Complex control of seed germination timing by ERF50 involves RGL2 antagonism and negative feedback regulation of DOG1.

Seed dormancy governs germination timing, with both evolutionary and applied consequences. Despite extensive studies on the hormonal and genetic control of these processes, molecular mechanisms directly linking dormancy and germination remain poorly understood. By screening a collection of lines overexpressing Arabidopsis transcription factors, we identified ERF50 as a key gene to control dormancy and germination. To study its regulation, we measured seed-related physiological parameters in loss-of-function mutants and carried out transactivation, protein interaction and ChIP-PCR analyses. We found direct ERF50-mediated repression of DOG1 and activation of EXPA2 transcription, which results in enhanced seed germination. Although ERF50 expression is increased by DOG1 in dormant seeds, ERF50 germination-promoting activity is blocked by RGL2. The physiological, genetic and molecular evidence gathered here supports that ERF50 controls germination timing by regulating DOG1 levels to leverage its role as enhancer of seed germination, via RGL2 antagonism on EXPA2 expression. Our results highlight the central role of ERF50 as a feedback regulator to couple and fine-tune seed dormancy and germination.

S1 basic leucine zipper transcription factors shape plant architecture by controlling C/N partitioning to apical and lateral organs.

Plants tightly control growth of their lateral organs, which led to the concept of apical dominance. However, outgrowth of the dormant lateral primordia is sensitive to the plant's nutritional status, resulting in an immense plasticity in plant architecture. While the impact of hormonal regulation on apical dominance is well characterized, the prime importance of sugar signaling to unleash lateral organ formation has just recently emerged. Here, we aimed to identify transcriptional regulators, which control the trade-off between growth of apical versus lateral organs. Making use of locally inducible gain-of-function as well as single and higher-order loss-of-function approaches of the sugar-responsive S1-basic-leucine-zipper (S1-bZIP) transcription factors, we disclosed their largely redundant function in establishing apical growth dominance. Consistently, comprehensive phenotypical and analytical studies of S1-bZIP mutants show a clear shift of sugar and organic nitrogen (N) allocation from apical to lateral organs, coinciding with strong lateral organ outgrowth. Tissue-specific transcriptomics reveal specific clade III SWEET sugar transporters, crucial for long-distance sugar transport to apical sinks and the glutaminase GLUTAMINE AMIDO-TRANSFERASE 1_2.1, involved in N homeostasis, as direct S1-bZIP targets, linking the architectural and metabolic mutant phenotypes to downstream gene regulation. Based on these results, we propose that S1-bZIPs control carbohydrate (C) partitioning from source leaves to apical organs and tune systemic N supply to restrict lateral organ formation by C/N depletion. Knowledge of the underlying mechanisms controlling plant C/N partitioning is of pivotal importance for breeding strategies to generate plants with desired architectural and nutritional characteristics.

DOMAIN OF UNKNOWN FUNCTION581-9 negatively regulates SnRK1 kinase activity.

In plants, sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is a key energy sensor that orchestrates large-scale transcriptional reprograming to maintain cellular homeostasis under energy deficit. SnRK1 activity is under tight negative control, although the exact mechanisms leading to its activation are not well understood. We show that the Arabidopsis (Arabidopsis thaliana) DOMAIN OF UNKNOWN FUNCTION (DUF581) protein DUF581-9/FCS-like zinc finger 3 binds to the catalytic SnRK1.1 α subunit (KIN10) to inhibit its activation by geminivirus rep-interacting kinase (GRIK)-dependent T-loop phosphorylation. Overexpression of DUF581-9 in Arabidopsis dampens SnRK1 signaling and interferes with adaptation to dark-induced starvation. The presence of DUF581-9 significantly reduced SnRK1 activity in protoplasts and in vitro. This was accompanied by a reduction in T175 T-loop phosphorylation and also diminished KIN10 auto-phosphorylation. Furthermore, DUF581-9 reduced binding of the upstream activating kinase GRIK2 to KIN10, explaining the reduced KIN10 T-loop phosphorylation. Ectopically expressed DUF581-9 protein was rapidly turned over by the proteasome when Arabidopsis plants were subjected to starvation treatment, likely releasing its inhibitory activity on the SnRK1 complex. Taken together, our results support a model in which DUF581-9 negatively regulates SnRK1 activity under energy sufficient conditions. Turnover of the protein provides a rapid way for SnRK1 activation under energy deficit without the need of de novo protein synthesis.

