Effects Of Excessive Copper On Nitrogen Uptake And Assimilation In Rice And Its Relationship With Nitrogen Forms

Soil heavy metal contamination has become one of the urgent environmental problems.Accelerating the remediation and government of heavy metal contaminated soil has an important significance for ensuring economic benefits,food security and ecological security.Rice is the staple food in China,and its safe production is of great significance to the human health.As the most consumed fertilizer in the world,the application amount of nitrogen fertilizer and its forms play an important role in improving rice yield.Rice has a well-developed aerenchyma that can transport oxygen to the root system to participate in nitrification,thus,it grows in paddy soils with mixture of ammonium(NH4+)and nitrate(NO3-).As the mainly inorganic nitrogen(N)sources in the soil,NH4+and NO3-regulate the absorption,distribution and accumulation of heavy metals in plants and the resistance of plants to heavy metal stress.In the present study,we analyzed the changes of nitrogen assimilation and the mechanism of N forms regulating Cu accumulation in rice seedlings exposed to excess copper(Cu).Results are as follows:First,we analyzed the effects of different Cu concentrations on growth of Cu-tolerant rice variety(B1139)and Cu-sensitive rice variety(B1195),and the results showed that inhibition effect of excess Cu on NO3-concentration and NR activity in B1195 was significantly higher than B1139.Compared to 0.2 μmol L-1 Cu,the toxicity effect of 10μmol L-1 Cu on the growth of B1195 and B1139 was obviously higher in rice seedlings supplied with NO3-than those with NH4+.Under 10 μmol L-1 Cu,roots Cu concentration was higher in rice seedlings supplied with NO3-than those with NH4+,but shoots Cu concentration was less in plants supplied with NO3-than those with NH4+.RNA-seq technology was used to systematically analyze the impacts of 10 μmol L-1 Cu on gene expression in roots of rice seedlings supplied with 0.810 mmol L-1 NH4+or NO3nutrient.Total of 36,380 expressed genes and 64,671 expressed transcripts were annotated based on public databases.Using DESeq2 software,we obtained 4085 differentially expressed genes in roots of rice seedlings with different treatments.KEGG and GO enrichment analysis indicated that the differentially expressed genes were assigned to N metabolism,diterpene and phenylpropane biosynthesis,plant hormone signal transduction and GSH metabolism.GO enrichment analysis showed that N metabolism pathway we identified mainly focused on NO3-uptake and assimilation,and the diterpene and phenylpropane biosynthesis pathway mainly focused on the process of lignin biosynthesis.These results suggested that N forms can regulate the process of NO3-uptake and assimilation,Cu binding on the cell wall and antioxidant capacity by altering the expression of genes involved in nitrogen metabolism,diterpene and phenylpropane biosynthesis,thus,changing the tolerance of plants to copper stress.Excessive Cu has greater inhibitory effect on rice seedlings supplied with NO3-than those with NH4+,but Cu concnetration was less in shoots of rice seedlings supplied with NO3-than those with NH4+.The change of rhizosphere pH has no significant effect on Cu concentration in shoots of rice seedlings.In order to further study the mechanism of disturbed N assimilation under excessive Cu,the content of N-containing compounds and the activity of enzymes that involved in N assimilation in rice seedlings,as well as NO3-net uptake rate and the expression level of genes encoding NO3-transporter in roots were analyzed.Our results indicated that excessive Cu inhibited the expression of OsNPF2.2,OsNPF2.4 and OsNPF6.5 and NO3-uptake in roots,decreased the concentration of N-containing compounds and the activity of enzymes involved in N assimilation in rice seedlings.Increasement of NH4+or NO3-concentration in nutrient solution can effectively alleviate the inhibitory effect of excessive Cu on the growth of rice seedlings.These results suggested that excessive Cu inhibited rice growth through down-regulating the expression level of genes involved in NO3-uptake,leading to decreased net influx rate of NO3-in roots and the concentration of nitrogen compounds,thus,disturbing nitrogen homeostasis in plants.Increment of Fe2+ concentration in nutrient solution obviously enhanced the growth of rice seedlings exposed to 10 μmol L-1 Cu,especially when supplied with NO3-.The enhancement of Fe2+ concentration in nutrient solution increased Fe concentration in plants,promoted the formation of iron plaque on root surface and decreased Cu concentration in shoots of rice seedlings.Compared with NH4+,supplying with NO3-increased the amount of iron plaque and Cu concentration in iron plaque,but decreased shoot Cu concentration in rice seedlings exposed to excess Cu.The concentration of Cu in shoots of rice seedlings was significantly decreased with the increasement of Fe2+ concentration in nutrient solution under pH5.5 condition,whereas there was no significant difference under pH4.5 condition.In presence of 10 μmol L-1 Cu,the concentration of polysaccharides and lignin in cell wall and Cu concentration in cell wall components were higher in rice seedlings supplied with NO3-than with NH4+.These results indicated that the addition of NO3-increased the amount of Cu absorbed in iron plaque and root cell wall components and restricted the root to shoot transport of Cu,thus,reducing Cu content in the shoots under excessive Cu treatment.