BBX21 integrates brassinosteroid biosynthesis and signalling in the inhibition of hypocotyl growth under shade.

B-Box-containing zinc finger transcription factors (BBX) are involved in light-mediated growth, affecting processes such as hypocotyl elongation in Arabidopsis thaliana. However, the molecular and hormonal framework that regulates plant growth through BBX proteins is incomplete. Here, we demonstrate that BBX21 inhibits the hypocotyl elongation through the brassinosteroid (BR) pathway. BBX21 reduces the sensitivity to 24-epiBL, a synthetic active BR, principally at very-low concentrations in simulated shade. The biosynthesis profile of BRs showed that two active BR -brassinolide (BL) and 28-homobrassinolide (28-homoBL)- and 8 of 11 intermediates can be repressed by BBX21 under white light (WL) or simulated shade. Furthermore, BBX21 represses the expression of CYTOCHROME P450 90B1 (DWF4/CYP90B1), BRASSINOSTEROID-6-OXIDASE 1 (BR6OX1, CYP85A1) and BR6OX2 (CYP85A2) genes involved in the BR biosynthesis in WL while specifically promoting DWF4 and PHYB ACTIVATION TAGGED SUPPRESSOR 1 (CYP2B1/BAS1) expression in WL supplemented with far-red (WL+FR), a treatment that simulates shade. In addition, BBX21 represses BR signalling genes such as PACLOBUTRAZOL RESISTANCE1 (PRE1), PRE3 and ARABIDOPSIS MYB-LIKE 2 (MYBL2), and auxin-related and expansin genes, such as INDOLE-3-ACETIC ACID INDUCIBLE 1 (IAA1), IAA4 and EXPANSIN 11 (EXP11) in short-term shade. By a genetic approach we found that BBX21 acts genetically upstream of BRASSINAZOLE-RESISTANT 1 (BZR1) for the promotion of DWF4 and BAS1 gene expression in shade. We propose that BBX21 integrates the BR homeostasis and shade-light signalling allowing the fine-tuning of hypocotyl elongation in Arabidopsis.

Enhancer activation via TCP and HD-ZIP and repression by Dof transcription factors mediate giant cell-specific expression.

Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells.

BBX24 Interacts with JAZ3 to Promote Growth by Reducing DELLA Activity in Shade Avoidance.

Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

ORA47 is a transcriptional regulator of a general stress response hub.

Transcriptional regulators of the general stress response (GSR) reprogram the expression of selected genes to transduce informational signals into cellular events, ultimately manifested in a plant's ability to cope with environmental challenges. Identification of the core GSR regulatory proteins will uncover the principal modules and their mode of action in the establishment of adaptive responses. To define the GSR regulatory components, we employed a yeast-one-hybrid assay to identify the protein(s) binding to the previously established functional GSR motif, termed the rapid stress response element (RSRE). This led to the isolation of octadecanoid-responsive AP2/ERF-domain transcription factor 47 (ORA47), a methyl jasmonate inducible protein. Subsequently, ORA47 transcriptional activity was confirmed using the RSRE-driven luciferase (LUC) activity assay performed in the ORA47 loss- and gain-of-function lines introgressed into the 4xRSRE::Luc background. In addition, the prime contribution of CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) protein in the induction of RSRE was reaffirmed by genetic studies. Moreover, exogenous application of methyl jasmonate led to enhanced levels of ORA47 and CAMTA3 transcripts, as well as the induction of RSRE::LUC activity. Metabolic analyses illustrated the reciprocal functional inputs of ORA47 and CAMTA3 in increasing JA levels. Lastly, transient assays identified JASMONATE ZIM-domain1 (JAZ1) as a repressor of RSRE::LUC activity. Collectively, the present study provides fresh insight into the initial features of the mechanism that transduces informational signals into adaptive responses. This mechanism involves the functional interplay between the JA biosynthesis/signaling cascade and the transcriptional reprogramming that potentiates GSR. Furthermore, these findings offer a window into the role of intraorganellar communication in the establishment of adaptive responses.

Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis.

The basic helix-loop-helix (bHLH) transcription factor PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) is a central regulator that promotes stem growth by activating growth-related gene expression during shade-avoidance responses. Studying the co-factors of PIF7 can facilitate understanding of the mechanism of PIFs and light signal transduction. Here, we describe the identification of two bHLH transcription factors, bHLH48 and bHLH60 (bHLH48/bHLH60), as essential partners for PIF7-dependent modulation of hypocotyl elongation and function downstream of phytochrome B. These two bHLH factors display DNA binding activity and interact with PIF7. Genetic analysis indicated that bHLH48/bHLH60 and PIF7 are interdependent in promoting hypocotyl elongation. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis identified the substantially overlapping downstream targets of bHLH60 and PIF7. Biochemical analysis revealed that bHLH48/bHLH60 enhance the DNA binding ability of PIF7. These results provide evidence that bHLH48/bHLH60 act as positive partners of PIF7 for mutual benefit in the regulation of hypocotyl elongation.

