A novel family of PLAC8 motif-containing/PCR genes mediates Cd tolerance and Cd accumulation in rice
Cd is one of the highly toxic heavy metals to most organisms, including humans and plants, and Cd-contaminated rice from China has become a global food safety issue. The early prediction of OsPCR (the plant cadmium resistance protein) which contained a PLAC8 domain was related with the accumulation of Cd in rice. To further understand the biological function of the OsPCR genes on the Cd tolerance and Cd accumulation in rice, we used a low grain-Cd-accumulating rice (xiushui 11) and a high grain-Cd-accumulating rice (xiushui 110) varieties to analyze the relationship between the expression levels of the two most abundant expression genes (OsPCR1 and OsPCR3) and the Cd concentrations in different tissues at different growth periods during Cd stress, and transgenic experiments of OsPCR1 and OsPCR3 were carried out.
OsPCR1 and OsPCR3 were closely related with Cd accumulation. Overexpression of OsPCR1 and OsPCR3 could not only increase the Cd tolerance, but also decrease the Cd accumulation obviously in different parts of the transgenic rice plants (especially in the rice grains), while the RNAi expression plants showed the opposite results.
These results indicate that OsPCR1 and OsPCR3 play critical roles in Cd accumulation in rice, which provides a theoretical basis for the safe production of rice.
KeywordsRice OsPCR1 OsPCR3 Transgenic rice plants Cd accumulation
the plant cadmium resistance
the natural resistance-associated macrophage protein
heavy metal ATPase
low-affinity ion transporter
fruit weight 2.2
cell number regulator
OsPCR: OsPCR overexpression transgenic plants
OsPCR1: OsPCR1 interference expression transgenic plants
wheat cell number regulator 2
Heavy metals have a serious impact if released into the environment even in trace quantities which can enter into the food chain from aquatic and agricultural ecosystems and threaten human health indirectly . Cd is one of the most toxic heavy metals in the soil; it has strong chemical activity and can be absorbed by plants easily [2, 3]. Rice is one of the most important food crops in the world. The problem of Cd pollution has been highly valued by the government departments of all countries. Therefore, how to reduce the Cd content in rice grain and clarify its accumulation rule to realize the production of low FAO/WHO and the national standard of rice grain in the heavy metal contaminated soil is of great significance.
Many studies have been carried out on the molecular mechanisms of Cd accumulation in rice, and several genes involved in Cd translocation and accumulation have been identified [4, 5]; for example, phytochelatin synthase genes (OsPCS1 and OsPCS2) , the natural resistance-associated macrophage protein (Nramp) family genes (OsNRAMP1 and OsNRAMP5) [7, 8], heavy metal ATPase gene (OsHMA2) , and low-affinity ion transporter gene (OsLCT1) , the Fe transporters (OsIRT1 and OsIRT2) . OsLCD gene was expressed in the phloem of vascular bundle and leaf in rice root which was involved in the Cd accumulation of rice ; OsLCT1 protein was a membrane protein, involved in the transport process of Cd from the cell to the outside world ; Ueno reported that heavy metal ATP enzyme (OsHMA3) in rice could decrease Cd transport to the shoot [10, 13]; and OsCCX2 was reported as a node-expressed transporter participated in Cd accumulation in rice grain of rice . But the uptake, translocation and accumulation of Cd in rice seedlings were still not clear, and the discovery of Cd-accumulation-related genes was still very poor.
The plant cadmium resistance protein (PCR) which belonged to a membrane protein family should contain CCXXXXCPC or CL/FXXXXCPC conserved amino acid sequences to be effective, and a PLAC8 domain was reported as associate with cadmium resistance in plants [15, 16, 17, 18]. Many functions have been reported for PLAC8 domain-containing proteins of plants; Arabidopsis thaliana plant cadmium resistance 2 (AtPCR2) acts as a Zn efflux transporter and related with Zn resistance . The similar proteins such as Solanum lycopersium (tomato) fruit weight 2.2 (fw2.2), Zea mays (maize) cell number regulator (ZmCNR1) and an animal protein (onzin) were also contain the PLAC8 domain, but they all played important roles in the control of cell growth [20, 21, 22], and only onzin was involved in the pathogen defense and autothagy [23, 24]. Brassica juncea plant cadmium resistance 1 protein (BjPCR1) facilitated the radial transport of calcium in the root and so on . Most studies suggested that the CCXXXXCPC motif was likely to take part in the binding of divalent cations, then complete the transport of the divalent cations. In our previous research work, we predicted that OsPCR1 (Loc_Os02g0578900) was important in Cd accumulation in rice . And similar results were shown by Song et al. about the function of OsPCR genes in rice. Their recent reports suggested that OsPCR (Loc_Os10g02300) could influence Zn accumulation and grain weight in rice, and the OsPCR-knockdown rice seedlings showed lower Cd concentrations than the control rice grain which indicated that OsPCR played an important role in Cd accumulation in rice grain . Recently, Xiong et al.  reported a FW2.2-like family gene in rice (OsFWL4, Os03g614440) which contained a PLAC8 that could not only regulate grain size and plant height, but also involved in Cd translocation form roots to shoots in rice.
