Tobacco Transcription Factor Research Articles

Co-Expression of JcNAC1- and JcZFP8-Improved Agronomic Traits of Tobacco and Enhanced Drought Resistance through NbbHLH1 and NbbHLH2.

Previous studies have identified numerous transcription factors involved in drought response, each of which play different roles in plants. The objective of the present study was to evaluate the effectiveness of two transcription factors on drought response in Jatropha curcas L., JcNAC1 and JcZFP8. The overexpression of these transcription factors in tobacco (Nicotiana benthamiana L.) improved drought resistance, but JcZFP8 delayed germination and JcNAC1 reduced biomass and yield. By constitutively co-expressing these two genes in tobacco, drought resistance was improved, and the negative effects of each of them were overcome. The transgenic plants with double-gene co-expression showed stronger drought tolerance with 1.76-fold greater accumulation of proline and lower H2O2 and malondialdehyde (MDA) content to 43 and 65% of wildtype (WT) levels, respectively. The expression levels of NbbHLH1 and NbbHLH2 genes upregulated linearly with the increased drought tolerance of double genes co-expression plants. In drought conditions, the leaf water contents of bhlh1, bhlh2, and bhlh1bhlh2 deletion mutants obtained by CRISPR-CAS9 knockout technique were maintained at 99%, 97%, and 97% of WT. The bhlh1bhlh2 was found with lower germination rate but with higher reactive oxygen levels (1.64-fold H2O2 and 1.41-fold MDA levels). Thus, the co-expression of two transcription factors with different functions overcame the adverse traits brought by a single gene and enhanced the shared drought-tolerant traits, which can provide guidance on theory and selection of gene combinations for the application of multi-gene co-expression in agriculture in the future.

Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence.

Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites' regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

MYB41, MYB107, and MYC2 promote ABA-mediated primary fatty alcohol accumulation via activation of AchnFAR in wound suberization in kiwifruit

Wound damage triggers the accumulation of abscisic acid (ABA), which induces the expression of a large number of genes involved in wound suberization in plants. Fatty acyl-CoA reductase (FAR) catalyzes the generation of primary fatty alcohols by the reduction of fatty acids in suberin biosynthesis. However, the regulatory effects of transcription factors (TFs) on AchnFAR in response to ABA are unexplored. In this study, kiwifruit AchnFAR displayed a biological function analogous to that of FAR in transiently overexpressed tobacco (Nicotiana benthamiana) leaves. The positive role of TFs, including AchnMYB41, AchnMYB107, and AchnMYC2, in the regulation of AchnFAR was identified. The three TFs could individually bind to the AchnFAR promoter to activate gene transcription in yeast one-hybrid and dual-luciferase assays. Transient overexpression of TFs in tobacco leaves resulted in the upregulation of aliphatic synthesis genes (including FAR) and the increase in aliphatics, including primary alcohols, α,ω-diacids, ω-hydroxyacids, and fatty acids. Moreover, exogenous ABA treatment elevated TF-mediated AchnFAR expression and the accumulation of primary alcohols. Conversely, fluridone, an inhibitor of ABA biosynthesis, suppressed the expression of AchnFAR and TF genes and reduced the formation of primary alcohols. The results indicate that AchnMYB41, AchnMYB107, and AchnMYC2 activate AchnFAR transcription to promote ABA-mediated primary alcohol formation in wound suberization in kiwifruit.

