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Quantifying the Capacity of Phloem Loading in Leaf Disks with [14C]Sucrose
用[14C]蔗糖定量叶盘韧皮部装载能力   

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Plant Physiology
Jan 2016

Abstract

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (this protocol), (2) the relative rates of carbon loading and transport through the phloem (Yadav et al., 2017a), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (Yadav et al., 2017b). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation (Dasgupta et al., 2014; Khadilkar et al., 2016). In this protocol, Arabidopsis leaf disks isolated from mature rosette leaves are infiltrated with a buffered solution containing [14C]Suc. Suc transporters (SUCs or SUTs) load Suc into the phloem and excess, unloaded Suc in the leaf disk is then washed away. Loading of labeled Suc into the veins is visualized by autoradiography of lyophilized leaf disks and quantified by scintillation counting. Results are expressed as disintegration per minute per unit of leaf disk fresh weight or area.

Keywords: Arabidopsis (拟南芥), Phloem loading (韧皮部装载), Phloem transport (韧皮部运输), [14C]Suc uptake ([14C]蔗糖摄取), Leaf disks (叶盘)

Background

Transport of photoassimilates from source to sink organs is essential for normal growth and maintenance of whole plants. Phloem loading in leaves is the delivery of photoassimilate synthesized in mesophyll cells to the companion cells (CC) and sieve elements (SE) of the phloem vasculature system. Three distinct loading mechanisms are recognized. Two of these expend energy to accumulate high concentrations of sugar in the CC and SEs and generate a high hydrostatic pressure in source-leaf phloem. The first is apoplastic phloem loading, in which Suc (and/or sugar alcohols in some species) is loaded across the plasma membrane from the cell wall space (i.e., the apoplast) into the CCs at the expense of the proton motive force (Giaquinta, 1983). The second is polymer trapping, in which Suc diffuses into the phloem through specialized plasmodesmata and is converted to oligosaccharides that are too large to diffuse back out (Turgeon, 1996). The third mechanism is passive loading, in which the highest solute concentrations are in the mesophyll cells and plasmodesmata provide an open path for passive movement into the CCs and SEs (Rennie and Turgeon, 2009).

Due to the central role of phloem loading and transport to plant physiology and productivity, it is desirable to have reliable methods to identify and quantify the contents of the phloem and the rates of transport. In addition, phloem loading and transport are targets for biotechnology and metabolic engineering to enhance productivity (Ainsworth and Bush, 2011; Cao et al., 2013; Dasgupta et al., 2014; Zhang et al., 2015; Yadav et al., 2015). Quantitatively assessing the rates and capacity of phloem loading and transport in natural and engineered systems is difficult for several reasons. As examples, CCs and SEs are imbedded in surrounding tissues and are very narrow relative to surrounding non-phloem cells; the phloem is under high pressure and seals rapidly when damaged; collected phloem sap is usually contaminated with the content of other cells; because transport is a dynamic process, estimates of phloem content do not indicate rates of transport (Turgeon and Wolf, 2009; Dinant and Kehr, 2013; Tetyuk et al., 2013). C isotope 11C, 13C and 14C, in CO2 or labeled sugars, have provided, and continue to provide, critical information on phloem loading mechanisms (Sovonick et al., 1974; Turgeon and Gowan, 1990; Turgeon and Medville, 1998), as well as quantitative information on the rates of loading and transport (Thorpe and Minchin, 1988; Karve et al., 2015; Dersch et al., 2016).

Arabidopsis loads Suc from the apoplast with the Suc transporter (SUT) encoded by AtSUC2 (Truernit and Sauer, 1995; Gottwald et al., 2000; Srivastava et al., 2008). AtSUC2 and other transporters have been assessed in Saccharomyces cerevisiae and Xenopus laevis oocytes as heterologous systems to establish Michaelis Menten kinetic parameters (reviewed in [Ayre, 2011]). While valuable for comparing the activities and affinities among transporters, these do not inform on the activity of the transporters in planta, or on the overall impact on plant growth and productivity. As an example of this, SUTs from Solanaceae species with roughly the same kinetic properties as AtSUC2 did not rescue an Arabidopsis Atsuc2 mutant, while an AtSUC2 cDNA and ZmSUT1 cDNA from Zea mays did rescue the mutant (Dasgupta et al., 2014). In this protocol, and two that follow (Yadav et al., 2017a and 2017b), we detail the use of [14C]CO2 and [14C]Suc to quantitatively assess phloem loading and carbon transport in living explants and intact plants. These methods are used routinely in our laboratory, and recently contributed to our demonstration that plants over expressing certain SUTs in CCs showed increased phloem loading and transport, despite having stunted growth (Dasgupta et al., 2014) and, in a separate study, over expression of a H+-pumping pyrophosphatase enhanced Suc loading and transport without a corresponding increase in SUT expression levels (Khadilkar et al., 2016).

In the procedure described here (Figure 1), Arabidopsis leaf disks isolated from mature rosette leaves are vacuum infiltrated with a buffered solution containing [14C]Suc. SUTs load the Suc into CCs, and excess Suc is washed away. The disks are quickly frozen in powdered dry ice, lyophilized, and autoradiography is used to visualize loading into the veins. Rapid freezing and lyophilization prevent the highly soluble labeled sugar from diffusing away from the sites of loading. Scintillation counting then quantifies the loaded label. We generally pool four leaf disks into one replicate, and perform 6 biological replicates for each control and experimental system. This protocol was adapted for Arabidopsis (Dasgupta et al., 2014; Khadilkar et al., 2016) from procedures described by Turgeon and colleagues (Turgeon and Wimmers, 1988; Turgeon and Gowan, 1992; Turgeon and Medville, 1998; Haritatos et al., 2000; Goggin et al., 2001), and can be further adapted for other plants. Typical autoradiography results for Arabidopsis, tobacco, and wheat are shown in Figure 2; representative quantitative results of loading in different transgenic Arabidopsis plants are published (Dasgupta et al., 2014; Khadilkar et al., 2016).

