Effects of Interaction between Cadmium (Cd) and Selenium (Se) on

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Effects of interaction between cadmium (Cd) and selenium (Se) on grain yield and Cd and Se accumulation in a hybrid rice (Oryza sativa) system Baifei Huang, Junliang Xin, Hongwen Dai, and Wenjing Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03316 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Journal of Agricultural and Food Chemistry

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Effects of interaction between cadmium (Cd) and selenium (Se)

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on grain yield and Cd and Se accumulation in a hybrid rice

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(Oryza sativa) system

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Baifei Huang, Junliang Xin*, Hongwen Dai, Wenjing Zhou

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Research Center for Environmental Pollution Control Technology, School of Safety

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and Environmental Engineering, Hunan Institute of Technology, Hengyang 421002,

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China

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*

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Tel.: +86-734-3452399

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Corresponding author

E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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A pot experiment was conducted to investigate the interactive effects of cadmium (Cd)

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and selenium (Se) on their accumulation in three rice cultivars, which remains unclear.

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The results showed that Se reduced Cd-induced growth inhibition, and increased and

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decreased Se and Cd concentrations in brown rice, respecially. Cadmium

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concentrations in all tissues of the hybrid were similar to those in its male parent yet

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significantly lower than those in its female parent. Selenium reduced Cd accumulation

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in rice when Cd concentration exceeded 2.0 mg kg-1; however Se accumulation

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depended on the levels of Cd exposure. Finally, Cd had minimal effect on Se

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translocation within the three cultivars. We concluded that Cd concentration in brown

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rice is a heritable trait, making crossbreeding a feasible method for cultivating

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high-yield, low-Cd rice cultivars. Selenium effectively decreased the toxicity and

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accumulation of Cd, and Cd affected Se uptake but not translocation.

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Keywords: Cadmium; Selenium; Rice (Oryza sativa); Translocation; Hybridization

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INTRODUCTION

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Cadmium (Cd) is a non-essential metal, and is highly mobile and toxic to living

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organisms.1 Cadmium in soil is easily taken up by crop roots and rapidly transported

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to the edible parts. Cadmium enters agricultural soil mainly from anthropogenic

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sources, such as industrial discharge and emissions, sewage used for irrigation, and

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phosphate fertilizers, and can threaten human health through bioaccumulation and

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biomagnification.2 Currently, approximately 7.0% of the land area of China has been

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contaminated by Cd at different levels.3 Therefore, the need for reducing Cd

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accumulation in agricultural products is urgent.

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Rice (Oryza sativa L.) is the predominant cereal crop in China, and additionally, the

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staple food for over 60% of the Chinese population. Undoubtedly, the yield and

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quality of rice are closely related to social stability, economic development, and

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human health. Previously, several physical, chemical, and biological methods have

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been used to minimize Cd accumulation in rice, including soil removal and

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replacement,4 chemical washing,5 phytoremediation,6 application of soil amendments,

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7

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cost-ineffectiveness and time-consumption nature, ion imbalance in the soil, low grain

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yield, and inconvenience.9 Although transgenic approaches could similarly reduce Cd

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accumulation in rice grains,10-12 they are less publicly acceptable owing to concerns

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over genetically modified crops, as demonstrated by the recent “Golden Rice”

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controversy.13 By comparison, the selection and breeding of low-Cd rice cultivars is

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becoming an increasingly popular method for reducing Cd accumulation in grains, as

and water management.8 The major limitations of these measures are their

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there are already substantial differences in the uptake and accumulation of Cd among

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cultivars.14 However, the inherited patterns of Cd concentration in rice are still unclear,

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and additionally, relevant information is quite limited.

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In recent years, researchers have discovered that selenium (Se), which is an essential

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micronutrient for humans, plays an important role in the enhancement of immunity,

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protection against cancer, and reduction of heavy metal toxicity.15, 16 Nevertheless,

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approximately 72% of the total land area in China is Se-deficient,17 and consequently,

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Se concentration in the grains of the rice cultivated in this region is 20 µg kg-1 on

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average.18 Therefore, average daily Se intake by the Chinese is only 28–40 µg, which

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is less than the recommended daily intake of 55–85 µg for adults,19 and often results

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in diseases or problems related to Se deficiency.20 Furthermore, at low levels, Se is

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beneficial to plants,21, 22 and biofortification of rice using Se fertilizers not only

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increases grain Se concentration, but also reduces grain Cd concentration.23, 24

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Consequently, the application of Se to slightly-to-moderately Cd-contaminated paddy

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soils may be a cost-effective approach to the production of Cd-deficient, Se-rich rice

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grains.24 Se application to paddy soils not only decreases Cd concentration in brown

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rice, but also reduces Cd accumulation in other tissues.24 However, Chen et al.

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observed that Se did not reduce Cd accumulation in rice roots.25 Consequently, the

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effects of Cd-Se interactions on the uptake, translocation, and accumulation of Cd in

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rice, especially in hybrid rice systems, are not yet well understood, and require further

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investigation.