Transcriptome analysis of tobacco root response to different concentrations of nitrate

AbstractWe aimed to explore genes and transcription factors (TFs) in tobacco (Nicotiana tabacum L.) that were closely related to responses to different concentrations of nitrate (NO3−) in tobacco root. Cultivar K326 was used in this study. The RNA sequence data generated from K326 treated with high or low concentration of NO3− supplied at different time points (0, 6, 12, and 24 h) were analyzed to determine the transcriptome response of tobacco root to NO3− supply. The expression of numerous genes was altered after high and low NO3− treatment. Further analysis revealed 1981 and 3573 differentially expressed genes (DEGs) from low and high NO3− treatment groups, respectively. Comparative analysis identified 567 low‐NO3−‐specific, 2159 high‐NO3−‐specific, and 1414 commonly expressed genes. Combining short time‐series expression miner (STEM) and weighed gene co‐expression network analysis (WGCNA) results, 136 and 416 DEGs were identified from low and high NO3− groups, respectively, among which, 12 were TFs. In the constructed protein−protein interaction (PPI) network, three DEGs in high NO3− group (LOC107807186, LOC107792457, and LOC107786314) exhibited highest degrees. Within low NO3− group, LOC107798206, LOC107815159, and LOC107779796 had highest degrees. Among the 12 TFs, LOC107778750, LOC107810552, and LOC107804260 had the highest degrees. Our data provides valuable information about genes, especially the 12 TFs, which were induced by different concentrations of NO3− in tobacco root. These findings may be useful for identification of high or low NO3− treatment‐induced genes and TFs, as well as for understanding potential gene regulation mechanisms in response to NO3− supply in tobacco.

NAC Family Transcription Factors in Tobacco and Their Potential Role in Regulating Leaf Senescence.

The NAC family is one of the largest families of plant-specific transcription factors (TFs) and NAC proteins play important regulatory roles in a variety of developmental and stress response processes in plants. Members of the NAC family TFs have been shown to be important regulators of leaf senescence in a number of plant species. Here we report the identification of the NAC family in tobacco (Nicotiana tabacum) and characterization of the potential role of some of the tobacco NAC TFs in regulating leaf senescence. A total of 154 NAC genes (NtNACs) were identified and clustered together with the Arabidopsis NAC family into fifteen groups (a-o). Transcriptome data analysis followed by qRT-PCR validation showed that the majority of the senescence-up-regulated NtNACs fall into subgroups NAC-b and f. A number of known senescence regulators from Arabidopsis also belong to these two subgroups. Among these senescence-up-regulated NtNACs, NtNAC080, a close homolog of AtNAP, is a positive regulator of leaf senescence. Overexpression of NtNAC080 caused early senescence in Arabidopsis leaves and NtNAC080 mutation induced by Cas9/gRNA in tobacco led to delayed leaf senescence.

Tobacco Transcription Factor NtbHLH123 Confers Tolerance to Cold Stress by Regulating the NtCBF Pathway and Reactive Oxygen Species Homeostasis.

Cold stress is a major environmental factor that impairs plant growth and development, geographic distribution, and crop productivity. The C-repeat binding factor (CBF) regulatory pathway has an essential role in response to cold stress. Here, we characterized a bHLH transcription factor from Nicotiana tabacum, NtbHLH123, in response to cold stress (4°C). Overexpression of NtbHLH123 enhanced cold tolerance in transgenic tobacco plants. Based on yeast one-hybrid, chromatin immunoprecipitation PCR, and transient expression analysis assays, NtbHLH123 binds directly to the G-box/E-box motifs in the promoter of the NtCBF genes and positively regulates their expression. Furthermore, NtbHLH123-overexpressing plants showed lower electrolyte leakage, reduced malondialdehyde contents, H2O2 and reactive oxygen species (ROS) accumulation under cold stress, which contributed to alleviating oxidative damage to the cell membrane after cold stress treatment. And NtbHLH123 increased stress tolerance by improving the expression of a number of abiotic stress-responsive genes to mediate the ROS scavenging ability and other stress tolerance pathways. Taken together, we present a model suggesting that NtbHLH123 is a transcriptional activator that functions as a positive regulator of cold tolerance by activating NtCBF, ROS scavenging-related, and stress-responsive genes.

Overexpression of SbSI-1, A Nuclear Protein from Salicornia brachiata Confers Drought and Salt Stress Tolerance and Maintains Photosynthetic Efficiency in Transgenic Tobacco.

