Skip to content

Advertisement

  • Original Article
  • Open Access

Arabidopsis and rice showed a distinct pattern in ZIPs genes expression profile in response to Cd stress

Botanical StudiesAn International Journal201859:22

https://doi.org/10.1186/s40529-018-0238-6

  • Received: 9 May 2018
  • Accepted: 17 September 2018
  • Published:

Abstract

Background

Plant ZIP genes represent an important transporter family involved in metal transport. Evidence has implied that some ZIPs may contribute to plant Cd uptake, but a genome-wide examination of ZIPs’ role in Cd tolerance and uptake has rarely been reported. In this study, a genome-wide bioinformatic screening of candidate ZIP genes in Arabidopsis and rice was performed, followed by a systematic determination of their expression profile in response to Cd stress. Typical up-regulated ZIPs genes were then expressed in yeast cells to examine their effect on hosts’ Cd uptake.

Results

A total of 27 ZIP genes in Arabidopsis and rice were screened out based on sequence similarity. In Arabidopsis, Cd exposure strongly impacted the expression of most ZIPs, among which AtIRT1, AtIRT2, AtIRT4 AtZIP9, AtZIP10 and AtZIP12 were sharply up-regulated and AtIRT3, AtIRT5 were significantly down-regulated in root. In rice, all tested genes in shoot except for OsIRT1 and OsIRT12 were sharply up-regulated, while OsIRT1 and OsZIP1 in root were significantly down-regulated. Interestingly, some genes like AtIRT3, AtZIP5, AtZIP12, OsIRT1 and OsZIP1 showed converse expression regulation when subject to the tested Cd stress. When expressed in yeast cells, three ZIPs, AtIRT1, OsZIP1 and OsZIP3, caused a substantial increase in Cd sensitivity and Cd accumulation of the host cells.

Conclusions

In conclusion, this study revealed a distinct pattern in ZIPs family genes expression between Arabidopsis and rice in response to Cd stress. Arabidopsis mainly up-regulated root ZIPs genes, while rice mainly up-regulated shoot ZIPs genes. Three genes, AtIRT1, OsZIP1 and OsZIP3, conferred an increased Cd accumulation and sensitivity to Cd stress when expressed in yeast cells, further implying their roles in Cd uptake in plants.

Keywords

  • ZIP family
  • Cd
  • Metal cation transporter
  • Gene expression
  • Cd uptake

Background

The zinc(Zn)-regulated/iron(Fe)-regulated transporter-like family proteins (ZIPs) are membrane-located proteins for cations transport (Eng et al. 1998; Guerinot 2000). They have been found to exist broadly in prokaryotic cells, fungi, plants and mammalians. In plants, ZIPs have been identified in both dicots and monocots, such as Arabidopsis (Grotz et al. 1998; Milner et al. 2013), rice (Chen et al. 2008), maize (Li et al. 2013), medicago (Lopez-Millan et al. 2004; Stephens et al. 2011) and barely (Tiong et al. 2015). Grotz et al. identified five ZIP genes (IRT1, ZIP1-4) in Arabidopsis (Grotz et al. 1998), and later up to 11 ZIP genes from Arabidopsis were detected bioinformatically (Guerinot 2000). Roles of ZIP1-12 from Arabidopsis in Zn transport were explored experimentally (Milner et al. 2013). More recently, 18 ZIPs from Arabidopsis and 16 ZIPs from rice were annotated (Ivanov and Bauer 2017).

In Arabidopsis and rice, only a small number of ZIPs have been examined for biological functions in plant till now. Arabidopsis IRT1 is a well-studied ZIP gene first identified as a crucial transporter for plant Fe uptake (Varotto et al. 2002; Vert et al. 2002). Arabidopsis IRT1 can be induced by iron deficiency (Korshunova et al. 1999; Connolly et al. 2002), and may play a role in Mn/Zn transport as well (Korshunova et al. 1999; Rogers et al. 2000; Connolly et al. 2002). Biological functions in Zn/Fe transport of AtIRT2 (Vert et al. 2001, 2009), AtZIP1/2 (Grotz et al. 1998; Wintz et al. 2003; Milner et al. 2013), OsIRT1 (Nakanishi et al. 2006; Lee and An 2009; Ishimaru et al. 2006; Bughio et al. 2002) and OsZIP4/5/8 (Ishimaru et al. 2005; Chen et al. 2008; Lee et al. 2010a, b; Yang et al. 2009) have also been examined in the past decade.

