Probing plasmodesmata function with biochemical inhibitors

Chapter 14 Probing Plasmodesmata Function with Biochemical Inhibitors Rosemary G. White Abstract To investigate plasmodesmata (PD) function, a useful technique is to monitor the effect on cell-to-cell transport of applying an inhibitor of a physiological process, protein, or other cell component of interest. Changes in PD transport can then be monitored in one of several ways, most commonly by measuring the cell-to-cell movement of fluorescent tracer dyes or of free fluorescent proteins. Effects on PD structure can be detected in thin sections of embedded tissue observed using an electron microscope, most commonly a Transmission Electron Microscope (TEM). This chapter outlines commonly used inhibitors, methods for treating different tissues, how to detect altered cell-to-cell transport and PD structure, and important caveats. Key words Arabidopsis thaliana, Cell-to-cell transport, Fluorescence microscopy, Fluorescent tracer dyes, Green fluorescent protein, Microinjection, Transmission electron microscopy 1 Introduction Plasmodesmata play a key role in transmission of cytoplasmic signals from cell to cell, yet there are large gaps in our knowledge of their composition, function, and regulation. Proteomic analyses have identified a large suite of proteins potentially located within or associated with PD, but most of these await experimental confirmation of both their location and function (e.g. [1, 2], reviewed in ref. 3). Mutant analyses are beginning to reveal an additional set of proteins not located at PD but which are essential to regulate PD behavior (e.g. [4–6]). For example, mutations in the thioredoxin GAT1, a plastid-located regulator of redox status essential in PD function, tissue viability, and embryo development, cause reduced cell–cell transport [4] and mutations in ISE1, a mitochondrial DEAD-box RNA helicase [5] or ISE2, a DEVH box RNA helicase [6], cause increased cell–cell transport. Such analyses can demonstrate the role of a single gene by knockout and subsequent complementation studies, but this is difficult if the RNA or protein Manfred Heinlein (ed.), Plasmodesmata: Methods and Protocols, Methods in Molecular Biology, vol. 1217, DOI 10.1007/978-1-4939-1523-1_14, © Springer Science+Business Media New York 2015 199 200 Rosemary G. White product is vital to cell function since mutants may be lethal at the embryo or young seedling stage (e.g. [7]). Even for proteins known to be PD components, such as myosin, analysis of nonlethal mutants or green fluorescent protein- (GFP-) tagged lines has been less informative than expected (e.g. [8–10]). For these reasons, it is still common to use well-characterized chemical inhibitors of biological processes to probe PD function. For example, the role of microtubules in cell-to-cell movement of transcription factors was shown using a combination of microtubule mutants with moderate phenotypes together with chemical treatments [11]. This approach is based on the assumption that the chosen inhibitors are specific, in that they target a single enzyme or small class of enzymes, or target a specific molecular substructure within a class of proteins, lipids, or carbohydrates. For example, inhibitors of ATP activity or energy metabolism have been used to show that regulation of PD requires energy from the cell (e.g. [12– 14]). Herbicides, such as alloxan, that induce callose (β-1,3 glucan) synthesis were used to show that additional callose restricts transport via PD [4], and treatment with the callose synthesis inhibitor, 2-deoxy-D-glucose (DDG; [15, 16]) showed that reducing callose causes PD opening [17, 18]. Furthermore, new, more targeted inhibitors of specific proteins are constantly under development (e.g. small molecule inhibitors of actin; [19–21]), and could be used in plants after suitable testing. Finally, the phenotypic effects of inhibitors can be compared with similar effects of partial inhibition or knockout of proteins to suggest a role for the inhibited protein in the cell process under study. This chapter summarizes current methods for using biochemical inhibitors, proteins, and antibodies to alter PD permeability, with considerations about how to detect effects on PD, which will affect the type of plant material to use, together with known artifacts and how to avoid or ameliorate them. 1.1 Monitoring Effects on Plasmodesmata The effects of biochemicals on PD can be examined either by functional analyses or by detecting structural changes. To elucidate the role of a putative PD regulator in transport dynamics, effects on cell-to-cell transport need to be monitored, and for this, both the appropriate type of plant material and assessment protocol must be chosen (Subheading 1.2). Most experiments involve monitoring the spread of fluorescent tracer molecules from the cytoplasm of one cell into the cytoplasm of an adjacent cell or cells. These observations require a fluorescence microscope, in which shorter wavelengths of light (excitation light) are projected onto the tissue, causing emission of fluorescence from the tracer molecules at somewhat longer wavelengths of light. The appropriate optical filters must be used to select excitation and emission wavelengths for detection of specific tracer molecules, whether dyes or fluorescent proteins. In some cases, a dissecting microscope equipped for fluorescence imaging can provide sufficient resolution to discriminate Biochemical Inhibition of Plasmodesmata 201 effects of biochemical modulators on cell-to-cell transport via PD. More often, either a regular fluorescence compound microscope or a confocal laser scanning microscope (CLSM) is required to provide sufficient detail, intensity of excitation light and sensitivity of detectors to the fluorescence emission in order to locate low levels of tracers that have moved from the source cell into neighboring cells. Assessment of changes in structure generally requires imaging at sufficient magnification to resolve the small size of PD, which have cross-section diameters of the order of 40–50 nm. This can be obtained using an electron microscope, usually a Transmission Electron Microscope (TEM). The tissue must be prepared to withstand observation under an electron beam in a vacuum, and this necessitates fixation (chemically cross-linking proteins to lock them in place) of the plant material, dehydration in solvent to enable penetration of water-immiscible resins, infiltration with liquid resin, polymerization of the resin (usually by heat or UV light), ultrathin sectioning, and staining with electron-dense heavy metals. Both short- and long-term structural effects can be monitored which may require slightly different fixation protocols. Tissue processing also varies depending on the specific tissue to be examined and whether external (in the cell wall) or internal (within the cytoplasmic sleeve) details of PD structure are of interest. Instantaneous, rapid structural changes are best seen in rapidly frozen and freeze-substituted material. However, ultrastructural details within larger tissue pieces are usually poorly preserved by rapid freezing techniques, and more conventional chemical fixation can often reveal changes caused by biochemical treatments. For example, reduction in callose synthesis after treatment with the callose synthesis inhibitor DDG is easily seen in chemically fixed tissues [17, 18]. In some cases, structural modifications associated with PD maturation, e.g. in Arabidopsis leaves, can be detected by their ability to incorporate fluorescently tagged viral proteins [22]. 1.2 Choice of Plant Material 1.2.1 Studying PD Dynamic Behavior The key requirement for studying PD dynamics is usually that a fluorescent tracer can be detected moving from one cell or tissue to the next. These tracers (listed in Table 1) include fluorescent dyes, such as carboxyfluorescein diacetate (CFDA), which can be esterloaded into cells without injection (e.g. [23–26]), or loaded into the phloem via application to source tissue (e.g. [24, 27, 28]), to follow movement via PD into surrounding tissue. More conventionally, we can follow the movement of fluorescent tracers into neighboring cells after they have been microinjected or bombarded into single cells (e.g. [29, 30] and references therein [31, 32]). The latter techniques do involve some cell damage and such wound responses will affect cell–cell transport [18, 33], and quantitation is not straightforward (e.g. [34]). For application of chemical inhibitors, tissues ideally need to have external cell walls that allow 202 Rosemary G. White Table 1 Properties of fluorescent tracers used for tracking cell-to-cell transport via plasmodesmataa Fluorescencec Referenced Reference Ex, Em Use in inhibitor studies Use in other analyses Tracer mw (kDa)b Small fluorescent probes Fluoresceine Carboxyfluorescein (CF) 0.332 0.376 494, 521 492, 517 [30, 38, 60] [34, 45–47, 65] 0.443 425, 528 [12, 13, 18, 38, 69, 70] 0.524 403, 454 0.547 578, 594 [13, 18, 