As noted in the previous paper (Kalaji et al. 2014a), at wavelengths longer than 700 nm PSI fluorescence emission contributes considerably to F
O
. For commercial fluorimeters this contribution may be as high as 30–35% for C3 plants and 50–60% for C4 plants (Genty et al. 1990b; Adams et al. 1990b; Pfündel 1998; Peterson et al. 2001). The stronger contribution of PSI fluorescence (F
PSI) in C4 plants is due to a higher PSI/PSII ratio (Edwards and Walker 1983; Ku et al. 1991) and to higher levels of spillover of excitation energy from PSII to PSI (Pfündel and Pfeffer 1997). The question whether PSI emits variable fluorescence at room temperature has been studied as well. It is often assumed that the fluorescence yield of open and closed RCs of PSI is the same (Butler 1978; Kyle et al. 1983; Savikhin 2006). Byrdin et al. (2000) reported a 12% increase of the fluorescence yield of PSI of Synechococcus elongatus on closing. If F
PSI is 30% of the F
O
fluorescence emission, then 12% more would be equal to 4% of F
O
, and, since F
M
is 5–6 times F
O
, this would represent 1% or less of the total variable fluorescence. In other words, even if there is some PSI variable fluorescence, this amount is so small that it can be ignored. This is further supported by several kinetic experiments. In leaves or intact chloroplasts, in the presence or absence of DCMU, the F
M
is the same (Schreiber and Krieger 1996; Tóth et al. 2005b) despite the fact that in the absence of DCMU P700 is reduced at F
M
and in its presence is oxidized (Schansker et al. 2005). In a variation on this experiment Peterson et al. (2014) showed that during fluorescence induction (F
O
to F
M
) the relationship between F(680) (more PSII fluorescence) and F(750) (more PSI fluorescence) did not show an oscillation related to the P700 oxidation and reduction kinetics occurring during OJIP fluorescence rise. Peterson et al. (2014) concluded that variable PSI fluorescence was less than 0.8% of F
V
. In contrast, theoretical simulations performed by Lazár (2013), based on known values of rate constants of PSI reactions and considering the reported PSII/PSI stoichiometry, yielded an OJIP simulation with approximately correct kinetics. On the basis of these results Lazár concluded that the contribution of PSI variable Chl a fluorescence to total F
V
could be 8–17%. However, a close link between PSI kinetics and the OJIP rise can also be explained on the basis of the PSII conformational change hypothesis (Schansker et al. 2014).
Several authors have studied methods to correct parameters like F
V
/F
M
for the contribution of PSI fluorescence, but, so far, this has not led to a simple formula that can be applied. It is important to note that the PSI contribution is instrument sensitive. Pfündel (1998) wrote that a special PAM instrument that detects the fluorescence emission at wavelengths shorter than 710 nm shows very little, or at least much less, contribution of PSI fluorescence.
Pfündel (1998) showed for a set of C3, C3–C4 and C4 plants that there is a linear relationship between the parameter F
M
/F
V
determined at room temperature and the parameter F735/F685 determined at 77 K, with a slope m and an intercept of the Y axis b. In the model of Pfündel (1998):
$$ F_{M} /F_{V} = \, b \, + \, m \, \times {\text{ F}}735/{\text{F}}685 $$
For the data set of Pfündel (1998) this gave a regression coefficient of 0.963. On the basis of this approach, a corrected F
V
/F
M
value of about 0.88 was obtained. To use this approach, it would be necessary to construct a calibration curve, like Pfündel (1998) did, for each instrument used and then to determine for the samples of interest both the F
O
and F
M
at room temperature measured on leaves and the 77 K fluorescence emission spectrum of diluted leaf powder, which in most cases is impractical.
Franck et al. (2002) approached the topic in a different way, developing a method for the resolution of the PSII and PSI contributions to the fluorescence emission spectrum. The authors noted that, for diluted PSII particles, the F
M
/F
O
was wavelength independent. On that basis, they concluded that the wavelength dependence of F
M
/F
O
observed for leaves was due to the presence of PSI. Furthermore, they assumed that the PSI and PSII spectra do not change and, therefore, that these spectra can be scaled to obtain the F
O
and F
M
spectra. After correction by this method, the authors obtained a F
V
/F
M
value of 0.83 instead of 0.81. This difference is considerably smaller than the correction found by Pfündel (1998).
The quantum yield of PSII can also be determined on the basis of time-resolved (ps) fluorescence measurements. Wientjes et al. (2013a) acclimated Arabidopsis plants to 20, 100 and 800 µmol photons m−2 s−1. Under such conditions the PSII antenna size decreased as the light intensity increased. The quantum yields derived from the time-resolved fluorescence measurements were 0.84, 0.89 and 0.91, respectively. The F
V
/F
M
values (corrected for the PSI contribution) determined for the same plants were 0.83, 0.87 and 0.86, respectively. Since the first set of data is measured on thylakoid membranes and the second set of data on leaves, there are several possible explanations for the observed discrepancies.