A few studies have also implied that ZIPs may be involved in Cd transport. Yeast cells expressing AtIRT1 showed increased Cd sensitivity (Rogers et al. 2000; Vert et al. 2001), and IRT1-dependent Fe/Mn/Zn uptake was inhibited by excess Cd (Eide et al. 1996; Korshunova et al. 1999). The Arabidopsis IRT1 knock-out mutant irt1-1 exhibited reduced Cd sensitivity and Cd accumulation (Vert et al. 2002; Fan et al. 2014), while overexpression of AtIRT1 increased Cd sensitivity in Arabidopsis (Connolly et al. 2002). AtIRT2, phylogenetically similar to AtIRT1, increased Cd uptake when overexpressed in Arabidopsis (Vert et al. 2009), though the yeast cells expressing AtIRT2 exhibited no altered Cd sensitivity (Vert et al. 2001). In rice, expression of OsIRT1 and OsIRT2 made the cells more sensitive to Cd and increased Cd accumulation (Nakanishi et al. 2006; Lee and An 2009). Nonetheless, we still know little about the roles of most of the ZIPs genes in Cd stress response in Arabidopsis and rice.

In this study, genome-wide ZIPs identification in Arabidopsis and rice was performed with rigorous evolutional analysis. A comparative examination of genome-wide expression profile of ZIPs in Arabidopsis and rice in response to Cd stress were carried out. Their role in Cd uptake of typical ZIPs responding to Cd stress was further tested by expressing them in yeast. As expected, most identified ZIPs gene expression responded remarkably to Cd stress, while unexpectedly it was found that Arabidopsis and rice showed a distinct pattern in ZIPs genes expression profile. These results may help to elucidate the plants’ genetic basis for Cd translocation via a ZIPs-dependent pathway.

Materials and methods

Bioinformatics

Genomic query of Arabidopsis and rice ZIP family genes was performed online using the PLAZA database (http://bioinformatics.psb.ugent.be/plaza/). The sequences of 27 ZIP genes were retrieved manually from the TAIR database (http://www.arabidopsis.org/index.jsp) and the TIGR database (http://rice.plantbiology.msu.edu/index.shtml).

TM regions and other domains of the identified ZIPs gens were predicted through the TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and UniProtKB database (http://www.uniprot.org/), following a routine procedure.

Experimental design

Arabidopsis thaliana ecotype Col-0 and Oryza sativa ssp. japonica (cv. Taichung65) were subject to Cd inhibition test. For Arabidopsis, plants were germinated on Murashige and Skoog (MS; pH 5.7) solid medium containing 1% (w/v) sucrose. A total of 60 1-week-old seedlings were transferred to MS (control) or MS with 300 μM CdCl2 (Cd stress treatment) solid medium, and grown for 3 days in a controlled chamber environment under a 16/8 h photoperiod at 22 °C. For rice, seedlings were germinated hydroponically in distilled water. A total of six 10-day-old seedlings were then subject to a hydroponic culture in distilled water (control) or 300 μM CdCl2 solution (Cd stress treatment) for 3 days under 16/8 h photoperiod at 25 °C. The Cd concentration used in this study was selected based on our pilot experiment.

After Cd stress treatment, the shoot and root tissues were harvested and frozen immediately in liquid nitrogen. Total RNA was isolated from the tissues using Trizol reagent (Invitrogen, Corp., Carlsbad, CA, USA) and treated with DNase I (Promega, Madison, WI, USA). A total of 5 μg RNA was used for reverse transcription with PrimeScript™ RT reagent Kit (Takara Biotechnology Co. Ltd., Dalian, China) following the manufacturer’s protocol.

Quantitative Real-Time PCR (qPCR) was performed in a Bio-Rad CFX Connect™ Real-Time PCR Detection System (Hercules, CA, USA) using a SYBR Green Premix Ex Taq (Takara). The PCR parameters were set as: 95  °C for 5 min, followed by 40 cycles of 95  °C for 10 s and 60  °C for 30 s. Arabidopsis ACTIN gene (GenBank accession number NM_179953) and rice ACTIN gene (GenBank accession number XM_015774830) were used as internal references. Relative gene expression levels were detected using the 2−ΔΔCT method (Livak and Schmittgen 2001). Gene expression level was normalized using shoot expression level of each gene in the controls as a calibrator. All primer sequences are listed in the Additional file 1: Table S1.

Cd sensitivity analysis was performed using drop assay. Full-length coding sequence (CDS) was obtained via PCR amplification (see primers in Additional file 1: Table S2), and ligated into pCEV-G1-Km vector under the PGK1 promoter. The recombinant plasmids were then introduced into Saccharomyces cerevisiae (strain AH109) using a lithium acetate-based method. Transformed cells were cultured in Yeast Extract Peptone Dextrose (YPD) media with 300 μg/mL geneticin (G418), harvested by centrifugation, and resuspended in water (OD600 = 1.0), followed by a serial dilution. A total of 5 μL of each dilution was inoculated onto the YPD plates containing 300 μg/mL G418 and 50 μM CdCl2. Cells harbouring empty pCEV-G1-Km were used as a negative control. The plates were incubated at 28 °C for 5 days and the growth of the colonies was subsequently observed.