The evolutionarily conserved kinase SnRK1 orchestrates resource mobilization during Arabidopsis seedling establishment.

The onset of plant life is characterized by a major phase transition. During early heterotrophic seedling establishment, seed storage reserves fuel metabolic demands, allowing the plant to switch to autotrophic metabolism. Although metabolic pathways leading to storage compound mobilization are well-described, the regulatory circuits remain largely unresolved. Using an inducible knockdown approach of the evolutionarily conserved energy master regulator Snf1-RELATED-PROTEIN-KINASE1 (SnRK1), phenotypic studies reveal its crucial function in Arabidopsis thaliana seedling establishment. Importantly, glucose feeding largely restores growth defects of the kinase mutant, supporting its major impact in resource mobilization. Detailed metabolite studies reveal sucrose as a primary resource early in seedling establishment, in a SnRK1-independent manner. Later, SnRK1 orchestrates catabolism of triacylglycerols and amino acids. Concurrent transcriptomic studies highlight SnRK1 functions in controlling metabolic hubs fuelling gluconeogenesis, as exemplified by cytosolic PYRUVATE ORTHOPHOSPHATE DIKINASE (cyPPDK). Here, SnRK1 establishes its function via phosphorylation of the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63), which directly targets and activates the cyPPDK promoter. Taken together, our results disclose developmental and catabolic functions of SnRK1 in seed storage mobilization and describe a prototypic gene regulatory mechanism. As seedling establishment is important for plant vigor and crop yield, our findings are of agronomical importance.

Perturbations in plant energy homeostasis prime lateral root initiation via SnRK1-bZIP63-ARF19 signaling.

Plants adjust their energy metabolism to continuous environmental fluctuations, resulting in a tremendous plasticity in their architecture. The regulatory circuits involved, however, remain largely unresolved. In Arabidopsis, moderate perturbations in photosynthetic activity, administered by short-term low light exposure or unexpected darkness, lead to increased lateral root (LR) initiation. Consistent with expression of low-energy markers, these treatments alter energy homeostasis and reduce sugar availability in roots. Here, we demonstrate that the LR response requires the metabolic stress sensor kinase Snf1-RELATED-KINASE1 (SnRK1), which phosphorylates the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) that directly binds and activates the promoter of AUXIN RESPONSE FACTOR19 (ARF19), a key regulator of LR initiation. Consistently, starvation-induced ARF19 transcription is impaired in bzip63 mutants. This study highlights a positive developmental function of SnRK1. During energy limitation, LRs are initiated and primed for outgrowth upon recovery. Hence, this study provides mechanistic insights into how energy shapes the agronomically important root system.

HEXOKINASE1 signalling promotes shoot branching and interacts with cytokinin and strigolactone pathways.

Plant architecture is controlled by several endogenous signals including hormones and sugars. However, only little information is known about the nature and roles of the sugar signalling pathways in this process. Here we test whether the sugar signalling pathway mediated by HEXOKINASE1 (HXK1) is involved in the control of shoot branching. To test the involvement of HXK1 in shoot branching and in the hormonal network controlling this process, we modulated the HXK1 pathway using physiological and genetic approaches in rose, pea and arabidopsis. Mannose-induced HXK signalling triggered bud outgrowth in rose and pea. In arabidopsis, both HXK1 deficiency and defoliation led to decreased shoot branching and conferred hypersensitivity to auxin. Complementation of the HXK1 knockout mutant gin2 with a catalytically inactive HXK1, restored shoot branching to the wild-type level. HXK1-deficient plants displayed decreased cytokinin levels and increased expression of MAX2, which is required for strigolactone signalling. The branching phenotype of HXK1-deficient plants could be partly restored by cytokinin treatment and strigolactone deficiency could override the negative impact of HXK1 deficiency on shoot branching. Our observations demonstrate that HXK1 signalling contributes to the regulation of shoot branching and interacts with hormones to modulate plant architecture.

