Cd hyperaccumulator Sedum alfredii in response to zinc and cadmium

Chemosphere 164 (2016) 190e200

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Transcriptional up-regulation of genes involved in photosynthesis of the Zn/Cd hyperaccumulator Sedum alfredii in response to zinc and cadmium Lu Tang a, Aijun Yao b, Ming Yuan a, Yetao Tang a, c, **, Jian Liu d, Xi Liu a, Rongliang Qiu a, c, * a

School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou, PR China Department of Land Resource and Environment, School of Geography and Planning, Sun Yat-Sen University, Guangzhou, PR China Guangdong Provincial Key Lab of Environmental Pollution Control and Remediation Technology (Sun Yat-sen University), Guangzhou, PR China d MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, PR China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Zn/Cd up-regulated photosynthesisrelated genes in hyperaccumulator Sedum alfredii.
Zn/Cd supported better morphological parameters and growth of S. alfredii leaves.
Leaf chlorophyll fluorescence parameters responded to Cd much as it did to Zn.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 May 2016 Received in revised form 25 July 2016 Accepted 4 August 2016

Zinc (Zn) and cadmium (Cd) are two closely related chemical elements with very different biological roles in photosynthesis. Zinc plays unique biochemical functions in photosynthesis. Previous studies suggested that in some Zn/Cd hyperaccumulators, many steps in photosynthesis may be Cd tolerant or even Cd stimulated. Using RNA-seq data, we found not only that Cd and Zn both up-regulated the CA1 gene, which encodes a b class carbonic anhydrase (CA) in chloroplasts, but that a large number of other Zn up-regulated genes in the photosynthetic pathway were also significantly up-regulated by Cd in leaves of the Zn/Cd hyperaccumulator Sedum alfredii. These genes also include chloroplast genes involved in transcription and translation (rps18 and rps14), electron transport and ATP synthesis (atpF and ccsA), Photosystem II (PSBI, PSBM, PSBK, PSBZ/YCF9, PSBO-1, PSBQ, LHCB1.1, LHCB1.4, LHCB2.1, LHCB4.3 and LHCB6) and Photosystem I (PSAE-1, PSAF, PSAH2, LHCA1 and LHCA4). Cadmium and Zn also up-regulated the VAR1 gene, which encodes the ATP-dependent zinc metalloprotease FTSH 5 (a member of the FtsH family), and the DAG gene, which influences chloroplast differentiation and plastid development, and the CP29 gene, which supports RNA processing in chloroplasts and has a potential role in signal-dependent co-regulation of chloroplast genes. Further morphological parameters (dry biomass, cross-sectional thickness, chloroplast size, chlorophyll content) and chlorophyll fluorescence parameters confirmed

Handling Editor: Caroline Gaus Keywords: Cadmium (Cd) Hyperaccumulator Photosynthesis RNA-seq Sedum alfredii Zinc (Zn)

* Corresponding author. School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou, PR China. ** Corresponding author. School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou, PR China. E-mail addresses: [email protected] (Y. Tang), [email protected] (R. Qiu). http://dx.doi.org/10.1016/j.chemosphere.2016.08.026 0045-6535/© 2016 Elsevier Ltd. All rights reserved.

L. Tang et al. / Chemosphere 164 (2016) 190e200

191

that leaf photosynthesis of S. alfredii responded to Cd much as it did to Zn, which will contribute to our understanding of the positive effects of Zn and Cd on growth of this plant. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Zinc (Zn) and cadmium (Cd) are two closely related chemical elements in group IIB that, nevertheless, have very different biological roles in higher plants (Das et al., 1997; Broadley et al., 2007; Chaney, 2010). Zinc is an essential nutrient for all plants, and Cd is considered to be one of the most toxic heavy metals in contaminated soils. The chemical similarity between Zn and Cd allows Cd to substitute for Zn relatively easily in biological systems (Das et al., 1997; Chaney, 2010; Tang et al., 2014). A number of previous studies have demonstrated the biological relationships between Cd and Zn, and especially the effects of Cd on Zn-related proteins (Aravind and Prasad, 2004; Sandalio et al., 2001; Küpper and Kochian, 2010; Tang et al., 2014). Numerous chloroplast proteins are identified as Zn-binding (Wedel et al., 1997; Broadley et al., 2007; Marri et al., 2010). During photosynthesis, Zn has been implicated in maintaining the energy spillover and fluorescence reactions from photosystem II (PSII) to photosystem I (PSI) in the light stage (Chen et al., 2008; Rehman et al., 2012). Many metalloproteases targeted to the chloroplasts (e.g., the FtsH family), which may mediate a variety of plastid activities, are Zn-dependent (Clemens, 2010). Zinc can also modulate proliferation and expansion of differentiating cells through affecting auxin metabolism or related transcription factors (Broadley et al., 2007; Choi et al., 2013). Moreover, Zn is required in plants not only as a catalytic factor in enzymes, but also as a necessary structural component in hundreds of proteins (Berg and Shi, 1996; King, 2011). Particularly, a large number of transcription factors in the nucleus are known to contain Zn fingers and similar domains, whose functions range from regulation of DNAtranscription and RNA-processing to protein-protein interactions (He et al., 2005; Broadley et al., 2007). Photosynthetic reaction may be the main site of inhibition in higher plants under Cd stress (Küpper et al., 2007). However, certain plant species called Zn/Cd hyperaccumulators have evolved the ability to accumulate extraordinarily high level of Cd in the aerial parts without resulting in serious toxicity with regard to some photosynthetic parameters (Baker, 1987; Zhou and Qiu, 2005; Jin et al., 2008; Ying et al., 2010; Liu et al., 2010; Tang et al., 2013). For the Zn/Cd hyperaccumulator Sedum alfredii, relatively normal chloroplasts with an intact ultrastructure were observed in plants exposed to nutrient solution containing 100 mM Cd and the value of Fv/Fm (potential efficiency of PSII photochemistry) was nearly unchanged even in the presence of 400 mM Cd (Zhou and Qiu, 2005; Jin et al., 2008). Similarly, Ying et al. (2010) and Tang et al. (2013) suggested that Zn/Cd hyperaccumulator Picris divaricate was capable of protecting chloroplast ultrastructure and PSII reaction centers from Cd. In addition, in the case of S. alfredii, Liu et al. (2010) showed that 50 to 100 mg1 kg Cd increased the dry biomass of shoots by 1.6e3.2 times and diminished the visible sublethal chlorophyll deficiency symptoms such as chlorosis observed in control plants. Moreover, for Noccaea caerulescens and P. divaricata, Cd addition significantly increased the activity of carbonic anhydrase (CA), a ubiquitous photosynthetic enzyme in C3 and C4 plants (Liu et al., 2008; Ying et al., 2010; Ludwig, 2011). All of these results suggest that in some Zn/Cd hyperaccumulators, many steps in photosynthesis may be Cd tolerant or even Cd stimulated. Gene expression profiles can reflect physiological responses in plants more quickly and in greater detail than do phenotypic

