Improved Cd, Zn and Mn tolerance and reduced Cd accumulation in grains with wheat-based cell number regulator TaCNR2

Soil microelement deficiency and heavy metal contamination affects plant growth and development, but improving trace element uptake and reducing heavy metal accumulation by genetic breeding can help alleviate this. Cell number regulator 2 (TaCNR2) from common wheat (Triticum aestivum) are similar to plant cadmium resistance proteins, involved with regulating heavy metal translocation. Our aim was to understand the effect of TaCNR2 on heavy metal tolerance and translocation. In this study, real-time quantitative PCR indicated TaCNR2 expression in the wheat seedlings increased under Cd, Zn and Mn treatment. Overexpression of TaCNR2 in Arabidopsis and rice enhanced its stress tolerance to Cd, Zn and Mn, and overexpression in rice improved Cd, Zn and Mn translocation from roots to shoots. The grain husks in overexpressed rice had higher Cd, Zn and Mn concentrations, but the brown rice accumulated less Cd but higher Mn than wild rice. The results showed that TaCNR2 can transport heavy metal ions. Thus, this study provides a novel gene resource for increasing nutrition uptake and reducing toxic metal accumulation in crops.

that it does not change the nature of the soil. However, it takes time and is laborious, and, most importantly, it does not fundamentally reduce the heavy metal concentrations in crops. At present, heavy metal transporters have been used in plants to improve their ion balance. As a result, an increasing number of heavy metal transporters have been isolated and studied. Metal-tolerance proteins, members of the cation diffusion facilitator family, are highly specific for transporting Zn, but they can also transport Co 2+ , Fe 2+ and Cd 2+ 17-19 . Heavy metal ATPase can transport heavy metals across membranes 11,14,20 , and plays an important role in transporting Zn/Cd from plant roots to shoots. The natural resistance-associated macrophage protein is a major Mn transporter 21 and also participates in Fe 2+ , Zn 2+ and Cd 2+ transportation [22][23][24] . Plant cadmium resistance proteins (PCR) are involved with transporting Zn 2+ and Cd 2+ and in Arabidopsis one PCR, AtPCR1, a Cd-efflux transporter, improves the tolerance of Cd by exporting Cd out of the cell and decreasing its concentration 25 . Additionally, AtPCR2 is a Zn-efflux transporter, which can regulate Zn redistribution in plants 26 . However, the use of these transporters cannot meet the current demand for heavy metal treatments.
Previously, fruit-weight 2.2 (FW2.2) from tomato was located through quantitative trait locus, and regulated plant growth and development 27 . Maize cell number regulator 1 (ZmCNR1), a maize homolog of FW2.2, decreased fruit weight of transgenic maize 28 . Two of them were reported to regulate cell numbers and organ size 27,28 . It was found that the sequence of CNR and PCR were highly similar, as they both contain placenta-specific 8 (PLAC8) domains 29 . Furthermore, CNR and PCR both contained the CC/LXXXXCPC conserved motif. However, a specific CNR, ZmCNR2 from Zea mays, had been suggested to be involved in Cd tolerance and chelation 28 . Therefore, we wanted to investigate whether other CNRs could regulate heavy metal transport. In this study, a CNR2 from common wheat (Triticum aestivum) was isolated (TaCNR2), and its expression was analyzed under Cd, Zn and Mn stresses by real-time quantitative PCR. The overexpression of TaCNR2 in yeast, Arabidopsis and rice was used to determine stress tolerance to Cd, Zn and Mn. Heavy metal content was measured in the seedlings of overexpressed Arabidopsis and rice, grains of brown rice and husks of mature rice. This study identifies a potential transporter for cultivation by genetic breeding, which is useful for improving crop yields and food security, and managing heavy metal contamination in soil.

