Identification of novel rice (Oryza sativa) HPP and HIPP genes tolerant to heavy metal toxicity

Ecotoxicology and Environmental Safety 175 (2019) 8–18

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Identification of novel rice (Oryza sativa) HPP and HIPP genes tolerant to heavy metal toxicity

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Irfan ullah khan, Justice Kipkoir Rono, Bai Qing Zhang, Xue Song Liu, Meng Qi Wang, Lei Lei Wang, Xue Chun Wu, Xi Chen, Hong Wei Cao, Zhi Min Yang∗ Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China

A R T I C LE I N FO

A B S T R A C T

Keywords: HPP/HIPP proteins Isoprenylation Detoxification Heavy metal stress Rice

HPP (heavy metal associated plant protein) and HIPP (heavy metal associated isoprenylated plant protein) are a group of metal-binding metallochaperones playing crucial roles in metal homeostasis and detoxification. Up to now, only few of them have been functionally identified in plants. Here, we identified 54 HPP and HIPP genes in rice genome. Analysis of the transcriptome datasets of the rice genome exposed to cadmium (Cd) revealed 17 HPP/HIPP genes differentially expressed, with 11 being upregulated (> 2 fold change, p < 0.05). Comprehensive analysis of transcripts by qRT-PCR showed that both types of genes displayed diverse expression pattern in rice under excess manganese (Mn), copper (Cu) and Cd stress. Multiple genomic analyses of HPPs/ HIPPs including phylogenesis, conserved domains and motifs, genomic arrangement and genomic and tandem duplication were performed. To identify the role of the genes, OsHIPP16, OsHIPP34 and OsHIPP60 were randomly selected to express in yeast (Saccharomyces cerevisiae) mutants pmrl, cup2, ycf1 and zrc1, exhibiting sensitivity to Mn, Cu, Cd and Zn toxicity, respectively. Complementation test showed that the transformed cells accumulated more metals in the cells, but their growth status was improved. To confirm the functional role, two mutant oshipp42 lines defective in OsHIPP42 expression were identified under metal stress. Under normal condition, no difference of growth between the oshipp42 mutant and wild-type plants was observed. Upon excess Cu, Zn, Cd and Mn, the oshipp42 lines grew weaker than the wild-type. Our work provided a novel source of heavy metal-binding genes in rice that can be potentially used to develop engineered plants for phytoremediation in heavy metal-contaminated soils.

1. Introduction Homeostasis of essential heavy metals such manganese (Mn) and copper (Cu) is critical for plant growth, development and against various environmental stresses (Marschner, 1995). While concentrations of the metals need to be under tight control, some other non-essential metals such as cadmium (Cd) and mercury (Hg) must be reduced to a minimal level because of their high toxicity to animals and humans through food chains (Di Toppi and Gabbrielli, 1999; Chen and Yang, 2012). Toxic heavy metals are transferred to human beings and accumulate in vital organs, affecting public health, such as impaired nutrition balance and disorder of immune system (Yousefi et al., 2018; Midhat et al., 2019). Mediation of metal ion levels in plants relies on the homeostatic mechanisms (Hall, 2002). One of them concerns the lowmolecule weight (LMW) compounds which bind to metal ions in cytoplasm to make them detoxified (Freisinger, 2011). A good example of the LMW compounds is the small cysteine-rich peptides called

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phytochelatins (PCs) with general structures of (γ-Glu-Cys)n-Gly (n = 2–7)(Cobbett and Goldsbrough, 2002). PCs are substantially generated upon excess metal influx to cells (Clemens, 2006). Meanwhile, excess metals in the cells can be detoxified by a group of metalloproteins or metallochaperone-like proteins (Robinson and Winge, 2010). Metallochaperones are a large protein family that can bind metal ions and make them transfer to intracellular compartments through membrane-located metal transporters (Huffman and Halloran, 2001; Tehseen et al., 2010). Because metallochaperones are also enriched with cysteine residues, they function as metal-detoxified mediators (Suzuki et al., 2002; Barth et al., 2009; Freisinger, 2011). The typical metallochaperone proteins contain a heavy metal-binding domain (HMA, pfam00403.6) with a highly conserved CysXXCys (X: any amino acid) motif comprising a Beta-Alfa-Beta-Alfa fold shape for binding heavy metals such as Cd, Cu or Zn ions (Hung et al., 1998; Zhang et al., 2018e). According to the structure, metallochaperones can be classified into two subfamilies: one contains only 1–2 HMA domain(s) and is

Corresponding author. E-mail addresses: [email protected], [email protected] (Z.M. Yang).

