cd

404

accumulative species, the differences between low- and high-Cd cultivars in TFs of

405

Cd were larger than those of BFs. It is thus suggested that the cultivar dependent Cd

406

accumulation of Chinese kale mainly relied on cultivar difference of Cd translocation

407

from roots to shoots, which can be well explained by the differences of Cd subcellular

408

distributions between the low- and high-Cd cultivars.

409

The cell wall had been reported to be the predominant sink for Cd and the first

410

barrier for Cd entry into plants, because it can bind considerable quantity of Cd to

411

limit it entering the protoplasm.43 It will result in a retention effect which helps to

412

reduce the Cd transport from roots to shoots, and protects organelles from the damage

413

of Cd toxicity.14,44 In the present study, it was found that the Cd restricting effect in

20


Page 20 of 46

Page 21 of 46


414

cell wall of roots could be enhanced by the Cd stresses and the higher concentrations

415

as well as proportions of Cd in root cell wall of low-Cd cultivar than in high-Cd

416

cultivar were clearly induced by Cd stresses. It is considered there are some

417

mechanisms that responded to Cd which should be different between low- and

418

high-Cd cultivars. It has been reported that expressions of miRNA397 between low-

419

and high-Cd cultivars of water spinach were much different and it seemed to be Cd

420

stress relevant. The high-Cd cultivars had a Cd induced high expression of

421

miRNA397 and low expression of laccase gene which is one of the target genes of

422

miRNA397, and similar results were found in Chinese flowering cabbage.

423

laccase is a key enzyme relating to cell wall biosynthesis which is involved in

424

synthesis and structure of lignin. However, the Cd induce change of the miRNA397

425

expression was not observed in the low-Cd cultivar of water spinach.

426

perhaps an important molecular mechanism that leads to the cultivar dependent ability

427

in the Cd retention in cell wall and then the root-to-shoot translocation of Cd in many

428

crops and further investigations are eagerly expected.

32,46

45

The

This is

429

In this study, it was found that the Cd concentrations and proportions in

430

trophoplast of roots all increased under Cd stresses, especially in high-Cd cultivar,

431

which was in accordance with water spinach,22 while was different to pakchoi

432

watercress.49 These results indicated that trophoplast might contribute to Cd

433

detoxification in root or Cd translocation from roots to shoots. However, it is not clear

434

and needed further investigations if the Cd binding to trophoplast played certain role

21


16

and


Page 22 of 46

435

in Cd translocation and detoxification in plants and can enhance the transfer ability of

436

Cd from roots to shoots.

437

In Chinese kale, the lower Cd concentrations of F3-Cd both in shoots and roots

438

in low-Cd cultivar than in high-Cd cultivar were distinctly induced by Cd stresses.

439

Similar results were found in pakchoi under 0.7 mg kg-1 Cd stress

440

under 1 mg L-1 Cd stress 18 and spinach under 5 mg kg-1 Cd stress 48. It was suggested

441

that the protection of membrane organelle in the low-Cd cultivars was more effective

442

than in the high-Cd cultivars.

16

, water spinach

443

Peng et al. 47 had reported that the vacuolar sequestration capacity (VSC) of root

444

vacuole had an important influence on the retention of Cd in root and the increase of

445

the VSC size could enhance this effect. Since Cd concentrations as well as

446

proportions in root vacuole of low-Cd cultivar were all higher than high-Cd cultivar,

447

it is considered that compartmentalization of Cd into root vacuole is another

448

important mechanism that brings about the cultivar dependent Cd accumulation in

449

Chinese kale. However, different to the Cd in cell wall fraction, the proportions of Cd

450

in vacuole were negatively responded to Cd stress levels, which was consistent with

451

water spinach

452

watercress (Nasturtium officinale L.)

453

vacuole may play different roles in different crop species when dealing with

454

root-to-shoot translocation of Cd. The Cd detoxification via vacuole way should also

455

be important. Differing from roots of Chinese kale where Cd detoxification should be

22

and Chinese flowering cabbage,5 but differed from spinach,48 49

and pakchoi.16 These suggested that the

22


Page 23 of 46


456

mainly realized through the way of Cd retention in cell wall, detoxification of Cd in

457

shoots of Chinese kale may be achieved mainly by transporting Cd into vacuole

458

where the Cd could be inactivated. Some results from some previous studies 16, 18, 22, 48

459

relating to the different distribution patterns of Cd in vacuole fractions under Cd stress

460

among different species suggested an inter-species differentiation in Cd detoxification

461

mechanisms.