Materials and Reagents

  1. Potting containers (The HC Companies, catalog number: IJT06060 ;Jumbo Insert)
  2. Potting mixture (Sun Gro Horticulture, catalog number: Fafard 3B Mix ; or similar)
  3. 24-well culture plates (Greiner Bio-One International, catalog number: 662160 )
  4. 5 ml transport vials (Stockwell Scientific, catalog number: 3205 )
  5. Nylon window screen (available at most hardware stores)
  6. Cyanoacrylate (Super Glue, or equivalent)
  7. Razor blades (Double-edged PERSONNA) (Electron Microscopy Sciences, catalog number: 72000 )
  8. Petri dishes (100 x 25 mm) (Fisher Scientific, catalog number: FB0875711 )
  9. Filter paper (Fisher Scientific, catalog number: 09-795C )
  10. Glass beads 4 mm (Water Stern, catalog number: 100E )
  11. Dow Corning high vacuum grease
  12. Thick, smooth paper (e.g., Bristol board or manila folder)
  13. Scintillation vials (Fisher Scientific, catalog number: 12383317 )
  14. Wax paper (Reynolds CUT-RITE)
  15. Aluminum foil envelopes (70 x 70 mm–homemade from standard aluminum foil)
  16. Autoradiography film (Kodak BioMax MR Film, Eastman Kodak, catalog number: 870 1302 )
  17. Plant material (e.g., Arabidopsis thaliana Col-0, control and experimental material), 10 to 12 healthy plants for each treatment
  18. Developer and fixer solutions (Eastman Kodak, catalog numbers: 190 0984 and 190 2485 )
  19. Sodium hypochlorite (NaClO) (Commercial bleach)
  20. Ecolume scintillation fluid (MP Biomedicals, catalog number: 0188247004 )
  21. 95% ethanol (Pharmco-AAPER, catalog number: 111000200 )
  22. 2(N-morpholino) ethane-sulfonic acid [MES] (Fisher Scientific, catalog number: BP300-100 )
  23. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 223506 )
  24. Potassium hydroxide (KOH) (Merck, catalog number: PX1480-1 )
  25. Sucrose (Fisher Scientific, catalog number: BP220-212 )
  26. [14C]Sucrose (100 µCi ml-1, > 350 mCi mmol-1) (MP Biomedicals, catalog number: 011113791 )
  27. Powdered dry ice (preferred) or liquid nitrogen
  28. Drierite desiccant (W A Hammond Drierite, catalog number: 13001 )
  29. MES/CaCl2 buffer (see Recipes)
  30. 1 mM Suc in MES/CaCl2 buffer (see Recipes)
  31. [14C]Suc in 1 mM Suc and MES/CaCl2 buffer (see Recipes)

Equipment

  1. Personal safety equipment: lab coat, nitrile gloves (or similar), and eye protection
  2. Growth chamber for Arabidopsis, 12 h light/12 h dark at ~100 µmol photons m-2 sec-1
  3. Cork borer (Carolina Biology, catalog number: 712202 )
  4. Cover glass forceps or similar (Fine Science Tools, catalog number: 11073-10 )
  5. Fine scissors (Fisher Scientific, catalog number: 08-951-20 )
  6. Vacuum bell jar (Kimble, catalog number: 31200-150 )
  7. Vacuum pump (Franklin Electric, catalog number: 1112585400 )
  8. Rotary shaker (Orbital Shaker Variable) (BioExpress, GeneMate, catalog number: S-3200-LS )
  9. Balance (METTLER TOLEDO, model: AE100 )
  10. Geiger counter (Ludlum Measurements, model: Model 3 )
  11. Lyophilizer (The VIRTIS company, catalog number: 10-030 )
  12. Mesh basket for lyophilizer chamber (homemade from metal window screen)
  13. Heavy duty bench vise (Kobalt 4” Vise)
  14. Two flat-surface, metal plates (~15 x 3 cm)
  15. Autoradiography cassettes (Exposure Cassette Kodak®) (Sigma-Aldrich, catalog number: E9010 )
  16. Autoradiography developing trays (Commercial plastic trays ~35 x 25 x 8 cm)
  17. Scintillation counter (Beckman Counter, model: LS 6000IC )