parameters. One exception to the general rule of Cd toxicity is the utilization of Cd in photosynthetic CO2 assimilation by the marine diatom Thalassiosira weissflogii, which has been shown to substitute Cd for Zn as a catalytic factor in CA, synthesizing the specific cadmium enzyme Cd-CA (Lane et al., 2005; Xu et al., 2008). At the transcriptional level, Lane et al. (2005) indicated that expression of the CDCA1 gene in T. weissflogii may be up-regulated with increased Cd concentration. However, there have been no reports demonstrating that Cd addition can induce over-expression of the CA gene or other photosynthesis-related genes in Zn/Cd hyperaccumulators. Moreover, little work has focused on the expression of genes associated with photosynthesis in the Zn/Cd hyperaccumulators under Zn or Cd exposure. RNA-seq is a highly sensitive and dynamic tool for plant transcriptome studies, which can provide an overall picture of plant biological pathways (Yu et al., 2012; Gao et al., 2013; McGettigan, 2013; O'Rourke et al., 2013). With the recent advances in sequencing and bioinformatic technologies, researchers have begun to move to non-model plants to study molecular mechanisms that are responsible to heavy metals and other environmental stresses. For example, comparative transcriptome analysis was employed for low- and high-cadmium-accumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress (Zhou et al., 2016). Gao et al. (2013) demonstrated the transcriptomic analysis of cadmium stress response in S. alfredii, the only hyperaccumulating species in Crassulaceae family. Also, using Arabidopsis thaliana database (TAIR10, http://www.arabidopsis.org/ ) as a reference, Tsukagoshi et al. (2015) demonstrated the RNA-Seq analysis of the effects of high salinity to the Mesembryanthemum crystallinum (ice plant), which is a halophyte that switches from C3 photosynthesis to Crassulacean acid metabolism (CAM) under high salinity and drought stress. Here, we used RNA-seq data combined with Chlorophyll fluorescence parameters to show how photosynthetic processes in leaves of the Zn/Cd hyperaccumulator S. alfredii grown hydroponically respond to Cd and Zn at the transcriptional level. Specifically, we looked at genes up-regulated by both Zn and Cd and our goal was to identify overexpressed genes potentially associated with photosynthesis and we compared the effects of Cd and Zn on gene expression levels. 2. Materials and methods 2.1. Plant material S. alfredii plants containing as little as possible Zn and Cd were used. In order to minimize the internal metal concentrations, plants were pre-cultured for several generations. Originally, mother seedlings were obtained from an old lead (Pb)/Zn mining area in Zhejiang Province, China. They were cultured in potting soil through cutting propagation under controlled greenhouse conditions with natural lighting at a temperature of 21  C (Liu et al., 2010). Subsequently, healthy and equally sized S. alfredii cuttings were grown hydroponically in deionized water in a growth chamber with day/night temperatures of 25/18  C and relative humidity of 75%. A 16 h: 8 h photoperiod with an average light intensity of 200 ± 8 mmol m2s1 (n ¼ 10; determined by a quantitherm light meter, Hansatech instrument, Norkfold, UK) was supplied by an assembly of fluorescent lamps (specially designed

192

L. Tang et al. / Chemosphere 164 (2016) 190e200

fluorescent tubes, Xintian company, Jiangsu, China). After rooting and acclimation for one week in deionized water, the plants were cultured with one-quarter strength modified 20% Hoagland solution containing the following composition (in mM): Ca(NO3)2$4H2O 800, NH4H2PO4 50, MgSO4$7H2O 420, KNO3 1,300, H3BO3 9.0, MnSO4$H2O 0.50, CuSO4$5H2O 0.08, (NH4)6Mo7O24 0.02, H2SO4 0.002, and Fe-EDTA (ethylenediaminetetraacetic acid ferric sodium salt) 25, with pH maintained at 5.8 ± 0.2 with 2 mM MES-KOH (2morpholinoethanesulphonic acid-KOH). After two months, the plants had produced multiple tillers. Tillers of equal size were cut from the same plant, transferred to 0.9-L plastic pots, rooted and acclimated for two weeks in deionized water and full-strength nutrient solution, and prepared for being treated with Zn and Cd. Plant leaves before treatment (subsamples, n ¼ 10) contained 156 ± 10 mg Zn kg1 DW and 0.72 ± 0.08 mg Cd kg1 DW, respectively. All chemicals were analytical grade and purchased from the Dongzheng company (Guangzhou, China) except for FeEDTA, which was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Germany) connected to a computer with data acquisition software (ImagingWin v2.0 m, Walz) was used to determine the chlorophyll fluorescence parameters (Li et al., 2012a). Leaves were adapted in darkness for at least 15 min for precise determination of minimal and maximal fluorescence levels in the dark (Fo and Fm, respectively). Fm was obtained by exposing the leaf sample to a high intensity (8000 mmol m2s1) short pulse (0.8 s). Fv/Fm was defined as [Fm e Fo]/Fm. The actual PS II efficiency (FPSII ¼ [Fm0 eF]/Fm0 ), the non-photochemical quenching (NPQ ¼ [FmeFm0 ]/Fm0 ), the photochemical quenching (qP ¼ [Fm0 eF]/Fv0 ¼ [Fm0 eF]/[Fm0 eFo0 ]) were automatically calculated by the ImagingWin software (Walz) in the presence of actinic illumination (for 204 mmol m2 s1, which was close to our growth conditions). Fm0 and Fo0 were the maximum and minimal light-adapted fluorescence, respectively; and F was the steady-state fluorescence in the light. Images of the fluorescence were displayed with the help of a false color code ranging from 0.000 (black) to 1.000 (purple).

2.2. Plant treatment

Total RNA was isolated and purified using TRIzol LS (Invitrogen, Carlsbad, CA, USA) combined with Pure RNA Mini Kit (New England Biolabs, Inc. Ipswich, MA, USA). mRNA was extracted from total RNA using Dynabeads mRNA Purification Kit. cDNA library preparation and sequencing reactions were carried out according to the manufacturer's recommendations (Ion PGM™ Sequencing). Using Ion PGM high-throughput sequencing, 4.3e5.1 million >50 bp pairedend mRNA reads were generated in the analyzed samples. The goal of the RNA-seq data processing was to identify a set of genes potentially involved in Zn whose expression levels were increased by both 5 mM Zn and 5 mM Cd addition. We selected the genome of Arabidopsis as a reference, considering that A. thaliana is a model plant with abundant genetic information regarding Zn function and plant growth. For obtaining the most efficient and accurate mapping, Bowtie2 was used as a tool (Langmead and Salzberg, 2012) for mapping the paired-end mRNA sequence tags to the Arabidopsis (A. thaliana) genome database (TAIR10_peptide), with parameters set as -t, -p 28, very-sensitive, -N 1, -L 10, L,0.7,0.7. The overall alignment rates in the control, Zn5, and Cd5 treatments were 11.42%, 8.16%, and 9.18%, respectively. Cufflinks was used to analyze the differentially expressed genes. Gene expression levels within a given sample were normalized using values for a gene's uniquely aligned read counts per kilobase of exon model per million reads (RPKM). To be considered differentially expressed, the transcript must have had RPKM 3 in at least one sample, fold-change  2, and P  0.05 (O'Rourke et al., 2013). Heat maps illustrating expression patterns of various differentially expressed genes were created using the HeatMap tool (http://www.mathworks.cn/cn/ help/bioinfo/ref/heatmap.html). Principal component analysis (PCA) of the gene expression data was performed using the Matlab software. Gene ontology (GO) annotation was performed using the GO-TermFinder tool. Six photosynthesis-related S. alfredii genes were cloned and sequenced using the primers listed in Table S1. The six cloned S. alfredii genes were homologs of the A. thaliana genes PSBM, PSBK, atpF, CAB2, LHB1B1 and LHCB2.1. Gene information for these six S. alfredii genes we sequenced has been submitted to the NCBI database with the accession numbers KF700925 (PSBM), KF700924 (PSBK), KF700923 (atpF), KF700926 (CAB2/LHCB1.1), KF700927 (LHB1B1) and KF700928 (LHCB2.1). Their amino acid sequence alignments, phylogenetic trees, and identity scores with other plant species were obtained using ClustalW2 and BLAST tools. Quantitative reverse transcription PCR (qRT-PCR) was carried out according to Yu et al. (2012) using the primers listed in

