ZOU Wenli , LI Chang , ZHU Yajun CHEN Jingguang HE Haohua YE Guoyou

(1Group of Crop Genetics and Breeding, School of Agriculture Science, Jiangxi Agricultural University, Nanchang 330045, China;2CAAS-IRRI Joint Laboratory for Genomics-Assisted Germplasm Enhancement, Agricultural Genomics Institute in Shenzhen,Chinese Academy of Agricultural Sciences, Shenzhen 518057, China; 3College of Resources and Environment, Henan Agricultural University, Zhengzhou 450002, China; #These authors contributed equally to this work)

Abstract: P1B-type heavy metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. We here reported the characterization of rice OsHMA6, a member of the P1B-type ATPase family. Phylogenetic tree analysis showed that OsHMA6 belonged to the Cu/Ag subgroup of the HMA family and had a close evolutionary relationship with OsHMA9. Amino acid sequence alignment showed 82.78% consistency between OsHMA6 and OsHMA9. OsHMA6 expressed in all organs at different growth stages, including spikelet, and abundant in leaf blades, however,OsHMA9 most strongly expressed in roots, but very low in spikelet. Excessive Cu2+ can up-regulate the expression of OsHMA6 and OsHMA9 in rice seedlings. The heterologous expression in yeast showed that OsHMA6 can significantly rescue the growth of yeast strain CM52 when supplied with 3 or 6 mmol/L Cu2+. Compared with the empty vector pYES2, the Cu concentration in OsHMA6-pYES2 decreased by 23.4% and 30.3% under 3 or 6 mmol/L Cu2+, respectively. Subcellular localization revealed that OsHMA6 was located in the plasma membrane. These results suggested that OsHMA6, similar to OsHMA9, is likely a copper efflux protein located in the plasma membrane.

Key words: OsHMA6; P1B-type ATPase; copper; cadmium; efflux; rice; subcellular localization

Copper (Cu) is an essential micronutrient, which plays a key role in many plant processes such as respiration,photosynthesis, oxidative stress, nitrogen and carbon metabolism, hormone perception and cell wall synthesis(Pilon et al, 2006; Hansch and Mendel, 2009). Cu deficiency in plants can lead to growth retardation,yellowing of young leaves and development defects(Klaumann et al, 2011). Cu deficiency also impairs pollen fertility and reduces growth rate, seed-setting rate and yield (Huang et al, 2016). Cu also plays an important role in human health (Collins and Klevay,2011; Gulec and Collins, 2014). Cu deficiency causes anaemia and immune defects (Collins and Klevay,2011). At present, more than two billion people worldwide suffer from micronutrient deficiencies,such as Cu, iron (Fe) and zinc (Zn) (White and Broadley, 2009).

Similarly, excessive Cu is toxic to plants (Abdel-Ghany et al, 2005; Andrés-Colás et al, 2006) and gives rise to plant root growth inhibition, biomass reduction and plant necrosis (Navari-Izzo et al, 2006;Lequeux et al, 2010; Huang et al, 2016). Cu toxicity can yellow leaves and reduce photosynthetic rate of plants (Alaoui-Sossé et al, 2004; Bernal et al, 2006).Cu toxicity also reduces iron uptake by plants(Alaoui-Sossé et al, 2004). Thylakoid membranes in chloroplasts are also major targets of Cu toxicity(Bernal et al, 2004).

Both deficiency and excess of copper ions are harmful to plants, so plants have established a precise mechanism to balance the concentration of copper ions in the system and cells (Burkhead et al, 2009). Cu transporters play an important role in the key steps of absorption, chelation, compartmentalization and metabolic utilization of Cu2+(Li et al, 2018). Cu uptake related proteins mainly include copper transporter (COPT)protein family (Kampfenkel et al, 1995; Sancenón et al,2004; Andrés-Colás et al, 2010; Yuan et al, 2011;Jung et al, 2012), zinc-regulated transporter (ZIP)protein family (Sancenón et al, 2003; Wintz et al,2003; Colangelo and Guerinot, 2006) and yellow stripe-like (YSL) protein family (Waters et al, 2006;Ueno et al, 2009; Zhang et al, 2018).