For the determination of Cd concentration in transformed yeast cells, cells expressing ZIPs were harvested after 12 h with 50 μM CdCl2 treatment. Cd was determined using a flame atomic absorption spectrometry (F-AAS) quantitative method. In Brief, cells in the liquid culture were harvested by centrifugation at 4000×g and washed three times with 3% NaCl solution. The cells were then oven-dried, weighed and digested using 4 mL 65% HNO3. The digested mixture was dissolved in 3 mL Millipore® water and subject to Cd determination using a Zeenit 700 P Atomic Absorption Spectrometer (Analytik Jena, Germany) equipped with a flame atomizer. CRM Laver (GWB10023, certified by IGGE) was used as a standard reference material for Cd determination.

Data analysis

Phylogenetic analysis was performed using MEGA 7 (Kumar et al. 2016). The model of ZIP gene structure was constructed using Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/).

Statistical analysis was performed using SPSS 21.0 (IBM, New York, USA). Unpaired two-tailed t test was performed for comparison between the controls and the Cd stress group.

Results and discussion

In this study, 15 candidate ZIP genes from Arabidopsis and 12 from rice were screened out based on sequence similarity. The number of ZIPs identified here was similar to previous studies (Ivanov and Bauer 2017; Guerinot 2000). Evolutionary analysis further indicated that all of these ZIP genes contain 1–3 introns (Additional file 1: Figures S1 and S2), whose protein precursors comprise eight TM regions (~ 20 aa length), one variable region with a conserved HG repeat and a typical signal peptide (SP) located on the N-terminal (Fig. 1). AtZIP13 and OsZIP13, which were previously annotated as putative Zn transporter (Ivanov and Bauer 2017), contain more TM domains. AtZTP29, AtIAR1, OsIAR1, OsZIP11 and OsZIP12 contain more than 10 exons. These ZIP-like genes seem to be phylogenetically distant from SpZRT1 and AtIRT1 and were not tested in this study. Phylogenetic clustering of the tested 27 ZIPs identified three subgroups, which is similar to previous study (Ivanov and Bauer 2017), namely the seed plant-specific group, the mixed plant group, and the mixed group1/2 (Fig. 1).
Fig. 1
Fig. 1

Phylogenetic relationship of identified ZIPs in Arabidopsis and rice. The Neighbor-Joining tree was generated using MEGA7 with 1000 bootstrap replicates, and rooted to the AtNRAMP1. Topological structure was predicted using UniProtKB and TMHMM. Black boxes indicate TM regions. Pink boxes indicate extracellular regions. Blue boxes represent cytoplasmic regions. Gray boxes represent luminal regions. Yellow boxes represent SP region. Uncharacteristic SPs are represented by the white boxes

Most previous studies on ZIPs’ biological functions focused on Zn/Fe/Mn/Cu uptake in yeast cells (Table 1), and expression profile of most ZIPs (except for AtIRT1/2 and OsIRT1/2) in response to Cd remained unknown. In this study, the genome-wide expression profile of ZIP genes in response to Cd stress in Arabidopsis and rice were quantified using qPCR. To induce a substantial stress response, 300 μM Cd in culture medium was applied based on our pilot experiment. The 3 days’ treatment obviously inhibited seedling growth and root elongation of both Arabidopsis and rice, and rice seedling height was also reduced (Fig. 2a). It was reported that even moderate Cd exposure can cause toxic symptoms and increased Cd accumulation in Arabidopsis (Fan et al. 2014) and rice (Rafiq et al. 2014). The Cd level used here significantly reduced the root length and seedling dry weight (Additional file 1: Figure S3), and was thus supposed to induce rapid expressional changes in the tested plants.
Table 1

Locations and known functions of ZIP proteins in Arabidopsis and rice

Gene name

Locus

Complementation of yeast metal uptake mutants (y/n)

Validated location in plant

Experimental evidence for potential function in Cd uptake

References

AtIRT1

At4g19690

fet3fet4 (Y); ∆zrt1zrt2 (Y); ∆ctr1 (N); ∆smf1 (Y)

Early endosome, vacuole, trans-Golgi network and cell membrane; root epidermis, flower

Increased Cd sensitivity of overexpression plant/yeast

Reduced Cd sensitivity of irt1

Inhibited IRT1-dependent Fe/Mn/Zn uptake by Cd in yeast

Reduced Cd uptake of irt1

Eide et al. (1996), Vert et al. (2002, 2001), Korshunova et al. (1999), Rogers et al. (2000), Connolly et al. (2002), Varotto et al. (2002), Henriques et al. (2002), Nishida et al. (2011), Shin et al. (2013), Potocki et al. (2013), Fan et al. (2014), Barberon et al. (2014), Blum et al. (2014)

AtIRT2

At4g19680

fet3fet4 (Y); ∆zrt1zrt2 (Y); ∆smf1 (N)

Intracellular vesicles; root epidermis

No altered Cd sensitivity of overexpression yeast

Increased Cd uptake and IRT1 expression of overexpression plant

Vert et al. (2001, 2009), Wintz et al. (2003), Varotto et al. (2002)

AtIRT3

At1g60960

Spzrt1 (Y); ∆zrt1zrt2 (Y); ∆fet3fet4 (Y); ∆smf1 (N)