462

Supporting Information Available:

463

Table S1: Two-way ANOVA for shoot Cd concentrations in Exp-1.

464

Table S2: Shoots and roots Cd concentrations of 28 cultivars in Exp-1.

465

Table S3: Two-way ANOVA for shoots and roots Cd concentrations for

466 467

verification in Exp-2. Table S4: Two-way ANOVA for total Cd concentrations of shoots and roots for

468

subcellular distribution in Exp-2.

469

Funding Sources

470

This study was supported by grants from the National Natural Science Foundation of

471

China (Grant No. 21277178 and No. 21777195) and the Chang Hungta Science

472

Foundation of Sun Yat-sen University.

23



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(39) Nocito, F. F.; Lancilli, C.; Dendena, B.; Lucchini, G.; Sacchi, G. A. Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation. Plant Cell Environ. 2011, 34, 994-1008. (40) Dai, H. W. Variations of Cd and Pb accumulation in different Chinese leaf mustard (Brassica juncea var. foliosa) cultivars and its mechanisms. Ph.D. dissertation, Sun Yet-Sen University, Guangzhou, China. 2011, 45-46. (41) Zhang, K. Genotype variations in Cd and Pb accumulations and rapid screening methods for low Cd accumulative cultivars in two leaf using vegetable. Ph.D. dissertation, Sun Yet-Sen University, Guangzhou, China. 2011, 34-36, 65-67. (42) Zhang, K.; Wang, J.; Yang, Z.; Xin, G.; Yuan, J.; Xin, J.; Huang, C. Genotype variations in accumulation of cadmium and lead in celery (apium graveolens l.) and screening for low cd and pb accumulative cultivars. Front. Env. Sci. Eng. 2013, 7, 85-96. (43) Kubo, K.; Kobayashi, H.; Fujita, M.; Ota, T.; Minamiyama, Y.; Watanabe, Y.; Nakajima, T.; Shinano, T. Varietal differences in the absorption and partitioning of cadmium in common wheat (triticum aestivum, l.). Environ. Exp. Bot. 2016, 124, 79-88. (44) Zhang, H.; Chen, J.; Zhu, L.; Yang, G.; Li, D. Transfer of cadmium from soil to vegetable in the pearl river delta area, south china. Plos One. 2014, 9, e108572. (45) Zhou, Q.; Yang, Y. C.; Shen, C.; He, C. T.; Yuan, J. G.; Yang, Z. Y. Comparative analysis between low- and high-cadmium-accumulating cultivars of brassica

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31



Figure captions  Figure 1. Biomass responses to stress (BRS) of shoots of the tested cultivars under LCd and HCd treatments in Exp-1 Notes: LCd, low Cd treatment (soil Cd = 0.543 mg kg-1); HCd, high Cd treatment (soil Cd = 0.925 mg kg-1); ns, not significant difference; *, significant difference at p < 0.05 level; **, significant difference at p < 0.01 level.

Figure 2. Shoot Cd concentrations of 28 cultivars under CK, LCd and HCd exposures in Exp-1 Notes: Values are mean ± standard error (n = 3). LCd, low Cd treatment (soil Cd = 0.543 mg kg-1); HCd, high Cd treatment (soil Cd = 0.925 mg kg-1); CAC, the maximum level for Cd in leafy vegetables according to Codex Alimentarius Commission Standard; different small letters indicate significant difference at p < 0.05 level across different cultivars in the same soil treatment.

Figure 3. Correlations of shoot Cd concentrations (mg kg-1, FW) between LCd and HCd treatments (a), and correlations of Cd concentrations between soil and edible parts of low-Cd cultivars (b) and high-Cd cultivars (c) in Exp-1 Notes: LCd, low Cd treatment (soil Cd = 0.543 mg kg-1); HCd, high Cd treatment (soil Cd = 0.925 mg kg-1); ◆, the selected candidates of low-Cd cultivar; ▲, the selected candidates of high-Cd cultivar.

32


Page 32 of 46

Page 33 of 46


Figure 4. Biomass responses to stress (BRS) of shoots of the 8 cultivars under LCd and HCd treatments in Exp-2. Notes: LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); ns, not significant difference with CK; *, significant difference at p < 0.05 level with CK; **, significant difference at p < 0.01 level with CK.

Figure 5. Cd concentrations of the 8 selected cultivars in shoots and roots under CK, LCd and HCd treatments. Notes: Values are mean ± standard error (n = 3). LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); different small letters indicate significant difference at p < 0.05 level across different cultivars in the same soil treatment.