We compared leaves of plants supplied with 5 mM Cd (treatment Cd5) or 5 mM Zn (treatment Zn5) with those of control plants grown without adding either Zn or Cd to the hydroponic culturing medium. Zinc and Cd were provided as the chloride forms ZnCl2 and CdCl2, respectively. For Zn, this concentration has been normally used in the culture medium of S. alfredii (Tian et al., 2011); For Cd, this concentration is commonly found in heavy metal-rich habitats and is one at which we have found that S. alfredii growth was stimulated in a pre-experiment (Supplementary material Fig. S1). Two parallel experiments were conducted, in which plants were harvested after 15 days and 3 months, respectively. The former experiment focused on the measurement of physiological and molecular parameters such as chlorophyll fluorescence parameters, RNA-seq, and element concentration. The latter experiment enabled extended observation of plant growth characteristics and determination of growth-related parameters such as biomass, leaf thickness, and chloroplast size. Each treatment was replicated in six vessels. The nutrient solution was renewed every three days and aerated continuously. Whole plants were thoroughly washed three times with deionized water and blotted dry with tissue paper at harvest. Leaves, stems, and roots were separated and weighed fresh. Aliquot parts were oven-dried at 75  C for water content calculation. The remaining fresh plant material was frozen in liquid nitrogen and stored at 80  C for subsequent analysis. 2.3. Leaf morphological and chlorophyll fluorescence parameters Leaf chlorophyll content was determined spectrophotometrically in 80% acetone according to Arnon (1949). Leaf cross-sections and isolated chloroplasts were investigated and pictured by scanning electron microscope (SEM, Model Quanta 400, FEI, Holland). Size measurements were carried out manually using Adobe Photoshop 7.0 software. Intact chloroplasts were isolated using the method of Phee et al. (2004) with minor modifications. Extracted chloroplasts and small pieces of leaf were both fixed in 2.5% glutaraldehyde in 0.1 mM phosphate buffer (pH 7.2) for 8 h. After dehydration in an ethanol series (30, 50, 70, 90, 100%), tertiary butyl alcohol was used to exchange ethanol. The cryo-fractured specimens were dried using freeze-drier, coated with carbon and prepared for observation by SEM. A portable version of an ImagingPAM Chlorophyll Fluorometer (PAM-MINI, Walz, Effeltrich,

2.4. RNA-seq

L. Tang et al. / Chemosphere 164 (2016) 190e200

Supplementary material Table S2. 2.5. Elemental composition Dried plant parts were ground and the powder was digested using HNO3:HClO4 (5:1, v/v) mixture. The metals in the digests were determined by inductively coupled plasma-optima emission spectrometry (ICP-OES, Optima 5300DV). Microscopic imaging of Zn in the leaves of S. alfredii was conducted according to the method of Tian et al. (2009). After 15 days, fresh leaf slices were cut (thickness < 0.5 mm). Plant samples were then immersed in a 10 mM solution of the Zn fluorescent indicator Zinpyr-1 Sigmaaldrich (St. Louis, MO, USA) in 10 mM MES buffer, pH 6.1. The samples immersed in the solution were vacuum-infiltrated for 10 min, and incubated in the same solution for a further 20 min. Plant samples were kept in the dark during this procedure. After washing, images were taken on a fluorescent microscope (Zeiss Imager Z1, Germany) using filters S484/15 for excitation and S517/ 30 for emission (Gu et al., 2012). Exposure time (50 ms) was uniform for all samples. No autofluorescence was observed in plant sections without dye treatment in the control, Zn5, or Cd5 treatments. The Zn signal in leaves in the Zn5 treatment was also performed using a Zeiss laser confocal scanning microscope (LCSM; LSM510/ConfoCor2, Carl-Zeiss, Jena, Germany; Li et al., 2012b), which was visualized with excitation at 488 nm and emission at 500e550 nm using a band pass filter, and chloroplast autofluorescence (488 nm excitation) was visualized at 650 nm with a long pass filter. Zinc distribution in leaf cross-sections using synchrotron X-ray fluorescence (SXRF) microprobe was performed at beamline LU15 at the Shanghai Institute of Applied Physics, Chinese Academy of Science. Sample preparation and experiment performance were according to the method of Tian et al. (2009) with minor modifications. Sample preparation details for the synchrotron mXRF measurements are as follows: 1) Wash the leaves three times each with deionized water and use paper towel to remove residual water from the surfaces of the leaves; 2) The washed fresh leaves were cut into small pieces (3 cm*5 cm) using a stainless steel blade; 3) Quickly plunge freeze the stripped leaves into liquid nitrogen-cooled propane for about 3e4 s before transferring to the liquid nitrogen-precooled aluminium utensils; 4) Through the observation of dissecting microscope, cut the frozen leaves into small pieces (thickness ¼ 100 mm 500 mm) with precooled stainless steel blade; 5) Freeze-dry the samples for three days with Labconco FeeZone (4.5 L) at 103 mbar and 50  C; 6) Through the observation of dissecting microscope and SEM, pick up the qualified samples for testing. The characteristics of the SXRF microprobe apparatus at beamline LU15 were reported in detail by Zheng et al. (2011).

