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Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

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.

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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

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.

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.

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 H 2 O 2 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., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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 ., (© 2020 The Author(s).)

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., (© 2020 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

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 FLC haplotypes. 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., Competing Interests: JH, RA, KB, CS, ET, BY, DC, BC, JI, MH, TS, SH, CD No competing interests declared, (© 2020, Hepworth et al.)

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., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

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., Competing Interests: Declaration of competing interest No potential conflict of interest is disclosed., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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&#95;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.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities 1 . 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.

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.

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.

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.

Aluminium (Al) toxicity is a major limiting factor for plant productivity in acidic soils. Calcium (Ca) is an essential element and participates in various physiological responses to environmental stress. Here, the aim of this work was to study the role of exogenous Ca in alleviating Al toxicity in Arabidopsis thaliana. For that we used the methods of physiology and proteomics. Results showed that Ca alleviated Al-induced growth inhibition and decreased Al accumulation. Proteomic analyses showed that 75 differentially expressed protein spots, including those related to organic acid metabolism, cell wall components, cellular transport, signal transduction and antioxidant activity, transcription and protein metabolism were identified during the response of Arabidopsis to Ca alleviated Al toxicity. Ca regulated tricarboxylic acid (TCA) cycle-related protein abundances and affected organic acid concentrations and related enzyme activities under Al stress. Vacuolar and mitochondrion adenosine triphosphate (ATP) synthase, and cell wall component-related proteins played important roles in Ca-alleviated Al toxicity. Ethylene-insensitive 3 (EIN3) participated in Ca-alleviated Al toxicity. Glutathione S-transferase (GST6) and glutathione S-transferase tau 19 (ATGSTU19) were associated with antioxidant activities induced by Ca under Al stress. Our results may contribute to an understanding of the functional mechanism by which Ca alleviates Al stress in plants. SIGNIFICANT: Our results elucidated how Ca alleviate the effects of Al toxicity on the inhibition of plant growth and Al accumulation in plants using the proteomics and physiological methods, which may contribute to a better understanding of the molecular mechanism of Ca alleviation Al stress in plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis ( Arabidopsis thaliana ), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Physiological acclimation of plants to an everchanging environment is governed by complex combinatorial signaling networks that perceive and transduce various abiotic and biotic stimuli. Reactive oxygen species (ROS) serve as one of the second messengers in plant responses to hyperosmotic stress. The molecular bases of ROS production and the primary cellular processes that they target were investigated in the Arabidopsis ( Arabidopsis thaliana ) root. Combined pharmacological and genetic approaches showed that the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway and an additional pathway involving apoplastic ascorbate and iron can account for ROS production upon hyperosmotic stimulation. The two pathways determine synergistically the rate of membrane internalization, within minutes after activation. Live superresolution microscopy revealed at single-molecule scale how ROS control specific diffusion and nano-organization of membrane cargo proteins. In particular, ROS generated by RBOHs initiated clustering of the PLASMA MEMBRANE INTRINSIC PROTEIN2;1 aquaporin and its removal from the plasma membrane. This process is contributed to by clathrin-mediated endocytosis, with a positive role of RBOH-dependent ROS, specifically under hyperosmotic stress., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate iron‑sulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a 158 CGFS signature., Methods: The biochemical properties (redox activity, sensitivity to oxidation, pK a of cysteine residues, midpoint redox potential) of Arabidopsis thaliana GRXS16 were investigated by coupling oxidative treatments to alkylation shift assays, activity measurements and mass spectrometry analyses., Results: Activity measurements using redox-sensitive GFP2 (roGFP2) as target protein did not reveal any significant glutathione-dependent reductase activity of A. thaliana GRXS16 whereas it was able to catalyze the oxidation of roGFP2 in the presence of glutathione disulfide. Accordingly, Arabidopsis GRXS16 reacted efficiently with oxidized forms of glutathione, leading to the formation of an intramolecular disulfide between Cys 158 and the semi-conserved Cys 215 , which has a midpoint redox potential of - 298 mV at pH 7.0 and is reduced by plastidial thioredoxins (TRXs) but not GSH. By promoting the formation of this disulfide, Cys 215 modulates GRXS16 oxidoreductase activity., Conclusion: The reduction of AtGRXS16, which is mandatory for its oxidoreductase activity and the binding of Fe-S clusters, depends on light through the plastidial FTR/TRX system. Hence, disulfide formation may constitute a redox switch mechanism controlling GRXS16 function in response to day/night transition or oxidizing conditions., General Significance: From the in vitro data obtained with roGFP2, one can postulate that GRXS16 would mediate protein glutathionylation/oxidation in plastids but not their deglutathionylation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The circadian oscillator, an internal time-keeping device found in most organisms, enables timely regulation of daily biological activities by maintaining synchrony with the external environment. The mechanistic basis underlying the adjustment of circadian rhythms to changing external conditions, however, has yet to be clearly elucidated. We explored the mechanism of action of nicotinamide in Arabidopsis thaliana, a metabolite that lengthens the period of circadian rhythms, to understand the regulation of circadian period. To identify the key mechanisms involved in the circadian response to nicotinamide, we developed a systematic and practical modeling framework based on the identification and comparison of gene regulatory dynamics. Our mathematical predictions, confirmed by experimentation, identified key transcriptional regulatory mechanisms of circadian period and uncovered the role of blue light in the response of the circadian oscillator to nicotinamide. We suggest that our methodology could be adapted to predict mechanisms of drug action in complex biological systems., Competing Interests: The authors have declared that no competing interests exist.