Procedure

  1. Working with any radionuclide requires special consideration and approval from the appropriate institutional office. Clearance can take a long time (months or more) and the application process should be started early, or collaborations should be established with groups that already have approvals in place.
  2. Grow Arabidopsis plants under short-day conditions with potting mix (Fafard 3B mix, or similar) to achieve well separated, large, and healthy rosettes; larger shade leaves are easier to work with than small sun leaves: 12 h light/12 h dark at ~100 µmol photons m-2 sec-1 works well. Select source leaves for labeling from each rosette that are fully expanded but not shaded or beginning to senesce (e.g., leaves 8 through 12 on ~25-d old rosettes) (Figure 1A). We generally use 32 leaf disks per treatment (8 pools of 4 disks, see below), and prepare 10 to 12 plants for each treatment to ensure sufficient healthy material.
  3. This procedure requires numerous (3 or 4) washing steps, which are made easier with small baskets constructed to fit inside the wells of a 24-well culture plate (shown in Figure 1C). We constructed baskets from polypropylene vials (5 ml transport vials, Stockwell Scientific). The top and bottom of each vial was cut off, leaving ¾ inches of open cylinder. Nylon window screen was attached to the bottom opening with cyanoacrylate (Super Glue, or equivalent) and excess screen trimmed away with a razor blade. These baskets maximize use of the space in the culture plate wells while still moving freely in and out of the wells during washing. Other forms of tubing will undoubtedly work, but investigators are cautioned that standard commercial tubing with 5/8 inch outer diameter and 1/2 inch inner diameter will likely not move freely in the wells.
  4. Cut the petioles near the stem and transfer the leaves to a Petri dish lined with three layers of filter paper and MES/CaCl2 buffer (20 mM MES, 2 mM CaCl2, pH 5.5 with KOH) to a depth of a few mm to ensure leaf disks are completely submerged. Under buffer, cut out a leaf disk with a #5 cork bore (~8 mm diameter, or smaller, depending on the size of the source leaves) (Figure 1B). Transfer the leaf disks abaxial side down into a basket in the well of a 24-well microtiter plate containing 1 ml MES/CaCl2 buffer (Figure 1D); pool 4 disks in each basket in each well and gently cover with 4 mm glass beads to keep them submerged (Figure 1E). We generally use 6 (minimum) to 8 (preferred) replicates of 4 pooled disks, so 24 to 32 disks are required for each treatment. The disks are randomized among the pools.
    Note: All solutions should be at temperatures approximating leaf temperature. Cold solutions, such as from a refrigerator, will inhibit phloem loading.
  5. Once all the baskets in the wells are prepared, lift the baskets (with leaf disks and glass beads) out of the culture plates and transfer to the wells of fresh plate pre-filled with 0.5 ml MES/CaCl2 buffer supplemented with 1 mM Suc solution (1 mM unlabeled Suc supplemented with 0.81 µCi (3 x 104 Bq) ml-1 [14C]Suc) (Figure 1F). The leaf disks should be submerged owing to the glass beads; more solution can be added to each well if necessary.
  6. Vacuum infiltrate the MES/CaCl2 with [14C]Suc solution into the leaf disks. In a bell jar and with a vacuum pump, pull a strong vacuum on the microtiter plate until bubbles no longer emerge from the leaf disks and then release the vacuum (Figure 1G). This pulls air out of the leaf air space and replaces it with labeling [14C]Suc solution. Imbibed sections of the disks leaves will look darker than non-imbibed sections; repeat the vacuum infiltration until the disks are completely imbibed and saturated.
    Note: It is important to keep the disks submerged in MES/CaCl2 with [14C]Suc solution for effective infiltration, otherwise, air will be pulled back into the disks when the vacuum is released, rather than solution.
  7. Cover the culture plate and incubate the vacuum-infiltrated discs for 20 min with gentle shaking on rotating platform (shaker) at room temperature.
  8. Lift the baskets (with labeled disks and glass beads), letting the MES/CaCl2 with [14C]Suc solution drain, and transfer the baskets to fresh culture plates pre-filled with 1 ml MES/CaCl2 buffer (Figure 1H). Incubate with gentle rotation on a rotary platform for 10 min.
  9. Repeat the above wash step another two times. Treat all wash solutions as a radioactive, [14C] containing liquid waste as it contains a significant amount of [14C]Suc. Process wells as quickly as practical to minimize deviations in incubation times between samples.
    Note: The MES/CaCl2 with [14C]Suc solution can be reused, if desired, since only a small portion of the [14C]Suc is absorbed into the leaf disks. However, this increases the risk of contamination by enzymes such as invertase and microorganisms. We recommend filter sterilizing the solution and freezing promptly, with appropriate labels and storage for isotopes, if it is to be reused.
  10. Blot the leaf disks on absorbent filter paper (Figure 1I) and place them in perforated aluminum foil envelopes (70 x 70 mm), prepared and labeled in advance. To make envelopes, fold a piece of aluminum foil in half and then fold in two sides to leave one side open; perforate the foil with a tack. We put the four disks representing one repetition into a single envelope. Place each envelope immediately on powdered dry ice and cover the envelope with more powered dry ice, repeat for each envelope, keep them frozen until all samples are processed.
    Note: Liquid nitrogen can also be used, but layering the envelopes in powdered dry ice is preferred since it causes less cracking than submerging in liquid nitrogen: intact leaf disks are much easier to work with during autoradiography.
  11. Lyophilize the frozen disks in a lyophilizer for 48 h. It is imperative that they remain frozen to prevent movement of the highly soluble [14C]Suc (Figure 1J).
    Note: A lyophilizer with a sample chamber cooled to well below the freezing point (~-30 °C) of the tissue works best; lyophilizers with external sample vessels attached via a manifold work poorly because the disks thaw sufficiently to allow diffusion of the label. In Figure 1J, the aluminum foil envelopes are placed inside a homemade metal mesh basket (made from metal window screen) that fits snuggly within the coiling coils of our lyophilizer. The coiling coils themselves are the coldest part of the system, so water sublimates from the leaf disks and condenses on the coils, while the entire chamber stays well below the freezing point of the samples.
  12. After lyophilizing, transfer the envelopes with leaf disks to a bell jar with Drierite desiccant: it is important to prevent condensation and exposure to humid conditions to avert diffusion of the [14C]Suc.
  13. Autoradiography and scintillation counting
    1. Visualizing phloem loading into leaf veins by autoradiography
      1. Visualizing phloem loading by autoradiography is based on the veins accumulating [14C]Suc from the apoplast while it is washed out of the areoles. Because 14C emits weak β particles and the signal disperses with distance from the source, the best autoradiograms are obtained with flat, thin leaf disks placed directly on single emulsion, high-resolution film, such as Kodak BioMax MR Film.
      2. Press the leaf disks flat between two metal plates (15 x 3 cm) in a heavy duty bench vise. On top of one metal plate (Figure 1K), place a piece of paperboard (Bristol paper or manila folder paper) and a piece of wax paper (~12.5 x 2.5 cm). Arrange the lyophilized leaf disks abaxial side up on the wax paper, keeping track of which disks belong to each repetition by labeling either the wax paper or the underlying paperboard. Cover the disks with another piece of paperboard and then the second metal plate. Insert the ‘sandwich’ into a heavy duty bench vise and compress as flat as possible (Figure 1L). Disassemble the sandwich but do not separate the disks from the wax paper; after compression, the wax paper provides a slightly tacky surface to help keep the disks in place during autoradiography. Repeat until all the disks are pressed flat.
      3. Arrange the bottom pieces of paperboard, wax paper and loosely adhered leaf disks (abaxial side up) in an autoradiography cassette (Figure 1M). In a darkroom licensed for work with isotopes, and under safe lights, place a piece of Kodak BioMax MR Film (or similar) with the emulsion side down so that it contacts the leaf disks.
      4. Depending on the strength of the [14C]Suc labeling, expose the autoradiography film to the leaf disks for 24 to 48 h. We generally start with a 48 h exposure, and after developing we make a decision if a second exposure, for a shorter or longer period of time, is warranted.
      5. In a dark room, remove the autoradiography film from the cassette taking care that the leaf disks stay adhered to the wax paper. Keeping the disks organized is important if a second exposure will be done. Develop the autography film by hand with freshly prepared developer and fixer (Kodak GBX) according to the manufacturer’s instructions. In brief, place the film in a tray with developer solution and incubate at room temperature with gentle shaking for 5 min. Transfer the film to a tray with water for 30 sec and then transfer to a tray with fixer solution for 5 min. Rinse with running water for 15 min and hang the film to drip and air dry.
      6. A stereo microscope equipped with a digital camera works well for photographing the autoradiographs.
    2. Quantifying phloem loading into leaf veins by scintillation counting
      1. After autoradiography, remove the leaf disks from the wax paper and place the disks from each replicate in a scintillation vial (Figure 1N).
      2. To extract the pigments and solutes, add 500 µl 80% ethanol and shake gently for 20 min. Add 500 µl commercial bleach and shake gently for 20 min to destroy the pigments.
      3. Add 5 ml of biodegradable scintillation cocktail suitable for aqueous solutions. Mix thoroughly by shaking the vial to ensure the scintillation cocktail permeates the leaf disk and creates a monophasic solution. Include a negative control vial containing ethanol, bleach and scintillation cocktail, but without leaf disks.
      4. Perform scintillation counting with a program suitable for 14C with a scintillation counter.


