Using the Pressure Bomb
Midday, from about 1:00 pm to 3:00 pm (daylight savings time) is the best time to measure stem water potential, because it corresponds to the time of maximum plant water stress (maximum water deficit). Midday is usually the time when weather conditions cause the maximum rate of water loss from the plant. This measurement is called midday stem water potential.
We will assume that a leaf or leaflet is being used for the pressure chamber measurement, and that the leaf has been covered to prevent water loss, either for a long time while it is still attached to the plant, or just before removal from the plant. When a leaf is covered for a long time (we generally recommend about 2 hours) the measured water potential is called "stem water potential." In a variety of tree and vine crops it appears that stem water potential is the most sensitive of the two methods, so that method will be described here.
Sometime in the morning, a lower canopy leaf that is close to the trunk or a main scaffold is covered with a plastic/foil envelope (Fig.2 image gallery: Pressure Chamber). The recommended minimum time between covering and measuring in the pressure chamber is 10 minutes, but longer times (hours or days) are not a problem, as long as the leaf remains dry and undamaged. The leaf must be dry, in shade at the time of covering and should remain in shade until the time of sampling (Fig.3). Direct sunlight will cause the envelope to heat up, and can cause water condensation on the inside, which can artificially hydrate the leaf.
It is most convenient to pick the leaf from the plant by gently snapping the leaf off at its connection to the spur or shoot, and then re-cut the leaf petiole with a sharp razor, at an angle, so that it easily slips through the seal. [For the pressure tank style it must be cut flat]. The leaf petiole is inserted through the sealing ring (Fig.4) of the chamber and then re-cut flush with the lid (Fig.5) after the seal is tightened. [For the pressure tank style, the leaf petiole is inserted through the seal so that after tightening there is at most 1 mm of petiole exposed outside of the chamber.]
Removal of the petiole by re-cutting has no influence on the pressure chamber measurement, nor does the degree of seal tightening, as long as the petiole remains intact and the endpoint can be clearly observed in both directions (see below). It is best that the minimum of petiole is left outside of the seal, however, because the more the petiole outside of the seal, the higher the pressure required to see the endpoint. If this is a small effect for your particular conditions then it will not be important, but may make it difficult for you to compare your values to published reference values or values obtained by others, and can generate unnecessary variation in your measurements. The leaf is then placed in the chamber (Fig.6) and lid sealed to the chamber. The details of how the lid is locked in place will depend on the chamber design.
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The rate of pressure increase itself does not influence the measurement, unless it is so fast that the time taken to stop the pressurization or read the gauge causes overshoot. You should get nearly the same value if you re-measure the same leaf, especially when measuring stem water potential. This is essentially what you are doing when you reduce the pressure to see that water disappears into the petiole, and increase the pressure again until you see the endpoint. You should also get nearly the same value (typically within 0.3 bar) when you measure adjacent leaves on the same spur or shoot, so this is a good way to check your reproducibility or compare the effects of different operators or techniques.
There are two common problems that can make the endpoint difficult to detect: bubbling and the appearance of non-xylem water. If there are breaks in the leaf inside the chamber, then air can be forced through the xylem and come out of the cut end. If this air pushes some water out, or if there is a little fluid from the cells at the cut surface, then the air coming out can bubble through the water, and it can look like there is water coming out when in fact it is just the same water being bubbled around. If this happens you can temporarily stop pressurization and dry the cut surface with a cloth or cotton swab. This should stop the bubbling, and allow you to continue the pressure increase. If the cut surface re-wets and starts bubbling immediately after being dried, then you are at (or may have past) the endpoint.
Non-xylem water can occur when you squeeze the petiole in the seal and water is physically squeezed out the cut end. [For the pressure tank style, sometimes this can happen if you re-tighten the seal during a measurement.] If you think it is the endpoint, note the pressure, then dry off the cut end and raise the pressure a bit. If more water comes out of the cut surface, then it probably was the endpoint, but if it remains dry, then it probably was non-xylem water. Some species of plants have resins or other materials that can come out of the petiole when the leaf is pressurized, but these typically come out of tissue other than the xylem, so a good knowledge of the leaf anatomy can help the operator to discern the difference between the correct endpoint (water from the xylem) and the appearance of these other fluids.
Two or more leaves on the same tree should give almost identical readings, i.e., within about 0.2 bar. It is good practice for beginners to sample more than one leaf per tree to check for reproducibility of measurement. With experience, only 1 leaf per tree is necessary. You should also get nearly the same value if you re- measure the same leaf. This is done once you see the first endpoint by reducing the pressure enough that water disappears into the petiole, and then increasing the pressure until you see the endpoint again. Different trees can give different readings, however, and these will reflect real differences in water potential, so it is important to keep track of each tree separately.