Dr Verena Kriechbaumer

Abstract

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of ‘humanising’ proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems.

For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae.

Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins.

Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells.

In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

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 organised smooth endoplasmic reticulum (OSER). However, these membrane  structures formed by ER proliferation are poorly characterised and this hampers their potential development for plant synthetic biology. Here we characterise a range of ER-derived membranous  compartments in tobacco and show how the nature of the polyproteins introduced into the ER  membrane affect the final compartment morphology. We show that a cytosol-facing oligomerisation  domain is an essential component for compartment formation. Using FRAP, 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.

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.

Abstract: 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.

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.

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 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 summarize the most recent advances in this field, and discuss the mechanism and biological relevance of these essential links in plants.

Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer together 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 eGFP (donor molecule) and 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 or less 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 as well as 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 as well as 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.

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 stabilising the physical connection between ER and ERES/Golgi cisternae, but may also be involved in cargo transport from the ER to Golgi bodies.

Plant reticulon proteins (RTN) are capable of constricting membranes and 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 was 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 localise 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 6 in a membrane yeast-2-hybrid assay, co-immunoprecipitation and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy (FRET-FLIM). Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localisation and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins.

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.

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 and this 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 as well as a 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.

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.

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 A. thaliana. These AtLPAATs, display a specific enzymatic activity converting lysophosphatidic acid (LPA) to PA and are located in the endomembrane system. We investigate a putative role of the AtLPAATs 3, 4 and 5 in the secretory pathway of root cells through genetical (knock-out 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 showed 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.​​​​​​​

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 adaptors. Recent discoveries also revealed alternative pathways that influence ER movement, through a microtubule-based machinery. Therefore, plants utilize both cytoskeletal components to drive ER dynamics, a process that is likely to be dependent on the cell type and the developmental stages. On the other hand, the ER membrane also has a direct effect towards the organization of the cytoskeletal network and disrupting the tethering factors at the ER-PM interface also rearranges the cytoskeletal structure. However, the influence of the ER network on the cytoskeleton organization has not been studied. In this review, we will provide an overview of the ER-cytoskeleton network in plants, and discuss the most recent discoveries in the field.

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 the ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of the ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Finally, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome-structure axis, with implications in biotechnology and medicine. 

In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton [1-7] . Strong co-alignment between EPCS and the cytoskeleton is observed in plants [1, 8], but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss of function mutants, net3a/NET3C RNAi, klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis.

Distributing proteins to the correct sub-cellular compartments and organelles is crucial for the proper functionality of the proteins as well as for the general function of eukaryotic cells. Cellular targeting is best understood in the case of endoplasmic reticulum (ER) proteins targeted co-translationally via the signal recognition particle (SRP)-mediated pathway but various targeting mechanisms, signals and pathways are in place depending on organism, organelle and protein types to allow for specificity and efficiency of protein targeting.

This review aims to give an overview on membrane targeting sequences taking into account the connected targeting mechanism and co-factors. It focusses on primary targeting to membranes of the endoplasmic reticulum, chloroplast, mitochondrion, peroxisome, nucleus and tonoplast.

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 in the interface between host and parasite. Glycans can modify immune responses by binding to carbohydrate-binding receptors on immune cells and are able to elicit potent antibody responses. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids, but little is known on how these complex glycans contribute to parasitism. Up to 20 different fucosyltransferase (FucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. In order to unravel the synthesis of highly complex fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes successfully localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs that are involved in core α1,3- and α1,6-fucosylation, the synthesis of antennary Lewis X, LDN-F and F-LDN-F. This knowledge can now be used to specifically knock-out the synthesis of specific N-glycan structures in the parasite to investigate the role of N-glycans during parasitism. The functionally characterized SmFucTs can also directly be applied to synthesize complex helminth N-glycan structures on recombinant proteins in order to study their contribution to immunomodulation. Furthermore, this expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

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 (TMDs) 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 localisation 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 localises 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.

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 arrays. In plants, these structures were first described in certain specialised tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow.

In this review we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating the evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

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. The ER structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review, we report on the findings that contributed toward the understanding of the ER morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.

The availability of quantification methods for sub-cellular organelle dynamic analysis has increased rapidly over the last 20 years. The application of these techniques to contiguous sub-cellular structures that exhibit dynamic re-modelling over a range of scales and orientations is challenging as quantification of ‘movement’ rarely corresponds to traditional, qualitative classifications of types of organelle movement. The plant endoplasmic reticulum represents a particular challenge for dynamic quantification as it itself is an entirely contiguous organelle that is in a constant state of flux and gross remodelling, controlled by the actinomyosin cytoskeleton.

Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism. 

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 dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is a novel amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1.

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 temperature, water stress, herbivory and pathogens. At present, the best defined pathway of auxin biosynthesis is the TAA/YUC route, in which the tryptophan aminotransferases TAA and TAR and YUCCA flavin-dependent monooxygenases produce the auxin indole-3-acetic acid from tryptophan. This review highlights recent advances in our knowledge of TAA/YUC-dependent auxin biosynthesis focussing on membrane localisation of auxin biosynthetic enzymes, differential regulation in root and shoot tissue, and auxin biosynthesis during abiotic stress.

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 central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.

complex network of a multiplicity of potential auxin biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complicated system of auxin function. This raises the question how such a complex and multifaceted system producing such a powerful and important molecule as auxin can be effectively organised and controlled. Here we report that a subset of auxin biosynthetic enzymes in the TAA/YUC route of auxin biosynthesis is localised to the endoplasmic reticulum (ER). ER microsomal fractions also contain a significant percentage of auxin biosynthetic activity. This could point toward a model of auxin function using ER membrane location and subcellular compartmentation for supplementary layers of regulation. Additionally we show specific protein-protein interactions between some of the enzymes in the TAA/YUC route of auxin biosynthesis.

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 ER by dimer- and oligomerization, creating localised ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmal (PD) proteome (Fernandez-Calvino et al., 2011) and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD (Knox et al., 2015). Here we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis (Knox et al., 2015). Co-immunoprecipitation of GFP-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localised proteins as well as plasma-membrane proteins with putative membrane anchoring roles. FRET-FLIM assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modelling, may play important roles in linking the cortical ER to the plasma membrane.

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over more than sixty 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 complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here we show that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localised to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the endoplasmic reticulum could be necessary to bring auxin biosynthesis in closer proximity to ER-localised factors for transport, conjugation and signalling and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore it might provide a link to ethylene action and be a factor in hormonal crosstalk as all five ethylene receptors are ER-localised.

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 during cell-plate formation. Here we show that members of the reticulon (RTNLB) family of ER-tubulating proteins may play a role in formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant co-localisation at PD with the viral movement protein of tobacco mosaic virus while super-resolution imaging (3D-SIM) of primary PD revealed the central desmotubule to be labelled by RTNLB6. FRAP studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its centre where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation

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 and dynamics, its relationship with closely associated organelles, and its possible function as a highway for the distribution and delivery of a diverse range of structures from metabolic complexes to viral particles.

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 therapeutic proteins and tools.In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers.
Results: Here a modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise.
To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact.
Conclusions: The actin-chromobody technique presented in this study provides a novel option for in vivo labelling ofthe actin cytoskeleton in comparison to conventionally used probes that are based on actin binding proteins.
The actin-chromobody is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin filaments.

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 polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

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 specific to the precursor, which makes TA proteins a model system for investigating post-translational protein targeting. Bioinformatics approaches have previously been used to identify potential TA proteins in yeast and humans, yet little is known about TA proteins in plants. The identification of plant TA proteins is important for extending the post-translational model system to plastids, in addition to general proteome characterization, and the identification of functional homologues characterized in other organisms. We identified 454 loci that potentially encode TA proteins in Arabidopsis, and combined published data with new localization experiments to assign localizations to 130 proteins, including 29 associated with plastids. By analysing the tail anchor sequences of characterized proteins, we have developed a tool for predicting localization and estimate that 138 TA proteins are localized to plastids.

Background: In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole- 3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB.

Results: In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto

uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP), and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α- subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves.
 

Conclusion: It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase

complex and ZmTSA functions as α-subunit in this complex.

Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.

A number of pathways starting from tryptophan have been proposed for the biosynthesis of the auxin indole-3-acetic acid (IAA), a key regulator of plant development. Many aspects of auxin metabolism have been investigated in the model species Arabidopsis thaliana that, in addition to IAA, synthesizes tryptophan-derived indole glucosinolates and camalexin as defence compounds. This results in a complex metabolic network, which makes it difficult to assign specific enzymatic functions and biosynthetic genes to IAA biosynthesis. In this review, the
Arabidopsis system is compared to the maize system, where the branch point between IAA and secondary metabolite biosynthesis occurs prior to tryptophan.