The Arabidopsis xylosyltransferases, XXT3, XXT4, and XXT5, are essential to complete the fully xylosylated glucan backbone XXXG-type structure of xyloglucans.

Although most xyloglucans (XyGs) biosynthesis enzymes have been identified, the molecular mechanism that defines XyG branching patterns is unclear. Four out of five XyG xylosyltransferases (XXT1, XXT2, XXT4, and XXT5) are known to add the xylosyl residue from UDP-xylose onto a glucan backbone chain; however, the function of XXT3 has yet to be demonstrated. Single xxt3 and triple xxt3xxt4xxt5 mutant Arabidopsis (Arabidopsis thaliana) plants were generated using CRISPR-Cas9 technology to determine the specific function of XXT3. Combined biochemical, bioinformatic, and morphological data conclusively established for the first time that XXT3, together with XXT4 and XXT5, adds xylosyl residue specifically at the third glucose in the glucan chain to synthesize XXXG-type XyGs. We propose that the specificity of XXT3, XXT4, and XXT5 is directed toward the prior synthesis of the acceptor substrate by the other two enzymes, XXT1 and XXT2. We also conclude that XXT5 plays a dominant role in the synthesis of XXXG-type XyGs, while XXT3 and XXT4 complementarily contribute their activities in a tissue-specific manner. The newly generated xxt3xxt4xxt5 mutant produces only XXGG-type XyGs, which further helps to understand the impact of structurally deficient polysaccharides on plant cell wall organization, growth, and development.

A dive into the cell wall with Arabidopsis.

One of the many legacies of the work of Michel Caboche is our understanding of plant cell wall synthesis and metabolism thanks to the use of Arabidopsis mutants. Here I describe how he was instrumental in initiating the genetic study of plant cell walls. I also show, with a few examples for cellulose and pectins, how this approach has led to important new insights in cell wall synthesis and how the metabolism of pectins contributes to plant growth and morphogenesis. I also illustrate the limitations of the use of mutants to explain processes at the scale of cells, organs or whole plants in terms of the physico-chemical properties of cell wall polymers. Finally, I sketch how new approaches can cope with these limitations.

L'un des nombreux héritages des travaux de Michel Caboche est notre compréhension de la synthèse et du métabolisme des parois cellulaires végétales grâce à l'utilisation de mutants d'Arabidopsis. Je décris ici comment il a joué un rôle déterminant dans le lancement de l'étude génétique des parois cellulaires végétales. Je montre également, avec quelques exemples pour la cellulose et les pectines, comment cette approche a conduit à de nouvelles connaissances importantes sur la synthèse de la paroi cellulaire et comment le métabolisme des pectines contribue à la croissance et à la morphogenèse des plantes. J'illustre également les limites de l'utilisation de mutants pour expliquer des processus à l'échelle de cellules, d'organes ou de plantes entières en termes de propriétés physico-chimiques de polymères de parois cellulaires. Enfin, j'esquisse comment de nouvelles approches peuvent faire face à ces limitations.

An automatic method to quantify trichomes in Arabidopsis thaliana.

Trichomes are unicellular or multicellular hair-like appendages developed on the aerial plant epidermis of most plant species that act as a protective barrier against natural hazards. For this reason, evaluating the density of trichomes is a valuable approach for elucidating plant defence responses to a continuous challenging environment. However, previous methods for trichome counting, although reliable, require the use of specialised equipment, software or previous manipulation steps of the plant tissue, which poses a complicated hurdle for many laboratories. Here, we propose a new fast, accessible and user-friendly method to quantify trichomes that overcomes all these drawbacks and makes trichome quantification a reachable option for the scientific community. Particularly, this new method is based on the use of machine learning as a reliable tool for quantifying trichomes, following an Ilastik-Fiji tandem approach directly performed on 2D images. Our method shows high reliability and efficacy on trichome quantification in Arabidopsis thaliana by comparing manual and automated results in Arabidopsis accessions with diverse trichome densities. Due to the plasticity that machine learning provides, this method also showed adaptability to other plant species, demonstrating the ability of the method to spread its scope to a greater scientific community.

Analysis of cellulose synthase activity in Arabidopsis using spinning disk microscopy.

We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021.

Genomic impact of stress-induced transposable element mobility in Arabidopsis.

