In plants, non-essential heavy metals cause toxicity and eventually inhibit plant growth and development (Balen et al., 2011). Cadmium (Cd), a heavy metal widespread in the environment, damages the photosynthetic apparatus, affects the respiratory and nitrogen metabolism, and alters the balance of water and nutrient uptake (Herbette et al., 2006; Balen et al., 2011). Cd absorbed by plants can be introduced into the food chain (McLaughlin et al., 1999). Consumption, either directly or indirectly, of these parts could be a human health concern (Grant et al., 2008). Therefore, the Cd concentrations in several safe cereal grains were limited below 0.2 mg/Kg. In contrast, zinc (Zn) is an essential metal for plant growth. It has been/being widely utilized to reduce the Cd toxicity, although the effect varies with genotypes, the dose and duration of the Zn and Cd exposure (Rizwan et al., 2016). Excess Zn also limits the plant growth and causes a strong toxicity (Zhao et al., 2005; Wang et al., 2009). Plants therefore need to prevent damage from non-essential metals and ensure the proper homeostasis of essential metals (Lin and Aarts, 2012).

Cd and Zn, co-existed in the soil, cause various synergistic and antagonistic interactions according to the species, external metal concentrations, tissues, and developmental stages (Cataldo et al., 1983; Nan et al., 2002; Hassan et al., 2005; Sun et al., 2005; Balen et al., 2011; Cherif et al., 2011; Tkalec et al., 2014). In soybean, the uptake of Cd/Zn exhibits competitive inhibition (Cataldo et al., 1983). In durum and bread wheat, Cd and Zn mutually inhibit their uptake in the roots, stems and leaves (Hart et al., 2002, 2005). In tomato (Cherif et al., 2011) and Lemna minor (Balen et al., 2011), Zn inhibits the Cd uptake. However, the Cd/Zn interactions are not always antagonistic. Synergistic interactions were observed in both wheat and corn under field conditions (Nan et al., 2002). In tobacco, Zn promotes the Cd uptake, while Cd inhibits the Zn uptake in the roots and leaves (Tkalec et al., 2014). In rice, Zn increases the Cd concentration in the shoots, but inhibits the Cd uptake in the roots (Hassan et al., 2005). However, all these studies focused on the transport and biochemical responses by measuring plant growth, metal concentration, pigment content, and antioxidant content (Cataldo et al., 1983; Nan et al., 2002; Hassan et al., 2005; Sun et al., 2005; Cherif et al., 2011; Balen et al., 2011; Tkalec et al., 2014). Meanwhile, studies revealed the changes of transcriptomic profiles were focused on Cd or Zn alone (Herbette et al., 2006; Di Baccio et al., 2011; Lin et al., 2013). The changes of transcriptomic profiles for Cd/Zn interactions were not revealed.

Transcriptomes of Triticum turgidum (2n = 4x = 28, AABB) and common wheat (2n = 6x = 42, AABBDD) using RNA-sequencing (RNA-Seq) have been reported (Duan et al., 2012; Schreiber et al., 2012; Krasileva et al., 2013). Recently, the transcriptomic profiles of the developing starchy endosperm and the grain filling of bread wheat, and the dwarfism of dwarf Polish wheat were revealed using RNA-Seq (Pont et al., 2011; Pellny et al., 2012; Wang et al., 2016a). Although the molecular responses to Cd or Zn in plants have been widely investigated (Herbette et al., 2006; Di Baccio et al., 2011; Lin et al., 2013), the similar study in wheat using RNA-Seq is not processed. Based on genetic analysis and taxonomical classification, Polish wheat (2n = 4x = 28, AABB, Triticum polonicum L.) presents a low level of genetic similarity with T. durum, T. turgidum, and T. aestivum (Wang et al., 2013; Michalcová et al., 2014). Due to the high thousand kernel weights and high Zn, Fe, and Cu concentrations in the seeds (Wiwart et al., 2013), and the dwarfing gene (Kang et al., 2012), Polish wheat has attracted the interest of producers and breeders (Wiwart et al., 2013). Dwarf polish wheat (DPW, Triticum polonicum L.) which collected from Tulufan, Xingjiang, China, shows high tolerance to Cd and Zn. Therefore, it is a desirable material for studying Cd/Zn interactions. Previously proteomic study revealed that many proteins mainly participated in sucrose, glutathione (GSH), S-adenosyl-l-methionine (SAM), organic acids metabolisms and oxidation-reduction process were response to the Cd/Zn interactions on two days after treatments (Wang et al., 2016b). However, results of transtriptomic and proteomic analysis are very low overlay. It is interesting to investigate that what kinds of genes response to the Cd/Zn interactions when prolonged the treated time. In the present study, our aims are therefore to investigate the transcriptome responses under Cd, Zn and Cd+Zn stresses, finally reveal the molecular mechanisms of Cd/Zn interactions in the DPW roots on 5 days after treatments.

