66
5. It is common to use the youngest fully mature leaf blade for diagnosis of
deficiencies in plants
(Reuter and Robinson 1997)
6. The duration of the saturation pulse should be between 0.5 seconds and
1.5 seconds
for higher plants, and 25 to 50 milliseconds for Phytoplankton and
cyanobacteria. (Schreiber 2006). Times outside these ranges increase the error in Fv/Fm
measurements. Shorter durations prevent complete saturation of PSII regardless of the
light intensity. 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. The default value is 0.8 seconds, a figure that works well for almost all higher
plants.
7. Saturation pulse intensity.
Dark adapted leaves saturate easily with lower saturation
pulse intensities. It may take a few hundred
:
mols to saturate shade leaves and sun leaves
will saturate below 1,500
:
mols. Lower values may not fully saturate PSII, and provide
an error. Higher values always work.
8. Far-red pre-illumination
. Some Fv/Fm fluorometers have the ability to pre-
illuminate dark adapted leaves with far-red light. When this feature is used for five to ten
seconds before an Fv/Fm measurement takes place, it activates PSI, and ensures that all
electrons have been drained from PSII before the measurement of Fo. While this feature
ensures that PSII is completely re-oxidized, it does not relax the xanthophyll cycle, state
transitions, or photoinhibition. Time is still required in a darkened environment to relax
all forms of NPQ and to obtain a reliable Fv/Fm measurement.
9.
Overlap of PSI fluorescence
-Part of the minimum fluorescence, the Fo parameter, in
Fv/Fm or (Fm – Fo)/Fm, contains PSI fluorescence as well as PSII fluorescence. With
Fv/Fm, one is trying to measure the maximum variable fluorescence of PSII in a dark-
adapted state. PSI fluorescence is not variable, but the low fluorescent signal from PSI
does overlap with PSII. This produces an error. In C
3
plants, about 30% of Fo
fluorescence is due to PSI, and in C
4
plants about 50% of Fo fluorescence is due to PSI
fluorescence. PSI produces about 6% of the fluorescence found in Fm in C
3
plants, and
about 12% of Fm in C
4
plants (Pfundle 1998). This not a problem when comparing
Fv/Fm measurements for plant stress because PSI fluorescence does not change. It
remains constant.
The best experiments are ones that take these issues into account
. PSI fluorescence is
involved in all measurements. It does not vary with light level or plant stress (Schreiber
2004). With this in mind, comparing samples with similar light histories allows
comparison of many types of plant stress. The Plant Stress guide provided by Opti-
Sciences references papers that deal with specific types of plant stress and limitations of
different chlorophyll fluorescence parameters for measuring plant stress.
Measurement parameter selection for different types of plant stress.
Fv/ Fm is not a
sensitive test for water stress, heat stress, nitrogen stress, nickel stress, sulfur stress, zinc
stress, some herbicides, and salt stress in some types of plants (Opti-Sciences Plant
Stress Guide 2010). It may be used effectively in most other types of plant stress. For
specific research results on specific types of plant stress, see the Plant Stress Guide. If the
