New Biofortification Tool: Wheat TaCNR5 Enhances Zinc and

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Biotechnology and Biological Transformations

A new biofortification tool: wheat TaCNR5 enhances zinc and manganese tolerance and increases zinc and manganese accumulation in rice grains Kun Qiao, Fanhong Wang, Shuang Liang, Hong Wang, Zhangli Hu, and Tuanyao Chai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04210 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Journal of Agricultural and Food Chemistry

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A new biofortification tool: wheat TaCNR5 enhances zinc and manganese tolerance and

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increases zinc and manganese accumulation in rice grains

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Kun Qiao a, b, Fanhong Wang b, Shuang Liang b, Hong Wang b, Zhangli Hu b,, Tuanyao Chai a, c, d,

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e,*

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a College

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b

of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, China

Shenzhen Key Laboratory of Marine Bioresource & Eco-environmental Science, Guangdong

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Engineering Research Center for Marine Algal Biotechnology, College of Life Science and

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Oceanography, Shenzhen University, Shenzhen, 518060, China

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c

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100101, China d

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Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing,

Southeast Asia Biodiversity Research Institute, Chinese Academy of Science, Yezin, Nay Pyi Taw 05282, 650223, Myanmar

e

The Innovative Academy of Seed Design (INASEED), Chinese Academy of Sciences, Beijing, 100101, China

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*Corresponding Authors

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Tuanyao Chai Ph.D.

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Phone: +86 10 88256343. Fax: +86 10 88256343. E-mail: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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Heavy metal contaminants and nutrient deficiencies in soil negatively affect crop growth and

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human health. The plant cadmium resistance (PCR) protein transports heavy metals. The

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abundance of PCR is correlated with that of cell number regulator (CNR) protein, and the two

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proteins have similar conserved domains. Hence, CNR might also participate in heavy metal

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transport. We isolated and analyzed TaCNR5 from wheat (Triticum aestivum). The expression

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level of TaCNR5 in the shoots of wheat increased under cadmium (Cd), zinc (Zn), or manganese

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(Mn) treatments. Transgenic plants expressing TaCNR5 showed enhanced tolerance to Zn and Mn.

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Overexpression of TaCNR5 in Arabidopsis increased Cd, Zn, and Mn translocation from roots to

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shoots. The concentrations of Zn and Mn in rice grains were increased in transgenic plants

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expressing TaCNR5. These roles of TaCNR5 in the translocation and distribution of heavy metals

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mean that it has potential as a genetic biofortification tool to fortify cereal grains with

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micronutrients.

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KEYWORDS Triticum aestivum; TaCNR5; Zn; Mn; grains

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INTRODUCTION

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Heavy metal microelements, including zinc (Zn), iron (Fe), manganese (Mn), and copper (Cu), are

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essential for plant growth and human development.1 Among the many trace elements, Zn is a

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cofactor for many enzymes and is involved in protein binding, signal transduction, and the

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regulation of enzyme activity, transcription, and transformation.2-3 In plants, Zn deficiency can 2


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result in reduced height, slow growth, and impaired photosynthesis. In humans, a lack of Zn may

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lead to anemia, hepatitis, cancer, and other diseases.4 Similar to Zn, Mn is a cofactor for several

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crucial catalytic reactions in various cellular processes and metabolic pathways, including the

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synthesis of fatty acids and carbohydrate metabolism.5 Manganese deficiency can affect human

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reproduction, intellectual development and cause neurasthenia syndrome.6 Thus, for plants and

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humans, Mn and Zn are necessary microelements and their deficiencies can result in serious health

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problems and economic losses.

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In contrast, cadmium (Cd) is a heavy metal that is highly poisonous to plants.7-8 Excess Cd

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can lead to reduced plant height, yellow leaves, and restricted root growth, which seriously affect

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crop quality and yield.9 In humans, Cd can accumulate in the body through food-chain transfer,

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resulting in kidney, neuralgic, and endocrine disorders.10 Because Cd directly threatens human

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health, an effective strategy to reduce Cd accumulation in crops should be developed.