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Currently, brown rice constitutes a considerable part of human diets because of its

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high nutritional value. We previously observed that brown rice contains more

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nutrients than polished rice; however, it also has higher concentrations of heavy

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metals (Table S1). In this study, three rice cultivars, including a female parent

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(male-sterile line), a male parent (restorer line), and their F1 hybrid were grown in

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nine soils with different concentrations of Cd and Se. The aims were to compare the

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accumulation of Cd and Se among the tested cultivars to investigate the inherited

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characteristics of grain Cd accumulation, and effects of Se-Cd interactions on the

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translocation of Cd and Se within the rice plants. We hypothesized that: (1) Se

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application substantially reduces Cd accumulation in all tissues; (2) grain Se

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concentration is not affected by soil Cd levels. The results of this study would help in

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further understanding the feasibility of reducing grain Cd concentration by

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crossbreeding and the mechanisms of Cd-Se interactions underlying grain Cd

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accumulation.

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MATERIALS AND METHODS

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Plant cultivation

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Two rice cultivars, Zhongjiu A (female parent, F) and Huazhan R (male parent, M),

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and their F1 hybrid (H) were used in this study. The seeds of the three cultivars were

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surface-sterilized for 15 min with 0.5% NaClO solution, rinsed with deionized water

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for 10 min, and then, germinated in moist, sterile quartz sand at 30°C. The

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germination time of F was delayed by 25 days to synchronize its flowering time with

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that of M. Three weeks after germination, uniform-sized seedlings were transferred to

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soil-filled plastic pots.

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Experimental site and soil

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A pot experiment was conducted in the greenhouse of the Hunan Institute of

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Technology (26°52′N, 112°41′E), Hunan Province, China at 28–35°C. Experimental

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soil was collected from a nearby farmland, air-dried, ground, and passed through a

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5-mm sieve. The physical and chemical properties of the soil were evaluated using the

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analytical methods described by Lu.26 The soil pH, cation-exchange capacity, organic

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matter content, and contents of total N, available P, available K, total Cd, and total Se

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were 6.40, 89.2 mmol kg-1, 19.5 g kg-1, 1.5 g kg-1, 112 mg kg-1, 124 mg kg-1, 1.23 mg

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kg-1, and 0.58 mg kg-1, respectively. According to the Farmland environmental quality

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evaluation standards for edible agricultural products (HJ 332-2006), the maximum

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level (ML) of Cd in soil should be 0.3 mg kg-1; however, the standards do not specify

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any limitation for Se concentration. Therefore, the tested soil was considered as

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Cd-contaminated, and served as a treatment group (C1S1) in this experiment. Eight

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other treatment groups (C2S1, C3S1, C1S2, C2S2, C3S2, C1S3, C2S3, and C3S3)

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contained soil with target Cd concentrations of 2.0 and 4.0 mg kg-1 and/or Se

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concentrations of 0.9 and 1.2 mg kg-1, which were generated by mixing C1S1 soil

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with appropriate quantities of Cd in the form of Cd(NO3)2·4H2O and Se in the form of

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Na2SeO3. Each of the eight soil groups was placed in a large plastic basin, watered,

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and allowed to equilibrate in the greenhouse for approximately 6 months. This period

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allowed for the attainment of balance between the various sorption mechanisms in the

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soils.27 The final total Cd concentrations were 2.01 mg kg-1 for C2S1, C2S2, and

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C2S3, and 4.16 mg kg-1 for C3S1, C3S2, and C3S3. The final total Se concentrations

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were 0.86 mg kg-1 for C1S2, C2S2, and C3S2, and 1.13 mg kg-1 for C1S3, C2S3, and

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C3S3.

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Experimental design

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Plastic pots (diameter at the top, 25 cm; diameter at the base, 18 cm; height, 26 cm)

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were filled with 6.0 kg (dry weight, dw) of prepared soil. For each cultivar, three

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replicates of each treatment group (n = 3 pots) were cultivared. The prepared rice

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seedlings were transplanted into the pots (two plants per pot) on April 28 (M and H)

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and May 23, 2016 (F). The pots with the M and F seedlings were arranged alternately

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to facilitate effective cross-fertilization between them. All the pots were submerged,

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and a water depth of approximately 3 cm above the soil surface was maintained

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throughout the cultivation period. A solid compound fertilizer (N:P:K = 15:15:15) was

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applied to the pots at a rate of 3.5 g pot−1 every 2 weeks. Owing to male sterility in F,

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manual pollination was performed thrice to increase the seed-setting rate of F during

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the flowering period of M and F.

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Sampling and chemical analysis

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Each plant was harvested entirely at maturity. Grains, shoots (including leaves and

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stems), and roots were rinsed separately with tap water to remove dust and soil, and

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then, the roots were desorbed for 15 min in ice-cold 5 mM CaCl2 solution (5 mM

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MES-Tris, pH 6.0). Thereafter, all the plant samples were washed thoroughly with

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deionized water, and dried at 70°C to attain constant weight. The grains were dehulled

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using a motorized dehusker (JLGJ4.5, TZYQ, Zhejiang, China) to yield brown rice.

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The dried samples were ground, passed through a 0.149-mm sieve, and digested with

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HNO3-H2O2 (10:3, v/v) using a microwave digester (XT-9900 A, Shanghai Xintuo

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Analytical Instruments Co., Ltd., China). The concentrations of Cd and Se were

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determined using ICP-MS (Agilent-7500, Agilent Technologies Co. Ltd, Palo Alto,

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CA, USA). Certified Reference Materials (CRM) for plants, GBW07605 and soil,

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GBW07410 (provided by the National Research Center for CRM, China) were used

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for quality assurance and quality control of the analysis of Cd and Se. The detection

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limits of Cd and Se in the plant samples were 0.002 and 0.003 mg kg-1 dw,

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respectively. The recovery rates of Cd in the plant and soil samples were 96–105%

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and 93–112%, respectively, and those of Se were 94–106% and 92–105%,

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respectively.

