Make It Shine: Labelling the ER for Light and Fluorescence Microscopy.

The ER is a highly dynamic network of tubules and membrane cisternae. Hence, imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labelling the native plant ER using fluorescent proteins and lipid dyes as well as methods for immunolabelling on plant tissue.

Preparation of ER Microsomes from Arabidopsis thaliana.

Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER functions such as protein and lipid synthesis in vitro.Here we describe a protocol to isolate ER-derived microsomes Arabidopsis thaliana seedlings and exemplify the use of these purified microsomes in enzyme assays with the auxin precursors tryptophan (Trp) or indole-3-pyruvic acid (IPyA) to quantify auxin synthetic capacity in microsomal and cytosolic fractions.

Quantitation of ER Morphology and Dynamics.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

Immunoprecipitation and FRET-FLIM to Determine Metabolons on the Plant ER.

Metabolons are protein complexes that contain all the enzymes necessary for a metabolic pathway but also scaffolding proteins. Such a structure allows efficient channeling of intermediate metabolites form one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify metabolons via protein-protein interaction methodology: immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and potential metabolon components and FRET-FLIM to test for and quantify protein-protein interactions in planta.

Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER.

Single-particle tracking (SPT) of biomolecules in the plant endoplasmic reticulum has the potential to inform on the formation of protein-protein complexes, metabolons, and the transport of molecules through both the ER membrane and lumen. Plant cells are particularly challenging for observing and tracking single molecules due to their unique structure, size, and considerable autofluorescence. However, by using variable-angle or highly inclined epifluorescence microscopy (VAEM) and transient expression in tobacco, it is possible to observe single-particle dynamics in the ER. Selecting the appropriate fluorophore, and ensuring the correct fluorophore density in the ER, is essential for successful SPT. By using tuneable fluorophores, which can be photoconverted and photoactivated, it is possible to vary the density of visible fluorophores in the ER dynamically. Here we describe methods to prepare plant samples for VAEM and two methods for determining and analyzing single-particle tracks from VAEM time series.

Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using "2A" Self-Cleaving Peptides.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or "gene stacking") have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

Observing ER Dynamics over Long Timescales Using Light Sheet Fluorescence Microscopy.

The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock.

Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

Recombinant expression and subcellular targeting of the particulate methane monooxygenase (pMMO) protein components in plants.

Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein-protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

In Depth Topological Analysis of Arabidopsis Mid-SUN Proteins and Their Interaction with the Membrane-Bound Transcription Factor MaMYB.

Mid-SUN proteins are a neglected family of conserved type III membrane proteins of ancient origin with representatives in plants, animals, and fungi. Previous higher plant studies have associated them with functions at the nuclear envelope and the endoplasmic reticulum (ER). In this study, high-resolution confocal light microscopy is used to explore the localisation of SUN3 and SUN4 in the perinuclear region, to explore topology, and to study the role of mid-SUNs on endoplasmic reticulum morphology. The role of SUN3 in the ER is reinforced by the identification of a protein interaction between SUN3 and the ER membrane-bound transcription factor maMYB. The results highlight the importance of mid-SUNs as functional components of the ER and outer nuclear membrane.

An Interplay between Mitochondrial and ER Targeting of a Bacterial Signal Peptide in Plants.

Protein targeting is essential in eukaryotic cells to maintain cell function and organelle identity. Signal peptides are a major type of targeting sequences containing a tripartite structure, which is conserved across all domains in life. They are frequently included in recombinant protein design in plants to increase yields by directing them to the endoplasmic reticulum (ER) or apoplast. The processing of bacterial signal peptides by plant cells is not well understood but could aid in the design of efficient heterologous expression systems. Here we analysed the signal peptide of the enzyme PmoB from methanotrophic bacteria. In plant cells, the PmoB signal peptide targeted proteins to both mitochondria and the ER. This dual localisation was still observed in a mutated version of the signal peptide sequence with enhanced mitochondrial targeting efficiency. Mitochondrial targeting was shown to be dependent on a hydrophobic region involved in transport to the ER. We, therefore, suggest that the dual localisation could be due to an ER-SURF pathway recently characterised in yeast. This work thus sheds light on the processing of bacterial signal peptides by plant cells and proposes a novel pathway for mitochondrial targeting in plants.

