Analysis of Chromatin Accessibility, Histone Modifications, and Transcriptional States in Specific Cell Types Using Flow Cytometry.

The past two decades in biomedical research have experienced an explosion of cell type-specific and single-cell studies, especially concerning the concomitant dissection of regulatory and transcriptional landscapes of those under investigation. Additionally, leveraging next-generation sequencing (NGS) platforms efforts have been undertaken to evaluate the effects of chromatin accessibility, histone modifications, or even transcription factor binding sites. We have shown that Fluorescence-Activated Nuclear Sorting (FANS) is an effective means to characterize the transcriptomes of nuclei from different tissues. In light of our own technical and experimental developments, we extend this effort to combine FACS/FANS with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) for profiling individual cell types according to their chromatin and transcriptional states.

Two-step regulation of centromere distribution by condensin II and the nuclear envelope proteins.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.

The integral spliceosomal component CWC15 is required for development in Arabidopsis.

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

Transcriptomic Profiling of the Arabidopsis Embryonic Epidermis Using FANS in Combination with RNAseq.

The fundamental mechanisms of cell identity and tissue establishment are important already from the very beginning of a plant's life and reiterate later during development. In order to unravel and understand the underlying mechanisms to generate differences that in turn lead to cell or tissue types, plant cells have to be separated and their transcriptional setup analyzed. We have previously demonstrated that fluorescence-activated nuclear sorting (FANS) is a powerful tool to generate nuclear transcriptomic profiles of the most inaccessible embryonic tissues. In this protocol, we extend this effort to combine FANS with next generation RNA sequencing (RNA-seq) to achieve early embryonic transcriptomes of Arabidopsis epidermis precursor tissue (protoderm) and the inner tissue counterpart.

Constitutive signaling activity of a receptor-associated protein links fertilization with embryonic patterning in Arabidopsis thaliana.

In flowering plants, the asymmetrical division of the zygote is the first hallmark of apical-basal polarity of the embryo and is controlled by a MAP kinase pathway that includes the MAPKKK YODA (YDA). In Arabidopsis, YDA is activated by the membrane-associated pseudokinase SHORT SUSPENSOR (SSP) through an unusual parent-of-origin effect: SSP transcripts accumulate specifically in sperm cells but are translationally silent. Only after fertilization is SSP protein transiently produced in the zygote, presumably from paternally inherited transcripts. SSP is a recently diverged, Brassicaceae-specific member of the BRASSINOSTEROID SIGNALING KINASE (BSK) family. BSK proteins typically play broadly overlapping roles as receptor-associated signaling partners in various receptor kinase pathways involved in growth and innate immunity. This raises two questions: How did a protein with generic function involved in signal relay acquire the property of a signal-like patterning cue, and how is the early patterning process activated in plants outside the Brassicaceae family, where SSP orthologs are absent? Here, we show that Arabidopsis BSK1 and BSK2, two close paralogs of SSP that are conserved in flowering plants, are involved in several YDA-dependent signaling events, including embryogenesis. However, the contribution of SSP to YDA activation in the early embryo does not overlap with the contributions of BSK1 and BSK2. The loss of an intramolecular regulatory interaction enables SSP to constitutively activate the YDA signaling pathway, and thus initiates apical-basal patterning as soon as SSP protein is translated after fertilization and without the necessity of invoking canonical receptor activation.

ARR22 overexpression can suppress plant Two-Component Regulatory Systems.

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

Staining and Clearing of Arabidopsis Reproductive Tissue for Imaging of Fluorescent Proteins.

Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins. 2,2'-thiodiethanol (TDE) has recently been introduced as a clearing agent that allows the imaging of fluorescent proteins in a cleared plant tissue. Here, we describe a simple protocol that combines TDE-based tissue clearing with cell wall staining to outline cells that enable deep tissue imaging in reproductive structures of Arabidopsis thaliana.

Evolutionarily diverse SYP1 Qa-SNAREs jointly sustain pollen tube growth in Arabidopsis.

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans-SNARE complexes. Qa-SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen-specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen-specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa-SNARE proteins to sustain their growth-promoting membrane dynamics during the reproductive process.

Cell Type-Specific Gene Expression Profiling Using Fluorescence-Activated Nuclear Sorting.

Fluorescence-activated cell sorting (FACS) is a powerful method for the analysis of cell type-specific transcriptome profiles, DNA or histone modifications, and chemical compounds. In plants, it has been employed mainly with root and shoot tissue in combination with cell wall digestion on cellular and nuclear content. However, many tissues are recalcitrant to cell separation and are therefore not readily accessible for FACS analysis. Here, we lay out a detailed protocol for the generation of transcriptional profiles from fluorescently labeled nuclei. The protocol described in this chapter has been used successfully to generate a transcriptional map of the early Arabidopsis thaliana embryo.