A novel Salicornia brachiata Salt Inducible (SbSI-1) gene was isolated and overexpressed in tobacco for in planta functional validation subjected to drought and salt stress. SbSI-1 is a nuclear protein. The transgenic tobacco overexpressing SbSI-1 gene exhibited better seed germination, growth performances, pigment contents, cell viability, starch accumulation, and tolerance index under drought and salt stress. Overexpression of SbSI-1 gene alleviated the build-up of reactive oxygen species (ROS) and curtailed the ROS-induced oxidative damages thus improved the physiological health of transgenic tobacco under stressed conditions. The higher activities of antioxidant enzymes, lower accumulation of ROS, higher membrane stability, relative water content, and polyphenol contents indicated the better survival of the transgenic tobacco than wild-type (WT) tobacco under stressed conditions. Transgenic tobacco had a higher net photosynthetic rate, PSII operating efficiency, and performance index under drought and salt stress. Higher accumulation of compatible solutes and K+/Na+ ratio in transgenic tobacco than WT showed the better osmotic and redox homeostasis under stressed conditions. The up-regulation of genes encoding antioxidant enzymes (NtSOD, NtAPX, and NtCAT) and transcription factors (NtDREB2 and NtAP2) in transgenic tobacco under stressed conditions showed the role of SbSI-1 in ROS alleviation and involvement of this gene in abiotic stress tolerance. Multivariate data analysis exhibited statistical distinction among growth responses, physiological health, osmotic adjustment, and photosynthetic responses of WT and transgenic tobacco under stressed conditions. The overexpression of SbSI-1 gene curtailed the ROS-induced oxidative damages and maintained the osmotic homeostasis under stress conditions thus improved physiological health and photosynthetic efficiencies of the transgenic tobacco overexpressing SbSI-1 gene.

High Temperature Induces Expression of Tobacco Transcription Factor NtMYC2a to Regulate Nicotine and JA Biosynthesis.

Environmental stress elevates the level of jasmonic acid (JA) and activates the biosynthesis of nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) by up-regulating the expression of putrescine N-methyltransferase 1 (NtPMT1), which encodes a putrescine N-methyl transferase that catalyzes nicotine formation. The JA signal suppressor JASMONATE ZIM DOMAIN 1 (NtJAZ1) and its target protein, NtMYC2a, also regulate nicotine biosynthesis; however, how these proteins interact to regulate abiotic-induced nicotine biosynthesis is poorly understood. In this study, we found that high-temperature (HT) treatment activated transcription of NtMYC2a, which subsequently stimulated the transcription of genes associated with JA biosynthesis, including Lipoxygenase (LOX), Allene oxide synthase (AOS), Allene oxide cyclase (AOC), and 12-oxophytodienodate reductase (OPR). Overexpression of NtMYC2a increased nicotine biosynthesis by enhancing its binding to the promoter of NtPMT1. Overexpression of either NtJAZ1 or proteasome-resistant NtJAZ1ΔC suppressed nicotine production under normal conditions, but overexpression only of the former resulted in low levels of nicotine under HT treatment. These data suggest that HT induces NtMYC2a accumulation through increased transcription to activate nicotine synthesis; meanwhile, HT-induced NtMYC2a can activate JA synthesis to promote additional NtMYC2a activity by degrading NtJAZ1 at the post-transcriptional level.

Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.

Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.

Tobacco drought stress responses reveal new targets for Solanaceae crop improvement.