At present, the identified copper-excreting proteins are mainly from the members of the P1B-type ATPases family known as heavy metal ATPases (HMAs).HMAs play roles in plant metal transport (Takahashi et al, 2012). By hydrolyzing ATP, HMAs mainly export metal cations (Argüello et al, 2007; Morel et al,2009). During plant growth, HMAs can selectively uptake and transport not only essential metal ions such as Zn2+and Cu2+, but also non-essential ions such as plumbum (Pb2+) and cadmium (Cd2+) (Takahashi et al,2012; Migocka et al, 2015a). When expressed in yeast,CsHMA5.1 and CsHMA5.2 are located in the vacuole membrane, and help to detoxify Cu2+by yeast(Migocka et al, 2015b). SvHMA5I and SvHMA5II are localized in the vacuole membrane and endoplasmic reticulum, respectively, and their expression alone can improve the Cu tolerance of Arabidopsis thaliana (Li et al, 2017). Huang et al (2016) reported that OsHMA4 is located in the vacuole membrane, which can store Cu in the vacuoles of root cells and reduce the accumulation of Cu in rice seeds. Arabidopsis AtHMA5, rice OsHMA5 and OsHMA9 are located in the plasma membrane, which are associated with the efflux of Cu from cells (Andrés-Colás et al, 2006; Lee et al, 2007; Kobayashi et al, 2008; Deng et al, 2013).In this study, we studied the function of OsHMA6 and found that OsHMA6 may be a Cu2+efflux protein localized into the plasma membrane.

MATERIALS AND METHODS

Rice materials and growth conditions

The OsHMA6 open reading frame (ORF) sequence was amplified from total cDNA isolated from Oryza sativa L. ssp. japonica cv. Wuyunjing 7 using primers 5′-ATGGCCCATCTCCAGCTCACGCCGC-3′ and 5′-TCACTCTACAGTTATTTGTAACAGG-3′, which were designed based on the sequence of Os02g0172600 from The Rice Annotation Project Database (https://rapdb.dna.affrc.go.jp/index.html). The PCR amplification product was ligated into a pMD19-T vector (TaKaRa Bio, Shiga, Japan) independently and sequenced.

The rice cultivar Wuyunjing 7 was used to study the expression patterns of OsHMA6 and OsHMA9 and dose-responses of OsHMA6 and OsHMA9 to Cu and Cd. The expression patterns were investigated by taking samples of different tissues at different growth stages grown in a paddy field at the Experimental Station of Nanjing Agricultural University in Nanjing,Jiangsu Province, China. To investigate the doseresponses of OsHMA6 and OsHMA9 expression to Cu and Cd, 14-day-old seedlings grown in a growth room with a 14 h light (8:00-22:00) at 30 °C/10 h dark(22:00-8:00) at 22 °C photoperiod and 60% relative humidity were exposed to various Cu concentrations(0.2, 2, 20, 50, 100, 200 and 500 μmol/L) or Cd concentrations (0, 20, 50, 100 and 200 μmol/L) for 3 d.

RNA extraction and real-time PCR (qRT-PCR)

Total RNAs were extracted using the TRIzol reagent(Vazyme Biotech Co. Ltd, Nanjing, China). DNaseItreated total RNAs were subjected to reverse transcription (RT) with the HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co. Ltd, Nanjing, China). Triplicate quantitative assays were performed using the 2× T5 Fast qPCR Mix (SYBRGreenI) kit (Vazyme Biotech Co. Ltd,Nanjing, China). The primers for qRT-PCR are given in Supplemental Table 1.

Heterologous expression of OsHMA6 in yeast

The wild-type yeast strain CM52 was used as a host strain. The full-length cDNA of OsHMA6 was amplified using OsHMA6-pMD19-T vector as a template and primers 5′-ttCGGATCCGATGGCCCATCTCCAGCT CACGCCGC-3′ and 5′-gcCTCTAGAGTCACTCTAC AGTTATTTGTAACAGG-3′. The PCR product was cleaved using BamHI and XbaI and ligated into a pYES2 vector with correct direction.