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Safety standards and statistical analysis

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According to the Chinese National Food Safety Standard for Maximum Levels of

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Contaminants in Food (GB 2762-2017,

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http://bz.cfsa.net.cn/staticPages/D5921FFE-BD08-4D34-AE26-CF9CA4FEB001.html

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), the maximum recommended Cd level in brown rice is 0.2 mg kg-1 (dw). This

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standard was used to evaluate the safety of the tested rice cultivars. Translocation

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factors (TFs) of Cd and Se from roots to shoots and from shoots to brown rice were

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calculated using the following equation:

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TFa to b = Cb-x/Ca-x

(1)

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where a represents roots or shoots; b represents shoots or brown rice; x represents Cd or Se; Cb-x represents the concentration of Cd or Se in b; and Ca-x represents the

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concentration of Cd or Se in a.

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Data were analyzed for homogeneity of variances by Levene’s test, and showed no

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heteroscedasticity. All data were analyzed statistically by three-way analysis of

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variance (ANOVA) at p < 0.05. When the cultivar treatment was significant, the

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differences in the same cultivar among the three Se concentrations at the same Cd

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concentration and among the three Cd concentrations at the same Se concentration

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were further evaluated by one-way ANOVA with Tukey's HSD (Honest Significant

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Difference) test. All tests were performed using Microsoft Excel 2003 (Microsoft

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Corp., Redmond, WA, USA) and SPSS 13.0 (IBM, Armonk, NY, USA). All

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correlations were assessed using Pearson product-moment correlation.

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RESULTS

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Grain Biomass in Response to Cd and Se Treatments

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Rice grain dw was significantly affected by cultivar type, Se treatment, Cd treatment,

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and the interaction among the three factors (Figure 1), indicating that grain biomass

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was determined not only by genetic factors, but also by Cd and Se levels in the soil.

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The H plants invariably had the highest grain dw, followed by M and F in each

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treatment group (Figure 1), showing heterosis in yield. Overall, the grain dw of M and

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H increased significantly with increasing Se concentration in the treatment groups at

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the same Cd concentration (Figure 1); therefore, Se may help promote grain biomass

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accumulation. Additionally, the grain biomass of each cultivar did not decrease

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significantly; rather it increased when soil Cd concentration increased to 2.01 mg kg-1;

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however, it decreased significantly afterward, especially for H (Figure 1), implying

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that the rice cultivars showed moderate tolerance to Cd toxicity.

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Cadmium Accumulation in Response to Cd and Se Treatments

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Generally, Cd accumulation in the brown rice, shoots, and roots was significantly

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affected by the cultivar type, Se treatment, Cd treatment, and the interactions among

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these factors (Figure 2A, B, and C). This demonstrated that Cd accumulation in rice is

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determined by genetic factors, as well as exposure to Cd and Se. The ability of H to

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accumulate Cd was similar to that of M, and significantly lower than that of F (Figure

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2A), suggesting that the trait of high Cd accumulation of F was not inherited by H.

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For each cultivar, Cd concentration in the brown rice increased significantly with

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increasing Cd concentration in the treatment groups at the same Se concentration,

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except in C2S3, which was similar to C1S3. In the treatment groups without

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additional Cd2+ (i.e., C1S1, C1S2, and C1S3), Cd concentration in the brown rice did

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not change significantly, and increased in F with increasing soil Se concentrations.

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However, Se application significantly decreased the Cd concentration in the brown

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rice in the treatment groups with additional Cd2+ (Figure 2A). Additionally, Cd

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concentration in the H brown rice exceeded the maximum level (0.2 mg kg-1)

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recommended by the Chinese food standard in C3S1; although not when Se was

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added to the Cd-contaminated soils (i.e., C3S2 and C3S3).

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Cadmium concentrations in the shoots and roots of each cultivar increased with

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increasing Cd levels (Figure 2B and C). Furthermore, there was no significant

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difference in Cd accumulation in the shoots and roots between M and H, and Cd

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accumulation was invariably higher in F than the other two cultivars. In the treatment

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groups without additional Cd2+, Cd concentrations in the shoots did not change

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significantly in the two parents; however, they increased significantly in H with

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increasing soil concentration of Se. Nevertheless, the increased Se levels in other

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higher Cd-contaminated soils significantly decreased Cd concentrations in the shoots

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in all cultivars. Cadmium concentrations in the roots of the three cultivars in the

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treatment groups without additional Cd2+ increased significantly with increasing soil

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concentration of Se. Contrastingly, root Cd concentrations in the three cultivars

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decreased with increasing Se levels in the other higher Cd-contaminated soils;

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however, this decrease was statistically significant only in F. Overall, Cd

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concentrations in the brown rice showed significant positive correlations with Cd

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concentrations in the shoots and roots (Figure 3A and B), demonstrating that low Cd

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levels in the latter may give rise to low Cd levels in the former.