38] [26, 31] 490, 525g 490, 525g [45, 47, 66, 71] [45, 47, 66, 71] [12, 13, 18, 67, 70, 72, 73] [30] [30] [4, 5, 7, 31, 33] 488, 509 [4, 14, 38] [6, 7, 32, 68, 74, 75] 488, 509 [11] Lucifer yellow carbohydrazide (LYCH) 8-Hydroxypyrene-1,3,6trisulfonic acid (HPTS) Sulforhodamine 101, sulforhodamine G, lissamine rhodamine B Fluorescent-tagged amino acidsf FITC-single amino acid 0.464–0.594 FITC-amino acid polymers 0.639–1.678 Fluorescent dextrans (dextran 1–40 mw without fluorescent tag) Fluorescent proteins, tagged proteins GFP (or photoactivatable 27 GFP) or RFP SHR-GFP 86 h [23–25, 27, 31, 37, 66–68] [23, 25, 33, 42] [7, 26, 27, 67] a Fluorescent proteins are included here since they can be expressed in specific tissues, for example in phloem companion cells, and spread into adjacent cells if PD are open (e.g. spread into the root tips of Arabidopsis thaliana; [70]) b mw = molecular weight of free dye in cell cytoplasm, whether injected as dye or derived from acetate conjugate c Fluorescence excitation (Ex) and emission (Em) maxima from company catalogues d Selected references in which fluorescent probes were used together with inhibitors to analyze PD transport. Other references using injected, bombarded, or expressed fluorescent probes may be found with a literature search e From injected FITC (fluorescein isothiocyanate) or caged fluorescein (fluorescein bis-[5-carboxymethoxy-2-nitrobenzyl] ether) f Properties (charge, polarity, hydrodynamic radius) of amino acid and other probes are given in refs. 30, 31, 45; details of synthesis in ref. 76 g Ex and Em will depend on the attached fluorochrome, in this case, FITC h Ex and Em will depend on the attached fluorochrome penetration of the inhibitor, which then must be able to cross the plasma membrane if a protein in the cytoplasmic sleeve of PD is the target. Some way of assessing inhibitor penetration into cells is essential, for example, being able to see the slowing or cessation of cytoplasmic streaming. If streaming is not affected, a method of assessing treatment effects is critical and may require preliminary experiments to work out the minimum inhibitor concentration and duration of treatment needed to affect the tissue, as with any chemical treatment. For example, a dye with similar chemical Biochemical Inhibition of Plasmodesmata 203 properties to the inhibitor may be used to assess penetration in preliminary experiments, or a transgenic line expressing a visible reporter protein responsive to inhibitor-generated changes in calcium, pH, or membrane potential may be used. Since in most cases effects on transport are monitored by cellto-cell movement of visible tracers, the tissue used must be transparent enough to see the earliest signs of the tracer in adjacent cells. The roots of Arabidopsis thaliana and other small plants are useful as they are fairly transparent and cytoplasmic streaming can be monitored in root hairs. They are also very sensitive to growing conditions and uniformity of growth is essential for reasonable quantitation of transport in treated and control tissues. Examples of quantitative comparisons of Arabidopsis root growth after application of different metabolic and cytoskeleton inhibitors are shown in [35, 36]. In addition, ref. 34 presents an elegant method to rapidly quantify tracer dye flux and show dynamic behavior in timeframes similar to that measured by electrophysiological techniques (see below). Although the method is limited somewhat by currently available image capture speeds, this will be more amenable as more sensitive and faster instruments become more routinely available. Plants expressing GFP or other fluorescent protein in just one or a few cell types are very useful as they can give insights into PD regulation between developmental domains. Least traumatic to the plant and the experimental system are expressed fluorescent proteins, such as GFP (e.g. [37]). GFP is relatively large (27 kDa) compared to many signals that move through PD, although the photoconvertable forms of GFP eliminate the physical trauma of microinjection (e.g. [38, 39]). There is now a selection of smaller expressed proteins, such as iLOV (10 kDa), engineered from the light, oxygen, and voltage-sensing (LOV) domain of the blue light receptor phototropin [40] and other small flavin-based fluorescent proteins [41]. Whether monitoring injected tracers or spread of expressed proteins, in all cases, maintenance of rapid growth by optimal growing conditions is essential. Before and after application of inhibitors, cell viability should be monitored by observing cytoplasmic streaming and membrane integrity (exclusion of propidium iodide, for example). Epidermal peels, especially from Allium species, are commonly used for PD transport studies (e.g. [42–44] and references therein), because the epidermal layer is readily detached from underlying tissue for imaging. For repeatability, elongating tissue from leaves, e.g. from Allium porrum (leek), is best (e.g. [44]) since the growth and storage history of Allium cepa (onion) bulbs purchased at the supermarket is usually unknown. Alternative two-dimensional tissues are the fronds of water plants, such as Elodea canadensis [31] or Egeria densa [45] with highly permeable outer cell walls which usually lack a hydrophobic cuticle. 204 Rosemary G. White Detection of fluorescent tracer movement is most straightforward in single cell files, as found in stamen hairs of Tradescantia virginiana ([13, 29] and references therein) or Setcreasea purpurea (e.g. [30, 46, 47]), which requires maintenance of these plants in a warm greenhouse with long daylength. The multicellular trichomes of Nicotiana species are also useful (e.g. N. tabacum, [38]) since their tip cells can be loaded with tracer dye, or alternatively, they can be readily transformed to express fluorescent protein tracers, either by transient or constitutive expression. In all of these tissues, the permeability of the cell walls may be an issue and young tissues are desirable for chemical treatments. One aspect of PD behavior that is hard to monitor by following the movement of tracer dyes or proteins is their very fast dynamic behavior in response to certain stimuli or inhibitors. For example, application of calcium causes rapid closure of PD within 5 s, followed by reopening within a further 5–10 s, and the sharp opening and closing profiles can be detected only by monitoring electrical signals [48]. Here, considerable skill is required to insert microelectrodes into adjacent cells with the least cell damage or effects on PD. If a series of cells is to be studied, electrodes must be inserted into each cell of the series (e.g. [49, 50]). We assume that there is less PD damage than when tracer dyes are injected since cell membrane voltage is monitored, the electrodes are small, and there is no addition of a relatively undefined volume of tracer dye to a small volume of cytoplasm. Electrophysiology appears rarely in recent PD methods, but will be needed in future studies to understand subtle details of PD protein function. 1.2.2 Analysis of PD Structure A much greater range of tissues is used to analyze effects on PD structure. As for dynamic studies, penetration of the inhibitor must be assessed to ensure that it is affecting the tissue under study. Often, this involves baseline analysis of all cell structures, including PD, likely to be modified by inhibitor treatment, for example, noting loss of actin microfilaments in tissue treated with an actin inhibitor (e.g. [51, 52]) or loss of microtubules in tissue treated with a microtubule inhibitor (e.g. [11, 53]). A further requirement is development of a fixation, embedding, staining, and imaging protocol that allows both qualitative and quantitative assessment of inhibitor effects. Short-term treatments may affect the usual range of PD morphologies seen within a given tissue (e.g. effects of cytochalasin on young PD, [54]), and somewhat longer-term treatments (similar to effects of genetic mutations) may affect the usual change in PD morphologies seen during maturation of the tissue, or tissue interface [22]. Because the distribution, density, branching, and cell wall structures are likely to be different at each cell wall interface, and will vary with growing conditions, baseline PD structural information must be collected in each case. Biochemical Inhibition of Plasmodesmata 205 1.3 Recognizing Artifacts of Treatment or Handling A major caveat to the use of chemical inhibitors is that they are often applied to the entire tissue under study, and will affect all targeted proteins in the tissue, not just those in PD. A second reservation is that inhibitors commonly are less specific than claimed and may interfere with a number of other cell processes. With these caveats in mind, additional artifacts are common to all chemical treatments, and involve the numerous steps required to incubate tissue in the inhibitor, introduce a tracer dye into the cells, either before or after treatment, then observe the tissue for some time under fluorescence illumination. Apart from chemical side-effects, tissue preparation generally requires transfer of the tissue into the test solution in a chamber for observation or microinjection or placement onto the test agar for treatment followed by a second transfer for microinjection. Controls must be carefully monitored to quantify the effects on and/or allow for any inhibition of PD caused by handling during the experiment. Plants are surprisingly sensitive to mechanical handling, which will generate callose at PD and affect assessment of cell-to-cell transport ([17] and references therein), which can be ameliorated by treatment with DDG [17, 18, 33]. DDG is added to block callose synthesis, and does this by trapping nucleotides, here UDP, into a metabolically unusable form [55]. Inside the cell, DDG is phosphorylated to DDG-6-phosphate and then converted into UDP-2-DDG, which competes with UDP-glucose for dolichol phosphate forming dolichol-phosphate-DDG, which cannot be incorporated into β-glucans, such as callose [15, 56–58]. The experimental tissue needs to be carefully monitored to ensure that, although specific functions are inhibited or blocked by each treatment chemical, the cells examined are still alive and relatively healthy. 