The empty vector pYES2 and the constructed vector OsHMA6-pYES2 were introduced into CM52 yeast cells, respectively, using the Yeast Transformation kit according to the manufacturer’s protocol (Beijing Kulaibo Technology Co. Ltd, China), and transformants were selected on synthetic dextrose media without uracil (SD-U). Positive clones were cultured in SD-U liquid medium until the early logarithmic phase, and then concentrated and washed three times with sterile distilled water. After sequential 10-fold dilution, 8 μL of the cell suspension were spotted on SD-U plates containing 0, 3 and 6 mmol/L CuSO4, or 0, 10 and 20 μmol/L CdCl2, respectively. The plates were incubated at 30 °C for 3 d before the growth phenotypes were evaluated.

The growth of CM52 yeast strain transformed with various plasmids in liquid SD-U media containing various concentrations of Cu2+or Cd2+was determined.Overnight yeast cells were prepared and the optical density (OD) at 600 nm was adjusted to 0.5 with sterile distilled water. Then, 20 μL of cell suspension was added to 20 mL liquid SD-U media containing 0,3, 6 mmol/L CuSO4or 20 μmol/L CdCl2in each bottle.The OD values at 600 nm were determined at indicated time. The cells of strains in liquid media for 30 h were collected by centrifugation, and washed three times with sterile deionized water. All samples were dried at 80 °C for 3 d, and then the metal concentration was determined by an inductively coupled plasma mass spectrometry (ICP-MS).

For the determination of OsHMA6 subcellular localization in yeast, OsHMA6 was fused to YFP by PCR and then the fused fragment was inserted into the pYES2 vector. The derived plasmids were then transformed into the CM52 yeast strain. The YFP fluorescence was observed with a confocal laser scanning microscopy (LSM410, Carl Zeiss, Germany).

Subcellular localization of OsHMA6

The coding sequence of OsHMA6 for subcellular localization was amplified using the OsHMA6-pMD19-T vector as a template and primers 5′-ccCCCGGGAT GGCCCATCTCCAGCTCACGCCGC-3′ and 5′-gcCG GATCCGTCACTCTACAGTTATTTGTAACAGG-3′.

The PCR product was cleaved using SmaI and BamHI and ligated into pSAT6A-GFP with correct direction.The OsHMA6:GFP vector was transformed into Arabidopsis mesophyll protoplasts and detected by a laser scanning microscope (LSM410, Carl Zeiss, Germany).Plasma membrane dye FMTM4-64FX (Invitrogen, Life Technologies, China) was used as a marker.

RESULTS

Sequence alignment of OsHMA6 and HMA family members

To study the evolution history of OsHMA6 gene in the HMA family, we conducted the phylogenic analysis of OsHMA and AtHMA families. Seventeen HMA genes were subdivided into two clades. The first clade had 10 genes and belonged to the Cu/Ag subgroup,while the second clade contained 7 genes and was the Zn/Cd/Co/Pb subgroup (Fig. 1). OsHMA6 was in the Cu/Ag subgroup and had a close evolutionary relationship with OsHMA9 (Fig. 1). The results of amino acid sequence alignment are given in Table 1.The amino acid sequence of OsHMA6 had the highest similarity (82.78%) to that of OsHMA9 (Table 1).

OsHMA6 and OsHMA9 have similar DNA structures,with nine exons and eight introns (Supplemental Fig.1-A). The CDS of OsHMA6 is 3 039 bp and encodes 1 013 amino acids, while the CDS of OsHMA9 is 3 012 bp and encodes 1 004 amino acids. The transmembrane topology model of OsHMA6 protein is similar to that of OsHMA9 as well. They both have seven transmembrane structures (Supplemental Fig. 1-B).

Expression patterns of OsHMA6 and OsHMA9 genes

OsHMA6 and OsHMA9 may have similar biological functions. OsHMA6 expressed in all tissues of rice atdifferent growth stages, especially in leaf blades,followed by roots, it was also expressed in spikelets(Fig. 2). OsHMA9 expressed most strongly in roots,followed by basal stems, leaf sheaths, and less expressed in leaf blades. The expression level was very low in spikelets (Fig. 2).

