Inflorescences of flowering plants adopt diverse genetically programmed and environmentally tuned architectures. By contrast, continued maintenance of the stem cell pool within the apical meristem is unresponsive to environmental cues. Through a combination of modeling and experimentation in Arabidopsis, we reveal a negative feedback loop that buffers environmental signals. This loop comprises the determinacy-promoting pioneer transcription factor LEAFY (LFY) and the indeterminacy-promoting transcriptional corepressor TERMINAL FLOWER1 (TFL1). At the transition to the flower-producing reproductive phase, LFY directly and quantitatively up-regulates expression of TFL1. TFL1 in turn negatively feeds back on LFY to prevent LFY overaccumulation. This blocks inflorescence termination even under strong florally inductive signals. Our work uncovers a mechanism for robust environmental buffering involving differential responses of two cell populations to the same environmental stimulus.

Adult mammalian stem cells typically maintain stem cell identity through proliferative quiescence. In contrast, we demonstrate that stem cell maintenance in Arabidopsis bud precursor cells requires active cell-cycle progression. Inhibiting division silences the shoot meristem marker gene SHOOT MERISTEMLESS ( STM ) and promotes differentiation. Whereas proliferation dilutes H3K27me3 levels to counteract silencing. Meanwhile, we identified two classes of transcription factors recruiting polycomb repressive complex 2 (PRC2) to epigenetically silence STM . The balance between these forces establishes a cell cycle-coupled epigenetic “Sisyphus” mechanism that maintains pluripotency. This cell fate switch is bistable; modeling and experimental data confirm that prolonged quiescence triggers irreversible differentiation. We propose that sequence-dependent PRC2 recruitment in plants enables precise silencing of fate-determining genes, while cell proliferation sustains pluripotency by resetting epigenetic marks. ### Competing Interest Statement The authors have declared no competing interest.

Cis-regulatory elements (CREs) are essential for regulating gene expression, yet their evolutionary dynamics in plants remain elusive. Here we constructed a single-cell chromatin accessibility atlas for Oryza sativa from 103,911 nuclei representing 126 cell states across nine organs. Comparative genomics between O. sativa and 57,552 nuclei from four additional grass species (Zea mays, Sorghum bicolor, Panicum miliaceum and Urochloa fusca) revealed that chromatin accessibility conservation varies with cell-type specificity. Epidermal accessible chromatin regions in the leaf were less conserved compared to other cell types, indicating accelerated regulatory evolution in the L1-derived epidermal layer of O. sativa relative to other species. Conserved accessible chromatin regions overlapping the repressive histone modification H3K27me3 were identified as potentially silencer-like CREs, as deleting these regions led to up-regulation of gene expression. This study provides a comprehensive epigenomic resource for the rice community, demonstrating the utility of a comparative genomics approach that highlights the dynamics of plant cell-type-specific CRE evolution.

The nectaries of flowers in angiosperms are typically located at the base of the stamens and secrete nectar, composed primarily of sugars and various secondary metabolites, either actively or passively. Although nectaries develop in specific locations and perform specialized functions, the regulatory mechanisms governing their formation are unclear. Through a pseudotime trajectory analysis based on single-nucleus RNA-seq data and newly performed spatial transcriptomics, we uncover a spatiotemporal regulatory network governing nectary development. We establish CRABS CLAW (CRC) as a master regulator that integrates temporal transcriptional regulation with spatial auxin transport, orchestrating nectary development through phytohormone signaling, sugar transport and terpene biosynthesis pathways. CRC interacts to MYB21, MYB24 and MYB57, with the CRC–MYB57 complex binding the 5′ and 3′ regulatory regions of the MACCHI-BOU 4 (MAB4) locus, facilitating the formation of a chromatin loop that enhances MAB4 expression. This loop is necessary for the development of properly sized nectaries. MAB4 reinforces PIN-FORMED 1 (PIN1)-mediated auxin transport to establish auxin maxima at the nectary tip and regulates PIN6 to maintain auxin gradients. These findings reveal how CRC integrates transcriptional regulation and auxin transport to coordinate nectary development, linking metabolic output with organogenesis.