1.4 Selection of Biochemical Inhibitors A wide range of chemical agents, antibodies, and injected proteins has been used to modify PD function, outlined in Table 2. Also shown are references in which each inhibitor has been used, the range of concentrations applied and length of treatments. Selection of inhibitor depends upon the process under investigation, background information about the tissue and inhibitor and their interactions, and experience of the investigator. Many of these agents are not readily soluble in aqueous solutions and are first prepared and then stored as stock solutions in the appropriate nonaqueous solvent, which is often dimethylsulfoxide (DMSO). Each needs to be monitored when diluted to working concentration in aqueous solution to ensure they do not precipitate out of solution. Furthermore, since some of these nonaqueous solvents have unanticipated effects on either the action of the inhibitor, the activities of cell enzymes, or the integrity of membranes, constant monitoring of membrane integrity is Table 2 Application of biochemical inhibitors and their reported effects on cell–cell transport or PD structure Compound, mode of action, stock solution Cytochalasin B: Actin inhibitor 4.2 or 42 mM in DMSO at −20 °C Concentration (in bathing solution unless stated otherwise) 21, 42 or 104 μM for up to 90 min 42 μM for 20 min 0.21 mM in 1 % DMSO for 60 min 0.63, 6.3 or 63 μM for up to 30 min Cytochalasin D: Actin inhibitor 0.5–42 mM in DMSO at −20 °C 53 mM plus 0.5 % DMSO in buffer co-injected with dye 10 μM in 2 % DMSO co-injected with dye 2 μM co-injected with dye 2 μM for 22 days in growth medium 20 μM for 24 h in growth medium 1 mM for 1 or 6 h in growth medium Effects on transport or PD structure; specific tissue tested Detection method Reference Chloride transport [77] Transport of 5 FITC-tagged amino acid conjugates TEM analysis [45] [54] Cell–cell electrical conductance [78] No effect on transport, Setcreasea purpurea stamen hairs CF dye transport monitored for 5 min [65] Increased transport, Nicotiana tabacum mesophyll Increased transport, Nicotiana tabacum mesophyll No effect on spread of silencing signal in Arabidopsis Little effect on transport; Arabidopsis roots No effect on transport; Arabidopsis root tissues FITC-dextran transport [72] FITC-dextran transport [73] Small RNA transport [68] SHR-GFP movement between stele and endodermis Transport of injected dyes, GFP export from phloem [11] Reduced transport due to reduced cytoplasmic streaming; Chara sp. No effect (non-sig. reduction in transport); Egeria densa leaf tissues Widening of PD neck region; Nephrolepis exaltata rhizomes No effect on PD structure; Hordeum vulgare roots, Azolla pinnata roots Increased or decreased transport depending on low or high initial cell–cell conductance, respectively; Salvinia auriculata trichomes a Latrunculin A: Actin polymerization inhibitor 1 or 2 mM in DMSO at −20 °C 0.1 μM for 30 min prior to injection of virus movement protein ± dye Increased transport, Nicotiana tabacum mesophyll FITC-dextran transport [73] Latrunculin B: Actin polymerization inhibitor 1 or 2 mM in DMSO at −20 °C 10 μM for 2 h, 6 h or 3 days 25 μM in 0.5 % DMSO for 15 min 50 nM for 60 min Reduced transport (smaller diameter virus spread), N. tabacum mesophyll No effect on transport; N tabacum leaf trichomes No effect on transport; Tradescantia virginiana stamen hairs No effect on spread of silencing signal in Arabidopsis Little effect on transport; Arabidopsis roots No effect on transport; Arabidopsis root tissues Virus transport [79] LYCH transport [38] Transport of injected dyes [13] Small RNA transport [68] SHR-GFP movement between stele and endodermis Transport of injected dyes, GFP export from phloem [11] FITC dextran transport [72] FITC-dextran transport [73] RNA transport [68] GFP export from phloem a FITC-dextran transport [72] 1 μM for 22 days in growth medium 50 nM for 24 h in growth medium 25 μM in 0.5 % DMSO for 15 min Phalloidin: Actin filament stabilizer 10 μM in methanol at −20 °C 6.6 μM co-injected with dye 2 μM co-injected with dye No effect on transport; N. tabacum mesophyll Prevented increased transport due to virus movement protein, N. tabacum mesophyll Jasplakinolide: Actin filament stabilizer 1 mM in DMSO at −20 °C 1 μM for 22 days in growth medium 25 μM in 0.5 % DMSO for 15 min No effect on spread of silencing signal in Arabidopsis No effect; Arabidopsis root tissues Profilin: Sequesters actin monomers maintained in 20 mM Tris–HCI, pH 7.4, 150 mM KCI, 0.2 mM DTT 60 μM co-injected with dye Increased transport; N. tabacum mesophyll a (continued) Table 2 (continued) Compound, mode of action, stock solution 2,3 Butanedione 2-monoxime (BDM): Myosin inhibitor 300–500 mM aqueous (made fresh) N-Ethyl maleimide (NEM): Myosin inhibitor 50–100 mM aqueous (made fresh) Myosin antibodies: Block myosin function Concentration (in bathing solution unless stated otherwise) 0.1–30 mM for 1 h 1 mM for 2 h, 6 h, 3 days 1 and 30 mM for 60 min 2.5 mM for 22 days in growth medium 1 and 30 mM for 60 min 0.1 mM for up to 50 min 1 mM in for 60 min 50 μM for 22 days in growth medium 0.1–10 mM for 1–2 h in bathing solution or growth medium 0.2 mg/μl coinjected with dye 0.5 mg/ml aqueous coinjected with dye 0.5 mg/ml aqueous coinjected with dye Effects on transport or PD structure; specific tissue tested Narrowing of PD neck region; Zea mays roots No effect on transport; N. tabacum mesophyll Increased transport; T. virginiana stamen hairs No effect on spread of silencing signal in Arabidopsis Reduced transport; Arabidopsis root tissues, Elodea canadensis leaf epidermis Irreversible cessation of transport; Nitella translucens Reduced transport; T. virginiana stamen hairs Increased movement of silencing signal in Arabidopsis Increased transport; Arabidopsis root tissues, E. canadensis leaf epidermis Increased transport; Arabidopsis root epidermis, N. tabacum mesophyll Increased transport; T. virginiana stamen hairs Reduced transport; Arabidopsis root epidermis Detection method Reference TEM analysis [80] Virus transport [79] Dye transport [13] Small RNA transport [68] Transport of injected dyes, GFP export from phloem a 11 [81] C and 14C transport Dye transport [13] Small RNA transport [68] Dye transport, GFP transport a LYCH or FITC-dextran transport, respectively Dye transport [70] Dye transport a [13] Colchicine: Microtubule inhibitor Aqueous (made fresh) 0.05 mM for 20 min No effect on transport; Nitella translucens 11 [81] Oryzalin: Microtubule inhibitor 20 mM in DMSO 0.3–3.0 μM for 12 h, 1.0 μM for 6 h in growth medium Reduced transport; Arabidopsis roots SHR-GFP movement between stele and endodermis [11] Tamoxifen: Microtubule stabilizer 10 mM in DMSO 1.0 μM for 12 h, or 10 μM for 6 h in growth medium No effect on transport; Arabidopsis roots SHR-GFP movement between stele and endodermis [11] Alloxan, paraquat: Herbicides inducing H2O2 or O2− respectively Aqueous (made fresh) 1.5 mM or 1 μM, respectively, for 7 days in growth medium Reduced transport; Arabidopsis roots GFP export from root phloem [4] H2O2: Metabolic inhibitor 60 mM aqueous (made fresh each day) 0.6 mM for 2 h in growth medium Increased transport; Arabidopsis root tissue Dye transport [34] 6 mM for 2 h in growth medium Completely blocked transport; Arabidopsis root tissue Dye transport [34] 1 mM for 5 min Reduced transport in oat coleoptile parenchyma No effect, S. purpurea stamen hairs Electrical coupling [82] CF dye transport [65] Transport of FITC-tagged amino acids Transport of fluorescent dextrans LYCH transport [47] [12] Dye transport [13] NaN3: Metabolic poison 50 mM or 10 % aqueous (made fresh) 10 mM added to bathing solution immediately after dye injection 10 mM co-injected 