Keep in contact: multiple roles of endoplasmic reticulum-membrane contact sites and the organelle interaction network in plants.

Functional regulation and structural maintenance of the different organelles in plants contribute directly to plant development, reproduction and stress responses. To ensure these activities take place effectively, cells have evolved an interconnected network amongst various subcellular compartments, regulating rapid signal transduction and the exchange of biomaterial. Many proteins that regulate membrane connections have recently been identified in plants, and this is the first step in elucidating both the mechanism and function of these connections. Amongst all organelles, the endoplasmic reticulum is the key structure, which likely links most of the different subcellular compartments through membrane contact sites (MCS) and the ER-PM contact sites (EPCS) have been the most intensely studied in plants. However, the molecular composition and function of plant MCS are being found to be different from other eukaryotic systems. In this article, we will summarise the most recent advances in this field and discuss the mechanism and biological relevance of these essential links in plants.

intER-ACTINg: The structure and dynamics of ER and actin are interlinked.

The actin cytoskeleton is the driver of gross ER remodelling and the movement and positioning of other membrane-bound organelles such as Golgi bodies. Rapid ER membrane remodelling is a feature of most plant cells and is important for normal cellular processes, including targeted secretion, immunity and signalling. Modifications to the actin cytoskeleton through pharmacological agents such as Latrunculin B and phalloidin, or disruption of normal myosin function also affect ER structure and/or dynamics. Here, we investigate the impact of changes in the actin cytoskeleton on structure and dynamics on the ER as well as in return the impact of modified ER structure on the architecture of the actin cytoskeleton. By expressing actin markers that affect actin dynamics, or expressing of ER-shaping proteins that influence ER architecture, we found that the structure of ER-actin networks is closely inter-related; affecting one component is likely to have a direct effect on the other. Therefore, our results indicate that a complicated regulatory machinery and cross-talk between these two structures must exist in plants to co-ordinate the function of ER-actin network during multiple subcellular processes. In addition, when considering organelle structure and dynamics, the choice of actin marker is essential in preventing off-target organelle structure and dynamics modifications.

FRET-FLIM to Determine Protein Interactions and Membrane Topology of Enzyme Complexes.

Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer with fluorescence lifetime imaging microscopy (FRET-FLIM) is an advanced imaging methodology that allows for the quantitative detection of protein-protein interactions. In this method, proteins of interest for interaction studies are fused to different fluorophores such as enhanced green fluorescent protein (eGFP; donor molecule) and monomeric red fluorescent protein (mRFP; acceptor molecule). Energy transfer between the two fluorophore groups can only occur efficiently when the proteins of interest are in close physical proximity, around ≤10 nm, and therefore are most likely interacting. FRET-FLIM measures the decrease in excited-state lifetime of the donor fluorophore (eGFP) with and without the presence of the acceptor (mRFP) and can therefore give information on protein-protein interactions and the membrane topology of the tested protein. Here we describe the production of fluorescent protein fusions for FRET-FLIM analysis in tobacco leaf epidermal cells using Agrobacterium-mediated plant transformation and a FRET-FLIM data acquisition and analysis protocol in plant cells. These protocols are applicable and can be adapted for both membrane and soluble proteins in different cellular localizations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein expression in tobacco leaf cells via transient Agrobacterium-mediated plant transformation Basic Protocol 2: FRET-FLIM data acquisition and analysis.

On the nature of the plant ER exit sites.

