Arabidopsis Transcription Factor Research Articles

Cold-inducible Gene Expression Regulated by CAMTA Transcription Factors in Arabidopsis

Cold-inducible Gene Expression Regulated by CAMTA Transcription Factors in Arabidopsis

Regulation of pollen lipid body biogenesis by MAP kinases and downstream WRKY transcription factors in Arabidopsis.

Signaling pathways that control the activities in non-photosynthetic plastids, important sites of plant metabolism, are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Loss of function of WRKY2/WRKY34 results in reduced GPT1 expression, and concomitantly, reduced accumulation of lipid bodies in mature pollen, which leads to compromised pollen viability, germination, pollen tube growth, and male transmission in Arabidopsis. Pollen-specific overexpression of GPT1 rescues the pollen defects of wrky2 wrky34 double mutant. Furthermore, gain-of-function activation of MPK3/MPK6 enhances GPT1 expression; whereas GPT1 expression is reduced in mkk4 mkk5 double mutant. Together, this study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction.

Overexpression of OsAP2 and OsWRKY24 in Arabidopsis results in reduction of plant size.

Recently, two rice genes, OsAPETALA2 (OsAP2) and OsWRKY24 have been reported to be positive regulators involved in increased lamina inclination and grain size through cell elongation. Here, we found that the two genes have tightly linked expression patterns and functional convergence in rice, and are also likely to play an opposite role in Arabidopsis. Overexpression of the two rice transcription factors in Arabidopsis caused smaller plant size with reduced cell size, and the expression of a series of genes encoding expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) involved in cell elongation was reduced. However, transgenic Arabidopsis expressing OsWRKY24-SRDX as a synthetic chimeric repressor displayed indistinguishable phenotypes from wild-type plants. Moreover, the subcellular localization pattern of OsWRKY24 in Arabidopsis was different from that in rice. Thus, we demonstrate an example of transcription factors from one species playing distinct roles in different plant species.

DNA recognition by Arabidopsis transcription factors ABI3 and NGA1

B3 transcription factors constitute a large plant-specific protein superfamily, which plays a central role in plant life. Family members are characterized by the presence of B3 DNA-binding domains (DBDs). To date, only a few B3 DBDs were structurally characterized; therefore, the DNA recognition mechanism of other family members remains to be elucidated. Here, we analyze DNA recognition mechanism of two structurally uncharacterized B3 transcription factors, ABI3 and NGA1. Guided by the structure of the DNA-bound B3 domain of Arabidopsis transcriptional repressor VAL1, we have performed mutational analysis of the ABI3 B3 domain. We demonstrate that both VAL1-B3 and ABI3-B3 recognize the Sph/RY DNA sequence 5'-TGCATG-3' via a conserved set of base-specific contacts. We have also solved a 1.8 Å apo-structure of NGA1-B3, DBD of Arabidopsis transcription factor NGA1. We show that NGA1-B3, like the structurally related RAV1-B3 domain, is specific for the 5'-CACCTG-3' DNA sequence, albeit tolerates single base pair substitutions at the 5'-terminal half of the recognition site. Employing distance-dependent fluorophore quenching, we show that NGA1-B3 binds the asymmetric recognition site in a defined orientation, with the 'N-arm' and 'C-arm' structural elements interacting with the 5'- and 3'-terminal nucleotides of the 5'-CACCTG-3' sequence, respectively. Mutational analysis guided by the model of DNA-bound NGA1-B3 helped us identify NGA1-B3 residues involved in base-specific and DNA backbone contacts, providing new insights into the mechanism of DNA recognition by plant B3 domains of RAV and REM families. DATABASES: RCSB Protein Data Bank, accession number 5OS9.

Arabidopsis HD-Zip II proteins regulate the exit from proliferation during leaf development in canopy shade.