BackgroundThe Solanaceae are an economically important family of plants that include tobacco (Nicotiana tabacum L.), tomato, and potato. Drought is a major cause of crop losses.ResultsWe have identified major changes in physiology, metabolites, mRNA levels, and promoter activities during the tobacco response to drought. We have classified these as potential components of core responses that may be common to many plant species or responses that may be family/species-specific features of the drought stress response in tobacco or the Solanaceae. In tobacco the largest increase in any metabolite was a striking 70-fold increase in 4-hydroxy-2-oxoglutaric acid (KHG) in roots that appears to be tobacco/Solanaceae specific. KHG is poorly characterized in plants but is broken down to pyruvate and glyoxylate after the E. coli SOS response to facilitate the resumption of respiration. A similar process in tobacco would represent a mechanism to restart respiration upon water availability after drought. At the mRNA level, transcription factor gene induction by drought also showed both core and species/family specific responses. Many Group IX Subgroup 3 AP2/ERF transcription factors in tobacco appear to play roles in nicotine biosynthesis as a response to herbivory, whereas their counterparts in legume species appear to play roles in drought responses. We observed apparent Solanaceae-specific drought induction of several Group IId WRKY genes. One of these, NtWRKY69, showed ABA-independent drought stress-inducible promoter activity that moved into the leaf through the vascular tissue and then eventually into the surrounding leaf cells.ConclusionsWe propose components of a core metabolic response to drought stress in plants and also show that some major responses to drought stress at the metabolome and transcriptome levels are family specific. We therefore propose that the observed family-specific changes in metabolism are regulated, at least in part, by family-specific changes in transcription factor activity. We also present a list of potential targets for the improvement of Solanaceae drought responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1575-4) contains supplementary material, which is available to authorized users.

A Region Containing an as-1 Element of Dahlia Mosaic Virus (DaMV) Subgenomic Transcript Promoter Plays a Key Role in Green Tissue- and Root-Specific Expression in Plants

A subgenomic transcript (Sgt) promoter was isolated from the genomic clone of dahlia mosaic virus (DaMV), which is a double-stranded DNA virus of the Caulimoviridae family. The DaMVSgt promoter, which is linked to the heterologous β-glucuronidase (GUS) reporter gene, was characterized in transient protoplasts and in transgenic tobacco, as well as in Arabidopsis plants. The 5′- and 3′-deletion analysis of a 591-bp DaMVSgt promoter fragment indicated that a 441-bp promoter fragment (−372 to +69 from the transcription start site; TSS) was sufficient for maximal promoter activity. A 141-bp promoter fragment (−72 to +69 from TSS) was the minimal promoter region that also showed relatively strong activity. The three activation sequence-1 (as-1) elements and the border regions were primarily responsible for the promoter activity, as revealed by a finer internal deletion and mutation analysis of the cis-elements and of the immediate border sequence of the activation domain. Electrophoretic mobility shift assay (EMSA), supershift EMSA, DNase I footprinting, Southwestern blotting, and UV cross-linking studies demonstrated the binding of a tobacco transcription factor, TGA1a, that correlated with 2,4-dichlorophenylacetic acid (2,4D)-induced transcriptional activity of the DaMVSgt promoter. Histological GUS staining and the GUS enzymatic assay demonstrated that the 441-bp DaMVSgt4 promoter and 141-bp minimal DaMVSgt4F are 5.5 and 4.6 times, respectively, stronger than the CaMV 35S promoter. The minimal DaMVSgt4F promoter is more active than CaMV 35S in all types of green tissues and roots, without any detectable expression in reproductive tissues and seeds. The DaMVSgt4F promoter may be useful for transgene containment applications.

Stress-induced expression of NICOTINE2-locus genes and their homologs encoding Ethylene Response Factor transcription factors in tobacco