Cell membrane; broadly expressed

No altered Cd sensitivity of overexpression yeast

Lin et al. (2009), Talke et al. (2006), Shanmugam et al. (2011), Hammes et al. (2005)

AtZIP1

At3g12750

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (Y)

Vacuolar; predominantly root stele and leaf vasculature

Inhibited ZIP1-dependent Zn uptake by Cd in yeast, to a less extent

Grotz et al. (1998), Milner et al. (2013)

AtZIP2

At5g59520

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (Y); ∆ctr1 (Y)

Cell membrane; predominantly mature root stele

Inhibited ZIP2-dependent Zn uptake by Cd in yeast

Grotz et al. (1998), Milner et al. (2013), Wintz et al. (2003)

AtZIP3

At2g32270

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (N)

Predominantly root

Inhibited ZIP3-dependent Zn uptake by Cd in yeast, to a less extent

Grotz et al. (1998), Talke et al. (2006), Milner et al. (2013)

AtZIP4

At1g10970

zrt1zrt2 (N); ∆fet3fet4 (N); ∆ctr1 (Y)

Root and leaf

N/A

Grotz et al. (1998), Talke et al. (2006), Wintz et al. (2003)

AtZIP5

At1g05300

zrt1zrt2 (N); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (Y)

Root

N/A

Milner et al. (2013), Wintz et al. (2003)

AtZIP6

At2g30080

zrt1zrt2 (N); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (Y)

Root

N/A

Milner et al. (2013), Hammes et al. (2005)

AtZIP7

At2g04032

zrt1zrt2 (Y); ∆fet3fet4 (Y); ∆ctr1ctr3 (N); ∆smf1 (Y)

N/A

N/A

Milner et al. (2013)

AtZIP8

At5g45105

zrt1zrt2 (N); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (N)

N/A

N/A

Milner et al. (2013)

AtZIP9

At4g33020

zrt1zrt2 (N); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (Y)

Root and shoot

N/A

Talke et al. (2006), Milner et al. (2013), Wintz et al. (2003), Inaba et al. (2015)

AtZIP10

At1g31260

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (N)

N/A

N/A

Milner et al. (2013)

AtZIP11

At1g55910

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (N)

N/A

N/A

Milner et al. (2013)

AtZIP12

At5g62160

zrt1zrt2 (Y); ∆fet3fet4 (N); ∆ctr1ctr3 (N); ∆smf1 (N)

Root

N/A

Milner et al. (2013), Inaba et al. (2015)

OsIRT1

LOC_Os03g46470

fet3fet4 (Y); ∆ctr1 (N); ∆zrt1zrt2 (N); ∆smf1 (N); ∆frt1fet4fre1(Y); ∆frt1fet1fre3(Y)

Cell membrane; mainly in root epidermis (the inner layer of the cortex, and the stele) and stems (companion cells)

Increased Cd sensitivity of overexpression plant

Increased Cd sensitivity and Cd uptake of overexpression yeast

Ishimaru et al. (2006), Bughio et al. (2002), Lee and An (2009), Nakanishi et al. (2006), Ishimaru et al. (2005)

OsIRT2

LOC_Os03g46454

fet3fet4 (Y); ∆ctr1 (N); ∆zrt1zrt2 (N); ∆smf1 (N)

Cell membrane

Increased Cd sensitivity and Cd uptake of overexpression yeast

Ishimaru et al. (2006), Nakanishi et al. (2006)

OsZIP1

LOC_Os01g74110

zrt1zrt2 (Y); ∆smf1 (Y); ∆fet3fet4 (N)

Broadly expressed

Increased Cd sensitivity of overexpression yeast

Inhibited ZIP1-dependent Zn uptake by Cd in yeast

Ramesh et al. (2003, Ishimaru et al. (2005), Chen et al. (2008)

OsZIP2

LOC_Os03g29850

zrt1zrt2 (N);

N/A

No altered Cd sensitivity of overexpression yeast

Ramesh et al. (2003)

OsZIP3

LOC_Os04g52310

zrt1zrt2 (Y); ∆smf1 (Y); ∆fet3fet4 (N)

Mainly induced by zinc deficiency to higher levels in roots

No altered Cd sensitivity of overexpression yeast

Mildly increased ZIP3-dependent Zn uptake by Cd in yeast

Ramesh et al. (2003, Ishimaru et al. (2005), Chen et al. (2008)

OsZIP4

LOC_Os08g10630

zrt1zrt2 (Y); ∆frt1fet1fre3(N)

Cell membrane; phloem cells of leaves, roots and meristem

N/A

Ishimaru et al. (2005), Chen et al. (2008)

OsZIP5

LOC_Os05g39560

zrt1zrt2 (Y)

Cell membrane; mainly panicle

N/A

Chen et al. (2008), Lee et al. (2010a)

OsZIP6

LOC_Os05g07210

N/A

Root, shoot and panicle

Increased Cd uptake of overexpression cells

Chen et al. (2008)

