Comparative Analysis Of Tobacco Roots Dynamic Expression Profile Following Topping Reveals Putative Shoot-to-root Communication For Nicotine Biosynthesis In Nicotiana Tabacum

Decapitation/topping(removal of the inflorescence and adjacent young leaves)is an conventional culturing practice for agricultural production.Previous reports showed that diverse biological processes are changed in response to topping,such as hormonal balance,root development,source–sink relationship,ability of nicotine synthesis and stress tolerance.The topping induced that root nicotine synthesis ability increases and restrict upper leaf usability in industry.Except for smoking,nicotine is also widely used in biological pesticide and chemical products.So the topping induced regulatory mechanism of nicotine biosynthesis,Which is great significance.In order to elucidate the molecular mechanism of increased nicotine content,DGE of tobacco roots at 0,2,6,24 h after topping was analyzed with Solexa Illumina technology,and a putative regulatory mechanism of root development and nicotine biosynthesis was proposed.1.These DGE libraries generated over 3.6 million raw tags by RNA-sep.After filtering adaptor sequences,empty tags,low quality tags and one copy sequence,these DGE libraries generated over 3.4 million tags,and there were 0.3 to 0.4 million tags with distinct sequences.The number of total and distinct clean tags had a similar distribution patterns for all four DGE libraries.Tobacco Genome Survey Sequences was used reference gene database,and all clean tags were aligned to the reference gene database.There were about 57% to 62% of the distinct tags mapping uniquely to the reference gene database.The total of 158,626,153,034,151,455 and 145,294 tag-mapped transcripts were respectively obtained for the four libraries.2.Screening of topping-responsive genes in tobacco roots.Topping-responsive genes were screened from 2 h,6 h and 24 h libraries compared with 0 h library.The number of differentially expressed genes increased gradually from 0 h to 24 h.One hundred and forty-six genes,which were only differentially expressed at 2 h after topping,were identified as early topping-responsive genes.Correspondingly,770 genes,which were only differentially expressed at 24 h after topping,were identified as later topping-responsive genes.3.The pathway and GO enrichment analysis is helpful to better understand the differential genes function.Base on the analysis of the differentially expressed genes,178 known genes were found to be related to the response of tobacco to topping,including 73 genes related to stress,43 genes related to stimuli,12 genes related to secondary metabolism,28 genes related to plant signal transduction and 22 genes related to development.4.The q RT-PCR analysis of fifteen representative genes was performed to validate the expression of tag-mapped genes in tobacco roots.The fifteen genes were related to some biological process related to topping,such as the JA signaling pathway,IAA signaling pathway,defense response,plant development and secondary metabolism.The expressions of these genes were consistent with the tag-seq analysis results.5.Analysis of differentially expressed genes in the DGE libraries in combination with SSH libraries.In the DGE libraries,there were 49 tags that had mapping genes that were found to be the differential expressed genes from SSH analysis.These differentially expressed genes had different biological functions,including signaling,stress/defense and other metabolic processes.6.It had been reported that mi R164 a targets the NAC1 transcription factor(NAC1),which mediates auxin signaling to promote lateral root development.The results of DGE and q RT-PCR showed that Nta-mi R164 a was markedly repressed and Nt NAC-R1 was obviously induced by topping,indicating that Nt NAC-R1 and Nta-mi R164 a play important roles in the response of tobacco roots to topping.7.N t GRAS-R was predicted as the target gene of Nta-mi R171 d.In the present study,q RT-PCR results showed that Nta-mi R171 d was markedly induced and Nt GRAS-R1 was obviously repressed by topping,indicating that Nta-mi R171 d and Nt GRAS-R1 are involved in the response of tobacco roots to topping.when the expression of Nt GRAS-R1 increased,the expression of CLAVATA3(At CLV3)was down-regulated by induced,and a long root phenotype was induced.Therefore,Nta-mi R171 d and Nt GRAS-R1 are involved in the root development after topping.8.when the expression of Nt WRKY-R1 down-regulated,the expression of Nt PMT was up-regulated.WRKY transcription factors specially bind the cis-element 5?-TTGAC-C/T-3?(W-box).The promoter of Nt PMT contained three W-boxes.Therefore,it was deduced that Nt WRKY-R1 might directly bind the promoter of Nt PMT to negatively regulate the expression of Nt PMT.Nt WRKY-R1 was predicted as the target gene of Nta-mi R167 d.The expression of Nt WRKY-R1 decreased and the expression of Nta-mi R167 d increased in tobacco roots after topping,indicating that Nta-mi R167 d and Nt WRKY-R1 contributes to the increase of nicotine biosynthesis ability after topping.