1–10 mM in bathing solution 3 mM for 20 min 1 mM for 60 min Increased transport, S. purpurea stamen hairs Increased transport, wheat root epidermal cells Increased transport, N. tabacum leaf trichomes Increased transport; T. virginiana stamen hairs C and 14C transport [38] (continued) Table 2 (continued) Compound, mode of action, stock solution Concentration (in bathing solution unless stated otherwise) Effects on transport or PD structure; specific tissue tested Reference Electrical coupling [82] CF dye transport [65] KCN: Metabolic poison 50 mM aqueous 1 mM for 5 min Carbonyl CN trifluoromethoxyphenyl hydrazone: Metabolic poison 1 μM for 60 min Increased transport, Egeria densa leaf tissues Dye transport [45] Amiprophosmethyl: Metabolic poison 50 μM for 2 h, 6 h, 3 days No effect on transport; N. tabacum mesophyll Virus transport [78] 2-Dioxy-D-glucose (DDG): Callose synthesis inhibitor 10 mM aqueous 0.1 mM for 60 min Increased transport; T. virginiana stamen hairs Dye transport [13] Adenosine 5′-(β, γ-imido) triphosphate (AMPPNP): blocks Energy metabolism 1 mM for 2 h, 6 h, 3 days No effect on transport; N. tabacum mesophyll Virus transport [79] ATPγS: Blocks processes requiring ATP 12.5 mM co-injected with dye 0.5 mg/ml in water coinjected with dye Blocked transport; T. virginiana stamen hairs Reduced transport; Arabidopsis root epidermis Dye transport [13] Dye transport a 1 mM added to bathing solution immediately after dye injection Reduced transport in oat coleoptile parenchyma No effect on transport, S. purpurea stamen hairs Detection method a Adenosine triphosphate; ATP: alters energy balance 12.5 mM coinjected with dye Transient inhibition of transport; T. virginiana stamen hairs Dye transport [13] Ca-BAPTA: Calcium buffer 100 mM plus 50 mM Ca in buffer plus dye; co-injected with dye Reduced transport, S. purpurea stamen hairs CF dye transport [45] A23187: Calcium ionophore 100 μM for 10 min Reduced transport, Egeria densa leaf tissues Fluorescent amino acid transport [45] Trifluralin: Calcium ionophore 100 μM for 10 min Reduced transport, Egeria densa leaf tissues Fluorescent amino acid transport [45] Trifluoperazine: Calmodulin inhibitor 5 μM for 60 min Slight reduction in transport, Egeria densa leaf tissues Fluorescent amino acid transport [45] Metal cations: Ca2+, Mg2+, Sr2+ Concentrated aqueous solutions: modulate processes requiring divalent cations Co-injected with dye Slight reduction in transport, Egeria densa leaf tissues Fluorescent amino acid transport [45] RG White et al., unpublished data 212 Rosemary G. White essential to ensure the cell-to-cell movement is via PD rather than across the intervening plasma membranes and cell walls. Propidium iodide (PI) is a useful monitor of membrane integrity as it can be included in the bathing medium at very low concentrations (0.01 μg/ml) and is not fluorescent unless it binds to cell wall pectins or to DNA and other components within cells (e.g. [44]). If nuclei are fluorescent in the presence of PI, cell membrane integrity has been compromised. Although plant tissues may tolerate up to 1 % concentration of many of these solvents, it is advisable to use the lowest concentration possible (e.g. [35, 44]), and as with all other applied treatments, this must be tested with the particular tissue under study. Some tissues will tolerate up to 1 % solvent for a short time but will eventually show compromised membranes. Therefore as long as the chemical agent does not precipitate out of solution, the solvent concentration should be kept as low as possible. A literature search will show a wide range of inhibitor concentrations used on different tissues by different groups; it is best to use the lowest concentration to start with and constantly monitor cell viability. Some chemicals penetrate poorly and are best applied at a lower concentration for longer times. For example, the cytochalasins (actin inhibitors) diffuse relatively slowly into tissue and when used at moderate concentrations it can take more than 15 min for cytoplasmic streaming to cease, indicating disruption of actin filaments. By contrast, applications of cyanides or peroxides generally have immediate effects, indicating ready permeation of these small molecules throughout the tissue. 1.5 Application of Inhibitors to Different Tissues Readily water-soluble inhibitors can be dissolved in agar growth medium for treatment of Arabidopsis or other roots, or simply applied as drops of solution onto the tissue on a slide ready for imaging, or prepared for immersion of tissue in the treatment solution. Depending on the aim of the experiment, in our experience, a short treatment at relatively high concentration is preferable to a long treatment at low concentration. PD should respond rapidly, and a short treatment should reduce the chance of secondary effects via long-term inhibition of essential metabolic processes. For application to a local zone of tissue or to just a few cells, the inhibitor can be added to molten agarose, which is allowed to set as a thin sheet, and cut into 1 mm3 (or smaller/larger) cubes, which are then placed on the tissue of interest. Targeting of very few cells can be achieved by soaking resin beads with the inhibitor, as done for local application of pectin methyl esterase to Arabidopsis shoot apical meristems [59], followed by co-loading with a fluorescent tracer. Or the inhibitor can be loaded into one end of a trichome via a microinjection needle as for loading of fluorescent tracers [38]. Biochemical Inhibition of Plasmodesmata 213 Wounding artifacts can be ameliorated by co-incubation in DDG, but this can block other cell functions requiring UDPglucose, and a short treatment is preferable over a long treatment [17, 18]. This is especially true for microinjection experiments, in which application of DDG is useful to separate treatment effects from wound artifacts [17, 18, 33]. A little-used alternative to application of inhibitors to the entire tissue under study is uncaging of a caged inhibitor within a column of cells with single-photon or within a single cell using multiphoton confocal illumination of the appropriate wavelength, usually short UV (see Chapter 9). The inhibitor is only activated within a single cell and a caged dye, such as caged fluorescein (e.g. fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl) ether [CMNBfluorescein], [38, 60]), can be included to confirm the localisation of uncaging. Most chemicals can be caged, but there are relatively few publications in which caged compounds have been used as this usually requires the collaboration of a specialist organic chemistry lab to create the caged compound of interest (e.g. caged IP3 [61], caged hormones [62–64]). Several caged compounds are now available commercially and this will increase with future demand. This chapter outlines protocols for treatment of plant tissues with compounds known to alter PD permeability, together with methods for prior propagation and subsequent analysis of two species and tissues examined in the literature. Shown here are materials and protocols used by the author, but they can be modified for use with other plant tissues, and more recent protocols (e.g. Chapters 9, 16 and 17) can be substituted. 2 Materials 2.1 Materials for Plant Growth 2.1.1 MS Agar Plates Add 10 g agar to 900 ml distilled water. Add nutrients, usually Murashige and Skoog (MS) mixture (available commercially), as per supplier’s recommendation. Adjust nutrient type and strength as required for specific plant material. Autoclave. For Arabidopsis, add 20 g sucrose and 0.5 g MES, pH 6.5, dissolved in 100 ml distilled water, filter sterilize, and add to molten agar. Maintain agar at 60 °C in a waterbath until ready to dispense into 9 cm (or other) diameter petri dishes. Add inhibitors to the agar before dispensing into petri dishes (see Note 1). 1. Agar (final concentration 1 %). 2. Murashige and Skoog nutrient mix (available commercially). 3. Sucrose (final concentration 1–3 %). 4. 2-(N-Morpholino)ethanesulfonic acid (MES). 5. pH meter. 214 Rosemary G. White 6. Autoclave. 7. Sterile petri dishes, 9 cm diameter. 8. Laminar flow cabinet. 9. Household bleach (approx. 4 % NaOCl). 10. Micropore tape (1/2 or 1 in. wide). 11. Sterile distilled water. 2.2 Fluorescent Tracers and Dyes Fluorescent tracers, their molecular weights and fluorescence excitation and emission maxima are listed in Table 1. 1. Propidium iodide solution: 16 nM working solution, prepared from 16 μM stock solution in water (store in darkness at 4 °C). 2.3 Materials for Microinjection Make up all solutions using distilled water unless specified otherwise. Precise details of bathing solutions, dye loading solutions, and microinjection equipment may be varied according to the tissue under study and microinjection facilities available. 1. 0.05 % Triton X-100. 2. Incubation buffer: 0.5 mM HEPES ((N[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid]), 0.1 mM KCl, 0.1 mM CaCl2, 0.5 mM NaCl, pH 7.0. 