Darl adptation may require more time as in amaranthus or a variety of conditions that affect PQ pool. Transients reaction may reflect RC to antenna transfer buth all these are interesting sides but speculative. At end you may follow strictly your refs and try to vary actinic excitation. a further case in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504099/

Dear Lauri

For a detailed analysis in OJIP fluorescence rise kinetics you can use from the differential curves separately for main bands occurring during O to P transients: L, K, J, I, G and P bands. The curves for these bands can constructe by subtracting the normalize fluorescence values between O - K, K - J, J - I, and I – P phases. For example, the ChlF transients can double normalise between Fo (0.05 ms) and FK (0.3 ms), which can be expressed as WOK = (Ft −Fo)/(FK −Fo).

Please find the attached papers related to your request.

All the Best,

Ramin Lotfi

Thank you very much for the replies! I do not think the FJ-bump is due to insufficient dark-adaption, as the curve looks more or less the same in leaves that have been dark-adapted for several hours.

Ramin, I'm still quite new to this technique, so could you shortly explain what do these curves for the  O-K and K-J phases mean biologically? I browsed through the papers but couldn't find a very clear explanation of them.

What I did now was that I lowered the intensity of FR illumination slightly and changed its duration from 10s to 6s, because in WT (but interestingly not in my mutant line) the FR-preilluminated P phase was quite a lot lower than dark-adapted Fm. I read that this can be caused by the long FR illumination leading to activation of FNR. This change seems to have made the results more consistent...

Dear Lauri,

The fluorescence decrease that you observed around 2-3 ms may be a sign that the light intensity used for measurement is too high for your mutant line, as this fluorescence dip is known to increase with light intensity. You may try to use a lower light intensity, with the condition for this to still be a saturating light.

About using Vj to determine the PQ pool redox state, you have to determine also if there are no differences in the fraction of non reducing Qb centers between your samples, as these also increase the J level.

With best regards,

Sandra Stirbet

Dear Sandra,

Thanks for the reply. I think you were right about the MT intensity being high for the mutant line. I lowered it from around 8000uE to around 3000, and now fluorescence plateaus around 2-3 ms instead of dipping (see attached images of typical traces I get with these settings).

What method would you suggest to determine the fraction of non-reducing Qb centers?

Kind regards,

Lauri

Dear Lauri,

Please write to me at [email protected] to discuss these issues.

Sincerely, Sandra

Dear Lauri,

The dip you observed is due to a transient peak of P680+ in the S3 state. Reducing the light intensity broadens and lowers this peak making the dip disappear.

I do not agree with Sandra though that those in active centers affect the determination of the PQ-redox state in darkness. The idea of Toth et al. was to place the measured J-step between a minimum and a maximum value, showing that the PQ-redox state changes more or less linear with the J-step between these values. Those inactive centers are then in the off set (the fluorescence rise below J) which does not affect the calculation.

In 2005 (Schansker and Strasser Photosynth Res 2005) I published a paper in which I proposed a method to determine amount of those inactive centers. Lavergne and Leci in the nineties had listed all the problems with the quantification and I showed that by using far-red light to excite PSII you could reduce this to two factors and solve the problem. However since you use a modulated system with measuring light that is more actinic than far-red light we may assume that already at your F0 all inactive centers are closed.

You wrote that the literature suggests 2 ms for the J-step. I think we used consistently 3 ms and in Khalaji et al. (PRES in press) an argument is made why from a kinetic point of view the 3 ms point is better than the 2 ms point, but of course with a P680+ dip that gets complicated.

Best regards,

Gert Schansker

Dear Gert,

I appreciate your taking the time to reply, I will look into your paper. Now that I got rid of the p680+ dip by lowering the MT intensity, the difference between the lines in (FJ-FJ ox)/(Fm-FJ ox) stays the same whether I use the J value at 2 or 3 ms, but the absolute values will be lower at 3 ms. So would you say that using values at 2 ms overestimates the amount of dark-reduced PQ in general?

I used a 440 nm ML and MT to selectively excite the top layer of leaves, as suggested by Schreiber et al in their PAM application notes paper for the Multicolor PAM. Do you think that is a good idea for this purpose?

Sincerily,

Lauri

Dear Lauri,

If you would make a light intensity dependence of the J-step you would discover that the start of the step around 2 ms shifts, whereas the 3 ms is always on the step, it will only disappear if you reduce the light intensity too much. In your case the two pulses (with and without FR) are not kinetically equivalent. This means that at 2 ms there is an additional (kinetic) difference. The 3 ms points are not sensitive to this kinetic difference. The consequence, I am sorry, are smaller differences if you use the 3 ms point.

In general the top cell layers dominate the measured signal, simply because the light intensity decreases rapidly as the light penetrates into the leaf. It is true that this effect is less if you use green light, but since it is less effective you would need a much higher light intensity to induce similar fluorescence kinetics. In other words, I would also have a preference for either blue or red light.

Best regards,

Gert

Thank you, that clarifies the issue a lot. To minimize the effect of other differences between plant lines, it is then more reliable to use the 3 ms time point.

All the best,

Lauri

I agree with the answer of Ramin Lotfi.

Regards

SM Najim

Dear Dr Najim,

In the proceedings of the Brisbane Congress (Strasser et al. 2001) we showed that if you double normalize between O and J and you do this for a double pulse experiment with variable time between the pulses to get different redox states for the PQ-pool and then make these difference curves, it is possible as well to follow the re-oxidation of the PQ-pool. However, to determine an absolute value you need to oxidize the PQ-pool by a far-red pulse to obtain a minimum value. A far-red pulse increases the the F0 value by about 5% and this would affect the double normalization introducing an artefact. The method proposed in Toth et al. (2007) is based on the same principle, but more straightforward and does not suffer from the F0 artefact. 

Best regards,

Gert Schansker