搜索

Assessing Rates of Long-distance Carbon Transport in Arabidopsis by Collecting Phloem Exudations into EDTA Solutions after Photosynthetic Labeling with [14C]CO2
用[14C]CO2进行光合标记后通过EDTA溶液收集韧皮部渗出液来评估拟南芥长距离碳运输速率   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
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 (Yadav et al., 2017a), (2) the relative rates of carbon loading and transport through the phloem (this protocol), 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, [14C]CO2 is photoassimilated in source leaves and phloem loading and transport of photoassimilate is quantified by collecting phloem exudates into an EDTA solution followed by scintillation counting.

Keywords: Arabidopsis (拟南芥), Photosynthetic labeling (光合标记), 14C labeling (14C标记), Phloem transport (韧皮部运输), Phloem EDTA exudations (韧皮部EDTA渗出液), Carbon partitioning (碳分配)

Background

The allocation of reduced carbon and other compounds from photoautotrophic source tissues to heterotrophic sink tissues through the phloem is a crucial physiological process influencing growth and yield of plants. Because of this central role, there is interest in analyzing and quantifying phloem content from many areas of plant biology. However, collecting authentic phloem sap is difficult because the translocation stream is generally under high hydrostatic pressure and sieve elements have a rapid self-sealing mechanisms to prevent loss when damaged. Several collection techniques have emerged, but there is not currently a single or combination of methods that provide a complete and artifact-free measure of translocating phloem sap. Here, we briefly describe alternative techniques before detailing our approach to collecting phloem exudates into solutions containing low concentrations of ethylenediaminetetraacetic acid (EDTA) after photosynthetically labeling shoots with [14C]CO2. Turgeon and Wolf provide a comprehensive review of alternative techniques and their limitations (Turgeon and Wolf, 2009).

Phloem feeding insects, including aphids, scale insects, and planthoppers, evolved mechanisms to feed by directly drawing phloem sap from a plant’s vascular system and evade the self-sealing mechanism. Severing the feeding insect from a stylet penetrated into the phloem–referred to as stylectomy–can provide nanoliter to microliter quantities of sap that may most accurately reflect phloem content. Limitations of the technique are that it is technically challenging, works with only specific insect/plant combinations, the insects are selective for phloem with desired content, and insect saliva injected into the plant influences phloem content (Will et al., 2007; Hewer et al., 2011).

Another technique is to use plants that exude solution from cut stems without apparent sealing. Cucurbits, legumes, Ricinus communis, and some trees are well known for this and have become model systems for studying metabolites and signaling compounds in the phloem. However, the sap collected by this technique generally has low sugar concentrations, suggesting significant dilution and contamination from non-phloem sources and, particularly in the case of cucurbits, may be derived from specialized extrafascicular phloem elements rather than canonical fascicular phloem within vascular bundles (Zhang et al., 2010 and 2012).

The most common technique to sample phloem contents, and the one described in this protocol, is to collect phloem exudates from cut stems or petioles into solutions containing low concentrations of chelating agents, such as EDTA. Cations, particularly calcium (Ca2+), are involved in the rapid self-sealing mechanism of sieve tubes. Therefore, application of chelating agents to the cut ends of phloem-containing tissues limits sealing and permits phloem exudations for long periods from most, if not all, plants (King and Zeevaart, 1974; van Bel and Hess, 2008; Liu et al., 2012; Tetyuk et al., 2013). Although this method is most commonly used, it has important limitations and is not without controversy. Exudates are not collected directly, but are diluted into the EDTA solution during the collection period. This method therefore does not provide a measure of concentration, but rather a rate of exudation (i.e., unit of quantity per unit of time). As with the use of plants that naturally continue to exude, it is not clear how much the exudate is diluted by other sources in the plant or how much of the exudate is derived from the fascicular phloem involved in long distance translocation of photoassimilate in phloem sap. Enzymes, such as invertase from damaged cells, can enter the exudate solution and alter the profile of molecules exuded, as discussed at length in several articles (van Bel and Hess, 2008; Liu et al., 2012). A particularly important pitfall of this approach is that EDTA is toxic to cells and promotes membrane leakage which exacerbates contamination of the exudates with the contents of damaged cells and alteration of metabolite composition by leaked enzymes (Turgeon and Wolf, 2009). To minimize these impacts, we use the lowest EDTA concentration that still prevents sieve element sealing and as little tissue as possible is submersed in the EDTA solution (van Bel and Hess, 2008). In addition, we limit EDTA uptake via the xylem by conducting exudations in darkness and high humidity to promote stomatal closure and limit transpiration. Importantly, this procedure uses photosynthetic labeling of rosette leaves with [14C]CO2, and exudates are collected from cut petioles that have negligible photosynthetic activity and are well shaded by the mature rosette above. Therefore, counting 14C in the exudate solution is a quantitative representation of photoassimilate translocated from the leaves. When combined with other protocols (Yadav et al., 2017a and 2017b), this contributes to 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).

Materials and Reagents

  1. Labeling chambers derived from clear plastic deli containers (e.g., 16.5 cm long, 13 cm wide, and 5 cm deep ‘B16-Double Hot Dog Clamshell Case’ from Douglas Stephens Plastics, Paterson 640 NJ)
  2. Potting mixture (Sun Gro Horticulture, catalog number: Fafard 3B Mix ; or similar)
  3. 10 ml syringe (without a needle attached) (Luer-Lok Tip, BD, catalog number: 309604 )
  4. Dow Corning high vacuum grease
  5. Modeling clay (American Art Clay)
  6. Scotch Removable Mounting Putty
  7. 5 ml plastic syringe barrel and plunger (Luer-Lok Tip, BD, catalog number: 309646 )
  8. Syringe needle, 1.5-2.0 inches, 18 gauge (Monoject Needle, Covidien, catalog number: 1188818112 )
  9. High-humidity chambers derived from the clear plastic deli containers described above, lined with wet paper towel
  10. Petri dishes (100 x 25 mm) (Fisher Scientific, catalog number: FB0875711 )
  11. 24-well culture plates (Greiner Bio One International, catalog number: 662160 )
  12. Microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
  13. Microcentrifuge tubes, screw cap with O-rings (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3464 )
  14. 20 ml scintillation vials (Fisher Scientific, catalog number: 12383317 )
  15. Double edge razor blades (PERSONNA brand) (Electron Microscopy Sciences, catalog number: 72000 )
  16. Plant material (e.g., Arabidopsis thaliana Col-0, control and experimental material); > 12 healthy plants for each treatment with 3 WT and 3 of each experimental plant in each labeling chamber
  17. Sodium bicarbonate [14C]NaHCO3 (MP Biomedicals; catalog number: 0117441H ; 40-60 mCi/mmol; 2 mCi/ml; 5 mCi; 185 MBq)
  18. Lactic acid (85%) (Fisher Scientific, catalog number: A162-500 )
  19. Soda lime (LI-COR, catalog number: 9964-090 ) loosely packed to fill a column (e.g., Bio-Rad Econo-Column, 1.5 x 20 cm; Bio-Rad Laboratories, catalog number: 7371522 )
  20. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  21. Potassium hydroxide (KOH) for pH adjustment (Fisher Scientific)
  22. Ethanol absolute (Pharmco-AAPER, catalog number: 111000200 )
  23. Commercial bleach (Sodium hypochlorite, NaClO)
  24. Ecolume scintillation fluid (MP Biomedicals, catalog number: 0188247004 )
  25. 5 mM EDTA solution (see Recipes)

