Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

RuBisCO Depletion and Subcellular Fractionation for Enhanced Florigen Detection in Arabidopsis.

In plants, complex signaling networks monitor and respond to environmental cues to determine the optimal time for the transition from the vegetative to reproductive phase. Understanding these networks requires robust tools to examine the levels and subcellular localization of key factors. The florigen FLOWERING LOCUS T (FT) is a crucial regulator of flowering time and occurs in soluble and membrane-bound forms. At low ambient temperatures, the ratio of these forms of FT undergoes a significant shift, which leads to a delay in the onset of flowering. To investigate these changes in FT localization, epitope-tagged FT protein can be isolated from plants by subcellular fractionation and its localization examined by immunoblot analysis of the resulting fractions. However, the highly abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) can interfere with methods to detect and characterize low-abundance proteins such as FT. In this chapter, we present a method for analyzing the ratio of HA-tagged FT (HA:FT) in different subcellular fractions while mitigating the interference from RuBisCO by using protamine sulfate (PS) to deplete RuBisCO during protein purification, thereby enhancing HA:FT detection in fractionated samples.

Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

The CUL3A-LFH1-UBC15 ubiquitin ligase complex mediates SHORT VEGETATIVE PHASE degradation to accelerate flowering at high ambient temperature.

Ambient temperature affects flowering time in plants, and the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a crucial role in the response to changes in ambient temperature. SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature, but the details of SVP degradation are unclear. Here, we show that SVP degradation at high ambient temperature is mediated by the CULLIN3-RING E3 ubiquitin ligase (CRL3) complex in Arabidopsis thaliana. We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain. We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1 (LFH1). Single mutants of LFH1 or CULLIN3A (CUL3A) showed late flowering specifically at 27°C. LFH1 protein levels increased at high ambient temperature. We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP-CUL3A complex formation. Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature, suggesting that the CUL3-LFH1 complex functions in SVP degradation. Screening E2 ubiquitin-conjugating enzymes (UBCs) using RING-BOX PROTEIN 1 (RBX1), a component of the CRL3 complex, as bait identified UBC15. ubc15 mutants also showed late flowering at high ambient temperature. In vitro and in vivo ubiquitination assays using recombinant CUL3A, LFH1, RBX1, and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner. Collectively, these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A, LFH1, and UBC15.

Immaturity of immune cells around the dural venous sinuses contributes to viral meningoencephalitis in neonates.

High neonatal susceptibility to meningitis has been attributed to the anatomical barriers that act to protect the central nervous system (CNS) from infection being immature and not fully developed. However, the mechanisms by which pathogens breach CNS barriers are poorly understood. Using the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) to study virus propagation into the CNS during systemic infection, we demonstrate that mortality in neonatal, but not adult, mice is high after infection. Virus propagated extensively from the perivenous sinus region of the dura mater to the leptomeninges, choroid plexus, and cerebral cortex. Although the structural barrier of CNS border tissues is comparable between neonates and adults, immunofluorescence staining and single-cell RNA sequencing analyses revealed that the neonatal dural immune cells are immature and predominantly composed of CD206hi macrophages, with major histocompatibility complex class II (MHCII)hi macrophages being rare. In adults, however, perivenous sinus immune cells were enriched in MHCIIhi macrophages that are specialized for producing antiviral molecules and chemokines compared with CD206hi macrophages and protected the CNS against systemic virus invasion. Our findings clarify how systemic pathogens enter the CNS through its border tissues and how the immune barrier at the perivenous sinus region of the dura blocks pathogen access to the CNS.

Spike Optimization to Improve Properties of Ferroelectric Tunnel Junction Synaptic Devices for Neuromorphic Computing System Applications.