https://doi.org/10.1016/j.ecoenv.2019.03.040 Received 22 December 2018; Received in revised form 6 March 2019; Accepted 10 March 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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2 d. The young plants were then transferred to the half-strength Kimura B solution with pH maintained at 5.6–5.8 (Sasaki et al., 2014) and exposed to Mn, Zn, Cu and Cd at different concentrations (depending on the experiments). The growth and treatment solution was renewed every other day. All equipments were sterilized prior to use. After treatment, plant tissues were separately harvested and immediately stored in liquid nitrogen for following analysis.

known as heavy metal-associated plant proteins (HPPs) and the other subfamily members contain an additional element of a C-terminal isoprenylation motif, known as the heavy metal-associated isoprenylated plant proteins (HIPPs)(DeAbreu-Neto et al., 2013). Isoprenylation is a post-translational protein-modified process involved in the formation of a covalent thioether bond between cysteine and farnesyl- or geranylgeranyl-residues of proteins, which actually is a protein–protein or protein–membrane interaction (Crowell, 2000; Novelli and D'Apice, 2012). Although the HMA domain and isoprenylation are commonly occurring in many organisms from bacteria to humans, the presence of both interacted in the same protein was observed only in plants (Barth et al., 2009). Up to now, several metallochaperones have been functionally identified in Arabidopsis and rice (Andres-Colas et al., 2006; Banci et al., 2008; Zhang et al., 2018e). In Saccharomyces cerevisiae, the protein Ccc2a has been identified as a Cu transporter delivering Cu from the Cu chaperone to the Golgi network (Lin et al., 1997). Its homologs ATXl in Arabidopsis and rice are a small metal-binding protein that protects cells against reactive oxygen toxicity caused by excess or abnormal distribution of Cu ions (Huffman and Halloran, 2001; Banci et al., 2008; Zhang et al., 2018e). Therefore, ATX1 is proposed to involve the intracellular homeostasis and detoxification of Cu in roots (Andres-Colas et al., 2006; Puig et al., 2007). Ectopic expression of Arabidopsis HIPP20, HIPP22, HIPP26 and HIPP27 in yeast mutant ycf1 enhanced Cd tolerance in the cells, whereas the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type (Tehseen et al., 2010). AtCdi19 is a metal binding protein and is induced by Cd, Hg, Fe and Cu stress; AtCdi19 overexpression in Arabidopsis conferred plant tolerance to Cd (Suzuki et al., 2002). TaHIPP1 is a component playing multi-roles in defense responses; TaHIPP1 overexpression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under high Cu salt stresses (Zhang et al., 2015). Despite the fact that several HPP and HIPP genes have been functionally identified for metal detoxification in plants (Tehseen et al., 2010; DeAbreu-Neto et al., 2013), research on HPP and HIPP genes is still limited. Rice (Oryza sativa L.) is an excellent model plant species for environmental research owning to its abundant germplasm resources. Many genotypes and cultivars of rice are gifted with special traits contributing to accumulating heavy metals in metal-contaminated soils (Lu et al., 2013; Oono et al., 2014; Feng et al., 2016; Zhang et al., 2017; Mitra et al., 2018; Zhu et al., 2018). To date, dozens of metallochaperone protein genes in rice have been reported (Xu et al., 2013; Zhang et al., 2018a), but only a few of them have been functionally characterized. In this study we performed a genome-wide identification of rice HPP/HIPP genes encoding metallochaperone proteins with a HMA domain in response to excessive heavy metals such as Cd, Mn and Zn. Some of them were subjected to yeast complementary test, showing detoxifying capability. Two OsHIPP42 mutant lines were initially identified and displayed sensitive phenotype of metal exposure. Thus, the purpose of the study was to mine metallochaperone genes responding to excess heavy metals and to provide candidates for further functional characterization for potential application for phytoremediation.

