Verena Kriechbaumer

Oxford Brookes University · Department of Biological and Medical Sciences

Advances in Plant Auxin Biology

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 agg...

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...

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 explor...

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 th...

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 inter-connected network amongst various subcellular compartments, regulating rapid signal transduction and the ex...

Engineering of subcellular compartmentalisation is one of synthetic biology’s key challenges. Among different approaches, de novo construction of a synthetic compartment is the most coveted but also most difficult option. Restructuring the endoplasmic reticulum (ER), via the introduction of recombinant oligomerising ER-membrane resident proteins, i...

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 Golg...

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 interactio...

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 presen...

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 cytosk...

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-orient...

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...

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 cros...

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...

Plant endoplasmic reticulum (ER) remodelling is likely to be important for its function in targeted protein secretion, organelle interaction and signal exchange. It has been known for decades that the structure and movement of the ER network is mainly regulated by the actin cytoskeleton through actin motor proteins and membrane-cytoskeleton adaptor...

Phosphatidic acid (PA) and Lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (numbered 2,3,4 and 5) were identified in A. thaliana . These AtLPAATs, displaying an enzymatic activity specific for LPA to produce PA, are located in the endomembrane system. We focused on the putati...

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER s...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton i...

Membrane-targeting sequences, connected targeting mechanisms, and co-factors orchestrate primary targeting of proteins to membranes.

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fuc...

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains uncle...

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as 'organised smooth endoplasmic reticulum' (OSER), consisting of symmetrically stacked ER membrane array...

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...

The endoplasmic reticulum (ER) is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. T...

The availability of quantification methods for subcellular organelle dynamic analysis has increased rapidly over the last 20 years. The application of these techniques to contiguous subcellular structures that exhibit dynamic remodelling over a range of scales and orientations is challenging, as quantification of ‘movement’ rarely corresponds to tr...

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two indep...

The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on disperse...

The plant hormone auxin is essential for plant growth and development, controlling both organ development and overall plant architecture. Auxin homeostasis is regulated by coordination of biosynthesis, transport, conjugation, sequestration/storage, and catabolism to optimize concentration-dependent growth responses and adaptive responses to tempera...

The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its centra...

Fig. S1 Analysis of LNP transcript abundance in Arabidopsis amiRNA lines. Fig. S2 Microarray data in the eFP browser for AtLNP1 and AtLNP2. Fig. S3 Lunapark motif analysis in Embryophyta species. Fig. S4 OD expression series for AtLNP1 and AtLNP2 in tobacco epidermal leaf cells (addition to Fig. 4). Fig. S5 Raw data for protein–protein interact...

Fig. S7 Example movies for ER network persistency analysis in Fig. 10.

The plant secretory pathway is responsible for the production of the majority of proteins and carbohydrates consumed on the planet. The early secretory pathway is composed of Golgi bodies and the endoplasmic reticulum (ER) and is highly mobile in plants with rapid remodelling of the ER network. The dynamics of the ER and Golgi bodies is driven by t...

The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon proteins. Despite th...

Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. Using high resolutio...

The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon family of proteins....

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 pu...

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 from one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify...

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

This volume presents a range of different techniques that have been used to characterize the structure and function of the endoplasmic reticulum (ER) in higher plants. Chapters guide readers through application of modern microscopy techniques by fluorescence and electron microscopy, new protocols for analysing ER network structure, methods to purif...

Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to...

Auxin is a main plant growth hormone crucial in a multitude of developmental processes in plants. Auxin biosynthesis via the tryptophan aminotransferase of arabidopsis (TAA)/YUCCA (YUC) route involving tryptophan aminotransferases and YUC flavin-dependent monooxygenases that produce the auxin indole-3-acetic acid (IAA) from tryptophan is currently...

The family of reticulon proteins has been shown to be involved in a variety of functions in eukaryotic cells including tubulation of the endoplasmic reticulum (ER), formation of cell plates and primary plasmodesmata. Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which...

Auxin and cytokinin (CK) are both important hormones involved in many aspects of plant growth and development. However, the details of auxin biosynthesis and the interaction between auxin and CK are still unclear. Isolation and characterization of an auxin deficient mutant cytokinin induced root curling 2 (ckrc2) in this work reveal that CKRC2 enco...

Significance This study demonstrates, in vivo, that reticulon (RTN) proteins, responsible for the shaping and maintenance of endoplasmic reticulum (ER) membrane tubules, rely on a highly conserved C-terminal amphipathic helix (APH) for their morphogenic function. Previously it was thought that RTN could bend the ER membrane both by assuming a wedge...

The growth regulator auxin is involved in all key developmental processes in plants. A complex network of a multiplicity of potential biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complex and multifaceted system system for auxin function. This raises the question how such a system can be effecti...

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied (Darwin and Darwin, 1880). The auxin molecule in plants was first identified as indole-3-acetic acid (IAA) by Kögl et al. (1934). Active research over nearly a decade has shed light on many of the molecular mechanisms of its action but the complexity...

Auxin is one of the most important plant hormones as it diversely regulates growth and development. Because the action of auxin is often correlated with its local distribution and flux, quantitative analysis and monitoring of auxin is indispensable to understanding plant development. Great efforts have been made to detect, visualize, quantify and m...

The ER is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three way junctions, and back to tubules. Plant reticulon (RTNLB) proteins tubulate the E...

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The co...

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER dur...

The endoplasmic reticulum forms the first compartment in a series of organelles which comprise the secretory pathway. It takes the form of an extremely dynamic and pleomorphic membrane‐bounded network of tubules and cisternae which have numerous different cellular functions. In this review, we discuss the nature of endoplasmic reticulum structure a...

Certain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins a...

Background Certain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeuti...

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interact...

Illustration of the TIRE system with Langmuir-Schaefer cell deposition. Cr/Au coated slide with cells is placed into the ellipsometer cell which is flushed with the ligand/drug. The reflection of polarized light is measured and changes in layer thickness are detected. (TIF)

The general mechanisms of intracellular protein targeting are well established, and depend on a targeting sequence in the protein, which is recognized by a targeting factor. Once a membrane protein is delivered to the correct organelle its targeting sequence can be recognized by receptors and a translocase, leading to membrane insertion. However, t...

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in thei...

Auxin is a major growth hormone in plants, and recent studies have elucidated many of the molecular mechanisms underlying its action, including transport, perception and signal transduction. However, major gaps remain in our knowledge of auxin biosynthetic control, partly due to the complexity and probable redundancy of multiple pathways that invol...

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycat...

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to...

Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to the...

Tail-anchored (TA) proteins function in key cellular processes in eukaryotic cells, such as vesicle trafficking, protein translocation and regulation of transcription. They anchor to internal cell membranes by a C-terminal transmembrane domain, which also serves as a targeting sequence. Targeting occurs post-translationally, via pathways that are s...

Table S2: MS-signals yielding identification of sequence qualifying peptides of recombinant ZmTSA, ZmTSAlike, IGL, BX1, and ZmTSB1.

Table S3: LC/MSD TOF analysis of trypsin digested recombinant ZmTSA, ZmTSAlike, IGL, BX1, and TSB1; list of major signals.

Figure SF1: Amino acid sequence alignment of the maize TSA homologs (ClustalW). Transit peptides are depicted in italics. For ZmTSA and IGL they were predicted using TargetP [23], for BX1 it was determined experimentally [36].

Table S1: Transcript levels of ZmTSA and ZmTSAlike relative to GAPDH in RNA preparations from different tissues.