3. Chamber slides for microinjection: for each slide, attach a coverslip (with a nontoxic, flexible compound that remains adhesive under water and is easy to remove; we find Kemdent sticky dental wax useful for this purpose), over a 25 mm diameter hole drilled in a 75 × 50 mm glass slide, 1 mm thick. 4. Double-edged sharp razor blades. 5. Fine forceps (no. 5 or no. 7). 6. Pink dental wax. 7. 1 % ultra low temperature gelling agar (gel point approx. 37 °C). 8. Ice pack. 9. 2-Deoxy-D-glucose: 10 mM stock solution in distilled water. 10. Polydisperse 4 kDa FITC-dextran (see Note 2). 11. 3 kDa cutoff microconcentrator (e.g. microcon 3, Amicon) (see Note 2). 12. Quix-sep separator (e.g. Membrane Filtration Products) or similar to separate polydisperse fluorescent dextrans into different size classes—use according to manufacturer’s instructions (see Note 2). 13. 1 kDa dialysis membrane (see Note 2). 14. Kieselgel 60F thin layer chromatography sheets. Biochemical Inhibition of Plasmodesmata 215 15. Running solution: 150:30:100:120 n-butanol:acetic acid: pyridine: water. 16. Microcapillary tubes; 1.2 mm OD borosilicate glass with inner filament. 17. Very fine micropipettes for backfilling microinjection needles (e.g. MicroFil 28 gauge syringe, World Precision Instruments [WPI]). 18. 100 mM KCl. 19. Silver/silver chloride reference electrode (e.g. Sigma). 20. Micropipette holder micromanipulator. that will fit into the hydraulic 21. 1 mM aqueous solution of dye to be injected, prepared immediately before use. 22. Aniline blue stain: 0.05 % aniline blue in 0.067 M phosphate buffer, pH 8.5. 2.4 Materials for TEM Precise details of fixative, buffer, dehydration solvent, resin, and method of polymerization will need to be adjusted depending on the tissue under study and availability of materials and preparation facilities. 1. Sharp double-edged razor blades. 2. Fine forceps (no. 5 or no. 7). 3. Pink dental wax. 4. Fixative: 2.5 % glutaraldehyde in 0.025 M phosphate buffer, pH 7.2. 5. Buffer: 0.025 M phosphate buffer, pH 7.2. 6. 1 % aqueous osmium tetroxide. 7. 10, 20, 30, 50, 70, 90, 95 % acetone solutions. 8. 100 % dry acetone (dried over molecular sieve). 9. Spurr’s resin—prepare liquid resin according to supplier’s instructions (see Note 3). 10. LRWhite resin—prepare liquid resin according to supplier’s instructions (see Note 4). 11. Ultramicrotome, e.g. Leica Ultracut 6. 12. Knife glass for sectioning. 13. Knifemaker to make glass knives. 14. Diamond knife, e.g. Diatome. 15. TEM copper grids, 3.5 mm 400 mesh, coated or uncoated (see Note 5). 216 Rosemary G. White 16. Ethanolic uranyl acetate: 1.2 % uranyl acetate in ethanol (see Notes 6 and 7). 17. Reynolds lead citrate (see Notes 6 and 7). 18. Propane (see Note 8). 19. Plunge-freezing apparatus. 20. Freeze-substitution apparatus or −80 and −20 °C freezers plus 4 °C refrigerator. 21. 100 % dry ethanol or acetone (dried over molecular sieve) maintained at −80 °C. 2.5 Imaging Hardware (See Note 9) In each case, the appropriate excitation and emission filters for fluorescent tracers must be identified, sourced, and installed, if not already available on the instrument. 1. Dissecting microscope equipped for fluorescence imaging. 2. Fluorescence compound microscope. 3. Confocal laser scanning microscope. 2.6 Microinjection Hardware (See Note 10) 1. Fluorescence inverted microscope or upright microscope. 2. Hydraulic micromanipulator, e.g. Narashige OR-60. 3. Flaming/Brown type electrode puller, e.g. Sutter P67. 4. Iontophoresis apparatus, comprising Electrometer (e.g. S7071A, WPI) plus miniMainframe (WPI). 5. Multimeter to monitor membrane potential. 2.7 Plant Material (See Note 11) 1. Arabidopsis thaliana L. plants. Grow on standard MS agar plates (see Subheading 2.1.1). 2. Tradescantia virginiana L. Maintain under long daylength at 25 °C with minimal fertilization, and regular watering and pruning for vigorous growth and continued flowering. 2.8 Biochemical Inhibitors (See Note 12) Biochemicals that target different known or putative components or regulators of plasmodesmata are listed in Table 2. 1. Treatment solution 1 for Arabidopsis thaliana root epidermal cells: working concentration of the selected biochemical inhibitor as defined in Table 2 in MS nutrient mix made according to manufacturer’s recommendation, 2.5 mM MES, pH 6.5. 2. Treatment solution 2 for Tradescantia virginiana stamen hairs: working concentration of the selected biochemical inhibitor as defined in Table 2 in 0.5 mM HEPES, 0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl pH 7. Biochemical Inhibition of Plasmodesmata 3 217 Methods 3.1 Selection, Growth, and Chemical Treatment of Plant Material (See Note 12) 3.1.1 Arabidopsis thaliana Roots The treatments of two types of tissue, i.e. three-dimensional roots, and one-dimensional filaments, are detailed below (see Note 13). 1. Prepare MS agar plates (see Subheading 2.1.1). 2. Seed sterilization: (a) Place 30–100 Arabidopsis seeds in a 1.5–2.0 ml microfuge tube. Place tube in a vacuum desiccator in a fume hood. (b) Place 100 ml household bleach in a 250 ml beaker next to the Arabidopsis seeds in the desiccator. Add 3 ml concentrated HCl to the bleach. Immediately place the lid on the desiccator. (c) After 3.0–3.5 h, remove seeds from desiccator and transfer immediately into a laminar flow cabinet. Allow to sit for 5–10 min to ensure that residual Cl2 gas dissipates. (d) Scatter seeds sparsely on MS agar plates, seal with micropore tape. Place in the dark at 4 °C for 24–48 h to stratify. 3. Alternatively, in a laminar flow cabinet, surface sterilize Arabidopsis seeds in 30 % bleach containing 0.1 % Triton X-100 for 10 min followed by 4 × 10 min rinses in sterile distilled water. Dispense seeds individually onto agar using wide sterile pipette tips. Seal plates with micropore tape and place in the dark at 4 °C for 24–48 h to stratify. 4. After stratification, place plates on their sides in 22 °C growth room in constant light (or other light regime) so roots will grow vertically down along the agar surface. Roots can be used 5–7 days after germination. 5. Prepare agar plates containing inhibitor(s) (see Note 1). Either a) transfer Arabidopsis seedlings onto this agar or b) place a small cube, no greater than 1 mm on each side, of this agar onto each Arabidopsis root while still on MS agar (see Note 14). 6. Alternatively, incubate roots in treatment solution 1 with or without inhibitor for 1 h. Pretreatment with 1 mM DDG for 1 h will reveal whether the inhibitor stimulates production of wound-induced callose (also see Note 15). 3.1.2 Tradescantia virginiana L. Stamen Hairs 1. Selection of the appropriate size of flower bud is trial and error at first. The stamen hairs need to be young, or the outer walls become hydrophobic, virtually impervious to inhibitors and difficult to microinject. However, they need to be sufficiently mature to show vigorous cytoplasmic streaming (see Note 16). 218 Rosemary G. White 2. Remove an appropriately sized bud from the plant. Place into a drop of distilled water on a piece of dental wax. Slice off the pedicel, cutting through the base of the ovary. 3. Press gently on the top of the bud to squeeze out the remainder of the ovary and the two young stamens. 4. Carefully remove the pollen sacs from the filament, using a sharp razor blade. Dip each filament with attached stamen hairs into 0.05 % Triton X-100 for 1–2 s. This treatment greatly enhances penetration of treatment chemicals without apparent damage to the cells. 5. Transfer the filament with attached stamen hairs into treatment solution 2 for the length of time desired, less 15 min. With a 45 min incubation the tissue is allowed to recover for 15 min on the microscope before imaging and microinjection, giving a total of 1 h treatment. Bathe control filaments and hairs in the treatment solution 2 without inhibitor for an equivalent period of time (see Note 15). 3.2 Assessing PD Permeability This section outlines procedures for microinjection of dyes, imaging cell–cell dye transport, and methods to quantify transport (see Note 17). 3.2.1 Microinjection of Tracer Dyes Arabidopsis thaliana Root Epidermal Cells (See Note 11) 1. Cut Arabidopsis seedlings with supporting agar from the Petri dish and place vertically in treatment solution 1 (with or without inhibitor, see Note 15). Submerge just the tips of the roots in solution. Avoid disturbance to the roots by handling the supporting agar rather than the seedlings themselves. 2. If using dissected tissues, cut Arabidopsis roots from the cotyledons at least 10 mm away from the planned injection site. 3. After incubation in treatment solution 1, mount the roots horizontally in the well of a chamber slide 4. Hold roots in place by embedding in a thin layer of cool but still-molten 1 % ultra low temperature gelling agar (Type IX, Sigma), and set it by placing on ice (or use an ice-pack) for 5–10 s. 5. Immediately add treatment solution 1 (with or without inhibitor) to the well and allow the tissues to rest for 15 min prior to injection (see Note 15). 