Table 1. Percentage of amino acid sequence similarity among OsHMAs.%

Compared with the control (0.2 μmol/L Cu2+), high concentrations of Cu2+can significantly induce the expression of OsHMA6 and OsHMA9, and the expression level of OsHMA6 was the highest at 100 μmol/L Cu2+, 5.9 times as much as that of the control (Fig. 3-A), and OsHMA9 was the highest at 200 μmol/L Cu2+, 3.8 times as much as that of the control (Fig. 3-B). The expression level of OsHMA6 was not significantly affected by exogenous Cd2+addition (Fig. 3-C). OsHMA9 expression increased significantly at 200 μmol/L Cd2+,2.5 times higher than that of the control (Fig. 3-D).

Heterologous expression of OsHMA6 in yeast

To determine the Cu2+and Cd2+transport activity of OsHMA6, OsHMA6 expressed in yeast strain CM52 was compared with the empty vector pYES2. The results showed that OsHMA6 can significantly rescue the growth of yeast strain CM52 when supplied with 3 or 6 mmol/L Cu2+compared with the empty vector(Fig. 4-A), suggesting that OsHMA6 possessed high Cu2+transport activity. However, there was no significant difference in the growth of OsHMA6 and pYES2 on the media containing 10 or 20 μmol/L Cd2+(Fig. 4-B), suggesting that OsHMA6 may not have Cd2+transport activity.

To determine the sensitivity of OsHMA6 to Cu2+and Cd2+in yeast strain CM52, we measured the growth of transformed CM52 strains in the SD-U liquid media containing 0, 3, 6 mmol/L Cu2+or 20 μmol/L Cd2+. There was no significant difference in the growth of OsHMA6 and pYES2 under the control medium and the medium containing 20 μmol/L Cd2+(Fig. 5-A and -D). The growth of OsHMA6 was significantly higher than that of pYES2 in the media containing 3 or 6 mmol/L Cu2+after 15 h (Fig. 5-B and -C). To further confirm this result, the metal concentrations in the 30-hour strains were analyzed.Compared with pYES2, the concentration of Cu2+in OsHMA6 decreased by 23.4% and 30.3% under 3 or 6 mmol/L Cu2+treatment, respectively (Fig. 5-F).However, the Cd2+concentration was not significantly different between OsHMA6 and pYES2 under 20 μmol/L Cd2+treatment (Fig. 5-E).

OsHMA6 protein is localized in plasma membrane

In order to study the subcellular localization of OsHMA6 protein, a vector expressing OsHMA6:GFP fusion by cauliflower mosaic virus 35S promoter was constructed and transformed into A. thaliana protoplasts.After overnight transformation, the expression of OsHMA6:GFP in protoplasts was observed. OsHMA6:GFP fluorescence was completely merged with plasma membrane dye FMTM4-64FX (Fig. 6). We also transfected OsHMA6:YFP-pYES2 into the CM52 yeast strain.After galactose-induced expression, we observed that YFP fluorescence was also localized in the plasma membrane (Supplemental Fig. 2). Therefore, OsHMA6 was identified as a plasma membrane localization protein.

DISCUSSION

Cu is a necessary trace element for plant growth and development (Pilon et al, 2006; Klaumann et al, 2011;Huang et al, 2016). Plants absorb Cu from soil through roots and then transport it to shoots (Che et al,2018). Cu must pass through the concentric root cell layer of the root epidermis and cortex and be transported to the central stele (Barberon and Geldner,2014). Plants need to store excess Cu in vacuoles or transport it outside cells to ensure the normal growth,and transport to extracellular processes requires an efflux transporter (Che et al, 2018). Therefore, plants need not only a Cu transporter to absorb Cu into cells,but also an efflux transporter (Che et al, 2018). The concentration of Cu in paddy fields varies from micromolar to millimolar (Kong et al, 2018). Excessive Cu supply can produce toxic effects on plants(Abdel-Ghany et al, 2005; Andrés-Colás et al, 2006).

OsHMA6 and OsHMA9 belong to the Cu/Ag transporter group in the HMA family (Fig. 1),suggesting that they may be related to the transport of Cu. OsHMA6 and OsHMA9 have similar intron-exon structures and protein transmembrane topology models(Supplemental Fig. 1). The amino acid sequence alignments of OsHMA6 and OsHMA9 showed 82.78%consistency (Table 1). Compared with the control (0.2 μmol/L Cu2+), high concentrations of Cu2+can significantly induce the expression of OsHMA6 and OsHMA9 (Fig. 3-A). OsHMA9 is a Cu efflux protein located in the plasma membrane, and knockout of OsHMA9 affects the tolerance of rice to high Cu concentrations (Lee et al, 2007). We speculate that OsHMA6 may be a Cu efflux protein similar to OsHMA9 in function.

