Wheat Cell Number Regulator CNR10 Enhances the Tolerance

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Wheat Cell Number Regulator CNR10 Enhances the Tolerance, Translocation, and Accumulation of Heavy Metals in Plants Kun Qiao, Yanbao Tian, Zhangli Hu, and Tuanyao Chai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04021 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Environmental Science & Technology

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Wheat Cell Number Regulator CNR10 Enhances the Tolerance, Translocation, and

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Accumulation of Heavy Metals in Plants

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Kun Qiao, †,‡ Yanbao Tian, § Zhangli Hu *,† Tuanyao Chai, *, ‡, §, ※, ∏

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†

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, China

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‡

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§ Institute

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

※

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

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

∏The

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

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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Zhangli Hu Ph.D.

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Phone: +86 755 26557244. Fax: +86 755 26557244. E-mail: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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Heavy metal contamination affects crop growth and development, and can indirectly threaten

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human health. Therefore, improving the content of microelements and reducing the accumulation

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of toxic metals by genetic breeding in crops is an effective strategy to solve this environmental

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problem. Previous reports show plant cadmium resistance (PCR) protein can transport zinc (Zn)

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and cadmium (Cd). The cell number regulator (CNR) protein, which functions to regulate organ

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size, has high similarity to, and shares conserved motifs with, PCR. Therefore, CNR may be

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involved in regulating heavy metal translocation. We isolated TuCNR10 from diploid wheat,

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Triticum urartu. Real-time quantitative PCR showed TuCNR10 expression increased in the shoots

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and roots of seedlings under Cd, Zn, and manganese (Mn) stresses. Confocal imaging indicated

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TuCNR10 was localized at the plasma membrane. Overexpression of TuCNR10 in Arabidopsis

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and rice enhanced Cd, Zn, and Mn tolerance, and improved Cd, Zn, and Mn translocation from

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roots to shoots. Compared with wild-type rice, rice overexpressing TuCNR10 had lower Cd, and

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higher Zn and Mn contents in grains. These results indicated that TuCNR10 may be a transporter

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of Cd, Zn, and Mn. TuCNR10 may be a useful genetic resource for microelement fortification and

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reducing toxic metal accumulation in crops.

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INTRODUCTION

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Wheat is an important agricultural product and is one of the three major cereals grown worldwide.

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Up to 2010, wheat was the world's second-largest grain crop (651 million tons) after maize (844

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million tons). It contains large amounts of starch and protein and represents an important energy

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source for human consumption.1, 2 Therefore, scientific studies of wheat are required to generate 2


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good varieties, increase yield and quality, and improve resistance to abiotic stresses and pests.

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Rapid increases in industrialization and human activities have led to reductions in farmland

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area and the contamination of cultivated soil by heavy metals; this represents a serious threat to

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social stability and human security. Heavy metals including zinc (Zn) and manganese (Mn), are

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important trace elements in plant growth and development.3-5 Zn and Mn are both cofactors of

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many enzymes,6 are involved in the regulation of enzyme activity,7 influence the synthesis of

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proteins and carbohydrates, and regulate nitrogen metabolism.8 A lack of Zn and Mn hinders stem

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elongation, causes root apex necrosis, leaf chlorosis, internode shortening, and reduces protein and

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starch synthesis.7 Cadmium (Cd) is a non-essential element in plants and humans.9, 10 After soil

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pollution by Cd, excessive Cd can damage plant roots and inhibit plant growth. Cd is also a strong

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protease inhibitor that can lead to mitochondrial degeneration and mitotic abnormality in living

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organisms, thus inhibiting cell proliferation and cell division.11 Cd also can be enriched in human

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beings resulting in damage to the liver, kidneys, spleen, bones, and reproductive system.

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At present, decreases in the levels of beneficial trace elements and increases in toxic elements

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in soil pose a serious challenge to crop quality and yield and threaten the safety of animals and

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humans. Some researchers have investigated the use of phytoremediation technology to transfer

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and remove heavy metals in plants and soil while others have increased plant utilization rates of

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microelements by cross-breeding. However, these methods are expensive and time-consuming and

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produce only small benefits. Plant transgenic technology is widely used in ecological

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rehabilitation and improving the accumulation of microelements in plants. The acquisition of

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heavy metal transporters is the key to implement the technology. ZRT-IRT LIKE PROTEIN (ZIP)

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is a Zn transporter. In the absence of Zn, expression of ZIP can be up-regulated and can inhibit the 3



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absorption of excessive Zn.12-14 The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE

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PROTEIN (NRAMP) is a high-affinity Mn transporter. It regulates the ion balance of various

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heavy metals in plants.15,

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relieve toxic effects in Arabidopsis thaliana cells.17, 18 PLANT CADMIUM RESISTANCE (PCR)

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protein was involved in the translocation and redistribution of Zn or Cd. Arabidopsis AtPCR1 can

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transport Cd out of the cell, thereby reducing the concentration of Cd in the cell and improving Cd

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tolerance.19,

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However, the available transporter resources are still very limited so the identification of further

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crop-related functional proteins is required.