Equipment

  1. Environmental growth chambers for control and experimental plants
  2. Personal safety equipment: lab coat, nitrile gloves (or similar), and eye protection
  3. Lamp suitable for photosynthetic labeling, such as a 400 W metal halide lamp (SYLVANIA 64490 - 400 Watt - BT37 - Metal Halide)
  4. Fume hood with appropriate support for metal halide lamp (Figure 1)
  5. Light meter (LI-COR 1400)
  6. Cork borer (The Science Company, catalog number: NC-11269 )
  7. Slim line micro blowers for air circulation (optional; Exton PA, Pelonis Technologies, catalog number: RFB3004 ; powered by four 1.5 volt D-cell batteries in sequence to provide 6 volts)
  8. Small air pump with inlet and outlet (e.g., Airpo, Barcodable, catalog number: UPC 045635496699 )
  9. Clear plastic tubing (4 and 6 mm internal diameter)
  10. Scissors (Fisher Scientific, catalog number: 08-951-20 )
  11. Forceps (Fisher Scientific, catalog number: 22-327379 )
  12. Balance with readability to at least 1 mg (METTLER TOLEDO, model: AE100 )
  13. Pipettes (Bioexpress, GeneMate, P20-P1000)
  14. Microcentrifuge
  15. Rotary platform shaker (Orbital Shaker Variable, BioExpress, GeneMate, catalog number: S-3200-LS )
  16. Geiger counter (Ludlum Measurements, model: Model 3 )
  17. Scintillation counter (Beckman Counter, model: LS 6000IC )

Procedure

  1. Preparing a work area suitable for [14C]CO2 photoassimilation
    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. [14C]NaHCO3 stocks will release gaseous [14C]CO2. To minimize this, commercial stocks are buffered to pH 9.5. Stocks should be stored at 4 °C and not -20 °C: We recommend aliquoting stock into 1.5 ml screw cap microcentrifuge tubes with O-rings. Receipt and use of stocks should be recorded as required by the institute where the experiments are conducted.
    3. In this procedure, [14C]CO2 gas is created by acidification of [14C]NaHCO3. Establishing a work area in a dedicated fume hood is recommended (with appropriate traps for [14C]CO2; see below).
    4. Within the fume hood, a 400 W metal halide lamp is suspended 60 to 90 cm above the work area. We constructed a simple frame from 2 x 4 lumber inside our fume hood to suspend the lamp (Figure 1).
    5. The distance between the lamp and the working surface will impact the photon flux density to which the plants are exposed. Our lamp is 90 cm above the fume hood bench top, and we work on a 15 cm platform so the plants are 75 cm from the lamp. At this distance, we can expose six of the indicated clamshell containers with equal illumination of 130 µmol photons m-2 sec-1, as determined with a PAR (photosynthetically active radiation) sensor.


      Figure 1. A representation of a frame that fits inside a fume hood and allows a metal halide lamp to be supported above the labeling work surface. The frame is made of standard 2 x 4 lumber (orange), and the 400 W metal halide light fixture (red/yellow) is supported by a metal rod (black).

  2. Strategy to germinate and grow plants
    1. Germinate seeds in potting mixture in transparent, clamshell deli containers. Use a 6 mm cork borer to make drainage holes in the bottom of clamshell deli containers for aeration and drainage. Fill the bottom half of the clamshell container with potting mix, 3.5 cm deep and water the potting mix. Add seeds and germinate by standard procedures for growth on potting mixture (Dasgupta et al., 2014; Khadilkar et al., 2016).
      Notes:
      1. Each clamshell deli container will be an independent [14C]CO2 labeling chamber. Therefore, controls and experimental plants should be grown together in the same chamber. In the clamshell containers we use, nine plants can be grown without crowding for 21-28 days. We typically grow WT controls and two experimental lineages in a randomized design in a single container, and have six replicate containers.
      2. The hypocotyl will be cut and submerged in EDTA solution to collect exudates. Germinating seeds in darkness, such as in a light-tight cabinet, drawer or box, for 4 to 5 days will promote hypocotyl elongation and make it easier to work with.
    2. Grow plants under a 12 h light (22 °C)/12 h dark (20 °C) diurnal cycle at 130 μmol photons m-2 sec-1, or other conditions suitable to the specific experiment. Keep the lids of the chamber open and maintain watering and fertilizer levels as appropriate for plants grown on potting mix. Grow the plants for 21-28 days to produce rosettes 3 to 4 cm in diameter.
      Note: The transition from vegetative to reproductive growth impacts source/sink relationships. We plan our experiments so all plants are only vegetative during labeling and exudation.

  3. Preparation for [14C]CO2 photosynthetic labeling
    1. Photograph the plants on the day they are to be labeled. Turn on the 400 W metal halide light in the fume hood about an hour before labeling, so it reaches a stable intensity. Acclimate the plants under the metal halide lamp for about an hour before labeling, with the lid of the labeling chamber open.
      Notes:
      1. Photosynthesis, carbon partitioning into different metabolic pools, and long-distance transport to sink organs fluctuate through the diurnal cycle. For consistency, we generally label 6 h into the illuminated period.
      2. Although six clamshell containers will fit under the light and receive equal light intensity, it is impractical to process this number of plants in a single day (see time frame Table 1). With two people working together, we process three clamshell containers on one day, and the other three the following day. It is better to spread the experiment over two days and conduct labeling in the same phase of the diurnal cycle than it is to have the first clamshell container and the last clamshell container processed at significantly different times of day.

      Table 1. Sample time sheet (frame) to organize labeling and processing three chambers


    2. Prepare the chambers for labeling. Make two holes in the lid of each clamshell container: a small one in the front side to inject [14C]CO2, and one slightly bigger in the opposite side to remove remaining [14C]CO2 after labeling (see flow chart, Figure 2A). The end of a paper clip, heated with a Bunsen burner, works well. To improve the seal of the labeling chamber, fill a 10 ml syringe (without a needle attached) with vacuum grease and apply a bead around the internal margin of the cover, but do not close and seal the cover until just before labeling. Use a small ball of modeling clay to cover the inject and exhaust holes.