        Figure 1. Experimental procedure to study phloem loading using [14C]Suc in Arabidopsis leaf disks. A. Grow Arabidopsis thaliana Col-0 control and experimental plants on potting mixture under conditions suitable to the experiment. B. Cut healthy mature leaves at the petiole base, transfer leaves to a Petri dish lined with filter paper and while the leaves are submerged in MES/CaCl2 buffer, use a #5 mm cork borer to isolate leaf disks. C. Have prepared 24-well microtiter plates filled with 1 ml MES/CaCl2 buffer in each well, and mesh-bottom baskets for convenient washing in later steps. D. Pool 4 disks in each well and (E) gently cover with glass beads to submerge the leaf disks. F. Once all the baskets with leaf disks and glass beads are prepared, transfer the baskets to the wells of a fresh plate pre-filled with MES/CaCl2 buffer supplemented with 1 mM Suc solution (1 mM unlabeled Suc supplemented with 0.81 µCi (3 x 104 Bq) ml-1 [14C]Suc). G. In a bell jar, vacuum infiltrate the MES/CaCl2/[14C]Suc solution into the leaf disks making sure to imbibe the air-space with solution. H. Incubate the disks for 20 min with gentle shaking on a rotary platform, and wash disks three times with MES/CaCl2 solution without Suc. I. Blot the leaf disks on absorbent paper (I) and place them into premade and prelabeled, perforated aluminum foil envelopes. Layer the envelopes in powdered dry ice (preferred method) or submerge in liquid nitrogen; the perforations in the aluminum foil envelopes allow liquid N2 to enter and permit N2 gas to escape. J. Lyophilize the discs for 48 h such that they remain frozen while drying (shown are aluminum foil envelopes inside a metal mesh basket made to fit inside the coiling coils of the lyophilizer we use; temperature is maintained at ~-30 °C). After lyophilizing, move the aluminum foil envelopes to a desiccator to warm to room temperature without condensation forming. K. Carefully remove the disk from the envelopes and arrange, abaxial side up, on wax paper on Bristol paper on a flat-surface metal plate. Put a second sheet of Bristol paper (without wax paper) over the leaf disk and a second, flat surface metal plate. L. Transfer the sandwich to a heavy duty bench vise and press the leaf disks as flat as possible. M. The leaf disks will loosely adhere to the wax paper, and can be transferred to an autoradiography cassette for exposure to single emulsion, high-resolution autoradiography film. N. After satisfactory autoradiography, remove the leaf disks from the wax paper and quantify 14C label by scintillation counting.

Data analysis

Autoradiography is a qualitative visual assessment of loading whereas scintillation counting provides quantitative data. Representative autoradiograms of Arabidopsis, tobacco, and wheat are shown in Figure 2. The results of scintillation counting are expressed as disintegrations per minute (dpm) per unit of leaf area (mm2) or as a percent relative to controls: (experimental dpm/control dpm) x 100 (Khadilkar et al., 2016). For quantitative analysis, the mean is calculated for each of the 8 replicates of 4 pooled disks. Statistically significant differences in loading among treatments are determined by standard calculations such as Student’s t-test or analysis of variance (ANOVA) with post-hoc analysis.


Figure 2. Representative autoradiography of (A) Arabidopsis, (B) tobacco, and (C) wheat leaf segments prepared with this protocol

Recipes

  1. MES/CaCl2 buffer (500 ml)
    MES anhydrous 1.9524 g (FW 195.24) (20 mM)
    CaCl2·2H2O 0.147 g (FW 147.01) (2 mM)
    Adjust pH 5.5 with KOH (5 N)
  2. 1 mM Suc in MES/CaCl2 buffer (100 ml)
    Sucrose 0.034 g (FW 342.30) (1 mM) in 100 ml MES/CaCl2 buffer, or dilute appropriately from a more concentrated Suc stock
  3. [14C]Suc in 1 mM Suc and MES/CaCl2 buffer (10 ml, make fresh, calculate how much will be needed for the specific experiment and prepare ~10% extra)
    Commercial [14C]Suc stock, 100 µCi ml-1, > 350 mCi mmol-1
    Add 0.081 ml (3 x 104 Bq) of commercial stock to 9.92 ml of 1 mM Suc in MES/CaCl2 buffer
    Note: [14C]Suc specific activity may vary with different batches and suppliers. 0.081 ml of the stock described contributes 0.023 µmoles of Suc per 10 ml of 1 mM Suc and MES/CaCl2 buffer and increases the final Suc concentration to 1.002 mM, which we consider negligible.
    (0.081 ml) x (100 µCi ml-1)/(350 mCi mmol-1) = 0.023 µmoles
    0.023 µmoles/10 ml = 0.0023 mM

Acknowledgments

This protocol is based on methods published in Dasgupta et al. (2014) and Khadilkar et al. (2016). We thank John Evers for providing the plants contributing to Figure 2. Work on phloem loading and long distance transport in B.G. Ayre’s laboratory is/was supported by the National Science Foundation 0344088, 0922546, 1121819, and 1558012. The authors report no conflicts of interest or competing interests.

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  25. Yadav, U. P., Ayre, B. G. and Bush, D. R. (2015). Transgenic approaches to altering carbon and nitrogen partitioning in whole plants: assessing the potential to improve crop yields and nutritional quality. Front Plant Sci 6: 275.
  26. Yadav, U. P., Khadilkar, A. S., Shaikh, M. A., Turgeon, R. and Ayre, B. G. (2017a). Assessing rates of long-distance carbon transport in Arabidopsis by collecting phloem exudations into EDTA solutions after photosynthetic labeling with [14C]CO2. Bio Protoc 7(24): e2656.
  27. Yadav, U. P., Khadilkar, A. S., Shaikh, M. A., Turgeon, R. and Ayre, B. G. (2017b). Assessing long-distance transport from photosynthetic source leaves to heterotrophic sink organs with [14C]CO2. Bio Protoc 7(24): e2657.
  28. Zhang, L., Garneau, M. G., Majumdar, R., Grant, J. and Tegeder, M. (2015). Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids. Plant J 81(1): 134-146.