6. Prepare 1 mM dye solution for injection. If not using FITCdextrans, go to step 13. 7. Prepare FITC-dextrans from polydisperse 4 kDa FITC-dextran dissolved in water to a concentration of 20 mM and separated using a 3 kDa cutoff microconcentrator (see Note 2). Biochemical Inhibition of Plasmodesmata 219 8. Dilute the solution remaining in the filter to approximately 1 mM. This is the fraction larger than 3 kDa. 9. Dialyse the filtered solution (<3 kDa) in a separator using a 1 kDa dialysis membrane to give the 1–3 kDa fraction. 10. If required, check that free FITC has been removed from the purified dextrans by thin layer chromatography (TLC) using the protocol of [31]. 11. For TLC, separate the FITC-dextran solution on Kieselgel 60 F thin layer chromatography sheets using a running solution of 150:30:100:120 n-butanol:acetic acid:pyridine:water. Visualize the purified dextran with UV illumination; it should be seen as a single fluorescent spot. 12. Pull microinjection needles on a Flaming/Brown micropipette puller (or equivalent) (see Note 18). 13. Fill the tips of the needles with dye by capillary action using a very fine filament syringe (e.g. Microfil syringe, 28 gauge) that fits easily into the injection needle. 14. Back-fill the needles with 100 mM KCl. 15. Mount the needle on a silver chloride half-cell connected to an electrometer to detect membrane potential (e.g. Electrometer module with miniMainframe; WPI). 16. Use a return electrode filled with 3 mM KCl connected via a silver chloride half-cell. 17. Bring the tip of the injection needle up to a selected cell and tap the microscope gently so that the needle penetrates the cell wall. 18. Monitor cell membrane potential using a multimeter connected to the electrometer. A drop in this reading indicates penetration into the cytoplasm, but a substantial drop indicates that the needle has penetrated through into the vacuole or injury to the cell. Discard cells with vacuolar penetration or injury. 19. Inject dye by applying a brief, small (approx. 1 nA for 1–5 s, longer if required) current to the microinjection needle. Use a negative current for lucifer yellow and sulforhodamine 101. Use a positive current for the FITC-dextrans. 20. Test the viability of the injected tissue by releasing a small amount of dye into the microinjection buffer surrounding the tissue. If tissue is damaged it will take up dye from the medium, showing that the membranes have become leaky. 21. If assessing callose deposition, stain tissue with 0.05 % aniline blue in 0.067 M phosphate buffer, pH 8.5. Apply the stain immediately before observation on a fluorescence microscope. 220 Rosemary G. White Tradescantia virginiana Stamen Hairs 1. Prepare chamber slides by first attaching a coverslip over a 25 mm diameter hole drilled in a 75 × 50 mm glass slide. 2. Prepare and incubate stamen hairs in treatment solution 2 (see Note 15). 3. After incubation in treatment solution 2, mount the hairs in the well of a chamber slide. 4. Hold hairs in place by embedding in a thin layer of cool but still molten 1 % ultra low temperature gelling agar, and set the agar by placing on ice (or on an ice pack) for 5–10 s. 5. Immediately add fresh treatment solution 2 to the well and allow the tissues to rest for 15 min prior to injection. 6. Continue as described in steps 6–21 above for Arabidopsis roots. 3.3 Assessing Changes in PD Structure 3.3.1 PD Structure in Roots: Conventional Chemical Fixation Because tissue handling and noncryo fixation procedures will generate additional callose at the PD neck, either pretreatment with DDG, or its addition to the treatment solution, is very useful to separate fixation-induced changes in PD structure from treatmentinduced changes (e.g. [17, 18]). 1. Incubate live germinated seeds with root attached in 0.1 mM DDG for 1 h to prevent fixation-induced formation of callose (see Note 15). 2. Incubate live tissues in inhibitor in water (see Table 2), with or without 0.1 mM DDG, for 1 h. 3. Fix specimens in 2.5 % glutaraldehyde in 0.025 M phosphate buffer, pH 7.2, for 2 h. 4. Rinse in phosphate buffer, three times, 10 min each time. 5. Incubate in 1 % aqueous osmium tetroxide for 2 h. 6. Rinse in distilled water, two times, 10 min each time. 7. Dehydrate in a progressive acetone or ethanol series, 20 min each step (see Note 19). 8. Infiltrate slowly in liquid Spurr’s resin (see Notes 3, 4, and 20) over 2 days. 9. Polymerise at 50 °C overnight (see Note 21). 10. Collect ultrathin sections 80–90 nm thick on uncoated or coated copper grids. 11. Stain with 1.2 % ethanolic uranyl acetate for 10 min (see Note 6). 12. Rinse briefly in distilled water. 13. Stain with Reynold’s lead citrate for 8–12 min (see Note 6). 14. Rinse briefly with warm (approx. 30 °C) distilled water. 15. View on a TEM according to the manufacturer’s instructions. Biochemical Inhibition of Plasmodesmata 221 16. Take measurements of the neck regions of PD directly from negatives (or on-screen images) (see Note 22). 17. In most cases, comparison using the Student’s t-test (p < 0.01) is sufficient to assess the statistical significance of differences in PD dimensions [17]. 3.3.2 PD Structure in Roots: Cryofixation In general, cryofixation followed by freeze-substitution is optimal for faithful preservation of PD architecture. Even here, prior incubation in DDG will show the significance of tissue handling in generation of artefactual callose (see Note 15). 1. Incubate live germinated seeds with root attached in 0.1 mM DDG for 1 h to prevent handling-induced formation of callose. 2. Incubate in water 1 with or without inhibitor (see Table 2) for 1 h. 3. Immediately plunge-freeze in liquid propane, in a rapid-freezing apparatus, according to the manufacturer’s recommendations. 4. Freeze-substitute in a commercial freeze-substitution apparatus according to the manufacturer’s recommendations, or if this is unavailable, continue steps 5–9 below. 5. Transfer to dry ethanol or acetone (dried over molecular sieve) at −80 °C. 6. Allow to freeze-substitute for 1–3 days at −80 °C, change solvent once after the first 12 h. 7. Transfer to −20 °C freezer for 24 h. 8. Transfer to 4 °C refrigerator for 24 h. 9. Bring to room temperature and proceed as in steps 8–17 of Subheading 3.3.1. 4 Notes 1. Add the inhibitor to the agar before dispensing into petri dishes but after the agar has been autoclaved. In most cases the treatment agar does not need to be sterile since treatments are short. If sterility is essential, filter the drug solution through a 0.22 μm filter. Make a stock solution of ten times the final concentration and add 3 ml of this to 27 ml of agar solution to cover the base of one 9 cm diameter petri dish. 2. This protocol uses a 4 kDa polydisperse FITC-dextran mixture as starting material to prepare dextrans of different molecular size classes. For dextrans of larger size class, use a larger polydisperse fluorescent dextran mixture as starting material, with dialysis membranes of the required size cutoff to prepare dextrans of specific size-classes. Dextrans with different fluorescent tags, e.g. rhodamine, Texas Red, can be used also. Separate 222 Rosemary G. White the polydisperse dextrans into different size classes using a separator and dialysis membranes according to the manufacturers’ instructions. 3. The original formulation of Spurr’s resin is no longer available, and the new formulation, available from several suppliers, should be tested before use to ascertain the best component ratios, and infiltration and polymerization regimes for each plant tissue under study. 4. Although London Resin White (LR White) resin is somewhat less stable than Spurr’s resin under the electron beam, it is perfectly satisfactory if used with coated grids. LR White is considerably less viscous and generally requires shorter infiltration times. LR White Medium Grade is appropriate for most tissues. 5. Those experienced in TEM preparation techniques may prepare their own coated grids, but perfectly satisfactory ones are available from a number of suppliers. 6. Different TEM laboratories have different preferences for uranyl acetate and Reynolds lead citrate [83] staining which may differ from the formulations and staining times suggested here. Use the formulations recommended on-site and only modify if needed. 7. Spurr’s and LR White resin sections require somewhat different staining times with uranyl acetate and lead citrate, and these must be determined empirically. 8. A small (D-size) cylinder of propane gas will be required whether using a home-made liquid propane freezing device or a commercially available device. 