2.2. Analysis of sequence and phylogenesis The amino acid sequences of the proteins containing heavy metalassociated (HMA) (pfam 00403.19) domain and the BLAST Servers at NCBI were used to identify HPP/HIPP proteins from rice (http://rice. plantbiology.msu.edu) and Arabidopsis (http://www.arabidopsis.org) genomes. Phylogenetic trees were constructed by comparing the identified amino acid sequences using MEGA6.0 and the Neighbor-Joining (NJ) method (Number of bootstrap replications were 1000) (Zhang et al., 2018b). HMM (Hmmer 3.0 software) was applied to profile the heavy metal-associated domain pfam from the protein family database (http://pfam.xfam.org/family/). The sequence was used as a standard to identify all possible homologs in Oryza sativa through protein blast search (p < 0.001). The protein family and simple modular architecture tools (http://smart.embl-heidelberg.de/) were used to determine every possible protein as a part of the HMA domain gene family (Zhang et al., 2018d). 2.3. Determination of metals Fresh plant tissues and yeast cells were sampled and dried at 70 °C in an air-forced oven for 60 h. The dried samples were digested with nitric acid and hydrogen peroxide (HNO3: H2O2 = 3: 1, v/v). The metal concentrations in the samples were quantified using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Waltham MA, USA) (Zhang et al., 2018c). 2.4. Conserved motifs analysis and chromosome localization The conserved motif divergence of rice HPP/HIPP proteins was assessed by the way of MEME (http://meme-suite.org/tools/meme) (Bailey et al., 2009; Xuan et al., 2016). Analysis of the gene structure was carried out and displayed by comparing coding sequences and their corresponding genomic sequences through the Gene Structure Display Server 2 (GSDS2.0, http://gsds.cbi.pku.edu.cn) (Hu et al., 2015). For chromosomal indication, we retrieved the accession number of rice HPP/HIPP proteins from rice genome database (http://rice. plantbiology.msu.edu) and matched it manually on the corresponding chromosomes. Gene records were obtained from the Plaza (http:// bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_monocots/). The genomic indication data were isolated from database (http://rice. plantbiology.msu.edu). The protein 3D models were built up through the Phyre 2 server in depth mode and visualized via Pymol (Meng et al., 2017). 2.5. Heterologous expression of HPP/HIPP protein genes in yeast

2. Materials and methods Wild-type strain BY4741 and its mutant strains ycf1, cup2 and zrc1, which lack the ability to transport and detoxify Cd, Mn, Cu and Zn (Meng et al., 2017), were used for ectopic expression of selected genes. The open reading frames of the genes were amplified and inserted into the Nco1 and Sac1 sites of the yeast expression vector pYES2. The empty and gene-containing vectors were transformed into the corresponding mutants. The transformed cells were grown in yeast liquid medium containing 1.9% galactose and 0.65% YNB (sigma yeast nitrogen without amino acid). The suspension was shaken overnight. The dilution (1:10) culture was spotted into the agar medium with 75 μM Cd, 4 mM Mn, 60 μM Cu or8 mM Zn. The mutant lines converted with

2.1. Plant culture and treatment Rice (Oryza sativa L. Japonica, c.v. Nipponbare) was used in this study. The T-DNA insertion mutants OsHIPP42 (HY background) were ordered from Kyung Hee University, Korea. Seeds of rice were surfacedisinfected with 5% H2O2 solution, thoroughly washed and germinated in an incubator at 27 °C for two days under darkness. The uniform germinating seeds were sowed on a net floating in the 0.5 mM CaCl2 solution and grown under the condition of a 14/10 light/dark cycle at 28/25 ± 1 °C (day/night) and 200 μmol m−2 s−1 light intensity for 9

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may have diverse biochemical features and biological functions.

distinctive vectors were pre-cultured in a yeast liquid medium and diluted to OD600 of 0.79–1.00. The 250 μL cells were cultured onto the solid plates with the metals at the different concentrations. Cells on the plates were incubated at 27 °C for 3 d.

4.2. Chromosomal localization

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). The extracts were pre-treated with DNase I (Transgen, China). The integrity of the RNA was checked with one percent agarose gel stained by gold view and estimated for protein contamination (A260 nm/A280 nm ratios) and reagent contamination (A260 nm/A230 nm ratios) by a Nano drop ND 1000 spectrophotometer. The cDNA was synthesized by quantitative RT-PCR (qRT-PCR) with Easy Script First-Strand cDNA Synthesis Super Mix (Transgene, China) using Oligo dT Primer (mRNAs) and specific primers (Supplementary Data S1). The rice Actin gene was used as an internal control. Reactions were pre-incubated at 94 °C for 30 min, followed by 40 cycles of denaturation at 94 °C for 5 s, annealing at 60 °C for 1 min using iTaqTM Universal SYBR Green Super mix (BIO-RAD USA) in the 7500 Real-Time PCR System (Applied Bio systems). The PCR specificity was checked out by melting curve analysis and the data were analyzed (Shen et al., 2011).

