Molecular Screening Tools to Study Arabidopsis Transcription Factors

In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites

The Arabidopsis bZIP transcription factor family: an update

The basic (region) leucine zippers (bZIPs) are evolutionarily conserved transcription factors in eukaryotic organisms. Here, we have updated the classification of the Arabidopsis thaliana bZIP-family, comprising 78 members, which have been assorted into 13 groups. Arabidopsis bZIPs are involved in a plethora of functions related to plant development, environmental signalling and stress response. Based on the classification, we have highlighted functional and regulatory aspects of selected well-studied bZIPs, which may serve as prototypic examples for the particular groups

The Arabidopsis bZIP transcription factor family : an update

The basic (region) leucine zippers (bZIPs) are evolutionarily conserved transcription factors in eukaryotic organisms. Here, we have updated the classification of the Arabidopsis thaliana bZIP-family, comprising 78 members, which have been assorted into 13 groups. Arabidopsis bZIPs are involved in a plethora of functions related to plant development, environmental signalling and stress response. Based on the classification, we have highlighted functional and regulatory aspects of selected well-studied bZIPs, which may serve as prototypic examples for the particular groups

Regulation of Oncogene Expression in T-DNA-Transformed Host Plant Cells

Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation

In planta ORFeome analysis by large-scale over-expression of GATEWAY (R)-compatible cDNA clones: screening of ERF transcription factors involved in abiotic stress defense

Genomic approaches have generated large Arabidopsis thaliana open reading frame (ORF) collections. However, tools are required to functionally characterize this ORFeome. Here we describe a batch procedure to simultaneously recombine a population of GATEWAY((R))-tagged full-length cDNAs into a plant expression vector. A pool of agrobacteria carrying these constructs has been used in flower-dip transformation experiments to obtain a collection of transgenic lines that over-express HA-tagged ORFs. This AtTORF-Ex library can be used in various screening approaches to identify particular gene family members involved in plant development or stress responses. The feasibility of the approach was studied using a near-complete collection of the Arabidopsis ethylene response factor (ERF) transcription factor (TF) family. Quality control performed at each step of the procedure revealed that the complexity of the population is maintained, and that almost all members of the ORF collection are covered by the plant library. The frequency of multiple transformation events has been determined as approximately 4%. Significant transgene expression was detected at the RNA and protein level in more than 60% and 30% of the transgenic plants, respectively. Striking phenotypic alterations were observed in approximately 4% of the plants. Many ERF TFs have been shown to participate in plant stress responses. As a proof of principle, the AtTORF-Ex library has been used in a selection procedure to isolate TFs involved in enhanced abiotic stress tolerance. The corresponding TF gene can be easily polymerase chain reaction-amplified using GATEWAY((R))att site-specific primers. In summary, we describe here a method that can be generally applied for functional analysis of ORFeomes in planta

Bioinformatic <it>cis</it>-element analyses performed in <it>Arabidopsis</it> and rice disclose bZIP- and MYB-related binding sites as potential AuxRE-coupling elements in auxin-mediated transcription

<p>Abstract</p> <p>Background</p> <p>In higher plants, a diverse array of developmental and growth-related processes is regulated by the plant hormone auxin. Recent publications have proposed that besides the well-characterized Auxin Response Factors (ARFs) that bind Auxin Response Elements (AuxREs), also members of the bZIP- and MYB-transcription factor (TF) families participate in transcriptional control of auxin-regulated genes via bZIP Response Elements (ZREs) or Myb Response Elements (MREs), respectively.</p> <p>Results</p> <p>Applying a novel bioinformatic algorithm, we demonstrate on a genome-wide scale that singular motifs or composite modules of AuxREs, ZREs, MREs but also of MYC2 related elements are significantly enriched in promoters of auxin-inducible genes. Despite considerable, species-specific differences in the genome structure in terms of the GC content, this enrichment is generally conserved in dicot (<it>Arabidopsis thaliana</it>) and monocot (<it>Oryza sativa</it>) model plants. Moreover, an enrichment of defined composite modules has been observed in selected auxin-related gene families. Consistently, a bipartite module, which encompasses a bZIP-associated G-box Related Element (GRE) and an AuxRE motif, has been found to be highly enriched. Making use of transient reporter studies in protoplasts, these findings were experimentally confirmed, demonstrating that GREs functionally interact with AuxREs in regulating auxin-mediated transcription.</p> <p>Conclusions</p> <p>Using genome-wide bioinformatic analyses, evolutionary conserved motifs have been defined which potentially function as AuxRE-dependent coupling elements to establish auxin-specific expression patterns. Based on these findings, experimental approaches can be designed to broaden our understanding of combinatorial, auxin-controlled gene regulation.</p