Plants have evolved diverse defense metabolites as adaptations to biotic and abiotic stresses. The defense alkaloid nicotine is produced in Nicotiana tabacum (tobacco) and its biosynthesis is elicited by jasmonates in the roots. At least seven jasmonate-responsive genes that encode transcription factors of the Ethylene Response Factor (ERF) family are clustered at the nicotine-regulatory locus NICOTINE2 (NIC2) in the tobacco genome. A subset of the NIC2-locus ERFs and their homologs, including ERF189 and ERF199, have been shown to be most effective in controlling nicotine biosynthetic pathway genes. Herein reported is that the ERF genes of this group, other than ERF189 and ERF199, were strongly induced by NaCl in tobacco hairy roots, although salt stress had no effect on expression of nicotine biosynthesis genes. Abscisic acid and osmotic stress also increased expression of a subset of these NaCl-inducible ERF genes. Promoter expression analysis in transgenic tobacco hairy roots confirmed that while methyl jasmonate (MJ) activated the promoters of ERF29, ERF210 and ERF199, salt stress up-regulated the promoters of only ERF29 and ERF210, but not ERF199. The protein biosynthesis inhibitor cycloheximide induced expression of the ERFs, and simultaneous addition of MJ and cycloheximide showed synergistic effects. These results indicate that, after several gene duplication events, the NIC2-locus ERFs and possibly their homologs appear to have diverged in their responses to jasmonates and various environmental inputs, including salt stress, and may have evolved to regulate distinct metabolic processes and cellular responses.

Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation.

A molecular mechanism to ensure signaling specificity is a scaffold. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin feedback regulation. The 14-3-3 proteins negatively regulate RSG by sequestering it in the cytoplasm in response to gibberellins. The N. tabacum Ca2+-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. CDPKs are unique sensor responders of Ca2+ that are only found in plants and some protozoans. Here, we report a scaffolding function of CDPK. 14-3-3 proteins bound to NtCDPK1 by a new mode. Autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. NtCDPK1 formed a heterotrimer with RSG and 14-3-3. Furthermore, we found that NtCDPK1 transfers 14-3-3 to RSG after phosphorylation of RSG and that RSG dissociates from NtCDPK1 as a complex with 14-3-3. These results suggest that NtCDPK1 is an interesting scaffolding kinase that increases the specificity and efficiency of signaling by coupling catalysis with scaffolding on the same protein.

Analysis of Dahlia Mosaic Virus Full-length Transcript Promoter-Driven Gene Expression in Transgenic Plants

A 556-bp long full-length transcript promoter (DaMVFLt4-; −474 to +82 from transcription start site) with enhanced activity was characterized from the Dahlia mosaic virus (DaMV). The strength of the DaMVFLt4- promoter has been evaluated in transient systems and in transgenic plants using two different reporter genes like β-glucuronidase (GUS) and green fluorescent protein (GFP). The DaMVFLt4- promoter was found to be 4-fold and 5-fold stronger than the CaMV35S promoter in tobacco protoplast and transgenic tobacco plants, respectively. Electrophoretic mobility shift assay (EMSA) and supershift analysis confirmed the binding of tobacco transcription factor TGA1a to the enhancer region of the DaMVFLt4- promoter. TGA1a specially interacted with the activation sequence-1 (as-1) (−69 to –41 from transcription start site (TSS)) of DaMVFLt4- promoter as shown by DNaseI footprinting. UV cross-linking studies and Southwestern blot analysis clearly demonstrated that the purified TGA1a specifically bound to as-1 element, whereas in tobacco nuclear extract, two unknown transcription factors of about 41 kDa (putative TGA1a) and about 67 kDa were bound to the as-1 sequence of the DaMVFLt4- promoter. Expression studies with the DaMVFLt4-::β-glucuronidase (GUS) genes in tobacco protoplasts co-transfected with CaMV35S::TGA1a showed that expression of TGA1a resulted in approximately 3.7 times elevated levels of GUS activity. The DaMVFLt4- promoter is a constitutive promoter, and the expression level in tissues of transgenic tobacco plants was in the order root > leaf > stem. In addition, the DaMVFLt4- promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived DaMVFLt4- promoter would become an efficient tool for biotechnological application.