The shade avoidance response is mainly evident as increased plant elongation at the expense of leaf and root expansion. Despite the advances in understanding the mechanisms underlying shade-induced hypocotyl elongation, little is known about the responses to simulated shade in organs other than the hypocotyl. In Arabidopsis, there is evidence that shade rapidly and transiently reduces the frequency of cell division in young first and second leaf primordia through a non-cell-autonomous mechanism. However, the effects of canopy shade on leaf development are likely to be complex and need to be further investigated. Using combined methods of genetics, cell biology, and molecular biology, we uncovered an effect of prolonged canopy shade on leaf development. We show that persistent shade determines early exit from proliferation in the first and second leaves of Arabidopsis. Furthermore, we demonstrate that the early exit from proliferation in the first and second leaves under simulated shade depends at least in part on the action of the Homeodomain-leucine zipper II (HD-Zip II) transcription factors ARABIDOPSIS THALIANA HOMEOBOX2 (ATHB2) and ATHB4. Finally, we provide evidence that the ATHB2 and ATHB4 proteins work in concert. Together the data contribute new insights on the mechanisms controlling leaf development under canopy shade.

Actin Reorganization Triggers Rapid Cell Elongation in Roots.

Root growth is controlled by mechanisms underlying cell division and cell elongation, which respond to various internal and external factors. In Arabidopsis (Arabidopsis thaliana), cells produced in the proximal meristem (PM) elongate and differentiate in the transition zone (TZ) and the elongation/differentiation zone (EDZ). Previous studies have demonstrated that endoreplication is involved in root cell elongation; however, the manner by which cells increase in length by more than 2-fold remains unknown. Here, we show that epidermal and cortical cells in Arabidopsis roots undergo two modes of rapid cell elongation: the first rapid cell elongation occurs at the border of the proximal meristem and the TZ, and the second mode occurs during the transition from the TZ to the EDZ. Our previous study showed that cytokinin signaling promotes endoreplication, which triggers the first rapid cell elongation. Our cytological and genetic data revealed that the second rapid cell elongation involves dynamic actin reorganization independent of endoreplication. Cytokinins promote actin bundling and the resultant second rapid cell elongation through activating the signaling pathway involving the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE3 (AHK3) and AHK4 and the B-type transcription factor ARABIDOPSIS RESPONSE REGULATOR2. Our results suggest that cytokinins promote the two modes of rapid cell elongation by controlling distinct cellular events: endoreplication and actin reorganization.

Expression, purification and molecular characterization of a novel transcription factor KcCBF3 from Kandelia candel

Kandelia candel, a major species of mangrove in the tropical and subtropical area, is susceptible to low temperature in winter. K. candel was introduced into Zhejiang Province (the northern margin of South China) several decades ago, and suffered from low temperature causing growth retardation, in server cases, even death. To explore the molecular mechanisms of cold acclimation in K. candel, a novel C-repeat binding factor gene KcCBF3 (Genbank accession no. KF111715) of 729 bp open reading frame (ORF) encoding a protein of 242 amino acid residues was isolated, expressed, purified and characterized. Multiple sequence alignment analysis revealed that KcCBF3 contained a highly conserved AP2/EREBP DNA-binding domain which consisting of 79 amino acid residues, as well as two CBF signature sequences. Phylogenetic analysis indicated that KcCBF3 belonged to the A-1 subgroup of DREB subfamily based on the classification of AP2/EREBP transcription factors in Arabidopsis. Semi-quantitative RT-PCR showed that KcCBF3 transcripts were highly accumulated in roots and leaves, and could be induced by low temperature. Electrophoresis mobility shift assay (EMSA) demonstrated KcCBF3 could bind to the core sequence (CCGAC) of cis-acting element C-repeat (CRT)/dehydration-responsive element (DRE) in vitro. These results implied that KcCBF3 might participate in the adaptation of K. candel to low-temperature stress by binding to CRT/DRE element.

Nitric oxide responses in Arabidopsis hypocotyls are mediated by diverse phytohormone pathways.