Results
Identification and characterization of TaCNR2. The TaCNR2 gene was isolated from common wheat (Triticum aestivum), and the evolutionary relationship showed that it clustered with the CNR2 of Aegilops tauschii, Hordeum vulgare and Brachypodium distachyon, then clustered with the PCR2 of other species (Fig. 1). Therefore, the gene was named TaCNR2. In the yeast tolerance assay, TaCNR2 transgenic lines had better growth than the control pYES2 at 50 μM CdSO 4 and 5 mM MnSO 4 , but the growth was distinctly weaker than pYES2 under 200 μM ZnSO 4 (Fig. 2).
Expression characteristics of TaCNR2. The expression of TaCNR2 in different wheat tissues was determined. The leaf blade and flag leaf blade had the maximum expression of all wheat tissues, about 20-fold higher than the rachis, while the internode also had 14-fold higher expression than the rachis (Fig. 3). To verify whether TaCNR2 was induced by heavy metals, the expression was tested under Cd, Zn and Mn treatment. The untreated wheat seedlings served as control. Under 50 μM CdSO 4 , the seedling shoots had slightly higher expression than the control at 24 and 48 h, while the expression of roots was higher than the control at 12 and 24 h (p < 0.05, Fig. 4a,b). Expression of TaCNR2 in the shoots was 3-fold higher than the control at 48 h (p < 0.05, Fig. 4c), but the expression was no different in roots treated with 200 μM ZnSO 4 (Fig. 4d). The expression of TaCNR2 in the shoots did not change after 6 and 12 h, after they increased and reached a maximum at 24 h under 3 mM MnSO 4 The growth of overexpressed TaCNR2 rice was no different with wild-type rice (WO) in 1/2 HS liquid media without exposure to Cd, Zn and Mn stress (Fig. 6a-c). In samples treated with 30 μM CdSO 4 , the shoot length and fresh weight of transgenic lines (OE-1) were better than WO (p < 0.05, Fig. 6d,g,j). The shoot length of overexpressed lines was slightly higher than WT (Fig. 6e,f,h,i), and the fresh weight was significantly higher than WO at 100 μM ZnSO 4 and 3 mM MnSO 4 (p < 0.05, Fig. 6k,i).

TaCNR2 improved Cd, Zn and Mn translocation.
To understand the heavy metal transport influence of TaCNR2, the metal ion content of the seedlings of transgenic Arabidopsis and rice was determined after treatment with Cd, Zn and Mn. The three overexpressed Arabidopsis lines (OX-1, OX-2 and OX-3) had higher Cd and Zn concentrations in the shoots than WT at 30 μM CdSO 4 and 200 μM ZnSO 4 (p < 0.05, Fig. 7a,b). However, the Mn shoot content in overexpressing lines was not significantly different from WT, but over-accumulated in the roots (Fig. 7c).
In addition, overexpression of TaCNR2 in the shoots of rice resulted in distinctly higher Cd, Zn and Mn concentrations than WO under 30 μM CdSO 4 and, 200 μM ZnSO 4 and 3 mM MnSO 4 , respectively. The Cd content of the roots was lower than WO, but Zn and Mn were higher than in WO (p < 0.05, Fig. 7d-f).  TaCNR2 reduced Cd accumulation in grains of brown rice. To understand the influence of TaCNR2 on the accumulation of heavy metals, Cd, Zn and Mn content of brown rice and husks of overexpressed mature rice were measured. The husk Cd, Zn and Mn contents in overexpressed lines were all higher than WO (p < 0.05, Fig. 8a-c). The Mn content of overexpressed lines were obviously higher than WO in brown rice (p < 0.05, Fig. 8c). However, Zn content in brown rice from transgenic lines was no different than WO (Fig. 8b), and had lower Cd concentrations (p < 0.05, Fig. 8a).