In plants, the endoplasmic reticulum (ER) and Golgi bodies are not only in close proximity, but are also physically linked. This unique organization raises questions about the nature of the transport vectors carrying cargo between the two organelles. Same as in metazoan and yeast cells, it was suggested that cargo is transported from the ER to Golgi cisternae via COPII-coated vesicles produced at ribosome-free ER exit sites (ERES). Recent developments in mammalian cell research suggest, though, that COPII helps to select secretory cargo, but does not coat the carriers leaving the ER. Furthermore, it was shown that mammalian ERES expand into a tubular network containing secretory cargo, but no COPII components. Because of the close association of the ER and Golgi bodies in plant cells, it was previously proposed that ERES and the Golgi comprise a secretory unit that travels over or with a motile ER membrane. In this study, we aimed to explore the nature of ERES in plant cells and took advantage of high-resolution confocal microscopy and imaged ERES labelled with canonical markers (Sar1a, Sec16, Sec24). We found that ERES are dynamically connected to Golgi bodies and most likely represent pre-cis-Golgi cisternae. Furthermore, we showed fine tubular connections from the ER to Golgi compartments (ERGo tubules) as well as fine protrusions from ERES/Golgi cisternae connecting with the ER. We suggest that these tubules observed between the ER and Golgi as well as between the ER and ERES are involved in stabilizing the physical connection between ER and ERES/Golgi cisternae, but may also be involved in cargo transport from the ER to Golgi bodies.

Reticulons 3 and 6 interact with viral movement proteins.

Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two-hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins.

Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana.

The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/CCDC20 complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.

Expression of Clementine Asp-Rich Proteins (CcASP-RICH) in Tobacco Plants Interferes with the Mechanism of Pollen Tube Growth.

Low-molecular-weight, aspartic-acid-rich proteins (ASP-RICH) have been assumed to be involved in the self-incompatibility process of clementine. The role of ASP-RICH is not known, but hypothetically they could sequester calcium ions (Ca2+) and affect Ca2+-dependent mechanisms. In this article, we analyzed the effects induced by clementine ASP-RICH proteins (CcASP-RICH) when expressed in the tobacco heterologous system, focusing on the male gametophyte. The aim was to gain insight into the mechanism of action of ASP-RICH in a well-known cellular system, i.e., the pollen tube. Pollen tubes of tobacco transgenic lines expressing CcASP-RICH were analyzed for Ca2+ distribution, ROS, proton gradient, as well as cytoskeleton and cell wall. CcASP-RICH modulated Ca2+ content and consequently affected cytoskeleton organization and the deposition of cell wall components. In turn, this affected the growth pattern of pollen tubes. Although the expression of CcASP-RICH did not exert a remarkable effect on the growth rate of pollen tubes, effects at the level of growth pattern suggest that the expression of ASP-RICH may exert a regulatory action on the mechanism of plant cell growth.

Methods for Detection of Protein Interactions with Plasmodesmata-Localized Reticulons.

Plant reticulon family proteins (RTN) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Two RTN ER-shaping proteins have been found in the plasmodesmata (PD) proteome which could potentially contribute to the formation of the desmotubule, an ER-derived structure that crosses primary PD and physically connects the ER of two cells. Here we describe two methods used to identify partners of two PD-resident reticulon proteins, RTN3 and RTN6 that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum): immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and FRET-FLIM to test for and quantify direct protein-protein interactions in planta.

Lysophosphatidic acid acyltransferases: a link with intracellular protein trafficking in Arabidopsis root cells?

Phosphatidic acid (PA) and lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (LPAAT2, 3, 4, and 5) were identified in Arabidopsis thaliana. These AtLPAATs display a specific enzymatic activity converting lysophosphatidic acid to PA and are located in the endomembrane system. We investigate a putative role for AtLPAATs 3, 4, and 5 in the secretory pathway of root cells through genetical (knockout mutants), biochemical (activity inhibitor, lipid analyses), and imaging (live and immuno-confocal microscopy) approaches. Treating a lpaat4;lpaat5 double mutant with the LPAAT inhibitor CI976 produced a significant decrease in primary root growth. The trafficking of the auxin transporter PIN2 was disturbed in this lpaat4;lpaat5 double mutant treated with CI976, whereas trafficking of H+-ATPases was unaffected. The lpaat4;lpaat5 double mutant is sensitive to salt stress, and the trafficking of the aquaporin PIP2;7 to the plasma membrane in the lpaat4;lpaat5 double mutant treated with CI976 was reduced. We measured the amounts of neo-synthesized PA in roots, and found a decrease in PA only in the lpaat4;lpaat5 double mutant treated with CI976, suggesting that the protein trafficking impairment was due to a critical PA concentration threshold.