Techniques to measure plant cell water and solute relations
Using the pressure probe to measure single cell turgor pressure
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Collection of vacuolar sap
Microcapilaries attached to the pressure probe or mounted alone are inserted into vacuolate cells. The pressure in the cell forces the sap into the capillary and it can be collected and analysed. Volumes collected can range from a few picolitres to 100 picolitres depending on cell size, elasticity and turgor. A constriction pipette in use can be seen here. Some of these techniques were developed in the lab of Deri Tomos at University of Wales, Bangor.
Cutting aphid stylets to sample phloem sap
Phloem sieve elements are difficult
to sample, requiring cutting or techniques which may alter its composition. Aphids can
insert their stylets into the phloem and feed on the sieve element sap. Using radio
frequency microcautery we can cut the stylets and collect sap from phloem sieve elements
for subsequent analysis (ref).
Photosynthesis produces all the carbon on the planet but the regulation of translocation
through the phloem is poorly understood. Our studies are helping us understand the
regualtion of translocation and its modification by environmental stress.
Click here to see an avi movie of a stylet being cut, its BIG (and it doesn't exude!).It does work, an exuding stylet can be seen here
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Aphids feeding on barley leaf, as microcautery needle is introduced |
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Aphid feeding on barley root |
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Cut aphid stylet showing exuding sieve element sap |
Insects to sample the xylem
The insect Philaenus spumarius inserts its stylet into the xylem and can extract sap against large negative pressures (tensions) often present in the xylem. In the nymphs of P. spumarius the excess xylem sap is expelled and frothed up, forming the cuckoo spit often seen on plants in the spring. This can be collected and xylem composition analysed. The adult leaf hopper expels the excess sap, without frothing, as discrete droplets. This fluid can also be collected and analysed.
In collabration with Mike Malone at HRI Wellesbourne we are developing collection techniques to validate the method and apply it to understanding salt tolerance in plants (ref). We are also using aphids to sample the phloem to get a picture of whole plant responses to salt.
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| Cuckoo spit mass on barley leaf | Adult Philaenus expelling xylem sap |
Osmotic pressure measurement
Osmotic pressure is measured by freezing point depression of picolitre quantitiies of sap. Samples are frozen to -40oC to avoid supercooling and temperature is slowly raised. Temperature at which the last ice crystal melts is recorded. The osmometer is calibrated with NaCl standards of known osmotic pressure. An AVI of the melting ice on the osmometer stage can be seen here
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Frozen samples on the osmometer stage |
Inorganic ions are measured using EDAX analysis of flash-evaporated samples of extracted sap. Typical volumes used are around 10 picolitres. Calibration is facilitated by adding internal standards, usually RbFl. A study of maize roots using these techniques can be found in the publication list. An AVI of the pipetting of an EDAX grid can be seen here.
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| Pioloform coated EM copper grid | EM picture of flash-evaporated samples |
Organic solutes can be analysed using appropriate dehydrogenase enzymes linked to NAPH/NAPDH conversions. Changes in fluorescence are proportional to the solute under consideration and can be quantified using the confocal microscope.
Aphid feeding behaviour can be monitored using EPG. The aphid is linked into an electrical circuit. Different characteristic signals are generated depending which plant compartment (cell, xylem, phloem etc.) the aphid stylet is located in.