Biological processes consist of several consecutive and interacting steps as, for example, in signal transduction cascades or metabolic reaction chains. These processes are regulated by protein-protein interactions and the formation of larger protein complexes, which also occur within biological membranes. To gain a large-scale overview of complex-forming proteins and the composition of such complexes within the cellular membranes of Arabidopsis roots, we use the combination of size-exclusion chromatography and mass spectrometry. First, we identified complex-forming proteins by a retention shift analysis relative to expected retention times of monomeric proteins during size-exclusion chromatography. In a second step we predicted complex composition through pairwise correlation of elution profiles. As result we present an interactome of 963 proteins within cellular membranes of Arabidopsis roots. Identification of complex-forming proteins was highly robust between two independently grown root proteomes. The protein complex composition derived from pairwise correlations of coeluting proteins reproducibly identified stable protein complexes (ribosomes, proteasome, mitochondrial respiratory chain supercomplexes) but showed higher variance between replicates regarding transient interactions (e.g., interactions with kinases) within membrane protein complexes.

During dark adaptation, plant nuclei move centripetally toward the midplane of the leaf blade; thus, the nuclei on both the adaxial and abaxial sides become positioned at the inner periclinal walls of cells. This centripetal nuclear positioning implies that a characteristic cell polarity exists within a leaf, but little is known about the mechanism underlying this process. Here, we show that ANGUSTIFOLIA (AN) and ACTIN7 regulate centripetal nuclear positioning in Arabidopsis ( Arabidopsis thaliana ) leaves. Two mutants defective in the positioning of nuclei in the dark were isolated and designated as unusual nuclear positioning1 ( unp1 ) and unp2 In the dark, nuclei of unp1 were positioned at the anticlinal walls of adaxial and abaxial mesophyll cells and abaxial pavement cells, whereas the nuclei of unp2 were positioned at the anticlinal walls of mesophyll and pavement cells on both the adaxial and abaxial sides. unp1 was caused by a dominant-negative mutation in ACTIN7 , and unp2 resulted from a recessive mutation in AN Actin filaments in unp1 were fragmented and reduced in number, which led to pleiotropic defects in nuclear morphology, cytoplasmic streaming, and plant growth. The mutation in AN caused aberrant positioning of nuclei-associated actin filaments at the anticlinal walls. AN was detected in the cytosol, where it interacted physically with plant-specific dual-specificity tyrosine phosphorylation-regulated kinases (DYRKPs) and itself. The DYRK inhibitor (1 Z )-1-(3-ethyl-5-hydroxy-2(3 H )-benzothiazolylidene)-2-propanone significantly inhibited dark-induced nuclear positioning. Collectively, these results suggest that the AN-DYRKP complex regulates the alignment of actin filaments during centripetal nuclear positioning in leaf cells., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