简介

来自光合自养源的光合同化物的韧皮部装载和运输到异养宿主器官是必不可少的生理过程,其帮助植物的不同器官作为单一的统一生物体起作用。我们提出了三种方案,我们经常使用相互组合,以评估(1)蔗糖(Suc)加载到成熟叶片的韧皮部血管系统(本协议)的相对比率,(2)碳负荷和运输通过韧皮部(Yadav et al。,2017a),和(3)在长距离运输后碳卸载到异养汇器官特别是根中的相对速率(Yadav等人, / em>。,2017b)。我们建议,在实验和对照植物上进行所有三种方案提供了全植物碳分配的可靠比较,并且将与单独进行的单个方案相关联的歧义降至最低(Dasgupta等人,2014; Khadilkar 。,2016)。在该方案中,从成熟莲座叶中分离的拟南芥叶片用含有[14 C] Suc的缓冲溶液浸润。 Suc转运蛋白(SUCs或SUTs)将Suc载入韧皮部,并将多余的,卸载在叶片中的Suc洗掉。通过冻干叶盘的放射自显影显示标记的Suc加载到静脉中,并通过闪烁计数进行定量。结果表示为每单位叶盘鲜重或面积的每分钟崩解。

【背景】光合同化物从源头到宿主器官的运输对于整个植物的正常生长和维持至关重要。叶片中的韧皮部负载是将在叶肉细胞中合成的光合同化物递送至韧皮部脉管系统的伴生细胞(CC)和筛分元素(SE)。三种不同的加载机制被认可。其中两个消耗能量在CC和SEs中累积高浓度的糖,并在源叶韧皮部产生高静水压力。第一种是外源韧皮部装载,其中将Suc(和/或一些物种中的糖醇)穿过质膜从细胞壁空间(即,质外体)装载进入CC中的CC质子动力的牺牲(Giaquinta,1983)。第二种是聚合物捕获,其中Suc通过特化的胞间连丝(Botmodesmata)扩散到韧皮部,并转化为太大而不能扩散回来的寡糖(Turgeon,1996)。第三种机制是被动加载,其中叶肉细胞中的溶质浓度最高,胞间连丝为被动移入CC和SE提供了一个开放的途径(Rennie和Turgeon,2009)。
由于韧皮部装载和运输对植物生理和生产力的中心作用,所以希望有可靠的方法来鉴定和量化韧皮部的含量和运输速率。此外,韧皮部装载和运输是生物技术和代谢工程提高生产力的目标(Ainsworth和Bush,2011; Cao等人,2013; Dasgupta等人, 2014; Zhang等人,2015; Yadav等人,2015)。定量评估自然和工程系统中韧皮部装载和运输的速率和容量是困难的,原因有几个。例如,CC和SE嵌入周围组织,相对于周围的非韧皮部细胞非常狭窄;韧皮部处于高压状态,破损时密封迅速;采集的韧皮部汁液通常被其他细胞的内容物污染;因为运输是一个动态的过程,所以韧皮含量的估计并不表示运输速度(Turgeon and Wolf,2009; Dinant and Kehr,2013; Tetyuk et al。,2013)。在CO 2或标记的糖中,C同位素11 C,13 C和14 C已经提供了,并且继续提供有关韧皮部加载机制的重要信息(Sovonick et al。,1974; Turgeon and Gowan,1990; Turgeon and Medville,1998),以及关于加载和运输速率的定量信息(Thorpe和Minchin,1988; Karve等人,2015; Dersch等人,2016)。
用拟南芥AtSUC2编码的Suc转运蛋白(SUT)(Truernit和Sauer,1995; Gottwald et al。,2000),拟南芥从质外体载入Suc ; Srivastava等人,2008)。已经在酿酒酵母和非洲爪蟾卵母细胞中评估了AtSUC2和其他转运蛋白作为建立米氏动力学参数的异源系统(在[Ayre,2011]中综述)。尽管比较运输者之间的活动和亲和力是有价值的,但是这些并不告知运输者在植物中的活动或者对于植物生长和生产力的整体影响。作为一个例子,来自茄科的具有与AtSUC2大致相同的动力学性质的SUT没有拯救拟南芥突变体Atsuc2,而AtSUC2突变体来自玉米的cDNA和ZmSUT1 cDNA确实拯救了突变体(Dasgupta等人,2014)。在这个协议中,以及后面的两个(Yadav et al。,2017a和2017b),我们详细介绍了使用[14C] CO 2 >和[14C] Suc来定量评估活的外植体和完整植物中的韧皮部负载和碳运输。这些方法在我们的实验室中经常使用,并且最近有助于我们的证明,即在CC中超表达某些SUT的植物尽管发育受阻(Dasgupta等人,2014),但却表现出增加的韧皮部装载和运输在单独的研究中,H +泵式焦磷酸酶的过度表达增强Suc加载和转运,而SUT表达水平没有相应的增加(Khadilkar et al。,2016)。

在此处描述的过程(图1)中,从成熟莲座叶中分离的拟南芥叶片用含有[14 C] Suc的缓冲溶液真空渗入。 SUT将Suc加载到CC中,并将多余的Suc冲走。圆盘在粉状干冰中快速冷冻,冻干,并使用放射自显影来观察静脉中的加载情况。快速冷冻和冻干可防止高度可溶性标记糖从装载位点扩散开。闪烁计数然后量化加载的标签。我们通常将四片叶片集中在一个重复中,并为每个对照和实验系统进行6次生物重复。该方案适用于Turgeon及其同事描述的拟南芥(Dasgupta et al。,2014; Khadilkar et al。,2016) Turgeon和Wimmers,1988; Turgeon和Gowan,1992; Turgeon和Medville,1998; Haritatos等人,2000; Goggin等人,2001),并且可以是进一步适用于其他工厂。典型的拟南芥,烟草和小麦的放射自显影结果如图2所示;公布了在不同转基因拟南芥植物中加载的代表性定量结果(Dasgupta等人,2014; Khadilkar等人,2016)。

