The Se-rich rich is more sensitive to the application of Se than the Se-free rice variety
To reveal the physiological responses to Se, the seedlings of the Se-rich red-grain rice Z2057A/CR727 and the control Se-free rice CR727 were subjected to a range of exogenous Se treatments. The root growth in both rice variants was promoted in the presence of Se in a dose-dependent manner and impaired in the higher concentration of Se (80μM) (Fig. 1a and 1b). With the increasing of Se, the Se-induced increase and inhibition of root elongations occurred earlier in Z2057A/CR727 than in CR727, at 10 μMv.s. 20 μM and 40 μMv.s. 80 μM, respectively, indicating that the Se-rich rice is more sensitive to the application of Se than its Se-free counterpart. Similar response patterns were demonstrated in the observation of the leaf length, even though the overall leaf lengthofthe Se-rich rice are shorter than that of the Se-free rice in every conditions (Fig. 1a and 1c), demonstrating that there is a suitable range of exogenous Se to promote growth in a particular rice variety.
We next investigated whether the involvement of Se affects the content of chlorophyll. The results suggested that Se increase the accumulation of chlorophyll in a dose-dependent manner, which is in line with the phenotypes observed in the growth of roots and leaves (Fig. 1d).
To understand how the above physiological alterations occurred in the present of Se, the Se distributions in different organs after a 14 d exposure to exogenous Se were analyzed. With the increasing of Se, the internalization of Se accumulated accordingly in roots, stems, leaf tips and main leaves (Table 2). It was observed that the Se contents increased significantly with the increasing concentrations of sodium selenate in different plant parts. A similar ascending trend for Se accumulation was observed for Se-free rice and Se-rich rice for root and stem parts, while the trend for leaf tips and all leaf produce was different for material under investigation. Under the treatments of 20μM and 40μM Se, the accumulation of Se was maximum in the stem, leaf tips, and all leaf produce, while a high concentration of 80μM decreased the Se accumulation. The result indicated that the Se-rich rice was more sensitive to Se uptake than the Se-free rice.
The effects of exogenous Se on the aboveground architectures of rice seedlings
To assess the effects of Se on rice morphology, we observed the alterations of the aboveground architectures in response to the application of a gradient of Se concentrations for 2 d and 14 d. Regardless of the treatment duration and the presence or absence of Se, the plant heights of the Se-free rice were always higher than the Se-rich rice (Fig. 2). Nevertheless, in the comparison to the Se-free rice, the height of Se-rich was impaired clearly in presence of a high concentration of Se (80 μM) at both 2 d and 14 d, indicating that the Se-rich exhibits a fast and primary response to exogenous Se toxicity (Fig. 2). There is no change in leaf color or appearance during short-term exposure to a series of Se concentrations (Fig. 2a). On the contrary, although the plant height was still increasing, the long-term exposure under a high concentration of Se (80 μM) induced the yellowing of leave in both rice varieties which is in accordance with the decrease of chlorophyll (Fig. 1d and 2b) and inparticular, increasing senescence in leaves of the Se-rich rice were observed, demonstrating that the uptake of Se may result in cell death-related events.
The production of Reactive oxygen species in response to Se
ROS has been considered to mediate the primary response of plant resistance to environmental toxicity [22–24]. Thus, we reasoned that ROS signaling might be involved in rice response to Se toxicity during long-term exposure. To this end, we first investigated the production of the major ROS, superoxide (O2–) and hydrogen peroxide (H2O2) in response to Se by using histochemical staining methods, NBT staining and DAB staining, respectively. With the increasing of Se concentrations, the staining of NBT and DAB gradually spread to the entire leaf blade (Fig. 3a and 3b). Interestingly, the blast of accumulation of O2– and H2O2 began from 40 μM Se in leaves of Se-rich rice, while it occurred earlier in those of Se-free rice.
To quantify the endogenous oxidative stress in accompany with the generation of ROS, the activity of SOD and the content of MDA in leaves were measured. Interestingly, the Se-rich rice had higher SOD activity than the Se-free rice in the absence of exogenous Se. The activity of SOD increased with the increasing of Se until a threshold (80 μM) where it plummeted reversely (Fig. 3c), which is matched to a dose-dependent manner presented at the whole-seedling level (Fig. 1 and 2). As the consequence, the content of MDA, an indicator of lipid peroxidation in plant cells [25, 26], declined in an opposite tendency to the increasing of the activity of SOD induced by Se in the exception of leaves at 80 μM Se where the activity of SOD was strongly inhibited, supporting that the elimination of ROS would be activated once its generation (Fig. 3d). Conclusively, the above evidence supported that ROS signaling is involved in the primary rice response to Se toxicity during long-term exposure.
Loss of water is one of the responses in leaves to external stresses in plants . We next studied whether there are differences between Se-rich and Se-free rice, which may contribute to the diverse Se responses in these two varieties. The relative water contents (RWC) was determined. Intriguingly, while there are only slight changes of RWC in the Se-free rice, the values of RWC of the Se-rich rice in the presence of Se decreased remarkably at concentrations of 40 μM and 80 μM.
The gene expression patterns of Se uptake- and transport-related factors in the application of exogenous Se
To understand the underlying mechanism of distinct Se-mediated physiological responses in Se-rich and Se-free rice varieties, we investigated the gene expression patterns of some key Se uptake- and transport-related factors in the application of exogenous Se by using real-time quantitative PCR (RT-qPCR). The expression of OsPT2, which encodes phosphate transports, was consistently up-regulated in the application of increasing concentrations of Se, indicating that OsPT2 might play a key role in the transportation of Se (Fig. 4a). Interestingly, the expression levels of OsPT2 in the Se-rich rice were higher than in the Se-free rich in regardless of additions of Se (Fig. 4a). In absence of Se, there is no difference in the expression levels of the Si influx transporter encoding gene OsNIP2;1 in the two rice varieties, by contrast, its expression pattern differed in the presence of Se (Fig. 4b). Compared to the control, there was no change of expression until a dramatically decrease in the highest concentration of Se in the Se-free rice (Fig. 4b). Speaking of its expression pattern in response to Se in the Se-rich rice, an increasing and reversely declining of expression were sequentially demonstrated in lower concentrations of Se (10 μM and 20 μM) and higher concentrations (40 μM and 80 μM), respectively, with a peak expression at point of 10 μM when the seedlings began to respond to Se-induced physiological growth (Figs. 1, 2, and 4b). In line with the previous assumptions [27, 28], the unique expression patterns presenting in the two rice varieties support that OsNIP2;1 may serve as a main positive regulator in Se transportation. Although the result showed that Se continuously induces the expression of OsSultr1;2, a sulfate transporter in presence of all concentrations of Se, we observed no obvious difference between the two rice varieties in those scenarios (Fig. 4c). Notably, the Se-rich rice accumulated enhanced expression level of OsSultr1;2 than the Se-free rice in absence of Se (Fig. 4c), indicating that the activity of OsSultr1;2 may contribute to a relatively stunted shoot architecture of Se-rich rice (Figs. 1a and 2) and positively mediate its sensitiveness to exogenous Se. Interestingly, the expression of CAL1,which encodes a Cd relative transporter maintains stable in absence and presence of Se in the Se-free rice, whereas it has been inhibited in the Se-rich rice (Fig. 4d). Conclusively, the gene expression data demonstrated that the uptake and transport of Se rely on known ion transporters, connecting to the assimilation processes of other elements.