Mediating The Expression Of AtNRT1.1,AtNRT2.1and PdAMT1.1Genes Affects Nitrogen Absorption In Arabidopsis And Poplar

Nitrogen availability is a major environmental factor that regulates plant growth and development. Nitrate (NO3-) and ammonium (NH4+) represent the most readily available forms of nitrogen for root absorption from the soil. Based on recent studies, there are many genes from different nitrogen transporter families responsible for NH4+and NO3-absorption. NRT1.1and NRT2.1are two crucial genes in NO3-transport system, and AMT1.1is a key gene in NH4+transport system. Regulating the expression of these genes could improve the efficiency of nitrogen absorption in plants.We regulated the expression of nitrogen transporter genes AtNRT2.1, AtNRT1.1and PdAMT1.1by ethylene mediation, auxin mediation and transgenic technology, respectively, to improve the efficiency of nitrogen absorption in Arabidopsis and poplar.Gas chromatography, scanning ion-selective electrode (SIET), quantitative real-time PCR (qRT-PCR) and GUS reporter assay techniques were used to explore the molecular interaction between AtNRT2.1transcript levels and the ethylene signaling pathway under nitrate deficiency. We reported a low nitrate (LN) treatment-induced rapid burst of ethylene production and regulated expression of ethylene signalling components CTR1, EIN3and EIL1in wild-type Arabidopsis thaliana (Col-0) seedlings, and enhanced ethylene response reporter EBS:GUS activity in both Col-0and the ethylene mutants ein3-leill-1and ctr1-1. LN treatment also caused up-regulation of AtNRT2.1expression, which was responsible for an enhanced high-affinity nitrate uptake. Comparison of ethylene production and EBS:GUS activity between mi1.1, nrt2.1mutants and Col-0indicated that this up-regulation of AtNRT2.1expression caused a positive effect on ethylene biosynthesis and signaling under LN treatment. On the other hand, ethylene downregulated AtNRT2.1expression and reduced the high-affinity nitrate uptake. Together, these findings uncover a negative feedback loop between AtNRT2.1expression and ethylene biosynthesis and signalling under nitrate deficiency, which may contribute to finely tuning of plant nitrate acquisition during exploring dynamic soil conditions.In order to study the modulating effect of auxin signaling on nitrate uptake in plants, SIET and qRT-PCR techniques were used to determine the net plasma membrane NO3-fluxes and the expression of nitrate transporter AtNRT1.1in primary roots of wild-type Arabidopsis Col-0, auxin-overproducing mutant yucl-D, and auxin-insensitive mutant axrl-12. Moreover, we examined the net plasma membrane NO3-fluxes and the expression of AtNRT1.1in primary roots of Col-0and dual-affinity nitrate transporter mutant nrtl.l seedlings under the normal condition (CK), the treatment with exogenous IAA, or the treatment with the auxin efflux inhibitor2.3.5-triidobenzoid acid (TIBA). The nitrate uptake and the transcript level of AtNRT1.1in yucl-D seedlings were remarkably enhanced, whereas those in axrl-12seedlings declined compared with Col-0seedlings. The exogenous IAA treatment enhanced the nitrate uptake and the transcript level of AtNRT1.1in Col-0seedlings, while the TIBA treatment had an opposite effect. Furthermore, the differences of the nitrate uptake and the expression of AtNRT1.1in nrt1.1seedlings among CK, IAA and TIBA treatments were insignificant compared with Col-0seedlings, revealing the important role that AtNRT1.1gene played in nitrate uptake pathway response to auxin.We cloned a key gene for the high-affinity NH4+uptake, PdAMT1.1, from Populus nigra×(Populus deltoides×Populus nigra). After genetic sequencing and protein structure analysis, we found the PdAMT1.1cDNA is1,495bp in length and encodes498amino acids. PdAMT1.1protein encodes9transmembrane domains and one conserved domain. The tissue-specific expression analysis indicates PdAMT1.1was expressed more highly in roots than in stems, buds and leaves. The subcellular localization analysis indicates PdAMT1.1localized in membrane system. Transgenic Arabidopsis plants that constitutively expressed the PdAMT1.1gene were constructed. We found under nitrogen deficiency conditions, the physiological phenotypes such as leaf areas and plant height, and the NH4+influxes in transgenic OxPdAMT1.1seedlings were much better than other seedlings, while those in amtl.l seedlings were most inferior. The complemented line amt1.1/PdAMT1.1demonstrated similar phenotype and NH4+uptake efficiency as wild type Col-0seedlings and the empty vector transgenic line Col-O/V. These results suggest that PdAMTI.l gene plays an important role in plant growth and NH4+absorption. Then we transformed PdAMT1.1gene into triploid white poplar by leaf disk method. Six transgenic OxPdAMT1.1lines and three empty vector transgenic lines were checked by Chlorophenol-Red assay, GUS assay and PCR analysis. To study the biological functions of triploid white poplar wild type seedlings WT, the empty vector transgenic line WT/V and transgenic OxPdAMT1.1seedlings, we found OxPdAMT1.1seedlings have much better phenotypes and NH4+uptake efficiency than WT and WT/V seedlings. Thus we proved the overexpression of PdAMT1.1gene by transgenic technology could generate new poplar species which have high nitrogen absorption efficiency.