Preparation and Imaging of Specialized ER Using Super-Resolution and TEM Techniques.

The plant endoplasmic reticulum (ER) forms several specialized structures. These include the sieve element reticulum (SER) and the desmotubule formed as the ER passes through plasmodesmata. Imaging both of these structures has been inhibited by the resolution limits of light microscopy and their relatively inaccessible locations, combined with the fragile nature of the ER. Here we describe methods to view desmotubules in live cells under 3D-structured illumination microscopy (3D-SIM) and methods to fix and prepare phloem tissue for both 3D-SIM and transmission electron microscopy (TEM), which preserve the fragile structure and allow the detailed imaging of the SER.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Controlling intercellular flow through mechanosensitive plasmodesmata nanopores.

In plants, plasmodesmata (PD) are nanopores that serve as channels for molecular cell-to-cell transport. Precise control of PD permeability is essential to regulate processes such as growth and tissue patterning, photoassimilate distribution and defense against pathogens. Callose deposition modulates PD transport but little is known of the rapid events that lead to PD closure in response to tissue damage or osmotic shock. We propose a mechanism of PD closure as a result of mechanosensing. Pressure forces acting on the dumbbell-shaped ER-desmotubule complex cause it to be displaced from its equilibrium position, thus closing the PD aperture. The filamentous protein tethers that link the plasma membrane to the ER-desmotubule complex play a key role in determining the selectivity of the PD pore. This model of PD control compares favorably with experimental data on the pressure-generated closure of PD.

The Coumarin Glucoside, Esculin, Reveals Rapid Changes in Phloem-Transport Velocity in Response to Environmental Cues.

The study of phloem transport and its vital roles in long-distance communication and carbon allocation have been hampered by a lack of suitable tools that allow high-throughput, real-time studies. Esculin, a fluorescent coumarin glucoside, is recognized by Suc transporters, including AtSUC2, which loads it into the phloem for translocation to sink tissues. These properties make it an ideal tool for use in live-imaging experiments, where it acts as a surrogate for Suc. Here, we show that esculin is translocated with a similar efficiency to Suc and, because of its ease of application and detection, demonstrate that it is an ideal tool for in vivo studies of phloem transport. We used esculin to determine the effect of different environmental cues on the velocity of phloem transport. We provide evidence that fluctuations in cotyledon Suc levels influence phloem velocity rapidly, supporting the pressure-flow model of phloem transport. Under acute changes in light levels, the phloem velocity mirrored changes in the expression of AtSUC2 This observation suggests that under certain environmental conditions, transcriptional regulation may affect the abundance of AtSUC2 and thus regulate the phloem transport velocity.

Illuminating the translocation stream.

This review focuses on the recent development of a suite of fluorescent probes that can be used to trace phloem-transport rates in a range of diverse species. Some of these probes are loaded into the phloem by virtue of optimal physico-chemical properties for ion trapping in the high pH environment of the sieve element. However, others are clearly loaded by carrier-mediated transport, such as the blue-emitting probe, esculin, which is loaded into the Arabidopsis phloem by the sucrose transporter, AtSUC2, allowing it to be used as a surrogate for sucrose in phloem transport studies. We also describe additional chemical groups which, although highly charged (e.g. sulphonates), facilitate entry into the phloem. The addition of such 'mobilophores' to existing chemical groups has allowed us to expand the range of fluorophores that can be loaded into the phloem, and provides clues as to the nature of selectivity for phloem loading of xenobiotic compounds.

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new compounds to identify key structural features that specify recognition by AtSUC2. Our analysis of the structure-activity relationship revealed that the presence of a free hydroxyl group on the coumarin moiety is essential for binding by AtSUC2 and subsequent phloem mobility. Structural modeling of the AtSUC2 substrate-binding pocket explains some important structural requirements for the interaction of coumarin glucosides with the AtSUC2 transporter.

Sieve-element differentiation and phloem sap contamination.

