The cultivar variations in Cd toxicity and distribution in rice plants
In Fig. 1, for all used cultivars, root elongation varied only moderately with Cd treatment; by contrast, shoot extensions varied much more. The genotypic variation in Cd toxicity to rice seedlings would be related to the Cd distribution in plants and physiological regulations under Cd stress (Hsu and Kao 2003; Kuo and Kao 2004; Zhang et al. 2009; Singh and Shah 2014). In Fig. 2, the TF values, that almost all were less than 0.2, indicated that most of the Cd uptake by rice plants was accumulated in the root rather than being transferred to the shoot. Previous studies have emphasized that Cd uptake in the root dominates Cd accumulation in the overall rice plant (Dong et al. 2007; Takahashi et al. 2011; Ye et al. 2012). Zhang et al. (2009) suggested that the detoxification of Cd in rice plants could be referred to a succession of Cd retention in root cell walls (Xiong et al. 2009; Liu et al. 2016), compartmentalization of Cd into vacuoles (Ernst et al. 2008; Zhang et al. 2013), and suppressed Cd transportation from the root to shoot. The root Cd concentrations of TNG71 and TCS10 were significantly higher than 2000 mg kg− 1, but those of TY3 and TK9 were significantly lower. This revealed that the capability of rice roots to retain Cd varies with the rice cultivars.
In addition, TKW1, TKW3, TCS10, and TCS17, whose TF values were relatively high (i.e., > 0.15), showed serious injury in shoot extension with Cd treatment; however, TY3 and TK9 also had relatively high TF values but did not show severe injury. Both TY3 and TK9 show higher shoot extension under Cd treatment but may use other physiological regulations. This suggested that TY3 and TK9 were relatively non-sensitive to Cd stress. By contrast, TNG71 and KH145 with relatively low TF values (i.e., < 0.15) did not avoid serious injury in shoot extension with Cd treatment. The above findings of cultivar-specific differences in Cd toxicity and distribution in rice plants indicate that the plant growth responses to Cd stress are not simply and linearly related to Cd distributions in plants.
Nevertheless, root-to-shoot Cd translocation is the major process determining grain Cd accumulation (Uraguchi and Fujiwara 2012). Wang et al. (2015) emphasized that stem transportation may play an important role in grain Cd accumulation, which was combined with the expression level of the OsPCR1 gene. Lower Cd translocation from the root to the shoot may result in lower grain Cd accumulation. Therefore, TF values could be used as an index in a selection program for initially screening cultivars whose edible parts (e.g., grain or brown rice) potentially have low Cd contents (Zhang et al. 2014; Song et al. 2015; Liu et al. 2016). According to the TF values of used cultivars in Fig. 2, TNG71 and KH145 show the lower potential of grain Cd accumulation although they are not tolerant of Cd stress. Nevertheless, TY3 and TK9 not only tolerate Cd treatment such as 50 µM but also show the higher potential of grain Cd accumulation.
Characterization of the oxidative statuses in rice plants under Cd stress
In previous studies (Kuo and Kao 2004; He et al. 2014; Wang et al. 2015), the MDA concentrations in the rice shoot increased significantly with the treatment of high Cd levels (i.e., 100–500 µM). Xie et al. (2015) reported that the MDA observed in the shoot of rice seedlings treated with low Cd concentrations of ~ 40 µM for 15 days increased by up to two times compared with that in the control. In this study, rice seedlings were treated with 50 µM Cd for 7 days; however, they did not show a significant enhancement of lipid peroxides in the shoot, probably because the roots accumulated most of the Cd absorbed by rice seedlings to prevent translocation into the shoot. Thus, the Cd exposure level and duration did not result in high lipid peroxides in the shoot. Hsu and Kao (2007) suggested that the differences in H2O2 accumulation in leaves between different rice cultivars were related to the genotypic variation in Cd tolerance. The Cd treatment produced the greatest enhancements in shoot H2O2 concentration in TY3, TK9, and KH145, although their shoot H2O2 concentrations in CK were much lower, even being under 100 mg kg− 1. TKW1, TKW3, TCS10, and TCS17 showed moderately pronounced increases in shoot H2O2 concentrations with Cd treatment; however, their shoot H2O2 concentrations in CK were much higher than those of the other cultivars. A few studies showed that H2O2 accumulation and MDA content were positively correlated in rice plants with Cd treatment (Singh and Shah 2014; He et al. 2014; Wang et al. 2015). However, this was not in agreement with the results of the present study. It may be possible that significant increases in H2O2 accumulation in the shoot with Cd exposure have been found for the cultivars, but have not been directly related to their MDA measurements. That is, the Cd exposure (i.e. 50 µM Cd for 7 days) was high enough to result in significant increases of oxidative stress rather than in significant accumulation of lipid peroxides in the shoot.