Cd inhibits the growth of bread wheat (Sun et al., 2005) and durum wheat (Hart et al., 2005; Koleva-Valkova et al., 2012). However, it also stimulates or does not inhibit the plant growth of other types of bread wheat (Stolt et al., 2003; Zhao et al., 2005; Lin et al., 2007). In the present study, although DPW accumulated 992.29 ± 29.83 mg/Kg (dry weight, DW) Cd in the roots and 40.82 ± 13.70 mg/Kg (DW) Cd in the shoots, the lengths of root and shoot were not affected (Figures 1A,B), which validated that DPW seedlings had stronger Cd tolerance than other types of durum and bread wheat (Hart et al., 2002, 2005). Meanwhile, the growth was inhibited by Zn and Zn+Cd stresses when the leaf and root accumulated high Zn concentrations (Figure 1), which indicated that excess Zn could cause the toxicity in wheat seedling (Zhao et al., 2005).

Since Cd and Zn mutually inhibited their uptake in the roots (Figure 2), the Cd/Zn interactions in the DPW roots were antagonistic, which was same as the antagonists in bread and durum wheat (Hart et al., 2002, 2005; Sun et al., 2005). However, Zn promoted the Cd transport from the roots to shoots (Figure 2B), Cd did not affect Zn transport (Figure 2D), which indicated that the Cd/Zn interactions in the DPW leaves were synergistic. This result was different from that Zn inhibited the Cd transport from the roots to shoots on 2 days after treatments with the same metal treated concentrations (Wang et al., 2016b).

Our previous study revealed that various proteins participated in the Cd/Zn interactions on 2 days after treatment (Wang et al., 2016b). In the present study, the transcriptomic changes in the roots also indicated that some genes were involved in the Cd/Zn interactions (Figure 3, STables 2–8). On 5 days after treatments, the expression of 1,269, 1,254, and 820 unigenes was individually changed by Cd, Cd+Zn, and Zn. However, only 381 unigenes were co-regulated by these treatments (Figure 3). In addition to the specific unigenes individually induced by Cd, Zn, and Cd+Zn, the remaining DEGs classified into different subgroups were considered to participate in the Cd/Zn interactions. These results indicated that Cd, Zn, and Cd+Zn induced differential molecular responses, which ultimately resulted in the differential molecular responses for Zn and Cd stresses (Lin and Aarts, 2012). In the following discussion, some DEGs involved in several important processes were described.

Heavy metal transporters play important roles in the metal uptake, transport and distribution. In yeast, the expression of cadmium-transporting ATPase which is a cadmium-specific efflux pump enhanced Cd resistance by extruding intercellular Cd (Adle et al., 2007; Adle and Lee, 2008). In the DPW roots, the expression of cadmium-transporting ATPase and plant cadmium resistance 4 was specifically up-regulated by Cd+Zn (Table 1), suggesting that Cd might be extruded from roots, finally resulted in that the Cd concentration in the roots under Cd+Zn stress was significantly lower than that under Cd stress (Figure 2A). Therefore, Zn can enhance the Cd resistance by reducing the Cd accumulation (Rizwan et al., 2016).

Except of metal efflux pumps, other heavy metal transporters also play important roles in heavy metal detoxification. In Arabidopsis, AtABC25 (Kim D. Y. et al., 2006) and AtABCC1-3 (Bovet et al., 2003, 2005; Park et al., 2012; Brunetti et al., 2015) transported Cd into the vacuoles to increase Cd tolerance. Overexpression of a metal chelator, metallothionein (MT), increased Cd content in the roots and enhanced Cd tolerance (Sekhar et al., 2011). In this study, 6 ABC transporters (B, C, and D family members, Table 1) and a MT were up-induced by Cd, suggesting that most of Cd accumulated by root might be sequestrated into the vacuoles so that DPW seedling exhibited strong Cd tolerance under Cd stress (Figure 1). When these regulations were not observed under Cd+Zn stress (Table 1), the Cd accumulations in the roots should be reduced (Bovet et al., 2003, 2005; Sekhar et al., 2011; Park et al., 2012; Brunetti et al., 2015), finally resulted in that the Cd concentration in the roots under Cd stress was higher than that under Cd+Zn stress (Figure 2A). Meanwhile, most of Cd accumulated by the roots was not sequestrated into the vacuoles, but was uploaded into the xylems and then transported into the shoots (Figure 2B, Kim et al., 2007). Thus, these ABC transporters and MT participated in the Cd/Zn interactions for the Cd transport and accumulation.

In planta, the high accumulation of Fe in the shoots under Cd stress could alleviate Cd toxicity (Wu et al., 2012). Overexpression of nicotianamine synthase (NAS), vacuolar iron transporters (VIT), and metal-nicotianamine transporter YSL (YSL) increased the Fe content in the roots and shoots (Kim S. A. et al., 2006; Ishimaru et al., 2010; Wu et al., 2012). In the present study, Cd stress through up regulating NAS1, VIT, and YSL12 (Table 1) to increase the Fe content in the DPW roots and shoots (data not shown), which could alleviate Cd toxicity (Wu et al., 2012), and then enhanced the Cd tolerance in DPW seedlings (Figure 1). However, these regulations were not observed under Zn and Cd+Zn stresses (Table 1), suggesting that these up regulations were activated when the roots accumulating a certain amount of Cd.