Sieve elements (SEs) degrade selected organelles and cytoplasmic structures when they differentiate. According to classical investigations, only smooth ER, mitochondria, sieve element plastids, and, in most cases, P-proteins remain in mature SEs. More recent proteomics and immuno-histochemical studies, however, suggested that additional components including a protein-synthesizing machinery and a fully developed actin cytoskeleton operate in mature SEs. These interpretations are at odds with conventional imaging studies. Here we discuss potential causes for these discrepancies, concluding that differentiating SEs may play a role by 'contaminating' phloem exudates.

Preparation and Imaging of Specialized ER Using Super-Resolution and TEM Techniques.

The plant endoplasmic reticulum (ER) forms several specialized structures. These include the sieve element reticulum (SER) and the desmotubule formed as the ER passes through plasmodesmata. Imaging both of these structures has been inhibited by the resolution limits of light microscopy and their relatively inaccessible locations, combined with the fragile nature of the ER. Here we describe methods to view desmotubules in live cells under 3D-structured illumination microscopy (3D-SIM) and methods to fix and prepare phloem tissue for both 3D-SIM and transmission electron microscopy (TEM) which preserve the fragile structure and allow the detailed imaging of the SER.

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of 'funnel plasmodesmata'. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as 'batch unloading'. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots.

Lost in Transit: Long-Distance Trafficking and Phloem Unloading of Protein Signals in Arabidopsis Homografts.

In addition to moving sugars and nutrients, the phloem transports many macromolecules. While grafting and aphid stylectomy experiments have identified many macromolecules that move in the phloem, the functional significance of phloem transport of these remains unclear. To gain insight into protein trafficking, we micrografted Arabidopsis thaliana scions expressing GFP-tagged chloroplast transit peptides under the 35S promoter onto nontransgenic rootstocks. We found that plastids in the root tip became fluorescent 10 d after grafting. We obtained identical results with the companion cell-specific promoter SUC2 and with signals that target proteins to peroxisomes, actin, and the nucleus. We were unable to detect the respective mRNAs in the rootstock, indicating extensive movement of proteins in the phloem. Outward movement from the root protophloem was restricted to the pericycle-endodermis boundary, identifying plasmodesmata at this interface as control points in the exchange of macromolecules between stele and cortex. Intriguingly, signals directing proteins to the endoplasmic reticulum and Golgi apparatus from membrane-bound ribosomes were not translocated to the root. It appears that many organelle-targeting sequences are insufficient to prevent the loss of their proteins into the translocation stream. Thus, nonspecific loss of proteins from companion cells to sieve elements may explain the plethora of macromolecules identified in phloem sap.

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

The endoplasmic reticulum (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 family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy 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 modeling, may play important roles in linking the cortical ER to the plasma membrane.

New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth ß-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation.

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) 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 in Arabidopsis (Arabidopsis thaliana) may play a role in the 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 colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching 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 center, 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.

Multispectral phloem-mobile probes: properties and applications.

Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem. In wild-type plants, the fluorescence of esculin decayed to background levels about 2 h after phloem unloading, making it a suitable tracer for pulse-labeling studies of phloem transport. We identified additional probes, such as carboxytetraethylrhodamine, a red fluorescent probe that, unlike esculin, was stable for several hours after phloem unloading and could be used to study phloem transport in Arabidopsis lines expressing green fluorescent protein.

Preparative methods for imaging plasmodesmata at super-resolution.

Much of our knowledge of plasmodesmata has come from the ability to visualize them. Light microscopy is a popular tool for exploring subcellular structures but is limited in its resolving power due to the diffractive properties of light. At 50 nm in diameter plasmodesmata are below this limit and so cannot be resolved. Super-resolution microscopy operates beyond the limits of conventional light microscopy affording a more detailed view. Although lacking the ultrastructural resolving power of the electron microscope (EM), super-resolution microscopy helps to bridge the gap between conventional light microscopy and EM.Here we present three preparative methods for studying plasmodesmata at super-resolution using 3D-structured illumination microscopy (3D-SIM).

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata.

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5' end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.

Correlative imaging of fluorescent proteins in resin-embedded plant material.

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 µm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.