      Figure 2. A pictorial flow chart for photosynthetic labeling Arabidopsis plants with [14C]CO2 and collecting and quantifying 14C exudates into EDTA solutions. A. Grow Arabidopsis plants for 3-4 weeks in clear plastic deli containers containing potting mixture and with holes in the bottom for drainage and aeration, and two holes in the top to inject and exhaust [14C]CO2 (circles with arrows–blue for injection and red for exhaust). Acclimate the plants in the fume hood under the metal halide lamp for about an hour. Keep the lids open until just before labeling, and then seal by applying a bead of vacuum grease around the lid; place a small patch of modeling clay over the inject and exhaust holes. B. Prepare a syringe barrel for injecting [14C]CO2. A barrel cut to ~3 cm is easier to work with than an intact barrel. Place 2.5 µl of [14C]NaHCO3 in the barrel near the needle (indicated with yellow arrow) and a 15 µl of lactic acid ~1 cm away (indicated with gray arrow). C. Gently insert the plunger inside the barrel without disturbing the separate drops of [14C]NaHCO3 and lactic acid. D. Insert the needle through the inject hole and create [14C]CO2 by mixing [14C]NaHCO3 with lactic acid; pump the plunger 2-3 times to mix thoroughly and push the labeled gas into the chamber. E. Allow photosynthesis to proceed under the metal halide lamp for a 20 min ‘pulse’. F. Remove remaining unassimilated [14C]CO2 through the exhaust hole for 5 min using Tygon tubing connected to a vacuum pump and a column containing soda lime. G. Open the cover of labeling chamber and let photosynthesis continue for another 35 min ‘chase’. H. Gently cut the stem below the soil surface. I. Clean soil particles from the root surface, record the fresh weight. J. Recut through the hypocotyl under EDTA solution to prevent sieve element sealing. K. Transfer the shoot to a 24-well culture plate containing EDTA solution, such that only ~2 mm of hypocotyl is in the solution. Place the plate in a humidity chamber and then place in the dark for 10 min. Similarly collect subsequent exudations in 24-well culture plates with EDTA solution for the 1st and 2nd h. L. After collecting exudates, store the plates on ice to minimize enzyme activity until they can be conveniently processed further (image shows the plate on a bed of ice). Transfer phloem exudates into scintillation vials and add scintillation fluid (M) for scintillation counting. The labeled rosette should also be counted.

    3. Create a schedule (Table 1) to organize processing of three replicate containers.
    4. With three replicate containers arranged and acclimated under the light source, close the lid of the first chamber to be labeled and ensure a tight seal. To create [14C]CO2 for labeling, pipette 2.5 μl of [14C]NaHCO3 (2 μCi/μl) in a droplet near the syringe needle junction of a syringe barrel cut to ~3 cm (Figure 2B). Place a 15 µl droplet of 85% lactic acid in the barrel, being careful to keep this droplet separate from the droplet of [14C]NaHCO3. Gently insert the plunger just inside the barrel (Figure 2C). Insert the needle through the injection hole of the clamshell container, and arrange the modeling clay around it (Figure 2D and 2E) to seal the hole; make sure the exhaust hole is also covered. Keep the droplets of [14C]NaHCO3 and lactic acid separate during these steps. Push the plunger gently to mix the lactic acid with [14C]NaHCO3 and release the [14C]CO2 into the air space of the chamber. Move the plunger back and forth to pump the [14C]CO2 gas into the clamshell container; avoid injecting fluids into the chamber since the lactic acid can damage the plants. Remove the needle and cover the injection hole with modeling clay.
      Note: (Optional) For improved [14C]CO2 circulation, a small blower fan, such as one typically used for cooling small electronic equipment (e.g., Pelonis Technologies Cat. No. RFB3004), can be oriented inside the labeling chamber to blow air across the plants. We typically use Scotch Removable Mounting Putty to hold the blower to the top or side of the chamber, with the wires emerging through the vacuum grease used to seal top and bottom halves of the chamber. Power is provided by D-cell batteries.
    5. Allow plants to do photosynthesis in the presence of [14C]CO2 for 20 min. This is the ‘pulse’ phase (Figure 2E).
    6. While plants in the first chamber are being labeled, prepare and label the second chamber as indicated in Table 1, and then the third chamber, etc.
    7. 20 min after injecting [14C]CO2, use the larger hole to vent the chamber through soda lime and to capture unassimilated [14C]CO2 (Figure 2F). A column filled with soda lime connected with tubing to an air pump works well (Figure 3). After ~5 min of venting through soda lime, open the chamber lid (Figure 2G) to allow photosynthesis to continue for another 35 min. This provides 40 min of total ‘chase’ time.
      Note: To ensure effective capture of unassimilated CO2 by the soda lime, it should be fresh and well-sealed between uses. Old soda lime should be discarded as 14C labeled dry waste.


      Figure 3. Exhaust system to remove and capture unassimilated [14C]CO2. A column, such as a Bio-Rad chromatography column, is filled with soda lime to capture residual [14C]CO2. One end of the column has tubing (4 mm internal diameter) to insert into the labeling chamber (red arrow) to vent out the remaining [14C]CO2. The other end is connected to the air intake of a small air pump, and the outlet is vented into the exhaust stream of the fume hood. Both ends of the soda lime column are equipped with stopcocks to minimize soda lime exposure to ambient CO2 when the column is not in use.

  4. Collection of phloem exudates
    1. Prior to initiating labeling, prepare three 24-well culture plates for each clamshell labeling chamber. These will be used for the initial wash step, the 1st h of exudation, and the 2nd h of exudation, respectively. Fill nine, well-spaced wells with 500 µl 5 mM EDTA solution, and set aside until needed. Prepare high-humidity chambers: prepare clamshell containers (one for each labeling chamber) with a bead of vacuum grease as described above; line the bottom half of the containers with wet paper towels. During exudation the 24-well culture plates will be placed in these high-humidity chambers and the chambers will be placed in the dark to stimulate stomatal closure and limit transpiration.
    2. At T = 60 min, use small, sharp scissors to cut below the hypocotyls in the uppermost part of the root. Remove any soil clinging to the plant and record the rosette fresh weight (Figures 2H and 2I).
      Note: This weight will include a small portion of roots that are removed in the next step (D3), but we considered this small amount to be negligible and it is important to conduct Step D3 quickly without reweighing the tissue.
    3. Submerge the cut end of the stem in a Petri dish containing 5 mM EDTA. Keeping the stem submerged, use one half of a double-edged razor blade, snapped in half lengthways, to cleanly slice the stem in the hypocotyl ~5 mm up from the first cut (Figures 2I and 2J).
      Note: Double-edged razor blades are sharper than single edged blades. To work with them safely, fold the blade lengthways down the center while it is still in the protective wrapper and it will snap in half to provide a safe single edged blade.
    4. Transfer each rosette immediately to the first prepared 24-well culture plate containing 500 µl 5 mM EDTA solution so that ~2 mm of cut stem is submerged. Minimize EDTA exposure to the rest of the plant tissues (Figure 2K).
    5. Once all nine plants are processed, place the 24-well culture plate into the clamshell humidity chamber and close the chamber to maintain near 100% humidity (Figure 2K). Place the humidity chamber in complete darkness, such as a drawer or box, to promote stomatal closure.
      Notes:
      1. Steps D1 to D5 should be carried out carefully but efficiently. Table 1 allows for ~10 min to process the nine plants in each labeling chamber, and ~10 min for the wash step in EDTA solution. This initial wash step is intended to remove enzymes released from the cut and damaged surface.
      2. Leave the plants in high humidity and darkness until further processing. Throughout this wash step and subsequent collection steps, the plants should be in high humidity and darkness unless they are being actively worked on.
    6. Transfer each rosette to a second 24-well culture plate, submerging only ~2 mm of cut stem in the EDTA solution. Place this culture plate into a humidity chamber, seal the lid, and store in darkness for 1 h. If exudates from the first plate (i.e., the EDTA wash solution) are to be counted, store the plate on ice to minimize potential enzyme activity until it can be conveniently processed for scintillation counting (Figure 2L). Transfer the solutions to microcentrifuge tubes and store in the freezer for longer term storage.
    7. After 60 min exudation, transfer the rosettes to the third 24-well culture plate, submerging only ~2 mm of cut stem in the EDTA solution. Place this third plate in the humidity chamber and store in the dark for another 60 min. Place the second plate (i.e., exudations from the first hour) on ice to minimize potential enzyme activity until it can be conveniently processed for scintillation counting (Figure 2L). Transfer the solutions to microcentrifuge tubes and store in the freezer for longer term storage.
    8. After 60 min, remove the rosettes and chill this third plate (i.e., exudations from the second hour) on ice to minimize potential enzyme activity until it can be conveniently processed for scintillation counting (Figure 2L), or transfer the solutions to microcentrifuge tubes and store in the freezer for longer term storage.

  5. Analyzing phloem exudates by scintillation counting
    1. Transfer 400 µl of each EDTA solution to scintillation vials (Figure 2M) and add 5 ml scintillation fluid for aqueous samples. Mix thoroughly by shaking the vial to create a monophasic solution. Document counts per min (cpm) or disintegrations per min (dpm) with a scintillation counter and a program suitable for 14C. Calculate 14C in 500 µl exudations as a rate per rosette mass: cpm or dpm (fresh weight)-1 h-1. The first and second hour of exudations should be analyzed by scintillation counting and should give reasonably consistent counts. Remaining EDTA solution should be collected and disposed as 14C liquid waste.
    2. To assess the remaining quantity of labeled photoassimilate in shoots, submerge the rosette in 1 ml 80% ethanol, and gently agitate on a shaker for ~1 h to extract pigments and metabolites (a greater volume may be required for larger rosettes). Add 500 µl of commercial bleach and agitate for ~1 h to destroy the pigments. Add sufficient scintillation fluid and mix thoroughly to get a clear, single-phase solution; 5-10 volumes of scintillation fluid to 1 volume of ethanol/bleach solution should suffice.
    3. (Optional) To measure individual metabolites by HPLC or enzymatic assay, add 500 µl chloroform to exudates, mix and centrifuge to separate the phases. Recover the aqueous phase to fresh microcentrifuge tubes for polar metabolites. Retain the chloroform phase if required to analyze non-polar metabolites.
    4. The exudates can be stored at -20 °C for further analysis if desired, bearing in mind that exudates are isotopically labeled with 14C.

Data analysis

Experimental design for data analysis
Each chamber constitutes an independent labeling experiment. Values obtained from experimental plants in each plate should be standardized to a percent value of WT controls in the same chamber. Standardized values from separate chambers are then combined as independent replicates. This removes chamber to chamber variation in labeling efficiency to provide a more accurate representation of the differences in photoassimilation, distribution and transport between controls and experimental plants (Khadilkar et al., 2016).

Recipes

  1. EDTA solution (0.5 M stock 500 ml) (FW 292.24)
    Dissolve 73.06 g EDTA into 300 ml of ddH2O
    Adjust pH to 8.0 with 5 N KOH. Make the final volume 500 ml
    Autoclave in liquid cycle for 15 min
    Store at room temperature
    For working concentration, dilute 100x to prepare 5 mM of the required volume and adjust to pH 6.0

Acknowledgments

This protocol is based on methods published in (Dasgupta et al., 2014; Khadilkar et al., 2016). 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.

References

  1. Dasgupta, K., Khadilkar, A. S., Sulpice, R., Pant, B., Scheible, W. R., Fisahn, J., Stitt, M. and Ayre, B. G. (2014). Expression of sucrose transporter cDNAs specifically in companion cells enhances phloem loading and long-distance transport of sucrose but leads to an inhibition of growth and the perception of a phosphate limitation. Plant Physiol 165(2): 715-731.
  2. Hewer, A., Becker, A. and van Bel, A. J. (2011). An aphid's Odyssey--the cortical quest for the vascular bundle. J Exp Biol 214(Pt 22): 3868-3879.
  3. 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.
  4. King, R. W. and Zeevaart, J. A. (1974). Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiol 53(1): 96-103.
  5. Liu, D. D., Chao, W. M. and Turgeon, R. (2012). Transport of sucrose, not hexose, in the phloem. J Exp Bot 63(11): 4315-4320.
  6. Slewinski, T. L., Zhang, C. and Turgeon, R. (2013). Structural and functional heterogeneity in phloem loading and transport. Front Plant Sci 4: 244.
  7. Tetyuk, O., Benning, U. F. and Hoffmann-Benning, S. (2013). Collection and analysis of Arabidopsis phloem exudates using the EDTA-facilitated method. J Vis Exp (80): e51111.
  8. Turgeon, R. and Wolf, S. (2009). Phloem transport: cellular pathways and molecular trafficking. Annu Rev Plant Biol 60: 207-221.
  9. van Bel, A. J. E. and Hess, P. H. (2008). Hexoses as phloem transport sugars: the end of a dogma? J Exp Bot 59: 261-272.
  10. Will, T., Tjallingii, W. F., Thonnessen, A. and van Bel, A. J. (2007). Molecular sabotage of plant defense by aphid saliva. Proc Natl Acad Sci U S A 104(25): 10536-10541.
  11. Yadav, U. P., Khadilkar, A. S., Shaikh, M. A., Turgeon, R. and Ayre, B. G. (2017a). Quantifying the capacity of phloem loading in leaf disks with [14C]sucrose. Bio Protoc 7(24): e2658.
  12. 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 .
  13. Zhang, B., Tolstikov, V., Turnbull, C., Hicks, L. M. and Fiehn, O. (2010). Divergent metabolome and proteome suggest functional independence of dual phloem transport systems in cucurbits. Proc Natl Acad Sci U S A 107(30): 13532-13537.
  14. Zhang, C., Yu, X., Ayre, B. G. and Turgeon, R. (2012). The origin and composition of cucurbit "phloem" exudate. Plant Physiol 158(4): 1873-1882.

