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3. Y(II) values vary with light level and with temperature. The higher the light level,
the lower the Y(II) value. When measuring Y(II) in the field, it is extremely important to
measure leaf irradiation or light level at the leaf and leaf temperature. Comparing Y(II)
values taken at different light levels and different heat levels introduces a significant
error unless it is the change at different light levels and heat levels that is of interest. This
is commonly done with a PAR Clip.
4. Shade leaves vs. Sun leaves. – The Y(II) ratio will be higher on Sun leaves than on
shade leaves (Lichtenthaler 2004).
5. Field plants should only be compared to field plants and green house plants should
be compared to green houseplants due to light history. (Lichtenthaler 2004)
6. Leaf orientation. When making a yield measurement, with or without a PAR Clip, it
is important not
to change the orientation of the leaf. The leaf is at steady state
photosynthesis in its current orientation. Changing the orientation changes the amount of
light falling on the leaf, and the leaf will no longer be at steady state photosynthesis.
7. It is common to use the youngest fully mature leaf blade for diagnosis of
deficiencies in plants (Reuter and Robinson 1997)
8. The duration of the saturation pulse should be between 0.5 seconds and 1.5 seconds
for higher plants, and 25 to 50 m
illiseconds for Phytoplankton and cyanobacteria. Times
outside these ranges increase the error in Y(II) measurements. Shorter durations prevent
complete saturation of PSII regardless of the light intensity(Roseqvist & van Kooten
2006). Longer durations create a form of saturation pulse NPQ that rounds the tail end of
the pulse maximum value, and reduces the average maximum saturation pulse value
(Roseqvist & van Kooten 2006). Some fluorometers allow adjustment of this parameter,
and others are preset at the factory at either. 0.8 seconds, or 1.0 seconds for higher plants.
0.8 seconds is the default value on the OS1p and it will work well with almost all higher
plants.
9. Saturation pulse intensity. Saturation pulse intensity is more of an issue with Y(II)
than with Fv/Fm. When dark adapting, shade leaves will saturate at a few hundred :mols,
and sun leaves will usually saturate below 1,500:mols. However, a problem has been
found when measuring Y(II) at high light levels. It has been discovered that at high
actinic or sun light levels, leaves resist the complete closure of all PSII reaction centers
that is expected when using a saturation pulse. Even with a 6,400 :mol saturation pulse,
some reaction centers remain open. Up to a 41% error was found in Y(II) measurements
using standard techniques at high actinic light levels. To correct for this issue, multiple
saturation flashes are used, and the measured maximum fluorescence value for each flash
is entered into a linear regression analysis formula to determine the maximum
fluorescence intensity with an infinite saturation flash. The multiple saturation pulse
approach has been shown to work in multiple papers and posters. The resulting value has
been shown to correlate well with gas exchange carbon assimilation values. This multi-
flash method is available on the OS5p and OS1p fluorometers. (see the Multi-flash
section for more details).
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