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In 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.

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 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. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates. 

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 theArabidopsis thalianaLeaf 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 vitroandin vivo. Phenotypic analyses oflfr‐2mutant 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 ofAGAMOUS(AG), a C‐class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on theAGlocus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop atAGlocus is negatively correlated with theAGexpression level, and LFR‐SYD was functional to demolish theAGchromatin 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.

 

Abstract 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. 

Abstract 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. 

Abstract This year marks the 100 th anniversary of the experiments by Garner and Allard (Garner and Allard, 1920) 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 PEBP proteins in diverse plant species; here we will primarily highlight recent advances that enhance our understanding of the mechanism of action of PEBP proteins and discuss critical open questions.