Plants are often exposed to high levels of nitric oxide (NO) that affects development and stress-triggered responses. However, the way in which plants sense NO is still largely unknown. Here we combine the analysis of early changes in the transcriptome of plants exposed to a short acute pulse of exogenous NO with the identification of transcription factors (TFs) involved in NO sensing. The NO-responsive transcriptome was enriched in hormone homeostasis- and signaling-related genes. To assess events involved in NO sensing in hypocotyls, we used a functional sensing assay based on the NO-induced inhibition of hypocotyl elongation in etiolated seedlings. Hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis TFs were screened for NO-triggered hypocotyl shortening. These approaches allowed the identification of hormone-related TFs, ethylene perception and signaling, strigolactone biosynthesis and signaling, and salicylate production and accumulation that are essential for or modulate hypocotyl NO sensing. Moreover, NO inhibits hypocotyl elongation through the positive and negative regulation of some abscisic acid (ABA) receptors and transcripts encoding brassinosteroid signaling components thereby also implicating these hormones in NO sensing.

Overexpression of a chrysanthemum transcription factor gene DgNAC1 improves drought tolerance in chrysanthemum

The NAC transcription factor (TF) plays a pivotal role in resisting various abiotic stresses, and it has been proved that the overexpression of NAC transcription factors (TFs) in Arabidopsis is beneficial to increase drought tolerance. However, the function of NAC genes in chrysanthemum remains unclear. Here, we conducted physiological and molecular experiments to assess the role of DgNAC1 in response to drought stress of chrysanthemum. DgNAC1-overexpressed chrysanthemum was significantly more drought-resistant than wild type (WT). Especially, the transgenic chrysanthemum presented a greater survival rates (86.42, 88.89 and 87.65%, respectively) than WT (41.98%) under drought conditions. It also showed higher leaf water potential and relative water content; lower relative electrolyte conductivity; fewer accumulations of malondialdehyde, hydrogen peroxide (H2O2), superoxide anion (O2−); higher activities of superoxide dismutase, peroxidase, catalase; and more content of proline, chlorophyll, soluble protein, soluble sugar, glutathione (GSH), glutathione disulphide (GSSG), as well as higher ratio of GSH/GSSG than those of WT during drought stress. Moreover, stress-responsive genes in transgenic chrysanthemum showed a significant up-regulation than in WT under drought stress. Therefore, all the above results suggested that DgNAC1 served as an active regulator in chrysanthemum’s responses to drought stress, and it may have a significant impact on the molecular breeding of drought-resistant plants as well.

Arabidopsis Transcription Factor MYB102 Increases Plant Susceptibility to Aphids by Substantial Activation of Ethylene Biosynthesis.

Induction of ethylene biosynthesis by aphids increases the susceptibility of several plant species to aphids. Recent studies have indicated that some MYB transcription factors regulate the phloem-based defense against aphid infestation by modulating ethylene (ET) signaling. Arabidopsis MYB102 has previously been shown to be induced by wound signaling and regulate defense response against chewing insects. However, it remains unclear whether Arabidopsis MYB102 takes part in the defense response of plants to aphids. Here, we investigated the function of MYB102 in the response of Arabidopsis to aphid infestation. Arabidopsis MYB102 was primarily expressed in vascular tissues, and its transcription was remarkably induced by green peach aphids (GPA; Myzus persicae). The results of RNA-Sequencing revealed that overexpression of MYB102 in Arabidopsis promoted ET biosynthesis by upregulation of some 1-aminocyclopropane-1-carboxylate synthase (ACS) genes, which are rate-limiting enzymes of the ET-synthetic pathway. Enhanced ET levels led to reduced Arabidopsis resistance to GPA. Furthermore, dominant suppression of MYB102 inhibited aphid-induced increase of ET levels in Arabidopsis. In agreement with a negative regulatory role for ET in aphid defense responses, the MYB102-overexpressing lines were more susceptible to GPA than wild-type (WT) plants. Overexpression of MYB102 in Arabidopsis obviously repressed aphid-induced callose deposition. Conversely, overexpression of MYB102 failed to increase aphid susceptibility in both the ET-insensitive mutants and plants treated with inhibitors of ET signaling pathways, demonstrating that the ET was critical for promoting aphid performance conferred by overexpression of MYB102. Collectively, our findings indicate that the Arabidopsis MYB102 increases host susceptibility to GPA through the ET-dependent signaling pathways.

A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids.

Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a "NAC troika," govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NACs, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis.

SummaryThe flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5‐like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5‐like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1‐amino‐cyclopropane‐1‐carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5‐like TFs in the regulation of ethylene‐mediated petal development and growth.