193

chlorotic areas were gradually alleviated in the control, Zn5, and Cd5 treatments (Fig. 1a). However, on Day 15, leaves in the Zn5 and Cd5 treatments were both visibly greener than those of control plants (Fig. 1a). Total chlorophyll content was significantly increased by both the Zn5 and Cd5 treatments, with values that were higher than the control value by 207% and 155%, respectively, on Day 15 and by 42% and 71%, respectively, on Day 30 (P < 0.01; Fig. 1b). At 3 months, leaves in both the Zn5 and Cd5 treatments were much heavier and thicker than those in the control. Leaf dry weight was higher by 37% and 74% and cross-sectional thickness by 31% and 48% in the Zn5 and Cd5 treatments, respectively, compared to control values (P < 0.01; Figs. 1c,d, S2). The length and width of isolated chloroplasts were significantly greater in the Cd5 treatment but not in the Zn5 treatment, compared with the control values (P < 0.01; Figs. 1e, S2). 3.1.2. Chlorophyll fluorescence parameters The chlorophyll fluorescence experiments demonstrated that both the Zn5 and the Cd5 treatments stimulated the photochemical activity of PSII of S. alfredii leaves to a similar extent (Fig. 2). The false images of Fv/Fm, FPSII, NPQ/4 and qP of leaves after treatment with 5 mM Zn and 5 mM Cd were very different from those in the control on Day 15 (Fig. 2a). Values of Fv/Fm in the Zn5 and Cd5 treatments were 4% and 6% higher, respectively, than the control value, which represented significant increases, although they were still slightly lower than the normal value of 0.800 (P < 0.05; Fig. 2b). Values of the other chlorophyll fluorescence parameters were also increased, compared to the control, by the Zn5 and Cd5 treatments, with increases of 55% and 39% for FPSII, 46% and 35% for qP, and 33% and 32% for NPQ, respectively (P < 0.05; Fig. 2c,d,e). 3.2. Up-regulation of genes in leaves of S. alfredii plants in the Zn5 and Cd5 treatments Among the RNA-seq data mapped to the Arabidopsis genome (TAIR10) by Bowtie 2, only transcripts which had RPKM  3 in at least at one treatment, fold-change 2 between treatments, and P  0.05 were considered as differentially expressed. According to these criteria, in leaves of S. alfredii plants in the Zn5 and Cd5 treatments, 266 and 103 unique genes, respectively, were identified as up-regulated, and 161 genes were up-regulated in common (Fig. 3a; Supplementary material Table S3).

3.1. Effect of Zn5 and Cd5 treatments on leaves of S. alfredii plants

3.2.1. Gene Ontology Gene Ontology analyses of the 161 commonly up-regulated genes in the leaves of S. alfredii plants in the Zn5 and Cd5 treatments were performed. In total, 157 genes were matched to 141 GO items, which consisted of 93 items in Biological Process (BP), 44 items in Cellular Component (CC) and 4 items in Molecule Function (MF). Given the negative logarithm of the P Value, the major four biological processes identified for these genes were photosynthesis, generation of precursor metabolites and energy, the light reaction in photosynthesis, and metabolic processes (Fig. 3b; Supplementary material Table S4). The largest four cellular components associated with these genes were organelle subcompartment, chloroplast part, thylakoid, and chloroplast thylakoid (Fig. 3b; Supplementary material Table S4). The major four molecular functions for these genes were tetrapyrrole binding, chlorophyll binding, oxidoreductase activity, and binding (Fig. 3b; Supplementary material Table S4).

3.1.1. Visible growth symptoms Both the Zn5 and the Cd5 treatments were effective in supporting the growth of S. alfredii leaves (Fig. 1; Supplementary material Fig. S2). As leaves matured between Day 0 and Day 30,

3.2.2. Genes involved in Zn functions Among the 161 commonly up-regulated genes in leaves of S. alfredii plants in the Zn5 and Cd5 treatments, 62 were identified as being related to Zn and were classified into four clusters defined

2.6. Statistical analysis Statistical analyses were performed using the SPSS statistical package (version 11.0) for Windows Standard Version software package. All data were statistically analyzed using the one-way ANOVA followed by LSD test. Differences were considered significant at values of P < 0.01 or P < 0.05. 3. Results

194

L. Tang et al. / Chemosphere 164 (2016) 190e200

Fig. 1. Growth parameters of Sedum alfredii leaves in the control, 5 mM Zn (Zn5), and 5 mM Cd (Cd5) treatments. (a) Photos of visible growth of plants 0 days (Day 0), 15 days (Day 15) and 30 days (Day 30) after the beginning of treatments. Pictures represent a typical example. Bar ¼ 1 cm; white arrows indicate the same leaf in the different growing periods. (b) Total chlorophyll content of leaves in plants 0 days (white columns), 15 days (grey columns) and 30 days (black columns) after the beginning of treatments. (c) Leaf dry biomass, (d) leaf cross-sectional thickness, (e) length (white columns) and width (black columns) of isolated chloroplasts of plants 3 months after the beginning of treatments. Data are the mean ± SE, and different letters indicate statistically significant differences between treatments (LSD test, P < 0.01). The replicate number (n) in (b and c), (d), and (e) are 5e6, 28e57, and 66e76, respectively.

as follows: Cluster I, Zn-binding; Cluster II, photosynthesis; Cluster III, transcription and translation; and Cluster IV, cell proliferation and differentiation (Fig. 4; Supplementary material Table S3). Cluster I (Zn-binding) consisted of 9 genes which were found in the table (S1eS3) from Broadley et al. (2007) listing A. thaliana proteins containing putative domains with observed or predicted capabilities for binding Zn or involved in Zn function (Fig. 4a; Supplementary material Table S3). Three genes in this cluster were involved in photosynthesis: CA1 (AT3G01500), encoding carbonic anhydrase 1; CP12-1 (AT2G47400), encoding CP12 domaincontaining protein 1; and VAR1/FTSH5 (AT5G42270), encoding ATP-dependent zinc metalloprotease FTSH 5. Two genes in this cluster encoded Zn-finger binding proteins involved in transcription & translation: AT3G11110, encoding RING-H2 finger protein ATL66; and ING1 (AT3G24010), encoding PHD finger protein ING1. Other genes in this cluster were RABA2c (AT3G46830), encoding Ras-related protein RABA2c; AT1G57720, encoding elongation factor EF-1 gamma subunit; and two transport genes (NRAMP3 and IRT3). Cluster II (Photosynthesis) consisted of 20 genes, according to the chloroplast (plastid) genome in the model plant A. thaliana summarized by Allen et al. (2011) (Figs. 3c and 4b; Supplementary material Table S3). Most of their encoded proteins are located in the chloroplast thylakoid membrane where they are involved in:

electron transport and ATP synthesis (atpF and ccsA); PSII (PSBI, PSBM, PSBK, PSBZ/YCF9, PSBO-1, PSBQ, LHCB1.1, LHCB1.4, LHCB2.1, LHCB4.3 and LHCB6); and PSI (PSAE-1, PSAH2, PSAF, LHCA1 and LHCA4). Two genes in this cluster (rps18 and rps14) encode proteins involved in transcription and translation. Cluster III (Transcription & translation) consisted of 21 genes (Fig. 4c; Supplementary material Table S3). One of these genes, CP29, is photosynthesis-related, encoding chloroplast RNA-binding protein 29. Other genes in this cluster, according to previous reports, are thought to be involved in controlling leaf development and growth, including: histone family (AT3G09480, AT2G28720, AT5G59970, HIS4 and HTA5/H2AXA); 60S ribosomal proteins (AT4G26230, AT3G44590, RPL18AA); AGO4 (argonaute 4), transcription factors (AT1G05805, NF-YB8 and ERF3); DEAD-box ATP-dependent RNA helicase (RH8); conserved peptide upstream open reading frame (CPuORF10); Rac-like GTP-binding protein ARAC4 (ROP2); and others. Cluster IV consisted of 12 genes identified in previous reports as being involved in cell proliferation and differentiation (Fig. 4d; Supplementary material Table S3). Major genes in this cluster were IAA14 and IAA13 encoding auxin-responsive proteins, three tubulin-related genes (TUBG1, TUA6 and POR C), CKX3 encoding cytokinin dehydrogenase 3, and EXPA8 encoding expansin A8. Another two genes, CYCD4; 1 and AT1G32580, encode

