The Mechanisms Of NRT1.1and MPK6in Regulating The Tolerance Of Plant Against To Cadmium Stress

The rapid development of modern industry brings in great convenience to human life, but also it causes great damage to the natural environment. Soil heavy metal pollution is one of the most serious problems, Cadmium (Cd) contamination is much concerned due to its long life cycle, high toxicity, nonbiodegradation. Therefore, the search for an technology that could effectivly remediate soil heavy metal pollution is imminent. As we known, Nitrogen(N) management is an frequent agronomic measure, Previous studies have indicated that under Cd stress, the nitrate content of plants declines significantly and that the lack of nitrate can reduce the accumulation of cadmium significantly in the plant, The physiological and biochemical processes on how to change the nitrate content under cadmium stress remain unclear, and the roles of nitrate accumulation reduction play in Cd accumulation need to be verified in the experiment.The plants were grown in nutrient solution, we found that nitrate concentration of the wild-type Arabidopsis significantly decreased under Cd stress, Meanwhile, Cd treatment also inhibited the uptake rate of nitrate in roots, the activity of nitrate reductase and nitrate transport from roots to shoots, indicating that the inhibition of NO3-uptake was responsible for the Cd-decreased NO3-accumulation in roots. Furthermore, the transcriptome sequencing showed that cadmium treatment also inhibited the expression of NRT1.1significantly, while the expression of other genes responsible for nitrate uptake transporter was either not affected or up-regulated, indicating the inhibitioin of NRT1.1activity might be responsible for the inhibition of NCO3-uptake by Cd exposure. Histochemical GUS staining in roots of pNRT1.1::NRT1.1-GUS transgenic plants showed that Cd exposure triggered a great decrease in NRT1.1-GUS protein level in roots. By analyzing the NO3-uptake rate of chll.5and WT (Col-0), we found that the difference of the NO3-uptake rate of chll.5and WT was not obvious under Cd treatment. The result indicated that blocking NRT1.1-controlled NO3-uptake was responsible for the inhibition of NO3-uptake by Cd exposure. Further studies found that nrt1.1mutants (chll.5and nrt1.1-1) had decreased Cd concentrations and enhanced Cd tolerance compared with their wild type WT, Meanwhile, The concentrations of Fe、Ca、K were decreased compared with WT. Histochemical GUS staining showed that the NRT1.1protein level in roots was normal after1h growth in the NO3--omitted growth medium, by measuring Cd uptake rate, we found there was no difference in Cd uptake among these three plants in NO3-V-omitted growth medium, no difference in Cd concentration was observed in both WT and chll.5when we grew plants in NO3--omitted medium. Consequently, these results also validated that NO3-uptake was a necessary process for the negative regulations of both Cd resistance and Cd uptake by NRT1.1. By emphasizing the expression of the transporter genes related to acquisition of bivalent metal, we found chll.5mutant did not have a significant decrease in expression of either of these, as compared with WT plant in Cd-exposure treatment. Under N-Cd rotation treatment, The Cd concentration of WT was similar to chll.5and nrt1.1-1, but the concentrations of Fe、Ca、K were still higher than nrt1.1mutants, N-Cd rotation treatment eliminated the synchronous uptake of NO3-and Cd, but still maintained the synchronous uptake of NO3-and other cation nutrients. Therefore, the above observations validated that suppression of Cd entry into roots by blocking NRT1.1-controlled NO3--uptake was achieved through the ionic-balancing process.Nitrate Reductase plays an important role in changing the nitrate content of plants in vivo, Cd can activate mitogen-activated protein kinase MPK6activity, MPK6increased nitrate reductase activity by phosphorylating NIA2. Therefore, We hypothesize that MPK6plays an important role in the regulation of Cd tolerance. We used mpk6-2mutant to explore the regulation mechanism of MPK6in Cd tolerance. The results showed that the Cd tolerance of mpk6-2mutant was stronger than WT and that the NO content of mpk6-2mutant was lower than WT after Cd treatment, however, the results showed there was no difference between WT and mpk6-2mutant in Cd content, indicating the difference of cadmium tolerance between WT and mpk6-2mutant was not Cd absorption regulated by NO. Oxidative stress caused by reactive oxygen species is usually a follow-up response of toxic metal absorption. We found that the H2O2content of WT in roots increased more obvious than mpk6-2after Cd treatment; the membrane lipid oxidation extent and integrity of cell loss of WT was more serious than mpk6-2, this explained MPK6mutant Arabidopsis helped alleviate oxidative stress induced by Cd and ROS generated. In addition, We found that the extent of growth decline of WT and mpk6-2was weakened by foliar AsA application after Cd treatment, These above results suggested that the content of ROS and oxidative stress may be the reason to cause cadmium tolerance differences between WT and mpk6-2.