S-Acylation is increasingly being recognized as an important dynamic posttranslational modification of cysteine residues in proteins. Various approaches have been described for assaying protein S-acylation with acyl-switch approaches being the most common and accessible. However, these approaches can be time-consuming with low reproducibility as a result of multiple protein precipitation/resuspension cleanup steps. Here we present a faster, cleaner, and more sensitive acyl-switch approach for detecting the S-acylation state of any protein, from any cell or tissue type, that can be detected by western blotting. In the case of acyl-RAC, the procedure is now performed without protein precipitation, greatly increasing speed and improving sample handling in the assay. This also allows for more samples to be processed simultaneously and opens the way for medium-throughput assays. Overall, maleimide scavenging improves the reliability of determination and quantification of protein S-acylation state by acyl-switch methods.

Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.

Mathematical models are important tools in helping us to understand complex biological systems. Models of phytochrome-regulated systems in Arabidopsis thaliana have shown the importance of dimerization, nuclear transport, and thermal/dark reversion in mediating phytochrome activity and plant development. Here we go through the steps required to calculate the steady-state amounts of phytochrome subspecies relative to the total phytochrome molecule population. Starting from a simplified two-state system we expand and apply the technique to the extended phytochrome dimer model. Additionally, we provide a Python package that can automatically calculate the proportion of phytochrome B in a particular state given specific experimental conditions.

Nitrogen is an essential macronutrient for plant growth and crop productivity. The aim of this work was to further investigate the molecular events during plant adaptation to nitrogen stress. Here, we present a SWATH-MS (Sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in Arabidopsis seedlings following nitrogen starvation. In total, 736 proteins of diverse functions were determined to show significant abundance changes between nitrogen-supplied and nitrogen-starved Arabidopsis seedlings. Functional categorization revealed the involvement of nitrogen stress-responsive proteins in biological processes including amino acid and protein metabolism, photosynthesis, lipid metabolism and glucosinolate metabolism. Subsequent phospholipid profiling of Arabidopsis seedlings showed changes in phospholipid composition that may enhance membrane fluidity as a response to nitrogen starvation. Moreover, an Arabidopsis grf6 T-DNA insertion mutant was found to have a nitrogen stress-sensitive phenotype. GRF6 is a 14-3-3 protein with elevated abundance upon nitrogen starvation and it may function as a positive regulator during nitrogen stress adaptation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Phosphate (Pi) deficiency significantly limits plant growth in natural and agricultural systems. Accumulation of anthocyanins in shoots is a common response of Arabidopsis thaliana to Pi deficiency. To elucidate the mechanisms underlying Pi deficiency-induced anthocyanin accumulation, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ) to investigate protein expression profiles of Arabidopsis thaliana seedlings subjected to Pi deficiency for 7 days. In total, 5,106 proteins were identified, of which 156 displayed significant changes in abundance upon Pi deficiency. Bioinformatics analysis indicated that flavonoid biosynthesis was the most significantly elevated metabolic process under Pi deficiency. We further examined the potential role of the flavonoid biosynthetic pathway using a dihydroflavonol 4-reductase (DFR) mutant (tt3) and quantitative RT-PCR (qRT-PCR) analysis, and found that the tt3 mutant was deprived of transcriptional up-regulation of three genes related to anthocyanin biosynthesis, modification and transport under Pi deficiency. These results showed that Pi deficiency probably enhances the anthocyanin accumulation by promoting the flavonoid biosynthesis. The exact functions of these proteins remain to be examined. Nevertheless, our study increases the understanding of the mechanisms implicated in the anthocyanin accumulation induced by Pi deficiency and adaptive responses of plants to Pi starvation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We analyzed the metabolites and proteins contained in pure intact vacuoles isolated from Arabidopsis suspension-cultured cells using capillary electrophoresis-mass spectrometry (CE-MS), Fourier transform-ion cyclotron resonance (FT-ICR)-MS and liquid chromatography (LC)-MS. We identified 21 amino acids and five organic acids as major primary metabolites in the vacuoles with CE-MS. Further, we identified small amounts of 27 substances including well-known vacuolar molecules, but also some unexpected substances (e.g. organic phosphate compounds). Non-target analysis of the vacuolar sample with FT-ICR-MS suggested that there are 1,106 m/z peaks that could predict the 5,090 molecular formulae, and we have annotated 34 compounds in these peaks using the KNapSAck database. By conducting proteomic analysis of vacuolar sap, we found 186 proteins in the same vacuole samples. Since the vacuole is known as a major degradative compartment, many of these were hydrolases, but we also found various oxidoreductases and transferases. The relationships between the proteins and metabolites in the vacuole are discussed.