Cd inhibits the nitrate assimilation and transport in planta (Sanita di Toppi and Gabbrielli, 1999; Li et al., 2010). Cd regulates several nitrate-related genes, such as glutamate dehydrogenase and nitrate transporter (NRT) (Chaffei et al., 2004; Li et al., 2010), and also affects leaf nitrogen remobilization and root nitrogen storage (Chaffei et al., 2004). Down-regulation of AtNRT2.8 reduced the Cd accumulation in the roots and increased it in the shoots. Thus, AtNRT2.8-regulated nitrate distribution controls the Cd uptake and transport (Li et al., 2010). In the present study, Cd alone up regulated glutamate dehydrogenase and two NRTs (Table 1), suggesting that Cd might regulate the nitrate assimilation and transport in the DPW roots (Chaffei et al., 2004). Meanwhile, two high affinity nitrate transporters (HANTs) were specifically down-regulated by Cd+Zn (Table 1). HANTs participate in the nitrate uptake (Cerezo et al., 2001; Li et al., 2007). The inhibition of nitrate uptake reduces the Cd uptake and other essential metals (Mao et al., 2014). Thus, down regulation of HANTs might mutually inhibit the Cd/Zn uptake under Cd+Zn stress (Table 1). Due to different nitrate-related genes were induced, Cd and Cd+Zn might regulate different nitrate metabolism.

Although these heavy metal and nitrate transporters were not observed at proteomic level on two days after treatments (Wang et al., 2016b), unigenes participated in glutathione (GSH) metabolism, antioxidant enzymes and cell wall composition were induced at both transcriptomic (Table 1) and proteomic level (Wang et al., 2016b). GSH is a substrate for phytochelatin synthesis and crucial for detoxification of heavy meals (Freeman et al., 2004; Yadav, 2009). Formation of GSH-Cd or Zn complexes and then sequestration into the vacuoles is another mechanism of detoxification (Seth et al., 2012; Jozefczak et al., 2015). Genes involved in GSH metabolism, such as glutathione S-transferase (GST), hydroxyacylglutathione hydrolase (HGH), and glutaredoxin (Grx), were differentially regulated by heavy metals (Di Baccio et al., 2011; Lin et al., 2013; Jozefczak et al., 2015). As detoxifying enzymes present in all aerobic organisms, GSTs catalyze the nucleophilic attack of the sulfur atom of the tripeptide GSH on the electrophilic group of the substrate (Adamis et al., 2004), and also transport compound of GSH-cytotoxic substrates into the vacuoles for detoxification (Kumar et al., 2013). In this study, several unigenes of GSH metabolism, such as Grx, HGH, lactoyglutathione lyase, S-formylglutathione hydrolase, disulfide isomerase-like 1-4, and 5 GSTs, were induced and grouped into different interactions of Cd/Zn (Table 1), suggesting that GSH metabolism played different roles in the Cd/Zn interactions (Wang et al., 2016b).

Plants have established effective antioxidative systems to protect cells against damage from metal-induced oxidative threats (Di Baccio et al., 2011). Previous studies revealed that many Cd or Zn- induced genes participated in the defense against oxidative stress (Di Baccio et al., 2011; Lin et al., 2013). In this study, 13 peroxidases (POD) and 3 aldehyde dehydrogenases that removed the toxic aldehydes from lipid peroxidation were regulated by Cd, but were not regulated by Zn and Cd+Zn stresses (Table 1). Three genes (two ubiquinol oxidase 1as and NADPH: quinone oxidoreductase 1) were up-regulated by Zn but were not induced by Cd and Cd+Zn stresses (Table 1). Catalase (CAT) isozyme 2 was specifically up-regulated by Cd (Table 1). These results confirmed that antioxidant enzymes play different and important roles in adaptive response to Cd, Zn, or Cd+Zn stresses (Qiu et al., 2008; Zeng et al., 2011).

The plant cell wall is mainly composed of cellulose and polysaccharides (Cosgrove, 2005). It can be modified by Cd (Li et al., 2015; Shi et al., 2015). Therefore, modification of cell wall composition is associated with the Cd exclusion in the roots (Zhu et al., 2012). Additionally, exogenous glucose also alleviates the Cd toxicity by fixing Cd in the cell wall and sequestering it into the vacuoles (Shi et al., 2015). In this study, many carbohydrate (including glucose, cellulose, callose and mannose) metabolism-related genes were differentially regulated by Cd, Zn, or Cd+Zn (Table 1), which suggested that cellulose, glucose and polysaccharides play different roles in the Cd and Zn fixation, exclusion and sequestration in the roots (Li et al., 2015).

YW, XW, CW, and YZ conceived and designed research, and wrote the manuscript. YW, XW, CW, XX, FP, and RW conducted experiments. YW, XW, JZ, HK, XF, LS, and HZ analyzed data. All authors read and approved the manuscript.