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More helpful hints for setting test variable in quenching protocols.
Saturation intensity
The saturation pulse should be intense enough to completely close all PSII reaction centers.
To test this, one can try different saturation intensities and examine the saturation peak. This
can be done in the Y(II) protocol. After a measurement is made a partial graph of the
saturation pulse is viewed.
By touching the graph with a finger, the whole trace becomes visible. One must adjust the
intensity high enough so that the most of the top of the saturation pulse is flat. If it is rounded
at the middle of the top, more intensity is needed. According to studies done on the subject,
even the most intense saturation pulses do not damage plant tissue in a light adapted
environment. It has been found that damage can occur with night measurements if the
saturation pulses are made too frequently
(Rosenqvist and van Kooten 2006)
(Albert Porcar-
Castell 2008). With this in mind, one can use maximum intensity for saturation pulses in a
light adapted environment.
Saturation pulse frequency
By using the Stepped Actinic Test in the Kinetic Protocol, one can check to see if there is
enough time between pulses to allow complete relaxation of saturation pulse NPQ. If not,
then saturation pulse related NPQ would cause an error. Rosenqvist and van Kooten (2006)
state that a time of between one and two minutes is required for complete relaxation of
saturation pulse NPQ. When in doubt use two minutes.
If one wants to test for the minimum time required to eliminate saturation pulse NPQ, then
using the kinetic test with different times between saturation pulses will provide the answer.
The test should allow about twenty minutes for the plant to reach steady state photosynthesis
(Maxwell and Johnson 2000). If the Fm’ values continue to decline and the F’ values continue
to change, then the pulses are too close together. If you are looking for a safe time to use, two
minutes is suggested as the longest safe time by Rosenqvist and van Kooten (2006).
Saturation pulse duration
On the OS1p, the saturation pulse duration is set at the default value of 0.8 seconds. This is
generally ideal for higher land plants, however, times of 0.5 to 1.5 seconds have been used
Rosenqvist and van Kooten (2006). The ideal saturation duration is determined as the value
that completely saturates PSII without causing saturation pulse curvature at the trailing edge
of the saturation pulse. This is a form of saturation pulse NPQ, and it lowers the average value
of Fm, or Fm’ used in Fv/Fm quenching, and Y(II) measurements. The saturation pulse
duration can be set on the OS1p from 0.10 seconds to 2.00 seconds When in doubt, use 0.8
seconds. With modulated fluorometry, the value measured at Fm, or Fm’ represents multiple