Compared with the empty vector pYES2, OsHMA6-pYES2 can significantly rescue the growth of yeast strain CM52 on solid media with 3 or 6 mmol/L Cu2+(Fig. 4-A). The growth of CM52 with OsHMA6 was also significantly faster than that of pYES2 in liquid media containing 3 or 6 mmol/L Cu2+after 15 h (Fig.5-B and -C). These results suggested that OsHMA6 has high Cu2+transport activity. To further confirm this result, the metal concentrations of strains in liquid media for 30 h were analyzed. Compared with pYES2,the Cu2+concentrations in OsHMA6 decreased by 23.4%and 30.3% under 3 mmol/L or 6 mmol/L Cu2+,respectively (Fig. 5-F). The OsHMA6:YFP-pYES2 was transfected into yeast strains, and YFP fluorescence was localized in the plasma membrane (Supplemental Fig. 2). OsHMA6:GFP fusion vector was transformed into protoplasts of A. thaliana, and OsHMA6 was identified as a plasma membrane localization protein(Fig. 6). These results suggested that OsHMA6 is likely a Cu2+efflux protein located in the plasma membrane.

Although OsHMA6 and OsHMA9 shared high amino acid sequence identity and the similar function in Cu efflux, their functions in rice are different. The expression level of OsHMA6 was not significantly affected by exogenous Cd2+addition (Fig. 3-C), while the expression level of OsHMA9 was increased significantly at 200 μmol/L Cd2+, which was 2.5 times higher than that of the control (Fig. 3-D). Lee et al(2007) reported that the Cd concentration of OsHMA9 knockout mutants is significantly higher than that of the wild type in nutrient solution containing 500 μmol/L Cd2+and OsHMA9 also has Cd2+efflux function.We found that there was no significant difference in the growth of OsHMA6 and pYES2 yeast strains on solid media containing 10 or 20 μmol/L Cd2+(Fig.4-B) and liquid media containing 20 μmol/L Cd2+(Fig.5-D and -E). These results suggested that OsHMA6 has no Cd2+transport activity.

OsHMA6 expressed in all organs at different growth stages, including spikelets, and the highest expression was observed in leaf blades and roots (Fig. 2).OsHMA9 expressed most strongly in roots and very weakly in spikelets (Fig. 2). Root system is the main organ for the absorption and transport of heavy metal ions, and leaf blade is the main storage site for heavy metal ions (Ma et al, 2004). OsHMA9 plays an important role in the loading of root metal ions such as Cu2+and Cd2+into xylem (Lee et al, 2007). The strong expression of AtHMA2 and AtHMA4 in spikelets indicated that these two transporters play a special role in the transport of zinc to male reproductive tissues(Hussain et al, 2004). COPT1, a high affinity Cu transporter in A. thaliana, also highly expresses in spikelets, and the copt1 mutant is defective in pollen development due to blocked Cu transport (Sancenón et al, 2004). Athma5 expresses equally strongly in pollen, and plays a role in metal transport in Arabidopsis anthers (Andrés-Colás et al, 2006). Although both genes need more detailed analysis, these results suggested that OsHMA6 and OsHMA9 play different roles in the balance of Cu2+in vivo.

ACKNOWLEDGEMENTS

This work was supported by the Agricultural Science and Technology Innovation Program Cooperation and Innovation Mission (Grant No. CAAS-XTCX2016001),Shenzhen Science and Technology Projects (Grant No.JSGG20160608160725473), China Postdoctoral Science Foundation (Grant No. 2018M641558), and Fundamental Research Funds for Science, Technology and Innovation Commission of Shenzhen Municipality (Grant No.JCYJ20160530191619099).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/journal/16726308; http://www.ricescience.org.Supplemental Table 1. Primers used for qRT-PCR.

Supplemental Fig. 1. OsHMA6 and OsHMA9 have similar intron-exon structures and protein transmembrane topology models.

Supplemental Fig. 2. Subcellular localization of OsHMA6:YFP in yeast cells.