The continuous advancement of Artificial Intelligence (AI) technology depends on the efficient processing of unstructured data, encompassing text, speech, and video. Traditional serial computing systems based on the von Neumann architecture, employed in information and communication technology development for decades, are not suitable for the concurrent processing of massive unstructured data tasks with relatively low-level operations. As a result, there arises a pressing need to develop novel parallel computing systems. Recently, there has been a burgeoning interest among developers in emulating the intricate operations of the human brain, which efficiently processes vast datasets with remarkable energy efficiency. This has led to the proposal of neuromorphic computing systems. Of these, Spiking Neural Networks (SNNs), designed to closely resemble the information processing mechanisms of biological neural networks, are subjects of intense research activity. Nevertheless, a comprehensive investigation into the relationship between spike shapes and Spike-Timing-Dependent Plasticity (STDP) to ensure efficient synaptic behavior remains insufficiently explored. In this study, we systematically explore various input spike types to optimize the resistive memory characteristics of Hafnium-based Ferroelectric Tunnel Junction (FTJ) devices. Among the various spike shapes investigated, the square-triangle (RT) spike exhibited good linearity and symmetry, and a wide range of weight values could be realized depending on the offset of the RT spike. These results indicate that the spike shape serves as a crucial indicator in the alteration of synaptic connections, representing the strength of the signals.

Yttrium Doping Effects on Ferroelectricity and Electric Properties of As-Deposited Hf1-xZrxO2 Thin Films via Atomic Layer Deposition.

Hf1-xZrxO2 (HZO) thin films are versatile materials suitable for advanced ferroelectric semiconductor devices. Previous studies have shown that the ferroelectricity of HZO thin films can be stabilized by doping them with group III elements at low concentrations. While doping with Y improves the ferroelectric properties, there has been limited research on Y-HZO thin films fabricated using atomic layer deposition (ALD). In this study, we investigated the effects of Y-doping cycles on the ferroelectric and electrical properties of as-deposited Y-HZO thin films with varying compositions fabricated through ALD. The Y-HZO thin films were stably crystallized without the need for post-thermal treatment and exhibited transition behavior depending on the Y-doping cycle and initial composition ratio of the HZO thin films. These Y-HZO thin films offer several advantages, including enhanced dielectric constant, leakage current density, and improved endurance. Moreover, the optimized Y-doping cycle induced a phase transformation that resulted in Y-HZO thin films with improved ferroelectric properties, exhibiting stable behavior without fatigue for up to 1010 cycles. These as-deposited Y-HZO thin films show promise for applications in semiconductor devices that require high ferroelectric properties, excellent electrical properties, and reliable performance with a low thermal budget.

Chloroplasts prevent precocious flowering through a GOLDEN2-LIKE-B-BOX DOMAIN PROTEIN module.

The timing of flowering is tightly controlled by signals that integrate environmental and endogenous cues. Sugars produced by carbon fixation in the chloroplast are a crucial endogenous cue for floral initiation. Chloroplasts also convey information directly to the nucleus through retrograde signaling to control plant growth and development. Here, we show that mutants defective in chlorophyll biosynthesis and chloroplast development flowered early, especially under long-day conditions, although low sugar accumulation was seen in some mutants. Plants treated with the bleaching herbicide norflurazon also flowered early, suggesting that chloroplasts have a role in floral repression. Among retrograde signaling mutants, the golden2-like 1 (glk1) glk2 double mutants showed early flowering under long-day conditions. This early flowering was completely suppressed by constans (co) and flowering locus t (ft) mutations. Leaf vascular-specific knockdown of both GLK1 and GLK2 phenocopied the glk1 glk2 mutants. GLK1 and GLK2 repress flowering by directly activating the expression of B-BOX DOMAIN PROTEIN 14 (BBX14), BBX15, and BBX16 via CCAATC cis-elements in the BBX genes. BBX14/15/16 physically interact with CO in the nucleus, and expression of BBXs hampered CO-mediated FT transcription. Simultaneous knockdown of BBX14/15/16 by artificial miRNA (35S::amiR-BBX14/15/16) caused early flowering with increased FT transcript levels, whereas BBX overexpression caused late flowering. Flowering of glk1/2 and 35S::amiR-BBX14/15/16 plants was insensitive to norflurazon treatment. Taking these observations together, we propose that the GLK1/2-BBX14/15/16 module provides a novel mechanism explaining how the chloroplast represses flowering to balance plant growth and reproductive development.

Corrigendum: Polymerase II-associated factor 1 complex-regulated FLOWERING LOCUS C-Clade genes repress flowering in response to chilling.