A Gain-of-Function Screen Reveals Redundant ERF Transcription Factors Providing Opportunities for Resistance Breeding Toward the Vascular Fungal Pathogen Verticillium longisporum.

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.

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 C/S1 bZIP Network: A Regulatory Hub Orchestrating Plant Energy Homeostasis.

Sustaining energy homeostasis is crucial to every living being. To balance energy supply and demand, plants make use of an evolutionarily conserved management system consisting of two counteracting kinases, TOR (TARGET OF RAPAMYCIN) and SnRK1 (Snf1-RELATED PROTEIN KINASE 1). SnRK1 is involved in reorganizing enzymatic and transcriptional responses to survive energy-limiting conditions. Recently, members of the bZIP (basic leucine zipper) transcription factor family have been established as SnRK1 downstream mediators. We review here current knowledge on the functional impact of these group C and S1 bZIPs, and analyze their regulation by environmental and endogenous cues. Given their specific homo- and heterodimerization, the so-called C/S1 bZIP network is proposed to act as a signaling hub that coordinates plant development and stress responses.

Snf1-RELATED KINASE1-Controlled C/S1-bZIP Signaling Activates Alternative Mitochondrial Metabolic Pathways to Ensure Plant Survival in Extended Darkness.

Sustaining energy homeostasis is of pivotal importance for all living organisms. In Arabidopsis thaliana, evolutionarily conserved SnRK1 kinases (Snf1-RELATED KINASE1) control metabolic adaptation during low energy stress. To unravel starvation-induced transcriptional mechanisms, we performed transcriptome studies of inducible knockdown lines and found that S1-basic leucine zipper transcription factors (S1-bZIPs) control a defined subset of genes downstream of SnRK1. For example, S1-bZIPs coordinate the expression of genes involved in branched-chain amino acid catabolism, which constitutes an alternative mitochondrial respiratory pathway that is crucial for plant survival during starvation. Molecular analyses defined S1-bZIPs as SnRK1-dependent regulators that directly control transcription via binding to G-box promoter elements. Moreover, SnRK1 triggers phosphorylation of group C-bZIPs and the formation of C/S1-heterodimers and, thus, the recruitment of SnRK1 directly to target promoters. Subsequently, the C/S1-bZIP-SnRK1 complex interacts with the histone acetylation machinery to remodel chromatin and facilitate transcription. Taken together, this work reveals molecular mechanisms underlying how energy deprivation is transduced to reprogram gene expression, leading to metabolic adaptation upon stress.

High-Throughput Protoplast Trans-Activation (PTA) Screening to Define Transcription Factors in Auxin-Mediated Gene Regulation.

Genome sequencing and annotation studies clearly highlight the impact of transcriptional regulation in plants. However, functional characterization of the majority of transcriptional regulators remains elusive. Hence, high-throughput techniques are required to facilitate their molecular analysis. Here, we provide a detailed protocol to conduct a high-throughput protoplast trans-activation (PTA) screening, which enables simultaneous analysis of up to 95 individual transcription factor activities on a customizable promoter:LUCIFERASE reporter. This system is well suited to decipher complex transcriptional networks such as that triggered by the phytohormone auxin.

The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth.