Crucial to plant development, ambient temperature triggers intricate mechanisms enabling adaptive responses to temperature variations. The precise coordination of chromatin modifications in shaping cell developmental fate under diverse temperatures remains elusive. Our study, integrating comprehensive transcriptome, epigenome profiling, and genetics, demonstrates that lower ambient temperature (16°C) partially restores developmental defects caused by H3K27me3 loss in prc2 mutants by specifically depositing H2A.Zub at ectopically expressed embryonic genes in Arabidopsis, such as ABA INSENSITIVE 3 (ABI3) and LEAFY COTYLEDON 1 (LEC1). This deposition leads to downregulation of these genes and compensates for H3K27me3 depletion. Polycomb-repressive complex 1 (PRC1)-catalyzed H2A.Zub and PRC2-catalyzed H3K27me3 play roles in silencing transcription of embryonic genes for post-germination development. Low-temperature-induced reduction of TOE1 protein level decelerates H2A.Z turnover at specific loci, sustaining repression of embryonic genes and alleviating requirement for PRC2-H3K27me3 at post-germination stage. Our findings offer mechanistic insights into the cooperative epigenetic layers, facilitating plant adaptation to varying environmental temperatures.

Crucial to plant development, ambient temperature triggers intricate mechanisms enabling adaptive responses to temperature variations. The precise coordination of chromatin modifications in shaping cell fate under diverse temperatures remains elusive. Our study, integrating comprehensive transcriptome, epigenome profiling and genetics, unveils that lower ambient temperature (16°C) restores developmental defects caused by H3K27me3 loss in PRC2 mutants by specifically depositing H2A.Zub at ectopically expressed embryonic genes, such as ABI3 and LEC1 . This deposition leads to re-silencing of these genes and compensates for H3K27me3 depletion. PRC1-mediated H2A.Zub and PRC2-catalyzed H3K27me3 play roles in silencing transcription of these embryonic genes for post-germination development. Low temperature decelerates H2A.Z turnover at specific loci likely by attenuating the interaction between TOE1 and H2A.Z chaperone, sustaining repression of embryonic genes and alleviating requirement for PRC2-H3K27me3 at post-germination stage. Our findings offer mechanistic insights into the cooperative epigenetic layers facilitating plants adaptation to varying environmental temperatures. ### Competing Interest Statement The authors have declared no competing interest.

Cis-regulatory elements (CREs) are critical in regulating gene expression, and yet understanding of CRE evolution remains challenging. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa, integrating data from 103,911 nuclei representing 126 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species (Zea mays, Sorghum bicolor, Panicum miliaceum, and Urochloa fusca). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1-derived epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially silencer-like CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of plant cell-type-specific CRE evolution.

BackgroundIn the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported.ResultsWe adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol.ConclusionsThe whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.

AbstractIn the past few years, there has been an explosion in single-cell transcriptomics datasets, yetin vivoconfirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescencein situhybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported. We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol. The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are evolutionarily conserved, multi-subunit machinery that play vital roles in the regulation of gene expression by controlling nucleosome positioning and occupancy. However, little is known about the subunit composition of SPLAYED (SYD)-containing SWI/SNF complexes in plants. Here, we show that the Arabidopsis thaliana Leaf and Flower Related (LFR) is a subunit of SYD-containing SWI/SNF complexes. LFR interacts directly with multiple SWI/SNF subunits, including the catalytic ATPase subunit SYD, in vitro and in vivo. Phenotypic analyses of lfr-2 mutant flowers revealed that LFR is important for proper filament and pistil development, resembling the function of SYD. Transcriptome profiling revealed that LFR and SYD shared a subset of co-regulated genes. We further demonstrate that the LFR and SYD interdependently activate the transcription of AGAMOUS (AG), a C-class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on the AG locus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop at AG locus is negatively correlated with the AG expression level, and LFR-SYD was functional to demolish the AG chromatin loop to promote its transcription. Collectively, these results provide insight into the molecular mechanism of the Arabidopsis SYD-SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development.

