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Assessing Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [14C]CO2
利用[14C]CO2评估从光合源叶到异养库器官的远距离运输   

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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 (Yadav et al., 2017a), (2) the relative rates of carbon loading and transport through the phloem (Yadav et al., 2017b), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (this protocol). 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 the 14C label to heterotrophic sink organs, particularly roots, is quantified by scintillation counting. Using this protocol, we demonstrated that overexpression of sucrose transporters and a vacuolar proton pumping pyrophosphatase in the companion cells of Arabidopsis enhanced transport of 14C label photoassimilates to sink organs (Dasgupta et al., 2014; Khadilkar et al., 2016). This method can be adapted to quantify long-distance transport in other plant species.

Keywords: Arabidopsis (拟南芥), Photosynthetic labeling (光合标记), 14C labeling (14C标记), Long-distance phloem transport (远距离韧皮部运输), Photoassimilate partitioning (光合同化物分配), Source-sink relation (源库关系)

Background

Long-distance transport through the phloem from autotrophic source organs to heterotrophic sinks is fundamental to plant growth and yield. Based on its role and its location in the plant and its prevailing function in that location, the phloem network is commonly divided into the collection phloem, the transport phloem, and the release phloem (Ayre, 2011). The collection phloem is where sugars and other compounds are loaded into the phloem in preparation for transport. In established plants, the collection phloem is the minor veins of mature, photoautotrophic leaves where phloem loading occurs. Our first companion protocol (Yadav et al., 2017a) describes how [14C]Suc is used to quantify phloem loading capacity in leaf disks. The transport phloem connects source and sink tissues and represents the longest contiguous stretch of phloem in the long-distance-transport pathway. All photoassimilate destined for the heterotrophic sinks moves along the transport phloem, but the transport phloem is far from a simple pipe connecting regions where most of the loading and unloading occurs. Lateral tissues require nutrients and also act as transient storage reserves in stems and roots such that exchange between the transport phloem and adjacent tissue is highly dynamic (discussed at length in Ayre, 2011). Our second companion protocol (Yadav et al., 2017b) describes how 14C labeling with [14C]CO2 can be coupled with collecting phloem exudates into EDTA solutions to measure photoassimilate loaded into the collection phloem and moving through the transport phloem. The release phloem generally refers to that in terminal sink tissues at or very near the end of the phloem network where rates of unloading are highest. The release phloem is found in regions of rapid cell division and growth, or in storage organs, where resources are most strongly needed. The unloading mechanism from the phloem tissue itself is commonly through plasmodesmata into symplastic domains within the recipient tissue. Subsequent transport across membranes to the apoplast, followed by uptake into adjacent cells, occurs in some organs. Symplastic domains and apoplastic boundaries have been elegantly demonstrated with the green fluorescent protein, which moves readily through apical tips and ovule integuments when unloaded from the release phloem, but does not enter the filial tissue of seeds, which is symplastically isolated from maternal tissues (Stadler et al., 2005a; Stadler et al., 2005b). Here we describe a protocol for photosynthetically labeling source leaves with [14C]CO2 and measuring transport to heterotrophic sink organs, specifically roots. An advantage of this protocol is that it takes a whole-plant, holistic approach to quantifying source to sink relationships between control and experimental plants. A disadvantage is that it does not provide information on the individual steps of the transport process.

Materials and Reagents

  1. Microcentrifuge tubes, screw cap with O-rings (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3464 )
  2. Pasteur pipette
  3. [14C]CO2 labeling chambers derived from square Petri dishes (100 x 100 x 15 mm, Fisher Scientific, catalog number: FB0875711 A; or larger 120 x 120 mm, Fisher Scientific, catalog number: 07-000-330 )
  4. Sterile scalpel
  5. Circular deep-well Petri dishes for germinating seeds (100 x 25 mm) (Fisher Scientific, catalog number: FB0875711 )
  6. Surgical white porous tape (3M, catalog number: 1530-0 )
  7. 10 ml syringe (without a needle attached) (Luer-Lok Tip) (BD, catalog number: 309604 )
  8. 1 ml plastic syringe barrel (Luer-Lok Tip) (BD, catalog number: 309628 ), cut to 3 cm, and plunger
  9. Syringe needle, 1.5-2.0 inches, 18 gauge (Monoject Needle, Covidien, catalog number: 1188818112 )
  10. Scintillation vials (Fisher Scientific, catalog number: 03-337-15 )
  11. Double edge razor blades (PERSONNA brand, Electron Microscopy Sciences, catalog number: 72000 )
  12. Modeling clay (American Art Clay)
  13. Control and experimental plant material
  14. Sodium bicarbonate [14C]NaHCO3 (MP Biomedicals, catalog number: 0117441H ; 40-60 mCi/mmol; 2 mCi/ml; 5 mCi; 185 MBq)
  15. Sodium hypochlorite (NaClO) (Commercial bleach)
  16. Dow Corning high vacuum grease
  17. Lactic acid (85%) (Fisher Scientific, catalog number: A162-500 )
  18. Soda lime (LI-COR, catalog number: 9964-090 ) in a column (e.g., Bio-Rad Econo-Column, 1.5 x 20 cm, Bio-Rad Laboratories, catalog number: 7371522 )
  19. Ethanol absolute (Pharmco-AAPER, catalog number: 111000200 )
  20. Murashige and Skoog (MS) medium with Gamborg vitamins (PhytoTechnology Laboratories, catalog number: M404 ) or other synthetic, sterile medium suitable for plant growth in vertically-oriented Petri plates (see Recipes for seed germination and experiments)
  21. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  22. Potassium hydroxide (Fisher Scientific, catalog number: P250-500 )
  23. Gellan gum (PhytoTechnology Laboratories, catalog number: G434 )
  24. Ecolume scintillation fluid (MP Biomedicals, catalog number: 0188247004 )
  25. Half-strength Murashige and Skoog (MS) medium with Suc for seed germination (see Recipes)
  26. Half-strength Murashige and Skoog (MS) medium without Suc for experiments (see Recipes)

Equipment

  1. Glassware, balance, stir plates, pH meter, autoclave, water bath, clean bench, etc., for sterile media
  2. Desiccator (2.5 L) for sterilizing seeds (Kimble, catalog number: 31200-150 )
  3. Environmental growth chambers for growing control and experimental plants
  4. Personal safety equipment: lab coat, nitrile gloves (or similar), and eye protection
  5. Lamp suitable for photosynthetic labeling, such as a 400 W metal halide lamp (SYLVANIA 64490 - 400 Watt - BT37 - Metal Halide)
  6. Fume hood with appropriate support for metal halide lamp (see Figure 1, Yadav et al., 2017b,)
  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 vacuum pump with inlet and outlet (e.g., Airpo, Barcodable, catalog number: UPC 045635496699 )
  9. Geiger counter (Ludlum Measurements, model: Model 3 )
  10. Scissors (Fisher Scientific, catalog number: 08-951-20 )
  11. Scalpel handle (Fine Science Tools, catalog number: 10003-12 )
  12. Scalpel blades (Fine Science Tools, catalog number: 10011-00 )
  13. Forceps, fine, such as Dumont fine point No. 5 (Fine Science Tools, catalog number: 11251-10 )
  14. Balance (METTLER TOLEDO, model: AE100 )
  15. Microcentrifuge (GeneMate, catalog number: C-1301-PC )
  16. Rotary platform shaker (Orbital Shaker Variable, BioExpress, GeneMate, catalog number: S-3200-LS )
  17. Scintillation counter (Beckman Counter, model: LS 6000IC )
  18. Water bath

Procedure

  1. Preparing a work area suitable for [14C]CO2 photoassimilation
    1. Refer to Yadav et al., 2017b for instructions on preparing a work area suitable for labeling by [14C]CO2 photoassimilation.
    2. [14C]NaHCO3 stocks will release gaseous [14C]CO2. To minimize this, commercial stocks are supplied as basic solutions buffered to pH 9.5 since acidic pH promotes conversion to CO2. Stocks should be stored at 4 °C and not -20 °C to prevent freezing and localized concentrations of [14C]NaHCO3 among the water crystals. We recommend aliquoting stock into 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.

  2. Germination of experimental and control WT Arabidopsis thaliana seeds
    1. Aliquot 30-40 seeds (~1 mg) of each experimental and control line into separate 2 ml microcentrifuge tubes and sterilize by standard procedures with liquid bleach in the liquid or gas phase. Examples of experimental lines might be transgenic plants with modified transporter gene expression, in which case control lines would be wild type plants or preferably transgenic plants with the same T-DNA backbone but without modified transporter expression. We usually perform gas phase sterilization (Clough and Bent, 1998): in a fume hood, place open microcentrifuge tubes with seeds in a 2.5 L glass desiccator along with a beaker holding 40 ml commercial bleach. Quickly acidify the bleach with concentrated HCl (1-2 ml in a Pasteur pipette with rubber bulb) and seal the desiccator lid in place. Allow seeds to sterilize in the released chlorine gas for 4-5 h. Sprinkle the seeds immediately on germination medium (½ strength MS medium with Gamborg vitamins containing 1% sucrose with 5 g/L gellan gum).
      Notes:
      1. It is crucial to avoid uneven distribution of seeds on germination plates, since crowding can influence early seedling growth and development. We sprinkle seeds manually to achieve even distribution.
      2. Leaving the seeds in chlorine gas for too long can kill the seeds, and seeds sterilized by this method do not store well. Sucrose in the medium improves germination and consistent seedling establishment; later steps use medium without sucrose.
    2. Stratify the seeds for 3 days at 4 °C in darkness. For germination, transfer plates to the growth chamber under a 12 h light (22 °C)/12 h dark (20 °C) diurnal cycle at 130 μmol photons m-2 sec-1 in the vertical orientation. To avoid contamination, keep the plates sealed during germination.
    3. Prepare sterile ½ strength MS medium with Gamborg vitamins and 5 g/L gellan gum without sucrose in square culture plates for growth in the vertical orientation. Fill the plates half full with media and once solidified, use a sterile scalpel to aseptically remove 1 cm of medium from one edge of the plate. When placed vertically, the rosettes of each plant will be above this cut so they do not contact the medium, and the roots will lay along the surface of the medium. Medium without sucrose is used to best mimic physiological source and sink relationships.
    4. Approximately 4-5 days post germination, when roots are ~1 cm long, transfer young seedlings from the germination plates to the square plates. With fine forceps (e.g., Dumont #5), gently lift the seedlings from under the cotyledons without pinching or piercing the hypocotyl and without damaging the root. Lay the root down 3-4 cm below the cut edge of the medium in the square plates and drag the seedling up from under the cotyledons to straighten the roots. Position the seedling such that the root remains on the medium and the hypocotyl and rosette are above the cut edge of the medium.
      Note: Each square culture dish will be an independent [14C]CO2 labeling chamber. Therefore, control and experimental plants should be grown together in the same chamber. In 100 x 100 mm plates, we typically grow nine plants: 3 control plants and 3 each of 2 experimental lines with the order alternating in 6 replicate plates. Larger plates will accommodate more plants. Labeling chambers can also be made from containers, such as sterilized deli containers (Yadav et al., 2017b).
    5. Seal the square plates using porous surgical tape and arrange the plates near to vertical (~15° off vertical) to keep the roots growing down, but also in contact to the medium surface. Arrange the plates in a growth chamber with the same conditions described above. Depending on the experiment, labeling with [14C]CO2 will be performed in 4 to 7 days, when the roots have extended ~3 cm to cover ~75-80% the length of the medium, or later, if larger plates are used or if growth is slow. Labeling should be done before the roots reach the bottom of the plate.

