Characterize Of Roles Of MRNAs And MiRNAs In Responding To Drought Stress In Tobacco Seedling Roots

Tobacco is an important agricultural and economic crop in China, and its cultivation area and yield in China were the highest in the world. Moreover, Tobacco is an important part of Chinese fiscal revenue. Drought seriously impacts on tobacco yield and quality, which is one of main environmental stresses resulting in reduction of yield loss.To survive under drought stress in their rooted lifestyle, tobacco has evolved a considerable degree of drought stress response, which is a complex trait regulated at multiple levels including the adjustment of various biochemical and physiological processes and molecular adaption to gene regulatory network. The regulatory networks of gene expression under drought and cold stress are critical, and that particular transcription factors can be employed to enhance drought stress tolerance in plants.Decades of research into the effects of drought on model plant physiology and development have generated a wealth of information by molecular biology and genomics. It has identified some drought responsive genes (DRGs) under drought stress and some transcription factors (TFs) involved in regulation of these DRGs. Drought stress increases endogenous abscisic acid (ABA) levels and induces ABA-dependent and ABA-independent transcriptional regulatory networks. Many of these responses can be mimicked by external application of ABA. Drought-responsive transcriptional networks have been primarily developed from related studies in Arabidopsis, and some important promoter elements were confirmed experimentally, such as ABA-responsive elements (ABREs) and coupling elements (CE). However, the networks that underlie these responses in tobacco have not been extensively characterized. In addition to DRGs and these TFs mentioned above, microRNAs (miRNAs) are short (20-22nt), endogenously expressed, non-translated RNAs that function in posttranscriptional gene regulation. There is a complex interplay between transcriptional and posttranscriptional regulation of drought response in Arabidopsis, for instance, some miRNAs (miR159and miR169) play an important role in drought stress response. Unfortunately, it has not been extensively characterized in tobacco.In the study, the roots of a flue-cured tobacco (Nicotiana tabacum L.) cultivar, Honghua Dajinyuan (a drought-tolerant cultivar) were treated in time-course drought stress and then detected by physiological analyses. Only these samples at6h and48h treatment and normal sample were used for analyses of mRNA and miRNA expressing profiling, identification of their target regulated by specially-expressed miRNAs, developing and mapping the transcriptional and posttranscriptional gene regulatory network. We aim to unveil the roles of mRNAs and miRNAs in rapidly responding to drought in tobacco root cell, which will give sight to mRNA and miRNA regulation mechanism under drought stress and help breeder breed excellent drought resistance inbred lines by the marker-assisted selection of DRGs.1. To explore the optimal time point under drought stress for gene expression analysis, uniform seedlings of tobacco with six leaves were challenged to drought stress treatments at six time points (3,6,12,24,48, and96h) with20%PEG6000. We measure three physiological indexes, Superoxide dismutase (SOD) activities and proline (PRO) and malondialdehyde (MDA) contents. These results suggest that SOD activities, PRO, and MDA contents all significantly increased at6h and48h relative to3h and24h, respectively, and the optimal time point for drought stress assays are6h and48h. Our experiments using tobacco roots with experiments at two time points and control were named as NCK (control), N6H, and N48H, respectively.2. Total RNA was isolated from the frozen root samples and DGE library preparation was then performed in parallel by using the Illumina gene expression sample preparation kit. The sequence reads of all libraries ranged from3.33M (million) to3.39M, and averaged about3.37M. We examined the dynamics of gene expression under drought stress using DGE data, and only1,887out of21,128genes that were differentially expressed among NCK, N6H, and N48H, represented8.9%of the root transcriptome. A sample of22transcripts with significant differences in gene expression was randomly selected for validation via qRT-PCR, which were consistent with that obtained from DGE. We used Gene Ontology annotation to assign genes to functional categories and grouped genes by expression dynamics using the K-Means clustering algorithm. We identified six clusters which contained many genes that encode enzymes for fatty acid metabolism, transferase (transferring acyl groups), oxidoreductase, ethanol metabolism, primary alcohol metabolism and transferase, etc. In addition, biological pathways influenced by drought were evaluated by enrichment analysis of all differentially expressed genes. Significantly enriched metabolic pathways and signal transduction pathways were also identified. A total of17pathways, including contained glutathione metabolism, fatty acid elongation, stilbenoid, diarylheptanoid and gingerol biosynthesis, biosynthesis of secondary metabolites were affected based on the above mentioned six clusters (P＜0.05).3. A primary objective was to identify genes that encode TFs and resolve the dynamics of accumulation of TFs under drought stress in our DGE data. To test