Transcriptome profile analysis of cell proliferation molecular processes during multicellular trichome formation induced by tomato Wo (v) gene in tobacco.

Trichomes, developing from the epidermis of nearly all terrestrial plants, provide good protection from environmental stress. Regulation of trichomes in Rosids has been well characterized. However, little is known about the cell proliferation molecular processes during multicellular trichome formation in Asterids. Ectopic expression of Wo (v) in tobacco and potato induces much more trichome formation than wild type. To gain new insights into the underlying mechanisms during the processes of these trichomes formation, RNA-seq was employed for the young primary leaf tissues in Wo (v) transgenic and wild-type tobacco. We identified differentially expressed genes which are related to various biological processes and molecular functions. Here, we provide details of experimental methods, RNA-seq data (available at Gene Expression Omnibus database under GSE72310). Our data provide new insight into the molecular processes controlling multicellular formation in tobacco.

Direct link to deposited data

Deposited data can be found here: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72310.

Experimental design, materials and methods

Sample collection and RNA isolation

Wov transgenic tobacco plants and their WT species (N. tabacum) were grown in a naturally illuminated glasshouse in the experimental field of the Huazhong Agricultural University. Environmental control was implemented based on standard protocols [1]. Two independent Wov transgenic tobacco plants and two negative regenerated plants were used for transcriptomic analysis. All samples were immediately frozen in liquid nitrogen, and total RNA was isolated using TRIzol reagent (Invitrogen). The integrity of all RNA samples was examined by 1.2% agarose gel electrophoresis after treatment with RQ1 DNase (Promega). The quality and quantity of these RNA samples were further determined by measuring the absorbance at 260/280 and 260/230 nm by using SmartSpec Plus spectrophotometer (Bio-Rad).

RNA-seq and data processing

The materials used for RNA-seq analyses were three young primary leaves from the transgenic and control plants. Up to 10 μg of total RNA was sent to ABlife Inc. (Wuhan, China) where the libraries were produced. The cDNA libraries were then sequenced in BGI Inc. (Shenzhen, China) by using Illumina HiSeq™ 2000 by 100 nt pair-end sequencing. RNA-seq was performed as previously described [2]. Whole RNA-seq data were submitted to the Gene Expression Omnibus (series accession number GSE72310). The clean reads were aligned to the N. tabacum genome (ftp://anonymous@ftp.solgenomics.net/genomes/Nicotiana_tabacum/assembly/Ntab-K326_AWOJ-SS.fa.gz) using TopHat (2.0.12) software [3]. Based on the length of the gene and read counts uniquely mapped to this gene, gene expression levels were calculated using FPKM method.

Functional analysis of differentially expressed genes

We used the DESeq software to identify the differentially expressed genes between samples, which were specific for differential expression analysis of the RNA-Seq data with biological replicates [4]. Genes with P-value ≤ 0.05 and fold change ≥ 2 were regarded as differentially expressed. To characterize the putative functions of differentially expressed genes, Gene Ontology (GO) term analysis was also performed using GOseq based Wallenius non-central hyper-geometric distribution [5]. For further identification of pathways significantly influenced by the Wov gene, KEGG enrichment pathways analysis of DEGs was implemented by the KOBAS (2.0) software by a hypergeometric test and the Benjamini–Hochberg FDR correction (FDR ≤ 0.05) [6].

Specifications
Organism/cell line/tissue	Nicotiana tabacum
Sequencer or array type	Illumina HiSeq™ 2000-RNA sequencing
Data format	Raw and processed
Experimental factors	Wov transgenic and wild-type tobacco
Experimental features	RNA-seq dataset for gene expression profiling in young primary leaf in Wov transgenic and wild-type tobacco
Sample source location	Wuhan, China