Functional dissection of the ARGONAUTE7 promoter.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis thaliana has 10 ARGONAUTE (AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1,AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggered TAS3 pathway functions in timing and polarity.

Multi-level Modulation of Light Signaling by GIGANTEA Regulates Both the Output and Pace of the Circadian Clock.

Integration of environmental signals with endogenous biological processes is essential for organisms to thrive in their natural environment. Being entrained by periodic environmental changes, the circadian clock incorporates external information to coordinate physiological processes, phasing them to the optimal time of the day and year. Here, we present a pivotal role for the clock component GIGANTEA (GI) as a genome-wide regulator of transcriptional networks mediating growth and adaptive processes in plants. We provide mechanistic details on how GI integrates endogenous timing with light signaling pathways through the global modulation of PHYTOCHROME-INTERACTING FACTORs (PIFs). Gating of the activity of these transcriptional regulators by GI directly affects a wide array of output rhythms, including photoperiodic growth. Furthermore, we uncover a role for PIFs in mediating light input to the circadian oscillator and show how their regulation by GI is required to set the pace of the clock in response to light-dark cycles.

High-Throughput Yeast One-Hybrid Screens Using a Cell Surface gLUC Reporter.

Gene-centered yeast one-hybrid (Y1H) screens using arrayed genome-wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF-promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase-based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene-centered Y1H screens. Here, we described the procedure to perform high-throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

Interaction and Regulation Between Lipid Mediator Phosphatidic Acid and Circadian Clock Regulators.

Circadian clocks play important roles in regulating cellular metabolism, but the reciprocal effect that metabolism has on the clock is largely unknown in plants. Here, we show that the central glycerolipid metabolite and lipid mediator phosphatidic acid (PA) interacts with and modulates the function of the core clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) in Arabidopsis (Arabidopsis thaliana). PA reduced the ability of LHY and CCA1 to bind the promoter of their target gene TIMING OF CAB EXPRESSION1 Increased PA accumulation and inhibition of PA-producing enzymes had opposite effects on circadian clock outputs. Diurnal change in levels of several membrane phospholipid species, including PA, observed in wild type was lost in the LHY and CCA1 double knockout mutant. Storage lipid accumulation was also affected in the clock mutants. These results indicate that the interaction of PA with the clock regulator may function as a cellular conduit to integrate the circadian clock with lipid metabolism.

The 6xABRE Synthetic Promoter Enables the Spatiotemporal Analysis of ABA-Mediated Transcriptional Regulation.

The water stress-associated hormone abscisic acid (ABA) acts through a well-defined signal transduction cascade to mediate downstream transcriptional events important for acclimation to stress. Although ABA signaling is known to function in specific tissues to regulate root growth, little is understood regarding the spatial pattern of ABA-mediated transcriptional regulation. Here, we describe the construction and evaluation of an ABSCISIC ACID RESPONSIVE ELEMENT (ABRE)-based synthetic promoter reporter that reveals the transcriptional response of tissues to different levels of exogenous ABA and stresses. Genome-scale yeast one-hybrid screens complemented these approaches and revealed how promoter sequence and architecture affect the recruitment of diverse transcription factors (TFs) to the ABRE. Our analysis also revealed ABA-independent activity of the ABRE-reporter under nonstress conditions, with expression being enriched at the quiescent center and stem cell niche. We show that the WUSCHEL RELATED HOMEOBOX5 and NAC DOMAIN PROTEIN13 TFs regulate QC/SCN expression of the ABRE reporter, which highlights the convergence of developmental and DNA-damage signaling pathways onto this cis-element in the absence of water stress. This work establishes a tool to study the spatial pattern of ABA-mediated transcriptional regulation and a repertoire of TF-ABRE interactions that contribute to the developmental and environmental control of gene expression in roots.

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.

Decoys Untangle Complicated Redundancy and Reveal Targets of Circadian Clock F-Box Proteins.

Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.

ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis.

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light-dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.

A Localized Pseudomonas syringae Infection Triggers Systemic Clock Responses in Arabidopsis.

