Post-transcriptional Regulation Of Ammonium Transporter AtAMT1;1 In Arabidopsis

As one of the major nitrogen(N)forms in soil,ammonium is preferentially taken up by plant roots,while be toxic to plant in excess.Therefore,the tight controls of ammonium uptake process in roots are essential for plants to cope with changing external ammonium availability and internal N status.The previous study had revealed the mRNA stability of AtAMT1;1 a major contributor for high-affinity ammonium uptake in Arabidopsis roots,was regulated with organ-and N-dependent manner.However,the underlying molecular mechanism remains unknown to date.In this work,by molecular biology,genetics,and high-throughput sequencing approaches,we aimed to investigate the possible post-transcriptional regulation of AtAMT1;1 in Arabidopsis roots.The results will greatly extend our understanding on the regulatory mechanisms of N transport in roots,as well as provide valuable knowledge for the practices in nitrogen-efficient crop engineering.The main findings are summarized into two parts as follows:1.Post-transcriptional gene silencing of AtAMT1;1 in Arabidopsis rootsThe steady-state AtAMT1;1 transcript could not be detected in roots of 35S:AtAMT1;1-transformed Arabidopsis in atamt1;1-1(ko11)background,while AtAMT1;1-related siRNAs were accumulated massively.However,siRNAs was abolished by genetic cross with rdr6-11 mutant,indicating the RDR6-dependent post-transcriptional gene silencing(PTGS)involved in this process.The dependence of AtAMT1;1 PTGS in roots on its coding sequence was revealed by analyzing the transgenic plants expressing AtAMT1;1-ORF and GFP-AtAMT1;1-ORF fusion sequences.By the transgenic and crossing approaches,substitutions of either ko11 background or 35S promoter in 35S:AtAMT1;1-transformed-ko11 plants successively prevented PTGS,suggesting the trigger of PTGS by the interaction between ka11 background and the 35S promoter.Small RNA deep sequencing showed higher accumulation of siRNAs on AtAMT1;1 loci in the roots of silencing line,relating to its sequence features.In addition,mutations of two AtAMT1;1 sequence-specific sites could suppress the production of siRNAs and prevent the PTGS process for AtAMT1;1.These results indicate an RDR6-dependent aberrant RNA pathway in AtAMT1;1 PTGS in roots,which involves interactions of ko11 background and the 35S promoter,as well as AtAMT1;1 sequence features.2.N-dependent post-transcriptional regulation of AtAMT1;1 in Arabidopsis rootsDespite of the expected constitutive expression of AtAMT1;l in the "gain-of-function" mutant and UBQ:AtAMT1;1-transformed plants,higher accumulation of AtAMT1;1 transcript was evidenced under N-deficient condition(-N)than that under N-sufficient(+N)conditions,suggesting the existence of N-dependent post-transcriptional regulation of AtAMT1;1 in roots.Then a 207 bp sequence in the ORF 3'-end was shown to be essential for N-dependent post-transcriptional regulation as revealed by analysis of two T-DNA insertion lines expressing truncated forms of AtAMT1;1,and also by fusing with the GFP reporter gene.Using 5'-RACE analysis,an important site(+1458)located in 3'-end of AtAMT1;1 ORF was identified,as the major cleavage site of AtAMT1;1 transcript under +N condition.Site-directed mutagenesis of this site could prevent the N-dependent regulation.Although involvement of small RNAs was ruled out by deep sequencing analysis,a non-coding RNA(ncRNA)named S21 in length of 30-100 NT was detected using a probe specific to+1458 sequence.The expression of S21 was abundant under N-suffcient condition,of which AtAMT1;1 transcript was less accumulated reciprocally,suggesting a role of S21 in degrading AtAMTl;1 transcript.These results demonstrated the existence of N-dependent post-transcriptional regulation of AtAMT1;1,which probably mediated by the interaction between 207 bp from AtAMTl;1 3'-end and a putative ncRNA S21.Taken together,this work discovered the molecular mechanism underlying the post-transcriptional regulation of AtAMT1;1 in roots,and highlighted a novel epigenetic surveillance mechanism controlling ammonium transport process in planta.