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Selenium Accumulation in Response to Cd and Se Treatments

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Selenium accumulation in the brown rice, shoots, and roots was significantly affected

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by cultivar type, Se treatment, Cd treatment, and certain interactions among these

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factors (Figure 4A, B, and C), demonstrating that Se accumulation in rice is controlled

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by genetic factors, as well as exposure to Se and Cd. Generally, Se concentrations

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were higher in the brown rice and shoots of F than those of M and H, although there

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were no obvious differences between M and H (Figure 4A and B). Additionally, the Se

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concentration was slightly higher in the roots of F than those of M in all the treatment

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groups, except for C3S2 and C2S3; however, H showed no difference in root Se

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concentrations from M and F (Figure 4C). This indicated that the characteristics of Se

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accumulation in H were inherited from each parent.

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For each cultivar, Se concentrations in the brown rice, shoots, and roots increased

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significantly with increasing Se level in the treatment groups with the same Cd

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concentration (Figure 4A, B, and C). However, generally, Se concentrations in all the

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tissues first increased, and then, decreased dramatically with increasing Cd levels in

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the treatment groups with the same Se concentration, demonstrating the importance of

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Cd exposure for Se accumulation in rice. Additionally, Se concentration in the brown

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rice was significantly, positively correlated with that in the shoots and roots (Figure

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5A and B), implying that Se level in the brown rice depended on that in the shoots and

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roots.

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Cadmium Translocation within Rice Plants

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Cadmium translocation from the roots to the shoots and from the shoots to the brown

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rice varied greatly with cultivar type and levels of Cd and Se exposure (Table 1).

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Overall, the Cd translocation factors (TFs) from the roots to the shoots in F and H

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increased gradually with increasing Cd concentration; however, they were minimally

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affected by Se addition. In the treatment groups without additional Cd2+, the lowest

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Cd TFs from the roots to the shoots in M were observed in C1S1, and the increase in

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Se level significantly inhibited Cd translocation from the roots to the shoots. However,

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Cd translocation from the roots to the shoots in M did not changed noticeably with

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increasing Se level in the other higher Cd treatment groups. Additionally, M had the

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highest Cd TFs from the roots to the shoots, followed by F and H in C1S1, C1S2, and

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C1S3; however, the TF was slightly lower in M than F in the C1S1 (Table 1). There

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was no significant difference in the Cd TFs from the roots to the shoots among the

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three cultivars in the other higher Cd treatment groups excluding C3S1.

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For F, the Cd TF from the shoots to the brown rice was the highest in C2S3; however,

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it did not differ significantly from those in other treatment groups with the lowest Cd

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levels (Table 1). Furthermore, in the treatment groups with the same Cd levels,

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excluding C2S3, the ability of shoot-to-brown rice Cd translocation of F remained

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relatively stable with increasing Se concentration. It is noteworthy that the Cd TFs

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from the shoots to the brown rice in M and H were noticeably affected by Se levels in

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the highest Cd treatment groups (Table 1). Among the treatment groups without

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additional Se (i.e., C1S1, C2S1, and C3S1), there were no significant differences in

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the Cd TFs in either M or H. However, Cd translocation from the shoots to the brown

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rice in both cultivars decreased slightly when Se was applied to the Cd-contaminated

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soils. Additionally, the Cd TFs from the shoots to the brown rice were invariably,

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significantly higher in H and M than F, excluding C1S1 and C1S2.

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Selenium Translocation within the Rice Plants

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Generally, the Se TFs from the roots to the shoots in F did not change significantly

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with increasing Se level, and was not obviously affected by Cd application, except

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that the TF was significantly lower in C3S3 than C1S1 and C2S1 (Table 2). For M

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and H, Se translocation from the roots to the shoots was not significantly affected by

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the Se and Cd levels. Additionally, there were no significant differences in the

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root-to-shoot Se translocation among the three cultivars in any treatment group,

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except that the ability of Se translocation of F was significantly higher than that of M

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in C2S2 (Table 2).

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In the treatment groups with the lowest Se levels, the Se TFs from the shoots to the

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brown rice increased significantly in H with increasing Cd level (Table 2). Moreover,

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in other treatment groups with higher Se levels, the ability of shoot-to-brown rice Se

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translocation of the three cultivars did not change with increasing Cd level. A similar

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phenomenon occurred similarly in other treatment groups with the highest Se levels.

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However, the difference in Se translocation from the shoots to the brown rice among

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the three cultivars was observed only in C2S1, and the TF of H was similar to that of

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M; however it was significantly higher than that of F (Table 2).

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DISCUSSION

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Effects of Cadmium and Selenium on Grain Biomass

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In this study, the grain biomass of the three rice cultivars increased at different

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degrees when soil Cd concentration increased from 1.23 mg kg-1 (C1S1) to 2.01 mg

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kg-1 (C2S1). This suggested that the rice cultivars had high tolerance to the Cd levels

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detected in Chinese soils.3 The stimulatory effect of Cd on rice growth, which may

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represent hormesis, was similarly observed in other studies.28-30 However, grain dw

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decreased significantly in C3S1 (4.16 mg Cd kg-1 soil) compared with C1S1, possibly

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because of Cd toxicity-induced reduction in photosynthetic efficiency.31 It is

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noteworthy that grain dw increased significantly with increasing soil Se concentration

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when Cd concentration remained constant (4.16 mg kg-1). This demonstrated that

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exogenous Se addition can reduce the negative effects of Cd on rice growth, and

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enhance the activity of the photosynthetic system. Similarly, Zhang et al. observed

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that Se application enhances photosynthesis by increasing the net photosynthetic rate,

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intercellular CO2 concentration, and transpiration efficiency of rice, thereby

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increasing grain yield.32

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We similarly observed that grain biomass increased gradually with increased soil Se

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concentration without added Cd, excluding H in C1S2. Contrastingly, Liao et al.