In-gel digestion has been used as a standard method for the preparation of protein samples for mass spectrometry analysis for over 25 years. Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed "High Throughput in Gel digestion" (HiT-Gel), is based on a 96-well plate format which results in a drastic reduction in labour intensity and sample handling. Direct comparison revealed that HiT-Gel reduces technical variation and significantly decreases sample contamination over the conventional in-gel digestion method. HiT-Gel also produced superior results when a single protein band was excised from a gel and processed by in-gel digestion. Moreover, we applied Hit-Gel for a mass spectrometry analysis of Arabidopsis thaliana protein complexes separated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity of HiT-Gel facilitates large scale studies with high sample replication or detailed fractionation. Our method can easily be implemented as it does not require specialised laboratory equipment.

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research., (© 2018 American Society of Plant Biologists. All rights reserved.)

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a signaling peptide that regulates cell separation in Arabidopsis including floral organ abscission and lateral root emergence. IDA is highly conserved in dicotyledonous flowering plant genomes. IDA-like sequences were also found in the genomic sequences of root-knot nematodes, Meloidogyne spp., which are globally deleterious pathogens of agriculturally important plants, but the role of these genes is unknown. Exogenous treatment of the Arabidopsis ida mutant with synthetic peptide identical to the M. incognita IDA-like 1 (MiIDL1) protein sequence minus its N-terminal signal peptide recovered both the abscission and root architecture defects. Constitutive expression of the full-length MiIDL1 open reading frame in the ida mutant substantially recovered the delayed floral organ abscission phenotype whereas transformants expressing a construct missing the MiIDL1 signal peptide retained the delayed abscission phenotype. Importantly, wild-type Arabidopsis plants harboring an MiIDL1-RNAi construct and infected with nematodes had approximately 40% fewer galls per root than control plants. Thus, the MiIDL1 gene produces a functional IDA mimic that appears to play a role in successful gall development on Arabidopsis roots.

Spatial relocalization of proteins is crucial for the correct functioning of living cells. An interesting example of spatial ordering is the light-induced clustering of plant photoreceptor proteins. Upon irradiation by white or red light, the red light-active phytochrome, phytochrome B, enters the nucleus and accumulates in large nuclear bodies (NBs). The underlying physical process of nuclear body formation remains unclear, but phytochrome B is thought to coagulate via a simple protein-protein binding process. We measure, for the first time, the distribution of the number of phytochrome B-containing NBs as well as their volume distribution. We show that the experimental data cannot be explained by a stochastic model of nuclear body formation via simple protein-protein binding processes using physically meaningful parameter values. Rather modelling suggests that the data is consistent with a two step process: a fast nucleation step leading to macroparticles followed by a subsequent slow step in which the macroparticles bind to form the nuclear body. An alternative explanation for the observed nuclear body distribution is that the phytochromes bind to a so far unknown molecular structure. We believe it is likely this result holds more generally for other nuclear body-forming plant photoreceptors and proteins.

