Functional Characterization Of Ammonium Transporter PsAMT1.1 In Nitrogen Uptake And Utilization In Populus

Nitrogen is a component of nucleic acids,proteins,chlorophyll and enzymes,is an essential nutrient for plant growth and development,maintaining the normal material cycle and energy metabolism in plants.Plants mainly absorb inorganic nitrogen,and ammonium（NH₄⁺）is one of the main forms.The uptake of NH₄⁺by plants is mediated by ammonium transporters（AMTs）.The roots of plant rely on ammonium transporter proteins to take up ammonium from the soil and transport it to various organs and cells of the plant.Therefore,it is important to study the functions and regulation mechanisms of ammonium transporters to improve nitrogen utilization.Populus simonii is one of the major poplar species in northwest China because of its salinity tolerance,rapid growth and adaptability.In this study,we verified the function of ammonium transporter PsAMT1.1 in ammonium transport,nitrogen uptake and utilization in P.simonii and obtained PsAMT1.1 overexpression transgenic poplar.The PsAMT1.1 overexpression transgenic poplar lines were treated with different nitrogen levels（0.01 mM NH₄NO₃,1 mM NH₄NO₃,2 mM NH₄NO₃）.The activities of enzymes related to nitrogen uptake and metabolism and related physiological indicators such as NH₄⁺and NO₃^-were measured,and the effects of PsAMT1.1 on nitrogen uptake and metabolism were investigated in poplar.The main results were as follows:（1）The PsAMT1.1 gene was expressed in young leaves,mature leaves,old leaves,stems and root tissues of Populus simonii,with the highest expression in young leaves.The PsAMT1.1 gene was cloned from the root of P.simonii,and the length of coding sequence was1503bp,encoding 501 amino acids.The homology between PsAMT1.1 of P.simonii and PtrAMT1.1 of Populus trichocarpa was as high as 98.47%.（2）The PsAMT1.1 gene was transiently expressed in tobacco epidermal cells,and the PsAMT1.1 protein was localized on the plasma membrane,indicating that it is active at the membrane and is a membrane transporter protein.（3）Heterologous expression of the PsAMT1.1 gene in the yeast mutant 31019b,which is deficient in ammonium transporter,had the ability to transport NH₄⁺and restored yeast growth activity at low NH₄⁺concentrations;while heterologous expression of the PsAMT1.1 gene in the Arabidopsis quadruple deletion qko（deficient in AtAMT1.1;AtAMT1.2;AtAMT1.3;AtAMT2.1）,the NH₄⁺uptake kinetic curve conformed to a double regression curve fitted by the saturated Michaelis-Menten equation,with Km of 79.27±9.75μM,indicating that PsAMT1.1 is a high-affinity ammonium transporter.（4）Three PsAMT1.1 overexpression transgenic poplar lines were obtained and cultivated for 30 days at different nitrogen levels（0.01 mM NH₄NO₃,1 mM NH₄NO₃,2 mM NH₄NO₃）.The net photosynthetic rate,chlorophyll content and stem growth of the PsAMT1.1overexpression transgenic poplar significantly increased compared with the wild type under low and medium nitrogen conditions.The differences in stem and plant height growth and photosynthetic rate of transgenic poplars under high N conditions were not significant compared with the wild type.（5）Compared with the wild type,the accumulation of NO₃^-,NH₄⁺and total nitrogen contents in the roots and leaves of PsAMT1.1 overexpression poplar increased under low and medium nitrogen conditions,and NR,NiR,GS,GOGAT and GDH activities in the roots were enhanced to promote NH₄⁺assimilation and favor NH₄⁺metabolism;soluble sugar contents in the roots increased under low nitrogen conditions but not under medium nitrogen conditions.Under high nitrogen conditions,the roots of PsAMT1.1 overexpression poplar showed lower accumulation of NO₃^-and total nitrogen,increased NR,GS,GOGAT,and GDH activities,and increased soluble protein and soluble sugar content compared with wild type;the accumulation of NO₃^-and NO₂^-in leaves decreased,and the activities of NR and GS also reduced.In summary,PsAMT1.1 plays an important role in nitrogen uptake and utilization in poplar under low nitrogen conditions.