H2A.Z-containing nucleosomes are evicted to activate AtMYB44 transcription in response to salt stress

Transcripts of the Arabidopsis transcription factor gene, AtMYB44, accumulate rapidly to mediate a tolerance mechanism in response to salt stress. The AtMYB44 promoter is activated by salt stress, as illustrated in AtMYB44pro::GUS transgenic plants. Chromatin immunoprecipitation (ChIP) assays revealed that RNA polymerases were enriched on the AtMYB44 gene, especially on TSS-proximal regions, and nucleosome density was markedly reduced in the AtMYB44 gene-body region in response to salt stress. In addition, H2A.Z occupation was significantly decreased at the AtMYB44 promoter, transcription start site (TSS), and gene-body regions. Histone modifications including histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 and H4 acetylation (H3ac and H4ac) were not affected under the same stress conditions. We found a decrease in the number of AtMYB44 proteins bound to their own gene promoters in response to salt stress. These results suggest that salt stress induces the eviction of H2A.Z-containing nucleosomes from the AtMYB44 promoter region, which may weaken its affinity for binding AtMYB44 protein that acts as a repressor for AtMYB44 gene transcription under salt stress-free conditions.

Overexpression of HvCBF7 and HvCBF9 changes salt and drought tolerance in Arabidopsis

Crop yield losses due to the extreme environments have risen steadily over the past several decades, so it is quite imperative to develop the crop cultivars with high stress tolerance for ensuring global food security. Dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF) transcription factors are widely concerned as key regulators, specifically in abiotic tolerance of plants. In this study, we found that barley CBF7 and CBF9 were induced by salt and drought stress. Over-expression of HvCBF7 and HvCBF9 enhanced salt tolerance, showing increased survival rate of Arabidopsis seedlings under salt stress, but reduced drought and salt tolerance in adult plants before flowering stage, as reflected by higher malondialdehyde and proline contents. The altered phenotype was due to differently regulating stress response genes. In addition, we also found that many genes expressing differently under salt stress were reversely regulated by AtCBF3, a transcription factor in Arabidopsis.

Characterization and transcriptional regulation of chlorophyll b reductase gene NON-YELLOW COLORING 1 associated with leaf senescence in perennial ryegrass (Lolium perenne L.)

Chlorophyll (Chl) degradation leads to leaf senescence and adversely affects biomass production of forage grasses and aesthetic appearance of turfgrasses. The objectives of this study were to characterize the function of a Chl catabolic gene, NON-YELLOW COLORING 1 (LpNYC1), and to understand its transcriptional regulatory pattern involved in leaf senescence in perennial ryegrass (Lolium perenne L.). The LpNYC1 was initially cloned using the RACE-PCR method. Its encoded protein was localized in chloroplasts and its expression pattern was correlated with the progression of leaf senescence. LpNYC1 shared the same biological function with Arabidopsis NYC1, as overexpression of its encoding gene accelerated chlorophyll degradation in Nicotiana benthamiana and rescued the stay-green phenotype of the Arabidopsis nyc1 null mutant. A yeast one-hybrid cDNA library was prepared from senescent leaves of perennial ryegrass. Using the LpNYC1 promoter as bait, five putative transcription factors upstream of LpNYC1 were pooled out using this method, among which three (LpABI5, LpABF3, and LpEIN3) were orthologous to Arabidopsis transcription factors involved in the ABA and ethylene signaling pathways. The expression of LpNYC1 was highly inducible by ABA and ethephon (ethylene releasing reagent) but was suppressed by treatment with AVG (ethylene biosynthesis inhibitor). Furthermore, LpABI5, LpABF3, LpEIN3 directly activated the expression of LpNYC1 by binding to its promoter. The current result laid the groundwork for future in-depth analysis of the molecular regulation of LpNYC1 and Chl metabolism during leaf senescence in perennial grass species.