Directional growth of lateral roots is critical for radial expansion and soil coverage. Despite its importance, almost nothing is known about its molecular determinants. Initially, young lateral roots (LRs) grow away from the parental root, maintaining the angle acquired shortly after emergence. A second downwards bending response to gravity terminates the so-called plateau phase and thereby limits radial root expansion. Here, we show that the exit from the plateau phase correlates with an increase in auxin signalling at the tip of the LRs. Moreover, the increase in auxin levels induces the termination of the plateau phase, which requires PIN-FORMED (PIN) auxin efflux carriers. Our data suggests that the developmental increase in auxin triggers the preferential derepression of PIN7 in gravity-sensing columella cells. The subsequent polarization of PIN7 heralds the bending towards gravity and, hence, the exit from the plateau phase. This developmental framework reveals the distinct roles of PIN auxin efflux carriers in controlling the radial growth of root systems.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination., Competing Interests: The authors declare no conflicts of interest.

Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

Background: In plants, the shoot apical meristem (SAM) has two main functions, involving the production of all aerial organs on the one hand and self-maintenance on the other, allowing the production of organs during the entire post-embryonic life of the plant. Transcription factors, microRNA, hormones, peptides and forces have been involved in meristem function. Whereas phosphatidylinositol phosphates (PIPs) have been involved in almost all biological functions, including stem cell maintenance and organogenesis in animals, the processes in meristem biology to which PIPs contribute still need to be delineated., Results: Using biosensors for PI4P and PI(4,5)P 2 , the two most abundant PIPs at the plasma membrane, we reveal that meristem functions are associated with a stereotypical PIP tissue-scale pattern, with PI(4,5)P 2 always displaying a more clear-cut pattern than PI4P. Using clavata3 and pin-formed1 mutants, we show that stem cell maintenance is associated with reduced levels of PIPs. In contrast, high PIP levels are signatures for organ-meristem boundaries. Interestingly, this pattern echoes that of cortical microtubules and stress anisotropy at the meristem. Using ablations and pharmacological approaches, we further show that PIP levels can be increased when the tensile stress pattern is altered. Conversely, we find that katanin mutant meristems, with increased isotropy of microtubule arrays and slower response to mechanical perturbations, exhibit reduced PIP gradients within the SAM. Comparable PIP pattern defects were observed in phospholipase A3β overexpressor lines, which largely phenocopy katanin mutants at the whole plant level., Conclusions: Using phospholipid biosensors, we identified a stereotypical PIP accumulation pattern in the SAM that negatively correlates with stem cell maintenance and positively correlates with organ-boundary establishment. While other cues are very likely to contribute to the final PIP pattern, we provide evidence that the patterns of PIP, cortical microtubules and mechanical stress are positively correlated, suggesting that the PIP pattern, and its reproducibility, relies at least in part on the mechanical status of the SAM.

A large number of proteins in the vacuolar membrane (VM; tonoplast), including transporters and receptors, support the various functions of the vacuole. Molecular analysis of membrane proteins is an essential step in understanding how the vacuole operates but so far only a small number of tonoplast proteins have been identified at the molecular level. Accordingly, mutant lines with altered level of tonoplast proteins for characterizing their physiological roles have been developed sparsely. Also, detecting activities of tonoplast proteins remains difficult as it requires a certain degree of enrichment of this organelle fraction. In order to extend our understanding of the vacuole, several groups have turned to proteomic analysis of tonoplast membrane proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high so that its composition can be accurately analyzed with mass spectrometry. In this chapter, we describe a simple method for the isolation of intact vacuoles and subsequent proteome analysis of the VM fraction of cells from Arabidopsis suspension cultures.

The formation of longitudinal zonation patterns is important for normal root development and is regulated by the transcription factor PLETHORA (PLT). PLT proteins form a concentration gradient, and PLT protein levels determine root zonation. A peptide hormone root meristem growth factor (RGF) and its receptors (RGFRs) act as key regulators of root development by regulating the PLT protein expression pattern. Here, we describe a method for monitoring PLT protein gradient patterns in Arabidopsis with exogenous RGF treatment.