Early plant embryogenesis-dark ages or dark matter?

In nearly all flowering plants, the basic body plan is laid down during embryogenesis. In Arabidopsis, the crucial cell types are established extremely early as reflected in the stereotypic sequence of oriented cell divisions in the developing young embryo. Research into early embryogenesis was especially focused on the role of the infamous tryptophan derivative auxin in establishing embryo polarity and generating the main body axis. However, it is becoming obvious that the mere link to auxin does not provide any mechanistic understanding of early embryo patterning. Taking recent research into account, we discuss mechanisms underlying early embryonic patterning from an evolutionary perspective.

A Versatile Optical Clearing Protocol for Deep Tissue Imaging of Fluorescent Proteins in Arabidopsis thaliana.

Confocal microscopy is widely used to visualize gene expression patterns and developmental processes in plants. However, the imaging of plant tissue can be challenging due to its opacity, which often makes previous immersion in a clearing agent necessary. Many commonly-used chemicals suffer either from their incompatibility with fluorescent proteins or their complex and lengthy application. 2,2'-thiodiethanol (TDE) has recently been described as a clearing agent with an emphasis on high resolution microscopy due to its potential to adjust the refractive index. Here, we evaluate the use of TDE-based clearing for confocal as well as two-photon microscopy in various Arabidopsis thaliana tissue types. We demonstrate that tissue fixation is a mandatory prerequisite for the use of TDE, in order to preserve tissue integrity and fluorescent protein activity. TDE concentrations between 50-70% are a good compromise for imaging of technically challenging tissue to achieve good clearing without affecting fluorescent protein activity. TDE-based clearing is simple and rapid to use and allows for a flexible experimental setup while facilitating high quality imaging.

RALFL34 regulates formative cell divisions in Arabidopsis pericycle during lateral root initiation.

In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.

Profiling of embryonic nuclear vs. cellular RNA in Arabidopsis thaliana.

In Arabidopsis, various cell type-specific whole-genome expression analyses have been conducted. However, the vast majority of these were performed with cellular RNA from root tissues or other easily accessible cell types [1]. Nuclear RNA was neglected for a long time as not being representative for transcriptomic studies. In recent years, however, there have been reports describing the validity of nuclear RNA for these types of studies [2,3]. Here we describe the generation, quality assessment and analysis of nuclear transcriptomic data from Arabidopsis embryos published by Slane et al. (2014) [4]. Comparison of nuclear with cellular gene expression demonstrated the usefulness of nuclear transcriptomics.

Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo.

In multicellular organisms, cellular differences in gene activity are a prerequisite for differentiation and establishment of cell types. In order to study transcriptome profiles, specific cell types have to be isolated from a given tissue or even the whole organism. However, whole-transcriptome analysis of early embryos in flowering plants has been hampered by their size and inaccessibility. Here, we describe the purification of nuclear RNA from early stage Arabidopsis thaliana embryos using fluorescence-activated nuclear sorting (FANS) to generate expression profiles of early stages of the whole embryo, the proembryo and the suspensor. We validated our datasets of differentially expressed candidate genes by promoter-reporter gene fusions and in situ hybridization. Our study revealed that different classes of genes with respect to biological processes and molecular functions are preferentially expressed either in the proembryo or in the suspensor. This method can be used especially for tissues with a limited cell population and inaccessible tissue types. Furthermore, we provide a valuable resource for research on Arabidopsis early embryogenesis.

Early embryogenesis in flowering plants: setting up the basic body pattern.

Early embryogenesis is the critical developmental phase during which the basic features of the plant body are established: the apical-basal axis of polarity, different tissue layers, and both the root pole and the shoot pole. Polarization of the zygote correlates with the generation of apical and basal (embryonic and extraembryonic) cell fates. Whereas mechanisms of zygote polarization are still largely unknown, distinct expression domains of WOX family transcription factors as well as directional auxin transport and local auxin response are known to be involved in early apical-basal patterning. Radial patterning of tissue layers appears to be mediated by cell-cell communication involving both peptide signaling and transcription factor movement. Although the initiation of the shoot pole is still unclear, the apical organization of the embryo depends on both the proper establishment of transcription factor expression domains and, for cotyledon initiation, upward auxin flow in the protoderm. Here we focus on the essential patterning processes, drawing mainly on data from Arabidopsis thaliana and also including relevant data from other species if available.

Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis.

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion.