Tobacco Transcriptome Analysis Revealing The Role Of NtTTG2 In Regulating Expression Of Putative Auxin Response Factor Genes

Auxin regulates multiple aspects of plant development throught the function of auxin response factor (ARF) transcription factors in transcription regulation of auxin resposnse of plants. ARFs constitute a phylogenic protein family that comprises 10-43 members identified in different plant species but only a few members have been studied regarding their function in plant development. Plant development is also subject to regulation by TRANSPARENT TESTA GLABRA (TTG) proteins, such as NtTTG2 that we characterized in tobacco Nicotiana tabacum. Based on the biological effects of NtTTG2 gene silencing and overexpression, NtTTG2 is the key part of the plant growth and development. The NtTTG2 gene silencing causes repressive effects on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production. By contrast, the plant growth and development processes are promoted by NtTTG2 overexpression. To find the functional linkage between TTG and auxin in the regulation of plant development, the author performed de novo assembly of the tobacco transcriptome to identify candidates of NtTTG2-regulated ARF genes.1 The de novo assembly and analysis of transcription tobaccoThe WT plant of the tobacco (N. tabacum) variety NC89, the NtTTG2-overexpressing NC89 line, and the NtTTG2-silenced NC89 line were used in de nevo assembly of the transcriptome in the absence of genomic sequencing information. A total of 30,325,318 raw reads were produced by transcriptome sequencing with Illumina HiseqTM 2000 platform. Redundant reads were removed by Illumina RNA-Seq deep sequencing, and 111,207 transcripts were obtained after de novo assembly. In total,54,906 unigenes were identified in the transcriptome by dislodging redundant transcripts. There were 30,124 unigenes (54.86%) with annotated functions by searching in seven databases, and at least 8,024 unigenes (14.61%) assigned to plant growth and development. Evaluating the overall quality of RNA-seq and the expression level of gene in this transcriptome indicated that it has good overall quality of RNA-seq with a high abundance of transcripts and high the homogenization degree.2 Identification of putative ARFs gene regulated by NtTTG2 and transactivation assays of the ARF8 proteinNtTTG2 has been identified to regulate growth and development of tobacco, but the mechanism is not clear. Therefore, we performed de novo assembly of the tobacco transcriptome and classified unigenes with annotated functions. At least 8,024 unigenes (14.61%) were assigned to plant growth and development.40,39,15 and 33 unigenes were predicted to be ARF, Aux/IAA, GH3 and SAUR gene candidates, respectively. We found that NtTTG2 regulated the expression of 13 candidates in the 40 putative ARF genes by realtime RT-PCR analysis. Two of the 13 candidates were downregulated by NtTTG2. Conversely, the other 11 putative ARF genes were upregulated by NtTTG2. Full-length sequences of the putative ARF genes remain unknown except for comp42904_c0, which is 100% identical with the sequence of the ARF8 gene from the N. tabacum variety NC89. Transactivation assays of the ARF8 protein suggest that ARF8 could bind the GH3 promoter and be required for GH3 expression.