The data of Wientjes et al. (2013a) support the choice of a F
V
/F
M
value of 0.87 or 0.88 as a good approximation of the real F
V
/F
M
value, at least for C3 plants. Taking 0.88 as the real value of the parameter F
V
/F
M
of PSII (=ΦP0) of C3 and C4 plants, it can be used to estimate the contribution of PSI fluorescence:
$$ F_{\text{PSI}} = \left[ {\left( {\phi_{P0} \cdot \frac{{F_{m} }}{{F_{m} - F_{o} }} } \right) - 1} \right] \cdot F_{m} $$
(1)
when we take a typical F
V
/F
M
value for C3 plants (e.g., 0.836), we get F
PSI = ~5.2% of F
M
. When we take a typical value for C4 plants (e.g., 0.80), we get F
PSI = ~10% of F
M
. This calculation can, however, only be applied to F
O
and F
M
measurements on plants that are completely relaxed with respect to photoprotective dissipation mechanisms (no NPQ) and non-stressed (no photoinhibition). The data of Wientjes et al. (2013a) also suggest that 0.88 is too high for plants acclimated to shade conditions. Another approach will also have to be developed for the correction of the F
V
/F
M
values in the photosynthetic organisms in which the thylakoid stacking is hindered by the presence of phycobilisomes (cyanobacteria, red algae), or the thylakoids are appressed for their entire length (brown algae, diatoms, etc.), or display a not yet well-differentiated grana-intergrana arrangement (most green algae) (see Trissl and Wilhelm 1993; Solymosi 2012). Further, Peterson et al. (2014) described an additional contribution to F
O
in greening maize (up to 12–15% of F
M
at 680 nm) and sunflower (up to 8% of F
M
at 680 nm) leaves which was absent in mature leaves and correcting for which improved the analysis of the fluorescence data. The authors ascribed this fluorescence to emission by partially assembled PSII and could be the same fluorescence emission Srivastava et al. (1999) ascribed to free LHCII. Once F
PSI has been determined, it can be subtracted from all F
t
values and the resulting fluorescence data can be used for the calculation of all fluorescence parameters.
A correction of fluorescence measurements for the PSI contribution is especially relevant when fluorescence measurements are correlated with data obtained by other methods (e.g., gas exchange or absorbance measurements).
Strong red LEDs with a peak emission at ~650 nm were the first LEDs that became commercially available for a reasonable price. Instruments that use such LEDs need to measure fluorescence above 700 nm to avoid overlap with the emission of the red LEDs. This is the case for, e.g., classical PAM instruments and HandyPEAs. Using, e.g., blue LEDs it is possible to avoid the overlap problem and to measure fluorescence emission at ~685 nm, where the contribution of PSI fluorescence is very small (Krause and Weis 1991; Gitelson et al. 1998). However, Peterson et al. (2001, 2014) argued that in the end the fluorescence detected above 700 nm may be the better probe, because light around 680 nm is much more strongly absorbed by the leaf and, therefore, more a probe for the top cell layers of the leaf. Further, it should be noted that differences in filters and other specifications between instruments may affect the contribution of PSI to fluorescence measurements and can explain, at least to some extent, differences in the values of parameters like F
V
/F
M
between different fluorometers.
Part of the JIP test parameters (e.g., M
o
, Area, Sm, V
J
, V
I
, ψE
o
) only depend on variable fluorescence and are not affected by the contribution of PSI fluorescence. For measurements derived from OJIP measurements it may be noted that, as long as the PSII to PSI ratio does not change, PSI fluorescence causes a systematic error. This means that it does not affect the comparability of measurements of comparable samples. With respect to the quenching analysis, the effect of PSI fluorescence emission on the calculated parameters increases for measurements made at stronger actinic light intensities. Higher light intensities quench F
M
, and to a lesser extent F
O
, but are not expected to affect F
PSI, increasing the relative contribution of F
PSI. Pfündel et al. (2013) studied the effects of F
PSI under steady-state conditions. They noted that the method of Oxborough and Baker (1997) to calculate F
O
′ systematically produces values that are too low and they ascribed this to the fact that Oxborough and Baker (1997) did not take the contribution of F
PSI into account. Pfündel et al. (2013) also showed that correcting fluorescence data of maize for F
PSI makes the relationship between ΦPSI and ΦPSII more linear.
In summary, PSI fluorescence emission has only a significant effect on F
O
. Even a rough correction of fluorescence data for PSI fluorescence emission, assuming that the real F
V
/F
M
value is 0.88, will considerably improve the quality of the fluorescence data.