The bimolecular fluorescent complementation (BiFC) is a fluorescent complementation method largely used to investigate protein-protein interaction in living cells. This method is based on the ability of two nonfluorescent fragments to assemble forming a native fluorescent reporter with the same spectral properties of the native reporter. Such fragments are fused to putative protein partners that in case of interaction will bring the two halves in close proximity, allowing for the reconstitution of an active fluorescent reporter. The BiFC has been used to investigate protein-protein interaction in a number of different organisms, including plants. In plant cells, many essential pathways of protein trafficking and subcellular localization necessitate the intervention of several protein units organized in multisubunit complexes. It is well known that vacuolar sorting of many secretory soluble proteins require the intervention of specific transmembrane cargo receptors able to interact forming dimers. In this chapter we describe a BiFC method for the efficient visualization of RMR (Receptor Membrane RING-H2) type 2 dimerization in agro-infiltrated Nicotiana benthamiana leaves. Furthermore, this relatively simple method represents an optimal strategy to test protein-protein interaction using any other putative protein partners of interest in plant cells.

Eukaryotic cells comprise various organelles surrounded by the membrane. Each organelle is characterized by unique proteins and lipids and has its own specific functions. Single membrane-bounded organelles, including the Golgi apparatus, endosomes, and vacuoles are connected by membrane trafficking. Identifying the organelle localization of a protein of interest is essential for determining the proteins physiological functions. Here, we describe methods for determining protein subcellular localization using the inhibitors brefeldin A and wortmannin in Arabidopsis thaliana.

Chemical compounds are useful to perturb biological functions in the same way as classical genetic approaches take advantage of mutations at the DNA level to perturb gene function. The use of bioactive chemicals currently called chemical genetic is especially valuable for cell biology. Chemical genetic approaches allow perturbations of cellular processes post-germination in a given time window controlling the severity of the effect by modifying or modulating the dose and/or the period of the treatment. Additionally, compounds can be applied directly to different mutants and translational fluorescent reporters/marker lines, expanding the repertoire of experimental setups addressing cell biology research. In this chapter, we describe standard protocols to visualize vacuole morphology and trafficking to the vacuole and the use of bioactive compounds as a proxy to study these biological processes.

In plant cells, vacuoles are extremely important for growth and development, and influence important cellular functions as photosynthesis, respiration, and transpiration. Plant cells contain lytic and storage vacuoles, whose size can be different depending on cell type and tissue developmental stage. One of the main roles of vacuoles is to regulate the cell turgor in response to different stimuli. Thus, studying the morphology, dynamics, and physiology of vacuole is fundamentally important to advance knowledge in plant cell biology at large. The availability of fluorescent probes allows marking vacuoles in multiple ways. These may be fast, when using commercially available chemical dyes, or relatively slow, in the case of specific genetically encoded markers based on proteins directed either to the membrane of the vacuole (tonoplast) or to the vacuole lumen. Any of these approaches provides useful information about the morphology and physiology of the vacuole.

Chloroplasts are semiautonomous organelles found in plants and protists. They are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. The inner membrane surrounds a stroma, which is the site of the carbon chemistry of photosynthesis. Chloroplasts also contain an internal membrane system, or thylakoids, where the light phase of photosynthesis occurs. The thylakoid membranes themselves have a bipartite structure, consisting of grana stacks interconnected by stroma lamellae. These thylakoid membranes however form a continuous network that encloses a single lumenal space. Chloroplast-encoded or targeted proteins are thus addressed to various sub-compartments that turn out to be flexible systems and whose main functions can be modulated by alterations in the relative levels of their components. This article describes procedures developed to recover highly purified chloroplast membrane fractions (i.e., envelope, crude thylakoid membranes, as well as the two main thylakoid subdomains, grana and stroma lamellae), starting from Percoll-purified Arabidopsis chloroplasts. Immunological markers are also listed that can be used to assess the purity of these fractions and reveal specific contaminations by other plastid membrane compartments. The methods described here are compatible with chloroplast proteome dynamic studies relying on targeted quantitative proteomic investigations.