OJIP transients and P deficiency - can it be measured with the MultiSpeQ?
General Discussion
Anton Wasson
Aug 2018
Hello All, I am a newcomer to MultiSpeQ, PhotosynQ, and photosynthesis research in general. I work on root development and P deficiency. I recently read two papers which suggest that leaf P content and deficiency can be predicted from a multivariate analysis of the I step of an OJIP transient:
Carstensen, A., Herdean, A., Schmidt, S. B., Sharma, A., Spetea, C., Pribil, M., & Husted, S. (2018). The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain. Plant Physiology, 177(1), 271–284. https://doi.org/10.1104/pp.17.01624
Frydenvang, J., van Maarschalkerweerd, M., Carstensen, A., Mundus, S., Schmidt, S. B., Pedas, P. R., … Husted, S. (2015). Sensitive Detection of Phosphorus Deficiency in Plants Using Chlorophyll a Fluorescence. Plant Physiology, 169(1), 353–361. https://doi.org/10.1104/pp.15.00823
The measurement of the OJIP transients is described in Frydenvang et al as:
Chlorophyll a fluorescence transients (OJIP transients) were measured using a Handy PEA chlorophyll fluorometer (Hansatech Instruments). The midsection of the YFDL was dark adapted for at least 20 min before measuring, and a short, nonactinic light flash was used to adjust the gain of the detector. The actual measurement was conducted by illuminating the leaf with continuous actinic light at a saturating intensity (>3,000 μmol m–2 s–1) for 10 s from three red LEDs, optically filtered to a maximum wavelength of 690 nm. The fluorescence transients were recorded using a PIN photodiode and a filter to ensure only wavelengths greater than 700 nm were recorded.
The data was preprocessed:
As the observed effect of P deficiency affected the second and higher order components, all OJIP transients were normalized by F0 and subtracted by 1 to have origin at 0. The resulting fluorescence transients were then first-order corrected to minimize lower order variations by a method similar to multiplicative scatter correction and standard normal variate preprocessing (Geladi et al., 1985; Helland et al., 1995). This adjusted the overall offset and slope of all transients, yet did not affect the second and higher order components of the curves. All OJIP transients were subsequently differentiated, scaled, and smoothed using a Savitzky-Golay filter (Savitzky and Golay, 1964)
I can find very little about OJIP transients on the PhotosynQ website, and I am wondering whether it is possible to measure them with the MultiSpeQ, and if so, how it would be done? Any suggestions would be welcomed by this below-ground researcher who is blinking in the light!
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Rodrigo Gomez
Aug 2018
Hi Anton, I’m just a PhotosynQ user but I’ve been using the platform for a while so I’ll try to help you. As far as I know multispeQ is not intended to be use for OJIP measurements, its function relays on PAM (pulse amplitud mudulation) fluorometry using short light pulses to get a fluorescence signal used to calculate different parameters. These measurement pulses are given at a fix frequency (that you can change in the protocol editor) but the shorter time between to pulses is 1 millisecond (1000 microseconds). The OJIP transients need much more resolution, it means that the time between measuring pulses should be in the microseconds scale (some devices used to get OJIP use 5 to 10 us per point). OJIP points normally used are: O, florescence at the beginning of the transient, the J point about 2 to 3 ms, and the I point about 30 to 50 ms, and P, the maximum. So, if I’m not wrong you can’t use the MultispeQ device to get a complete OJIP transient, because of the resolution that OJIP needs. I tried to make OJIP with MultispeQ but they aren’t very reliable, mostly the J point at 2 ms. But! The O and P points (time 0 and maxima fluorescence) can be used. Maybe, if you just want to use the I point (30 ms) it would work. I’ve to check my old “OJIP” measurements using multispeQ to see the transient at the I point. In the case you want to compare the IP phase of the transient (the so call V(I), which is a normalization of the fluorescence at I point using the O and P points) it would work. If you need a protocol for this please let me know and I can send you the one I used. Unfortunately, this is the only help I can give you. Hopefully someone else in the forum could share more information about this issue. Good luck! Rodrigo
Rodrigo Gomez
Aug 2018
Yeah Anton, no problem, let me check the protocol and macro first. I'll send them as far as I can.
David M. Kramer
Aug 2018
Hi Anton and Rodrigo!
To be honest, until recently I have not been very positive about the OJIP approaches because the results were almost impossible to interpret in straightforward ways. However, I do note that sone recent work suggests that there are interesting relationships between P status and certain OJIP phases. I still think that the ECS signal would be a more direct probe of these, but it's interesting and I'd like to add this capacity.
OK, in principle the MultispeQ can make these measurements and it has (keep reading).
The current version only detects pulsed signals because the detector signal is EXTREMELY well filtered. This is why its can get amazingly high signal-to-noise ratios, but it limits the repetition rate for pulses.
To get an OJIP with this setup, one could use the measuring pulses as an actinic source. I've tried this, and gotten reasonable data with a rep rate of about 10 KHz (one pulse per 100 us). At this rate, the measuring pulse can be quite actinic and in principle it be used to produce an OJIP curve.
What you are looking for, Anton, is a higher rep rate. In this regard, I've built modified versions of the MultispeQ that can measure in DC-coupled mode (i.e. no AC filtering, just light OJIP instruments. These can also do DIRE or FRR or any of the variants. The microprocessor can get about 250KHz rep rate (one point per 4 us) and this is plenty to get any type of OJIP, FRR, FIRE etc.
So, should we build a version that can measure OJIP? Or better yet, an entire series of measurements, including OJIP, PAM and ECS? I think it would be very interesting to deploy such an instrument and really try to answer the question what these phases mean.
Thoughts?
Dave
Gonzalo Estavillo
Aug 2018
Dave, Thanks for your feedback about this. I am trying to help here (and learn in the process as I have not worked with OJIP curves before). The “RIDES” protocols we designed can measure ECS (electrochromic shift), associated with changes in thylakoid electric field. This could also be dependent on Pi pools and ATPase activity, I guess. WE could test the effect of “P” on this parameter alone. Alternatively, what protocol did you use to “use the measuring pulses as an actinic source at high frequency”? We could give it a go. Building a new “version that can measure OJIP, PAM and ECS” as you propose would be great, but if we could access to your “modified” MultispeQ that “that can measure in DC-coupled mode (for DIRE or FRR (fast repetition rate), FIRE (fluorescence induction and relaxation), and OJIP etc. we could run some measurements for Anton’s experiment as well. Happy to add this info to the forum if you prefer. Gonzalo
Rodrigo Gomez
Aug 2018
David M. Kramer: Hi Anton and Rodrigo! To be honest, until recently I have not been very positive about the OJIP approaches because the results were almost impossible to interpret in straightforward ways. However, I do note that sone recent work suggests that there are interesting relationships between P status and certain OJIP phases. I still think that the ECS signal would be a more dire...
Hi Dave, Thanks for your feedback. You are right about the OJIP complexity, but I think that is worth to get a chance to it. There are some pretty interesting methods based on OJIP that would be very useful. I'll try using the multispeQ as you suggest to get some curves. (Anton, as soon as I get a reasonable protocol and macro I'll send them to you.) About the multi-measurements multispeQ I totally agree with Gonzalo, I'd be great! Rodrigo