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Human activities have led to the contamination of the environment and increased toxic heavy

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metal levels in the soil. Meanwhile, deficiencies of necessary trace elements seriously affect crop

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growth and development. Therefore, there is a need to develop new crop varieties that can

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accumulate essential elements and remove toxic heavy metals from soil. Because conventional

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crop breeding is time consuming, laborious, and difficult, transgenic breeding technology is a

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viable alternative.11

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Genes encoding heavy metal transporters are important tools for transgenic breeding.

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Previous studies have shown that HEAVY METAL ATPASE 2 (TaHMA2) from wheat enhances

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Zn translocation in plants,12 and barley ZRT/IRT-LIKE PROTEIN (HvZIP) increases Zn

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concentrations in grains.4 Metal tolerance protein 11 in rice (OsMTP11) enhances Mn tolerance 3



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and reduces Mn accumulation in the shoots and roots under excess Mn conditions.13 Another Mn

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transporter,

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(OsNRAMP3), is expressed at high levels in the stem nodes of rice and participates in Mn

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redistribution.14 In Arabidopsis, PLEIOTROPIC DRUG RESISTANCE 8 (AtPDR8) increases Cd

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tolerance, and reduces Cd toxicity by transporting Cd out of the cell.15 Arabidopsis PLANT

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CADMIUM RESISTANCE PROTEIN 1 (AtPCR1) is another Cd-exporting transporter that

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increases Cd tolerance by transferring Cd, thereby decreasing the Cd concentration in cells.16

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Genes encoding these transporters have been used to breed transgenic plants.

NATURAL

RESISTANCE-ASSOCIATED

MACROPHAGE

PROTEIN

3

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Previous reports have shown that the cell number regulator (CNR) protein regulates cell

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number and fruit size. Maize ZmCNR1, Cherry PavCNR12, and PavCNR20 were shown to be

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involved in the regulation of organ size and cell number.17-18 Interestingly, CNR and PCR proteins

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show high sequence similarity, including the CXXXXXCPC conserved motif, and they both

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belong to a large protein family.19 The PCR proteins mainly participate in the transportation of Cd

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or Zn, and several studies have provided evidence for this role. For example, AtPCR1 decreased

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Cd uptake in yeast and Arabidopsis cells.16 AtPCR2 reduced the Zn concentration in yeast cells

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and enhanced Zn translocation from roots to shoots.20 Brassica juncea BjPCR1 increased Ca

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translocation in yeast and B. juncea.21 Overexpression of ZmCNR1 in maize decreased plant size

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by modulating cell number, while ZmCNR2 was suggested to participate in Cd translocation and

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chelation.17 Triticum urartu TuCNR10 improved Cd, Zn, and Mn translocation, and T. aestivum

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TaCNR2 enhanced Cd/Zn translocation from roots to shoots in Arabidopsis and rice.11, 22 These

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findings and observations suggest that CNRs similar to PCR may function as heavy metal

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transporters. 4


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To test this hypothesis, we isolated and identified the gene encoding CNR5 from wheat,

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Triticum aestivum (TaCNR5). The transcript level of TaCNR5 in wheat organs was detected by

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real-time quantitative PCR. We determined the effect of overexpression of TaCNR5 in planta on

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heavy metal tolerance. Inductively coupled plasma optical emission spectrometry (ICP-OES) was

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used to measure the contents of heavy metals in the transgenic seedlings and in the brown rice and

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husk of grains of mature transgenic rice expressing TaCNR5. These results help us to understand

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the role of TaCNR5 in heavy metal tolerance and translocation, and provide theoretical data for

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transgenic wheat breeding.

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MATERIALS AND METHODS

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Wheat Cultivation and Experimental Design

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The wheat seeds (T. aestivum, cultivar Chinese spring) were germinated in a glass dish with

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distilled H2O for 2 d, and then grown for 5 d under an 8-h light:16-h dark photoperiod at 23 °C

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with ½-Hoagland solution (HS).23 To detect TaCNR5 transcript levels under heavy metal stress,

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7-d-old seedlings were treated with ½HS containing 50 μM CdSO4, 200 μM ZnSO4, or 3 mM

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MnSO4. Samples were collected at 0, 6, 12, 24, and 48 h of these treatments. To measure the

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tissue-specific expression of TaCNR5, 7-d-old seedlings were kept at 4 °C for 10 d, and then

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grown in agricultural soil for 4 months under a 12-h light (25 °C)/12-h dark (22 °C) photoperiod.