9. Most research facilities will have one or more options for fluorescence imaging. Instruction in use of the instruments should be obtained on site. 10. The microinjection hardware currently available is highly integrated, such that microiontophoresis current generators and micropressure systems require no prior assembly from separate components. Both types of systems have built-in current detection for monitoring membrane potential during microneedle insertion and injection of tracers. In each case, instruction in use of specific instruments should be obtained on site. 11. Plant materials listed here have been used by the author. The protocols will need to be modified for application to other plant tissues, for example, seedling roots of other species, epidermal peels from Allium species (onion—A. cepa or leek— A. porrum), or fronds from aquatic plants such as Egeria densa or Elodea canadensis. 12. These protocols are appropriate for treatments with small molecules that can permeate throughout plant tissues. In addition, Biochemical Inhibition of Plasmodesmata 223 proteins and antibodies may be pressure-injected to assess their potential effects on PD. 13. For quantitative comparison of inhibitor effects, it is essential that plants can repeatedly be grown very uniformly. Rapid growth is another key criterion, as is membrane integrity during chemical treatment. Healthy cells with undamaged walls and membranes are essential. This can be tested by daily growth measurement of roots, and by incubation in dyes that are membrane impermeant, such as propidium iodide—entry of dye into the cells indicates that the plasma membrane has reduced integrity. Membrane integrity is critical to interpretation of results in which altered cell–cell transport via PD rather than altered cell–cell membrane transport is desired. 14. For treatment, transfer plants to agar containing the inhibitor. Protect the shoots from treatment by first placing a narrow strip of parafilm on the agar and ensuring the shoots lie on this strip. For treatment of a small section of tissue, place small inhibitor-soaked agar cubes or resin beads onto the tissue. 15. Manipulation of tissues during experiments and insertion of microinjection needles induces callose [18, 33] as does chemical fixation [17]. Pretreatment with 0.1 mM DDG in water or treatment solution with or without inhibitor is a useful additional control treatment. 16. A general rule is that the flower buds should be no more than 2–3 mm long and about 2–3 days before anthesis (see refs. 13, 18 and references therein). 17. It is essential to learn microinjection in a laboratory with proven success in using this technique. Each laboratory has their own set of instruments and protocols, and it is impossible to pick up essential technical skills without hands-on training. Dye flux can be quantitated using fluorescence recovery after photobleaching (FRAP) of a small tracer, CF, ester-loaded into root cells by incubating in CFDA [34]. Other quantification techniques for measuring very rapid changes in flux have generally involved measuring changes in electrical coupling (e.g. [48–50]). Although the latter technique has fallen from favor, it is indispensible for detecting fast changes in PD permeability, and to identify rapid PD opening and closing events. In unpublished observations, we have observed interactions between tracer dyes and certain inhibitors that alters their fluorescence intensity. This should be checked, for example, by applying dye to agar pre-soaked with either water or inhibitor. 18. Preparation of the correct-sized microinjection needle is determined empirically on the individual electrode puller. The needles 224 Rosemary G. White must be sharp enough to penetrate through the cell wall, but strong enough to penetrate without breaking. In most cases, needles can be used only once, because the tip breaks off as it traverses the cell wall, and after entry, wound responses in the cytoplasm block the tip, preventing further injection of dye or other components. 19. Both Spurr’s and LR White resins are soluble in either ethanol or acetone. While acetone often produces superior results when used as infiltration solvent, ethanol is less toxic and may be preferred for safety reasons. 20. Slow infiltration is critical to excellent preservation, and infiltration times must be longer for denser, larger tissues. 21. LR White resin requires polymerization in the absence of oxygen, either in an oven or under UV light. The capsules containing resin to be polymerised must either be sealed, to prevent entry of oxygen, or must be maintained in a nitrogen gas atmosphere during polymerization. 22. Measure the outer diameter at the narrower section of PD deep within the cell wall, from the outer layer of the plasma membrane on each side as reference. Then measure at the neck region, where PD open to the cell cytoplasm, also from the outer layer of the plasma membrane on each side. Differences may be obvious (e.g. [54]) or more subtle (e.g. [17, 80]). Acknowledgements Thanks to Janine Radford at Monash University; Robyn Overall, Terena Holdaway-Clarke, Debbie Barton, and other Sydney University colleagues; Mark Talbot, summer students, and others in my lab at CSIRO Plant Industry for tips, tricks, comments, and assistance along the way. References 1. Faulkner CR, Blackman LM, Cordwell SJ, Overall RL (2005) Proteomic identification of putative plasmodesmatal proteins from Chara corallina. Proteomics 5:2866–2875 2. Bayer EM, Bottrill AR, Walshaw J et al (2006) Arabidopsis cell wall proteome defined using multidimensional protein identification technology. Proteomics 6:301–311 3. Faulkner C, Maule A (2011) Opportunities and successes in the search for plasmodesmal proteins. Protoplasma 248:27–38 4. Benitez-Alfonso Y, Cilia M, San RA et al (2009) Control of Arabidopsis meristem devel- opment by thioredoxin-dependent regulation of intercellular transport. Proc Natl Acad Sci U S A 106:3615–3620 5. Stonebloom S, Burch-Smith T, Kim I et al (2009) Loss of the plant DEAD-box protein ISE1 leads to defective mitochondria and increased cell-to-cell transport via plasmodesmata. Proc Natl Acad Sci U S A 106: 17229–17234 6. Kobayashi K, Otegui MS, Krishnakumar S et al (2007) INCREASED SIZE EXCLUSION LIMIT2 encodes a putative DEVH box RNA helicase involved in plasmodesmata function Biochemical Inhibition of Plasmodesmata 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. during Arabidopsis embryogenesis. Plant Cell 19:1885–1897 Kim I, Hempel FD, Sha K et al (2002) Identification of a developmental transition in plasmodesmatal function during embryogenesis in Arabidopsis thaliana. Development 129: 1261–1272 Avisar D, Prokhnevsky AI, Dolja VV (2008) Class VIII myosins are required for plasmodesmatal localization of a closterovirus hsp70 homolog. J Virol 82:2836–2843 Golomb L, Abu-Abied M, Belausov E, Sadot E (2008) Different subcellular localizations and functions of Arabidopsis myosin VIII. BMC Plant Biol 8:3 Yokota E, Ueda S, Tamura K et al (2009) An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells. J Exp Bot 60: 197–212 Wu S, Gallagher KL (2013) Intact microtubules are required for the intercellular movement of the SHORTROOT transcription factor. Plant J 74:148–159 Cleland RE, Fujiwara T, Lucas WJ (1994) Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress. Protoplasma 178:81–85 Radford JE, White RG (2011) Inhibitors of myosin, but not actin, alter transport through Tradescantia plasmodesmata. Protoplasma 248: 205–216 Stonebloom S, Brunkard JO, Cheung AC et al (2012) Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata. Plant Physiol 158: 190–199 Gale EF, Wayman F, Orlean PA (1984) The action of 2-deoxy-D-glucose on the incorporation of glucose into (1-3)-ß-glucan in stationary phase cultures of Candida albicans. J Gen Microbiol 130:3303–3311 Jaffe MJ, Leopold AC (1984) Callose deposition during gravitropism of Zea mays and Pisum sativum and its inhibition by 2-deoxyD-glucose. Planta 161:20–26 Radford JE, Vesk M, Overall RL (1998) Callose deposition at plasmodesmata. Protoplasma 201:30–37 Radford JE, White RG (2001) Effects of tissuepreparation-induced callose synthesis on estimates of plasmodesma size exclusion limits. Protoplasma 216:47–55 Fenteany G, Zhu S (2003) Small-molecule inhibitors of actin dynamics and cell motility. Curr Top Med Chem 3:593–616 Baggett AW, Cournia Z, Han MS et al (2012) Structural characterization and computer- 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 225 aided optimization of a small-molecule inhibitor of the Arp2/3 complex, a key regulator of the actin cytoskeleton. ChemMedChem 7: 1286–1294 Bonello T, Coombes J, Schevzov G et al (2012) Therapeutic targeting of the actin cytoskeleton in cancer. In: Cytoskeleton and human disease. Humana, Totowa, NJ, pp 181–200 Fitzgibbon J, Beck M, Zhou J et al (2013) A developmental framework for complex plasmodesmata formation revealed by large-scale imaging of the Arabidopsis leaf epidermis. Plant Cell 25:57–70 Duckett CM, Oparka KJ, Prior DAM et al (1994) Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development. Development 120:3247–3255 Oparka KJ, Duckett CM, Prior DAM, Fisher DB (1994) Realtime imaging of phloem unloading in the root tip of Arabidopsis. Plant J 6:759–766 Wang N, Fisher DB (1994) The use of fluorescent tracers to characterize the postphloem transport pathway in maternal tissues