L. Tang et al. / Chemosphere 164 (2016) 190e200

195

Fig. 2. Chlorophyll fluorescence parameters of Sedum alfredii leaves in the control, 5 mM Zn (Zn5), and 5 mM Cd (Cd5) treatments. (a) False images of leaf chlorophyll fluorescence (Fv/Fm, FPSII, qP and NPQ/4) displayed with the help of a false color code ranging from 0.000 (black) to 1.000 (purple). Bar ¼ 1 cm. Pictures represent a typical example. The values of Fv/Fm, FPSII, qP, and NPQ are shown in (b), (c), (d), and (e), respectively. Data are the mean ± SE (n ¼ 6), and different letters indicate statistically significant differences between treatments (LSD test, P < 0.05). Fv/Fm, maximum potential PS II efficiency; FPSII, actual PS II efficiency; qP, photochemical quenching value; NPQ, non-photochemical quenching value. The values are calculated for the presence of actinic illumination of 204 mmol m2 s1, which is close to our growth conditions (about 200 mmol m2 s1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

cyclin-D4-1 and putative plastid developmental protein DAG, respectively. Three other genes in this cluster were APC8, encoding anaphase-promoting complex subunit 8; AGL92, encoding agamous-like MADS-box protein AGL92; and AFO, encoding axial regulator YABBY 1. 3.2.3. Gene expression profiles The 161 S. alfredii genes that were up-regulated in both the Zn5 and Cd5 treatments showed similar changes in expression profile in response to Zn or Cd, based on PCA and relativity analysis of their RPKM values (Fig. 5). Principal component analysis showed that principal components 1, 2 and 3 accounted for 99.80, 0.16 and 0.04% of the variance, respectively. The coefficient matrices of Component 1 in the Zn5 and Cd5 treatments were similar, with values of 0.7667 and 0.5885, respectively, compared to a value of 0.2567 in the control treatment (Fig. 5a). Moreover, relativity analysis showed that there were strong similarities between the expression levels of the 161 commonly up-regulated genes in the Zn5 and Cd5 treatments (Fig. 5b). This was further confirmed by the expression level changes of the seven genes (PSBI, PSBM, PSBK, atpF, CAB2, LHB1B1, LHCB2.1) which were selected for confirmation of RNA-Seq expression profiles with q-PCR (Fig. 5c).

was distributed primarily in the epidermis and was almost undetectable in mesophyll cells, according to SRXF microprobe analysis and fluorescence microscopy using a Zn probe (Fig. 6b; Supplementary material Figs S3 and S4). Leaf total Zn concentration was 61 and 51 mg kg1 DW in the control and Cd5 treatments, respectively (Fig. 6a). In comparison with the other two treatments, the Zn5 treatment significantly increased leaf Zn concentration, to 731 mg kg1 DW, and it also increased the amount of Zn patchily distributed in mesophyll cells (P < 0.01; Fig. 6; Supplementary material Figs S3eS5). The Cd5 treatment had no significant effect on leaf Zn concentration, but it significantly increased leaf Cd concentration to 1161 mg kg1 DW, compared to the control in which no Cd was detectable (P < 0.01; Fig. 6; Supplementary material Figs S3 and S4). Leaf concentrations of Cu, Fe, K and Mg were not affected by either the Zn5 or Cd5 treatments (P < 0.01; Supplementary material Fig. 6a). Compared to the control, leaf Mn concentration decreased and Ca concentration increased in the Cd5 treatment (P < 0.01; Supplementary material Fig. 6a). In contrast, the Zn5 treatment had no effect on Mn and Ca concentrations, while it decreased leaf P concentration significantly (P < 0.01; Supplementary material Fig. 6a). 4. Discussions

3.3. Elemental concentrations in leaves of S. alfredii plants in the Zn5 and Cd5 treatments Zinc in S. alfredii leaves in both the control and Cd5 treatments

Cadmium is generally toxic to photosynthesis in higher plants. Previous studies have suggested that certain Zn/Cd hyperaccumulators were capable of protecting some photosynthetic

196

L. Tang et al. / Chemosphere 164 (2016) 190e200

Fig. 3. Genes differentially up-regulated in Sedum alfredii leaves in the 5 mM Zn (Zn5) and 5 mM Cd (Cd5) treatments. (a) Number of differentially up-regulated genes. To be considered differentially expressed, the transcript must have had RPKM 3 in at least one sample, fold-change  2, and P  0.05. Zn-unique (blue color) and Cd-unique (green color) indicate that 266 and 103 genes were up-regulated uniquely in the Zn5 and Cd5 treatments, respectively. Zn,Cd-common (yellow color) indicates that 161 genes were up-regulated in both the 5 mM Zn and 5 mM Cd treatments. See also Supplementary material Table S3. (b) The major four biological processes, molecular functions, and cellular components identified for the 161 commonly up-regulated genes in the leaves of Sedum alfredii plants in the Zn5 and Cd5 treatments, according to Gene Ontology (GO) analysis and the negative logarithm of the P value. See also Supplementary material Table S4. (c) 20 genes up-regulated in both the Zn5 and Cd5 treatments that are associated with a wide variety of biological pathways in the photosynthetic reaction inside chloroplasts and the thylakoid membrane: electron transport and ATP synthesis (atpF and ccsA); photosystem II (PSBI, PSBM, PSBK, PSBZ/YCF9, PSBO-1, and PSBQ); LHCII (LHCB1.1, LHCB1.4, LHCB2.1, LHCB4.3, and LHCB6); photosystem I (PSAE-1, PSAH2, and PSAF); LHCI (LHCA1 and LHCA4), and transcription and translation (rps18 and rps14). The picture is drawn according to the chloroplast (plastid) genome in the model plant Arabidopsis thaliana summarized by Allen et al. (2011). PSII, photosystem II; PSI, photosystem I; LHCII, light harvesting complex II; LHCI, light harvesting complex I. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

functions (e.g., carbon assimilation enzymes, chloroplast ultrastructure and chlorophyll fluorescence parameters) from Cd toxicity (Jin et al., 2008; Liu et al., 2008, 2010; Ying et al., 2010; Tang et al., 2013). Presently, looking at phenotype effects, the stimulating effect of 5 mM Cd addition on S. alfredii leaf growth and

development was equal to or greater than that of 5 mM Zn with respect to morphological parameters (dry biomass, cross-sectional thickness, chloroplast size, chlorophyll content) and chlorophyll fluorescence parameters (Fv/Fm, FPSII, qP, NPQ) (Figs. 1 and 2; Supplementary material Fig. S2). Very surprising was the similar