Tobacco Transcription Factor NtWRKY12 Interacts with TGA2.2 in vitro and in vivo

The promoter of the salicylic acid-inducible PR-1a gene of Nicotiana tabacum contains binding sites for transcription factor NtWRKY12 (WK-box at position −564) and TGA factors (as-1-like element at position −592). Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1, tga256, and tga2356 mutant plants revealed that NtWRKY12 alone was able to induce a PR-1a::β-glucuronidase (GUS) reporter gene to high levels, independent of co-expressed tobacco NtNPR1, TGA2.1, TGA2.2, or endogenous Arabidopsis NPR1, TGA2/3/5/6. By in vitro pull-down assays with GST and Strep fusion proteins and by Fluorescence Resonance Energy Transfer assays with protein–CFP and protein–YFP fusions in transfected protoplasts, it was shown that NtWRKY12 and TGA2.2 could interact in vitro and in vivo. Interaction of NtWRKY12 with TGA1a or TGA2.1 was not detectable by these techniques. A possible mechanism for the role of NtWRKY12 and TGA2.2 in PR-1a gene expression is discussed.

Tobacco transcription factor WRKY4 is a modulator of leaf development and disease resistance

The role of tobacco transcription factor WRKY4 in leaf development and biotic stress tolerance was studied using RNAi suppressed transgenic plants. The leaves were more numerous and wider in NtWRKY4 RNAi suppressed transgenic lines compared to the vector control, while the levels of miRNA166 and miRNA396 were reduced in suppressed lines. NtWRKY4 expression was markedly induced in response to salicylic acid (SA), but not to abiotic stresses. When infected by tobacco mosaic virus (TMV), the leaves of the transgenic plants were more twisted and displayed a more obvious mosaic pattern compared to those of vector-transgenic plants. Less TMV viral RNA accumulated in vector-transformed plants than in transgenic plants. The results indicate that NtWRKY4 is involved in leaf morphogenesis and antiviral defense, which is seldom seen in WRKY family members.

Preferential Up-Regulation of G2/M Phase-Specific Genes by Overexpression of the Hyperactive Form of NtmybA2 Lacking Its Negative Regulation Domain in Tobacco BY-2 Cells

Many G2/M phase-specific genes in plants contain mitosis-specific activator (MSA) elements, which act as G2/M phase-specific enhancers and bind with R1R2R3-Myb transcription factors. Here, we examined the genome-wide effects of NtmybA2 overexpression, one of the R1R2R3-Myb transcription factors in tobacco (Nicotiana tabacum). We used a custom-made 16-K cDNA microarray for comparative transcriptome analysis of transgenic tobacco BY-2 cell lines that overexpress NtmybA2 or its truncated hyperactive form. The microarray was also used to determine the transcript profile during the cell cycle in synchronized cultures of BY-2 cells. Combined microarray data from transgenic lines and synchronized cells revealed that overexpression of the truncated hyperactive form of NtmybA2, but not its full-length form, preferentially up-regulated many G2/M phase-specific genes in BY-2 cells. We determined promoter sequences of several such up-regulated genes and showed that all contain MSA-like motifs in the proximal regions of their promoters. One of the up-regulated genes, NtE2C, encoding for cyclin-specific ubiquitin carrier proteins, contained a single functional MSA-like motif, which specifically controlled the expression of a reporter gene in the G2/M phase in BY-2 cells. Furthermore, a genomic footprint experiment showed that the MSA element in the NtE2C promoter interacted with nuclear proteins in vivo. Therefore, we propose that the transcription of many G2/M phase-specific genes in tobacco is positively regulated by NtmybA2, in most cases through direct binding to the MSA elements.