Therefore, it was urgent to understand the information of the functional OsPCRs genes in rice, and clarify the expression patterns of OsPCR genes in different rice cultivars. This study selected the two most abundantly expressed genes (OsPCR1 and OsPCR3(LOC_Os02g52550.1)) predicted by bioinformatics as the target genes. The relationship between the expression levels and the Cd concentrations in different tissues at different growth periods during Cd stress in xiushui110 (high-Cd in grains) and xiushui11 (low-Cd in grains) (Oryza sativa L.) was analyzed, and the transgenic rice plants of OsPCR1 and OsPCR3 genes response to Cd stress were studied, respectively. The results of this study will provide a reference to guide future experiments that focus on the function and mechanism of OsPCRs genes in the growth and development of rice, and provide a theoretical basis for the study on the molecular mechanisms of Cd tolerance and accumulation in rice.
Materials and methods
Plant materials and treatments
The pre-screened low grain-Cd-accumulating rice (xiushui 11) and high grain-Cd-accumulating rice (xiushui 110) seeds were used in these experiments. The rice seed germination and seedling cultivation with nutrient solutions were according to Wang et al. . The uniform 5th-leaf stage rice seedlings were treated with 2 µM CdCl2 (determination of Cd accumulation) or 10 µM CdCl2 (determination of physiological indicators); then different rice samples (leaf, stem, root, flag leaf, and panicle) of the two rice cultivars were collected at different growth development for the further experiments.
Expression patterns of OsPCR1 and OsPCR3 in response to Cd stress
Primer sequences of OsPCR1 for qRT-PCR
Forward sequence (5′ → 3′)
Reverse sequence (5′ → 3′)
Construction of transgenic rice plants
PCR amplification of OsPCR gene and its interference fragment sequence primer information
Sequence length (bp)
Estimation of hydrogen peroxide and lipid peroxidation
The difference of peroxidation level induced by Cd stress between wild-type (WT) xiushi 11 and transgenic rice seedlings was estimated by the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA). H2O2 and MDA determinations were carried out by the methods of Wang et al.  and Wang et al. , respectively.
Extraction and determination of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity
SOD activity, CAT activity and POD activity were assayed according to Ma et al.  and Wang et al. . 0.3 g fresh rice leaves samples were ground with liquid nitrogen, and then added with 3 ml cold buffer (50 mM PBS pH 7.0, 3 mM DTT, 1 mM EDTA-Na2, 5% PVP). The homogenate was centrifuged at 15,000g for 20 min, and the supernatant was used for analyzing the enzyme activities. SOD activity was measured by the inhibition of the photochemical reduction of β-nitroblue-tretrazolium chloride (BNT), CAT activity was analyzed with the rate of decrease in H2O2 absorbance at 240 nm, POG activity was assayed by the reaction of oxidation of guaiacol at 470 nm. All operations were performed at 4 °C.
Cd determination in rice samples
0.2 g rice samples were used in the digestion, and the specific steps refereed to Liu et al. . Except brown rice was digested in a HNO3/HF mixture (3:1) in a microwave digester (CEM-MARS: Boston, MA, USA); other rice samples were all digested with 6 ml HNO3. Cd concentrations in different samples were detected by AA-7000 (SHIMADZU: Kyoto, Japan).
The Excel 2003 and SPSS (Product and Service Solutions Statistical, 18.0) were used to analyze the data, and the experiments in this manuscript were performed at least three repetitions and all the data reported in this paper were means of three replicates.
Results and analysis
Expression patterns of OsPCR1 and OsPCR3 in different Cd accumulating rice
Different distribution of Cd in xiushui 11 and xiushui 110
Expression analysis of OsPCR1 and OsPCR3 in different tissues at different growth stages of rice with qRT-PCR
Effect of different expression levels of OsPCR1 and OsPCR3 on H2O2 accumulation, lipid peroxidation and antioxidant enzymes’ activities
Thus, the results indicated that the interference expression of OsPCR1 or OsPCR3 could cause higher membrane oxidation and higher Cd sensibility under Cd stress, while the overexpression of OsPCR1 or OsPCR3 could induce higher antioxidant activities to arise the higher Cd tolerance.
Overexpression of OsPCR1 and OsPCR3 decreased Cd accumulations in rice
Cd is a highly toxic heavy metal to all forms of life including plants and humans. Cd is absorbed by plant roots and accumulated in plant tissues, when grown slightly or moderately Cd-polluted soil, plant growth and development may not be substantially affected but the accumulated Cd can enter the food chain and cause harmful effects to human health. In recent years, many experiments have shown genotypic differences in Cd accumulation among rice varieties [8, 35]. The Cd accumulations in indica subspecies were more and easier than the japonica subspecies. In this study, low grain-Cd-accumulating rice (xiushui 11) and a high grain-Cd-accumulating rice (xiushui 110) varieties were used as experimental materials. Owing to the genotypic difference, the accumulation of Cd in xiushui110 was obviously higher than that of xiushui11. Furthermore, the Cd accumulation in rice grain was correlated with the uptake of Cd by roots, and the root-to-shoot or shoot-to-grain translocation abilities.