  3. Photosynthetic labeling
    1. Photograph the plates to record growth 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 of ~130 μmol photons m-2 sec-1 at the working surface.
    2. Acclimate the plants under the metal halide lamp for about 30 min before labeling. Remove the surgical tape, but leave the lids in place.
      Note: 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. Six plates can be arranged under the light and receive equal light intensity, but care must be taken to ensure that the number of plates labeled does not exceed the ability to process the plant material efficiently in subsequent steps. Table 1 provides a scheduling template.
      For each plate to be labeled, prepare a fresh lid to be used during labeling: make two holes in the top of each lid but at opposite ends to inject and exhaust the labeling [14C]CO2. The inject hole should be at top near the rosettes and the exhaust hole should be at the bottom near the tips of the roots. The end of a paper clip, heated with a Bunsen burner, works well. With a syringe barrel filled with vacuum grease and without a needle, apply a bead of vacuum grease inside each lid to seal the labeling chambers. Use a small ball of modeling clay to cover the inject and exhaust holes (Figure 1B).
      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. Circulation is more important in larger chambers with more plants, and we do not use blowers in 100 x 100 mm plates.

      Table 1. Schedule template to organize labeling and processing six chambers for long-distance phloem transport from source to sink organs


    3. Remove the lid of the first plate to be labeled and replace it with a lid equipped with inject and exhaust holes, and apply a bead of vacuum grease. Confirm by visual inspection that the vacuum grease seal is complete.
    4. To create [14C]CO2 for labeling, pipette 2.5 μl of [14C]NaHCO3 (2 μCi/μl, 2 mCi/ml) in a droplet near the syringe needle junction of a syringe barrel cut to ~3 cm. Place a 15 µl droplet of 80% 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 1C). Insert the needle through the injection hole of the culture plate to be labeled (remove the clay plug or push to the side), and arrange the modeling clay around it; a needle that is bent 45° works well (Figure 1D). The location of the injection hole and the size of the clay plug should not block light reaching the rosettes. 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. Move the plunger back and forth to pump the [14C]CO2 gas into the labeling chamber; 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.
    5. Allow plants to do photosynthesis in the presence of [14C]CO2 for 20 min. This is the ‘pulse’ phase (Figure 1E).
    6. While plants in the first chamber are being labeled, replace the lid of the second culture dish with a pre-prepared lid with inject and exhaust holes and a vacuum grease bead, and make sure the seal is complete by visual observations (i.e., repeat Steps C3 and C4), and label as described in Step C5. Repeat for the third culture plate, etc.
    7. 20 min after injecting [14C]CO2, use the exhaust hole to vent the chamber through soda lime and capture unassimilated [14C]CO2 (Figure 1F). A column filled with soda lime and attached to a small air pump works well. After ~5 min of venting through soda lime, remove the lid used during labeling and replace with the original lid to allow gas exchange with unlabeled air. Do not try to reseal the lid with medical tape, but the plants should remain covered since they were grown in the high humidity of the culture dish. Allow photosynthesis to continue to provide 40 min of total ‘chase’ time.
      Note: To ensure effective capture of unassimilated [14C]CO2 by the soda lime, it should be fresh and kept well-sealed between uses. Old soda lime should be discarded as 14C-labeled dry waste