The circadian clock drives daily rhythms of many plant physiological responses, providing a competitive advantage that improves plant fitness and survival rates [1-5]. Whereas multiple environmental cues are predicted to regulate the plant clock function, most studies focused on understanding the effects of light and temperature [5-8]. Increasing evidence indicates a significant role of plant-pathogen interactions on clock regulation [9, 10], but the underlying mechanisms remain elusive. In Arabidopsis, the clock function largely relies on a transcriptional feedback loop between morning (CCA1 and LHY)- and evening (TOC1)-expressed transcription factors [6-8]. Here, we focused on these core components to investigate the Arabidopsis clock regulation using a unique biotic stress approach. We found that a single-leaf Pseudomonas syringae infection systemically lengthened the period and reduced the amplitude of circadian rhythms in distal uninfected tissues. Remarkably, the low-amplitude phenotype observed upon infection was recapitulated by a transient treatment with the defense-related phytohormone salicylic acid (SA), which also triggered a significant clock phase delay. Strikingly, despite SA-modulated circadian rhythms, we revealed that the master regulator of SA signaling, NPR1 [11, 12], antagonized clock responses triggered by both SA treatment and P. syringae. In contrast, we uncovered that the NADPH oxidase RBOHD [13] largely mediated the aforementioned clock responses after either SA treatment or the bacterial infection. Altogether, we demonstrated novel and unexpected roles for SA, NPR1, and redox signaling in clock regulation by P. syringae and revealed a previously unrecognized layer of systemic clock regulation by locally perceived environmental cues.

Novel cell surface luciferase reporter for high-throughput yeast one-hybrid screens.

Gene-centered yeast one-hybrid (Y1H) screens provide a powerful and effective strategy to identify transcription factor (TF)-promoter interactions. While genome-wide TF ORFeome clone collections are increasingly available, screening protocols have limitations inherent to the properties of the enzymatic reaction used to identify interactions and to the procedure required to perform the assay in a high-throughput format. Here, we present the development and validation of a streamlined strategy for quantitative and fully automated gene-centered Y1H screens using a novel cell surface Gaussia luciferase reporter.

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis.

Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis.

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

Plant Stress Tolerance Requires Auxin-Sensitive Aux/IAA Transcriptional Repressors.

The Aux/IAA proteins are auxin-sensitive repressors that mediate diverse physiological and developmental processes in plants [1, 2]. There are 29 Aux/IAA genes in Arabidopsis that exhibit unique but partially overlapping patterns of expression [3]. Although some studies have suggested that individual Aux/IAA genes have specialized function, genetic analyses of the family have been limited by the scarcity of loss-of-function phenotypes [4]. Furthermore, with a few exceptions, our knowledge of the factors that regulate Aux/IAA expression is limited [1, 5]. We hypothesize that transcriptional control of Aux/IAA genes plays a central role in the establishment of the auxin-signaling pathways that regulate organogenesis, growth, and environmental response. Here, we describe a screen for transcription factors (TFs) that regulate the Aux/IAA genes. We identify TFs from 38 families, including 26 members of the DREB/CBF family. Several DREB/CBF TFs directly promote transcription of the IAA5 and IAA19 genes in response to abiotic stress. Recessive mutations in these IAA genes result in decreased tolerance to stress conditions, demonstrating a role for auxin in abiotic stress. Our results demonstrate that stress pathways interact with the auxin gene regulatory network (GRN) through transcription of the Aux/IAA genes. We propose that the Aux/IAA genes function as hubs that integrate genetic and environmental information to achieve the appropriate developmental or physiological outcome.

Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome.

Genetic and molecular approaches revealed that the circadian clock network structure is comprised of several interlocked positive and negative transcriptional feedback loops. The network evolved to sense and integrate inputs from environmental cues to adjust daily rhythms in physiological processes. Compiling evidence indicates that part of this regulation happens at the transcriptional level through subtle adjustments in the expression of core clock genes. Thus, to better understand the network and identify the molecular mechanisms of clock input pathways, it is imperative to determine how core clock genes are regulated. For this purpose we developed reagents for an unbiased approach to identify transcription factors (TFs) interacting with the promoters of core clock genes. At the center of this approach lies the yeast one-hybrid (Y1H) assay in which a pool of proteins fused to the GAL4 transcriptional activation domain are tested for their ability to interact with a selected promoter fragment in yeast cells. Taking advantage of the fact that Arabidopsis TF genes are well annotated, we generated a comprehensive TF clone collection (TF ORFeome) and used it to replace the standard cDNA pool strategy traditionally used in Y1H screens. The use of this TF clone collection substantially accelerates the comprehensive discovery of promoter-specific DNA binding activities among all Arabidopsis TFs. Considering that this strategy can be extended to the study of the promoter interactome of any Arabidopsis gene, we developed a low throughput protocol that can be universally implemented to screen the ~2000 TF clone library.