[This corrects the article DOI: 10.3389/fpls.2022.817356.].

FLOWERING LOCUS M isoforms differentially affect the subcellular localization and stability of SHORT VEGETATIVE PHASE to regulate temperature-responsive flowering in Arabidopsis.

Temperature is an important environmental cue that affects flowering time in plants. The MADS-box transcription factor FLOWERING LOCUS M (FLM) forms a heterodimeric complex with SHORT VEGETATIVE PHASE (SVP) and controls ambient temperature-responsive flowering in Arabidopsis. FLM-ß and FLM-δ, two major splice variants produced from the FLM locus, exert opposite effects on flowering, but the molecular mechanism by which the interaction between FLM isoforms and SVP affects temperature-responsive flowering remains poorly understood. Here, we show that FLM-ß and FLM-δ play important roles in modulating the temperature-dependent behavior, conformation, and stability of SVP. Nuclear localization of SVP decreases as temperature increases. FLM-ß is required for SVP nuclear translocation at low temperature, whereas SVP interacts with FLM-δ mainly in the cytoplasm at high temperature. SVP preferentially binds to FLM-ß at low temperature in tobacco leaf cells. SVP shows high binding affinity to FLM-ß at low temperature and to FLM-δ at high temperature. SVP undergoes similar structural changes in the interactions with FLM-ß and FLM-δ; however, FLM-δ likely causes more pronounced conformational changes in the SVP structure. FLM-δ causes rapid degradation of SVP at high temperature, compared with FLM-ß, possibly via ubiquitination. Mutation of lysine 53 or lysine 165 in SVP causes increased abundance of SVP due to reduced ubiquitination of SVP and thus delays flowering at high temperature. Our findings suggest that temperature-dependent differential interactions between SVP and FLM isoforms modulate the temperature-responsive induction of flowering in Arabidopsis.

In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions.

Protein-lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein-lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid-protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

Advanced Atomic Layer Deposition: Ultrathin and Continuous Metal Thin Film Growth and Work Function Control Using the Discrete Feeding Method.

The ultrathin and continuous ruthenium (Ru) film was deposited through an improved atomic layer deposition (ALD) process with a discrete feeding method (DFM), called DF-ALD, employing a cut-in purge step during the precursor feeding. The excess precursor molecules can be physically adsorbed onto the chemisorbed precursors on the substrate during precursor feeding, which screens the reactive sites on the surface. Using DF-ALD, surface coverage of precursors was enhanced because the cut-in purge removes the physisorbed precursors securing the reactive sites beneath them; thus, nucleation density was greatly increased. Therefore, the grain size decreased, which changed the microstructure and increased oxygen impurity concentration. However, a more metallic Ru thin film was formed due to thermodynamic stability and improved physical density. Consequently, DF-ALD enables the deposition of the ultrathin (3 nm) and continuous Ru film with a low resistivity of ∼60 µΩ cm and a high effective work function of ∼4.8 eV.

Polymerase II-Associated Factor 1 Complex-Regulated FLOWERING LOCUS C-Clade Genes Repress Flowering in Response to Chilling.