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reported that rice grain yield was reduced by 12.81% and 9.16% when Se was added

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to the soil at 0.1 and 1 mg kg-1, respectively. 23 These inconsistent results may be

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related to the rice cultivars, Se species, experimental conditions, or levels of Se

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exposure. Additionally, one major reason for the lower grain yield of F than those of

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M or H was the lower seeding rate; although H invariably had the highest grain dw,

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suggesting heterosis in grain yield.

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Effects of Selenium on Cadmium Accumulation in Rice

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Cadmium accumulation was lower in H, similar to M, than F, indicating that this

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characteristic of low Cd accumulation in rice is heritable, and probably controlled by

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dominant gene(s). Similarly, Cd accumulation in durum wheat grains and water

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spinach shoots is largely controlled by a single locus of multiple genes with low Cd

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accumulation dominance.33, 34 It is noteworthy that OsNramp5 (natural

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resistance-associated macrophage protein 5) is a major transporter responsible for Cd

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uptake in rice,11 and OsHMA3 (heavy metal ATPase 3) is closely related to Cd

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translocation.12 OsHMA3 in a low-Cd cultivar (Nipponbare) functionally limits

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root-to-shoot Cd translocation by sequestering Cd in root vacuoles; however, the

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transporter in a high-Cd cultivar (Anjana Dhan) loses its function.12 Further, Miyadate

312

et al. observed that OsHMA3 is dominant in controlling the phenotype of low

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root-to-shoot Cd translocation.35 Moreover, the ability of low Cd accumulation in H

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and M was not influenced by soil Se levels, and was stable. Therefore, it is feasible to

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breed rice cultivars with low Cd accumulation and high yield using conventional

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hybridization techniques.

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In this study, Se application significantly decreased Cd concentrations in all the

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tissues of the rice plants exposed to exogenous Cd. One reason may be that selenite is

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reduced to Se2- in acidic soil, and further forms Se-Cd complexes in the rhizosphere,

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which are unavailable to the plants.36 Selenite may also chelate with Cd2+ to form

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CdSeO3 complexes, thus, reducing Cd bioavailability in the soil.37 In any case,

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selenite limits root growth, and decreases the proportion of fine roots,38 which further

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reduces Cd uptake. Therefore, Se is involved in antagonistic processes with Cd in rice

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plants, and inhibits Cd uptake by the abovementioned mechanisms. Additionally, we

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observed that Se application did not significantly affect root-to-shoot Cd translocation

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in most cases, which is similar to the finding by Hu et al.24 Most of the selenite

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absorbed by plant roots is rapidly assimilated into organic forms, such as SeOMet,

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MeSeCys, and SeMet,39 which may not compete with Cd2+ in the xylem during

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transport from roots to shoots. The difference in root-to-shoot Cd translocation among

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the three cultivars appeared to depend on the levels of Cd and Se exposure. However,

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the difficulty in Cd translocation from shoots to brown rice increased with increasing

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soil Se levels in M and H, yet not in F. Hu et al. similarly observed that Se application

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did not significantly affect Cd translocation from shoots to grains.24 This

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demonstrated that the effect of Se on Cd translocation from shoots to grains may be

335

related to genotype. Furthermore, Cd was more easily transported to the brown rice

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from the shoots in F than in M and H. Selenium was reported to significantly increase

337

leaf glutathione (GSH) levels in plants cultivated in the soil;40 further, GSH is a

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substrate for phytochelatin (PC) synthesis, and crucial for the sequestration and

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detoxification of Cd. Cytosolic Cd can form complexes with S-containing ligands,

340

such as GSH and PCs, and subsequently be transported into vacuoles, resulting in Cd

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immobilization within the leaf.41 Therefore, further investigations are required to

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evaluate the synthesis of GSH and PCs in the shoots of the three cultivars.

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It is worth noting that by increasing Se levels, Cd accumulation increased in the rice

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tissues, especially roots, in the treatment groups without additional Cd, partially

345

confirming our first hypothesis. Fargašová et al. similarly discovered that Se increases

346

Cd accumulation in the roots of Sinapis alba L.42 Therefore, the interaction between

347

Se and Cd depends on the ratio of concentration of the two elements; thus, certain

348

stimulating effects of increased Se concentrations on Cd uptake may be expected in

349

addition. As Arvy et al. mentioned, selenite addition to the cultivation medium

350

increases the capacity of root cells to accumulate heavy metals.43 In our study, Se

351

application decreased root-to-shoot Cd translocation in M, yet not in H and F in the

352

treatment groups without additional Cd. Consequently, the interaction between Cd and

353

Se is dose-dependent and probably, cultivar-dependent.

354

Effects of Cadmium on Selenium Accumulation in Rice

17



355

Cadmium enters root cells via ZIP (zinc/iron-regulated transporter protein) or other

356

cation channels,44 while selenite is absorbed by plant roots via phosphate

357

transporters,35 silicon influx transporters,45 and aquaporins.46 Theoretically, there is no

358

competition between Cd and Se on root surfaces, as they are absorbed via different

359

transporters or channels. However, in this study, Se concentrations in all the tissues of

360

the three cultivars, including roots, shoots, and grains, initially increased to different

361

levels, and then, decreased rapidly as Cd concentrations increased from 1.23 mg kg-1

362

to 2.01 mg kg-1, and then, to 4.16 mg kg-1, thereby disproving our second hypothesis.