of developing wheat grains. Plant Physiol 104:17–27 Wang HL, Offler CE, Patrick JW, Ugalde TD (1994) The cellular pathway of photosynthate transfer in the developing wheat grain. I. Delineation of a potential transfer pathway using fluorescent dyes. Plant Cell Environ 17: 257–266 Wright KM, Oparka KJ (1996) The fluorescent probe HPTS as a phloem-mobile, symplastic tracer, an evaluation using confocal laser scanning microscopy. J Exp Bot 47:439–445 Wang X, Sager R, Cui W et al (2013) Salicylic acid regulates plasmodesmata closure during innate immune responses in Arabidopsis. Plant Cell 35:2315–2329 Tyree MT, Tammes PML (1975) Translocation of uranin in the symplasm of staminal hairs of Tradescantia. Can J Bot 53:2038–2046 Tucker EB (1982) Translocation in the staminal hairs of Setcreasea purpurea. I. A study of cell ultrastructure and cell-to-cell passage of molecular probes. Protoplasma 113: 193–201 Goodwin PB (1983) Molecular size limit for movement in the symplast of the Elodea leaf. Planta 157:124–130 Faulkner C, Petutschnig E, Benitez-Alfonso Y et al (2013) LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proc Natl Acad Sci U S A 110:9166–9170 Storms MHM, van der Schoot C, Prins M et al (1998) A comparison of two methods of 226 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. Rosemary G. White microinjection for assessing altered plasmodesmal gating in tissues expressing viral movement proteins. Plant J 13:131–140 Rutschow HL, Baskin TI, Kramer EM (2011) Regulation of solute flux through plasmodesmata in the root meristem. Plant Physiol 155: 1817–1826 Baskin TI, Bivens NJ (1995) Stimulation of radial expansion in arabidopsis roots by inhibitors of actomyosin and vesicle secretion but not by various inhibitors of metabolism. Planta 197:514–521 Baskin TI, Wilson JE (1997) lnhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules. Plant Physiol 113:493–502 Roberts AG, Cruz SS, Roberts IM et al (1997) Phloem unloading in sink leaves of Nicotiana benthamiana, comparison of a fluorescent solute with a fluorescent virus. Plant Cell 9:1381–1396 Christensen NM, Faulkner C, Oparka K (2009) Evidence for unidirectional flow through plasmodesmata. Plant Physiol 150:96–104 Urbanus SL, Dinh QK, Angenent GC, Immink RGH (2010) Investigation of MADS domain transcription factor dynamics in the floral meristem. Plant Signal Behav 5:1260–1262 Chapman S, Faulkner C, Kaiserli E et al (2008) The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proc Natl Acad Sci U S A 105: 20038–20043 Mukherjee A, Walker J, Weyant KB, Schroeder CM (2013) Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters. PLoS One 8(5):e64753 Palevitz BA, Hepler PK (1985) Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow. Planta 164:473–479 Scott AS, Wyatt S, Tsou P-L et al (1999) Model system for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26:1125–1132 Collings DA (2013) Subcellular localization of transiently expressed fluorescent fusion proteins. In: Chapter 16 in Legume genomics: methods and protocols. Methods in molecular biology, vol 1069. doi:10.1007/978-1-62703-613-9_16 Erwee MG, Goodwin PB (1983) Characterisation of the Egeria densa Planch. leaf symplast. Inhibition of the intercellular movement of fluorescent probes by group II ions. Planta 158(320):328 Tucker EB (1990) Calcium-loaded 1,2-bis(2aminophenoxy)ethane-N, N, N', N'-tetraacetic acid blocks cell-to-cell diffusion of carboxyfluo- 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. rescein in staminal hairs of Setcreasea purpurea. Planta 182:34–38 Tucker EB (1993) Azide treatment enhances cell-to-cell diffusion in staminal hairs of Setcreasea purpurea. Protoplasma 174:45–49 Holdaway-Clarke TL, Walker NA, Hepler PK, Overall RL (2000) Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata. Planta 210: 329–335 Spanswick RM (1972) Electrical coupling between cells of higher plants: a direct demonstration of intercellular communication. Planta 102:215–227 Overall RL, Gunning BES (1982) Intercellular communication in Azolla roots: II. Electrical coupling. Protoplasma 111:151–160 Staiger CJ (2000) Signaling to the cytoskeleton in plants. Annu Rev Plant Physiol Plant Mol Biol 51:257–288 Collings DA (2008) Crossed wires: interactions and cross-talk between the microtubule and microfilament networks in plants. In: Nick P (ed) Plant cell monographs: plant microtubules, development, and flexibility. Springer, Berlin, pp 47–82 Heinlein M, Padgett HS, Gens JS et al (1998) Changing patterns of localization of the Tobacco Mosaic Virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10: 1107–1120 White RG, Badelt K, Overall RL, Vesk M (1994) Actin associated with plasmodesmata. Protoplasma 180:169–184 Mooré D (1980) Effects of hexose analogues on fungi: mechanisms of inhibition and of resistance. New Phytol 87:487–515 Breier A, Crane AM, Kennedy C, Sokoloff L (1993) The effects of pharmacologic doses of 2-deoxy-D-glucose on local cerebral blood flow in the awake, unrestrained rat. Brain Res 618:277–282 Datema R, Schwartz RT, Rivas LA, PontLezica R (1983) Inhibition of β-1,4-glucan biosynthesis by deoxyglucose. Plant Physiol 71:76–81 Stone BA, Clarke AE (1992) Chemistry and biology of (1→3)-β-glucans. La Trobe University Press, Bundoora, VIC Peucelle A, Louvet R, Johansen JN et al (2008) Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins. Curr Biol 18:1943–1948 Martens HJ, Hansen M, Schulz A (2004) Caged probes: a novel tool in studying Biochemical Inhibition of Plasmodesmata 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. symplasmic transport in plant tissues. Protoplasma 223:63–66 Gilroy S, Read N, Trewavas AJ (1990) Elevation of cytoplasmic calcium by caged calcium or caged inositol trisphosphate initiates stomatal closure. Nature 346:769–771 Ward JL, Beale MH (1995) Caged plant hormones. Phytochemistry 38:811–816 Allan AC, Ward JL, Beale MH, Trewavas AJ (1998) Caged plant growth regulators. Methods Enzymol 291:474–483 Kusaka N, Maisch J, Nick P et al (2009) Manipulation of intracellular auxin in a single cell by light with esterase-resistant caged auxins. ChemBioChem 10:2195–2202 Tucker EB (1987) Cytoplasmic streaming does not drive intercellular passage in staminal hairs of Setcreasea purpurea. Protoplasma 137: 140–144 Tucker EB, Tucker JE (1993) Cell-to-cell diffusion selectivity in staminal hairs of Setcreasea purpurea. Protoplasma 174:36–44 Zhu T, Lucas WJ, Rost TL (1998) Directional cell-to-cell communication in the Arabidopsis root apical meristem. I. An ultrastructural and functional analysis. Protoplasma 203:35–47 Liang DC, White RG, Waterhouse PM (2012) Gene silencing in Arabidopsis spreads from the root to the shoot, through a gating barrier, by template-dependent, non-vascular, cell to cell movement. Plant Physiol 159:984–1000 Oparka KJ, Prior DAM (1988) Movement of lucifer yellow CH in potato tuber storage tissue: a comparison of symplastic and apoplastic transport. Planta 176:533–540 Volkmann D, Mori T, Tirlapur UK et al (2003) Unconventional myosins of the plant-specific class VIII: endocytosis, cytokinesis, plasmodesmata/pit-fields, and cell-to-cell coupling. Cell Biol Int 27:289–291 Erwee MG, Goodwin PB (1984) Characterisation of the Egeria densa leaf symplast: response to plasmolysis, deplasmolysis and to aromatic amino acids. Protoplasma 122: 162–168 Ding B, Kwon M-O, Warnberg L (1996) Evidence that actin filaments are involved in 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 227 controlling the permeability of plasmodesmata in tobacco mesophyll. Plant J 10:157–164 Su S, Liu Z, Chen C et al (2010) Cucumber Mosaic Virus movement protein severs actin filaments to increase the plasmodesmal size exclusion limit in tobacco. Plant Cell 22: 1373–1387 Stadler R, Wright KM, Lauterbach C et al (2005) Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots. Plant J 41:319–331 Imlau A, Truernit E, Sauer N (1999) Cell-tocell and long distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11:309–322 Simpson I (1978) Labelling of small molecules with fluorescein. Anal Biochem 89: 304–305 Bostrom TE, Walker NA (1975) Intercellular transport in plants I. The flux of chloride and the electric resistance in Chara. J Exp Bot 26: 767–782 Krasavina MS, Ktitorova IN, Burmistrova NA (2001) Electrical conductance of cell-to-cell junctions and the cytoskeleton of plant cells. Russ J Plant Physiol 48:741–748 Kawakami S, Watanabe Y, Beachy RN (2004) Tobacco mosaic virus infection spreads cell to cell as intact replication complexes. Proc Natl Acad Sci U S A 20:6291–6296 Radford JE, White RG (1998) Localization of a myosin-like protein to plasmodesmata. Plant J 14:743–750 Dale N, Lunn G, Fensom DS, Williams EJ (1983) Rates of axial transport of 11C and 14C in Characean cells: faster than visible streaming? J Exp Bot 34:130–143 Drake G (1979) Electrical coupling, potentials, and resistances in oat coleoptiles: effects of azide and cyanide. J Exp Bot 30:719–725 Reynolds ES (1963) Electron-opaque stain in electron microscopy. J Cell Biol 17:208–212