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,我们经常使用它们相互结合来评估(1)蔗糖(Suc)加载到成熟叶片的韧皮部血管系统中的相对比率(Yadav等人,2017a), (2)通过韧皮部的碳载量和转运的相对速率(本方案);(3)长距离运输后碳向异养池器官,特别是根部卸载的相对速率(Yadav等,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,并将与单独进行的单个方案相关的模糊度最小化(Dasgupta等人, 2014年;卡迪尔卡尔等人,2016年)。在该方案中,在源叶中光致同化[14C] CO 2 2-,并且通过将韧皮部流出物收集到EDTA溶液中,随后进行闪烁计数来量化光合同化物的韧皮部负载和转运。

【背景】通过韧皮部将光合自养源组织中的还原碳和其他化合物分配到异养池组织是影响植物生长和产量的关键生理过程。由于这一核心作用,有兴趣从植物生物学的许多领域分析和量化韧皮部含量。然而,收集真正的韧皮部汁液是困难的,因为易位流一般在高静水压力下,而且筛分元件具有快速的自密封机制以防止损坏时的损失。几种收集技术已经出现,但目前还没有一种方法或一组方法提供了一个完整的,无伪象的易位韧皮部液位测量方法。在这里,我们简要描述替代技术,然后详细描述我们的方法,收集韧皮部分泌物到含有低浓度乙二胺四乙酸(EDTA)的溶液中,然后用[14 C] CO 2 / sub >。 Turgeon和Wolf对替代技术及其局限性进行了全面的回顾(Turgeon和Wolf,2009)。

韧皮部的昆虫,包括蚜虫,蚧虫和飞虱,通过从植物的血管系统直接抽取韧皮部汁液并逃避自密封机制进化的机制。通过探针切断进食的昆虫,进入韧皮部(称为“方形切除术”),可以提供纳升至微升量的汁液,其可以最准确地反映韧皮部含量。该技术的局限在于技术上具有挑战性,仅与特定的昆虫/植物组合一起工作,昆虫对具有期望含量的韧皮部是选择性的,并且注入植物中的昆虫唾液影响韧皮部含量(Will >,2007; Hewer等人,2011年)。

另一种技术是使用从切茎渗出而没有明显密封的植物。葫芦科植物,豆科植物,蓖麻(Ricinus communis)和一些树木是众所周知的,已经成为研究韧皮部代谢物和信号传导化合物的模型系统。然而,采用这种技术收集的汁液一般糖浓度低,这表明来自非韧皮部源的明显稀释和污染,特别是在葫芦的情况下,可能来自特定的束外韧皮部元件而不是维管束内的典型束状韧皮部( Zhang等人,2010和2012)。

对韧皮部内容进行取样的最常用的方法是本方案中描述的方法,是将切茎或叶柄的韧皮部分泌物收集到含有低浓度螯合剂(如EDTA)的溶液中。阳离子,特别是钙(Ca2 +),参与了筛管的快速自封机理。因此,将螯合剂应用于含韧皮部组织的切割端限制了密封,并且允许来自大多数(如果不是全部的话)植物的长时间的韧皮部渗出(King和Zeevaart,1974; van Bel和Hess,2008; Liu等, et al。,2012; Tetyuk et al。,em。,2013)。虽然这种方法是最常用的,但是它有很大的局限性,并不是没有争议。分泌物不直接收集,但在收集期间稀释到EDTA溶液中。因此,该方法不提供浓度测量,而是提供渗出速率(即每单位时间的单位量)。与使用自然持续渗出的植物一样,尚不清楚植物中其他来源的渗出物被多少稀释,或多少渗出物来自涉及韧皮部汁液中光合同化物远距离移动的束状韧皮部。酶,如来自受损细胞的转化酶,可以进入渗出物溶液并改变渗出的分子的轮廓,如在几篇文章中详细讨论的(van Bel和Hess,2008; Liu等人,2012 )。这种方法的一个特别重要的缺陷是EDTA对细胞有毒,并且促进了膜的渗漏,加剧了受损细胞内容物的渗出物的污染以及由于泄漏的酶而改变代谢物组成(Turgeon and Wolf,2009)。为了尽量减少这些影响,我们使用最低的EDTA浓度仍然可以防止筛网元件密封,尽可能少的组织浸入EDTA溶液(van Bel和Hess,2008)。此外,通过在黑暗和高湿度下进行渗出来限制EDTA通过木质部吸收,促进气孔关闭和限制蒸腾。重要的是,这个过程使用光合作用标记莲座叶与[14C] CO 2,并且从具有可忽略的光合活性的切割叶柄收集渗出物,并且被以上的成熟玫瑰花。因此,在渗出液中计数14 C是光合同化物从叶子移位的定量表示。当与其他方案(Yadav等,2017a和2017b)结合时,这有助于全植物碳分配的可靠比较,并最小化与单独进行的单个方案(Dasgupta <等人,2014; Khadilkar等人,2016)。

关键字:拟南芥, 光合标记, 14C标记, 韧皮部运输, 韧皮部EDTA渗出液, 碳分配

材料和试剂

  1. 来自Douglas Stephens Plastics,Paterson 640 NJ的透明塑料熟食容器(例如,长16.5cm,宽13cm,深5cm的'B16-双热狗蛤壳壳')的标签室< br />
  2. 盆栽混合物(Sun Gro园艺,目录号:Fafard 3B Mix;或类似物)
  3. 10毫升注射器(没有连接针头)(Luer-Lok Tip,BD,目录号:309604)
  4. 道康宁高真空润滑脂
  5. 塑造黏土(美国艺术粘土)
  6. 苏格兰威士忌可拆卸安装油灰
  7. 5毫升塑料注射器筒和柱塞(Luer-Lok Tip,BD,目录号:309646)
  8. 注射针,1.5-2.0英寸,18号(Monoject Needle,Covidien,目录号:1188818112)
  9. 由湿纸巾内衬的上述透明塑料熟食容器产生的高湿度室
  10. 培养皿(100×25毫米)(Fisher Scientific,目录号:FB0875711)
  11. 24孔培养板(Greiner Bio One International,目录号:662160)
  12. 微量离心管(Fisher Scientific,目录号:05-408-138)
  13. 微量离心管,带O形环的螺旋盖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3464)
  14. 20ml闪烁瓶(Fisher Scientific,目录号:12383317)
  15. 双刃刀片(PERSONNA品牌)(电子显微镜科学,目录号:72000)
  16. 植物材料(例如,拟南芥Col-0,对照和实验材料); &GT;
    每个处理12个健康植物,每个标记室有3个WT和3个实验植物
  17. 碳酸氢钠[14 C] NaHCO 3(MP Biomedicals;目录号:0117441H; 40-60mCi / mmol; 2mCi / ml; 5mCi; 185MBq) br />
  18. 乳酸(85%)(Fisher Scientific,目录号:A162-500)
  19. 将碱石灰(LI-COR,目录号:9964-090)松散地填充以填充柱(例如Bio-Rad Econo-Column,1.5×20cm; Bio-Rad Laboratories,目录号:7371522)
  20. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  21. 用于pH值调整的氢氧化钾(KOH)(Fisher Scientific)
  22. 乙醇绝对(Pharmco-AAPER,目录号:111000200)
  23. 商业漂白剂(次氯酸钠,NaClO)
  24. Ecolume闪烁液(MP Biomedicals,目录号:0188247004)
  25. 5毫米EDTA溶液(见食谱)