      Figure 1. Experimental procedure for labeling plants grown in culture plates with [14C]CO2. A. Arabidopsis plants on sterile ½ strength MS without sucrose solidified with 5 g/L gellan gum approximately 7 days after transfer from germination medium; the first three are one experimental line, the middle three are controls, and the last three are a second experimental line. Replicate plates will have a different order. Note that 1 cm of medium is removed and the plants were arranged so the shoots and hypocotyls are above the medium and the roots grow down the surface of the medium. The plate was grown in a vertical position at 15°. B. To convert the culture plate to a labeling chamber, a second lid with two holes for [14C]CO2 for injection (top) and exhaust (bottom) is prepared with a bead of vacuum grease to seal it with the bottom of the culture dish. C. A 1 ml syringe barrel cut to ~3 cm (cut end not shown) with a needle bent 45° and a small droplet of [14C]NaHCO3 (indicated by upper white arrow) and a larger droplet of 85% lactic acid (lower white arrow). The plunger is not yet inserted. D. Needle is inserted through the upper inject hole of the labeling chamber with the plunger inserted and the [14C]NaHCO3 and lactic acid mixed. Note that both the inject hole and the exhaust hole are sealed. E. Allow the plants to photosynthesize for 20 min. F. Remove the clay from both inject and exhaust holes and use Tygon tubing connected to a column of soda lime and a small air pump to exhaust the chamber for 5 min. G. Put the original plate back in place, but do not seal. Allow plants to do photosynthesis in regular air and transport photoassimilate for a 40 min ‘chase’ (5 min of exhausting the chamber and 35 min in regular air). H. Mark root length from root tip to shoot (e.g., 0.0-1.0 cm, 1.0-2.0 cm, etc.). I. Use a razor blade (shown) or fine scissors to cut the sections and transfer to separate scintillation vials containing 80% ethanol to stop metabolic reactions and extract metabolites. Alternatively, sections from each genotype can be pooled together into one scintillation vial for scintillation counting. Each plate should be considered a separate labeling experiment (i.e., one replicate), and the experimental samples should be standardized to controls before performing statistics on replicates.

  4. Collection of tissues (Roots and Shoots) and scintillation counting
    1. Use fine scissors or a razor blade to cut the roots into sections as desired for the experiment. We usually cut the whole root from the shoot as a single heterotrophic organ, or we section the root into three regions: 0.0-1.0 cm from the tip (this is most actively growing and the strongest sink), 1.0-2.0 cm, and the remaining root (which will also include emerging lateral roots). Retain the shoot for scintillation counting.
    2. Collect the different sections of root tissues and the labeled shoot into 500 μl 80% ethanol in scintillation vials to terminate metabolism and extract the metabolites. The root sections and shoot of each plant can be collected and counted separately (preferred, but uses up a lot of scintillation vials) or tissues from each line in each plate can be pooled, and samples from each plate counted separately. Gently agitate the scintillation vials on a shaker for ~1 h to extract pigments and metabolites; greater volumes of 80% ethanol (~1 ml or more) may be required for larger rosettes. Add 500 μl commercial bleach to destroy pigments that may impede scintillation counting. 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. Measure counts or disintegrations per minute in a scintillation counter.

Data analysis

Each square culture plate constitutes an independent labeling experiment and photosynthetic incorporation of 14C[CO2] can vary between plates due to effects that are difficult to control. For example, since the plants in the plates are continuing to do photosynthesis while they are being prepared for labeling, the amount of atmospheric ‘cold’ CO2 in the vessel can vary from plate to plate when the 14C[CO2] is injected. This changes the 14C[CO2] specific activity during the pulse phase of the experiment. To control for this, control plants are included in each labeling chamber, and 14C[CO2] incorporation into experimental plants is expressed relative to these controls. That is, values obtained from experimental plants in each plate are standardized to a percent value of controls in the same chamber. Standardized values from separate plates are then combined as independent replicates. This removes the plate to plate variation in labeling efficiency to provide a more accurate representation of the differences in photoassimilation and transport between controls and experimental plants. Furthermore, to control for microenvironment effects in the plates, the order of control and experimental plants are altered among replicate chambers.

Recipes

  1. Half-strength Murashige and Skoog (MS) medium with sucrose for seed germination
    Dissolve 2.22 g of MS into 800 ml of ddH2O
    Add 10 g of sucrose, allow it to dissolve completely
    Make the final volume 1,000 ml
    Adjust the pH to 5.8 with 1 N KOH
    Add 5 g gellan gum, mix the solution
    Autoclave in liquid cycle for 20 min
    Allow the medium to cool in a 55 °C water bath for 15-20 min
    Pour into 100 x 25 mm circular Petri dishes
  2. Half-strength Murashige and Skoog (MS) medium for experiments
    Dissolve 2.22 g of MS into 1,000 ml of ddH2O
    Adjust the pH to 5.8 with 1 N KOH
    Add 5 g gellan gum, mix the solution
    Autoclave in liquid cycle for 20 min
    Allow the medium to cool in a 55 °C water bath for 15-20 min
    Pour into 100 x 100 mm square plates

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. Ayre, B. G. (2011). Membrane-transport systems for sucrose in relation to whole-plant carbon partitioning. Mol Plant 4(3): 377-394.
  2. Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735-743.
  3. 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.
  4. 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.
  5. Stadler, R., Lauterbach, C. and Sauer, N. (2005a). Cell-to-cell movement of green fluorescent protein reveals post-phloem transport in the outer integument and identifies symplastic domains in Arabidopsis seeds and embryos. Plant Physiol 139(2): 701-712.
  6. Stadler, R., Wright, K. M., Lauterbach, C., Amon, G., Gahrtz, M., Feuerstein, A., Oparka, K. J. and Sauer, N. (2005b). 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(2): 319-331.
  7. 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.
  8. Yadav, U. P., Khadilkar, A. S., Shaikh, M. A., Turgeon, R. and Ayre, B. G. (2017b). 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.

