- responsive non-coding RNAs in tobacco roots ( Nicotiana tabacum. - The number of circRNAs identified were higher than that of miRNAs and lncRNAs, but only two circRNAs were present in the co-expression network. - LncRNAs appear to be the most active ncRNAs based on their numbers presented in the co-expression network, but none of them seems to be an eTM (endogenous Target Mimicry) of miRNAs. - Integrated with analyses of sequence interaction, several mRNA-circRNA-miRNA interaction networks with a potential role in the regulation of nicotine biosynthesis were uncovered, including a QS-circQS-miR6024 interaction network. - The findings reported here indicate that ncRNAs appear to form interaction complex for the regulation of stress response forming regulation networks with transcripts involved in nicotine biosynthesis in tobacco.. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - 6 Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China Full list of author information is available at the end of the article. - Wang et al. - The pyridine alkaloid nicotine is the signature com- pound of Nicotiana species, especially in tobacco, re- sponsible for the addiction of smoking and functioning as one of the most effective plant defense metabolites in nature. - Gao et al. - Recently, Li et al. - In our previous study, a new miRNA, nta-miRX27, was shown to target QPT2 , one of the key genes involved in nicotine biosynthesis. - Nevertheless, it is still unclear whether or not circRNAs are also one of the components of the RNA network related to nicotine bio- synthesis in tobacco roots.. - On the base of identification of the four types of RNAs, their expression levels and abundance were quantified and compared based on a unified normalization approach. - A co-expression net- work for the four types of RNAs was established and a few mRNA-circRNA-miRNA interaction networks were iden- tified, including QS-circQS-miR6024, involved in the regulation of nicotine biosynthesis. - RNA-seq, circRNA-Seq, small RNA-Seq and ssRNA-Seq were performed using roots from both topping-treated (i.e., removal of the floral head and upper young leaves) and untreated tobacco plants. - The number of the four types of RNA mole- cules identified and used in this study were shown in. - More than 90% of the 85,570 gene models anno- tated by Sierro (2014) were supported by transcripts found in our RNA-seq data, with 77,849 and 78,467 genes expressed in the topping-treated and untreated roots, re- spectively. - The majority predicted targets of the conserved miRNAs were transcription factors (TF). - Only about 10% of the lncRNA loci were identified in both datasets (Additional file 2: Fig. - A much higher proportion of single exon transcripts was found in the ssRNA-seq datasets transcripts) compared to the poly(A) RNA-seq datasets transcripts) (Additional file 2:. - S3B) probably due to vari- able sensitivity of the prediction tools towards different types of circRNAs [27]. - The general features of the identified to- bacco circRNAs consist with those identified in other plants [28, 29], such as more than 80% of exonic circRNAs har- bored 1–4 exons (Additional file 3: Figure S3C) and most genes produced only one isoform of circRNAs (Additional file 3: Fig. - Expression profiling of coding RNAs and three types of ncRNAs in tobacco roots. - In the topping-treated sample, 1058 significantly differentially expressed genes (DEGs) (|log2 (fold-changes)|. - As expected, most genes, such as QPT2 , QS and PMT2, of the nicotine biosynthesis pathway were significantly up-regulated in response to top- ping. - Besides, the differential expression patterns of 6 of the 7 selected miRNAs, including 3 target- ing nicotine biosynthesis genes, were also verified by qRT- Table 1 Summary of raw sequencing data (Gb) from the. - Table 2 Number of coding and three types of non-coding RNAs identified in tobacco. - For the circRNAs, the topping-induced expression patterns were verified for 9 out of the 17 selected ones, just as their topping-induced parental genes (Fig. - In order to effectively compare the expression level of the four types of RNAs, a unified normalization standard is required. - The expression level of circRNA and miRNA was converted into FPKM and plotted FPKM of the four types of RNAs based on their length (Fig. - 1 Expression profiling of mRNAs, circRNAs, miRNAs and lncRNAs in tobacco. - 19.324) transcripts in the topping-treated sample were lncRNAs. - The transcript length of the expressed RNAs was also compared. - Co-expression of the four types of RNA molecules responsive to topping in tobacco roots. - To identify potential roles