Transposable elements (TEs) have long been known to be major contributors to plant evolution, adaptation and crop domestication. Stress-induced TE mobilization is of particular interest because it may result in novel gene regulatory pathways responding to stresses and thereby contribute to stress adaptation. Here, we investigated the genomic impacts of stress induced TE mobilization in wild type Arabidopsis plants. We find that the heat-stress responsive ONSEN TE displays an insertion site preference that is associated with specific chromatin states, especially those rich in H2A.Z histone variant and H3K27me3 histone mark. In order to better understand how novel ONSEN insertions affect the plant's response to heat stress, we carried out an in-depth transcriptomic analysis. We find that in addition to simple gene knockouts, ONSEN can produce a plethora of gene expression changes such as: constitutive activation of gene expression, alternative splicing, acquisition of heat-responsiveness, exonisation and genesis of novel non-coding and antisense RNAs. This report shows how the mobilization of a single TE-family can lead to a rapid rise of its copy number increasing the host's genome size and contribute to a broad range of transcriptomic novelty on which natural selection can then act.

Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol.

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes.

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Natural variation in autumn expression is the major adaptive determinant distinguishing Arabidopsis FLC haplotypes.

In Arabidopsis thaliana, winter is registered during vernalization through the temperature-dependent repression and epigenetic silencing of floral repressor FLOWERING LOCUS C (FLC). Natural Arabidopsis accessions show considerable variation in vernalization. However, which aspect of the FLC repression mechanism is most important for adaptation to different environments is unclear. By analysing FLC dynamics in natural variants and mutants throughout winter in three field sites, we find that autumnal FLC expression, rather than epigenetic silencing, is the major variable conferred by the distinct Arabidopsis FLChaplotypes. This variation influences flowering responses of Arabidopsis accessions resulting in an interplay between promotion and delay of flowering in different climates to balance survival and, through a post-vernalization effect, reproductive output. These data reveal how expression variation through non-coding cis variation at FLC has enabled Arabidopsis accessions to adapt to different climatic conditions and year-on-year fluctuations.

Dual-color 3D-dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei.

To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using the roxy1 mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H2 O2 treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.

In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH).

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

Regulation of ABA-Non-Activated SNF1-Related Protein Kinase 2 Signaling Pathways by Phosphatidic Acid.

Phosphatidic acid (PA) is involved in the regulation of plant growth and development, as well as responses to various environmental stimuli. Several PA targets in plant cells were identified, including two SNF1-related protein kinases 2 (SnRK2s), SnRK2.10 and SnRK2.4, which are not activated by abscisic acid (ABA). Here, we investigated the effects of PA on various elements of ABA-non-activated SnRK2 signaling. PA 16:0/18:1 was found to modulate the SnRK2 structure and the phosphorylation of some SnRK2 targets. Conversely, phosphorylation by the ABA-non-activated SnRK2s, of one of such targets, dehydrin Early Responsive to Dehydration 14 (ERD14), affects its interaction with PA and subcellular localization. Moreover, PA 16:0/18:1 modulates the activity and/or localization of negative regulators of the ABA-non-activated SnRK2s, not only of the ABA insensitive 1 (ABI1) phosphatase, which was identified earlier, but also of another protein phosphatase 2C, PP2CA. The activity of both phosphatases was inhibited by about 50% in the presence of 50 µM PA. PA 16:0/18:1 also impacts the phosphorylation and subcellular localization of SnRK2-interacting calcium sensor, known to inhibit SnRK2 activity in a calcium-dependent manner. Thus, PA was found to regulate ABA-non-activated SnRK2 signaling at several levels: the activity, phosphorylation status and/or localization of SnRK2 cellular partners.

Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings.

Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein-protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts.

Living on the edge: the role of Atgolgin-84A at the plant ER-Golgi interface.

The plant Golgi apparatus is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors to mediate the physical connections between Golgi bodies and the ER network. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled-coil domains, which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. In animal cells, golgins are also implicated in specific recognition of cargo at the Golgi.Here, we investigate the plant golgin Atgolgin-84A for its subcellular localization and potential role as a tethering factor at the ER-Golgi interface. For this, fluorescent fusions of Atgolgin-84A and an Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84AΔ1-557) were transiently expressed in tobacco leaf epidermal cells and imaged using high-resolution confocal microscopy. We show that Atgolgin-84A localizes to a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins as well as by the tether protein AtCASP. Upon overexpression of Atgolgin-84A or its deletion mutant, transport between the ER and Golgi bodies is impaired and cargo proteins are redirected to the vacuole. LAY DESCRIPTION: The Golgi apparatus is a specialised compartment found in mammalian and plant cells. It is the post office of the cell and packages proteins into small membrane boxes for transport to their destination in the cell. The plant Golgi apparatus consist of many separate Golgi bodies and is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Specialised proteins called golgins have been suggested to tether Golgi bodies and the ER. Here we investigate the plant golgin Atgolgin-84A for its exact within the Golgi body and its potential role as a tethering factor at the ER-Golgi interface. For this, we have fused Atgolgin-84A with a fluorescent protein from jellyfish and we are producing this combination in tobacco leaf cells. This allows us to see the protein using laser microscopy. We show that Atgolgin-84A localises to a compartment between the ER and Golgi that is also labelled by the tether protein AtCASP. When Atgolgin-84A is produced in high amounts in the cell, transport between the ER and Golgi bodies is inhibited and proteins are redirected to the vacuole.