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运输的相对速率(Yadav等人,2017b),和(3)碳长期释放到异养池器官特别是根中的相对速率距离传输(这个协议)。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一方案相关联的歧义降至最低(Dasgupta等人,2014; Khadilkar 。,2016)。在该方案中,在源叶片和韧皮部装载和运输14 C标签到异养宿主器官中,[14 C] CO 2 2被光致同化,尤其是根,通过闪烁计数进行量化。使用该协议,我们证明在拟南芥的伴侣细胞中蔗糖转运蛋白和液泡质子泵激焦磷酸酶的过表达增强了14 C标记光合同化物向宿主器官的转运(Dasgupta <等人,2014; Khadilkar等人,2016)。这种方法可以适用于量化其他植物物种的长途运输。

【背景】通过从自养源器官到异养池的韧皮部的长途运输是植物生长和产量的基础。根据其在植物中的作用和位置及其在该地区的主要功能,韧皮部网络通常分为收集韧皮部,运输韧皮部和释放韧皮部(Ayre,2011)。收集韧皮部是糖和其他化合物装入韧皮部以准备运输的地方。在已建立的植物中,收集韧皮部是发生韧皮部负载的成熟,光自养叶子的小脉。我们的第一个伴侣协议(Yadav et al。,2017a)描述了如何使用Suc来量化叶盘中的韧皮部负载能力。运输韧皮部连接源和汇组织,代表长距离运输途径中最长的连续韧皮部。所有用于异养汇的光合同化物都沿着运输韧皮部运动,但运输韧皮部远离大部分装载和卸载发生的简单管道连接区域。侧向组织需要营养物质,也作为茎和根中的暂时储存储备,使得运输韧皮部和邻近组织之间的交换是高度动态的(在Ayre,2011中详细讨论)。我们的第二个伴侣协议(Yadav et al。,2017b)描述了如何用14 C标记CO 2,可以将韧皮部分泌物收集到EDTA溶液中,以测量载入韧皮部并通过运输韧皮部移动的光合同化物。释放韧皮部通常是指在卸载率最高的韧皮部网络末端或极端附近的末端汇组织中。释放韧皮部位于快速细胞分裂和生长的区域,或在最需要资源的储存器官中。来自韧皮部组织本身的卸载机制通常是通过胞间连丝(plasmodesmata)进入受体组织内的共质结构域。随后通过膜转运至质外体,随后摄入相邻细胞,发生在一些器官中。绿色荧光蛋白可以很好地表现出共质结构域和质外体的界限,绿色荧光蛋白在从释放韧皮部卸载时易于通过顶端尖端和胚珠外皮移动,但不进入与母体组织(Stadler) et al 。,2005a; Stadler et al 。,2005b)。在这里,我们描述了光合作用标记来源叶子[14C] CO 2和测量运输到异养宿主器官,特别是根的方案。这个协议的一个优点是,它需要一个整体的,整体的方法来量化源控制和实验植物之间的联系。缺点是它没有提供关于运输过程的各个步骤的信息。

关键字:拟南芥, 光合标记, 14C标记, 远距离韧皮部运输, 光合同化物分配, 源库关系

材料和试剂

  1. 微量离心管,带O形环的螺旋盖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3464)
  2. 巴斯德吸管
  3. (100×100×15mm,Fisher Scientific,产品目录号:FB0875711A;或更大的120×120mm,来自方形陪替氏培养皿的[14] C-CO 2 CO 2标记室) Fisher Scientific,目录号:07-000-330)
  4. 无菌手术刀
  5. 用于发芽种子的圆形深井培养皿(100 x 25 mm)(Fisher Scientific,产品目录号:FB0875711)
  6. 手术白色多孔胶带(3M,目录号:1530-0)
  7. 10毫升注射器(不带针头)(Luer-Lok Tip)(BD,目录号:309604)
  8. 1毫升塑料注射器筒(Luer-Lok Tip)(BD,目录号:309628),切成3厘米和柱塞。
  9. 注射针,1.5-2.0英寸,18号(Monoject Needle,Covidien,目录号:1188818112)
  10. 闪烁瓶(Fisher Scientific,目录号:03-337-15)
  11. 双刃剃须刀(PERSONNA品牌,电子显微镜科学,目录号:72000)
  12. 塑造黏土(美国艺术粘土)
  13. 控制和实验植物材料
  14. 碳酸氢钠[14 C] NaHCO 3(MP Biomedicals,目录号:0117441H; 40-60mCi / mmol; 2mCi / ml; 5mCi; 185MBq) br />
  15. 次氯酸钠(NaClO)(商业漂白剂)
  16. 道康宁高真空油脂
  17. 乳酸(85%)(Fisher Scientific,目录号:A162-500)
  18. (例如Bio-Rad Econo-Column,1.5×20cm,Bio-Rad Laboratories,目录号:7371522)中的碱石灰(LI-COR,目录号:9964-090)
  19. 乙醇绝对(Pharmco-AAPER,目录号:111000200)
  20. 适用于垂直取向培养皿中的植物生长的Murashige和Skoog(MS)培养基(PhytoTechnologyLaboratories,目录号:M404)或其他合成的无菌培养基(参见种子发芽和实验的配方)
  21. 蔗糖(Sigma-Aldrich,目录号:S0389)
  22. 氢氧化钾(Fisher Scientific,目录号:P250-500)
  23. 结冷胶(PhytoTechnology Laboratories,目录号:G434)
  24. Ecolume闪烁液(MP Biomedicals,目录号:0188247004)
  25. 含有Suc的半强度Murashige和Skoog(MS)培养基,用于种子发芽(见食谱)