L. Tang et al. / Chemosphere 164 (2016) 190e200

197

Fig. 4. Heat-map illustrating the expression profiles of the 62 genes in Sedum alfredii leaves up-regulated in both the 5 mM Zn (Zn5) and 5 mM Cd (Cd5) treatments that are identified as being related to Zn and which are classified into four clusters. (a) Cluster I (Possibly related with Zn): 9 genes from tables S1eS3 in Broadley et al. (2007). That list Arabidopsis thaliana proteins containing putative domains with observed or predicted capabilities for binding Zn or involved in Zn function. (b) Cluster II (Photosynthesis): 20 genes homologous to genes from the chloroplast (plastid) genome in the model plant Arabidopsis thaliana summarized by Allen et al. (2011). (c) Cluster III (Transcription & Translation): 21 genes thought to be involved in transcription and translation according to previous reports. (d) Cluster IV (Cell proliferation & differentiation): 12 genes identified as being involved in cell proliferation and differentiation according to previous reports. Black indicates low expression (value of 0.0) and red indicates high expression (value of 0.6). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

patterns of the expression levels of the 161 commonly up-regulated genes in the Cd5 and Zn5 treatments, based on PCA and relativity analysis of their RPKM values (Fig. 5a,b). For these genes, photosynthesis, generation of precursor metabolites and energy, and the light reaction in photosynthesis are the major three biological processes (Fig. 3b; Supplementary material Table S4). Our present results showed that 5 mM Cd addition stimulated S. alfredii photosynthesis and leaf growth as much or more than 5 mM Zn did, but without affecting (compared to the control treatment) the amount of Zn in the leaf, at least based on the facts at the transcriptional and phenotypical level (Fig. 6; Supplementary material Figs. S3 and S4). Carbonic anhydrase has been cited as a typical Zn-requiring photosynthetic enzyme involved in the Calvin cycle of higher plants by Liu et al. (2008). In the present study, we found not only that 5 mМ Cd increased expression of the CA1 gene (homolog of AT3G01500), which encodes a b class carbonic anhydrase in chloroplasts, but that a large number of other Zn up-regulated genes in the photosynthetic pathway were also significantly up-regulated

by Cd (Fig. 4; Supplementary material Table S3). Among these were CP12-1 (homologous to AT2G47400), which encodes a small peptide found in the chloroplast stroma and which belongs to the CP12 gene family, thought to be associated with the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) as part of the Calvin cycle (Fig. 4a; Wedel et al., 1997; Marri et al., 2010). In addition to their positive effects on dark stage reactions, Cd and Zn also increased expression of genes which have been implicated in maintaining the energy spillover and fluorescence reactions from PSII to PSI in the light stage as well. According to the chloroplast (plastid) genome summarized for the model plant A. thaliana by Allen et al. (2011), these genes include chloroplast genes involved in transcription and translation (rps18 and rps14), electron transport and ATP synthesis (atpF and ccsA) and PSII (PSBI, PSBM, PSBK, PSBZ/YCF9) (Figs. 3c and 4b). Moreover, Zn and Cd upregulated twelve photosynthesis-related genes located in the nucleus but which produce the constituent protein complexes of PSII

198

L. Tang et al. / Chemosphere 164 (2016) 190e200

Fig. 5. Similarity between the expression profiles of the 161 S. alfredii genes up-regulated in both the 5 mM Zn (Zn5) and 5 mM Cd (Cd5) treatments. (a) Principal component analysis (PCA) of the RPKM value (read counts per kilobase of exon model per million reads) in the control, Zn5 and Cd5 treatments. Components 1, 2, and 3 account for 99.8, 0.16 and 0.04% of the variance, respectively. The coefficients of Component 1 in the control, Zn5, and Cd5 treatments were 0.2567, 0.7667, and 0.5885, respectively. Red circles indicate the 161 genes. Blue circles indicate the control, Zn5, and Cd5 treatments. (b) Relativity analysis of the RPKM value in the Zn5 and Cd5 treatments. The regression equation is y (value in Zn5) ¼ 1.2967  (value in Cd5) e 7.9971 (R2 ¼ 0.9926). (c) Expression level changes of seven genes (PSBI, PSBM, PSBK, atpF, CAB2, LHB1B1, LHCB2.1) measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses in Sedum alfredii plants in the Zn5 (blue circles) and Cd5 (green circles) treatments compared with control levels, which are normalized to that of the ACTIN control gene. Data are the mean of three replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(PSBO-1, PSBQ, LHCB1.1, LHCB1.4, LHCB2.1, LHCB4.3 and LHCB6) and PSI (PSAE-1, PSAF, PSAH2, LHCA1 and LHCA4) in the chloroplast thylakoid membrane (Allen et al., 2011, Figs. 3c and 4b). Further qPCR results showed that Cd and Zn increased the expression levels of the seven genes (PSBI, PSBM, PSBK, atpF, CAB2, LHB1B1, LHCB2.1) (Fig. 5c). Our results clearly suggest that the Zn5 and Cd5 treatments both activated many normally Zn-dependent biochemical events related to photosynthesis or other chloroplast functions, through inducing the over-expression of the genes involved (Figs. 3c and 4; Supplementary material Table S3). These genes were associated with a wide variety of biological pathways in the photosynthetic reaction such as the Calvin cycle, the light reactions, electron transport, transcription and translation, and plastid development (Figs. 3c and 4). Most of these genes were located in cellular components including organelle subcompartment, chloroplast part, thylakoid, and chloroplast thylakoid (Fig. 3b; Supplementary material Table S4). Broadley et al. (2007) lists 2042 A. thaliana proteins containing putative Zn-binding domains, among which is VAR1 (AT5G42270), whose S. alfredii homolog was also up-regulated by Zn and Cd in our study (Fig. 4a). VAR1 encodes the ATP-dependent zinc metalloprotease FTSH 5, a member of the FtsH family, whose members localize in the thylakoid membrane with their ATPbinding domain and catalytic Zn-binding site facing the stroma, where they play a central role in chloroplast development, membrane fusion, the degradation of photodamaged D1 reaction center

proteins during the PSII repair cycle, and other functions (SinvanyVillalobo et al., 2004; Adam et al., 2006; Clemens, 2010). Two other genes up-regulated by Cd and Zn were DAG (homolog of AT1G32580), which influences chloroplast differentiation and plastid development, and CP29 (homolog of AT3G53460), which supports RNA processing in chloroplasts and has a potential role in signal-dependent co-regulation of chloroplast genes (Chatterjee et al., 1996; Kupsch et al., 2012, Fig. 4c,d). Our results further suggest that the up-regulated genes under Cd and Zn exposure can also be a large number of genes associated with modulating proliferation and expansion of differentiating chloroplast cells and plastids. Altogether, given the large number of photosynthetic genes affected by Zn and Cd, it is evident that Cd and Zn addition stimulated an entire network associated with a series of photosynthetic reactions such as Calvin cycle, the light reactions, chlorophyll fluorescence parameters, electron transport, and chloroplast development (Fig. 3c). Also, both Zn and Cd up-regulated a large number of genes encoding many proteins potentially involved in other Zn-dependent physiological processes, including transcription and translation, and cell proliferation and differentiation (Fig. 4). Moreover, in addition to the Zn-related genes cited above, there were approximately 100 other genes that were up-regulated by both Cd and Zn, 266 other genes that were up-regulated by Zn uniquely, and 103 other genes that were up-regulated by Cd uniquely (Fig. 3a; Supplementary material Tables S3 and S4). In the present study, we hope that these results will inspire further