Figure 6. Translocation factors (TFs, FW basis) for Cd of the 8 selected cultivars Notes: LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); different small letters indicate significant difference at p < 0.05 level across different cultivars in the same soil treatment.

Figure 7. Total Cd concentrations of shoots and roots in cultivar DX102 (low-Cd) and HJK (high-Cd) Notes: LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); ns, *, and ** indicate that the difference between the two 33



cultivars in the same treatment is not significant, significant at p < 0.05, and significant at p < 0.01, respectively.

Figure 8. Cd subcellular distributions in shoots and roots of cultivars DX102 (low-Cd) and HJK (high-Cd) (mg kg-1, FW) in Exp-2 (mean ± SD, n = 3). Notes: LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); F1, cell wall fraction; F2, chloroplast (for shoot) or trophoplast (for root) fraction; F3, membrane and organelle fraction; F4, vacuoles and cytoplasm fraction. ns, *, and ** indicate that the difference between the two cultivars in the same treatment is not significant, significant at p < 0.05, and significant at p < 0.01, respectively.

Figure 9. Proportions of Cd in subcellular fractions of shoots and roots of two typical cultivars under three Cd treatments Notes: Cd proportion (%) = Cd concentration in fraction / (sum of Cd concentrations in all fractions) × 100. LCd, low Cd treatment (soil Cd = 0.619 mg kg-1); HCd, high Cd treatment (soil Cd = 1.407 mg kg-1); F1, cell wall fraction; F2, chloroplast (for shoot) or trophoplast (for root) fraction; F3, membrane and organelle fraction; F4, vacuoles and cytoplasm fraction.

34


Page 34 of 46

Page 35 of 46


Tables  Table 1 The 28 tested cultivars of Chinese kale and their seed providers a) for Exp-1

a)

Cultivar

Provider

Cultivar

Provider

Cultivar

Provider

DX104

A

LJ-F1

E

JST

K

DX102

A

TWDX

F

JC

K

HJK

A

MH

F

YC-2

L

TJK

A

Yuan

G

ZJ

M

CHJK

A

HKBT

H

CD

N

TSD

B

JX

I

FZ

O

ZDH

B

HKGZ

I

HCD

P

JP400

B

SC

I

TWCY

Q

ATBJ

C

XC

J

GS

D

QS

J

Seeds providers: A, Shantou Chenghuashenghe Seeds Shop; B, Shantou Linong Seed Co., Ltd.;

C, Shenzhen Jialiang Seed Co., Ltd.; D, Beijing Fengmingyashi Technology Co., Ltd.; E, Jilin Changchun Zhengbang Gardending Co., Ltd.; F, Shouguang Xinxinran Gardening Co., Ltd.; G, Guangzhou Institute of Agricultural Sciences; H, Guangxi Liuzhou Qianjin Seeds Shop; I, Jieyang Bangfeng Seed Co., Ltd.; J, Guangdong Academy of Agricultural Sciences‘ Vegetable Research Institute; K, Shantou KingShire Seed Company, L, Shantou Yicai Seed Co., Ltd.; M, Guangzhou Changhe Seeds Shop; N, Foshan Nanhai District Dalimin’an Seeds Shop; O, Guangdong Heshan Shaping Hongtu Seed Store; P, Guangdong Jieyang Weida Seed Company; Q, Cuiyun Co., Ltd.

35



Page 36 of 46

Table 2 Properties of the tested soils in Exp-1 and Exp-2 (mean ± SD, n = 3) Exp-1

pH organic matter (%)

Exp-2

Control-soil

LCd-soil

HCd-soil

Control-soil

LCd-soil

HCd-soil

5.95 a

5.91 a

5.88 a

6.02 a

5.98 a

6.06 a

1.97 a

2.03 a

1.95 a

2.07 a

2.11 a

2.04 a

-1

1.08±0.14 a

1.05±0.06 a

1.11±0.09 a

0.91±0.10 a

0.85±0.04 a

0.98±0.04 a

-1

0.67±0.06 a

0.73±0.07 a

0.71±0.09 a

0.78±0.08 a

0.82±0.06 a

0.84±0.04 a

total N (g kg ) total P (g kg ) -1

total K (g kg )

4.48±0.52 a

4.62±0.16 a

4.53±0.39 a

5.27±0.15 a

5.13±0.17 a

5.07±0.12 a

-1

213.67±6.14 a

214.83±5.31 a

210.63±8.56 a

191.19±1.26 a

198.59±5.49 a

192.94±6.26 a

-1

available P (mg kg )

174.45±5.98 a

175.64±4.95 a

176.59±5.03 a

174.45±5.98 a

176.73±3.62 a

175.61±5.01 a

-1

available K (mg kg )

153.91±6.70 a

150.88±2.57 a

152.86±3.32 a

204.61±12.54 a

207.25±7.69 a

201.28±1.29 a

total Cd

0.215±0.041 c

0.543±0.018 b

0.925±0.009 a

0.143±0.019 c

0.619±0.129 b

1.407±0.109 a

available N (mg kg )

Note: Different letters within the same row indicate significance at p < 0.05 level.