  26. 不含Suc的半强度Murashige和Skoog(MS)培养基用于实验(见食谱)

设备

  1. 玻璃器皿,天平,搅拌板,pH计,高压灭菌器,水浴器,洁净台,等等。
  2. 用于杀菌种子的干燥器(2.5升)(Kimble,目录号:31200-150)
  3. 增长控制和实验工厂的环境增长分庭
  4. 个人安全设备:实验室外套,丁腈手套(或类似的)和护目镜
  5. 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)
  6. 通风橱适当的支持金属卤化物灯(参见图1,Yadav等人,2017b)
  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真空泵(例如,Airpo,Barcodable,目录号:UPC 045635496699)
  9. 盖革计数器(Ludlum测量,模型:模型3)
  10. 剪刀(Fisher Scientific,目录号:08-951-20)
  11. 手术刀(精细科学工具,目录号:10003-12)
  12. 手术刀片(精细科学工具,目录号:10011-00)
  13. 镊子,罚款,如杜蒙罚款点5号(精细科学工具,目录号:11251-10)
  14. 平衡(梅特勒 - 托利多,型号:AE100)
  15. 微量离心机(GeneMate,目录号:C-1301-PC)
  16. 旋转平台摇床(轨道摇床变量,BioExpress,GeneMate,目录号:S-3200-LS)
  17. 闪烁计数器(贝克曼计数器,型号:LS 6000IC)
  18. 水浴

程序

  1. 准备一个适合于[14C] CO_2光同化的工作区
    1. 请参考Yadav等人的文章“2017b”,了解如何制备适合用[14C] CO 2同化标记的工作区域。 />
    2. [14C] NaHCO 3储备将释放气态[14 C] CO 2 2。为了尽量减少这种情况,商业储备是作为缓冲到pH 9.5的碱性溶液提供的,因为酸性pH促进了转化为CO 2。股票应储存在4°C而不是-20°C,以防止水晶中冻结和[14C] NaHCO 3 3的局部浓度。我们建议用O型圈将样品分装到螺旋盖微量离心管中。
      按照所在实验所的要求,记录库存的接收和使用情况
  2. 实验和对照WT萌发拟南芥种子
    1. 将每个实验和对照线的30-40粒种子(〜1mg)分装到单独的2ml微量离心管中,并通过标准程序用液体漂白剂在液相或气相中灭菌。实验系的实例可以是具有修饰的转运蛋白基因表达的转基因植物,在这种情况下,对照系将是野生型植物或优选具有相同T-DNA主链但没有修饰的转运蛋白表达的转基因植物。我们通常进行气相灭菌(Clough和Bent,1998):在通风橱中,将开放的带有种子的微量离心管放置在2.5L玻璃干燥器中,同时装有40ml市售漂白剂的烧杯。用浓HCl快速酸化漂白剂(在带有橡皮球的巴斯德吸管中1-2ml)并密封干燥器盖。让种子在释放的氯气中消毒4-5小时。将种子立即撒在发芽培养基上(用含有1%蔗糖和5g / L结冷胶的Gamborg维生素的1/2强度MS培养基)。
      注意:
      1. 因为拥挤会影响早期幼苗生长和发育,所以避免种子在萌发板上分布不均是至关重要的。我们手动撒种子,以达到均匀分布。
      2. 将种子留在氯气中过久会杀死种子,用这种方法灭菌的种子储存不好。在培养基中的蔗糖提高了发芽和稳定的幼苗建立;以后的步骤使用不含蔗糖的培养基。
    2. 在4℃的黑暗条件下将种子分层3天。为了萌发,将板在12μm光照(22℃)/ 12小时黑暗(20℃)昼夜循环下在130μmol光子m 2 -2秒-1下转移到生长室在垂直方向。为了避免污染,在发芽过程中保持板子密封。
    3. 准备无菌½强度的MS培养基与甘美维生素和5克/升结冷胶不含蔗糖在方形培养板垂直方向的增长。用培养基填满培养皿一半,一旦凝固,使用无菌手术刀从培养皿的一个边缘无菌地移除1厘米的培养基。当垂直放置时,每个植物的玫瑰花结将在这个切口上方,以便它们不接触介质,并且根将沿着介质的表面放置。不含蔗糖的培养基被用来最好地模拟生理源和宿关系。
    4. 发芽后大约4-5天,当根长约1厘米时,将幼苗从发芽板转移到方板上。用细钳子(例如,杜蒙特#5)轻轻地将幼苗从子叶下面提起,而不会夹住或刺破下胚轴并且不损伤根部。将根部放置在方形平板中介质切割边缘下方3-4厘米的位置,并从子叶下拖曳幼苗以使根部伸直。将幼苗定位,使根部留在培养基上,下胚轴和花环高于培养基的切割边缘。
      注意:每个方形培养皿都是独立的[C] 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100×100mm平板中,我们通常生长9个植物:3个对照植物和3个每个2个实验线,顺序在6个重复平板中交替。较大的盘子将容纳更多的植物。标签室也可以由容器制成,例如经过消毒的熟食容器(Yadav等人 em>。em>。,2017b)。
    5. 使用多孔手术胶带密封正方形板,并将板放置在垂直(垂直〜15°)附近以保持根部向下生长,但也与介质表面接触。在与上述相同的条件下将平板放置在生长室中。取决于实验,用4-7天的CO 2标记将在4-7天内进行,当根已经延伸〜3cm以覆盖〜75-80介质的长度,或以后,如果使用较大的平板或生长缓慢。
      标签应该在根到达底部之前完成
  3. 光合标记
    1. 拍摄标签以记录标签发生的日期。在标记前一个小时,在通风橱中打开400W的金属卤化物灯,使其达到约130μmol光子m 2•s -1的稳定强度工作表面。
    2. 在标记之前使金属卤化物灯下的植物适应约30分钟。取下外科胶带,但将盖子留在原位。
      注:光合作用,碳分配到不同的代谢库,长途运输到汇器官在整个昼夜周期中波动。为了保持一致性,我们通常在光照期间标记6小时。可以在光照下安装六块板,获得相同的光照强度,但必须注意确保标记的板数不超过在后续步骤中有效处理植物材料的能力。表1提供了一个调度模板。
      对于每个要贴标签的印版,准备一个新标签在标签中使用:在每个盖子的顶部做两个孔,但是在相对的两端注入和排出标签[14C] CO 2 。注入孔应位于花环附近的顶部,排气孔应位于根部附近的底部。用本生灯加热的回形针末端效果很好。用注射器筒装满真空油脂并且没有针头,在每个盖子内部涂上一层真空润滑脂以密封标签室。使用一个造型粘土的小球来覆盖注入和排出孔(图1B)。
      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型电池供电。在有更多植物的大房间里,循环更重要,我们不使用鼓风机100 x 100毫米。

