Molecular And Physiological Mechanisms Underlying Amino Acids Uptake And Its Effects On Cadmium Enrichment In Populus × Canescens

Nitrogen(N)is quantitatively the most important nutrient for plant growth and development.Plants acquire N not only as NH4+ and NO3-of inorganic N forms,but also as amino acids of organic N forms.Amino acids,one of the most prevalent forms of organic nitrogen,and as a sole N source affect plant biomass partitioning and nitrogen use efficiency(NUE).Amino acids may also play an important role in plant response to heavy metal stress.It has been documented that Populus species can absorb and utilize amino acids as a sole N source for growth and development,but its preference to various amino acids and the specific molecular and physiological regulation mechanisms remains largely unknown.In addition,poplars have been regarded as potentially promising candidates in phytoextraction and phytoremediation of cadmium(Cd),but little is known about the influences of amino acids as sole N sources on the accumulation of Cd and its physiological and transcriptional regulations in poplars.The objectives of this study were to investigate which amino acids can be used as a sole N source for Populus × canescens(P.tremula × P.alba)growth and development,a preliminary experiment of screening amino acids was carried out,then poplar acquisition of15N-labeled N fertilizers was evaluated in P.× canescens.Subsequently,the physiological and transcriptional responses to organic and/or inorganic N of poplar were elucidated with the same N level.Finally,the effects of exogenous Phe on the uptake and accumulation of Cd was investigated in P.× canescens.The main results were as follow:To screen out which amino acids can be used as a sole N source for poplar growth,we initially used plantlets of P.× canescens supplied with inorganic N(NH4NO3)or one of protein-building amino acids to screen the target amino acid(Phe)as a sole N source for poplar growth,N was provided as 2 m M for 70 days.Plantlets of P.× canescens were rooted in the media containing NH4NO3 or one of amino acids(Phe,Gly,Asp,Gln,Glu,Arg,Ala,Ser,Cys,Asn,lys,Thr,Ile,Pro,His,Trp,Tyr)other than Leu,Val or Met.Particularly,poplars grown in the Phe-containing media displayed the highest root biomass,root length, root surface area and specific root length in comparison with those of plants supplied with NH4NO3,Gly or Asp.The concentrations of total free amino acids and Phe were also the highest in the roots of poplars grown in the Phe-containing.The fact that P.× canescens displayed the best growth performance in the Phe-versus other amino-acid-containing media indicates that Phe can serve as a sole N source for P.× canescens growth.And short term(24h)15N-labeling indicated that 15NH4+ was absorbed at the highest rate,followed by15N-labeled Phe,while15NO3-was absorbed at the lowest rate.And the15N-labeled Phe was mainly retained in the roots and only 3-17% of 15N-Phe was translocated to poplar aerial parts.To investigate the physiological and transcriptional responses of poplars to amino acids as sole N sources,P.× canescens was supplied with 6 m M N as one of nitrogen fertilizers(inorganic N(NH4NO3),phenylalanine(Phe),and the mixture of NH4NO3 and Phe)in sand culture for 42 days.A larger root system,and decreased leaf size and CO2 assimilation rate was observed in Phe-versus NH4NO3-treated poplars.Consistently,a greater root biomass and a decreased shoot growth were detected in Phe-supplied poplars.Decreased enzymatic activities of NR,GOGAT and GDH,and elevated PAL activity were found in Phe-treated roots.Accordingly,reduced concentrations of NH4+,NO3-and total N,increased concentrations of starch,total free amino acids and endogenous Phe in Phe-treated roots.Phe as a sole N source promoted root growth and enhanced NUE in P.× canescens.Moreover,the transcript levels of putative Phe transporters ANT1 and ANT3 were upregulated,and the PAL1/3 were overexpressed in the Phe-supplied leaves indicates that transcriptional regulation of PAL1/3 plays a role in Phe-derived N re-cycling in poplar leaves.And the m RNA levels of NR,GS2,NADH-GOGAT and GDH were downexpressed in Phe-treated roots and/or leaves.The downregulation of these genes involved in uptake and assimilation of NH4+and NO3-in Phe-supplied poplars is probably play an important role in adapting organic N to poplars.To investigate the effects of exogenous Phe on the uptake and accumulation of Cd and its physiological and transcriptional regulations in P.× canescens,saplings of P.×canescens were treated with low(1 m M),normal(6 m M)or high(12 m M)levels of Phe and other 0 or 100 ?M Cd Cl2 for 30 days.The low and high levels of exogenous Phe decreased the concentrations of total free amino acids and endogenous Phe in the roots,reduced the concentrations of NH4+,NO3-and total N in the roots and leaves.But elevated GOGAT and PAL activities were found in low level Phe-treated roots.Moreover,the transcript levels of AAP1,ANT1 and ANT3 were upregulated in the roots,and the PAL3 were overexpressed in the roots and leaves under low level Phe-supplied indicated that the low level of Phe enhanced amino acids uptake and metabolism in P.× canescens.High level of Phe inhibited the activity of PAL in the roots and down-regulated the expression of PAL3 in the roots and leaves.The low level of exogenous Phe promoted the metabolism of amino acids in the roots,enhanced the expression of ANT3,ABCC1 or HA2.1,increased the concentrations of amino acids in the leaves,and elevated levels of malic acid and citric acid,which resulted in reduced Cd accumulation in the roots,increased Cd translocation to poplar aerial parts.But the high level of exogenous Phe significantly reduced Cd concentrations in the aerial parts of P.×canescens.Furthermore,the concentrations of malic acid,citric acid,soluble phenolics and total thiols in the roots of P.× canescens under Cd treatment were increased in low Phe supplied,and the transcript levels of PCS and MTP1 related to the Cd tolerance,were enhanced in the roots and leaves of P.× canescens.Low level of exogenous Phe enhanced the chelation and reduced the toxicity of Cd,alleviated the inhibition of photosynthesis under Cd stress,and increased the Cd tolerance.In summary,these results indicate that P.× canescens roots can acquire Phe as a N source and transport it to the aerial parts to support plant growth and development;Phe as a sole N source stimulated root growth and enhanced NUE in poplar;and the low level of Phe increased Cd translocation to the poplar aerial parts and enhanced the chelation of Cd,increased Cd tolerance.These results have important theoretical and practical significance for improving NUE and Cd enrichment in poplars and other woody plants.