关键字:拟南芥, 韧皮部装载, 韧皮部运输, [14C]蔗糖摄取, 叶盘

材料和试剂

  1. 灌封容器(慧聪公司,目录号:IJT06060; Jumbo Insert)
  2. 盆栽混合物(Sun Gro园艺,目录号:Fafard 3B Mix;或类似物)
  3. 24孔培养板(Greiner Bio-One International,目录号:662160)
  4. 5毫升运输小瓶(斯托克韦尔科学,目录号码:3205)
  5. 尼龙窗纱(大多数五金商店都有)
  6. 氰基丙烯酸酯(超级胶水或相当的)
  7. 剃刀片(双刃PERSONNA)(电子显微镜科学,目录号:72000)
  8. 培养皿(100×25毫米)(Fisher Scientific,目录号:FB0875711)
  9. 滤纸(Fisher Scientific,目录号:09-795C)
  10. 玻璃珠4毫米(水S,目录号:100E)
  11. 道康宁高真空油脂
  12. 厚实,光滑的纸张(例如,布里斯托尔板或马尼拉文件夹)
  13. 闪烁瓶(Fisher Scientific,目录号:12383317)
  14. 蜡纸(Reynolds CUT-RITE)
  15. 铝箔信封(70 x 70毫米,由标准铝箔制成)
  16. 放射自显影胶片(Kodak BioMax MR Film,Eastman Kodak,产品目录号:870 1302)
  17. 植物材料(例如拟南芥Col-0,对照和实验材料),每种处理10-12个健康植物。
  18. 开发商和定影剂解决方案(伊士曼柯达,产品目录号:190 0984和190 2485)
  19. 次氯酸钠(NaClO)(商业漂白剂)
  20. Ecolume闪烁液(MP Biomedicals,目录号:0188247004)
  21. 95%乙醇(Pharmco-AAPER,目录号:111000200)
  22. 2(N-吗啉代)乙磺酸[MES](Fisher Scientific,目录号:BP300-100)
  23. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:223506)
  24. 氢氧化钾(KOH)(Merck,目录号:PX1480-1)
  25. 蔗糖(Fisher Scientific,目录号:BP220-212)
  26. 蔗糖(100μCiml-1,> 350mCi mmol-1)(MP Biomedicals,目录号:011113791)
  27. 粉状干冰(优选)或液氮
  28. 干燥剂干燥剂(W A Hammond Drierite,目录号:13001)
  29. MES / CaCl 2 缓冲液(见食谱)
  30. 在MES / CaCl 2缓冲液中的1mM Suc(见食谱)
  31. 在1mM Suc和MES / CaCl 2缓冲液中的[14S] Suc(见食谱)

设备

  1. 个人安全设备:实验室外套,丁腈手套(或类似的)和护目镜
  2. 用于拟南芥的生长室,在〜100μmol光子下12小时光照/ 12小时黑暗下,mS / 2秒-1
  3. 软木钻(卡罗莱纳州生物学,目录号:712202)
  4. 盖玻璃镊子或类似的(精细科学工具,目录号:11073-10)
  5. 精细剪刀(Fisher Scientific,目录号:08-951-20)
  6. 真空钟罩(金布尔,目录号:31200-150)
  7. 真空泵(富兰克林电气,目录号:1112585400)
  8. 旋转摇床(Orbital Shaker Variable)(BioExpress,GeneMate,产品目录号:S-3200-LS)
  9. 平衡(梅特勒 - 托利多,型号:AE100)
  10. 盖革计数器(Ludlum测量,模型:模型3)
  11. 冻干机(VIRTIS公司,目录号:10-030)
  12. 用于冻干箱的网篮(由金属窗纱制成)
  13. 重型台钳(Kobalt 4“老虎钳)
  14. 两个平面金属板(〜15×3厘米)
  15. 放射自显影盒(Exposure Cassette Kodak)(Sigma-Aldrich,目录号:E9010)
  16. 放射自显影显影盘(商用塑料托盘〜35×25×8厘米)
  17. 闪烁计数器(贝克曼计数器,型号:LS 6000IC)