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HEAVY METAL ATPASE (HMA) can store Cd in vacuoles to

These advances provide us with basic data resources for transgenic breeding.

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Previous research found that the cell number regulator (CNR) protein can regulate cell and

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organ size.21 The protein sequences of CNR and PCR had higher similarity and both belonged to

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the placenta-specific 8-domain (PLAC8) containing family.22-23 We speculated that CNR may

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function in heavy metal transport according to functional analysis of PCR.20, 23-25 To confirm this,

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cell number regulator 10 (TuCNR10) was isolated in this study from Triticum urartu and real-time

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quantitative PCR was used to detect TuCNR10 expression in wheat tissues and following

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induction with heavy metals. The root length and fresh weight of Arabidopsis and rice plants

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overexpressing TuCNR10 were measured in plants treated with Cd, Zn, and Mn. Localization of

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TuCNR10 protein was observed with confocal microscopy. The heavy metal concentrations in

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shoots and roots of overexpressing seedlings and the grains in mature rice were determined by

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inductively coupled plasma atomic emission spectrometry.

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MATERIALS AND METHODS 4


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Wheat Culture, Gene Cloning, and Treatments

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T. urartu (accession G1812) seeds were germinated in distilled water in Petri dishes for 3 d then

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transferred to ½-Hoagland's solution (1/2 HL, pH 6.0) for 6 d.26 The total RNA of seedlings was

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extracted using RNAiso plus reagent (TaKaRa, Japan), and cDNA was synthesized with HifairTM

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II 1st Strand cDNA Synthesis SuperMix (YEASEN, Shanghai, China) according to the

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manufacturer’s instructions. The wheat TuCNR10 (EMS50828.1) gene was retrieved from the

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NCBI database (http://www.ncbi.nlm.nih.gov) and cloned with the specific primers TuCNR10-F

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and TuCNR10-R (Table S1).

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To determine TuCNR10 expression levels in mature wheat, seedlings were vernalized at 4°C

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under an 8:16 h light/dark photoperiod for 50 d and then cultured in agricultural soil for 4 months.

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All tissues of mature wheat including root, internode, node I, leaf sheath, leaf blade, flag leaf

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sheath, flag leaf blade, peduncle, rachis, seed, and beard (awn of wheat) were collected,

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respectively. To analyze the expression level TuCNR10 under heavy metal treatments, the wheat

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seedlings were cultured in 1/2 HL liquid media supplemented with 50 μM CdSO4, 200 μM ZnSO4,

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or 3 mM MnSO4 for 0, 6, 12, 24, and 48 h. The shoots and roots were collected separately and

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ground into powder using liquid nitrogen.

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Quantitative Real-Time PCR (RT-qPCR)

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TuCNR10 gene transcript levels in different tissues of mature plants and those treated with heavy

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metal stress were detected using a Roche LightCycler 480 Real-time System (Roche, Switzerland).

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An actin gene from T. urartu was cloned using a pair of primers (Tuactin-F and Tuactin-R) for

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RT-qPCR. The specific primers TuCNR10-FSSDL and TuCNR10-RSSDL were used to amplify 5



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part of the TuCNR10 sequence. The RT-qPCR was performed in 20 μl reactions containing 10 µl

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HieffTM qPCR SYBR Green Master Mix (YEASEN), 1 μl cDNA, 0.5 μl of each primer (10 μM),

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and 8 μl ddH2O. The TuCNR10 transcript levels were calculated using the 2−ΔΔCT method.27 All

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primers were listed in Table S1.

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TuCNR10 Localization

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To determine TuCNR10 protein localization, the pBI121-TuCNR10-eGFP plasmid was

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constructed. TuCNR10 was amplified using the primers TuCNR10-BF and TuCNR10-KRV. The

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PCR fragment was ligated into the peGFP plasmid at the BamHI and KpnI sites. The

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peGFP-TuCNR10 plasmid was digested with BamHI and EcoRI and ligated into the pYES2

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vector. The pYES2-TuCNR10-eGFP plasmid was then digested using BamHI and XhoI and the

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resulting fragment inserted into the pBI121 vector. The pBI121-TuCNR10-eGFP plasmid was

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transformed into wild-type (WT) Arabidopsis (Col-0) by infection of transgenic Agrobacterium

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tumefaciens strain GV3101. GFP signals in transgenic Arabidopsis roots were detected using a

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laser-scanning confocal imaging system (Leica TCS SP5, Germany).