L. Tang et al. / Chemosphere 164 (2016) 190e200

199

Fig. 6. Elemental concentrations and zinc (Zn) distribution in Sedum alfredii leaves in the control, 5 mM Zn (Zn5), and 5 mM Cd (Cd5) treatments. (a) Leaf concentrations of total cadmium (Cd), zinc (Zn), copper (Cu), ion (Fe), manganese (Mn), potassium (K), calcium (Ca), magnesium (Mg) and phosphorus (P). Data are the mean ± SE (n ¼ 5e6); n.d., not detectable; different letters indicate statistically significant differences between treatments (LSD test, P < 0.01). (b) Skeleton diagram of Zn distribution in epidermis and mesophyll cells in Sedum alfredii leaf cross-sections in the control, 5 mM Zn (Zn5), and 5 mM Cd (Cd5) treatments, according to the results from Synchrotron X-ray fluorescence analysis (SXRF) and fluorescence microscopy using the Zn-fluorophore Zinpyr-1 which are shown in Figs. S3eS5 in the Supplementary material (Fig. S3, Fluorescence microscopy visualization; Fig. S4, Synchrotron X-ray fluorescence (SXRF) analysis; Fig. S5, Laser confocal scanning microscope (LCSM) visualization). Blue circles indicate zinc (Zn). Epidermis and mesophyll cells (including palisade cells and spongy cells) in leaves are the positions concerned. Our results indicate that the amount of Zn in mesophyll cells is almost undetectable in leaves of plants in the Cd5 and control treatments. Zn5 treatment significantly increased the amount of Zn patchily distributed in mesophyll cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

research on the possible evolution of Zn-like functions of Cd in Zn/ Cd hyperaccumulators, since additional evidence is needed to eliminate other possible explanations of our findings. Finally, of the RNA-seq data we obtained, we considered only genes up-regulated by both Zn and Cd that could be mapped to the A. thaliana genome. Studies on additional genes would add to our understanding of the positive effects of Zn and Cd on growth of the Zn/Cd hyperaccumulator S. alfredii.

5. Conclusion In conclusion, we demonstrated that Cd and Zn both increased the expression, compared to levels in control plants, of a large number of genes related to photosynthesis. These genes encode not only the Zn enzyme CA, but also many other Zn-dependent proteins involved in Calvin cycle. Moreover, Cd and Zn increased expression of genes which have been implicated in maintaining the energy

200

L. Tang et al. / Chemosphere 164 (2016) 190e200

spillover and fluorescence reactions from PSII to PSI in the light stage as well. Our additional results suggest that the up-regulated genes under Cd and Zn exposure can also be a large number of genes associated with modulating proliferation and expansion of differentiating chloroplast cells and plastids. Further morphological parameters (dry biomass, cross-sectional thickness, chloroplast size, chlorophyll content) and chlorophyll fluorescence parameters confirmed that leaf photosynthesis of S. alfredii responded to Cd much as it did to Zn, which will contribute to our understanding of the positive effects of Zn and Cd on growth of this plant. Acknowledgements We thank Dr. Xinxian Long (South China Agriculture University) for kindly providing the plant materials. We thank the Shanghai Institute of Applied Physics, Chinese Academy of Science, for providing technical support in relation to the synchrotron X-ray fluorescent microprobe. The present research is financially supported by Natural Science Foundation of China (No. 41225004, No. 41171374, No. 41301278) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20110171110018). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.08.026. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1e15. Aravind, P., Prasad, M.N.V., 2004. Carbonic anhydrase impairment in cadmiumtreated Ceratophyllum demersum L. (free floating freshwater macrophyte): toxicity reversal by zinc. J. Anal. At. Spectrom. 19, 52e57. Adam, Z., Rudella, A., van Wijk, K.J., 2006. Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts. Curr. Opin. Plant Biol. 9, 234e240. Allen, J.F., de Paula, W.B.M., Puthiyaveetil, S., Nield, J., 2011. A structural phylogenetic map for chloroplast photosynthesis. Trends Plant Sci. 16, 645e655. Baker, A.J.M., 1987. Metal tolerance. New Phytol. 106, 93e111. Berg, J.M., Shi, Y., 1996. The galvanization of biology: a growing appreciation for the roles of zinc. Science 271, 1081e1085. Broadley, M.R., White, P.J., Hammond, J.P., Zelko, I., Lux, A., 2007. Zinc in plants. New Phytol. 173, 677e702. Chatterjee, M., Sparvoli, S., Edmunds, C., Garosi, P., Findlay, K., Martin, C., 1996. DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus. EMBO J. 15, 4194e4207. Chen, W., Yang, X., He, Z., Feng, Y., Hu, F., 2008. Differential changes in photosynthetic capacity, 77 K chlorophyll fluorescence and chloroplast ultrastructure between Zn-efficient and Zn-inefficient rice genotypes (Oryza sativa) under low zinc stress. Physiol. Plant 132, 89e101. Chaney, R.L., 2010. Cadmium and zinc. In: Hooda, P.S. (Ed.), Trace Elements in Soils. Wiley, New York, pp. 409e429. Clemens, S., 2010. Zn e a versatile player in plant cell biology. In: Hell, R., Mendel, R.R. (Eds.), Cell Biology of Metals and Nutrients. Springer, Heidelberg, pp. 281e298. Choi, S.W., Tamaki, T., Ebine, K., Uemura, T., Ueda, T., Nakano, A., 2013. RABA members act in distinct steps of subcellular trafficking of the FLAGELLIN SENSING2 receptor. Plant Cell 25, 1174e1187. Das, P., Samantaray, S., Rout, G.R., 1997. Studies on cadmium toxicity in plants: a review. Environ. Pollut. 98, 29e36. Gu, H.H., Zhan, S.S., Wang, S.Z., Tang, Y.T., Chaney, R.L., Fang, X.H., Cai, X.D., Qiu, R.L., 2012. Silicon-mediated amelioration of zinc toxicity in rice (Oryza sativa L.) seedlings. Plant Soil 350, 193e204. Gao, J., Sun, L., Yang, X.E., Liu, J.X., 2013. Transcriptomic analysis of cadmium stress response in the heavy metal hyperaccumulator Sedum alfredii Hance. PLoS One 8, e64643. He, G.H.Y., Helbing, C.C., Wagner, M.J., Sensen, C.W., Riabowol, K., 2005. Phylogenetic analysis of the ING family of PHD finger proteins. Mol. Biol. Evol. 22, 104e116. Jin, X.F., Yang, X.E., Islam, E., Liu, D., Mahmood, Q., 2008. Effects of cadmium on ultrastructure and antioxidative defense system in hyperaccumulator and non hyperaccumulator ecotypes of Sedum alfredii Hance. J. Hazard Mater 156, 387e397. Küpper, H., Parameswaran, A., Leitenmaier, B., Trtílek, M., Setlík, I., 2007. Cadmium induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens. New Phytol. 175, 655e674.