程序

  1. 与任何放射性核素合作需要得到适当的机构办公室的特别考虑和批准。清关可能需要很长时间(几个月或更长时间),申请流程应该尽早开始,或者与已经批准的团体建立合作关系。
  2. 在短日照条件下用盆栽混合物(Fafard 3B混合物或类似物)培育拟南芥植物以获得分离良好,大而健康的玫瑰花结;较大的阴影叶比较小的阳光叶更容易处理:在〜100μmol光子m 2 -2秒-1的12h光/ 12h黑暗效果良好。选择来源于每个花环标记的来源完全展开但没有阴影或开始衰老的标记(例如,在〜25-d旧玫瑰花结上离开8到12)(图1A)。每次处理我们通常使用32片叶片(8个池,共4盘,见下文),每种处理准备10到12个植物,以确保足够的健康材料。
  3. 这个过程需要多次(3或4次)洗涤步骤,使用装在24孔培养板(图1C所示)的孔内的小篮子可以更容易地进行洗涤步骤。我们从聚丙烯小瓶(5毫升运输小瓶,斯托克韦尔科学)构建篮子。每个小瓶的顶部和底部被切断,留下3/4英寸的开放圆柱体。用氰基丙烯酸酯(超级胶水或等同物)将尼龙窗纱连接到底部开口,用剃刀刀片将多余的丝网切掉。这些篮子可以最大限度地利用培养板孔中的空间,同时在洗涤过程中仍能自由进出孔。其他形式的油管无疑是可行的,但调查人员注意到,外径5/8英寸,内径1/2英寸的标准商用油管可能不能在油井中自由移动。
  4. 切割茎附近的叶柄,并将叶转移到衬有三层滤纸和MES / CaCl 2缓冲液(20mM MES,2mM CaCl 2, ,用KOH调pH至5.5)至几毫米的深度,以确保叶盘完全浸没。在缓冲液下,用#5软木塞钻孔(〜8mm直径或更小,取决于来源叶的大小)切出叶盘(图1B)。将叶盘远轴侧向下转移到含有1ml MES / CaCl 2缓冲液(图1D)的24孔微量滴定板的孔中的篮中;在每个孔中的每个篮中共沉积4个盘并用4mm玻璃珠轻轻覆盖以保持其被淹没(图1E)。我们通常使用6个(最小)到8个(首选)4个池式磁盘的复制,因此每个处理需要24到32个磁盘。这些磁盘在池中被随机化。
    注意:所有解决方案应该在接近叶片温度的温度下。寒冷的解决方案,如从冰箱,将抑制韧皮部负载。
  5. 一旦准备好所有的篮筐,将篮(带有叶片和玻璃珠)从培养皿中取出并转移到预先装有0.5ml MES / CaCl 2 2 / sub的新鲜板的孔中(1mM未标记Suc,补充有0.81μCi(3×10 4 Bq)ml -1 /14μM)的缓冲液中, C] Suc)(图1F)。由于玻璃珠,叶盘应该被浸没;
    如果需要,可以在每个井中添加更多解决方案
  6. 用[14C] Suc溶液真空渗入MES / CaCl 2 2到叶盘中。在一个钟罩和一个真空泵,拉微量滴定板上的强真空,直到气泡不再从叶盘出来,然后释放真空(图1G)。这将空气从叶片空气空间中抽出,并用标签[14C] Suc溶液代替。吸入的叶片部分会比未吸入的部分看起来更暗;重复真空渗透,直到圆盘完全浸透饱和。
    注意:使用[ > 14 C] Suc解决方案有效的渗透,否则,当真空被释放,而不是解决方案时,空气将被拉回到磁盘中。
  7. 盖上培养板,孵育真空渗透圆盘20分钟,在旋转平台(摇床)上温和摇动,室温下。
  8. 提起篮子(带有标记的圆盘和玻璃珠),让含有[14C] Suc溶液的MES / CaCl 2溶液排出,并将篮子转移到新鲜的培养板上用1ml MES / CaCl 2缓冲液填充(图1H)。
    在旋转平台上温和旋转孵育10分钟
  9. 重复上述洗涤步骤两次。将所有洗涤溶液作为含有液体废物的含有放射性的14 C处理,因为它含有大量的[14 C] Suc。尽可能快地处理孔,以尽量减少样品间孵育时间的偏差。
    注意:MES / CaCl 2 如果需要的话,可以重复使用Suc解决方案,因为只有Suc的一小部分是吸收到叶盘中。但是,这增加了酶如转化酶和微生物污染的风险。如果要重复使用,我们建议过滤器对溶液进行消毒并迅速冷冻,并加上适当的标签和同位素储存。
  10. 在吸水滤纸上印上叶片(图1I),并将它们放入穿孔的铝箔信封(70×70毫米)中,预先准备并贴上标签。要制作信封,请将一片铝箔折成两半,然后折叠两面,使其一面敞开;用大头钉对箔片打孔。我们把代表一个重复的四个磁盘放在一个信封里。将每个信封立即放在粉末状的干冰上,用更多动力的干冰覆盖信封,重复每个信封,使其保持冷冻状态,直到所有样本都被处理。
    注意:也可以使用液氮,但将信封放入干粉冰粉是比较好的选择,因为与在液氮中浸没相比,它会引起更少的裂缝:在放射自显影过程中,完整的叶盘更容易处理。
  11. 在冷冻干燥器中冷冻干燥盘48小时。它们必须保持冷冻以防止高度可溶性[14 C] Suc的移动(图1J)。
    注意:将样品室冷却到远低于组织冰点(〜-30°C)的冷冻干燥器效果最好;通过歧管连接的外部样品容器的冻干机工作得不好,因为圆盘充分融化以允许标签扩散。在图1J中,铝箔信封放置在自制的金属网篮(由金属窗纱制成)内,该网篮紧贴在我们的冻干机的卷绕盘管内。盘绕盘管本身是系统中最冷的部分,所以水从盘片上升华并凝结在盘管上,而整个小室保持在低于样品的冰点以下。
  12. 冷冻干燥后,将带叶盘的信封转移到带有干燥剂干燥剂的钟罩中:防止冷凝和暴露在潮湿条件下以防止[14C] Suc扩散是重要的。
  13. 放射自显影和闪烁计数
    1. 通过放射自显影将韧皮部装载到叶脉中
      1. 通过放射自显影来可视化韧皮部负载是基于脉管从质外体积聚[14C] Suc,同时将其从球体中洗出。由于14 C发射微弱的β粒子,并且信号随离开源的距离而分散,所以获得最佳的放射自显影图,其中平坦,薄的叶片直接放置在单一乳剂,高分辨率膜如Kodak BioMax MR电影。
      2. 将两片金属板(15×3厘米)之间的叶片平放在重型台钳中。在一块金属板(图1K)的顶部,放置一块纸板(布里斯托尔纸或马尼拉折叠纸)和一块蜡纸(〜12.5×2.5厘米)。将冻干的叶盘背面朝上放在蜡纸上,通过标记蜡纸或底层纸板来跟踪哪些盘属于每次重复。用另一块纸板覆盖磁盘,然后用第二块金属板覆盖磁盘。将“三明治”插入重型台钳中并尽可能压平(图1L)。拆开三明治,但不要将蜡纸与蜡纸分开;在压缩之后,蜡纸提供稍微发粘的表面以在放射自显影期间帮助保持光盘在适当的位置。重复,直到所有的磁盘压平。
      3. 将底纸片,蜡纸和松散粘附的叶片(背面朝上)放置在放射自显影盒中(图1M)。在获准使用同位素工作的暗室中,在安全的灯光下,将一片柯达BioMax MR胶片(或类似物)与感光乳剂朝下放置,以使其接触到叶片。
      4. 根据[14C] Suc标记的强度,将放射自显影胶片暴露于叶圆片24至48小时。我们通常从48小时的曝光开始,在开发之后,如果第二次曝光时间较短或较长,则需要做出决定。
      5. 在黑暗的房间里,从磁带上取下放射自显影胶片,注意叶盘要粘在蜡纸上。如果第二次曝光将被完成,保持磁盘组织是重要的。根据制造商的指示,用新鲜制备的显影剂和定影剂(Kodak GBX)手动显影自动拍摄胶片。简而言之,将胶片放入带有显影剂溶液的托盘中,在室温下温和振荡5分钟。将胶片转移到带有水的托盘中30秒,然后转移到带有固定剂溶液的托盘中5分钟。
        用自来水冲洗15分钟,然后悬挂在膜上滴下,风干。
      6. 配有数码相机的立体显微镜可以很好地拍摄放射自显影照片。
    2. 通过闪烁计数量化韧皮部装载到叶脉中
      1. 放射自显影后,从蜡纸上取下叶片,并将每个重复样品的光盘置于闪烁瓶中(图1N)。
      2. 提取颜料和溶质,加入500μl80%乙醇,轻轻摇动20分钟。加500μL的商业漂白剂,轻轻摇晃20分钟,以消灭颜料。
      3. 加入5毫升适用于水溶液的可生物降解的闪烁鸡尾酒。通过摇动小瓶彻底混合,以确保闪烁鸡尾酒渗透叶盘,并创建一个单相的解决方案。包括一个阴性对照小瓶含有乙醇,漂白剂和闪烁鸡尾酒,但没有叶盘。
      4. 用闪烁计数器用适合于14 C的程序进行闪烁计数。