Plants have to tightly control their energy homeostasis to ensure survival and fitness under constantly changing environmental conditions. Thus, it is stringently required that energy-consuming stress-adaptation and growth-related processes are dynamically tuned according to the prevailing energy availability. The evolutionary conserved SUCROSE NON-FERMENTING1 RELATED KINASES1 (SnRK1) and the downstream group C/S1 basic leucine zipper (bZIP) transcription factors (TFs) are well-characterised central players in plants' low-energy management. Nevertheless, mechanistic insights into plant growth control under energy deprived conditions remains largely elusive. In this work, we disclose the novel function of the low-energy activated group S1 bZIP11-related TFs as regulators of auxin-mediated primary root growth. Whereas transgenic gain-of-function approaches of these bZIPs interfere with the activity of the root apical meristem and result in root growth repression, root growth of loss-of-function plants show a pronounced insensitivity to low-energy conditions. Based on ensuing molecular and biochemical analyses, we propose a mechanistic model, in which bZIP11-related TFs gain control over the root meristem by directly activating IAA3/SHY2 transcription. IAA3/SHY2 is a pivotal negative regulator of root growth, which has been demonstrated to efficiently repress transcription of major auxin transport facilitators of the PIN-FORMED (PIN) gene family, thereby restricting polar auxin transport to the root tip and in consequence auxin-driven primary root growth. Taken together, our results disclose the central low-energy activated SnRK1-C/S1-bZIP signalling module as gateway to integrate information on the plant's energy status into root meristem control, thereby balancing plant growth and cellular energy resources.

Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins.

13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins.

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.

Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots.

Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity.

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.

The Arabidopsis transcription factor bZIP11 activates auxin-mediated transcription by recruiting the histone acetylation machinery.

In higher plants, the hormone auxin orchestrates a diverse array of developmental and environmental responses mainly exerted via transcriptional control. In its absence, auxin-mediated transcription is postulated to be repressed by histone deacetylases, which convert chromatin into a highly packed inactive state. Here we present a converse mechanism where Arabidopsis bZIP11-related basic leucine zipper (bZIP) transcription factors interact via an amino-terminal activation domain with ADA2b adapter proteins to recruit the histone acetylation machinery to specific auxin-responsive genes. Gain, loss-of-function and pharmacological approaches as well as chromatin immunoprecipitation experiments addressing various components of the recruitment and acetylation machinery substantiate the proposed mechanism. Importantly, G-box-related cis-elements, frequently found in auxin-induced promoters, are shown to bind bZIP11-related bZIPs and to function as quantitative modulators of auxin-induced transcription. In conclusion, we describe a regulatory activation mechanism that serves as a rheostat to modulate auxin-mediated responses.

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

BACKGROUND: 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. RESULTS: 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 (Arabidopsis thaliana) and monocot (Oryza sativa) 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. CONCLUSIONS: 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.

Heterodimers of the Arabidopsis transcription factors bZIP1 and bZIP53 reprogram amino acid metabolism during low energy stress.

Control of energy homeostasis is crucial for plant survival, particularly under biotic or abiotic stress conditions. Energy deprivation induces dramatic reprogramming of transcription, facilitating metabolic adjustment. An in-depth knowledge of the corresponding regulatory networks would provide opportunities for the development of biotechnological strategies. Low energy stress activates the Arabidopsis thaliana group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 by transcriptional and posttranscriptional mechanisms. Gain-of-function approaches define these bZIPs as crucial transcriptional regulators in Pro, Asn, and branched-chain amino acid metabolism. Whereas chromatin immunoprecipitation analyses confirm the direct binding of bZIP1 and bZIP53 to promoters of key metabolic genes, such as ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE, the G-box, C-box, or ACT motifs (ACTCAT) have been defined as regulatory cis-elements in the starvation response. bZIP1 and bZIP53 were shown to specifically heterodimerize with group C bZIPs. Although single loss-of-function mutants did not affect starvation-induced transcription, quadruple mutants of group S1 and C bZIPs displayed a significant impairment. We therefore propose that bZIP1 and bZIP53 transduce low energy signals by heterodimerization with members of the partially redundant C/S1 bZIP factor network to reprogram primary metabolism in the starvation response.

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 thalianaTF 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 transactivation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.