of the ncRNAs, a co-expression network for the four types of RNA mole- cules was constructed by calculating Pearson’s correl- ation coefficient value (the absolute value of PCC >. - 3, after applying stringent filtering steps 6 conserved miRNAs were found in the co-expression net- work. - Two miRNAs (miR482a and miR482d) of the 6 miR- NAs identified belong to the miR482 family. - However, a larger number of lncRNAs (18) was presented in the network and showed a co-expression pattern with 106 mRNAs as well as the aforementioned 6 miRNAs and 2 circRNAs. - Closer examination of the genes of the nicotine biosyn- thesis pathway showed that A622 and PMT were present in this co-expression network and that miR394 showed a nega- tive correlation with these two genes. - circCHMP4B and circEIF4G1 were present in the network, but their parental genes ( CHMP4B and EIF4G1 ) were not related to nicotine metabolism, thus no putative circRNA-containing network related to nicotine biosynthesis was identified in this study.. - Nevertheless, it was possible to find 5 nicotine biosynthesis related co-expression networks containing miRNA, mRNA and lncRNA (i.e. - 1) pattern of the RNAs involved and their physical interaction potential were checked.. - Majority of these miRNAs are conserved ones, for example, 40 of the 114 miRNAs involved in the interaction between miRNA and mRNA,. - and 23 of the 36 miRNAs involved in the interaction be- tween miRNA and circRNA were conserved. - As for lncRNA-miRNA interaction, differentially-expressed miRNA and lncRNA that was predicted to be eTM of the miRNA were ana- lyzed, and no statistical relationship could be concluded.. - 4b), implying that many parental genes of the. - The yellow block in the middle of the network indicated the RNAs involved in nicotine biosynthesis. - differentially expressed circRNAs in the topping-treated tobacco roots are involved in synthesis of secondary me- tabolites, including nicotine.. - The regulation network of the three types of RNA molecules involved in the nicotine biosynthesis pathway Based on BLASTp search using known genes [31], 27 to- bacco genes related to nicotine biosynthesis and metab- olism were identified (Fig. - For the nicotine biosynthesis related miRNAs, in addition to those identified in our previous study [22], one conserved (miR6024) and 6 novel miRNAs were newly found to target 5 of the 27 genes related to nicotine biosynthesis (Fig. - Fourteen (14) out of the 27 genes related to nicotine biosynthesis were significantly up-regulated after the topping treatment (Fig. - The differential expression pattern was verified by qRT-PCR for 9 of the 16 circRNAs (generated from 4 parental genes). - on the expression analysis and identification of the inter- action sites, three networks involving mRNA, miRNA and circRNA were uncovered. - QS , that contributes to both the nicotine and NAD pathways, was involved in the network QS-circQS-miR6024 (Fig. - Of the components of the network AO2-circAO2-miRX282, AO2 gene was significantly up-regulated after the top- ping treatment, while circAO2 and miRX282 were slightly down-regulated (Fig. - Two (circQPT2.1 and circQPT2.2) were only identified in the control while the third circRNA (circQPT2.3) was only found in the topping treatment.. - Of the three networks, QS-circQS-miR6024 showed the highest expression levels of its components (Fig. - 6, coding genes were not the only determinant in nicotine biosynthesis in tobacco root, the interaction with non-coding RNAs, such as circRNAs and miRNAs, also extensively involved. - The present work investigated the dynamic changes of mRNAs, miRNAs, circRNAs and lncRNAs, and their interactions in response to topping treatment in tobacco, with a focus on coding and non- coding RNAs related to nicotine biosynthesis. - Our re- sults provide evidence for the involvement of ncRNAs and their interactions with coding genes in nicotine bio- synthesis and the foundation for further functional characterization of the networks involving both coding and non-coding RNAs in tobacco.. - The most abundant transcripts in tobacco were coding RNAs. - This difference could partially be the result of the many different bioinformatics tools having been used in cir- cRNA identification in A. - As the limitation of the circRNA enrichment technology and no single method dominated on all of the metrics such as preci- sion and sensitivity to date, use of different bioinformatic tools would compensate the drawbacks of each individual tool to get comprehensive results. - of the miRNAs identified in this study were conserved ones (Additional file 1: Figure S1A). - Nevertheless, most of