Transcriptome profile of NO-induced Arabidopsis transcription factor genes suggests their putative regulatory role in multiple biological processes

TFs are important proteins regulating plant responses during environmental stresses. These insults typically induce changes in cellular redox tone driven in part by promoting the production of reactive nitrogen species (RNS). The main source of these RNS is nitric oxide (NO), which serves as a signalling molecule, eliciting defence and resistance responses. To understand how these signalling molecules regulate key biological processes, we performed a large scale S-nitrosocysteine (CySNO)-mediated RNA-seq analysis. The DEGs were analysed to identify potential regulatory TFs. We found a total of 673 (up- and down-regulated) TFs representing a broad range of TF families. GO-enrichment and MapMan analysis suggests that more than 98% of TFs were mapped to the Arabidopsis thaliana genome and classified into pathways like hormone signalling, protein degradation, development, biotic and abiotic stress, etc. A functional analysis of three randomly selected TFs, DDF1, RAP2.6, and AtMYB48 identified a regulatory role in plant growth and immunity. Loss-of-function mutations within DDF1 and RAP2.6 showed compromised basal defence and effector triggered immunity, suggesting their positive role in two major plant defence systems. Together, these results imply an important data representing NO-responsive TFs that will help in exploring the core mechanisms involved in biological processes in plants.

PHB3 Maintains Root Stem Cell Niche Identity through ROS-Responsive AP2/ERF Transcription Factors in Arabidopsis

The root stem cell niche, which is composed of four mitotically inactive quiescent center (QC) cells and the surrounding actively divided stem cells in Arabidopsis, is critical for growth and root development. Here, we demonstrate that the Arabidopsis prohibitin protein PHB3 is required for the maintenance of root stem cell niche identity by both inhibiting proliferative processes in the QC and stimulating cell division in the proximal meristem (PM). PHB3 coordinates cell division and differentiation in the root apical meristem by restricting the spatial expression of ethylene response factor (ERF) transcription factors 115, 114, and 109. ERF115, ERF114, and ERF109 mediate ROS signaling, in a PLT-independent manner, to control root stem cell niche maintenance and root growth through phytosulfokine (PSK) peptide hormones in Arabidopsis.

Generation of Inducible Transgenic Lines of Arabidopsis Transcription Factors Regulated by MicroRNAs.

Transcription factors play key regulatory roles in all the life processes across kingdoms. In plants, the genome of a typical model species such as Arabidopsis thaliana encodes over 1500 transcription factors that regulate the expression dynamics of all the genes in time and space. Therefore, studying their function by analyzing the loss and gain-of-function lines is of prime importance in basic plant biology and its agricultural application. However, the current approach of knocking out genes often causes embryonic lethal phenotype, while inactivating one or two members of a redundant gene family yields little phenotypic changes, thereby making the functional analysis a technically challenging task. In such cases, inducible knock-down or overexpression of transcription factors appears to be a more effective approach. Restricting the transcription factors in the cytoplasm by fusing them with animal glucocorticoid/estrogen receptors (GR/ER) and then re-localizing them to the nucleus by external application of animal hormone analogues has been a useful method of gene function analysis in the model plants. In this chapter, we describe the recent advancements in the GR and ER expression systems and their use in analyzing the function of transcription factors in Arabidopsis.

Construction of Arabidopsis Transcription Factor ORFeome Collections and Identification of Protein-DNA Interactions by High-Throughput Yeast One-Hybrid Screens.

Identification of transcription factor (TF)-promoter interactions is key to understanding the basic molecular underpinnings of gene regulation. The complexity of gene regulation, however, is driven by the combined function of several TFs recruited to the promoter region, which often confounds the discovery of transcriptional regulatory mechanisms. Genome sequencing enabled the construction of TF-specific ORFeome clone collections that can be used to study TF function with unprecedented coverage. Among the recently developed methods, gene-centered yeast one-hybrid (Y1H) screens performed with these ORFeome collections provide a simple and reliable strategy to identify TF-promoter interactions. Here, we describe high-throughput cloning protocols used to generate a gold standard TF ORFeome collection for the model organism Arabidopsis thaliana. Furthermore, we outline the protocol to build a daughter clone collection suitable for the Y1H assay and a high-throughput Y1H screening procedure that enables rapid assessment of thousands TF-promoter interactions using a robotic platform. These protocols can be universally adopted to build ORFeome libraries and thus expand the usage of gene-centered Y1H screens or other alternative strategies for discovery and characterization of TF functions.