363

Lin et al. observed that Cd2+ (50 µM) addition significantly decreased Se

364

accumulation in rice cultured in a nutrient solution containing 3 µM Se (Na2SO3).47

365

Meanwhile, the effects of Cd on Se accumulation in cucumber depended on the

366

concentrations of Cd and Se in the medium, and Cd addition strongly promoted Se

367

translocation from roots to shoots.48 Therefore, the effects of Cd-Se interactions on Se

368

accumulation in plants are related not only to the levels of Cd and Se exposure, but

369

also to plant species or cultivar types. The main reason for the Cd-promoted increase

370

in Se concentrations in the rice tissues may be that rice absorbs more Se to detoxify

371

Cd, since Se can reduce Cd-induced oxidative stress by increasing the accumulation

372

of proline and GSH.49 Furthermore, -SeH can replace the -SH group in GSH and PCs,

373

and protect against Cd toxicity by increasing thiol concentrations.36 Notably, Se

374

translocation from roots to shoots and from shoots to brown rice was minimally

375

affected by the levels of Cd exposure, particularly when soil Cd concentration

376

increased from 2.01 mg kg-1 to 4.16 mg kg-1. Thus, the obvious decrease in Se

18


Page 18 of 38

Page 19 of 38


377

concentrations in the three cultivars when the level of Cd exposure increased to 4.16

378

mg kg-1 was mainly due to the decrease in Se uptake via root surfaces. Possibly, high

379

Cd toxicity inhibited the activities of the transporters or water channels involved in Se

380

uptake; however, this requires further investigation of the mechanisms controlling Se

381

uptake in rice.

382

In conclusion, our results showed that F invariably had significantly higher grain Cd

383

concentration and higher shoot-to-brown rice Cd translocation than M or H. The trait

384

of low Cd accumulation in rice may be dominant, and thus, it is feasible to develop

385

new low-Cd, high-yield rice cultivars through crossbreeding. Selenium

386

biofortification not only increases grain Se concentration, which helps overcome Se

387

deficiency in humans, but also significantly decreases grain Cd concentration when

388

soil Cd concentration exceeds 2.0 mg kg-1. Additionally, these results suggested that

389

Se can reduce Cd-induced growth inhibition, improve photosynthesis, and increase

390

grain yield in rice. Furthermore, the effects of Cd-Se interactions on the accumulation

391

of Cd and Se in rice are related to the levels of Cd and Se exposure. Selenium

392

application inhibited shoot-to-brown rice Cd translocation in M and H; however, Cd

393

affected Se translocation in the rice plants negligibly.

394

Notes

395

The authors declare no competing financial interest.

396

ACKNOWLEDGMENTS

397

This study was supported by the Hunan Provincial Natural Science Foundation of

398

China (Grant 2016JJ5017) and the National Natural Science Foundation of China

19



Page 20 of 38

399

(Grants 41101303 and 41201320).

400

Supporting Information

401

Table S1 Concentrations of different elements in rice husk, brown rice, and polished

402

rice

403

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404

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Page 27 of 38


Figure captions Figure 1 Effects of cadmium (Cd) and selenium (Se) on the grain biomass of three rice cultivars. Error bars represent the standard deviation (n = 3). ns, not significant; *, significant at the p < 0.01 level. For the same cultivar, different lower-case letters indicate significant differences (p < 0.05) among different Se treatment groups with the same Cd concentration; different upper-case letters indicate significant differences (p < 0.05) among different Cd treatment groups with the same Se concentration. See the Materials and Methods section for the abbreviations.

Figure 2 Effects of selenium (Se) on the accumulation of cadmium (Cd) in brown rice (A), shoots (B), and roots (C) of three rice cultivars. Error bars represent the standard deviation (n = 3). *, significant at the p < 0.05 level; **, significant at the p < 0.01 level. For the same cultivar, different lower-case letters indicate significant differences (p < 0.05) among different Se treatment groups with the same Cd concentration; different upper-case letters indicate significant differences (p < 0.05) among different Cd treatment groups with the same Se concentration. See the Materials and Methods section for the abbreviations.

Figure 3 Correlations between brown rice cadmium (Cd) concentration and Cd concentrations in the shoots (A) and roots (B) of three rice cultivars.

Figure 4 Effects of cadmium (Cd) on the accumulation of selenium (Se) in brown rice

27



(A), shoots (B), and roots (C) of three rice cultivars. Error bars represent the standard deviation (n = 3). ns, not significant; *, significant at the p < 0.05 level; **, significant at the p < 0.01 level. For the same cultivar, different lower-case letters indicate significant differences (p < 0.05) among different Se treatment groups with the same Cd concentration; different upper-case letters indicate significant differences (p < 0.05) among different Cd treatment groups with the same Se concentration. See the Materials and Methods section for the abbreviations.

Figure 5 Correlations between brown rice grain selenium (Se) concentration and Se concentrations in the shoots (A) and roots (B) of three rice cultivars.