SWITCH deficient SUCROSE NONFERMENTING (SWI/SNF) class chromatin remodeling complexes (CRCs) use the energy derived from ATP hydrolysis to facilitate access of proteins to the genomic DNA for transcription, replication, and DNA repair. Uniquely, SWI/SNF CRCs can both slide the histone octamer along the DNA or eject it from the DNA. Given their ability to change the chromatin status quo, SWI/SNF remodelers are critical for cell fate reprogramming with pioneer and other transcription factors, for responses to environmental challenges, and for disease prevention. Recent cryo-electron microscopy and mass spectrometry approaches have uncovered different subtypes of SWI/SNF complexes with unique properties and functions. At the same time, tethering or rapid depletion and inactivation of SWI/SNF have provided novel insight into SWI/SNF requirements for enhancer activity and into balancing chromatin compaction and accessibility in concert with Polycomb complexes. Given their importance, SWI/SNF recruitment to genomic locations by transcription factors and their biochemical activity is tightly controlled. This review focuses on recent advances in our understanding of SWI/SNF CRCs in animals and plants and discusses the multiple nuclear and biological roles of SWI/SNF CRCs and how SWI/SNF activity is altered by complex subunit composition, posttranslational modifications, and the chromatin context to support proper development and response to extrinsic cues.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is used widely to identify both factor binding to genomic DNA and chromatin modifications. Analysis of ChIP-seq data is impacted by regions of the genome which generate ultra-high artifactual signals. To remove these signals from ChIP-seq data, ENCODE developed blacklists, comprehensive sets of regions defined by low mappability and ultra-high signals for human, mouse, worm, and flies. Currently, blacklists are not available for many model and non-model species. Here we describe an alternative approach for removing false-positive peaks we called “greenscreen”. Greenscreen is facile to implement, requires few input samples, and uses analysis tools frequently employed for ChIP-seq. We show that greenscreen removes artifact signal as effectively as blacklists in Arabidopsis and human ChIP-seq datasets while covering less of the genome, dramatically improving ChIP-seq data quality. Greenscreen filtering reveals true factor binding overlap and of occupancy changes in different genetic backgrounds or tissues. Because it is effective with as few as three inputs, greenscreen is readily adaptable for use in any species or genome build. Although developed for ChIP-seq, greenscreen also identifies artifact signals from other genomic datasets including CUT&RUN. Finally, we present an improved ChIP-seq pipeline which incorporates greenscreen, that detects more true peaks than published methods. One Sentence Summary A facile method for removing artifact signal from ChIP-seq that improves downstream analyses ### Competing Interest Statement The authors have declared no competing interest.