Discussion
Common wheat is an important staple food for humans and is one of the three major cultivated grains. In 2013, heavy metals were detected in wheat from more than 20 provinces in China, and Cd concentrations exceeded the national food standards by >20 times. An important source of these heavy metals is contaminated soil. Hence, preventing and controlling heavy metal pollution in crops is an urgent and difficult challenge. The Chinese spring wheat is a very important variety of common wheat, and is widely used in wheat genetic research. Although TaCNR2 from Triticum aestivum has a closely evolved relationship with CNR2, it also has a high similarity to PCR2 (Fig. 1). Furthermore, TaCNR2 has the same AtPCR1, AtPCR2 and OsPCR1 containing CCXXXXCPC motif (data not shown), which was reported to be involved in Zn and Cd tolerance and transportation 25,26,30 .
The maximum expression of TaCNR2 in different wheat tissues was found in the leaf blade, flag leaf blade and internode. The results suggested that TaCNR2 may participate in the transportation of water and inorganic salts from the internode to the leaf. The expression level was induced to increase in the wheat seedlings under Cd, Zn and Mn (Fig. 2), indicating TaCNR2 may be involved in the binding and transport of heavy metals. In our experiments, genetically modified yeast, Arabidopsis and rice were used to analyze TaCNR2 function. Yeast with TaCNR2 were more sensitive to Zn stress, which may cause excess Zn to be absorbed in the cells, resulting in zinc poisoning that leads to weak growth. However, TaCNR2 may prevent the uptake of Cd and Mn, or export Cd and Mn out of the cell, which would reduce the concentration of those heavy metals within the cell. Arabidopsis AtPCR1 can enhance Cd tolerance in yeast, which reduces the Cd concentration in yeast cells and decreases Cd toxicity 25 .
Overexpressed TaCNR2 Arabidopsis and rice both had tolerance to Cd, Zn and Mn, and enhanced Cd and Zn translocation from roots to shoots, but Mn translocation was prevented in Arabidopsis (Figs 5-7), suggesting that TaCNR2 may enhance the translocation of Cd, Zn, and Mn to tolerate the stress of heavy metals. Overexpression of AtPCR1 also improved the tolerance to Cd, and removed Cd from Arabidopsis protoplast 25 . AtPCR2 had strong tolerance to Cd and Zn, and the mutant atpcr2 was more sensitive to Cd and Zn than WT. The Zn concentration in overexpressed AtPCR2 Arabidopsis roots was significantly lower than WT, suggesting AtPCR2 decreased Zn toxicity by excreting it from the roots 26 . The Cd and Zn tolerance in overexpressed TaCNR2 Arabidopsis and rice was stronger than WT (Figs 5 and 6), a similar result to AtPCR2. However, Zn concentrations in overexpressed TaCNR2 Arabidopsis and rice shoots and roots were higher than WT; presumably, excess Zn was transported to the shoots to relieve the Zn concentration in the roots. Overexpression of TaCNR2 in Arabidopsis led to better Mn tolerance, but not Mn translocation, suggesting excess Mn was not transported to the shoots, which reduced Mn toxicity. The growth of overexpressed TaCNR2 rice was slightly better than WO when treated with Mn, and the shoots and roots had more Mn than WO (Fig. 7). The results indicated that excess Mn was transported into the shoots, and may inhibit rice growth. The Zn content of brown rice and grain husks in OsPCR1 knockout mutants was higher than WT rice 30 . In our study, Cd, Zn and Mn content in husks of TaCNR2-transgenic rice were all higher than WO, while these heavy metals in brown rice of overexpressed TaCNR2 lines had lower Cd content, but Zn concentrations showed no distinction from WO (Fig. 8); however, the Mn concentrations were higher than WO, illustrating that the metal ion supply to the brown rice and husks is through two different transport channels. These indicated that TaCNR2 was involved in the transport of heavy metals (Cd, Zn and Mn) to grains, but it hindered the translocation and accumulation of Cd in brown rice.
The tolerance to Cd, Zn and Mn in TaCNR2-transgenic yeast, Arabidopsis and rice slightly differed, and the translocation of Cd, Zn and Mn in transgenic Arabidopsis and rice was similar, suggesting TaCNR2 had different tolerances and transport mechanisms in different organisms. However, the TaCNR2 in transgenic yeast, Arabidopsis and rice enhance Cd tolerance and translocation from roots to shoots.
In summary, the expression of TaCNR2 increased under Cd, Zn and Mn treatments. TaCNR2 overexpressed in Arabidopsis and rice exhibited Cd, Zn and Mn tolerance, and strong translocation of Cd, Zn and Mn in rice. TaCNR2 can reduce the Cd accumulation in brown rice, and enhance Mn content in husks. The results of this study suggest a feasible heavy metal transporter, and could play an important role in maintaining the ion balance of plants. These aspects could help us improve crop yield and quality, and maintain food security.  The shoot and root samples were separated and collected after 0, 6, 12, 24 and 48 h. The seedlings were vernalized at 4 °C for 10 days and cultivated in agricultural soil for four months to obtain different wheat tissues, including roots, internodes, node I, leaf sheaths, leaf blades, flag leaf sheaths, flag leaf blades, peduncles, rachises and seeds. All samples were ground into powder after application of liquid nitrogen. All primer sequences used for the PCR reactions are listed in Table 1.
Gene cloning. Total RNA was isolated from 6 day old wheat seedlings using RNAiso Plus (TaKaRa, Japan), and the cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer's instructions. TaCNR2 sequences were cloned using the pair primers TaCNR2-F and  TaCNR2-R   The treatment group was transferred into Kimura B containing 10 μM CdSO 4 , 100 μM ZnSO 4 and 3 mM MnSO 4 for 7-14 days. The length and fresh weight of the plants from both groups were measured after the experiment. statistical analysis. All data are presented as mean ± standard error (SE) from three independent experiments. Statistical analysis was performed using the software programs Office 2010 and SPSS 13.0. The one-way ANOVA and t-test were used to compare the mean values (p < 0.05).