The spatial arrangement of HPPs and HIPPs genes within the Oryza sativa genome was investigated by mapping the loci to the virtual chromosomes using the Chromosome Map Tool (viewer.shigen.info/ oryzavw/map tool/Map Tool). By analyzing localization of the genes on the rice chromosomes, we found that chromosome number three (Chr. 3) had the largest number of genes with ten, followed by Chr. 4 with nine (Fig. 2). Eight genes were present in Chr. 1, seven genes in Chr. 2, five genes in Chr. 10, four genes in Chr. 8, and three genes were present in Chr. 7 (Fig. 2). Each of Chr. 5, Chr. 9 and Chr. 12 had two genes, while the remaining two chromosomes, Chr. 6 and Chr. 11 had a single gene. A tandem HPP and HIPP genes OsHIPP36/42 were observed in Chr. 3. Additionally, several other tandems including OsHIPP59/ATX1 and OsHIPP46/HPP07 in Chr. 8 and OsHIPP37/35 and OsHIPP25/40 in Chr. 10 were detected. These results suggested that segmental duplication events have played a significant role in the expansion of HPP and HIPP genes in the rice genome. Finally, examination of all HPP and HIPP genes on the genome revealed that they have diverse gene structures concerning the number of their introns and exons (Data S3).

3. Statistical analysis

4.3. HPP and HIPP genes differentially responded to metal stress in rice

All results are represented as mean ± standard deviation of at least three independent replications. Each treatment contained 10–15 plants. The significant difference between treatments was statistically tested by one way analysis of variance. The normality and homogeneity of variance were examined. The corresponding results met the statistical significance requirement with the least significant difference (LSD, method, at p < 0.05). Data were analyzed using statistical software package SPSS 22.0.

Using our previous transcriptome datasets of rice exposed to Cd (80 μM) (Feng et al., 2016), we identified 17 differentially expressed genes (> 2 fold change, p < 0.05) encoding HPP and HIPP proteins. Of these, 11 genes were upregulated, 5 were down-regulated and one gene OsHPP03 remained unchanged under Cd stress (Fig. 3). To show whether these genes respond to other excessive heavy metals, 4 OsHPPs and 5 OsHIPPs were randomly selected for qRT-PCR analysis. Two week-old young rice plants were exposed to Mn (0, 100, 250, 500 μM), Cd (0, 1, 80, 160 μM) and Cu (0, 10, 30, 60 μM) for 4 h. Expression of OsHPP01 in shoots and roots was upregulated under 100 and 250 μM Mn condition, and further increasing Mn concentration up to 500 μM led to the decline of expression (Fig. 4A). Treatments with Cd and Cu generally induced upregulation of OsHPP01 in shoots and roots although under the high levels the transcripts slightly decreased (Fig. 4B and C). A similar inducible gene expression was detected for OsHPP05 under Mn, Cu and Cd (Fig. 4D–F). For example, expression of OsHPP05 in roots and shoots was upregulated at the high level of Cd, with more than 5 fold transcript abundance being detected under 160 μM Cd (Fig. 4E). OsHPP06 expression in shoots decreased with the concentrations of Mn, while its transcripts in roots increased under the same Mn stress condition (Fig. 4G). Like OsHPP05, the OsHPP06 expression with Cd and Cu was induced in roots and shoots (Fig. 4H and I). Compared to the control, expression of OsHIPP07 in shoots constantly decreased under Mn stress (Fig. 4J), while it was upregulated under Mn, Cd and Cu in roots (Fig. 4J-L). We further assessed the transcripts of OsHIPP. Expression of OsHIPP16 in shoots and roots was generally upregulated by Cd, Mn and Cu, except some points where no differences between the treatments and control were detected (Fig. 5A–C). The OsHIPP28 expression level after Mn treatment substantially increased in shoots but decreased in roots compared to the control (Fig. 5D). Similarly, OsHIPP28 under Cd stress had significantly higher expression level in shoots but lower in roots (Fig. 5E). Under Cu exposure, the expression level in shoots was significantly higher at 10 and 30 μM of Cu, whereas the expression in roots was always lower (Fig. 5F). OsHIPP34 expression in roots was significantly induced under Mn, Cd and Cu stress (Fig. 5G–I). However, while the shoot expression of OsHIPP34 decreased under Mn stress, it increased under Cd and Cu stress. Expression of OsATX1 in roots under 100 and 250 μM Mn stress was significantly higher than the control, but treatment with 500 μM led to severe inhibition of OsATX1 expression (Fig. 5J). A similar expression