OsZIP7

LOC_Os05g10940

N/A

Root, shoot and panicle

N/A

Chen et al. (2008), Yang et al. (2009)

OsZIP8

LOC_Os07g12890

N/A

Cell membrane; mainly root and panicle

N/A

Chen et al. (2008), Lee et al. (2010b), Yang et al. (2009)

OsZIP9

LOC_Os05g39540

N/A

Root, shoot and panicle

N/A

Chen et al. (2008)

OsZIP10

LOC_Os06g37010

N/A

N/A

N/A

N/A

N/A represents not available

Fig. 2
Fig. 2

Expression profiles of ZIP genes of Arabidopsis and rice in response to Cd stress. a 1-week-old and 10-day-old seedlings of Arabidopsis and rice were treated with 300 μM CdCl2 for 3 days. Scale bars indicate 1 cm. b Changes in the expression of the 26 ZIP genes in response to Cd exposure Gene expression level was normalized using shoot expression level of each gene in the controls as the calibrator. (n = 3, Student t test, *indicates P < 0.05, **indicates P < 0.01)

In Arabidopsis, Cd exposure impacted the expression of all ZIPs significantly. Strikingly, AtIRT1 was induced with a 525-fold increase in shoot and a 22-fold increase in root (Fig. 2b). As abovementioned, some evidence already pointed to the Cd transport role of AtIRT1 in yeast cells (Korshunova et al. 1999; Rogers et al. 2000; Vert et al. 2001; Eide et al. 1996) and in Arabidopsis (Fan et al. 2014; Connolly et al. 2002; Vert et al. 2002). Considering that AtIRT1 is mainly expressed in root (Vert et al. 2002), AtIRT1 may function as a pump absorbing Cd from soil into root under sever Cd stress. A sharp increase of AtIRT1 expression in shoot was also observed, indicating its potential role in Cd transport in shoot. Indeed, overexpression of AtIRT1 in yeast increased the hosts’ sensitivity substantially (Fig. 3). Cd accumulation of yeast cells expressing AtIRT1 was also increased by 40.1%, compared with the control (Additional file 1: Figure S4). Taken together, the results here further confirmed the role of AtIRT1 in plant Cd uptake implied in previous studies (Rogers et al. 2000).
Fig. 3
Fig. 3

Drop assay for Cd sensitivity of yeast cells (S. cerevisiae AH109) expressing representative ZIPs tested in this study. The transformed cells expression ZIPs were subjected to a serial dilution (0–10−4) drop assay on YPD plates. 300 μg/mL G418 was added to maintain the vectors. Plates containing 50 μM CdCl2 were incubated at 28 °C for 5 days and growth state was subsequently observed. This experiment was performed three times

Like AtIRT1, AtIRT2 was induced with a 1452-fold increase in shoot and a fourfold increase in root (Fig. 2b). Previous studies showed that AtIRT2 overexpression increased Cd uptake of transgenic Arabidopsis, probably through the induction of AtIRT1 expression (Vert et al. 2001, 2009). In this study, while both AtIRT2 and AtIRT1 were coincidently sharply induced when subject to Cd stress, overexpression of AtIRT2 caused no significant changes in neither Cd sensitivity nor Cd accumulation (Fig. 3 and Additional file 1: Figure S4). It is thus very likely that AtIRT2 worked indirectly and synergistically with AtIRT1 in response to the Cd stress.

It was also highlighted that the expression of AtZIP9 was significantly increased by ninefolds in shoot and 57-folds in root after Cd stress (Fig. 2b). Till now no evidence showed any role of AtZIP9 in Cd uptake. The strong induction by Cd stress may imply its role in Cd transport, and its overexpression in yeast cells moderately increased hosts’ sensitivity to Cd. Conversely, expression of AtZIP9 did not increase the Cd accumulation of host cells (Additional file 1: Figure S4). As a hypothetic transmembrane ion transporter, AtZIP9 might affect the growth of host cells by a Cd-independent way. In addition, AtIRT3, AtZIP4, AtZIP5, AtZIP11 and AtZIP12 showed converse expression regulation when subject to the tested Cd stress, and AtZIP7 was reduced in shoot and was under the detection limit in root (Fig. 2b). Their potential roles in Cd transport merit a further investigation.

In rice, homologous ZIPs responded differently from Arabidopsis to the Cd stress. Unlike in Arabidopsis, Cd stress increased the expression of most rice ZIPs in shoot but not root. These results imply that all these Cd-induced ZIPs involve in plant response to Cd. Except for OsIRT2, all ZIPs were significantly induced in rice shoot (Fig. 2b). Like AtIRT3, expression changes of OsIRT1 and OsZIP1 were converse in shoot and root (Fig. 2b). The positive role of OsIRT1 and OsZIP1 was demonstrated in the response of yeast and/or plant to Cd stress (Nakanishi et al. 2006; Lee and An 2009; Ramesh et al. 2003). Rice over-expressing OsIRT1 showed reduced plant height and increased Cd accumulation under 300 μM Cd stress (Lee and An 2009), and the growth of OsZIP1-expressing yeast cells was inhibited by 10 μM Cd stress. In this study, the expression regulation of OsIRT1 and OsZIP1 in response to Cd stress was contrary between root and shoot. Rice might have a feedback regulation of OsIRT1 and OsZIP1 in root to prevent increasing Cd uptake from soil.