According to the above discussion, the oxidative statuses of rice plants with Cd treatment are associated with the configuration of H2O2 distributed in the shoot and root. Based on the variations in H2O2 concentrations in the shoot and root among the rice cultivars, they could be classified into the two groups. One includes TY3, TK9, TNG71, and KH145, whose H2O2 concentrations in both the root and the shoot increased dramatically with Cd treatment; the other includes TKW1, TKW3, TCS10, and TCS17, whose H2O2 concentrations in the shoot are more than those in the root with Cd treatment (Fig. 3).
The antioxidative enzymes in rice plants under Cd stress
In Fig. 4, the enhancements of APX activity with Cd treatment were more pronounced in the root than in the shoot. All cultivars could be divided into two groups according to the profile APX activities as well as based on the H2O2 concentrations in the shoot and root (Fig. 3). One group includes TY3, TK9, TNG71, and KH145, whose APX activities in the root were less than 1 unit g− 1 and those in the shoot were less than 2 unit g− 1. The other group includes TKW1, TKW3, TCS10, and TCS17, whose APX activities in the root were higher than 1 unit g− 1 and those in the shoot were higher than 4 unit g− 1. After Cd treatment, the APX activities in both the root and the shoot for TY3, TK9, TNG71, and KH145 were much lower than those for TKW1, TKW3, TCS10, and TCS17 (Fig. 4). Also, the SOD activities of the group including TY3, TK9, TNG71, and KH145 were enhanced more significantly by Cd treatment than those for the other one including TKW1, TKW3, TCS10, and TCS17.
It is well known that SOD catalyzes the conversion of superoxide anion to less toxic H2O2 and shows promise as the first line of defense against oxidative stress (Shah et al. 2001). The major sources of H2O2 in cells are mitochondria and chloroplasts, in which peroxisomes and glyoxysomes contain SOD that is responsible for H2O2 production (Jiménez et al. 1997; Dixit et al. 2001). APX is the most important enzyme in chloroplast for scavenging H2O2 by ascorbate converting into H2O and O2 (Sidhu et al. 2016). The APX activities in the root and leaf could be stimulated by Cd treatment to remove H2O2 (Dixit et al. 2001), and they would be involved in some additive function in the metal tolerance mechanism in plants. For the group including TY3, TK9, TNG71, and KH145, H2O2 concentrations in the root and shoot increased greatly with Cd treatment owing to relatively low increases in APX activity. For the other one including TKW1, TKW3, TCS10, and TCS17, Cd treatment resulted in relatively high increases in APX activities in the root. Thus, their H2O2 concentrations with Cd treatment in the root did not increase dramatically as much as those seen for TY3, TK9, TNG71, and KH145. Based on the catalyzation mechanism of SOD in the dismutation of superoxide into H2O2 (Cruz de Carvalho 2008), the more the enhancement of SOD activity, the more is the increase in H2O2 concentration. Thus, H2O2 concentrations in the shoot being higher than those in the root for almost all rice cultivars by Cd treatment (Fig. 3) would be due to the SOD activities in the shoot being higher than those in the root for all the cultivars by Cd treatment (Fig. 4).