2.6. cDNA synthesis and quantitative RT-PCR analysis

4. Results 4.1. Identification of HPP and HIPP genes from rice genome The genomic database search on Oryza sativa (http://rice. plantbiology.msu.edu) was conducted to identify putative HPP and HIPP sequences. We also retrieved the sequences of the orthologs from Arabidopsis thaliana (http://www.arabidopsis.org). By identifying sequences of HPP and HIPP amino acids, a total of 54 rice proteins were identified (Data S2). Phylogenetic analyses showed that all rice HPP/ HIPP proteins could be clustered into five distinct groups (Fig. 1A). Group I consisted of OsHPP1, 5–7, OsHIPP38-48, 60, OsATX1 and OsCCH. Goup II contained OsHIPP28-37. Group III comprised OsHIPP50-59. Group IV consisted of OsHIPP16, 21–22, 24–27, 49 and OsHPP2-4, and the last one was made up of OsHIPP5-6, 8–9 and OsCCS. The 54 rice HPP/HIPP proteins showed 28–90% identity to each other. We further analyzed the relationship of HPP/HIPP proteins between rice and Arabidopsis. All HPP/HIPP proteins from both rice and Arabidopsis were also divided into five groups (Fig. 1B), suggesting that the proteins are conserved in both plant species. Based on the number of the HMA domains, the 54 rice HPP/HIPP proteins were classified into 4 subfamilies (Fig. 1C). The first had 39 proteins with only one type of HMA domain whereby most of HIPP fell into this group. The second group consisted of 9 proteins with 2 types of HMA domains, while the third group had 5 with SCOP (structural classification of proteins) domain. The last group had a single protein containing HMA-Sod Cu (heavy metal associated copper chaperone) domain. The conserved motifs were analyzed using the MEME database (http://meme.nbcr.net/meme/). This would help understand potential functions of each HPP and HIPP. The number of motifs in the HPP and HIPP proteins varied significantly (Fig. 1D). While HIPP24 contained only 2 types, most of them contained 3–6 motifs, implying that they 10

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Fig. 1. Phylogenetic trees of HPP and HIPP proteins constructed from sequences of rice and Arabidopsis, and identification of the domains and motifs of HPP and HIPP proteins. A: rice only and B: rice and Arabidopsis. The tree was prepared based on the HMA domains using the Clustal X algorithm. Confidence of groupings was estimated using 1000 bootstrap replicates. Numbers next to the branching point indicate the percentage of replicates supporting each branch. Red dots show the Arabidopsis HIPP and HPP gene and the pink dot show the rice HIPP and HPP genes. C: The domains names were given according to Pfam. D: the conserved motifs analysis of rice HPP and HIPP proteins, which was performed by the MEME program online. Different colors of boxes represent different motifs in the corresponding position of each gene. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 2. Chromosomal mapping of HPP and HIPP genes in rice. The chromosome number is indicated at the top of each chromosome. The genes occurring in tandem pairs and tandem array are captioned with red and blue color, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

in the OsHIPP60-transformed cells increased compared to the control (Fig. 6D). Examining the response of the OsHIPP60-transformed cells to excess Mn, Cu and Cd revealed no difference of the growth compared to the control cells (Data S4, S5). When subjected to Cd and Zn stress, the cellular population of OsHIPP16 transformants grew better than the control cells (Fig. 6E, G). The Cd and Zn concentrations in the OsHIPP16 transformed cells increased compared to the wild-type (Fig. 6F, H), suggesting that expression of OsHIPP16 in yeast cells enhanced its ability to detoxify Cd and Zn. Expression of OsHIPP16 in yeast did not affect the cell response to Mn and Cu stress (Data S4, S5). The structures of the three representative HIPP proteins with 3D models were predicted. All proteins display topological and conformational variations depending on their family type. OsHIPP34 proteins with two types of HMA domains had 4 α-helices and 6 β-sheets. It displayed a varied number of β-turns without the regular coil (Fig. 6I). While OsHIPP60 had 2 α-helices together with three β-sheets, OsHIPP16 showed the tertiary structure consisting of 4 α-helices and 8 β-sheets (Fig. 6J and K).