this, we retrieved putative orthologs of tobacco genes based on information from the EnsemblCompara gene trees at solgenomics.net, plantgdb.org, and http://www.ncbi.nlm.nih.gov/. We then queried known plant TFs in the Plant Transcription Factor Database (v2.0, http://planttfdb.cbi.edu.cn/) and identified609tobacco TFs with sequence similarities to known plant TFs. Furthermore, all TFs can be detected in roots of tobacco seedlings responding to drought stress. Of these TFs in root tissue,82were differentially expressed during time-points and belong to24TF families. These TFs associated with functions in drought tolerance (MYB, NAC, and ERF), while others played roles in development and meristem maintenance or identity (HD-ZIP, NF-YA, NAC, GRAS, and TCP), defense/stress signaling pathways (HSP, WRKY, and bZIP), hormone-mediated or stress-mediated signaling by auxin (AUX/IAA). The abundance of most of these TFs (53%) was at the highest levels in NCK (G1), whereas only10%was at the highest levels in N6H (G2). The reminder (37%) indicated peak expression in N48H (G3). We also identified family-specific expression trends. Members of the C2H2(3genes), MYB (3), bHLH (4), WRKY (3), ERF (13) and Dof (3) families of transcriptional regulators were highly expressed in NCK. Several GATA (1), MYB (3) and ERF (3) TFs accumulated to the highest levels during the stress-response phase from NCK to N6H. Transcriptional regulators including MYB (6), NAC (6), MYB-related (2), NF-YA (2), HD-ZIP (2) and ERF (2) were preferentially expressed in N48H.4. We identified276candidate DRGs in tobacco with sequence similarity to known genes and Nicotiana benthamiana annotation. Interestingly, about40%(110out of276genes) were TFs including WRKY, NAC, ERF, and bZIP families. In the present study, we also investigated the roles of these candidate DRGs, and found46differentially expressed DRGs under drought stress. Out of the54differentially expressed DRGs,21(46%) were TFs which belonged to NAC (6), MYB (4), ERF (10), and bZIP (1) families. Other DRGs, such as GRF6, ABF1, APX2, SIPK, and ZPT2, have different expression patterns in response to drought stress.5. The samples of our small RNA libraries were used based on the result of physiological index measurement as follows:equal quantities (10ug) of total RNA isolated from tobacco roots treated with two time points (6and48h) were mixed together to construct the drought-treated small RNA library (Root-treat), and total RNA prepared from the control roots sample was used to construct the control small RNA library (Root-ck). Here,122tobacco miRNAs were detected in our sequencing datasets. Conserved miRNAs were far more abundant than non-conserved miRNAs in our libraries as reported previously. MiR166and miR168were the most abundant miRNA families which accounted for about57%and16%of the total sequence reads from the known miRNAs datasets, respectively. However,44experimentally identified tobacco miRNA families (33miRNAs) were not detected in our dataset. Comparison of the normalized sequence reads of the miRNAs between the two libraries indicated that five known tobacco miRNA families had relative changes (log2root-ck/root-treat) greater than two and thus might be differentially or extremely differentially expressed. However, miR159, miR169, miR402, and miR408sequence reads displayed no meaningful changes between two libraries even though their expression had been reported to be affected by drought stress treatments in other plants.6. MiRNAs regulate gene expression at the posttranscriptional level by repressing mRNA expression, and some miRNA families were experimentally verified to be responsive to salt or drought stress in plants in recent research. To understand the functions of drought-responsive miRNAs in tobacco, we got the complete list of targets of tobacco miRNAs identified by degradome sequencing in a recent study, and only27target_GSS sequences can be mapped to the tobacco reference genome and correspond to87transcripts. Unfortunately, the targets of only two drought-responsive miRNAs (miR160and miR395) were obtained.7. With the availability of regulatory networks of gene expression in drought and cold stress responses, an integrated gene regulatory network has been proposed for the molecular mechanisms of the response of tobacco roots to drought stress using differentially expressed DRGs, the changed expression profiles of miRNAs and subsequent target transcripts as a basis. Two pathways (ABA-dependent and ABA-independent) can shed light into cell mechanisms involved in stress signaling and/or adaptation at transcriptional regulation. In the ABA-dependent pathway, NCED1was involved in rapid and emergency responses to drought stress. Left parts of Figure7show transcription cascades that were involved in slow and adaptive processes in stress responses, such as those involving AREB/ABF, MYB, bZIP, NAC and CBF/DREB1. SnRK2.6protein kinases were also involved in ABA signaling. In the ABA-independent pathway, unknown proteins were thought to function as an osmo-sensor and function upstream of the ERF system. In addition, the responsive miRNAs (miR160, miR162, miR394, miR395, and miR827) also showed a transitory expression model. Right parts of Figure7show proposed regulation cascades after drought in tobacco roots. This network analysis will also serve as a reference for future studies on tobacco responses to various stresses, such as to drought, cold and heavy metals.