Inferential understandings for the physiological regulation in rice plants
According to the abovementioned physiological traits (i.e., MDA, H2O2, SOD, and APX) in rice plants with Cd treatment (Figs. 3 and 4), the physiological patterns responding to Cd stress in the root were different from those in the shoot. The TF values of MDA, H2O2, SOD, and APX were used (Eq. [3]) to associate the responses to Cd stress in the root and shoot. In CK, TF observations showed the steady-state conditions in which a delicate balance exists between the production of reactive oxygen species and the cellular antioxidative defense machinery (Gill and Tuteja 2010; Srivastava et al. 2015). Compared with the TF values in CK, those under Cd treatment indicated the disturbances in stationary oxidative statuses for rice seedlings. In Table 1, with Cd treatment, the TF values of both SOD and APX for all cultivars were much higher than 1. This indicated that the antioxidative machinery was pronounced more in the shoot than in the root; this agreed with a previous study by He et al. (2014). The fact that with Cd treatment the TF values of H2O2 concentration for all cultivars except TY3 and TNG71were higher than 1 revealed that H2O2 would be present more in the shoot than in the root. However, the TF values of MDA were less than 1 for all cultivars except TKW1. That is, MDA preferentially accumulated in the root with Cd treatment. Nevertheless, the conflict between MDA and H2O2 distributed in the root and shoot arises owing to the relatively high SOD and APX activities in the shoot (Fig. 4). The high APX activity in the shoot could scavenge H2O2 to reduce lipid peroxidation in the shoot (He et al. 2014), even though there was higher H2O2 accumulation in the shoot owing to the high SOD activity to transform more superoxide anion into H2O2.
To illustrate the physiological regulation in rice plants under Cd stress, which should be based on the steady-state of physiological symptoms with CK, the TI values, which indicate the tendency of changes in the physiological traits (i.e. MDA, H2O2, SOD, and APX) toward shoot by Cd treatment (Eq. [4]), for the rice cultivars were shown in Table 1. All cultivars except TKW1 had the TI values of MDA being lower than 0.5. However, almost all cultivars had the TI values of H2O2 being higher than 0.5, except for TY3. This suggested that the rice plants under Cd stress would preferentially enhance MDA toward the root and regulate H2O2 accumulation toward the shoot. The TI values of SOD for the cultivars except TY3 and TCS10 were close to or higher than 1. Also, most cultivars excluding TKW3, TCS10, and TCS17 had the TI values of APX were relatively near and higher than 1. Therefore, the Cd treatment promoted most cultivars to preferentially regulate SOD and APX toward the shoot.
Table 2 shows the correlation coefficients (r) between the plant growth factors, RRE, and RSE and the plant Cd concentrations in the root and shoot versus the TI values of SOD, APX, H2O2, and MDA. The RRE and RSE were not significantly correlated with the TI values of SOD, APX, H2O2, and MDA except for the relationship between RRE and TI of H2O2. The positive correlation between RRE and TI of H2O2 (r = 0.77) was significant. This revealed that under Cd stress, the rice cultivars regulated H2O2 accumulation toward shoot and their root could be with gradually keeping growth. The cultivars such as TK9 and TKW1 had relatively high TI values of H2O2 (i.e. 1.74 and 1.31), and their root elongations were consistent with their TI values of H2O2 to be higher than other cultivars’ (Fig. 1). In addition, the root Cd content was significantly positively correlated with the TI of SOD (r = 0.65); however, the shoot Cd content was significantly negatively correlated with the TI of APX (r = -0.73). Recall Fig. 2 to find that TNG71’s Cd concentration in root was the highest and KH145’s Cd concentration in the shoot was approaching to the lowest. Their TF values of Cd concentration were the lowest. Also, we found that TNG71 had the highest TI value of SOD (i.e., 2.49); KH145 had the highest TI value of APX (i.e., 2.64). These findings indicated that Cd preferentially accumulated in the root would be involved in the regulation tendency of SOD toward the shoot; the regulation tendency of APX toward the shoot would be related to less translocation of Cd into the shoot.