pattern for OsATX1 in roots exposed to Cd was detected (Fig. 5K). Different from roots, the rice shoots had a decrease in OsATX1 transcripts under Mn and Cd stress. Expression of OsATX1 in roots increased with the concentration of Cu; however, treatment with 10, 30 and 60 μM Cu did not affect the expression of OsATX1; only under 60 μM Cu, the shoot expression level decreased (Fig. 5L). The shoot OsHIPP60 appeared sensitive to Mn, Cd and Cu because its expression was progressively declining with the metal concentration (Fig. 5M–O). In roots, OsHIPP60 had a similar expression showing an up and down pattern under Mn and Cd stress. The induced transcripts of OsHIPP60 were found in roots only exposed to 60 μM Cu (Fig. 5O). Overall, most of the tested HIP and HIPP genes could be differentially expressed in roots and shoots under the different metal stresses. 4.4. Expression of OsHIPP genes in yeast enhanced cell tolerance to metal stress To get an insight into the role of OsHIPPs in detoxification, three genes OsHIPP34, OsHIPP60 and OsHIPP16 were selected for functional test in yeast (Saccharomyces cerevisiae) cells. The entire open readingframes of the genes were cloned into the vector pYES2 and transformed into the corresponding mutants pmr1, ycf1, cup2 and zrc1 defective in Mn, Cd, Cu and Zn tolerance, respectively (Meng et al., 2017). The transformed cells were grown in the Yeast Nitrogen Base solid medium containing 75 μM Cd, 60 μM Cu, 8 mM Zn or 4 mM Mn. Under the normal Cu supply condition, there was no difference of cell growth between the OsHIPP34-expressing cells and wild-type (Fig. 6A). When 60 μM Cu was added to the growth medium, a better growth was shown for the transgenic cells which had a higher level of Cu over the control (Fig. 6B). We did not see any growth change in the transformed cells in response to Mn, Zn and Cd (Data S4, S5). Like OsHIPP34, the growth of the OsHIPP60-transformed yeast was similar to that of the empty vector cells; when subjected to Zn, the OsHIPP60-expressing cells grew better than the control cells (Fig. 6C). We also found that the Zn concentration

4.5. Mutation of OsHIPP42 led to weak growth under excess metal stress To validate the role of HIPPs in regulating plant response to the metal stress, two OsHIPP42 mutant lines were ordered. Two week-old young rice plants of wild-type and the mutant lines were exposed to 25 μM Cu, 8 μM Mn, 1 μM Cd and 100 μM Zn, respectively. Our analysis showed that under normal metal supply conditions, no difference of growth response between the oshipp42 lines and wild-type was observed (Fig. 7A). When the plants were subjected to 25 μM Cu, both shoot and root elongation were negatively affected (Fig. 7B and C). The shoot and root lengths of the mutant lines were only 57.1–64.2% and 68.4–73.6% of the wild-type, respectively (Fig. 7D and E). The biomass of the oshipp42 shoot was also significantly reduced, with 62.5–83.3% of the wild-type (Fig. 7F). However, one of the mutant root lines in biomass was slightly but not significantly lowered compared to the 12

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promoting root-to-shoot Cu translocation and distribution to the development tissues by interacting with the heavy metal transporters (Zhang et al., 2018e). The HPP and HIPP proteins can be phylogenetically divided into five distinct clades which are further divided into four subfamilies based on the domains (Tehseen et al., 2010). The presence of HIPPs in all clades suggests the occurrence of the allele duplication and diversification events (Zhang et al., 2018a). Gene duplication allows an organism to expand its genome in the course of evolution, thus in favor of adaptation to various environmental stresses (Lynch and Conery, 2000; Zhang et al., 2018b). Our analysis showed that most of the HPP and HIPP genes are located in chromosome number 1, 2, 3 and 4. The tandem genes were observed in chromosomes 2, 3, 4, 8 and 10. The presence of either HPP or HIPP gene or both in all 12 chromosomes indicated that they are dispersed throughout the rice genome. The segmental duplication events suggested the HPP and HIPP expansion in the rice genome. Gene duplication activities with tandem and segmental duplication play a critical role in genomic realignment and expansion (Lynch and Conery, 2000). Tehseen et al. (2010) concluded that the rice genome undergoes a minimum three rounds of genomeextensive duplication and experienced more than one segmental and tandem duplications and transposition activities, which have the retro role and replicative transposition. The rice HPP and HIPP genes have distinctive numbers of exons that perform primary and diverse functions following a long period of evolution of the gene family (Xu et al., 2012). The current study showed that the genomic structures of HPP and HIPP genes varied in terms of the size and number of introns and exons and would be classified into five major groups, which is consistent with the previous report (Tehseen et al., 2010). It has been suggested that increase or decrease in the exon/introns number is attributed to the combination and realignment of gene fragments (Xu et al., 2012). The exon/intron diversification of gene family members suggests critical roles in evolution of multiple gene families when challenged by environmental stress (Laloum et al., 2018). This study showed that a set of HPP/HIPP genes transcriptionally responded to heavy metals Cd, Mn, and Cu stress. Their expression varied in roots and shoots. Some were upregulated in both roots and shoots, whereas others displayed upregulation in either root or shoot. In particular, the OsHIPP16, 28, 34 and 60 and OsATXI were highly expressed in either roots or shoots, implying that these HIPPs would be involved in response to heavy metal stress. However, expression of HIPPs was dependent on the concentration of the metals supplied. For instance, OsHIPP28 was upregulated in shoots but downregulated in roots when exposed to Mn. This is not surprising because most of HMA domain-containing proteins or transporters such as OsHMA family members and others in rice were also reported to be differentially expressed (Feng et al., 2016; Hung et al., 1998). Ectopic expression of genes in yeast is a fast and effective way widely used for identifying basic function of genes under metal stress (Meng et al., 2017). Our yeast complemental analysis showed that OsHIPP34, OsHIPP60 and OsHIPP16 were involved in heavy metal tolerance. The high growth rate of the transformed yeast under Zn, Cu or Cd in contrast with the controls indicated that these proteins were able to confer heavy metal tolerance in the cells. Furthermore, most of the transformed cells had a high concentration of the respective metals, suggesting that the yeast cells expressing the genes could detoxify the metals. These results were in agreement with the previous research that expression of HIPP genes can enhance the tolerance to heavy metal stress (Tehseen et al., 2010). For example, in Arabidopsis AtHIPP06 transcripts were enhanced by heavy metals such as Cd, Hg, Fe and Cu and AtHIPP26 transcription could be also promoted by Cd and Zn (Barth et al., 2009; DeAbreu-Neto et al., 2013). Overexpression of AtHIPP06 and AtHIPP26 conferred Cd tolerance to transgenic plants, whereas the triple knockout mutant AtHIPP20/21/22 gained greater sensitivity to Cd than the wild-type of Arabidopsis (Tehseen et al., 2010). To verify the role of rice HIPPs in tolerance to metal stress, we