OsZIP1-10 were subject to Cd sensitivity and Cd accumulation tests. The expression of OsZIP1 and OsZIP3 in yeast caused an increased Cd sensitivity and Cd accumulation (Fig. 3 and Additional file 1: Figure S4), suggesting their potential roles in Cd uptake. This result is different from those by Ramesh et al. (2003), where yeast ZHY3 strains were used and different culture medium was applied. It was also noticed that OsZIP6 did not caused an obvious increasing in Cd sensitivity (Fig. 3). This is not consistence with previous report, in which Xenopus laevis oocytes was used to test the Cd sensitivity (Kavitha et al. 2015). Different host and micro-environment may cause the altered conformation and activity of tested proteins. Expression of OsZIP5-10 failed to alter Cd sensitivity and Cd accumulation of host cells obviously, implying that these ZIPs probably did not uptake Cd individually. Considering that AtIRT2 involves in indirect Cd uptake in Arabidopsis, these Cd-induced ZIPs may also play roles in Cd uptake or transport indirectly. Their potential roles under Cd stress need further investigation using transgenic plants.

Indeed, this study showed that many ZIPs were significantly induced by Cd stress even the growth of seedling was inhibited obviously, and some of them increased hosts’ Cd sensitivity or Cd accumulation. These results will help to elucidate the genetic basis for Cd accumulation via a ZIP-dependent pathway in plants. Further analysis using transgenic plants will clarify the biological function of these ZIPs in plant Cd uptake and transport.

Conclusions

In conclusion, this study revealed a distinct pattern in ZIPs genes expression regulation in response to Cd stress between Arabidopsis and rice. Arabidopsis mainly up-regulated root ZIPs genes, while rice mainly up-regulated shoot ZIPs genes. Interestingly, some genes like AtIRT3, AtZIP5, AtZIP12, OsIRT1 and OsZIP1 showed contrary expression regulation when subject to the tested Cd stress. Three genes, AtIRT1, OsZIP1 and OsZIP3, conferred an increased sensitivity to Cd stress and more Cd accumulation when expressed in yeast cells, implying a role in direct Cd uptake in plants.

Abbreviations

ZIPs: 

zinc(Zn)-regulated/iron(Fe)-regulated transporter-like family proteins

Cd: 

cadmium

TM: 

transmembrane

Zn: 

zinc

Fe: 

iron

Mn: 

manganese

Cu: 

copper

Declarations

Authors’ contributions

XZ and XL initiated the project. XZ designed the experiment. XZ and LC carried out the experiments and analyzed the data. All authors wrote and revised the manuscript. All authors read and approved the final mansucript.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Not applicable.

Consent for publication

Authors agree to the terms of the Springer Open Copyright and License Agreement.

Ethics approval and consent to participate

Not applicable.

Funding

This work was supported by the Pioneer “Hundred Talents Program” of the Chinese Academy of Sciences (Y726012203), the National Key Research and Development Plan (2018YFD0800306) and the Hebei Science Fund for Distinguished Young Scholars (D2018503005).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Key Laboratory for Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang, 050021, Hebei, People’s Republic of China
(2)
CMLR, Sustainable Minerals Institute, The University of Queensland, Brisbane, QLD, 4072, Australia