      表1.组织标记和处理六个室的时间表模板,用于远距离从韧皮部到源器官的韧皮运输


    3. 取下要贴标签的第一块板的盖子,并用装有注入孔和排气孔的盖子替换,并涂上一层真空脂。目视确认真空油脂密封完成。
    4. 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,2mCi / ml)在注射器针筒的注射器针接头附近的液滴中切割成〜3cm。将15μl80%的乳酸液滴放入桶中,注意将液滴与[14C] NaHCO 3液滴分开。轻轻地将柱塞插入桶内(图1C)。将针穿过培养板的注射孔进行标记(去除粘土塞或推到
      侧面),并在其周围安排造型粘土;弯曲45°的针效果良好(图1D)。注入孔的位置和泥塞的大小不应阻挡光线到达玫瑰花结。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, /子>。将柱塞来回移动,将[14C] CO 2气体泵入贴标签室;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。
    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1E)。
    6. 当第一个室中的植物被贴上标签时,用预先准备好的带有注入和排出孔的盖子以及一个真空油脂珠子来替换第二个培养皿的盖子,并通过肉眼观察确认密封完成。 ,重复步骤C3和C4),并按照步骤C5中的描述进行标记。重复第三个培养皿, etc 。
    7. 注入14C] CO 2 20分钟后,使用排气孔通过碱石灰排出室,并捕获未同化的[14 C] CO 2 (图1F)。一个充满了苏打石灰和连接到一个小型气泵的柱子效果很好。在通过碱石灰通气5分钟后,取下标签过程中使用的盖子,并更换原始盖子,以便与未标记的空气进行气体交换。不要试图用医用胶带重新盖上盖子,但植物应该保持被覆盖,因为它们是在培养皿的高湿度下生长的。让光合作用继续提供40分钟的“追逐”时间。
      注:为确保苏打石灰有效地捕获未同化的[14C] CO 2,它应该是新鲜的并在使用之间保持良好密封。旧苏打石灰应作为14 C标记的干废物
      丢弃

      图1.标记在[14C] CO 2/2培养平板上生长的植物的实验程序A.拟南芥 从发芽培养基转移大约7天后,用5g / L结冷胶固化的无蔗糖强度MS的无蔗糖固体;前三个是一个实验线,中间三个是控制线,最后三个是第二个实验线。复制板将有不同的顺序。请注意,1厘米的培养基被删除和植物的排列,使芽和下胚轴高于培养基和根生长在培养基的表面。该板在15°的垂直位置上生长。 B.为了将培养板转化为贴标签室,用于注射(顶部)和排气(底部)的具有两个用于14C] CO 2的孔的第二盖是准备一个真空脂的珠子,用培养皿的底部密封。 C.将1毫升注射器筒切成约3厘米(切割端未显示),弯曲45°的针和一小滴[14 C] NaHCO 3(由上部白色箭头指示)和更大的85%乳酸液滴(较低的白色箭头)。柱塞尚未插入。 D.插入穿过贴标签腔的上部注射孔的针,插入柱塞并混合[14 C] NaHCO 3和乳酸。请注意,注入孔和排气孔都是密封的。 E.让植物光合作用20分钟。 F.从注入孔和排气孔中去除粘土,并使用连接到一个碱石灰柱和一个小型空气泵的聚乙烯管来排空该腔室5分钟。 G.把原板放回原位,但不要密封。允许植物在常规空气中进行光合作用,运输光合同化物40分钟(在室内抽空5分钟,在常规空气中抽35分钟)。 H.从根尖到根的长度标记(例如,0.0-1.0厘米,1.0-2.0厘米,等等)。 I.使用剃刀刀片(如图所示)或精细剪刀切割切片,并转移到含有80%乙醇的闪烁小瓶中以停止代谢反应并提取代谢物。或者,可以将来自每种基因型的切片汇集在一起用于闪烁计数的闪烁小瓶中。每个平板应该被认为是一个单独的标记实验(即一次重复),并且实验样品应该在对重复进行统计之前对照对照进行标准化。