Fig. 3. Genome-wild identification of HPP and HIPP transcripts in rice exposed to Cd using one of our previous transcriptome datasets. A: phylogenetic tree constructed for the differentially expressed HPP and HIPP transcripts from Cdexposed rice. B: hierarchical clustering display HPP and HIPP genes differentially expressed and determined by mRNA-seq for the Cd exposed (+Cd) rice relative to the control (-Cd). The gene normalized signal intensities are shown in the heat maps and the data are represented as the ratio of log2 [Fragments Per kb per Million reads (FPKM) values (T + Cd/T-Cd)]. The differentially expressed HPP and HIPP genes are framed by red boxes. Arrows with up and down directions indicate the upregulation and down regulation of the genes (≥2 fold changes, p < 0.05), respectively. Star means no different change. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

wild-type (Fig. 7G). We further determined the response of oshipp42 lines to Zn stress. In general, the oshipp42 lines grew weaker, manifested by reduced shoot and root elongation and biomass, but in some cases, there was no significant difference of root biomass between the oshipp42 lines and wild-type (Fig. 7H-M). Identification of the growth response to Mn and Cd stress revealed a similar consequence (Data S6). These results indicated that OsHIPP42 is required for rice tolerance to the metal stress. 5. Discussion HPPs and HIPPs are a group of special metallochaperones which can bind, accumulate, tolerate or detoxify heavy metals in plants (DeAbreuNeto et al., 2013). To date, only a few of plant HPPs and HIPPs family members have been functionally described. Therefore, it is imperative to characterize the HPP and HIPP genes in plants. The present study identified 54 rice protein genes by exhaustive genome-wide research on publicly available databases. Of these, 44 and 10 genes encode HIPPs and HPPs, respectively. All these proteins have been strictly identified to have at least one HMA domain, indicating that these HPPs/HIPPs presented here were reliable. The identified HPP/HIPP proteins are comparable to the number (40) from Arabidopsis. The high number of HIPP genes identified in rice is implicated in the functional importance and diversification of the genes in the plant. Isoprenylation is a characteristic post-translational modification of many regulatory proteins, which attaches a hydrophobic side chain to the protein, enabling specific interaction with either endomembrane system or other transport proteins (Crowell, 2000; DeAbreu-Neto et al., 2013). For example, the rice chaperone OsATX1 has been reported to play an important role in 13

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Fig. 4. Expression of OsHPP01, OsHPP05, OsHPP06 and OsHPP07 genes in rice exposed to excess Mn, Cd and Cu by qRT-PCR. Two week-old rice plants were exposed to Mn (0, 100, 250, 500 μM), Cd (0, 1, 80, 160 μM) and Cu (0, 10, 30, 60 μM) for 4 h. CK: control. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate the mean values that are significantly different between the treatment and control (p < 0.05).