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Wheat tissues, including the seeds, peduncle, rachis, flag leaf blade, flag leaf sheath, node I,

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internode, leaf blade, leaf sheath, and roots, were collected separately during the ripening stage.

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Each wheat sample was ground in liquid nitrogen before extracting total RNA. The sequence

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information of all primers used for the PCR reactions were listed in Table S1.

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Gene Cloning and Analysis

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Total RNA was extracted from wheat samples, and cDNA was synthesized using Hifair™ II 1st

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Strand cDNA Synthesis SuperMix for qPCR (YEASEN, Shanghai, China) according to the

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manufacturer’s instructions. The sequence of AtPCR1 was used to search for and retrieve the

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full-length sequence of TaCNR5 (GenBank ID: CDM80498.1) from the NCBI database

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(http://www.ncbi.nlm.nih.gov). Special open reading frame (ORF) primers were designed,

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namely, TaCNR5-Forward and TaCNR5-Reverse. The 20-μL PCR mixture consisted of 1 μL

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cDNA, 1 μL Ex Taq (TaKaRa, Japan), and 0.5 μL primer (10 μM). The ORF sequence and the

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amino acid sequence were analyzed using DNASTAR Lasergene v7.1 software. Sequences of

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TaCNR5, CNR5, and PCR10 proteins from other species were retrieved from the NCBI database

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(http://www.ncbi.nlm.nih.gov) and aligned using ClustalX 8.1 software. Sequence similarity

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between TaCNR5 and other sequences was analyzed using the neighbor-joining (NJ) method.

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Real-Time Quantitative PCR (RT-qPCR)

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Total RNAs were isolated from heavy metal-treated seedlings and different tissues of mature

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wheat using the RNAiso reagent (TaKaRa). The cDNA was synthesized using Hifair™ II 1st

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Strand cDNA Synthesis SuperMix for qPCR. A pair of primers, Taactin-Forward and

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Taactin-Reverse, was used to amplify a fragment of the Actin gene as a control. The RT-qPCR

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mixture for amplification of TaCNR5 contained 0.5 μL each of the two specific primers,

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TaCNR5-ForwardSSDL and TaCNR5-ReverseSSDL; 1 μL cDNA; and 10 μL Hieff™ qPCR

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SYBR Green Master Mix (YEASEN). A Roche LightCycler 480 Real-time System (Roche, Basel,

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Switzerland) was used to detect the transcript level of TaCNR5.

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Preparation of Transgenic Plants Expressing TaCNR5 and Evaluation of Stress Tolerance

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To obtain the plasmid pBI121-TaCNR5, TaCNR5 was amplified using the primers

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TaCNR5-XbForward and TaCNR5-SacReverse and ligated into plasmid pBI121 via the XbaI and

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SacI sites. The recombinant plasmid strain was transferred into Agrobacterium tumefaciens, which

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was then infected into wild-type (WT) Arabidopsis (Col-0).24 TaCNR5 was amplified with the

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specific primers pUN1301-TaCNR5-Forward and pUN1301-TaCNR5-Reverse, and the PCR

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product was inserted into the pUN1301 plasmid using the Clone Express® II One-Step Cloning

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Kit according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd, Nanjing, China).

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Experiments on TaCNR5-transgenic rice (Oryza sativa L. japonica. cv. Nipponbare) were

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conducted at the Hangzhou Biogle Biotech, Co., Ltd. (Hangzhou, Jiangsu, China).

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For heavy metal treatments, WT and TaCNR5-expressing Arabidopsis and seeds of

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transgenic rice (homozygous T3 generation) expressing TaCNR5 were sterilized with ethanol and

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NaClO, and then germinated and grown on ½-Murashige and Skoog (MS) solid medium

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supplemented with 30 μM CdSO4, 200 or 300 μM ZnSO4, or 1 or 3 mM MnSO4 for 7-14 d. The

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plates were kept at 22±1 °C under an 8-h light:16-h dark photoperiod. The rice seeds were

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cultivated on ½-MS solid medium for 3 d in the dark, and then grown with Kimura B solution25

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for 5 d (control group) at 25 °C under an 16-h light: 8-h dark photoperiod. Seedlings with the

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same initial plant length were selected for use in the heavy metal treatments. These seedlings were

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treated with Kimura B liquid medium containing 10 μM CdSO4, 100 μM ZnSO4, or 1 mM MnSO4

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for 7-14 d. The plant lengths and fresh weight of treated and control plants were determined, and

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photographs were taken (Sony DSC-TX20, Japan) to record plant growth.