References

  1. Barberon M, Dubeaux G, Kolb C, Isono E, Zelazny E, Vert G (2014) Polarization of iron-regulated transporter 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc Natl Acad Sci USA 111(22):8293–8298. https://doi.org/10.1073/pnas.1402262111 View ArticlePubMedGoogle Scholar
  2. Blum A, Brumbarova T, Bauer P, Ivanov R (2014) Hormone influence on the spatial regulation of IRT1 expression in iron-deficient Arabidopsis thaliana roots. Plant Signal Behav 9(4):e28787View ArticlePubMed CentralGoogle Scholar
  3. Bughio N, Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S (2002) Cloning an iron-regulated metal transporter from rice. J Exp Bot 53(374):1677–1682View ArticleGoogle Scholar
  4. Chen WR, Feng Y, Chao YE (2008) Genomic analysis and expression pattern of OsZIP1, OsZIP3, and OsZIP4 in two rice (Oryza sativa L.) genotypes with different zinc efficiency. Russ J Plant Physl+ 55(3):400–409. https://doi.org/10.1134/s1021443708030175 View ArticleGoogle Scholar
  5. Connolly EL, Fett JP, Guerinot ML (2002) Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell 14(6):1347–1357View ArticlePubMed CentralGoogle Scholar
  6. Eide D, Broderius M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 93(11):5624–5628View ArticleGoogle Scholar
  7. Eng BH, Guerinot ML, Eide D, Saier MH Jr (1998) Sequence analyses and phylogenetic characterization of the ZIP family of metal ion transport proteins. J Membr Biol 166(1):1–7View ArticleGoogle Scholar
  8. Fan SK, Fang XZ, Guan MY, Ye YQ, Lin XY, Du ST, Jin CW (2014) Exogenous abscisic acid application decreases cadmium accumulation in Arabidopsis plants, which is associated with the inhibition of IRT1-mediated cadmium uptake. Front Plant Sci 5:721. https://doi.org/10.3389/fpls.2014.00721 View ArticlePubMedPubMed CentralGoogle Scholar
  9. Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D (1998) Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci USA 95(12):7220–7224View ArticleGoogle Scholar
  10. Guerinot ML (2000) The ZIP family of metal transporters. Biochim Biophys Acta 1465(1–2):190–198View ArticleGoogle Scholar
  11. Hammes UZ, Schachtman DP, Berg RH, Nielsen E, Koch W, McIntyre LM, Taylor CG (2005) Nematode-induced changes of transporter gene expression in Arabidopsis roots. Mol Plant Microbe Interact 18(12):1247–1257. https://doi.org/10.1094/MPMI-18-1247 View ArticlePubMedGoogle Scholar
  12. Henriques R, Jasik J, Klein M, Martinoia E, Feller U, Schell J, Pais MS, Koncz C (2002) Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects. Plant Mol Biol 50(4–5):587–597View ArticleGoogle Scholar
  13. Inaba S, Kurata R, Kobayashi M, Yamagishi Y, Mori I, Ogata Y, Fukao Y (2015) Identification of putative target genes of bZIP19, a transcription factor essential for Arabidopsis adaptation to Zn deficiency in roots. Plant J 84(2):323–334. https://doi.org/10.1111/tpj.12996 View ArticlePubMedGoogle Scholar
  14. Ishimaru Y, Suzuki M, Kobayashi T, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2005) OsZIP4, a novel zinc-regulated zinc transporter in rice. J Exp Bot 56(422):3207–3214. https://doi.org/10.1093/jxb/eri317 View ArticlePubMedGoogle Scholar
  15. Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2006) Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. Plant J 45(3):335–346. https://doi.org/10.1111/j.1365-313X.2005.02624.x View ArticlePubMedGoogle Scholar
  16. Ivanov R, Bauer P (2017) Sequence and coexpression analysis of iron-regulated ZIP transporter genes reveals crossing points between iron acquisition strategies in green algae and land plants. Plant Soil 418(1–2):61–73. https://doi.org/10.1007/s11104-016-3128-2 View ArticleGoogle Scholar
  17. Kavitha PG, Kuruvilla S, Mathew MK (2015) Functional characterization of a transition metal ion transporter, OsZIP6 from rice (Oryza sativa L.). Plant Physiol Biochem 97:165–174. https://doi.org/10.1016/j.plaphy.2015.10.005 View ArticleGoogle Scholar
  18. Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB (1999) The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Mol Biol 40(1):37–44View ArticleGoogle Scholar
  19. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054 View ArticlePubMedGoogle Scholar
  20. Lee S, An G (2009) Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell Environ 32(4):408–416. https://doi.org/10.1111/j.1365-3040.2009.01935.x View ArticlePubMedGoogle Scholar
  21. Lee S, Jeong HJ, Kim SA, Lee J, Guerinot ML, An G (2010a) OsZIP5 is a plasma membrane zinc transporter in rice. Plant Mol Biol 73(4–5):507–517. https://doi.org/10.1007/s11103-010-9637-0 View ArticlePubMedGoogle Scholar
  22. Lee S, Kim SA, Lee J, Guerinot ML, An G (2010b) Zinc deficiency-inducible OsZIP8 encodes a plasma membrane-localized zinc transporter in rice. Mol Cells 29(6):551–558. https://doi.org/10.1007/s10059-010-0069-0 View ArticlePubMedGoogle Scholar
  23. Li S, Zhou X, Huang Y, Zhu L, Zhang S, Zhao Y, Guo J, Chen J, Chen R (2013) Identification and characterization of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) gene family in maize. BMC Plant Biol 13:114. https://doi.org/10.1186/1471-2229-13-114 View ArticlePubMedPubMed CentralGoogle Scholar