28


Page 28 of 38

Page 29 of 38


Table 1 Changes in cadmium (Cd) translocation from roots to shoots, and from shoots to brown rice at different levels of Cd and selenium (Se) exposure Treatment group

F

M

H TF root to shoot

C1S1

3.17 ± 0.40

d

A

2.78 ± 0.52

c

AB

2.20 ± 0.12

d

B

C1S2

3.79 ± 0.51

cd

B

6.34 ± 0.84

a

A

2.60 ± 0.04

cd

C

C1S3

3.89 ± 0.36

bcd B

5.29 ± 0.25

ab

A

3.03 ± 0.21

bcd C

C2S1

3.92 ± 0.55

bcd

4.09 ± 1.09

bc

3.71 ± 0.72

abc

C2S2

4.21 ± 0.74

bcd

3.63 ± 0.73

bc

4.15 ± 0.28

ab

C2S3

4.54 ± 0.33

abc

4.26 ± 0.48

bc

4.11 ± 0.32

ab

C3S1

4.66 ± 0.10

abc

3.44 ± 0.14

c

3.93 ± 0.55

abc B

C3S2

5.46 ± 0.26

a

4.08 ± 0.05

bc

4.91 ± 0.96

a

C3S3

5.11 ± 0.36

ab

4.18 ± 0.51

bc

4.57 ± 0.28

a

8.84 ± 1.80

ab

B

12.70 ± 2.14

a

A

A

B

TF shoot to brown rice C1S1

6.95 ± 0.84

ab

C1S2

6.52 ± 0.39

abc

C1S3

6.35 ± 1.78

abcd B

11.02 ± 0.91 abc A

11.49 ± 3.10

ab

A

C2S1

4.35 ± 0.69

cde

C

6.59 ± 0.63

d

B

9.00 ± 0.85

ab

A

C2S2

4.80 ± 0.52

bcde B

9.95 ± 2.31

bc

A

10.70 ± 2.07

ab

A

C2S3

7.93 ± 0.71

a

B

13.29 ± 1.07 a

A

12.78 ± 2.69

a

A

C3S1

4.30 ± 0.17

de

C

8.38 ± 0.50

cd

A

6.94 ± 0.51

b

B

C3S2

3.99 ± 0.19

e

B

12.09 ± 0.81 ab

A

12.58 ± 1.23

a

A

C3S3

4.31 ± 0.22

cde

C

9.82 ± 1.16

B

11.78 ± 0.70

ab

A

B

8.27 ± 0.38

cd

7.88 ± 0.90

cd

bc

Notes: Values shown are the mean ± SD (n = 3). Different lower-case letters in the same column indicate significant (p < 0.05) difference among treatment groups. Different upper-case letters in the same row indicate significant (p < 0.05) difference among cultivars. See the Materials and Methods section for the abbreviations.

29



Page 30 of 38

Table 2 Changes in selenium (Se) translocation from roots to shoots, and from shoots to brown rice at different levels of cadmium (Cd) and Se exposure Treatment group

F

M

H TF root to shoot

C1S1

4.59 ± 0.69

a

5.38 ± 0.75

4.89 ± 0.55

C2S1

4.49 ± 0.31

a

4.81 ± 1.32

4.97 ± 0.51

C3S1

4.24 ± 0.30

ab

4.95 ± 0.87

4.50 ± 0.43

C1S2

4.09 ± 0.33

ab

4.58 ± 0.65

4.47 ± 0.20

C2S2

4.38 ± 0.17

ab

C3S2

3.27 ± 0.71

ab

4.69 ± 0.81

4.67 ± 1.19

C1S3

3.33 ± 0.60

ab

4.13 ± 0.92

5.19 ± 1.14

C2S3

3.47 ± 0.39

ab

5.58 ± 1.15

4.60 ± 1.03

C3S3

3.07 ± 0.41

b

4.39 ± 0.82

4.04 ± 0.74

B

5.55 ± 0.76

A

4.67 ± 0.20

AB

TF shoot to brown rice C1S1

0.94 ± 0.08

ab

0.95 ± 0.07

ab

0.87 ± 0.05

b

C2S1

0.81 ± 0.12

b

1.10 ± 0.17

a

1.31 ± 0.10

a

C3S1

1.28 ± 0.23

a

1.19 ± 0.13

a

1.27 ± 0.13

a

C1S2

0.67 ± 0.08

b

0.59 ± 0.12

c

0.65 ± 0.10

b

C2S2

0.65 ± 0.13

b

0.62 ± 0.09

c

0.81 ± 0.16

b

C3S2

0.64 ± 0.10

b

0.76 ± 0.04

bc

0.69 ± 0.20

b

C1S3

0.77 ± 0.18

b

0.58 ± 0.04

c

0.54 ± 0.15

b

C2S3

0.59 ± 0.10

b

0.53 ± 0.04

c

0.61 ± 0.11

b

C3S3

0.70 ± 0.05

b

0.58 ± 0.10

c

0.72 ± 0.17

b

B

A

A

Notes: Values shown are the mean ± SD (n = 3). Different lower-case letters in the same column indicate significant (p < 0.05) difference among treatment groups. Different upper-case letters in the same row indicate significant (p < 0.05) difference among cultivars. See the Materials and Methods section for the abbreviations.