The nuclear localized RIN13 induces cell death through interacting with ARF1.

Resistance to Pseudomonas syringae pv. Maculicola 1 (RPM1) is a crucial immune receptor conferring plant enhanced resistance to pathogenic bacteria. RPM1-interacting protein 13 (RIN13) enhances RPM1-mediated disease resistance through interacting with the central domain of RPM1 in Arabidopsis, while the underlying mechanism remains elusive. Here, we report the subcellular localization and function of RIN13 using the Nicotiana benthamiana (N. benthamiana) transient expression system. Our results showed that RIN13 is exclusively localized in the nucleus, and RIN13 (231-300) fragment is responsible for its nuclear localization. Transient expression of RIN13 in N. benthamiana leaves can accelerate leaf senescence and cell death, and affect the activities of ROS-scavenging enzymes, and the C-terminus of RIN13 is crucial for its function. Furthermore, we identified a RIN13-interacting protein, Auxin Response Factor 1 (ARF1), and found that similar to RIN13, ARF1 can also promote leaf senescence and cell death. In addition, expression of RIN13 in N. benthamiana leaves can facilitate the translocation of ARF1 into the nucleus. Collectively, our study revealed a possible mechanism of RIN13 in accelerating leaf senescence and cell death by changing the subcellular localization of ARF1.

An improved extraction method enables the comprehensive analysis of lipids, proteins, metabolites and phytohormones from a single sample of leaf tissue under water-deficit stress.

Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl-tert-butyl-ether) phase, eliminated the need for solid-phase extraction for sample clean-up, and therefore reduces both sampling time and cost. As a proof-of-concept analysis, Arabidopsis thaliana plants were subjected to water-deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty-acid derivatives and diverse jasmonates in the course of adaptation to water-deficit stress.

Functional Divergence of Microtubule-Associated TPX2 Family Members in Arabidopsis thaliana.

TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.

Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection.

Lipid droplets (LDs) have classically been viewed as seed storage particles, yet they are now emerging as dynamic organelles associated with developmental and stress responses. Nevertheless, their involvement in plant immunity has still been little studied. Here, we found LD accumulation in Arabidopsis thaliana leaves that induced a hypersensitive response (HR) after Pseudomonas infection. We established a protocol to reproducibly isolate LDs and to analyze their protein content. The expression of GFP fusion proteins in Nicotiana benthamiana and in transgenic Arabidopsis lines validated the LD localization of glycerol-3-phosphate acyltransferase 4 (GPAT4) and 8 (GPAT8), required for cutin biosynthesis. Similarly, we showed LD localization of α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3), involved in the synthesis of fatty acid derivatives, and that of phytoalexin-deficient 3 (PAD3), which is involved in camalexin synthesis. We found evidence suggesting the existence of different populations of LDs, with varying protein contents and distributions. GPAT4 and GPAT8 were associated with LDs inside stomata and surrounding cells of untreated leaves, yet they were mainly confined to LDs in guard cells after bacterial inoculation. By contrast, α-DOX1 and PAD3 were associated with LDs in the epidermal cells of HR-responding leaves, with PAD3 mostly restricted to cells near dead tissue, while CLO3 had a more ubiquitous distribution. As such, the nature of the proteins identified, together with the phenotypic examination of selected mutants, suggests that LDs participate in lipid changes and in the production and transport of defense components affecting the interaction of plants with invading pathogens.

Mass-spectrometry-based draft of the Arabidopsis proteome.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

Visualizing Protein Associations in Living Arabidopsis Embryo.

Protein-protein interactions (PPI) are essential for a plethora of biological processes. These interactions can be visualized and quantified with spatial resolution using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) technology. Currently, FRET-FLIM is routinely used in cell biology, and it has become a powerful tool to map protein interactions in native environments. However, implementing this technology in living multicellular organism remains challenging, especially when dealing with developing plant embryos where tissues are confined in multiple cell layers preventing direct imaging. In this chapter, we describe a step-by-step protocol for studying PPI using FRET-FLIM of the two transcription factors SCARECROW and SHORTROOT in Arabidopsis embryos. We provide a detailed description from embryo isolation to data analysis and representation.