Tobacco transcription factors: novel insights into transcriptional regulation in the Solanaceae

Abstract

Tobacco (Nicotiana tabacum) is a member of the Solanaceae, one of the agronomically most important groups of flowering plants. We have performed an in silico analysis of 1.15 million gene-space sequence reads from the tobacco nuclear genome and report the detailed analysis of more than 2,500 tobacco transcription factors (TFs). The tobacco genome contains at least one member of each of the 64 well-characterized TF families identified in sequenced vascular plant genomes, indicating that evolution of the Solanaceae was not associated with the gain or loss of TF families. However, we found notable differences between tobacco and non-Solanaceae species in TF family size and evidence for both tobacco- and Solanaceae-specific subfamily expansions. Compared with TF families from sequenced plant genomes, tobacco has a higher proportion of ERF/AP2, C2H2 zinc finger, homeodomain, GRF, TCP, zinc finger homeodomain, BES, and STERILE APETALA (SAP) genes and novel subfamilies of BES, C2H2 zinc finger, SAP, and NAC genes. The novel NAC subfamily, termed TNACS, appears restricted to the Solanaceae, as they are absent from currently sequenced plant genomes but present in tomato (Solanum lycopersicum), pepper (Capsicum annuum), and potato (Solanum tuberosum). They constitute approximately 25% of NAC genes in tobacco. Based on our phylogenetic studies, we predict that many of the more than 50 tobacco group IX ERF genes are involved in jasmonate responses. Consistent with this, over two-thirds of group IX ERF genes tested showed increased mRNA levels following jasmonate treatment. Our data are a major resource for the Solanaceae and fill a void in studies of TF families across the plant kingdom.

Figure 1.

Tobacco ERF genes. Shown is an unrooted phylogenetic tree of the ERF domains constructed using the neighbor-joining method. Each gene is designated by an arbitrary number, and published genes are represented by their published names. Roman numerals indicate previously defined subgroups, and a line separates the ERF and DREB subfamilies. Group V is found as two separate clades.

Figure 2.

Tobacco WRKY genes. Shown is an unrooted phylogenetic tree of the WRKY domains constructed using the neighbor-joining method. Each gene is designated by an arbitrary number, and published genes are represented by their published names. Arabidopsis domains are included for identification purposes and indicated by the name of their group. Group I WRKY genes normally have two WRKY domains, and both were used individually to construct the phylogenetic tree. INT and ICT denote the N-terminal and C-terminal domains from these group I genes.

Figure 3.

Tobacco bHLH genes. Shown is an unrooted phylogenetic tree of the bHLH domains constructed using the neighbor-joining method. Each tobacco gene identified in the GSR data set is designated by an arbitrary number. Large letters indicate previously defined subfamilies. To identify subfamilies, the bHLH domain from one representative gene of each subfamily was included and is indicated by the small letter within the phylogenetic tree. The Arabidopsis gene groups used were as follows: A, AtbHLH087 (At3g21330); B, AtbHLH139 (At5g43175); C, AtbHLH44 (At1g18400) and AtbHLH31 (At1g59640); D, AtbHLH130 (At2g42280); E, AtbHLH26 (At1g02340) and AtbHLH73 (At5g67110); F, AtbHLH103 (At4g21340); G, AtbHLH135 (At1g74500); H, AtbHLH150 (At3g05800); I, AtbHLH142 (At5g64340); K, AtbHLH12 (At4g00480); L, AtbHLH102 (At1g69010); M, AtbHLH34 (At3g23210); N, AtbHLH6 (At1g32640); O, AtbHLH95 (At1g49770); P, AtbHLH96 (At1g72210); R, AtbHLH22 (At4g21330); S, AtbHLH20 (At2g22770); T/R, AtbHLH1 (At5g41315); U, AtbHLH41 (At5g56960); V, AtbHLH30 (At1g68810); W, AtbHLH117 (At3g22100); X, AtbHLH55 (NM_101125.3); and Y, AtbHLH91 (At2g31210). The bHLH domain from PIF4 (At2g43010) is also included for comparison.

Figure 4.

Tobacco NAC genes. Shown is an unrooted phylogenetic tree of the NAC domains constructed using the neighbor-joining method. Each tobacco gene identified in the GSR data set is designated by an arbitrary number. Six clades (1–6) are found in tobacco and other plant species, and three clades, designated TNAC A to TNAC C, are found in tobacco and other Solanaceae species. EST sequences from TNAC genes of potato (CV505554), tomato (BI422367), and pepper (U204177) are included in TNAC clade C. The published tobacco NAC gene TERN (AB021178) is indicated.

Figure 5.

Amino acid sequence comparisons of the N-terminal half of tobacco NAC domains. A, Amino acid sequences of the N-terminal half of tobacco NAC domains from the TNAC A, B, and C clades and other NAC genes. Areas of significant difference are indicated by red boxes at the bottom of the alignment, and missing/additional amino acids are indicated by blue boxes. Three EST sequences from other Solanaceae TNACs are also included: tomato (BI422367), potato (CV505554), and pepper (U204177). B, Consensus sequences of NAC and the three TNAC clades. Blue letters show amino acids that are conserved in NAC domains but absent from TNAC domains. Red letters show amino acids that are characteristic of TNAC clades. Amino acids that are conserved in the consensus sequence for all four are highlighted in black boxes.

Figure 6.

Phylogenetic analysis of the complete NAC gene families from Arabidopsis, poplar, and rice together with NAC and TNAC genes from tobacco. TNAC genes are highlighted with red dots and tobacco NAC genes are highlighted with blue dots. The phylogeny contains over 450 genes and shows that the TNACs form a separate clade. All rice, poplar, and Arabidopsis NAC genes are indicated by their accession numbers.

Figure 7.

Phylogenetic analysis of tobacco and Arabidopsis BES genes. A, Unrooted phylogenetic tree of tobacco and Arabidopsis BES genes constructed using the neighbor-joining method. Each tobacco gene is designated by an arbitrary number, and five clades are circled. Arabidopsis BES genes are indicated by accession numbers, except for BES1 (At1g19350) and BZR1 (At1g75080), which are indicated by their published names. A small number of tobacco BES sequences that do not contain complete domains (e.g. NtBES1, NtBES8, and NtBES19) are omitted from the analysis. B, Multiple sequence alignment of the bHLH-like domains from the tobacco and Arabidopsis BES genes. The alignment was produced with ClustalW and displayed using Jalview. The bHLH domain is indicated, and the black arrows show amino acids that are conserved within the tobacco-specific clade. The red arrow indicates the members of the tobacco-specific clade.

Figure 8.

Phylogenetic analysis of SAP genes from tobacco, Arabidopsis, and poplar. Shown is a phylogenetic tree of SAP genes from tobacco, Arabidopsis (At5g35770), and poplar (fgenesh4_pg.C_LG_XIV001059) constructed using the neighbor-joining method. Numbers indicate bootstrap values from 1,000 replications.

Figure 9.

Analysis of MeJA induction of tobacco group IX ERF genes. A, Results of RT-PCR analysis of the expression levels of 13 ERF genes in tobacco BY-2 cells following treatment with 50 μm MeJA. Numbers represent the time of treatment in hours (0, 1–24) or minutes (30). The NtPMT1a gene, a well-documented MeJA-induced transcript in tobacco, was included as a positive control, and actin was used as a non-MeJA-induced negative control. PCR products were separated by electrophoresis on 2% agarose gels. Experiments were replicated at least three times, and representative data are shown. B, Phylogenetic tree of group IX tobacco ERF genes. Genes that are inducible by MeJA in BY-2 cells are indicated by green circles. [See online article for color version of this figure.]