TOBFAC: the database of tobacco transcription factors

BackgroundRegulation of gene expression at the level of transcription is a major control point in many biological processes. Transcription factors (TFs) can activate and/or repress the transcriptional rate of target genes and vascular plant genomes devote approximately 7% of their coding capacity to TFs. Global analysis of TFs has only been performed for three complete higher plant genomes – Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa) and rice (Oryza sativa). Presently, no large-scale analysis of TFs has been made from a member of the Solanaceae, one of the most important families of vascular plants. To fill this void, we have analysed tobacco (Nicotiana tabacum) TFs using a dataset of 1,159,022 gene-space sequence reads (GSRs) obtained by methylation filtering of the tobacco genome. An analytical pipeline was developed to isolate TF sequences from the GSR data set. This involved multiple (typically 10–15) independent searches with different versions of the TF family-defining domain(s) (normally the DNA-binding domain) followed by assembly into contigs and verification. Our analysis revealed that tobacco contains a minimum of 2,513 TFs representing all of the 64 well-characterised plant TF families. The number of TFs in tobacco is higher than previously reported for Arabidopsis and rice.ResultsTOBFAC: the database of tobacco transcription factors, is an integrative database that provides a portal to sequence and phylogeny data for the identified TFs, together with a large quantity of other data concerning TFs in tobacco. The database contains an individual page dedicated to each of the 64 TF families. These contain background information, domain architecture via Pfam links, a list of all sequences and an assessment of the minimum number of TFs in this family in tobacco. Downloadable phylogenetic trees of the major families are provided along with detailed information on the bioinformatic pipeline that was used to find all family members. TOBFAC also contains EST data, a list of published tobacco TFs and a list of papers concerning tobacco TFs. The sequences and annotation data are stored in relational tables using a PostgrelSQL relational database management system. The data processing and analysis pipelines used the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The computationally intensive data processing and analysis pipelines were run on an Apple XServe cluster with more than 20 nodes.ConclusionTOBFAC is an expandable knowledgebase of tobacco TFs with data currently available for over 2,513 TFs from 64 gene families. TOBFAC integrates available sequence information, phylogenetic analysis, and EST data with published reports on tobacco TF function. The database provides a major resource for the study of gene expression in tobacco and the Solanaceae and helps to fill a current gap in studies of TF families across the plant kingdom. TOBFAC is publicly accessible at .

14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1

Tobacco DBP1 is the founding member of a novel class of plant transcription factors featuring sequence-specific DNA binding and protein phosphatase activity. To understand the mechanisms underlying the function of this family of transcriptional regulators, we have identified the tobacco 14-3-3 isoform G as the first protein interacting with a DBP factor. 14-3-3 recognition involves the N-terminal region of DBP1, which also supports the DNA binding activity attributed to DBP1. The relevance of this interaction is reinforced by its conservation in Arabidopsis plants, where the closest relative of DBP1 in this species also interacts with a homologous 14-3-3 protein through its N-terminal region. Furthermore, we show that in planta 14-3-3 G is directly involved in regulating DBP1 function by promoting nuclear export and subsequent cytoplasmic retention of DBP1 under conditions that in turn alleviate DBP1-mediated repression of target gene expression.

Tobacco Transcription Factor WRKY1 Is Phosphorylated by the MAP Kinase SIPK and Mediates HR-Like Cell Death in Tobacco

The salicylic acid-induced protein kinase (SIPK) of tobacco, which is a mitogen-activated protein kinase (MAPK), is activated by various biotic and abiotic treatments. Overexpression of SIPK has been shown to trigger cell death. In this study, a targeted yeast two-hybrid approach identified the tobacco transcription factor WRKY1 as a potential substrate. SIPK phosphorylated WRKY1, which resulted in enhanced DNA-binding activity of WRKY1 to its cognate binding site, a W box sequence from the tobacco chitinase gene CHN50. SIPK-mediated enhancement of WRKY1 DNA-binding activity was inhibited by staurosporine, a general kinase inhibitor. Co-expression of SIPK and WRKY1 in Nicotiana benthamiana led to more rapid cell death than expression of SIPK alone, suggesting that WRKY1 is involved in the formation of hypersensitive response-like cell death and may be a component of the signaling cascade downstream of SIPK.