Dear Editor, In multicellular eukaryotes,Polycomb repression heritably si-lences gene expression programs not needed or detrimental for a given developmental stage or tissue(Schuettengruber et al.,2017).During cell fate reprogramming,Polycomb silencing can be overcome by the combined activity of multiple Trithorax group(TrxG)proteins(Wu et al.,2012;Liang et al.,2015;Schuettengruber et al.,2017).TrxG proteins are genetically defined as suppressors of homeotic defects caused by loss of Polycomb function and have diverse enzymatic activities(Schuettengruber et al.,2017).We used a genetic enhancer screen to identify candidate TrxG proteins and uncovered TrxG activity for components of the SWR1 chromatin remodeling complex,which deposits the histone variant H2A.Z(Deal et al.,2007;March-Diaz et al.,2008).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to identify factor binding to genomic DNA and chromatin modifications. ChIP-seq data analysis is affected by genomic regions that generate ultra-high artifactual signals. To remove these signals from ChIP-seq data, the Encyclopedia of DNA Elements (ENCODE) project developed comprehensive sets of regions defined by low mappability and ultra-high signals called blacklists for human, mouse (Mus musculus), nematode (Caenorhabditis elegans), and fruit fly (Drosophila melanogaster). However, blacklists are not currently available for many model and nonmodel species. Here, we describe an alternative approach for removing false-positive peaks called greenscreen. Greenscreen is easy to implement, requires few input samples, and uses analysis tools frequently employed for ChIP-seq. Greenscreen removes artifactual signals as effectively as blacklists in Arabidopsis thaliana and human ChIP-seq dataset while covering less of the genome and dramatically improves ChIP-seq peak calling and downstream analyses. Greenscreen filtering reveals true factor binding overlap and occupancy changes in different genetic backgrounds or tissues. Because it is effective with as few as two inputs, greenscreen is readily adaptable for use in any species or genome build. Although developed for ChIP-seq, greenscreen also identifies artifactual signals from other genomic datasets including Cleavage Under Targets and Release Using Nuclease. We present an improved ChIP-seq pipeline incorporating greenscreen that detects more true peaks than other methods.

Transcriptional regulation underlies many of the growth and developmental processes that shape plants as well as their adaptation to their environment. Key to transcriptional control are transcription factors, DNA-binding proteins that serve two essential functions: to find the appropriate DNA contact sites in their target genes; and to recruit other proteins to execute transcriptional transactions. In recent years, protein structural, genomic, bioinformatic, and proteomic analyses have led to new insights into how these central functions are regulated. Here, we review new findings relating to plant transcription factor function and to their role in shaping transcription in the context of chromatin.

Master transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet little is known about pioneer transcription factors in this kingdom. Here, we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1. Upon binding, LFY 'unlocks' chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between LFY and animal pioneer transcription factor.

This year marks the 100th anniversary of the experiments by Garner and Allard that showed that plants measure the duration of the night and day (the photoperiod) to time flowering. This discovery led to the identification of Flowering Locus T (FT) in Arabidopsis and Heading Date 3a (Hd3a) in rice as a mobile signal that promotes flowering in tissues distal to the site of cue perception. FT/Hd3a belong to the family of phosphatidylethanolamine-binding proteins (PEBPs). Collectively, these proteins control plant developmental transitions and plant architecture. Several excellent recent reviews have focused on the roles of PEBPs in diverse plant species; here we will primarily highlight recent advances that enhance our understanding of the mechanism of action of PEBPs and discuss critical open questions.

Polycomb repressive complex (PRC)-mediated gene silencing is vital for cell identity and development in both the plant and the animal kingdoms. It also modulates responses to stress. Two major protein complexes, PRC1 and PRC2, execute conserved nuclear functions in metazoans and plants through covalent modification of histones and by compacting chromatin. While a general requirement for Polycomb complexes in mitotically heritable gene repression in the context of chromatin is well established, recent studies have brought new insights into the regulation of Polycomb complex activity and recruitment. Here, we discuss these recent advances with emphasis on PRC2.

Plants monitor seasonal cues to optimize reproductive success by tuning onset of reproduction and inflorescence architecture. TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) and their orthologs antagonistically regulate these life history traits, yet their mechanism of action, antagonism and targets remain poorly understood. Here, we show that TFL1 is recruited to thousands of loci by the bZIP transcription factor FD. We identify the master regulator of floral fate, LEAFY (LFY) as a target under dual opposite regulation by TFL1 and FT and uncover a pivotal role of FT in promoting flower fate via LFY upregulation. We provide evidence that the antagonism between FT and TFL1 relies on competition for chromatin-bound FD at shared target loci. Direct TFL1-FD regulated target genes identify this complex as a hub for repressing both master regulators of reproductive development and endogenous signalling pathways. Our data provide mechanistic insight into how TFL1-FD sculpt inflorescence architecture, a trait important for reproductive success, plant architecture and yield.