the significantly differentially expressed circRNAs. - Only two circRNAs represented in the co- expression network, largely due to the low expression level of circRNAs and their unique to one of the two samples used. - Both A662 and PMT2 are not target genes of miR394, suggesting that co- expression of miR394 with A662 or PMT2 could be achieved through the lncRNAs of the modules. - The majority nodes of the co-expression network were mRNAs, ncRNAs occupied only a small number of. - 6 The potential interaction of RNA molecules involved in the nicotine biosynthesis pathway in tobacco root. - The differential expressed circRNAs which generated from parental genes involved in nicotine biosynthesis pathway were presented. - The nicotine biosynthesis pathway diagram drew based on previous studies in tobacco. - nodes of the co-expression network, suggesting that ncRNAs-mediated regulation of cellular processes might be mainly achieved by interacting with mRNAs rather than with other ncRNA molecules.. - Because each miRNA has the identical targets in the circRNA and its parental gene, binding of the miRNA to each of its two targets would be competitive. - In view of the negative correlation between miRNA and its targets, it is expected that binding of the miRNA to cir- cRNA would relax the miRNA-mediated cleavage of its tar- get mRNA. - therefore, the role of the circRNA is similar to that of eTM, i.e. - In summary, our comprehensive analyses of mRNAs and ncRNAs (miRNAs, lncRNAs, circRNAs) in tobacco roots and identification of coding and non-coding RNAs and their interaction networks in responsive to topping treatment enabled us to draw an overall picture of the transcriptome profile of all the four types of RNA mole- cules and offered us an insight into the crosstalk amongst these RNAs in tobacco.. - At least three 50-d-old (days after seeding) plants were used for topping treatment (i.e., re- moval of the floral head and the upper young leaves of the tobacco plants), and plants of a similar size without topping treatment were used as control. - Prediction of miRNAs in tobacco. - All transcriptomes were then merged based on the annotation file of the reference genome to gener- ate a final transcriptome using Cuffmerge. - Quantifications of the expression level of genes, circRNAs and miRNAs were performed using FPKM (fragments per kilobase of transcripts per million mapped reads), SRPBM (number of circular reads/number of mapped reads (units in billion)/read length), and RPM (reads count of miRNAs/. - Differentially expressed mRNAs, cir- cRNAs, miRNAs and lncRNAs were defined by different standard in terms of the adjusted P-value and the absolute log2 (fold-changes) as shown in Additional file 8: Table S4, Additional file 9: Table S5, Additional file 10: Table S6, Additional file 11: Table S7. - To compare the expression level of the four types of RNA molecules in a unified man- ner, they were normalized as FPKM mRNA (number of mRNA reads/number of mapped reads (units in billion)/. - Pearson’s correlation coefficient was used to measure the strength of the linear dependence of two vari- ables. - Annotation of nicotine biosynthesis and catabolism related genes. - 50% were used in BLASTp search of homolo- gous genes related to nicotine biosynthesis and catabol- ism [50].. - The Oligo (dT) and random hexamers supplied in the kit were used in generation of the 1st strand cDNA for quantification of mRNAs and lncRNAs, respectively. - In this study, 688 miRNAs, 7423 lncRNAs and 12,414 cir- cRNAs were identified in tobacco roots. - mRNA-miRNA-circRNA) related to nicotine biosynthesis were uncovered. - Taken together, the study enhanced our under- standing of the RNA landscape in tobacco and will facilitate functional characterization of mRNAs and ncRNAs in bi- otic/abiotic responses in tobacco.. - Genome-wide identification of miRNAs in tobacco.. - Genome-wide identification of lncRNAs in tobacco.. - Features of circRNAs identified in tobacco and compared with other plants.. - All the authors read and approved the final version of the paper.. - During the execution of these experiments, all plant materials collected were destroyed at the conclusion of the work. - Recruitment of a duplicated primary metabolism gene into the nicotine biosynthesis regulon in tobacco. - NtERF32: a non-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco. - Regulation of nicotine biosynthesis by an endogenous target mimicry of MicroRNA in tobacco.. - Wild tobacco genomes reveal the evolution of nicotine biosynthesis. - Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice
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