Photon Systems Instruments AquaPen-C AP 110-C, Instruction Manual

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5.1.4 N
ON
-
PHOTOCHEMICAL QUENCHING
(NPQ)
PROTOCOLS
The NPQ protocol is used to quantify photochemical and non-photochemical quenching. It should be performed with dark-adapted
samples. The NPQ protocol starts with a measurement of minimal level of fluorescence
F
0
during a dark period. A short saturating flash
of light is then applied to reduce the plastoquinone pool and measure maximum fluorescence in the dark-adapted state, F
m
. After a short
dark relaxation, the sample is exposed to actinic irradiance for tens to hundreds of seconds to elicit a transient called the Kautsky effect.
A sequence of saturating flashes is then applied during the exposure to the actinic light to probe the non-photochemical quenching
NPQ
and effective quantum yield of photosynthesis QY in light adapted state. After exposure to continuous illumination, the relaxation of
non-photochemical quenching is determined by means of saturating pulses applied in dark. This sequence of the protocol is illustrated
in Fig. 8.
The AquaPen comes with three predefined NPQ protocols, NPQ1, NPQ2 and NPQ3. The protocols differ in the duration of the light
exposure and the dark recovery phase, in the number and interval between pulses. See Table 1 NPQ Protocols.Table 1:
Phase Duration # of pulses 1st pulse Pulse interval
NPQ1 Light 60 s 5 7 s 12 s
Dark recovery 88 s 3 11 s 26 s
NPQ2 Light 200 s 10 10 s 20 s
Dark recovery 390 s 7 20 s 60 s
NPQ3 Light 200 s 10 11 s 21 s
Dark recovery 60 s 2 20 s 21 s
Table 1 NPQ Protocols.
The NPQ protocols include the following measured and calculated parameters:
Abbreviation Explanation
F
0
minimum fluorescence in dark-adapted state
F
m
maximum fluorescence in dark-adapted state, measured during the first saturation flash after dark
adaptation
F
p
fluorescence in the peak of fast Kautsky induction
F
m
_Ln, Lss, D, Dn 1 maximum fluorescence
QYmax 2 maximum quantum yield of PSII in dark-adapted state - F
v
/F
m
QY_Ln, Lss, D, Dn
1,3
effective quantum yield of PSII
NPQ_Ln, Lss, D, Dn 1,4 non-photochemical chlorophyll fluorescence quenching
Qp_Ln, Lss, D, Dn
1,5
coefficient of photochemical quenching, an estimate of open PSII reaction centers
1 L - indicates light adapted parameters; D - refers to dark recovery phase after switching of the actinic illumination; n - represents a
sequential number of light phases; ss - steady state
2 Calculated as (F
m
– F
0
) / F
m
3 Calculated as (F
m
_Ln – F
t
_Ln) / F
m
_Ln or of corresponding steady state or dark recovery parameters
4 Calculated as (F
m
– F
m
_Ln) / F
m
_Ln or of corresponding ss, Dn or Dss parameters
5 Calculated as (F
m
_Ln – F
t
_Ln) / (F
m
_Ln – F
0
_Ln) or of corresponding ss, Dn or Dss parameters