Many functions have been reported for PLAC8 domain-containing proteins of plants (such as Arabidopsis thaliana, Solanum lycopersium, Zea mays, Brassica juncea) which showed that these proteins including the CCXXXXCPC or CLXXXXCPC motif were reported as associate with Cd resistance in plants [15, 16, 17]. Two members of this family, AtPCR1 and AtPCR2, played an important role in the Cd tolerance and transport of Zn, the CPC motif had a much more important role in the function of the AtPCR proteins than CC motif in Cd stress . BjPCR1 protein also had a hydrophobic domain composed of CC-CPC, which was a Ca2+ efflux transporter in mustard . In this study, we demonstrated the functions of OsPCR1 and OsPCR3 which were homologous to AtPCR2, and found that the expression levels of OsPCR1 and OsPCR3 were closely related with the Cd contents in rice grains (Figs. 2 and 3). Moreover, OsPCR3 overexpression transgenic plants (OE:OsPCR1 and OE:OsPCR3) enhanced Cd tolerance, while the interference expression transgenic plants (Ri:OsPCR1 and Ri:OsPCR3) suffered higher Cd injure (Fig. 4). The results were similar to the reports on the heterologous expression of the OsFWL4 which also belonged to PLAC8 domain-containing proteins in yeast cells .
One mechanism for regulating metal ion uptake and transport is through alteration of gene expression. As Cd is a non-essential ion, there are no specific Cd transporters. However, many transporters for divalent transition metals (Mn, Fe, and Zn) can absorb Cd. For example, the expression of the root ZIP transporter IRT1, which plays a critical role in the uptake of Fe and non-essential heavy metals including Cd, is highly regulated at the transcriptional level [36, 37]. OsNramp5 was found contribute to Mn, Cd and Fe transport [7, 38, 39]. OsHMA2 was revealed to play a pivotal role in Zn and Cd accumulation in rice [9, 40]. As we predicted, OsPCR genes take effect as a transporter which may play an important role in the transport of Cd from roots to shoots in rice. In our experiments, we found overexpression of OsPCR1 and OsPCR3, both, could decrease the Cd contents in brown rice, as well as roots, stems, leaves and husks. However, the interference expression of the two genes would increase the Cd contents in brown rice, as well as roots, stems and leaves (Figs. 11 and 12). Due to the grain-ripening stage is a critical period for grain Cd accumulation [30, 41], the lower Cd accumulation in rice grain of the OsPCR1 or OsPCR3 overexpression transgenic rice plants may be due to the reduction of Cd uptake and transportation in rice.
Many studies have been carried out on the molecular mechanisms of Cd accumulation in rice. Several genes involved in Cd translocation and accumulation have been identified, but the uptake, translocation and accumulation of Cd in rice seedlings were still not clear, and the discovery of Cd-accumulation-related genes was still very poor. These results elaborated that there was a negative correlation between the expression levels of OsPCR1 or OsPCR3 and Cd accumulation in rice. Lower Cd accumulation in the overexpression transgenic rice plants may due to the decrease of Cd uptake and transport in rice; the overexpression of OsPCR1 or OsPCR3 in rice could increase Cd tolerance by enhancing antioxidant levels in vivo. These results indicated that OsPCR1 and OsPCR3 play critical roles in Cd tolerance and accumulation in rice, which provides a theoretical basis for the safe production of rice. However, many questions remain to be answered. Are OsPCR1 and OsPCR3 involved in the regulation of gene expression in response to Cd? Do OsPCR1 and OsPCR3 contain any other recognizable domains which contribute to Cd tolerance and what are the mechanisms by which OsPCR1 and OsPCR3 confer Cd tolerance? How do OsPCR1 and OsPCR3 interact with Cd transporters and regulatory approaches? Therefore, more mechanisms of OsPCR1- and OsPCR3-mediated Cd tolerance and low-Cd-accumulation in rice should be studied in the future research.
This work was supported by the Natural Science Foundation of Zhejiang Province, China (Grant No. Y17C020020), the Key Research and Development Project of Zhejiang Province, China (Grant No. 2015C03020-4), the National Nature Science Foundation of China (Grant No. 31401356), Jinhua Science and Technology Project (Grant No. 2015-2-012), and the National Training Program for College Students to Innovate and Start Enterprise (Grant No. 201710356013).
This study was designed by CZ, ZC and FW. Construction of transgenic rice plants was conducted by XH and YD. The other experiments and the data analyze were performed by HT, JH, YZ and FW. FW wrote this manuscript, CZ and ZC polished the manuscript. All authors contributed equally to this work. All authors read and approved the final manuscript.
The Natural Science Foundation of Zhejiang Province, China (Grant No. Y17C020020), the Key Research and Development Project of Zhejiang Province, China (Grant No. 2015C03020-4), the National Nature Science Foundation of China (Grant No. 31401356), Jinhua Science and Technology Project (Grant No. 2015-2-012), and the National Training Program for College Students to Innovate and Start Enterprise (Grant No. 201710356013).
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The authors declare that they have no competing interests.
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