RNA polymerase II-associated factor 1 complex (PAF1C) regulates the transition from the vegetative to the reproductive phase primarily by modulating the expression of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M [FLM, also known as MADS AFFECTING FLOWERING1 (MAF1)] at standard growth temperatures. However, the role of PAF1C in the regulation of flowering time at chilling temperatures (i.e., cold temperatures that are above freezing) and whether PAF1C affects other FLC-clade genes (MAF2-MAF5) remains unknown. Here, we showed that Arabidopsis thaliana mutants of any of the six known genes that encode components of PAF1C [CELL DIVISION CYCLE73/PLANT HOMOLOGOUS TO PARAFIBROMIN, VERNALIZATION INDEPENDENCE2 (VIP2)/EARLY FLOWERING7 (ELF7), VIP3, VIP4, VIP5, and VIP6/ELF8] showed temperature-insensitive early flowering across a broad temperature range (10°C-27°C). Flowering of PAF1C-deficient mutants at 10°C was even earlier than that in flc, flm, and flc flm mutants, suggesting that PAF1C regulates additional factors. Indeed, RNA sequencing (RNA-Seq) of PAF1C-deficient mutants revealed downregulation of MAF2-MAF5 in addition to FLC and FLM at both 10 and 23°C. Consistent with the reduced expression of FLC and the FLC-clade members FLM/MAF1 and MAF2-MAF5, chromatin immunoprecipitation (ChIP)-quantitative PCR assays showed reduced levels of the permissive epigenetic modification H3K4me3/H3K36me3 and increased levels of the repressive modification H3K27me3 at their chromatin. Knocking down MAF2-MAF5 using artificial microRNAs (amiRNAs) in the flc flm background (35S::amiR-MAF2-5 flc flm) resulted in significantly earlier flowering than flc flm mutants and even earlier than short vegetative phase (svp) mutants at 10°C. Wild-type seedlings showed higher accumulation of FLC and FLC-clade gene transcripts at 10°C compared to 23°C. Our yeast two-hybrid assays and in vivo co-immunoprecipitation (Co-IP) analyses revealed that MAF2-MAF5 directly interact with the prominent floral repressor SVP. Late flowering caused by SVP overexpression was almost completely suppressed by the elf7 and vip4 mutations, suggesting that SVP-mediated floral repression required a functional PAF1C. Taken together, our results showed that PAF1C regulates the transcription of FLC and FLC-clade genes to modulate temperature-responsive flowering at a broad range of temperatures and that the interaction between SVP and these FLC-clade proteins is important for floral repression.

cis-prenyltransferase 3 and α/ß-hydrolase are new determinants of dolichol accumulation in Arabidopsis.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for post-translational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis-the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/ß-hydrolase-is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Florigen sequestration in cellular membranes modulates temperature-responsive flowering.

Plants respond to temperature changes by modulating florigen activity to optimize the timing of flowering. We show that the Arabidopsis thaliana mobile florigen FLOWERING LOCUS T (FT) interacts with the negatively charged phospholipid phosphatidylglycerol (PG) at cellular membranes and binds the lipid bilayer. Perturbing PG biosynthesis in phloem companion cells leads to temperature-insensitive early flowering. Low temperatures facilitate FT sequestration in the cellular membrane of the companion cell, thus reducing soluble FT levels and delaying flowering. A mutant in PHOSPHATIDYLGLYCEROLPHOSPHATE SYNTHASE 1 accumulates more soluble FT at lower temperatures and exhibits reduced temperature sensitivity. Thus, cellular membranes sequester FT through their ability to bind the phospholipid PG, and this sequestration modulates the plant's response to temperature changes.

Nonsense-mediated mRNA decay modulates Arabidopsis flowering time via the SET DOMAIN GROUP 40-FLOWERING LOCUS C module.

The nonsense-mediated mRNA decay (NMD) surveillance system clears aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. Although loss of the core NMD proteins UP-FRAMESHIFT1 (UPF1) and UPF3 leads to late flowering in Arabidopsis, the underlying mechanism remains elusive. Here, we showed that mutations in UPF1 and UPF3 cause temperature- and photoperiod-independent late flowering. Expression analyses revealed high FLOWERING LOCUS C (FLC) mRNA levels in upf mutants; in agreement with this, the flc mutation strongly suppressed the late flowering of upf mutants. Vernalization accelerated flowering of upf mutants in a temperature-independent manner. FLC transcript levels rose in wild-type plants upon NMD inhibition. In upf mutants, we observed increased enrichment of H3K4me3 and reduced enrichment of H3K27me3 in FLC chromatin. Transcriptome analyses showed that SET DOMAIN GROUP 40 (SDG40) mRNA levels increased in upf mutants, and the SDG40 transcript underwent NMD-coupled alternative splicing, suggesting that SDG40 affects flowering time in upf mutants. Furthermore, NMD directly regulated SDG40 transcript stability. The sdg40 mutants showed decreased H3K4me3 and increased H3K27me3 levels in FLC chromatin, flowered early, and rescued the late flowering of upf mutants. Taken together, these results suggest that NMD epigenetically regulates FLC through SDG40 to modulate flowering time in Arabidopsis.

