The Mechanisms For Inhibition Of Root Growth By Ammonium And Ammonium-chloride Salt Combined Stresses

In recent years,with the increase of nitrogen-fertilizer input and dry deposition of ammonia,the occurrence of NH₄⁺toxicity in plants tends to be more frequent.Additionally,unreasonable application of N fertilizer in coastal saline soil and facility agriculture will result in the exposure of plants with NH₄⁺and salt combined stress.In this study,we used Arabidopsis thaliana to investigate the response mechanisms in plants to alone stress with NH₄⁺and combined stress with NH₄⁺and chloride salt.The main findings are as follows:NH₄⁺ stress significantly stimulates iron(Fe)accumulation in the stele of Arabidopsis thaliana roots.However,decrease of the iron level in growth medium confers an insensitive phenotype of primary root growth in response to NH₄⁺stress,indicating that Fe is crucial for the NH₄⁺-induced root growth inhibition.Comparisons between Col-0,lpr1-1,and lpr2-1 regarding their root growth under NH₄⁺supply indicate that the LPR2 but not the LPR1 is involved in the NH₄⁺-induced root growth inhibition.Comparative transcriptome analysis between Col-0 plants and lpr2-1 mutants revealed that oxidation processes may be critical in the root developmental response to NH₄⁺stress under different Fe levels.Noteworthy,a large number of peroxidase genes were expressed differently between Col-0 plants and lpr2-1 grown in the NH₄⁺medium.In situ localization of peroxidase activity also showed that the accumulation of apoplastic Fe induced the peroxidase activity in Col-0 roots stele,which participates in ROS generation and Fe redox cycling.Inhibition of peroxidase activity by Methimazole was able to rescue the root elongation under NH₄⁺stress,indicating that induction of peroxidase activity by LPR2-meidated excess iron deposition in stele should be a mechanism for root growth inhibition by NH₄⁺stress.In accordance with the ROS over-accumulation in the root stele,we also observed an over-production of callose in stele in an LPR2-dependent manner under NH₄⁺stress.Moreover,NH₄⁺inhibits the expression of PIN1 in an LPR2-dependent manner,which in turn further reduced auxin transport.Accordingly,root growth inhibition by NH₄⁺can be partially restored by exogenous addition of IBA,which showed that inhibition of auxin transport by callose deposition and by lower PIN1 expression is another way that NH₄⁺inhibited root growth by Fe accumulation.We then investigate the effect of the NH₄⁺and chloride salt combined stress on root growth.The results showed that salt stress inhibited root growth regardless of nitrogen forms used.But compared with NO₃^--fed plants,the inhibition of root growth was significantly more pronounced in NH₄⁺-fed plants,which indicates that NH₄⁺can exacerbate salt stress in plants.Interestingly,NH₄⁺-conferred salt hypersensitivity was abolished by removing the Cl^-,but not affected by the removal of Na⁺,suggesting that the excess accumulation of Cl^-rather than Na⁺is involved in NH₄⁺-conferred salt hypersensitivity.Because the NO₃^-and Cl^-have similar chemical properties,we further investigated a role of NO₃^-uptake transporters in mediating the sensitivity to NH₄⁺and chloride salt combined stress.We found that the expression of NRT1.1 in the roots was enhanced by NH₄⁺.Besides,loss function of NRT1.1 in plants showed improved tolerance to mild Na Cl stress in the NH₄⁺medium.We,therefore,compared the Cl^-uptake between Col-0 and chl1-5 using a non-invasive technique and showed that NRT1.1 has a Cl^-uptake activity in plants.Therefore,The NH₄⁺-enrichment of the growth environment aggravates salt stress in A.thaliana plants as a result of the excess accumulation of Cl^-,which is attributed to the increased Cl^-acquisition by NRT1.1 and lack of competition from NO₃^-.