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Determination of Heavy Metal Concentrations

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Arabidopsis seedlings were grown on ½MS solid medium for 7 d, and then in ½HS liquid medium

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for 40 d. Then, these samples were transferred into ½HS supplemented with 30 μM CdSO4, 100

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μM ZnSO4, or 3 mM MnSO4 and grown for 2 d. The culture conditions were 8-h:16-h light/dark

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at 23±1 °C. Seven-day-old rice seedlings were cultured in Kimura B solution containing 10 μM

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CdSO4, 200 μM ZnSO4, or 3 mM MnSO4 for 7 d under a 16 h-light: 8 h-dark photoperiod at 25 °C.

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Arabidopsis and rice seedlings and organs (shoots and roots) were collected separately.

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To detect the accumulation of heavy metals in the grains of transgenic rice overexpressing

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TaCNR5, 10-day-old rice seedlings were transplanted into agricultural soil which was weighed

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and packed in square plastic baskets. The transgenic rice seedlings and wild-type rice (WS) were

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grown for 4 months (to the heading stage) under a 16-h light (28 °C)/8-h dark (25 °C) photoperiod,

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and then subjected to heavy metal treatments by cultivation in soil containing 5 mg CdSO4/kg soil,

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600 mg ZnSO4 /kg soil, or 600 MnSO4 mg/kg soil for 40 d. The brown rice and husk of rice grains

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were collected separately. The residual metal ions were removed from the plant samples by

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washing in 10 mM EDTA for 30 min, and then the samples were dried at 80 °C for 3 d. All plant

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samples were weighed and then digested with HNO3 and H2O2 using a microwave digestion

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instrument (Milestone, Sorisole, Italy). The Cd, Zn, and Mn concentrations were measured by

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ICP-OES (Perkin Elmer, Waltham, MA, USA).

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Statistical Analyses

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All reported data are the mean ± standard error from three replicates in independent experiments.

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The RT-qPCR data were subjected to analysis of variance (ANOVA) using SPSS 13.0 software 8


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(SPSS Inc. Chicago, IL, USA). The t-test in Microsoft Office 2010 software was used to analyze

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the significance of differences between WT/WS and transgenic lines. Column charts were

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produced using Origin 2017 software.

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RESULTS

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The cDNA ORF sequence of TaCNR5 was 555 bp, encoding a polypeptide of 184 amino acids.

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The putative protein sequence contained the CCXXXXCPC conserved motif (data not shown).

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Previous reports have documented that CNR and PCR belong to a large PLAC8 motif-containing

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protein family, and that PCRs also contain the CCXXXXCPC conserved motif. However, the NJ

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phylogenetic tree showed that TaCNR5 was most closely related to AtCNR5 (Aegilops tauschii)

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and HvCNR5 (Hordeum vulgare), and then clustered with PCR10s of other species (Figure 1).

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Although TaCNR5 may be involved in heavy metal tolerance and translocation, the evolutionary

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relationship analyses showed that the sequence of TaCNR5 had higher similarity to CNR5 than to

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PCR10. Therefore, the protein was designated as TaCNR5.

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Expression of TaCNR5 in Different Tissues

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The transcript levels of TaCNR5 in different tissues of mature wheat (seeds, peduncle, rachis, flag

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leaf blade, flag leaf sheath, node I, internode, leaf blade, leaf sheath, and roots) were determined

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by RT-qPCR analyses. The highest transcript level of TaCNR5 was at the internode, where its

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transcript level was 63-fold higher than that in the seeds (Figure 2).

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Expression of TaCNR5 Induced by Heavy Metal Treatments 9



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The transcript levels of TaCNR5 were detected in wheat seedlings under Cd, Zn, and Mn stress

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treatments. The TaCNR5 transcript levels in the shoots peaked at 6 h of 50 μM CdSO4 stress

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(p

New Biofortification Tool: Wheat TaCNR5 Enhances Zinc and

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