设备

  1. 控制和实验工厂的环境增长室
  2. 个人安全设备:实验室外套,丁腈手套(或类似的)和护目镜
  3. 适用于光合标记的灯,如400瓦金属卤化物灯(SYLVANIA 64490 - 400瓦 - BT37 - 金属卤化物灯)
  4. 通风橱适当支撑金属卤化物灯(图1)
  5. 测光表(LI-COR 1400)
  6. 软木钻(科学公司,目录号:NC-11269)
  7. 用于空气循环的超薄型微型鼓风机(可选; Exton PA,Pelonis Technologies,产品目录号:RFB3004;由四个1.5伏D型电池依次供电,以提供6伏电压)
  8. 带入口和出口的小型空气泵(例如,Airpo,Barcodable,产品目录号:UPC 045635496699)
  9. 清除塑料管(内径4和6毫米)
  10. 剪刀(Fisher Scientific,目录号:08-951-20)
  11. 镊子(Fisher Scientific,目录号:22-327379)
  12. 平衡可读性至少1毫克(梅特勒 - 托利多,型号:AE100)
  13. 移液器(Bioexpress,GeneMate,P20-P1000)
  14. 微量离心机
  15. 旋转平台摇床(轨道摇床变量,BioExpress,GeneMate,目录号:S-3200-LS)
  16. 盖革计数器(Ludlum测量,模型:模型3)
  17. 闪烁计数器(贝克曼计数器,型号:LS 6000IC)

程序

  1. 准备一个适合于[14C] CO_2光同化的工作区
    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. [14C] NaHCO 3储备将释放气态[14 C] CO 2 2。为了尽量减少这一点,商业库存缓冲到pH 9.5。库存应储存在4°C而不是-20°C:我们建议将样品分装到带有O型圈的1.5 ml螺旋盖微量离心管中。应按实验所在机构的要求记录库存的收货和使用情况。
    3. 在这个过程中,通过酸化[14 C] NaHCO 3 3产生[14 C] CO 2气体, 。建议在专用通风橱中建立一个工作区域(对[14C] CO 2 使用适当的陷阱;见下文)。
    4. 在通风柜内,400瓦金属卤化物灯悬挂在工作区域上方60至90厘米处。
      在我们的通风橱里,我们用2×4木材制作了一个简单的框架,用来悬挂灯(图1)
    5. 灯与工作表面之间的距离将影响植物所暴露的光子通量密度。我们的灯在通风橱台面上方90厘米,我们在15厘米的平台上工作,所以植物离灯泡75厘米。在这个距离,我们可以暴露六个指示的蛤壳式容器与130μmolphotons m 2 sup-1 -1的相同的照明,如PAR(光合有效辐射)传感器。


      图1.表示安装在通风橱内的框架,并允许金属卤化物灯支撑在标签工作表面的上方。框架由标准2 x 4木材(橙色)制成,和400瓦的金属卤化物灯(红/黄)由金属杆(黑色)支撑。

  2. 发芽和种植植物的策略
    1. 在透明的蛤壳式熟食容器中的盆栽混合物中发芽种子。使用一个6毫米的软木塞钻机在翻盖的熟食容器的底部设置排水孔进行曝气和排水。用盆栽混合物填充蛤壳式容器的下半部分,深度为3.5厘米,然后浇灌盆栽混合物。通过在盆栽混合物上生长的标准程序添加种子并发芽(Dasgupta等人,2014; Khadilkar等人,2016)。
      注意:
      1. 每个蛤壳式熟食容器将是一个独立的[14C] CO 2标签室。因此,控制和实验工厂应该在同一个房间里一起生长。在我们使用的蛤壳式容器中,可以种植九种植物而不会拥挤21-28天。我们通常在一个容器中随机设计生长WT对照和两个实验谱系,并有六个复制容器。
      2. 下胚轴将被切割并浸没在EDTA溶液中以收集渗出物。在黑暗的地方,如在一个不透光的柜子,抽屉或盒子里,种子发芽4至5天将促进下胚轴伸长,使其更容易处理。
    2. 在12μm光照(22℃)/ 12小时黑暗(20℃)昼夜循环中以130μmol光子m 2 / s -1或其它适合具体实验的条件。保持室的盖子打开,并保持灌溉混合物生长的植物适当的浇水和肥料水平。种植植物21-28天,以产生3至4厘米直径的玫瑰花结。
      注:从营养生长到生殖生长的转变影响源/汇关系。我们计划我们的实验,使所有的植物在标签和渗出过程中只有营养。

  3. 制备[14 C] CO 2 2-光合标记
    1. 在植物标记的那天拍摄植物。贴标前一个小时左右打开通风橱内的400W金属卤化物灯,达到稳定的强度。在金属卤化物灯下使植物适应大约一个小时,然后打开贴标签盖。
      注意:
      1. 通过昼夜循环,光合作用,碳分配到不同的代谢库,以及长途运输到宿主器官都是波动的。为了保持一致性,我们通常在光照期间标记6小时。
      2. 虽然六个蛤壳式容器可以安装在灯光下,并获得相同的光照强度,但在一天内处理这些植物数量是不切实际的(见表1的时间表)。两个人一起工作,我们一天处理三个蛤壳式容器,第二天另外处理三个。最好在两天的时间内分散实验,并在昼夜周期的同一阶段进行标记,而不是在一天中明显不同的时间处理第一个蛤壳式容器和最后一个蛤壳式容器。

      表1.组织标签和处理三个房间的样本时间表(框架)


    2. 准备标签的商会。在每个蛤壳式容器的盖子上做两个孔:一个在正面的一个小孔注入[14C] CO 2 ,另一个稍微大一些在标记后去除剩余的[ 14 C] CO 2 (参见流程图,图2A)。用本生灯加热的回形针末端效果很好。为了改善标签室的密封性,用真空润滑脂填充10毫升注射器(不带针头),并在封盖的内边缘周围涂上一个小珠,但是在贴标签之前不要关闭和密封封盖。
      使用一个塑料粘土的小球覆盖注入和排气孔