30


Page 31 of 38


210

Grain biomass (g, dw)

180

Cultivar F = 451.9* Cultivar × Se F = 1.1ns Se F = 41.5* Cultivar × Cd F = 1.5ns Cd F = 49.0* Se × Cd F = 1.7ns

150

C1S1 C2S1

C1S2 C2S2

C1S3 C2S3

C3S1

C3S2

C3S3 B

120

Cultivar × Se × Cd F = 3.2*

A

A

C

A

B

90

AB A

A A

AB

B

B

A

B

A

B

b a ab

b a a

B

60 b ab a

30

b ab a

b ab a

b b a

0 F

M Cultivar

Figure 1

31


H


A 1.0

C1S2

C1S3

C2S1

C2S2

C2S3

C3S1

C3S2

C3S3

Cultivar F = 362.3** Cultivar × Se F = 7.7** Se F = 95.8** Cultivar × Cd F = 42.9** Cd F = 228.8** Se × Cd F = 34.3** Cultivar × Se × Cd F = 4.5**

0.8

-1

Brown rice Cd concentration (mg kg dw)

0.9

C1S1

Page 32 of 38

0.7 0.6 C 0.5

B C

A B

B

A B

The maxmium level of Cd in brown rice (GB 2762-2017)

A

0.4 0.3

B

A B

0.2

C

A A

B

B B

A B

A

a b b

a b b

A A

A

a b b

a b b

0.1 0.0

b ab a

a b c

a b b

F

M

H

Cultivar

B

Cultivar F = 81.9** Cultivar × Se F = 2.8* Se F = 58.7** Cultivar × Cd F = 3.0* Cd F = 309.2** Se × Cd F = 33.0** Cultivar × Se × Cd F = 2.2*

3.5

-1

Shoot Cd concentration (mg kg dw)

3.0

2.5 C

B C

2.0

A B

B

C

A AB

A

B C

A B

C

B

A B

A

A C

A B

A

B

B

A

b a a

a b b

a b b

1.5

1.0

0.5

a ab b

a b b

a ab b

a b b

0.0 F

M Cultivar

32


H

Page 33 of 38


C

Cultivar F = 103.2** Cultivar × Se F = 3.2* Se F = 14.2** Cultivar × Cd F = 11.7** Cd F = 333.1** Se × Cd F = 21.8** Cultivar × Se × Cd F = 2.3*

15

-1

Root Cd concentration (mg kg dw)

12

9

C

B C

B

A B

B

C

A B

A

B B

B B

6

A B

A C

A

A B

A

B

B

b a a

a b b

A

3

b a a

a ab b

a ab b

b a a

0 F

M Cultivar

Figure 2

33


H

A


A

Root Cd concentration (mg kg dw)

2.5

-1

2.0

1.5 1.0 y = 3.2259x + 0.609 r = 0.877, n = 27, p < 0.01

0.5

B

14

-1

Shoot Cd concentration (mg kg dw)

3.0

Page 34 of 38

12 10 8 6 4

y = 16.148x + 2.0503 r = 0.895, n = 27, p < 0.01

2 0

0.0 0.0

0.2

0.4

0.6

0.0

0.8

0.2

0.4

0.6

0.8 -1

-1



Figure 3

34


Page 35 of 38


6

Cultivar F = 23.1** Se F = 482.0** Cd F = 102.5**

Cultivar × Se F = 2.7* Cultivar × Se × Cd F = 1.8ns Cultivar × Cd F = 5.1** C1S1 C2S1 Se × Cd F = 45.4**

5

C3S1

C1S2

C2S2

C3S2

C1S3

C2S3

C3S3

-1

Brown rice Se concentration (mg kg dw)

A

4

3

C

B B

2

A B

C

C

A B

A

C

B C

B C

B C

A

A B

C

A B

A

ab a b

a a b

A B

A

1

b a b

ab a b

b a b

b a b

a a b

b a c

0 F

M

H

Cultivar

B 3.0

Cultivar F = 27.9** Se F = 283.7** Cd F = 65.1**

Cultivar × Se F = 5.5** Cultivar × Se × Cd F = 0.6ns Cultivar × Cd F = 1.1ns Se × Cd F = 21.2**

-1

Shoot Se concentration (mg kg dw)

2.5

2.0 C 1.5

B C

A B

C

A B

A

C C

B B

A B

B

1.0

A AB

B B

A B

A

B

B

A

c a b

ab a b

ab a b

A

0.5 c a b

ab a b

b a b

b a ab

ab a b

0.0 F

M Cultivar

35


H


C

12

Cultivar F = 3.9* Se F = 417.1** Cd F = 167.3**

Page 36 of 38

Cultivar × Se F = 0.8ns Cultivar × Se × Cd F = 1.6ns Cultivar × Cd F = 0.4ns Se × Cd F = 45.6**

-1

Root Se concentration (mg kg dw)

10

8 B 6

A C

A B

B

C

A B

A

B C

A B

C

C

A B

A

B C

A B

C

4

A B

A

ab a b

b a c

2 b a b

a a b

b a b

b a ab

b a b

b a b

b a c

0 F

M Cultivar

Figure 4

36


H

Page 37 of 38


A

B

12

Root Se concentration (mg kg dw)

2.5

10

-1

-1

Shoot Se concentration (mg kg dw)

3.0

2.0 1.5 1.0

y = 0.5345x + 0.1521 r = 0.984, n = 27, p < 0.01

0.5

8 6 4 y = 2.1066x + 0.8548 r = 0.964, n = 27, p < 0.01 2 0

0.0 0

1

2

3

4

0

5

1

2

3

4 -1

-1



Figure 5

37


5


Effects of Cd-Se interaction on Cd and Se accumulation in rice 254x190mm (96 x 96 DPI)


Page 38 of 38

Effects of Interaction between Cadmium (Cd) and Selenium (Se) on

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