  4. 收集组织(根和芽)和闪烁计数
    1. 使用精细的剪刀或剃刀刀片根据需要切成根部分的实验。我们通常把整个根作为一个单一的异养器官从根部切下,或者把根切成三个区域:距尖端0.0-1.0厘米(这是最活跃生长和最强的水槽),1.0-2.0厘米,剩下的根(这也将包括新兴的侧根)。保留拍摄闪烁计数。
    2. 收集根部组织的不同部分和标记的射线到闪烁小瓶中的500μl80%乙醇中以终止代谢并提取代谢物。每个植物的根部和枝条可以分别收集和计数(优选的,但用尽大量的闪烁小瓶)或每个板的每行的组织可以汇集,每个板的样品分别计数。在振荡器上轻轻搅拌闪烁瓶约1小时以提取色素和代谢物;较大的玫瑰花瓣可能需要更大体积的80%乙醇(〜1毫升或更多)。添加500μL商业漂白剂,以破坏可能阻碍闪烁计数的颜料。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。测量每分钟的计数或分解

数据分析

每个方形培养板构成独立的标记实验,并且由于难以控制的效应,板之间的光合作用可以在板之间变化。例如,由于板材中的植物在准备标记时仍在继续进行光合作用,因此当板材中的大气“冷”CO 2的量随板材的不同而不同时,注入14 C [CO 2]。这在实验的脉冲阶段改变了特定的活性。为了控制这一点,在每个标记室中包括对照植物,并且相对于这些对照表达相对于实验植物的14C [CO 2:]掺入。也就是说,从每个平板中的实验植物获得的值被标准化为同一室中对照的百分比值。然后将来自不同平板的标准化值作为独立的重复进行组合。这消除了板的标记效率的板变化,以提供对照和实验植物之间的光同化和运输的差异更准确的代表。此外,为了控制板中的微环境效应,控制和实验植物的顺序在重复室之间改变。

食谱

  1. 半强度Murashige和Skoog(MS)培养基,含蔗糖,用于种子发芽 将2.22g MS溶于800ml ddH 2 O中 加10克蔗糖,使其完全溶解
    使最终量1000毫升

    用1N KOH调节pH值至5.8 加入5克结冷胶,混合溶液
    液体循环高压灭菌20分钟
    让介质在55°C水浴中冷却15-20分钟
    倒入100 x 25毫米的圆形培养皿
  2. 用于实验的半强度Murashige和Skoog(MS)培养基
    将2.22g MS溶于1000ml ddH 2 O中
    用1N KOH调节pH值至5.8 加入5克结冷胶,混合溶液
    液体循环高压灭菌20分钟
    让介质在55°C水浴中冷却15-20分钟
    倒入100×100毫米的方形板

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. Ayre,B.G。(2011)。 与全植物碳分配相关的蔗糖膜运输系统 Mol Plant 4(3):377-394。
  2. Clough,S.J。和Bent,A.F。(1998)。 花蘸:土壤杆菌介导的转化的简化方法> Arabidopsis thaliana 。 Plant J 16(6):735-743。
  3. 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。
  4. 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强生物量积累,韧皮部装载和长途运输。植物生理学 170(1):401-414。
  5. Stadler,R.,Lauterbach,C.和Sauer,N.(2005a)。 绿色荧光蛋白的细胞间移动揭示了韧皮部外韧皮部的外部运动,在拟南芥种子和胚胎中的共质结构域。植物生理学139(2):701-712。
  6. Stadler,R.,Wright,K.M.,Lauterbach,C.,Amon,G.,Gahrtz,M.,Feuerstein,A.,Oparka,K.J。和Sauer,N.(2005b)。 GFP融合蛋白在拟南芥伴侣细胞中的表达揭示了非特异性蛋白将其转变成筛分元素,并在根中鉴定出一种新的韧皮部后代结构域。 Plant J 41(2):319-331。
  7. Yadav,U. P.,Khadilkar,A. S.,Shaikh,M. A.,Turgeon,R.和Ayre,B. G.(2017a)。 量化[14C]蔗糖对叶片韧皮部负载的能力。 Bio Protoc 7(24):e2658。
  8. Yadav,U. P.,Khadilkar,A. S.,Shaikh,M. A.,Turgeon,R.和Ayre,B. G.(2017b)。 通过将韧皮部渗出物收集到EDTA溶液中评估拟南芥中远距离碳运输的速率光合作用用[14 C] CO 2 2标记。Bio Protoc 7(24):e2656。
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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 Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [14C]CO2. Bio-protocol 7(24): e2657. DOI: 10.21769/BioProtoc.2657.
  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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