      图2.光合标记拟南芥属植物的图示流程图,其中[14 C] CO 2 2和收集和定量的 2 将14 C的渗出物分散到EDTA溶液中。A.将植物拟南芥植物在透明塑料熟食容器中培养3-4周,所述塑料熟食容器包含盆栽混合物并在底部具有用于排水和曝气的孔顶部有两个孔,用于注入和排出[14C] CO 2(箭头形的圆形蓝色注射和红色的排气)。使金属卤化物灯下的通风橱中的植物适应大约一个小时。保持盖子打开直到贴标签为止,然后在盖子周围涂上一层真空润滑脂进行密封;在注入孔和排气孔上放一小块建模粘土。 B.准备用于注射[14 C] CO 2 2的注射器筒。一桶切到〜3厘米比一个完好的桶更容易工作。将2.5μl的[14 C] NaHCO 3 3置于针(用黄色箭头表示)附近的针筒中,并将15μl乳酸置于〜1cm处(用灰色箭头)。 C.轻轻地将活塞插入桶内而不干扰[14C] NaHCO 3和乳酸的单独滴。 D.将针穿过注射孔并通过混合[14 C] NaHCO 3 3而形成[14 C] CO 2 2,用乳酸;泵动柱塞2-3次以彻底混合并将标记的气体推入腔室。 E.让光合作用在金属卤化物灯下进行20分钟'脉冲'。 F.使用与真空泵连接的聚乙烯管和含有碱石的柱,通过排气孔除去剩余的未同化的[14 C] CO 2 5分钟。 G.打开贴标签的盖子,让光合作用继续进行35分钟的“追逐”。 H.轻轻切割土壤表面下的茎。 I.清除根面的土壤颗粒,记录鲜重。 J.在EDTA溶液下通过下胚轴切断以防止筛网元件密封。 K.将芽转移到含有EDTA溶液的24孔培养板中,使溶液中仅有〜2mm的下胚轴。将平板置于湿度室中,然后在黑暗中放置10分钟。同样采用EDTA溶液收集随后在24孔培养板中渗出的1s和2h。 L.收集分泌物后,将平板放置在冰上以使酶活性最小化,直到它们能被方便地进一步处理(图像显示在冰床上)。将韧皮部渗出液转移到闪烁瓶中,并添加闪烁液(M)进行闪烁计数。
      标记的花环也应计算在内。

    3. 创建一个时间表(表1)来组织三个复制容器的处理。
    4. 在光源下方放置三个重复的容器并使其适应环境,关闭第一个室的盖子,以确保密封。为了产生用于标记的[14 C] CO 2 2,移取2.5μl的[14 C] NaHCO 3( 2μCi/μl)在注射器针筒的注射器针接头附近的液滴中切割至〜3cm(图2B)。将15μl85%的乳酸液滴放入桶中,注意保持该液滴与[14 C] NaHCO 3液滴分离。轻轻地将柱塞插入桶内(图2C)。将针穿过蛤壳式容器的注入孔,并在其周围放置造型粘土(图2D和2E)以密封孔;确保排气孔也被覆盖。在这些步骤中保持[14 C] NaHCO 3和乳酸的液滴分离。轻轻推动柱塞,将乳酸与[14 C] NaHCO 3混合并释放[14 C] CO 2 2,进入腔室的空间。前后移动柱塞将[14C] CO 2气体泵入蛤壳式容器中;避免将液体注入腔内,因为乳酸会损害植物。
      拆下针头并用注塑粘土盖住注射孔。
      C 2 在小型电子设备(例如,Pelonis Technologies目录号RFB3004)中通常使用的小型鼓风机可以被定向在贴标签腔室内以将空气吹过工厂。我们通常使用Scotch Removable Mounting Putty将鼓风机固定在燃烧室的顶部或侧面,通过用于密封燃烧室顶部和底部半部分的真空润滑脂形成电线。电池由D-cell电池提供。
    5. 允许植物在[14 C] CO 2存在下进行光合作用20分钟。这是“脉冲”阶段(图2E)。
    6. 第一个房间的植物正在贴上标签,准备第二个房间,如表1所示,然后标记第三个房间,等等。
    7. 注入14C] CO 2 20分钟后,使用较大的孔通过碱石灰排出反应室并捕获未同化的[14 C] ] CO 2(图2F)。一个装满苏打石灰的管子连接着一个气泵,工作良好(图3)。在通过碱石灰通气5分钟后,打开室盖(图2G),使光合作用继续进行35分钟。这提供了40分钟的“追逐”时间。
      注意:为确保碱石灰有效捕获未被同化的CO 2,应在使用之间保持新鲜和良好的密封性。旧苏打石灰应作为14 C标记的干燥废物丢弃。


      图3.用于去除和捕获未同化的[14C] CO 2的排气系统。一列如Bio-Rad色谱柱,充满苏打石灰以捕获残留的[14 C] CO 2 2。柱子的一端有管子(内径4mm)插入贴标签室(红色箭头),以排出剩余的[14C] CO 2。另一端连接到小型气泵的进气口,出气口排入通风柜的排气管。苏打石灰塔的两端都装有活塞,以便在不使用塔时,将碱石灰暴露于环境CO 2下降到最低程度。

  4. 收集韧皮部分泌物
    1. 在开始标记之前,为每个蛤壳标记室准备三个24孔培养板。这些将被用于初始洗涤步骤,分别是渗出的第一个h和渗出的第二个h。用500μl5mM EDTA溶液填充9个很宽的孔,并放置直到需要。准备高湿室:如上所述用真空润滑脂珠制备蛤壳式容器(每个标签室一个);用湿纸巾将容器的下半部分排成一列。在渗出过程中,将24孔培养板置于这些高湿度的室中,室将放置在黑暗中以刺激气孔关闭并限制蒸腾作用。
    2. 在T = 60分钟时,使用小而锋利的剪刀剪下根部最上部的下胚轴。去除附着在植物上的任何土壤,并记录莲座鲜重(图2H和2I)。
      注意:这个重量将包括在下一个步骤(D3)中被移除的一小部分根部,但是我们认为这个小部分是微不足道的,重要的是快速地进行步骤D3而不重新组织重量。 em>
    3. 将茎的切割末端浸入含有5mM EDTA的培养皿中。保持茎淹没,使用一半的双刃剃刀刀片,纵向劈开一半,以干净切片从第一次切割(图2I和2J)〜5毫米的下胚轴茎杆。
      注:双刃刀片比单刃刀片锋利。安全地使用它们时,将刀片沿着中心纵向折叠,同时保持在保护包装中,并将其对齐,以提供安全的单刃刀片。
    4. 将每个花环立即转移到第一个准备好的含有500μl5mM EDTA溶液的24-孔培养板中,以使〜2mm的茎切下。
      最小化EDTA暴露于其余植物组织(图2K)。
    5. 一旦处理了所有九个植物,将24孔培养板置于蛤壳式湿度室中并关闭该室以维持接近100%的湿度(图2K)。将湿度室放在完全黑暗的地方,如抽屉或盒子,以促进气孔关闭。
      注意:
      1. 步骤D1至D5应该小心而有效地进行。表1允许〜10分钟处理每个标记室中的9个植物,并且在EDTA溶液中处理〜10分钟。这个初步的洗涤步骤旨在去除切割和损坏的表面释放的酶。
      2. 将植物置于高湿度和黑暗环境中直至进一步处理。在整个洗涤步骤和随后的收集步骤中,植物应该处于高湿度和黑暗环境中,除非它们正在积极地进行处理。
    6. 将每个玫瑰花结转移到第二个24孔培养板上,在EDTA溶液中仅浸渍〜2mm的茎杆。将此培养皿置于湿度室中,密封盖子并在黑暗中保存1小时。如果要计数来自第一个板(即EDTA洗涤溶液)的渗出物,将板储存在冰上以使潜在的酶活性最小化,直到可以方便地进行闪烁计数处理(图2L) 。将溶液转移到微量离心管中并储存在冷冻箱中以进行长期储存。
    7. 60分钟渗出之后,将玫瑰花结转移到第三个24孔培养板上,仅在EDTA溶液中浸没约2mm的茎梗。将第三块板放在湿度箱中,在黑暗中储存60分钟。将第二个板(即第一个小时的渗出物)置于冰上以使潜在的酶活性最小化,直到可以方便地进行闪烁计数处理(图2L)。将溶液转移到微量离心管中并储存在冷冻箱中以进行长期储存。
    8. 60分钟后,取出玫瑰花结,在冰上冷却第三块板(即第二小时的渗出液),以使潜在的酶活性最小化,直到可以方便地进行闪烁计数处理(图2L),或者将溶液转移到微量离心管中,并储存在冰箱中以进行长期储存。