  24. Lin YF, Liang HM, Yang SY, Boch A, Clemens S, Chen CC, Wu JF, Huang JL, Yeh KC (2009) Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter. New Phytol 182(2):392–404. https://doi.org/10.1111/j.1469-8137.2009.02766.x View ArticlePubMedGoogle Scholar
  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262 View ArticleGoogle Scholar
  26. Lopez-Millan AF, Ellis DR, Grusak MA (2004) Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula. Plant Mol Biol 54(4):583–596. https://doi.org/10.1023/B:PLAN.0000038271.96019.aa View ArticlePubMedGoogle Scholar
  27. Milner MJ, Seamon J, Craft E, Kochian LV (2013) Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. J Exp Bot 64(1):369–381. https://doi.org/10.1093/jxb/ers315 View ArticlePubMedPubMed CentralGoogle Scholar
  28. Nakanishi H, Ogawa I, Ishimaru Y, Mori S, Nishizawa NK (2006) Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+ transporters OsIRT1 and OsIRT2 in rice. Soil Sci Plant Nutr 52(4):464–469. https://doi.org/10.1111/j.1747-0765.2006.00055.x View ArticleGoogle Scholar
  29. Nishida S, Tsuzuki C, Kato A, Aisu A, Yoshida J, Mizuno T (2011) AtIRT1, the primary iron uptake transporter in the root, mediates excess nickel accumulation in Arabidopsis thaliana. Plant Cell Physiol 52(8):1433–1442. https://doi.org/10.1093/pcp/pcr089 View ArticlePubMedGoogle Scholar
  30. Potocki S, Valensin D, Camponeschi F, Kozlowski H (2013) The extracellular loop of IRT1 ZIP protein–the chosen one for zinc? J Inorg Biochem 127:246–252. https://doi.org/10.1016/j.jinorgbio.2013.05.003 View ArticlePubMedGoogle Scholar
  31. Rafiq MT, Aziz R, Yang XE, Xiao WD, Rafiq MK, Ali B, Li TQ (2014) Cadmium phytoavailability to rice (Oryza sativa L.) grown in representative Chinese soils. A model to improve soil environmental quality guidelines for food safety. Ecotox Environ Safe 103:101–107. https://doi.org/10.1016/j.ecoenv.2013.10.016 View ArticleGoogle Scholar
  32. Ramesh SA, Shin R, Eide DJ, Schachtman DP (2003) Differential metal selectivity and gene expression of two zinc transporters from rice. Plant Physiol 133(1):126–134View ArticlePubMed CentralGoogle Scholar
  33. Rogers EE, Eide DJ, Guerinot ML (2000) Altered selectivity in an Arabidopsis metal transporter. Proc Natl Acad Sci USA 97(22):12356–12360. https://doi.org/10.1073/pnas.210214197 View ArticlePubMedGoogle Scholar
  34. Shanmugam V, Lo JC, Wu CL, Wang SL, Lai CC, Connolly EL, Huang JL, Yeh KC (2011) Differential expression and regulation of iron-regulated metal transporters in Arabidopsis halleri and Arabidopsis thaliana–the role in zinc tolerance. New Phytol 190(1):125–137. https://doi.org/10.1111/j.1469-8137.2010.03606.x View ArticlePubMedGoogle Scholar
  35. Shin LJ, Lo JC, Chen GH, Callis J, Fu H, Yeh KC (2013) IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis. Plant Cell 25(8):3039–3051. https://doi.org/10.1105/tpc.113.115212 View ArticlePubMedPubMed CentralGoogle Scholar
  36. Stephens BW, Cook DR, Grusak MA (2011) Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume Medicago truncatula. Biometals 24(1):51–58. https://doi.org/10.1007/s10534-010-9373-6 View ArticlePubMedGoogle Scholar
  37. Talke IN, Hanikenne M, Kramer U (2006) Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol 142(1):148–167. https://doi.org/10.1104/pp.105.076232 View ArticlePubMedPubMed CentralGoogle Scholar
  38. Tiong J, McDonald G, Genc Y, Shirley N, Langridge P, Huang CY (2015) Increased expression of six ZIP family genes by zinc (Zn) deficiency is associated with enhanced uptake and root-to-shoot translocation of Zn in barley (Hordeum vulgare). New Phytol 207(4):1097–1109. https://doi.org/10.1111/nph.13413 View ArticlePubMedGoogle Scholar
  39. Varotto C, Maiwald D, Pesaresi P, Jahns P, Salamini F, Leister D (2002) The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana. Plant J 31(5):589–599View ArticleGoogle Scholar
  40. Vert G, Briat JF, Curie C (2001) Arabidopsis IRT2 gene encodes a root-periphery iron transporter. Plant J 26(2):181–189View ArticlePubMed CentralGoogle Scholar
  41. Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat JF, Curie C (2002) IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell 14(6):1223–1233View ArticlePubMed CentralGoogle Scholar
  42. Vert G, Barberon M, Zelazny E, Seguela M, Briat JF, Curie C (2009) Arabidopsis IRT2 cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells. Planta 229(6):1171–1179. https://doi.org/10.1007/s00425-009-0904-8 View ArticlePubMedGoogle Scholar
  43. Wintz H, Fox T, Wu YY, Feng V, Chen W, Chang HS, Zhu T, Vulpe C (2003) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis. J Biol Chem 278(48):47644–47653. https://doi.org/10.1074/jbc.M309338200 View ArticlePubMedGoogle Scholar
  44. Yang X, Huang J, Jiang Y, Zhang HS (2009) Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.). Mol Biol Rep 36(2):281–287. https://doi.org/10.1007/s11033-007-9177-0 View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2018

Advertisement