Küpper, H., Kochian, L.V., 2010. Transcriptional regulation of metal transport genes and mineral nutrition during acclimatization to cadmium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytol. 185, 114e129. King, J.C., 2011. Zinc: an essential but elusive nutrient. Am. J. Clin. Nutr. 94, 679Se684S. Kupsch, C., Ruwe, H., Gusewski, S., Tillich, M., Small, I., Schmitz-Linneweber, C., 2012. Arabidopsis chloroplast RNA binding proteins CP31A and CP29A associate with large transcript pools and confer cold stress tolerance by influencing multiple chloroplast RNA processing steps. Plant Cell 24, 4266e4280. Lane, T.W., Saito, M.A., George, G.N., Pickering, I.J., Prince, R.C., Morel, F.M.M., 2005. A cadmium enzyme from a marine diatom. Nature 435, 42. Liu, M.Q., Yanai, J.T., Jiang, R.F., Zhang, F., McGrath, S.P., Zhao, F.J., 2008. Does cadmium play a physiological role in the hyperaccumulator Thlaspi caerulescens? Chemosphere 71, 1276e1283. Liu, F.J., Tang, Y.T., Du, R.J., Yang, H.Y., Wu, Q.T., Qiu, R.L., 2010. Root foraging for zinc and cadmium requirement in the Zn/Cd hyperaccumulator plant Sedum alfredii. Plant Soil 327, 365e375. Ludwig, M., 2011. The molecular evolution of b-carbonic anhydrase in Flaveria. J. Exp. Bot. 62, 3071e3081. Li, Z., Xing, F.Q., Xing, D., 2012a. Characterization of target site of aluminum phytotoxicity in photosynthetic electron transport by fluorescence techniques in tobacco leaves. Plant Cell Physiol. 53, 1295e1309. Li, Z., Yue, H.Y., Xing, D., 2012b. MAP Kinase 6-mediated activation of vacuolar processing enzyme modulates heat shock-induced programmed cell death in Arabidopsis. New Phytol. 195, 85e96. Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357e359. Marri, L., Pesaresi, A., Valerio, C., Lamba, D., Pupillo, P., Trost, P., Sparla, F., 2010. In vitro characterization of Arabidopsis CP12 isoforms reveals common biochemical and molecular properties. J. Plant Physiol. 167, 939e950. McGettigan, P.A., 2013. Transcriptomics in the RNA-seq era. Curr. Opin. Chem. Biol. 17, 4e11. O'Rourke, J.A., Yang, S.S., Miller, S.S., Bucciarelli, B., Liu, J.Q., Rydeen, A., Bozsoki, Z., Uhde-Stone, C., Tu, Z.J., Allan, D., Gronwald, J.W., Vance, C.P., 2013. An RNA-seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants. Plant Physiol. 161, 705e724. Phee, B.K., Cho, J.H., Park, S., Jung, J.H., Lee, Y.H., Jeon, J.S., Bhoo, S.H., Hahn, T.R., 2004. Proteomic analyses of the response of Arabidopsis chloroplast proteins to high light stress. Proteomics 4, 3560e3568. Rehman, H., Aziz, T., Farooq, M., Wakeel, A., Rengel, Z., 2012. Zinc nutrition in rice production systems: a review. Plant Soil 361, 203e226. Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C., del Rio, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115e2126. Sinvany-Villalobo, G., Davydov, O., Ben-Ari, G., Zaltsman, A., Raskind, A., Adam, Z., 2004. Expression in multigene families. Analysis of chloroplast and mitochondrial proteases. Plant Physiol. 135, 1336e1345. Tian, S.K., Lu, L.L., Yang, X.E., Labavitch, J.M., Huang, Y.Y., Brown, P., 2009. Stem and leaf sequestration of zinc at the cellular level in the hyperaccumulator Sedum alfredii. New Phytol. 182, 116e126. Tian, S.K., Lu, L.L., Labavitch, J., Yang, X.E., He, Z.L., Hu, H.N., Sarangi, R., Newville, M., Commisso, J., Brown, P., 2011. Cellular sequestration of cadmium in the hyperaccumulator plant species Sedum alfredii. Plant Physiol. 157, 1914e1925. Tang, L., Ying, R.R., Jiang, D., Zeng, X.W., Morel, J.L., Tang, Y.T., Qiu, R.L., 2013. Impaired leaf CO2 diffusion mediates Cd-induced inhibition of photosynthesis in the Zn/Cd hyperaccumulator Picris divaricate. Plant Physiol. Bioch. 73, 70e76. Tang, L., Qiu, R.L., Tang, Y.T., Wang, S.Z., 2014. Cadmiumezinc exchange and their binary relationship in the structure of Zn-related proteins: a mini review. Metallomics 6, 1313e1323. Tsukagoshi, H., Suzuki, T., Nishikawa, K., Agarie, S., Ishiguro, S., Higashiyama, T., 2015. RNA-seq analysis of the response of the halophyte, Mesembryanthemum crystallinum (ice plant) to high salinity. PLoS One. http://dx.doi.org/10.1371/ journal.pone.0118339. Wedel, N., Soll, J., Paap, B.K., 1997. CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants. P. Natl. Acad. Sci. U. S. A. 94, 10479e10484. Xu, Y., Feng, L., Jeffrey, P.D., Shi, Y.G., Morel, F.M.M., 2008. Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms. Nature 452, 56e61. Ying, R.R., Qiu, R.L., Tang, Y.T., Hu, P.J., Qiu, H., Chen, H.R., Shi, T.H., Morel, J.L., 2010. Cadmium tolerance of carbon assimilation enzymes and chloroplast in Zn/Cd hyperaccumulator Picris divaricate. J. Plant Physiol. 167, 81e87. Yu, L.J., Luo, Y.F., Liao, B., Xie, L.J., Chen, L., Xiao, S., Li, J.T., Hu, S.N., Shu, W.S., 2012. Comparative transcriptome analysis of transporters, phytohormone and lipid metabolism pathways in response to arsenic stress in rice (Oryza sativa). New Phytol. 195, 97e112. Zhou, W.B., Qiu, B.S., 2005. Effects of cadmium hyperaccumulation on physiological characteristics of Sedum alfredii Hance (Crassulaceae). Plant Sci. 169, 737e745. Zheng, M.Z., Cai, C., Hu, Y., Sun, G.X., Williams, P.N., Cui, H.J., Li, G., Zhao, F.J., Zhu, Y.G., 2011. Spatial distribution of arsenic and temporal variation of its concentration in rice. New Phytol. 189, 200e209. Zhou, Q., Guo, J.J., He, C.T., Shen, C., Huang, Y.Y., Chen, J.X., Guo, J.H., Yuan, J.G., Yang, Z.Y., 2016. Comparative transcriptome analysis between low- and highcadmium-accumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress. Environ. Sci. Technol. 50 (12), 6485e6494.