36


Page 37 of 46


Table 3 The bioaccumulation factors (BF) of different vegetables Plants

Value

Ref.

0.81-3.07 (soil Cd: 0.22 mg kg-1)

Chinese kale (Brassica alboglabra L. H. Bailey)

5.70-17.71 (soil Cd: 0.54 mg kg-1) 9.55-22.49 (soil Cd: 0.93 mg kg-1)

The present study

1.95-7.55 (soil Cd: 0.40 mg kg-1)

water spinach (Nasturtium officinale R. Br.)

1.64-7.25 (soil Cd: 0.80 mg kg-1)

30

-1

1.41-4.64 (soil Cd: 1.60 mg kg ) 0.31-1.42 (soil Cd: 0.11 mg kg-1)

Chinese flowering cabbage (Brassica parachinensis L.)

0.42-2.63 (soil Cd: 0.67 mg kg-1)

35

0.39-2.02 (soil Cd: 1.13 mg kg-1) 3.71-7.78 (soil Cd: 0.31 mg kg-1)

Chinese leaf mustard (Brassica juncea var. foliosa)

3.04-8.10 (soil Cd: 0.71 mg kg-1)

40

-1


celery

8.63-16.35 (soil Cd: 0.43 mg kg-1)

(Apium graveolens L.)

41

-1


lettuce (Lactuca dolichophylla Kitam.)

2.27-9.88 (soil Cd: 0.35 mg kg-1) -1

3.18-10.75 (soil Cd: 1.25 mg kg )

37


41


Page 38 of 46

Figure graphics  Figure 1

140 120

**

100 *

BRS (%) under LCd

80

ns *

60

ns

40

* *

* *

20

ns ns ns ns ns ns

ns

0

ns ns ns ns

-20 -40

ns ns * *

-80

** **

ns

**

DX104 HKGZ LJ-F1 DX102 Yuan GS YC-2 TWDX CHJK ATBJ TSD HJK ZDH TWCY FZ ZJ TJK HKBT QS JP400 CD XC JX SC JST MH HCD JC

-60

Cultivars

160

**

140 100 80 60 40 20

** * ** ns

ns ns ns

ns ns

ns ns ns ns ns

0

ns ns ns ns

-20 -40

ns

ns ns ns

* * **

-60 -80

* **

-100

DX104 GS QS TWDX JP400 HJK YC-2 FZ TJK ZDH DX102 Yuan XC MH CHJK TSD HKBT JX JST JC TWCY ATBJ ZJ LJ-F1 CD SC HCD HKGZ

BRS (%) under HCd

120

Cultivars

38


HCD HJK ZJ TWDX HKGZ TSD LJ-F1 YC-2 HKBT SC XC JC JP400 Yuan CD TJK FZ ATBJ CHJK QS TWCY MH GS JX ZDH DX104 DX102 JST

Shoot Cd concentration (mg kg-1 , FW)

0.0

a a ab abc abc abcd abcd abcd abcd abcd abcde abcde abcde abcde abcde abcde abcde abcde abcde bcdef bcdef bcdef cdefg defg defg efg fg g

0.2

HJK HKBT TWDX TSD CHJK MH CD HCD Yuan ZDH JP400 ATBJ ZJ XC TJK SC FZ LJ-F1 HKGZ TWCY YC-2 JST QS DX104 JC GS DX102 JX

a b c cd cde cdef cdefg cdefg cdefgh cdefgh cdefghi cdefghi cdefghi defghi defghi efghij efghij fghijk fghijk ghijkl hijkl hijkl ijkl ijkl ijkl jkl kl l


HJK TJK JP400 DX104 Yuan TSD MH ATBJ HKBT TWDX CD ZJ SC ZDH CHJK JST JX QS HKGZ FZ HCD JC LJ-F1 YC-2 XC TWCY DX102 GS

a b b bc bcd bcd bcde bcde bcdef bcdef bcdef cdefg cdefg cdefg defg efg fgh ghi ghi ghi ghi ghi ghi ghi ghi hi i i