Nasal ciliated cells are primary targets for SARS-CoV-2 replication in the early stage of COVID-19.

The upper respiratory tract is compromised in the early period of COVID-19, but SARS-CoV-2 tropism at the cellular level is not fully defined. Unlike recent single-cell RNA-Seq analyses indicating uniformly low mRNA expression of SARS-CoV-2 entry-related host molecules in all nasal epithelial cells, we show that the protein levels are relatively high and that their localizations are restricted to the apical side of multiciliated epithelial cells. In addition, we provide evidence in patients with COVID-19 that SARS-CoV-2 is massively detected and replicated within the multiciliated cells. We observed these findings during the early stage of COVID-19, when infected ciliated cells were rapidly replaced by differentiating precursor cells. Moreover, our analyses revealed that SARS-CoV-2 cellular tropism was restricted to the nasal ciliated versus oral squamous epithelium. These results imply that targeting ciliated cells of the nasal epithelium during the early stage of COVID-19 could be an ideal strategy to prevent SARS-CoV-2 propagation.

Superior and stable ferroelectric properties of hafnium-zirconium-oxide thin films deposited via atomic layer deposition using cyclopentadienyl-based precursors without annealing.

HfO2-based ferroelectric thin films deposited via atomic layer deposition have been extensively studied as promising candidates for next-generation ferroelectric devices. The conversion of an amorphous Hf1-xZrxO2 film to the ferroelectric phase (non-centrosymmetric orthorhombic phase) has been achieved through annealing using a post-thermal process. However, in this study, we present the first report of ferroelectricity of hafnium-zirconium-oxide (HZO) thin films deposited via atomic layer deposition using cyclopentadienyl-based precursors without additional post-thermal processing. By increasing the deposition temperature using a cyclopentadienyl-based cocktail precursor, the conditions of the as-deposited HZO thin film to crystallize well with an orthorhombic phase were secured, and excellent ferroelectric properties with a large remanent polarization (2Pr ∼ 47.6 µC cm-2) were implemented without crystallization annealing. The as-deposited HZO thin film possessed very stable ferroelectric properties without a wake-up effect or significant fatigue up to 106 cycles. Futhermore, we demonstrated the applicability to devices using negative capacitance and non-volatile memory characteristics. This result suggests that a new strategy can be applied to ferroelectric devices where subsequent processing temperature constraints are required, such as back-end-of-line processes and ferroelectric-based flexible device applications.

Regulation of flowering time by ambient temperature: repressing the repressors and activating the activators.

Plants display remarkable developmental flexibility as they continuously sense and respond to changes in their environment. This flexibility allows them to select the optimal timing for critical developmental decisions such as when to flower. Ambient temperature is a major environmental factor that influences flowering; the mechanisms involved in ambient temperature-responsive flowering have attracted particular attention as a consequence of the effects of global climate change on temperature. PHYTOCHROME INTERACTING FACTOR 4 and alternative splicing of FLOWERING LOCUS M affect temperature-responsive flowering; however, the exact temperature-sensing mechanism in plants remains elusive. Further study of these molecular mechanisms will contribute to our understanding of how plants sense ambient temperature and respond via diverse biological signaling cascades.

Profiling Protein-DNA Interactions by Chromatin Immunoprecipitation in Arabidopsis.

In plant cells, transcription factors play an important role in the regulation of gene expression, which eventually leads to the formation of complex phenotypes. Although chromatin immunoprecipitation (ChIP) involves a lengthy process that requires up to 4 days to complete, it is a powerful technique to investigate the interactions between transcription factors and their target sequences in vivo. Here, we describe a detailed ChIP protocol, focusing on ChIP-qPCR, from material collection to data analyses. Moreover, we explain multiple checkpoints for the quality control of ChIP-qPCR data to ensure the success of this protocol. As this protocol is robust, it can be adapted to other plant materials and plant species, and it can be used for genome-wide profiling experiments, including ChIP-chip and ChIP-seq analyses. We believe that our ChIP-qPCR protocol facilitates research on the interactions between plant transcription factors and their target sequences in vivo.