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Stress Tolerance of Overexpression-TuCNR10 Plants

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To construct the pBI121-TuCNR10 plasmid, TuCNR10 was amplified by PCR using the primers

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TuCNR10-BF and TuCNR10-XR and the resulting fragment was inserted into pBI121 using the

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BamHI and XbaI sites. The plasmid pBI121-TuCNR10 was transformed into A. tumefaciens

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GV3101 strain, which was transformed into WT Arabidopsis using the floral dip method.29 Wild

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type rice (WR, Oryza sativa L. japonica. cv. Nipponbare) plants were also used for transformation. 6


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For construct the pUN1301-TuCNR10 plasmid, the pUN1301 empty vector was digested by

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restriction endonuclease (BamHI), and a pair of special primers (TuCNR10-1301-F and

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TuCNR10-1301-R) was designed to clone the TuCNR10 sequence. Each primer consists of a 21

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bp vector sequence and a 20 (forward)/22 (reverse) bp gene sequence. The ligase in the Hieff

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CloneTM Plus One Step Cloning Kit was used to combine pUN1301 vector with TuCNR10 gene

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fragment (YEASEN). The transgenic rice was generated by Hangzhou Biogle Biotech, Co., Ltd

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(Jiangsu, China).

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To determine the effect of TuCNR10 on stress tolerance, the transgenic Arabidopsis seeds

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were germinated on 1/2 Murashige and Skoog Stock (MS) solid agar plates supplemented with 30

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μM CdSO4, 3 mM MnSO4, and 100 μM ZnSO4, respectively, for 7-14 d; plants were cultivated

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upright at 22±1°C under an 8:16 h light/dark photoperiod. Root length and fresh weight were

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recorded. Additionally, transgenic rice seeds were germinated on 1/2 MS solid agar plates for 2 d

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in the dark at 37°C, then placed in a greenhouse at 25°C under a 16:8 h light/dark photoperiod for

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5 d (control group). The 7 d-old rice seedlings were transferred into Kimura B liquid media (a

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common nutrient solution for rice cultivation)29 supplemented with 10 μM CdSO4, 200 μM

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ZnSO4, and 3 mM MnSO4, respectively, for 7-14 d (treated group). Finally, the control and

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treatment groups of the length and fresh weight of rice seedlings were measured.

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Analysis of Heavy Metal Content

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The TuCNR10-overexpressing Arabidopsis and rice seeds were both germinated on 1/2 MS solid

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media. The Arabidopsis seedlings were placed in 1/2 HL liquid media for 40 d before being

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transferred into media with 30 μM CdSO4, 200 μM ZnSO4, or 3 mM MnSO4 for 2 d. Meanwhile, 7



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the rice seedlings were moved into liquid Kimura B for 7 d then transferred into solution

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supplemented with 10 μM CdSO4, 200 μM ZnSO4, or 3 mM MnSO4 for 7 d. The shoots and roots

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of seedlings were collected separately. For mature rice, 10-day-old rice seedlings were cultured in

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agricultural soil for 3 months, and placed in a greenhouse under a 16 h light (28°C): 8 h dark

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(25°C) photoperiod. At the grain-filling period, the plants were transferred into soil with 5 mg

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CdSO4/kg soil, 600 mg ZnSO4/kg soil, and 600 mg MnSO4/kg soil for 40 d, respectively. Brown

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rice and husk were isolated from the mature rice plants. All samples were cleaned with 10 mM

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EDTA for 30 min, and were dried at 80°C in an oven for 4 d. All samples were digested using a

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microwave digestion instrument (Milestone, Italy) with 8 ml HNO3 and 3 ml H2O2 for 60 min at

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180°C. The metal concentrations were determined by inductively coupled plasma atomic emission

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spectrometry (ICP-OES, Perkin Elmer, USA).

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Statistical Analysis

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All data, including RT-qPCR, root length, fresh weight, and metal content were presented as the

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mean ± standard error from three independent experiments. One-way ANOVA and the t-test were

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for statistical analysis (P

Wheat Cell Number Regulator CNR10 Enhances the Tolerance

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