        图1.研究拟南芥叶片中[14C] Suc的韧皮部负载的实验程序A.生长拟南芥 Col-0对照和实验植物在适合于实验的条件下对盆栽混合物进行处理。 B.在叶柄基部切下健康的成熟叶片,将叶片转移到衬有滤纸的培养皿中,并且当叶片浸没在MES / CaCl 2缓冲液中时,使用#5mm软木塞蛀虫以分离叶盘。 C.在每个孔中准备了装有1ml MES / CaCl 2缓冲液的24孔微量滴定板,以及网眼底部的篮子,以便在后面的步骤中便于洗涤。 D.在每个孔中沉积4个盘并(E)用玻璃珠轻轻地覆盖以浸没叶盘。 F.一旦制备了具有叶盘和玻璃珠的所有篮,将篮转移到预先填充有补充有1mM Suc溶液(1mM)的MES / CaCl 2缓冲液的新鲜板的孔中未标记的Suc,补充有0.81μCi(3×10 4 Bq)ml -1 [14 C] Suc)。 G.在钟罩中,真空渗入MES / CaCl 2 / [14 C] Suc溶液到叶盘中,确保用溶液吸入空气空间。 H.在旋转平台上温和振荡孵育20分钟,并用MES / CaCl 2溶液(不含Suc)洗碟3次。 I.在吸水纸(I)上印刷叶片,并将它们放入预先制好的和预贴标签的穿孔铝箔信封中。用干粉冰(优选方法)将信封层覆盖或浸没在液氮中;铝箔封套中的穿孔允许液体N 2进入并允许N 2气逸出。 J.将圆盘冻干48小时,使其在干燥时保持冷冻(如图所示,金属网篮内的铝箔信封装在我们使用的冻干机的卷曲盘内,温度保持在〜-30℃)。冷冻干燥后,将铝箔信封移至干燥器中升温至室温,不进行冷凝成型。 K.小心地将信封从信封中取出,并将其背面朝上放置在平面金属板上的布里斯托纸上的蜡纸上。把第二张布里斯托尔纸(没有蜡纸)放在叶盘和第二个平坦的表面金属板上。 L.将三明治转移到重型台虎钳上,并尽可能平坦地按压叶盘。 M.叶片松散地粘附在蜡纸上,并可转移到放射自显影盒中以暴露于单一乳剂,高分辨率放射自显影胶片。 N.在令人满意的放射自显影后,从蜡纸上取下叶盘并通过闪烁计数定量14 C标记。

数据分析

放射自显影是负荷的定性视觉评估,而闪烁计数提供了定量数据。图2中显示了拟南芥,烟草和小麦的代表性放射自显影图。闪烁计数的结果表示为每单位叶面积的分解速率(dpm)(mm 2 / s) )或作为相对于对照的百分比:(实验dpm /对照dpm)×100(Khadilkar et al。,2016)。对于定量分析,对于4个池化盘的8个重复中的每一个重复计算平均值。通过标准计算如Student's t-检验或用post-hoc分析的方差分析(ANOVA)来确定治疗中负荷的统计学显着差异。


图2.用这种方法制备的(A)拟南芥,烟草和(C)小麦叶片的代表性放射自显影协议

食谱

  1. MES / CaCl 2缓冲液(500毫升)
    无水MES 1.9524克(FW195.24)(20mM)
    CaCl 2•2H 2 O 0.147g(FW 147.01)(2mM)
    用KOH(5 N)调节pH 5.5
  2. 在MES / CaCl 2缓冲液(100ml)中的1mM Suc
    在100ml MES / CaCl 2缓冲液中的蔗糖0.034g(FW 342.30)(1mM),或从更浓缩的Suc储液中适当地稀释
  3. 在1mM Suc和MES / CaCl 2缓冲液中(10ml,制备新鲜的,计算需要多少特定实验并制备〜10 %额外)
    商业[14 C] Suc储备液,100μCiml -1, 350mCi mmol-1
    向MES / CaCl 2缓冲液中的9.92ml 1mM Suc中加入0.081ml(3×10 4 Bq)市售储备液。
    注:[ 14 C] Suc具体活动可能因不同的批次和供应商而异。所描述的0.081ml所述原料对每10ml 1mM Suc和MES / CaCl 2缓冲液贡献0.023μmolSuc,并且将最终的Suc浓度提高到1.002mM,我们认为这可以忽略不计。
    (0.081ml)×(100μCiml -1)/(350mCi mmol -1)=0.023μmol
    0.023微摩尔/ 10ml = 0.0023mM

致谢

该协议基于Dasgupta et al 中发布的方法。 (2014年)和Khadilkar 等人。 (2016)。我们感谢John Evers提供了图2所示的植物。B.G.在韧皮部装载和长途运输方面的工作Ayre的实验室由美国国家科学基金会0344088,0922546,1121819和1558012支持。作者报告没有利益冲突或利益冲突。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yadav, U. P., Khadilkar, A. S., Shaikh, M. A., Turgeon, R. and Ayre, B. G. (2017). Quantifying the Capacity of Phloem Loading in Leaf Disks with [14C]Sucrose. Bio-protocol 7(24): e2658. DOI: 10.21769/BioProtoc.2658.
  2. Khadilkar, A. S., Yadav, U. P., Salazar, C., Shulaev, V., Paez-Valencia, J., Pizzio, G. A., Gaxiola, R. A. and Ayre, B. G. (2016). Constitutive and companion cell-specific overexpression of AVP1, encoding a proton-pumping pyrophosphatase, enhances biomass accumulation, phloem loading, and long-distance transport. Plant Physiol 170(1): 401-414.
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