ABSTRACT: Drought stress response is a complex trait regulated at multiple levels. In the past few years, molecular and genomic studies have shown that several drought responsive genes (DRGs) with various functions are induced by drought stresses, and that various transcription factors (TFs) are involved in the regulation of stress-inducible genes. In addition to those DRGs mentioned above, microRNAs (miRNAs) are important regulators of gene expression at the posttranscriptional level by repressing mRNA expression. There is a complex interplay between transcriptional and post-transcriptional regulation of drought response that has not been extensively characterized in tobacco. In order to fully understand DRGs (including TFs) and different roles of miRNAs involved in the stress response, we sequenced and analysed three Digital Gene Expression (DGE) libraries in roots from drought treated tobacco plants, and four small RNA populations in roots, stems and leaves from control or drought treated tobacco plants. We identified 276 candidate DRGs in tobacco with sequence similarities to 64 known DRGs from model plants and crops and about 40% were TFs including WRKY, NAC, ERF and bZIP families. Furthermore, Out of these tobacco DRGs, 54differentially expressed DRGs included 21 TFs, which belonged to 24 TFs families such as NAC (6), MYB (4), ERF (10) and bZIP (1). Additionally, we confirmed expression of 39 known miRNA families (122 members) and five conserved miRNA families, which showed differential regulation under drought stress. Targets of miRNAs were further surveyed based on a recently published study, in which ten targets were DRGs. Finally, 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 their target transcripts as a basis base on previous studies. In general, our data provide valuable information for future studies of the molecular mechanisms underlying tobacco roots in response to drought resistance in tobacco and other plants. Three tobacco (Nicotiana tabacum L.) roots tag-based DGE libraries treated at three time points (0, 6 and 48 h) with 20% PEG6000 were generated using Illumina 1G technology. The samples for four small RNA libraries was used based on the result of physiological index measurement as follows: equal quantities (10 ug) of total RNA isolated from tobacco roots treated with two time points (6 and 48 h) 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). In addition, we also constructed another two libraries (stem and leaf) from leaves and stems of control plants, respectively. These libraries were constructed using the Small RNA Sample Prep Kit (Illumina, San Diego, CA) and then sent for Solexa sequencing.