  5. 闪烁计数分析韧皮部分泌物
    1. 将400μl的每种EDTA溶液转移到闪烁小瓶中(图2M)并加入5ml闪烁液用于含水样品。摇动小瓶以彻底混合,形成单相溶液。用闪烁计数器和适用于14C的程序记录每分钟计数(cpm)或每分钟崩解(dpm)。在500μl渗出物中计算每个玫瑰花丛质量中的14 C:cpm或dpm(鲜重)-1 h -1。应该通过闪烁计数来分析第一和第二小时的分泌物,并且应该给出合理的一致计数。剩余的EDTA溶液应该被收集并作为14 C液体废物处理。
    2. 为了评估标记的光合同化物在芽中的剩余数量,将玫瑰花结浸入1ml 80%的乙醇中,在振荡器上轻轻摇动约1小时以提取色素和代谢物(较大的玫瑰花结可能需要更大的体积)。加入500μL的商业漂白剂,搅拌〜1小时,以消灭颜料。加入足够的闪烁液并充分混合,得到清晰的单相溶液; 5-10体积的闪烁液到1体积的乙醇/漂白剂溶液应该足够了。
    3. (可选)要通过HPLC或酶分析来测量单个代谢物,向分泌液中加入500μl氯仿,混合并离心分离各相。回收水相到极性代谢物的新鲜微量离心管中。保留氯仿相,如果需要分析非极性代谢物。
    4. 如果需要的话,可将分泌物储存在-20℃下进一步分析,同时记住渗出物用14 C同位素标记。

数据分析

数据分析的实验设计:
每个室构成一个独立的标记实验。从每个平板中的实验植物获得的值应该被标准化为相同室中WT对照的百分比值。然后将来自分离室的标准化值作为独立的重复进行组合。这消除了标记效率的室间变化,以提供对照和实验植物之间在光同化,分布和运输方面的差异的更精确表示(Khadilkar et al。,2016)。

食谱

  1. EDTA溶液(0.5M储备500ml)(FW 292.24)
    将73.06g EDTA溶于300ml ddH 2 O中 用5N KOH调节pH至8.0。使最终卷500毫升
    液体循环高压灭菌15分钟
    在室温下储存
    对于工作浓度,稀释100倍准备5毫米所需的体积,并调整到pH 6.0

致谢

该协议基于(Dasgupta等人,2014; Khadilkar等人,2016年)中公布的方法。在B.G.上进行韧皮部装载和长途运输。 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。

参考

  1. Dasgupta,K.,Khadilkar,A. S.,Sulpice,R.,Pant,B.,Scheible,W. R.,Fisahn,J.,Stitt,M.和Ayre,B. G.(2014)。 在伴侣细胞中特异性表达蔗糖转运蛋白cDNA增强了韧皮部负载和蔗糖的长距离运输,但是导致到抑制生长和磷酸盐限制的感知。植物生理学 165(2):715-731。
  2. Hewer,A.,Becker,A。和van Bel,A.J。(2011)。 蚜虫的奥德赛 - 维管束的皮质探索 J Exp Biol 214 (Pt 22):3868-3879。
  3. Khadilkar,A. S.,Yadav,U. P.,Salazar,C.,Shulaev,V.,Paez-Valencia,J.,Pizzio,G.A.,Gaxiola,R.A。和Ayre,B.G。(2016) 编码质子泵送焦磷酸酶的AVP1的组成型和伴随细胞特异性过表达增加了生物量积累,韧皮部装载和长途运输。植物生理学 170(1):401-414。
  4. King,R.W。和Zeevaart,J.A。(1974)。 通过螯合剂增强从切叶柄的韧皮部渗出植物生理学 53(1):96-103。
  5. Liu,D.D。,Chao,W.M。和Turgeon,R。(2012)。 在韧皮部运输蔗糖而不是己糖。 J Exp Bot 63(11):4315-4320。
  6. Slewinski,T.L.,Zhang,C。和Turgeon,R。(2013)。 韧皮部装载和运输中的结构和功能异质性 <植物科学前沿 4:244.
  7. Tetyuk,O.,Benning,U.F。和Hoffmann-Benning,S。(2013)。 采用EDTA促进法收集和分析拟南芥韧皮部分泌物。 J Vis Exp (80):e51111。
  8. Turgeon,R.和Wolf,S.(2009)。 韧皮部转运:细胞通路和分子运输 Annu Rev Plant Biol 60:207-221。
  9. van Bel,A.J。E.和Hess,P.H。(2008)。 己糖作为韧皮部运输糖:教条的结束? J Exp Bot 59:261-272。
  10. Will,T.,Tjallingii,W.F.,Thonnessen,A。和van Bel,A.J。(2007)。 蚜虫唾液对植物防御的分子破坏 Proc Natl Acad Sci USA 104(25):10536-10541。
  11. Yadav,U. P.,Khadilkar,A. S.,Shaikh,M. A.,Turgeon,R.和Ayre,B. G.(2017a)。 量化[14C]蔗糖的叶片韧皮部装载量的能力。 Bio Protoc 7(24):e2658。
  12. Yadav,U. P.,Khadilkar,A. S.,Shaikh,M. A.,Turgeon,R.和Ayre,B. G.(2017b)。 评估长距离运输从光合来源叶到异养汇器官与[14C] CO 2 。 Bio Protoc 7(24):e2657。
  13. Zhang,B.,Tolstikov,V.,Turnbull,C.,Hicks,L.M。和Fiehn,O。(2010)。 发散的代谢组和蛋白质组提示葫芦双韧皮部运输系统的功能独立性。 > Proc Natl Acad Sci USA 107(30):13532-13537。
  14. Zhang,C.,Yu,X.,Ayre,B.G。和Turgeon,R。(2012)。 葫芦“韧皮部”渗出物的起源和组成植物生理学 158(4):1873-1882。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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). Assessing Rates of Long-distance Carbon Transport in Arabidopsis by Collecting Phloem Exudations into EDTA Solutions after Photosynthetic Labeling with [14C]CO2. Bio-protocol 7(24): e2656. DOI: 10.21769/BioProtoc.2656.
  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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。