Page 39 of 46 Journal of Agricultural and Food Chemistry

Figure 2 CK 0.08

0.06

0.04

0.02

0.00

Cultivars

LCd

1.0

0.8

0.6

CAC

0.4

0.0

Cultivars

HCd

2.0

1.5

1.0

CAC

0.5

Cultivars

39



Page 40 of 46

Figure 3 b

a

1.0

0.5

y = 0.522 x + 0.182 r = 0.998, p < 0.05, n=3 Soil Cd concentration (mg kg-1 , DW)

1.5

1.0

y = 1.046 x + 0.171 r = 0.995, p < 0.05, n=3

y = 0.9394x + 0.0848 (r=0.500, p < 0.01, n=28)

Soil Cd concentration (mg kg-1 , DW)

Cd concentration under HCd (mg kg-1, FW)

c 1.0

2.0

0.8

0.6

0.4

0.2

0.0

0.0 0.2

0.4

0.6

0.8

Cd concentration under LCd (mg kg-1, FW)

0.8

0.6

0.4

0.2

0.0

0.0

0.2

0.4

0.6


40


0.8

0.0

0.5

1.0

1.5


Page 41 of 46


Figure 4

60

LCd **

40 **

**

BRS (%)

** 20

HCd

ns

**

*

* ns

0 ns ns

ns -20

**

* **

** -40 DX104

JX

JST

DX102

HJK

TWDX

HKBT

HCD

Cultivars

Figure 5 DX104 HJK

4

Shoots

JX TWDX

JST HKBT

DX102 HCD a

3

Cd concentration (mg kg-1, FW)

b 2

a c

1 c cd d

d

a

b ab a

a bc cd

b

b

d cd b

c

b

b

b

a

d

d

d

cd

c

e

d

0 1 d 2

c

c f

3 b

b

4

b

b

e

de

cd

c

5

b a

6

a

Roots 7 CK

LCd

Treatments

41


HCd

c


Page 42 of 46

Figure 6 1.2

DX104 HJK

JX TWDX

JST HKBT

DX102 HCD

a

1.0

a 0.8

TF

a

b

b

a a

c

c c

0.6

d

b bc 0.4

bc

e

bc

d d d c

f g

e

0.2

0.0 CK

LCd

Treatments

42


HCd

bc

Page 43 of 46


Figure 7 DX102 4

HJK **

Shoots

Cd concentration (mg kg-1 , FW)

3 ** 2 *

1 0 1

ns

2 3

**

4 5 6

Roots

**

7 CK

LCd

Treatments

43


HCd


Page 44 of 46

Figure 8 F2

F1 DX102 2 *

1 ns 0 ns

1 2

**

3

DX102 0.8


**

Shoots


HJK

0.4

**

0.2

**

0.0 ns

0.2 0.4

*

0.6 0.8

**

Roots

*

1.0 CK

LCd

HCd

CK

LCd

F3 DX102

F4

HJK

0.8

DX102 1.0

Shoots


**

0.6

0.4 ** 0.2 ** 0.0 ns 0.2 Roots

ns

0.4 CK

LCd

HCd

Treatments

Treatments


HJK **

0.6

Roots 4

Shoots

HJK **

Shoots ** 0.5 * 0.0 ns

0.5

**

1.0 1.5 Roots

*

2.0

HCd

CK

LCd

Treatments

Treatments

44


** HCd

Page 45 of 46


Figure 9 Shoots Cd proportion in subcellular fractions

CK

LCd

HCd

100% 90% 80% 70%

F4

60%

F3

50%

F2

40%

F1

30% 20% 10% 0% DX102 HJK

DX102 HJK

DX102 HJK

Cultivars

Roots

Cd proportion in subcellular fractions

CK

LCd

HCd

100% 90% 80% 70%

F4

60%

F3

50%

F2

40%

F1

30% 20% 10% 0% DX102 HJK

DX102 HJK

Cultivars

45


DX102 HJK


TOC Cd minimized products for safe consumption Cd treatment Soil Biomass

BF

Cd concentration Plant tissues

Assistance for molecular breeding of Cd-PSCs

TF

Identification Low-Cd cultivars

Laws of Cd migration and detoxification

High-Cd cultivars

Comparison

Shoots

Cd subcellular distribution Roots

Cd was mainly transported into vacuole and inactivated Cd was mainly restricted among cell wall and immobilized

46


Page 46 of 46

Selection for Cd Pollution-Safe Cultivars of Chinese Kale (Brassica

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