tonoplast for sequestration in vacuoles (Clemens, 2006). Another role for HPP/HIPP proteins is to coordinate some types of metal transporters such as HMAs for binding and transporting heavy metals in plant cells (Zhang et al., 2015). In this type of situation, the HIPP proteins act as metal transporter chaperone to facilitate intracellular metal trafficking and help maintain homeostasis in plants. For example, Zhang et al. (2018e) recently have demonstrated that OsATX1 is a Cu chaperone with a major role in mediating the intracellular Cu trafficking to the P1B-ATPases HMA4, HMA5, HMA6, and HMA9 for Cu homeostasis in different rice tissues. These results indicate that OsATX1 is able to facilitate the root-to-shoot Cu translocation and the redistribution of Cu from old leaves to developing tissues and seeds in rice. In Arabidopsis, AtATX1 has been reported to be an essential efflux transporter that lowers Cd in the cytoplasm. AtHIPP26 can bind to Cu, Pb and Cd; overexpression of AtHIPP26 led to increased tolerance Cd stress (Barth et al., 2009).

functionally identified OsHIPP42 mutant lines. Our data showed that disruption of the gene resulted in sensitivity to excess Cu and Zn. In addition, several studies showed that some HIPP genes play a positive role in response to abiotic stress in plants such as drought, osmotic, temperature and heavy metal stress (Barth et al., 2009). Furthermore, HIPP3 was showed to be bind to Zn as an upstream regulator of the salicylic acid involved in the abiotic stress response (Zschiesche et al., 2015). Since Cd has a similar chemical property with divalent cations such as Cu and Zn, it can bind to proteins by metal replacement and interfere with the function of essential metals in plants (DeAbreu-Neto et al., 2013). Some HPP/HIPP proteins can chelate the free Cd ion to prevent its interaction with other proteins in plants and they are also capable of chelating excess essential metal ions to avoid metal toxicity. Possible mechanisms for the heavy metal homeostasis have been suggested. One of the mechanisms is the involvement of the metals going through the 14

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Fig. 5. Expression of OsHIPP16, OsHIPP28, OsHIPP34, OsATX1 and OsHIPP60 genes in rice exposed to excess Mn, Cd and Cu by qRT-PCR. Two week-old rice plants were exposed to Mn (0, 100, 250, 500 μM), Cd (0, 1, 80, 160 μM) and Cu (0, 10, 30, 60 μM) for 4 h. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate the mean values that are significantly different between the treatment and control (p < 0.05).

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Fig. 6. Expression of OsHIPP34, OsHIPP60 and OsHIPP16 in yeast to determine their activities under the stress of Cu, Zn and Cd. The corresponding mutants cup2, ycf1 and zrc1 expressing the HIPP or with empty vector were grown for 3 days in YNB medium supplemented with or without 60 μM Cu, 8 mM Zn and 75 μM Cd, respectively. The growth activity in response to metals was determined by comparing transformed and untransformed yeast. Clones described in the liquid cultures were diluted to OD 0.2 in fresh SD/ura medium. The metal concentration in the yeast was determined with ICP-MS after cells were collected. Vertical bars represent standard deviation of three replicates. Asterisks indicate that mean values are significantly different between the treatments and control (p < 0.05). The predicted 3D model of the three representative HIPP proteins was presented.

6. Conclusion

Cu, Mn and Zn stress. These genes hold potentials and may be applied to generating transgenic plants for phytoremediation.

The present study identified 54 HPP/HIPP genes from rice genome. Genome-wide transcript analysis showed 11 genes upregulated under Cd stress. qRT-PCR analysis revealed that 4 HPP and 5 HIPP genes were differentially expressed in roots and shoots under Mn, Cd and Cu stress. Transformation of 3 representative genes OsHIPP34, OsHIPP60 and OsHIPP16 in yeast cells showed that they played a functional role in plant tolerance to the metal stress. Furthermore, the genetic evidence was provided that mutation of OsHIPP42 significantly compromised rice tolerance to Cd, Mn, Zn and Cu. Thus, our work provided useful genes for the genetic improvement of plants to enhance tolerance to Cd,

Conflicts of interest The authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.03.040. 16

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Fig. 7. Identification of the growth response of oshipp42 mutant lines and wild-type (HY) under Cu and Zn stress. Two week-old rice plants were exposed to 25 μM Cu and 100 μM Zn for one week. After that, the shoot and root elongation and dry biomass were determined. Vertical bars represent standard deviation of three biological replicates. Asterisks indicate the mean values that are significantly different between the oshipp42 mutant lines and wild-type (p < 0.05).

Funding

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